Microorganisms and methods for the production of glycolic acid and glycine through a reverse glyoxylate shunt

文档序号：1894717 发布日期：2021-11-26 浏览：16次中文

阅读说明：本技术 通过逆向乙醛酸支路生产乙醇酸和甘氨酸的微生物和方法 (Microorganisms and methods for the production of glycolic acid and glycine through a reverse glyoxylate shunt ) 是由 D·J·科克 F·加尔泽莱尼 P·M·拉杜安亚历山德里诺 N·莫兰 J·M·弗朗索瓦于 2020-02-14 设计创作，主要内容包括：本发明提供用于通过逆向乙醛酸支路的生化途径、生产乙醛酸的重组微生物、以及生产和产率改善乙醇酸和/或甘氨酸的方法。所述逆向乙醛酸支路包含：催化磷酸烯醇丙酮酸(PEP)羧化为草酰乙酸(OAA)的酶、或催化丙酮酸羧化为草酰乙酸(OAA)的酶、或催化丙酮酸羧化为苹果酸的酶、或任一前述反应的组合；催化苹果酸转化为苹果酰辅酶A的酶；催化苹果酰辅酶A转化为乙醛酸和乙酰辅酶A的酶；以及任选的催化草酰乙酸(OAA)转化为苹果酸的酶。乙醛酸还原以生产乙醇酸。可选择的,将乙醛酸转化为甘氨酸。本发明的逆向乙醛酸支路途径可以与其它乙醇酸和/或甘氨酸生产途径协同利用以增加生产产率。(The present invention provides biochemical pathways for the production of glyoxylate through a reverse glyoxylate shunt, recombinant microorganisms that produce glyoxylate, and methods for producing and yield-improving glycolate and/or glycine. The retro-glyoxylic acid branch comprises: an enzyme that catalyzes the carboxylation of phosphoenolpyruvate (PEP) to Oxaloacetate (OAA), or an enzyme that catalyzes the carboxylation of pyruvate to malate, or a combination of any of the foregoing reactions; an enzyme that catalyzes the conversion of malic acid to maloyl-coa; an enzyme that catalyzes the conversion of malyl-coa to glyoxylic acid and acetyl-coa; and optionally an enzyme that catalyzes the conversion of Oxaloacetate (OAA) to malate. Glyoxylic acid is reduced to produce glycolic acid. Alternatively, glyoxylic acid is converted to glycine. The reverse glyoxylate shunt pathway of the invention can be utilized synergistically with other glycolate and/or glycine production pathways to increase production yield.)

1. A recombinant glyoxylate producing microorganism for the synthesis of Glycolic Acid (GA) and/or glycine comprising:

(a) a gene encoding malate dehydrogenase catalyzing the conversion of pyruvate to malate;

(b) a gene encoding malate thiokinase catalyzing the conversion of malate into malyl-coa; and

2. A recombinant glyoxylate producing microorganism for the synthesis of Glycolic Acid (GA) and/or glycine comprising:

(a) a gene encoding pyruvate carboxylase which catalyzes the conversion of pyruvate to Oxaloacetate (OAA), and/or a gene encoding phosphoenolpyruvate carboxylase which catalyzes the conversion of phosphoenolpyruvate to OAA, and/or a gene encoding phosphoenolpyruvate carboxykinase which catalyzes the conversion of phosphoenolpyruvate to OAA;

(b) a gene encoding malate dehydrogenase catalyzing the conversion of OAA to malate;

(d) a gene encoding a malyl-coa lyase that catalyzes the conversion of malyl-coa to glyoxylate and acetyl-coa, wherein acetyl-coa produced by the malyl-coa lyase is combined with OAA to increase the biosynthesis of GA and/or glycine.

3. A recombinant glyoxylate producing microorganism for the synthesis of Glycolic Acid (GA) and/or glycine comprising:

(b) a gene encoding malate thiokinase catalyzing the conversion of malate into malyl-coa; and

(c) a gene encoding a malyl-coa lyase that catalyzes the conversion of malyl-coa to glyoxylate and acetyl-coa, wherein the recombinant microorganism does not catalyze the conversion of oxaloacetate to malate.

4. The recombinant microorganism of any one of claims 1-3, wherein said recombinant microorganism does not produce isopropanol, ethanol, acetone, citric acid, itaconic acid, acetic acid, butyric acid, (poly) 3-hydroxybutyric acid, 3-hydroxyisobutyric acid, 3-aminoisobutyric acid, 2-hydroxyisobutyric acid, methacrylic acid, (poly) glutamic acid, arginine, ornithine, citrulline, leucine, isoleucine, or proline from acetyl-CoA produced by malyl-CoA lyase.

5. The recombinant microorganism of any one of claims 1 and 3-4, wherein acetyl-CoA produced by malyl-CoA lyase is combined with OAA to increase GA and/or glycine biosynthesis.

6. The recombinant microorganism of any one of claims 1-5, wherein said microorganism comprises a mutation in a gene encoding malate dehydrogenase, wherein said mutation results in partial or complete inhibition of the activity of malate dehydrogenase catalyzing the conversion of oxaloacetate to malate, malate to pyruvate, and/or malate to oxaloacetate.

7. The recombinant microorganism of any one of claims 1-6, wherein the microorganism comprises a gene encoding an NADH-dependent glyoxylate reductase that catalyzes the conversion of glyoxylate to glycolate or a gene encoding an NADPH-dependent glyoxylate reductase that catalyzes the conversion of glyoxylate to glycolate.

8. The recombinant microorganism of any one of claims 1-7, wherein the microorganism comprises: a gene encoding alanine-glyoxylate aminotransferase, a gene encoding glycine dehydrogenase, a gene encoding glycine aminotransferase, a gene encoding serine-glyoxylate aminotransferase, and/or a gene encoding glycine oxidase which catalyzes the conversion of glyoxylate to glycine.

9. The recombinant microorganism of any one of claims 1-8, wherein said malate dehydrogenase catalyzing the carboxylation of pyruvate to malate is from enzyme class (E.C.)1.1.1.38, E.C.1.1.1.39, or E.C.1.1.1.40.

10. The recombinant microorganism of any one of claims 2, 4, 5, 7, 8, and 9, wherein said malate dehydrogenase catalyzing the conversion of oxaloacetate to malate is from enzyme classification (E.C.) 1.1.1.37.

11. The recombinant microorganism of any one of claims 1-10, wherein said gene encoding malate dehydrogenase catalyzing the carboxylation of pyruvate to malate is selected from the group consisting of: maeA, maeB, dme, mez, mae1, and name-me 1 and name-me 2, or homologues thereof.

12. The recombinant microorganism of claim 10, wherein the gene maeA is from escherichia coli, pseudomonas, or bacillus; the gene maeB is from escherichia coli or salmonella; the gene dme is from rhizobia; the gene mez is from a mycobacterium; the gene mae1 is from saccharomyces cerevisiae (s.cerevisiae); and the gene, either nad-me1 or nad-me2, is from Arabidopsis thaliana (Arabidopsis thaliana).

13. The recombinant microorganism of claim 12, wherein said gene maeA is from bacillus subtilis (b.subtillis); the gene dme is from rhizobium meliloti (r.melilot); or the gene mez is from Mycobacterium tuberculosis (Mycobacterium tuberculosis).

14. The recombinant microorganism of any one of claims 2 and 4 to 13, wherein said gene encoding malate dehydrogenase catalyzing the conversion of oxaloacetate to malate is selected from the group consisting of: the gene mdh from Escherichia coli, Corynebacterium, Streptomyces, Yeast and Arabidopsis or homologues thereof.

15. The recombinant microorganism of claim 14, wherein said gene mdh is from streptomyces coelicolor (s.coelicolor) or said gene mdh1/2/3 is from saccharomyces cerevisiae.

16. The recombinant microorganism of any one of claims 1-15, wherein the gene encoding malate thiokinase is a sucCD and/or sucCD-2 and/or mtkAB from Methylobacterium (Methylobacterium sp.), Methylobacterium extorquens (Methylobacterium extorquens), escherichia coli, Thermus thermophilus (Thermus thermophilus), rhizogenes (Hyphomicrobium sp.), methanococcus jannaschii (methanococcus jannaschii), methanobacter thermoautotrophicum (methanotrophus thermothermophilus), Rhizobium (Rhizobium), Methylococcus capsulatus (Methylococcus capsulatus), or Pseudomonas pseudomonads (pseudomonads); or a homologue thereof.

17. The recombinant microorganism of any one of claims 1 to 16, wherein the gene encoding malyl-coa lyase is from methylobacterium extorquens, Rhodobacter sphaeroides (Rhodobacter sphaeroides), Streptomyces (Streptomyces), Rhodobacter thermophilus (Chloroflexus aurantiacaus), Nitrosomonas eurotia (nitrosomona europaea), Methylococcus capsulatus (methylcoccus capsulatus), Nereida ignova, pythium aphanidermatum (Hyphomicrobium methylvorum), Rhodobacter caldarius (thalmicus aculeus), Rhodobacter sphaeroides (r. sphaeroides), Rhodobacter sphaeroides (mycobaterium labrum), Rhodobacter sphaeroides (mycobacter sphaeroides littoralis), Rhodobacter denitrificus (hyphominis), Rhodobacter sphaeroides (mycoides) or Rhodococcus Rhodobacter sphaeroides (Rhodobacter sphaeroides) and/or Rhodobacter sphaeroides 1, or Rhodobacter sphaeroides, and/or Rhodobacter sphaeroides, or a.

18. The recombinant microorganism of any one of claims 2-17, wherein the gene encoding pyruvate carboxylase is PYC from Rhizobium phaseolus (Rhizobium etli), PYC1 or PYC2 from yeast, or PYC from bacillus subtilis; or a homologue thereof.

19. The recombinant microorganism of any one of claims 2 to 18, wherein the gene encoding phosphoenolpyruvate carboxylase is ppc from escherichia coli, ppc or pepC from Rhodothermus marinus (r. marinus), ppcA from methanotrophic bacillus thermoautotrophicus (m. thermotrophicus), pep1 from maize (z. mays), ppc1/2/3 from arabidopsis thaliana (a. thaliana), ppc from soybean (g.max), or is from Rhodothermus rubrum (Rhodothermus), corynebacterium, salmonella, filamentous microsclera, streptococcus, streptomyces, pantoea, bacillus, clostridium, pseudomonas pseudomonads, rhodopseudomonas, tobacco (Nicotiana tabacum), amaranthus sativa (amaranth pochondris), Triticum aestivum, or alfalfa (alfalfa); or a homologue thereof.

20. The recombinant microorganism of any one of claims 2 to 19, wherein the gene encoding phosphoenolpyruvate carboxykinase is pck or pckA from escherichia coli, pckA from Selenomonas ruminata (selenia ruminata), pckA from Salmonella typhimurium (Salmonella typhimurium), pckA from Klebsiella sp, pckA from Thermus sp, pckA from Ruminococcus albus (Ruminococcus albus) and Ruminococcus xanthina (Ruminococcus flavefaciens), pckA from Actinobacillus succinogenes (Actinobacillus succinogenes), pck or pckA from Streptococcus bovis (Streptococcus bovis), or rumen from bacillus, clostridium thermocellus (clostridium thermocellum), mycobacterium tuberculosis; or a homologue thereof.

21. The recombinant microorganism of any one of claims 1-20, wherein said microorganism comprises:

(a) a gene encoding citrate synthase that converts OAA and acetyl-coa produced by malyl-coa lyase to citrate;

(b) a gene encoding citrate hydrolase which converts citric acid into aconitic acid;

(d) a gene encoding an isocitrate lyase that converts isocitrate to succinate and glyoxylate;

(e) a gene encoding succinate dehydrogenase converting succinate into fumarate; and

(f) a gene encoding a fumarase enzyme which converts fumaric acid into malic acid.

22. The recombinant microorganism of any one of claims 1-21, wherein said microorganism comprises a loss of function mutation in a gene encoding malate synthase, or a deletion of said gene.

23. The recombinant microorganism of any one of claims 8-22, wherein the gene encoding glyoxylate reductase activity is selected from the group consisting of: ycdW and/or yiaE from E.coli, GOR1 from Saccharomyces cerevisiae, gyaR from Thermococcus litoralis and/or GLYR1 from Arabidopsis thaliana.

24. The recombinant microorganism of any one of claims 2-23, wherein said pyruvate carboxylase to convert pyruvate to OAA is from enzyme classification system number e.c. 6.4.1.1; phosphoenolpyruvate carboxylase to convert phosphoenolpyruvate to OAA is from e.c. 4.1.1.31; phosphoenolpyruvate carboxykinases that convert phosphoenolpyruvate to OAA are from e.c.4.1.1.32 and e.c. 4.1.1.49.

25. The recombinant microorganism of any one of claims 1-24, wherein said malate thiokinase that converts malate to malyl-coa is from enzyme classification system number e.c.6.2.1.4, e.c.6.2.1.5, e.c.6.2.1.9, or e.c. 6.2.1-; and/or the malyl-coa lyase for the conversion of malyl-coa to glyoxylate and acetyl-coa is from e.c.4.3.1.24 or e.c. 4.3.1.25.

26. The recombinant microorganism of any one of claims 1-25, wherein one or more genes are expressed heterologously.

27. The recombinant microorganism of any one of claims 1-26, wherein said microorganism comprises a deletion or modification that reduces the activity of one or more endogenous genes selected from the group consisting of:

(a) a gene encoding isocitrate dehydrogenase;

(b) a gene encoding pyruvate dehydrogenase, pyruvate oxidase, and/or pyruvate formate lyase;

(d) a gene encoding glycolate oxidase.

28. The recombinant microorganism of claim 22, wherein said gene encoding malate synthase is aceB and/or glcB from E.coli, or DAL7 and/or MLS1 from yeast.

29. The recombinant microorganism of claim 27, wherein the gene encoding isocitrate dehydrogenase is icd from escherichia coli, or IDP2 and/or IDH1/2 from yeast.

30. The recombinant microorganism of any one of claims 1-29, wherein said microorganism comprises a deletion or modification that reduces the activity of one or more endogenous genes selected from the group consisting of:

(a) a gene encoding glyoxylate aldehyde ligase;

(b) a gene encoding 2-oxo-4-hydroxyglutarate aldolase;

(d) a gene encoding a repressor of isocitrate lyase.

31. The recombinant microorganism of claim 22, wherein the gene encoding glyoxylate aldehyde ligase is gcl; the gene encoding 2-oxo-4-hydroxyglutarate aldolase is edA; the gene encoding glycolaldehyde reductase is fucO and/or gldA; and the gene encoding the repressor of isocitrate lyase is iclR.

32. The recombinant microorganism of any one of claims 1-31, wherein the expression levels of: a gene encoding alanine-glyoxylate aminotransferase, a gene encoding glycine dehydrogenase, a gene encoding glycine aminotransferase, a gene encoding serine-glyoxylate aminotransferase, and/or a gene encoding glycine oxidase.

33. The recombinant microorganism of any one of claims 1-32, wherein the expression level of the gene encoding alanine aminotransferase, and/or the gene encoding NADPH-dependent glutamate synthase is increased.

34. The recombinant microorganism of any one of claims 1-33, wherein said microorganism utilizes NADH and CO produced by other glycolate and/or glycine production pathways in a reaction catalyzed by malate dehydrogenase, pyruvate carboxylase, phosphoenolpyruvate carboxykinase, malate thiokinase, and malyl-coa lyase₂。

35. The recombinant microorganism of any one of claims 1-33, wherein said microorganism utilizes exogenously added CO in a reaction catalyzed by malate dehydrogenase, pyruvate carboxylase, phosphoenolpyruvate carboxykinase, malate thiokinase, and malyl-coa lyase₂Carbonic acid, and/or a reducing agent.

36. The recombinant microorganism of claim 35, wherein said reducing agent is hydrogen, electrons, and/or nad (p) H.

37. The recombinant microorganism of claim 35, wherein said reducing agent is from an external source.

38. The recombinant microorganism of any one of claims 1-34, wherein said microorganism utilizes NADH and CO produced based on the serine/hydroxypyruvate pathway in a reaction catalyzed by malate dehydrogenase, pyruvate carboxylase, phosphoenolpyruvate carboxykinase, malate thiokinase, and malyl-coa lyase₂。

39. The recombinant microorganism of any one of claims 1-34, wherein said microorganism utilizes NADH and CO produced by the glyoxylate shunt pathway in a reaction catalyzed by malate dehydrogenase, pyruvate carboxylase, phosphoenolpyruvate carboxykinase, malate thiokinase, and malyl-coa lyase₂。

40. The recombinant microorganism of any one of claims 1-34, wherein said microorganism utilizes NADH and CO produced by the D-erythrose to glycolaldehyde based pathway in a reaction catalyzed by malate dehydrogenase, pyruvate carboxylase, phosphoenolpyruvate carboxykinase, malate thiokinase, and malyl-coa lyase₂。

41. The recombinant microorganism of any one of claims 1-34, wherein said microorganism utilizes NADH and CO produced by the pentose derivative to glycolaldehyde based pathway in a reaction catalyzed by malate dehydrogenase, pyruvate carboxylase, phosphoenolpyruvate carboxykinase, malate thiokinase, and malyl-coa lyase₂。

42. The recombinant microorganism of any one of claims 1-41, wherein said microorganism is selected from the group consisting of bacteria, yeast, and fungi.

43. The recombinant microorganism of any one of claims 1-35, wherein said synthesis of glycolate and/or glycine is increased by increasing the expression level, or activity, or specificity of at least one enzyme selected from the group consisting of: pyruvate carboxylase, phosphoenolpyruvate carboxykinase, malate dehydrogenase, malate thiokinase, malyl-coa lyase, alanine-glyoxylate aminotransferase, glycine dehydrogenase, glycine aminotransferase, serine-glyoxylate aminotransferase, glycine oxidase, NADH-dependent glyoxylate reductase and NADPH-dependent glyoxylate reductase.

44. The recombinant microorganism of any one of claims 1-43, wherein said synthesis of glycolate and/or glycine is increased by decreasing the expression level, or activity, or specificity of at least one enzyme selected from the group consisting of: malate synthase, isocitrate dehydrogenase, pyruvate oxidase and/or pyruvate formate lyase, pyruvate kinase, glucose-6-phosphate isomerase, glyoxylate carboligase, 2-oxo-4-hydroxyglutarate aldolase, glycolaldehyde reductase and glycolate oxidase.

45. The recombinant microorganism of any one of claims 1-44, wherein said synthesis of glycolate and/or glycine is increased by decreasing the expression level of a gene encoding a repressor of isocitrate lyase.

46. A method of producing glycolic acid and/or glycine using the recombinant microorganism of any one of the preceding claims, wherein the method comprises culturing the recombinant microorganism in a culture medium containing a feedstock providing a carbon source until glycolic acid and/or glycine is produced.

47. The method of claim 46, wherein the carbon source is selected from the group consisting of: sugars, glycerol, alcohols, organic acids, alkanes, fatty acids, hemicellulose, lignocellulose, proteins, carbon dioxide and carbon monoxide.

48. The method of claim 46 or 47, wherein the carbon source is a hexose and/or pentose sugar.

49. The method of any one of claims 46 to 48, wherein the carbon source is glucose.

50. The method of any one of claims 46-48, wherein the carbon source is sucrose.

51. The method of claim 46 or 47, wherein the carbon source is CO₂Or carbonic acid.

52. A method of producing a recombinant microorganism that produces glycolate and/or glycine from glyoxylate, the method comprising introducing into the microorganism:

(a) a gene encoding malate dehydrogenase catalyzing the conversion of pyruvate to malate;

(b) a gene encoding malate thiokinase catalyzing the conversion of malate into malyl-coa; and

53. A method of producing a recombinant microorganism that produces glycolate and/or glycine from glyoxylate, the method comprising introducing into the microorganism:

(a) a gene coding for a pyruvate carboxylase which catalyzes the conversion of pyruvate to OAA, and/or a gene coding for a phosphoenolpyruvate carboxylase which catalyzes the conversion of phosphoenolpyruvate to OAA, and/or

A gene encoding phosphoenolpyruvate carboxykinase which catalyzes the conversion of phosphoenolpyruvate to OAA;

(b) a gene encoding malate dehydrogenase catalyzing the conversion of OAA to malate;

(d) a gene encoding a malyl-coa lyase that catalyzes the conversion of malyl-coa to glyoxylate and acetyl-coa, wherein acetyl-coa produced by the malyl-coa lyase combines with OAA to increase the biosynthesis of GA and/or glycine.

54. A method of producing a recombinant microorganism that produces glycolate and/or glycine from glyoxylate, the method comprising introducing into the microorganism:

(b) a gene encoding malate thiokinase catalyzing the conversion of malate into malyl-coa; and

Technical Field

The present application relates to the use of a recombinant microorganism for the biosynthesis of glycolic acid and/or glycine from glyoxylic acid using the retro-glyoxylic acid shunt and improving its yield, as well as to a method for producing said recombinant microorganism. The present application also relates to methods of producing glycolic acid and/or glycine from a carbon source (e.g., hexose or pentose feedstock) by a reverse glyoxylate shunt using a recombinant microorganism. The present application also relates to compositions comprising one or more of these compounds and/or recombinant microorganisms.

Statement regarding sequence listing

The sequence listing associated with this application is provided in textual format in place of the paper copy and is incorporated herein by reference. The name of the text file containing the sequence listing is BRSK-010_02WO _ st25. txt. This text file, about 45.5KB, was created at 12 days 2/2020 and was submitted electronically via EFS-Web.

Background

Glycolic acid and glycine are valuable raw materials for the production of a variety of compounds. For example, glycolic acid is an important raw material for the production of polyglycolic acid and other biocompatible copolymers and the like. Likewise, glycine has a variety of uses in the pharmaceutical and cosmetic industries, in the production of insecticides (pyrethroid insecticides), and in food and feed additives.

To develop an environmentally friendly process for the production of Glycolic Acid (GA) and glycine, researchers have engineered microorganisms with biosynthetic pathways to produce GA and/or glycine. For example, U.S. patent No. 9,034,615 and U.S. patent No. 8,945,888 disclose the production of glycolic acid via the Glyoxylate Shunt (GS) pathway. U.S. pre-grant publication No. 2014/0295510 discloses the GS pathway for the production of glycolic acid in eukaryotes, while patent documents such as WO2017/059236, WO2016/079440, US2016/0076061, and US2015/0147794 disclose the production of glycolic acid using pentose-based sugars. Although the biochemical pathways described in these and other patent documents were developed with the aim of providing high GA and glycine yields, since these pathways produce excess NADH and excess CO₂Resulting in loss of product yield, so the GA and glycine yields provided by these pathways are still not optimal.

The biosynthetic pathway for the production of glycolic acid and glycine provided by the present invention has a higher theoretical yield potential than existing metabolic pathways, which solves or partially reduces the problem of lost product yield potential. The present invention provides biosynthetic pathways in which a carbon-immobilized enzyme and a reverse glyoxylate shunt enzyme are coupled to produce and increase the yield of glycolic acid and glycine. The present invention also provides further improvements to prevent carbon loss from the previously described pathway and to facilitate carbon fixation coupled with the retro-glyoxylate branch.

The present invention also aims to further increase the theoretical yield of glycolic acid and glycine for the previously described pathways, in part by exploiting the CO released by these pathways₂And/or NAD (P) H, or by capturing an exogenously supplied carbon source (CO)₂、HCO₃ ^-Or other carbonic acid). The present invention additionally provides biosynthetic pathways that produce and improve the GA and glycine yield potential of the previously described pathways, by permitting carbon flow to be rerouted through carbon fixation in the pyruvate and/or phosphoenolpyruvate nodes to oxaloacetate, partially reducing or even eliminating carbon loss in the microbial intrinsic metabolic and enzymatic reactions.

Thus, the present invention allows the use of the same amount of starting carbon source (e.g., sugar) for higher production of GA and glycine and provides a method that increases the economic success rate of current methods.

Disclosure of Invention

The present disclosure provides recombinant microorganisms and uses thereof. The invention also provides a method for preparing the recombinant microorganism. In various embodiments, the recombinant microorganisms of the present disclosure produce Glycolic Acid (GA) and/or glycine via glyoxylic acid as an intermediate.

In some embodiments, provided herein is a recombinant glyoxylate-producing microorganism for the synthesis of Glycolic Acid (GA) and/or glycine, wherein the microorganism comprises: (a) a gene encoding malate dehydrogenase catalyzing the conversion of pyruvate to malate; (b) a gene encoding malate thiokinase catalyzing the conversion of malate into malyl-coa; and (c) a gene encoding malyl-coa lyase which catalyzes the conversion of malyl-coa to glyoxylate and acetyl-coa.

In some embodiments, provided herein is a recombinant glyoxylate-producing microorganism for the synthesis of Glycolic Acid (GA) and/or glycine, wherein the microorganism comprises: (a) a gene encoding pyruvate carboxylase which catalyzes the conversion of pyruvate to Oxaloacetate (OAA), and/or a gene encoding phosphoenolpyruvate carboxylase which catalyzes the conversion of phosphoenolpyruvate to OAA, and/or a gene encoding phosphoenolpyruvate carboxykinase which catalyzes the conversion of phosphoenolpyruvate to OAA; (b) a gene encoding malate thiokinase catalyzing the conversion of malate into malyl-coa; (c) a gene encoding a malyl-coa lyase that catalyzes the conversion of malyl-coa to glyoxylate and acetyl-coa, wherein acetyl-coa produced by the malyl-coa lyase combines with OAA to increase the biosynthesis of GA and/or glycine. In these embodiments, the recombinant microorganism has reduced phosphoglucose isomerase activity, or more preferably does not catalyze the conversion of glucose-6-phosphate to fructose-6-phosphate by the enzyme phosphoglucose isomerase. Furthermore, the recombinant microorganism may or may not comprise an overexpressed endogenous or exogenous enzyme: citrate synthase, isocitrate lyase and/or glyoxylate reductase. By reducing the activity of the phosphoglucose isomerase, or more preferably by deleting the gene encoding the phosphoglucose isomerase catalyzing the conversion of glucose-6-phosphate to fructose-6-phosphate (e.g., the gene pgi in E.coli), the carbon source can be diverted at least in part to the pentose-phosphate pathway (PPP) to provide the additional NADPH that may be required for optimal conversion of glyoxylic acid to glycolic acid. In some embodiments, CO generated by the PPP route₂It is possible to re-incorporate carboxykinase by using the carboxylase and carboxykinase enzymes suggested herein.

The recombinant microorganism in any of the embodiments described herein may produce isopropanol, ethanol, acetone, citric acid, itaconic acid, acetic acid, butyric acid, (poly) 3-hydroxybutyric acid, 3-hydroxyisobutyric acid, 3-aminoisobutyric acid, 2-hydroxyisobutyric acid, methacrylic acid, (poly) glutamic acid, arginine, ornithine, citrulline, leucine, isoleucine, or proline without acetyl-coa produced by malyl-coa lyase.

In the recombinant microorganisms of the present disclosure, acetyl-coa produced by malyl-coa lyase is expected to combine with OAA to increase biosynthesis of GA and/or glycine.

In some embodiments, any of the recombinant microorganisms described herein may comprise a deletion or loss-of-function mutation in the gene encoding malate dehydrogenase, wherein the mutation results in partial or complete inhibition of malate dehydrogenase activity that catalyzes the conversion of oxaloacetate to malate, malate to pyruvate, and/or malate to oxaloacetate.

In an embodiment of the recombinant microorganism for the production of glycolate, the recombinant microorganism comprises a gene encoding an NADH dependent glyoxylate reductase that catalyzes the conversion of glyoxylate to glycolate, and/or a gene encoding an NADPH dependent glyoxylate reductase that catalyzes the conversion of glyoxylate to glycolate.

In an embodiment where the recombinant microorganism produces glycine, the recombinant microorganism comprises a gene encoding alanine-glyoxylate aminotransferase, a gene encoding glycine dehydrogenase, a gene encoding glycine aminotransferase, a gene encoding serine-glyoxylate aminotransferase, and/or a gene encoding glycine oxidase to catalyze the conversion of glyoxylate to glycine.

In some embodiments, the recombinant microorganisms of the present disclosure can produce glycolic acid and glycine and comprise one or more of the genes described above for the conversion of glyoxylate to GA and/or glycine.

In some embodiments, the gene encoding glyoxylate reductase activity is selected from the group consisting of: ycdW and/or yiaE from escherichia coli, GOR1 from saccharomyces cerevisiae (s. cerevisiae), gyaR from thermus maritima (Thermococcus litoralis) and/or GLYR1 from arabidopsis thaliana (a. thaliana). The present disclosure also contemplates the use of homologs of these genes to catalyze the conversion of glyoxylate to glycolate.

In certain embodiments, the malate dehydrogenase catalyzing the carboxylation of pyruvate to malate in the recombinant microorganisms of the present disclosure is from enzyme class (E.C.)1.1.1.38, e.c.1.1.1.39, or e.c. 1.1.1.40.

In some embodiments, the malate dehydrogenase catalyzing the conversion of oxaloacetate to malate in the recombinant microorganisms of the present disclosure is from enzyme classification (E.C.) 1.1.1.37.

In some embodiments, the gene encoding malate dehydrogenase catalyzing the carboxylation of pyruvate to malate in the recombinant microorganism of the present disclosure is selected from the group consisting of: maeA, maeB, dme, mez, mae1, name-me 1, name-me 2, and homologs thereof. In these embodiments, the gene maeA may be from escherichia coli, pseudomonas, or bacillus; the gene maeB can be derived from Escherichia coli or salmonella; the gene dme can be from rhizobia; gene mez may be from mycobacterium; the gene mae1 can be derived from Saccharomyces cerevisiae; and the gene, nad-me1 or nad-me2, may be from Arabidopsis thaliana. For example, the maeA gene can be from bacillus subtilis (b.subtillis); gene dme may be from rhizobium melilote (r.melilot); or gene mez may be from Mycobacterium tuberculosis (Mycobacterium tuberculosis). The present disclosure also contemplates the use of homologues of these genes to catalyze the carboxylation of pyruvate to malate.

In some embodiments, the gene encoding malate dehydrogenase catalyzing the conversion of oxaloacetate to malate in the recombinant microorganism of the present disclosure is selected from the group consisting of: genes mdh from E.coli, Corynebacterium, Streptomyces, Yeast and Arabidopsis thaliana. For example, gene mdh may be from streptomyces coelicolor (s.coelicolor) or gene mdh1/2/3 from saccharomyces cerevisiae (s.cerevisiae). The present disclosure also contemplates the use of homologs of these genes to catalyze the conversion of oxaloacetate to malate.

In some embodiments, the malate thiokinase that converts malate to malyl-coa may be from enzyme classification system No. e.c.6.2.1.4, e.c.6.2.1.5, e.c.6.2.1.9, or e.c.6.2.1.

In one embodiment, the gene encoding a malate thiokinase in a recombinant microorganism of the present disclosure may be sucCD and/or sucCD-2 and/or mtkAB from Methylobacterium (Methylobacterium sp.), Methylobacterium extorquens (Methylobacterium extorquens), escherichia coli, Thermus thermophilus (Thermus thermophilus), rhizogenes (Hyphomicrobium sp.), methanococcus jannaschii (methanococcus jannaschii), methanobacter thermoautotrophicum (methanotrophus thermothermophilus), Rhizobium (Rhizobium), Methylococcus capsulatus (Methylococcus capsulatus) or Pseudomonas pseudomonads (pseudomonads), or homologues thereof.

In some embodiments, the malyl-coa lyase that converts malyl-coa to glyoxylate and acetyl-coa is from e.c.4.3.1.24 or e.c. 4.3.1.25.

In some embodiments, the gene encoding malyl-coa lyase in the recombinant microorganisms of the present disclosure may be from methylobacterium extorquens, Rhodobacter sphaeroides (Rhodobacter sphaeroides), streptomyces, phomophilus carotovorus (Chloroflexus aurantiacaus), Nitrosomonas eurotia (nitromonas europaea), methylococcus capsulatus, Nereida ignnavia, pythium aphanidermatum (Hyphomicrobium methylvorum), active deep sea bacteria (thalassivus), Rhodobacter marinus (roseobacterium litoralis), setaria denitrificans (hyphomobium densiticum), Rhodobacter sphaeroides (r.sphaericus), Mycobacterium smegmatis (mycoderma smegmatis) or Rhodococcus rhodochrous (Rhodococcus rhodochrous) and/or mcclol 1 or homologues thereof.

In some embodiments, the pyruvate carboxylase to convert pyruvate to OAA can be from enzyme classification system No. e.c.6.4.1.1; phosphoenolpyruvate carboxylase to convert phosphoenolpyruvate to OAA may be from e.c. 4.1.1.31; phosphoenolpyruvate carboxykinase, which converts phosphoenolpyruvate to OAA, can be from e.c.4.1.1.32 and e.c. 4.1.1.49.

In some embodiments, the gene encoding pyruvate carboxylase in the recombinant microorganisms of the present disclosure may be PYC from Rhizobium phaseolus (Rhizobium etli), PYC1 or PYC2 from yeast, or PYC from bacillus subtilis; or a homologue thereof.

In some embodiments, the gene encoding phosphoenolpyruvate carboxylase in the recombinant microorganism of the present disclosure may be ppc from escherichia coli, ppc or pepC from Rhodothermus marinus (r. marinus), ppcA from methanothermus thermoautotrophus (m. thermotrophicus), pep1 from corn (z. mays), ppc1/2/3 from arabidopsis thaliana, ppc from soybean (g.max), or from Rhodothermus rubrum (Rhodothermus), Corynebacterium (Corynebacterium), Salmonella (Salmonella), filamentous microbacterium (hygrophium), Streptococcus (Streptococcus) and streptomyces, Pantoea (Pantoea), Bacillus (Bacillus), Clostridium (Clostridium), Pseudomonas pseudomonads (Pseudomonas), Rhodopseudomonas (Rhodopseudomonas), Pseudomonas pseudoticum (arabidopsis), tobacco (Medicago sativa), alfalfa (alfalfa), or alfalfa seed (alfalfa); or a homologue thereof.

In some embodiments, the gene encoding phosphoenolpyruvate carboxykinase in the recombinant microorganisms of the present disclosure may be pck or pckA from escherichia coli, pckA from Selenomonas ruminata (selenia ruminata), pckA from Salmonella typhimurium (Salmonella typhimurium), pckA from Klebsiella (Klebsiella sp.), pckA from Thermus (Thermus sp.), pckA from Ruminococcus albus (Ruminococcus albus) or Ruminococcus flavus (Ruminococcus flavefaciens), pckA from Actinobacillus succinogenes (Actinobacillus succinogenes), pckA or pckA from Streptococcus bovis (Streptococcus bovis), or mycobacterium bovis, clostridium thermocellum (clostridium thermocellum), klonia; or a homologue thereof.

In some embodiments, the recombinant microorganisms of the present disclosure comprise: (a) a gene encoding citrate synthase that converts OAA and acetyl-coa produced by malyl-coa lyase to citrate; (b) a gene encoding citrate hydrolase which converts citric acid into aconitic acid; (c) a gene encoding D-threo isocitrate hydrolase or aconitase converting cis-aconitic acid into isocitric acid; (d) a gene encoding an isocitrate lyase that converts isocitrate to succinate and glyoxylate; (e) a gene encoding succinate dehydrogenase converting succinate into fumarate; and (f) a gene encoding a fumarase for converting fumarate into malate. In the same embodiment, the recombinant microorganism may have at least a portion of the malate dehydrogenase enzyme that catalyzes the conversion of malate to oxaloacetate retained. Alternatively, malate dehydrogenase, which catalyzes the conversion of malate to oxaloacetate, may be down-regulated or even inactivated in favor of malate thiokinase activity.

In some embodiments, a loss-of-function mutation in a gene encoding malate synthase, or deletion of the gene, may be included in a recombinant microorganism of the present disclosure. Exemplary genes encoding malate synthase include aceB and/or glcB from E.coli, or DAL7 and/or MLS1 from yeast (e.g., Saccharomyces cerevisiae).

The recombinant microorganism of any of the embodiments disclosed herein can include a deletion or modification (modification) that reduces the activity of one or more endogenous genes selected from the group consisting of: (a) a gene encoding isocitrate dehydrogenase; (b) genes encoding pyruvate dehydrogenase, pyruvate oxidase and/or pyruvate formate lyase; (c) a gene encoding pyruvate kinase; and (d) a gene encoding glycolate oxidase. Exemplary genes encoding isocitrate dehydrogenase include icd from E.coli, or IDP2 and/or IDH1/2 from yeast. Exemplary genes encoding pyruvate dehydrogenase include aceE and/or aceF from E.coli. Exemplary genes encoding pyruvate kinase include pykA and/or pykF from E.coli. Exemplary genes encoding glycolate oxidase include glcD, glcE, glcF and/or glcG from escherichia coli. Exemplary genes.

The recombinant microorganism of any of the embodiments disclosed herein can include deletions or modifications that reduce the activity of pyruvate dehydrogenase, prevent or at least attenuate the loss of the predominant carbon in the conversion from pyruvate to acetyl-coa, and facilitate carbon rerouting from pyruvate or phosphoenolpyruvate to oxaloacetate by the carboxylation activity of the enzyme candidates set forth herein.

The recombinant microorganism of any of the embodiments disclosed herein can include a deletion or modification that reduces the activity of pyruvate kinase and facilitates carbon fixation from phosphoenolpyruvate to oxaloacetate via carboxylation activity of the enzyme candidates set forth herein.

The recombinant microorganism of any of the embodiments disclosed herein can include a deletion or modification (modification) that reduces the activity of one or more endogenous genes selected from the group consisting of: (a) a gene encoding glyoxylate aldehyde ligase (carboligase); (b) a gene encoding 2-oxo-4-hydroxyglutarate aldolase; (c) a gene encoding glycolaldehyde reductase; and (d) a gene encoding a repressor of isocitrate lyase. An exemplary gene encoding glyoxylate aldehyde ligase is gcl. An exemplary gene encoding 2-oxo-4-hydroxyglutarate aldolase is edA. Exemplary genes encoding glycolaldehyde reductase include fucO and gldA. An exemplary gene encoding the repressor of isocitrate lyase is iclR.

In some embodiments, in the recombinant microorganisms of the present disclosure, the expression level of a gene encoding alanine-glyoxylate aminotransferase, a gene encoding glycine dehydrogenase, a gene encoding glycine aminotransferase, a gene encoding serine-glyoxylate aminotransferase, and/or a gene encoding glycine oxidase is increased.

In some embodiments, in the recombinant microorganisms of the present disclosure, the expression level of a gene encoding alanine aminotransferase, and/or a gene encoding NADPH-dependent glutamate synthase is increased.

In some embodiments, in the recombinant microorganisms of the present disclosure, the synthesis of glycolate and/or glycine is increased by increasing the expression level, or activity, or specificity of at least one enzyme selected from the group consisting of: pyruvate carboxylase, phosphoenolpyruvate carboxykinase, malate dehydrogenase, malate thiokinase, malyl-coa lyase, alanine-glyoxylate aminotransferase, glycine dehydrogenase, glycine aminotransferase, serine-glyoxylate aminotransferase, glycine oxidase, NADH-dependent glyoxylate reductase and NADPH-dependent glyoxylate reductase.

In some embodiments, in the recombinant microorganisms of the present disclosure, the synthesis of glycolate and/or glycine is increased by decreasing the expression level, or activity, or specificity of at least one enzyme selected from the group consisting of: malate synthase, isocitrate dehydrogenase, pyruvate oxidase and/or pyruvate formate lyase, pyruvate kinase, glyoxylate carboligase, 2-oxo-4-hydroxyglutarate aldolase, glucose-6-phosphate isomerase, glycolaldehyde reductase and glycolate oxidase.

In some embodiments, in the recombinant microorganisms of the present disclosure, the synthesis of glycolate and/or glycine is increased by decreasing the expression level of a gene encoding a repressor of isocitrate lyase.

In some embodiments, the recombinant microorganisms of the present disclosure can utilize NADH and CO produced by other glycolate and/or glycine production pathways in reactions catalyzed by malate dehydrogenase, pyruvate carboxylase, phosphoenolpyruvate carboxykinase, malate thiokinase, and malyl-coa lyase₂. For example, in some embodiments, a recombinant microorganism of the present disclosure can utilize NADH and/or CO produced based on the serine/hydroxypyruvate pathway in a reaction catalyzed by malate dehydrogenase, pyruvate carboxylase, phosphoenolpyruvate carboxykinase, malate thiokinase, and malyl-coa lyase₂. In some embodiments, the recombinant microorganisms of the present disclosure may utilize NADH and/or CO produced by the glyoxylate shunt pathway in a reaction catalyzed by malate dehydrogenase, pyruvate carboxylase, phosphoenolpyruvate carboxykinase, malate thiokinase, and malyl-coa lyase₂. In some embodiments, the recombinant microorganisms of the present disclosure can utilize NADH and/CO produced by the D-erythrose to glycolaldehyde based pathway in a reaction catalyzed by malate dehydrogenase, pyruvate carboxylase, phosphoenolpyruvate carboxykinase, malate thiokinase, and malyl-coa lyase₂. In some embodiments, the recombinant microorganisms of the present disclosure may be produced by malate dehydrogenase, pyruvateUtilization of NADH and/or CO generated by the pentose derivative-based pathway to glycolaldehyde in reactions catalysed by carboxylases, phosphoenolpyruvate carboxykinases, malate thiokinases and malyl-CoA lyases₂。

In some embodiments, the recombinant microorganisms of the present disclosure may utilize exogenously added CO in a reaction catalyzed by malate dehydrogenase, pyruvate carboxylase, phosphoenolpyruvate carboxykinase, malate thiokinase, and malyl-coa lyase₂Carbonic acid, and/or a reducing agent. The reducing agent may be hydrogen, electrons, and/or NAD (P) H.

Recombinant microorganisms provided by the present disclosure include bacteria, yeast, and fungi. In some embodiments, the recombinant microorganism of the present disclosure may be a bacterium selected from the group consisting of enterobacteriaceae, clostridiaceae, bacillaceae, streptomycetaceae, and corynebacteriaceae. In an exemplary embodiment, the recombinant microorganism of the present disclosure may be a species of escherichia, clostridium, bacillus, klebsiella, pantoea, salmonella, lactobacillus, or corynebacterium. For example, the recombinant microorganism of the present disclosure may be escherichia coli, Corynebacterium glutamicum (Corynebacterium glutamicum), Clostridium acetobutylicum (Clostridium acetobutylicum), or Bacillus subtilis (Bacillus subtilis).

In some embodiments, the recombinant microorganism of the present disclosure may be a yeast selected from the family saccharomyces. In exemplary embodiments, the recombinant microorganisms of the present disclosure can be of the yeast species. For example, the recombinant microorganism of the present disclosure may be Saccharomyces cerevisiae (Saccharomyces cerevisiae).

In the recombinant microorganisms of the present disclosure, any one of the genes described herein is heterologously expressed.

The present disclosure also provides methods of producing GA and/or glycine using the recombinant microorganisms described herein. In some embodiments, a method of producing glycolic acid and/or glycine using a recombinant microorganism described herein comprises culturing the recombinant microorganism in a culture medium containing feedstock that provides a carbon source until glycolic acid and/or glycine is produced.

In some embodiments, the carbon source used in the method for producing GA and/or glycine may be selected from: sugars, glycerol, alcohols, organic acids, alkanes, fatty acids, hemicellulose, lignocellulose, proteins, carbon dioxide and carbon monoxide. In exemplary embodiments, the carbon source is a hexose and/or pentose sugar. In exemplary embodiments, the carbon source is glucose. In another exemplary embodiment, the carbon source is sucrose. In another exemplary embodiment, the carbon source comprises a biomass hydrolysate comprising hemicellulose. In another exemplary embodiment, the carbon source is CO₂Or carbonic acid, e.g. HCO₃ ^-。

Also provided herein are methods of producing recombinant microorganisms that produce glycolate and/or glycine from glyoxylate.

In some embodiments, a method of producing a recombinant microorganism that produces glycolic acid and/or glycine comprises introducing into the microorganism: (a) a gene encoding malate dehydrogenase catalyzing the conversion of pyruvate to malate; (b) a gene encoding malate thiokinase catalyzing the conversion of malate into malyl-coa; and (c) a gene encoding malyl-coa lyase that catalyzes the conversion of malyl-coa to glyoxylate and acetyl-coa.

In some embodiments, a method of producing a recombinant microorganism that produces glycolic acid and/or glycine comprises introducing into the microorganism: (a) a gene encoding pyruvate carboxylase which catalyzes the conversion of pyruvate to OAA, and/or a gene encoding phosphoenolpyruvate carboxylase which catalyzes the conversion of phosphoenolpyruvate to OAA, and/or a gene encoding phosphoenolpyruvate carboxykinase which catalyzes the conversion of phosphoenolpyruvate to OAA; (b) a gene encoding malate dehydrogenase catalyzing the conversion of OAA to malate; (c) a gene encoding malate thiokinase catalyzing the conversion of malate into malyl-coa; and (d) a gene encoding a malyl-coa lyase that catalyzes the conversion of malyl-coa to glyoxylate and acetyl-coa, wherein acetyl-coa produced by the malyl-coa lyase combines with OAA to increase the biosynthesis of GA and/or glycine. In some of these embodiments, the method may comprise introducing into the microorganism a gene encoding a malate dehydrogenase that catalyzes the conversion of pyruvate to malate.

In some embodiments, a method of producing a recombinant microorganism that produces glycolic acid and/or glycine comprises introducing into the microorganism: (a) a gene encoding pyruvate carboxylase which catalyzes the conversion of pyruvate to Oxaloacetate (OAA), and/or a gene encoding phosphoenolpyruvate carboxylase which catalyzes the conversion of phosphoenolpyruvate to OAA, and/or a gene encoding phosphoenolpyruvate carboxykinase which catalyzes the conversion of phosphoenolpyruvate to OAA; (b) a gene encoding malate thiokinase catalyzing the conversion of malate into malyl-coa; and (c) a gene encoding a malyl-coa lyase that catalyzes the conversion of malyl-coa to glyoxylate and acetyl-coa, wherein the recombinant microorganism does not catalyze the conversion of oxaloacetate to malate.

In an exemplary embodiment, the gene encoding malate dehydrogenase heterologously introduced into the microorganism comprises a mutation resulting in partial or complete inhibition of malate dehydrogenase activity catalyzing the conversion of oxaloacetate to malate, or malate to pyruvate, or malate to oxaloacetate. In another exemplary embodiment, if a gene encoding malate dehydrogenase catalyzing the conversion of pyruvate to malate and/or oxaloacetate to malate is endogenously present in the recombinant microorganism, the method for producing a recombinant microorganism producing glycolate and/or glycine comprises introducing a mutation into the endogenous gene encoding malate dehydrogenase, wherein said mutation results in the partial or complete inhibition of malate dehydrogenase activity catalyzing the conversion of oxaloacetate to malate, or malate to pyruvate, or malate to oxaloacetate.

In some embodiments, the method of producing a recombinant microorganism that produces glycolic acid and/or glycine may further comprise introducing into the microorganism: (a) a gene encoding an NADH-dependent glyoxylate reductase that catalyzes the conversion of glyoxylate to glycolate; (b) a gene encoding an NADPH-dependent glyoxylate reductase that catalyzes the conversion of glyoxylate to glycolate; and/or c) a gene encoding alanine-glyoxylate aminotransferase, a gene encoding glycine dehydrogenase, a gene encoding glycine aminotransferase, a gene encoding serine-glyoxylate aminotransferase, and/or a gene encoding glycine oxidase which catalyzes the conversion of glyoxylate to glycine.

In some embodiments, the method of producing a recombinant microorganism that produces glycolate and/or glycin can further comprise introducing a loss of function mutation of the gene encoding malate synthase or deleting the gene into the microorganism.

In some embodiments, the method of producing a recombinant microorganism that produces glycolate and/or glycin may further comprise introducing into the microorganism a deletion or modification that reduces the activity of one or more enzymes encoded by a gene selected from the group consisting of: (a) a gene encoding isocitrate dehydrogenase; (b) genes encoding pyruvate dehydrogenase, pyruvate oxidase and/or pyruvate formate lyase; (c) a gene encoding pyruvate kinase; and (d) a gene encoding glycolate oxidase.

In some embodiments, the method of producing a recombinant microorganism that produces glycolate and/or glycin may further comprise introducing into the microorganism a deletion or modification that reduces the activity of one or more enzymes encoded by a gene selected from the group consisting of: (a) a gene encoding glyoxylate aldehyde ligase (carboligase); (b) a gene encoding 2-oxo-4-hydroxyglutarate aldolase; (c) a gene encoding glycolaldehyde reductase; and (d) a gene encoding a repressor of isocitrate lyase.

In some embodiments, the method of producing a recombinant microorganism that produces glycolic acid and/or glycine may further comprise introducing gain-of-function mutations into: a gene encoding alanine-glyoxylate aminotransferase, a gene encoding alanine-glyoxylate aminotransferase which converts glyoxylate to glycine, a gene encoding glycine dehydrogenase, a gene encoding glycine aminotransferase, a gene encoding serine-glyoxylate aminotransferase, and/or a gene encoding glycine oxidase which catalyzes the conversion of glyoxylate to glycine.

In some embodiments, the method of producing a recombinant microorganism that produces glycolate and/or glycin can further comprise introducing a gain-of-function mutation into the gene encoding alanine aminotransferase and/or the gene encoding NADPH-dependent glutamate synthase.

In embodiments where the gain-of-function mutation is introduced into a gene, the gain-of-function mutation may be introduced into an endogenous gene of the microorganism, or the gain-of-function mutation may be introduced into a heterologous gene, and the heterologous gene comprising the gain-of-function mutation introduced into the microorganism.

Microorganisms useful for producing the recombinant microorganisms of the present disclosure include bacteria, yeast, and fungi. Exemplary bacteria useful in the present disclosure include bacteria selected from the group consisting of: enterobacteriaceae, Clostridium, Bacillaceae, Streptomycetaceae, and Corynebacteriaceae. For example, the recombinant microorganism may be of the following species: escherichia (for example Escherichia coli), Clostridium (for example Clostridium acetobutylicum), Bacillus (for example Bacillus subtilis), Klebsiella, Pantoea, Salmonella, Lactobacillus, or Corynebacterium (for example Corynebacterium glutamicum).

Exemplary yeasts useful for producing the recombinant microorganisms of the present disclosure can be from the family saccharomyces. For example, the recombinant microorganism can be a yeast species, such as Saccharomyces cerevisiae.

Drawings

FIG. 1 shows a schematic representation of the production of Glycolic Acid (GA) and glycine (Gly) by the retro-glyoxylate shunt. SymbolIndicating that the enzyme will likely be down-regulated or inactivated/removed, i.e. the corresponding gene may be attenuated or deleted.

FIG. 2 shows a schematic representation of a production pathway that utilizes both known Glycolic Acid (GA) and glycine (Gly) and the reverse glyoxylate shunt pathway of the present disclosure. The dotted line shows the outline of the reaction. SymbolIndicating that the enzyme may be down-regulated or lostLive/removed, i.e., the corresponding gene may be attenuated or deleted.

FIG. 3 is a schematic diagram of a flux plot depicting the maximum theoretical production yield from glucose to GA using the hexokinase transport system, carboxylation via phosphoenolpyruvate carboxykinase (PEPCK), phosphoenolpyruvate carboxylase (PPC) or pyruvate carboxylase (PYC), and combining the Glyoxylate Shunt (GS) and the retro-glyoxylate shunt (rGS). Flux analysis was performed based on the enzyme candidates of NADPH-dependent glyoxylate reductase used. Flux values were normalized for glucose input.

FIG. 4 is a schematic diagram depicting a flux plot of the maximum theoretical production yield of GA from glucose using a Phosphotransferase (PTS) transport system, carboxylation by PEPCK, PPC or PYC, and combining GS and rGS. Flux assays were performed based on enzyme candidates using NADPH-dependent glyoxylate reductase. Flux values were normalized for glucose input.

Detailed Description

Definition of

The following definitions and abbreviations will be used to explain the present disclosure.

As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an enzyme" includes a plurality of such enzymes and reference to "the microorganism" includes reference to one or more microorganisms and the like.

As used herein, the terms "comprises," "comprising," "includes," "containing," "contains," "containing," "has," "contains," or any other variation thereof, are intended to cover a non-exclusive inclusion. A composition, mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Furthermore, unless expressly stated otherwise, "or" refers to an inclusive "or" and not to an exclusive "or".

The terms "about" and "approximately" as used herein to modify a numerical value denote a close proximity around the stated value. If "X" is such a value, "about X" and "about X" will refer to a value from 0.9X to 1.1X, or in some embodiments, from 0.95X to 1.05X. Any reference to "about X" or "about X" is at least explicitly indicative of the values X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Thus, "about X" and "about X" are intended to teach and provide written descriptive support for claim limitations such as "0.98X".

As used herein, the terms "microorganism (microbe)", "microbial organism (microbe)" and "microbial organism (microbe)" include any organism present as microscopic cells, which are comprised within the range of archaea, bacteria or eukaryotes, including yeast and filamentous fungi, protozoa, algae or higher protists. Thus, the term is intended to encompass prokaryotic cells, or eukaryotic cells, or organisms having microscopic dimensions, as well as including all species of bacteria, archaea, and eubacteria, as well as eukaryotic microorganisms, such as yeasts and fungi. Also included are cell cultures of any species that can be cultured for the production of chemicals.

As described herein, in some embodiments, the recombinant microorganism is a prokaryotic microorganism. In some embodiments, the prokaryotic microorganism is a bacterium. "bacterium (bacillia)" or "eubacterium (eubacterium)" refers to a range of prokaryotes. Bacteria comprise at least the following eleven distinct groups: (1) gram-positive (gram +) bacteria, of which there are mainly two subgroups (subdivisions): (1) high G + C group (actinomycetes, mycobacteria, micrococcus, etc.), (2) low G + C group (bacillus, clostridium, lactobacillus, staphylococcus, streptococcus, mycoplasma); (2) proteobacteria, such as purple photosynthetic + non-photosynthetic gram-negative bacteria (including most "common" gram-negative bacteria); (3) cyanobacteria, such as oxygenated phototrophic organisms; (4) spirochetes and related species; (5) planomycetes (Planctomyces); (6) bacteroides, flavobacterium; (7) a chlamydia; (8) a green sulfur bacterium; (9) green non-sulfur bacteria (also anaerobic phototrophic organisms); (10) radioresistant micrococcus and its related; (11) thermotoga (Thermotoga) and Thermosiphon thermophile (Thermosipho thermophiles).

"gram-negative bacteria" include cocci, non-enterobacteria and enterobacteria. Genera of gram-negative bacteria include, for example, Neisseria, Spirobacter, Pasteurella, Brucella, Yersinia, Francisella, Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella, Proteus, Vibrio, Pseudomonas, Bacteroides, Acetobacter, Aerobacter, Agrobacterium, Azotobacter, Spirobacter, Serratia, Vibrio, Rhizobium, Chlamydia, Rickettsia, Treponema and Clostridium.

"gram-positive bacteria" include cocci, non-spore bacilli and spore bacilli. The genus of gram-positive bacteria includes, for example, Actinomyces, Bacillus, Clostridium, Corynebacterium, erysipelas, Lactobacillus, Listeria, Mycobacterium, Myxococcus, Nocardia, Staphylococcus, Streptococcus and Streptomyces.

The terms "recombinant microorganism" and "recombinant host cell" are used interchangeably herein and refer to a microorganism that has been genetically modified to express or overexpress an endogenous enzyme, to express a heterologous enzyme, such as those enzymes contained in a vector, in an integrated construct, or to have altered expression of an endogenous gene. By "altering" is meant that the expression of a gene, or the level of an RNA molecule or equivalent RNA molecule encoding one or more polypeptides or polypeptide subunits, or the activity of one or more polypeptides or polypeptide subunits is up-regulated or down-regulated such that the expression, level or activity is greater or less than that observed in the absence of the alteration. It is to be understood that the terms "recombinant microorganism" and "recombinant host cell" refer not only to the particular recombinant microorganism, but also to the progeny or potential progeny of such a microorganism.

The term "expression" in relation to a gene sequence refers to the transcription of the gene and, where appropriate, the translation of the resulting mRNA transcript into protein. Thus, it is clear from the context that expression of a protein results from transcription and translation of open reading frame sequences. The expression level of the desired product in the host cell can be determined based on the amount of the corresponding mRNA present in the cell, or the amount of the desired product encoded by the selected sequence. For example, mRNA transcribed from a selected sequence can be quantified by qRT-PCR or Northern hybridization (see Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)). The protein encoded by the selected sequence may be quantified by various methods, such as by ELISA, by measuring the biological activity of the protein, or by using assays that are independent of such activity, such as western blots or radioimmunoassays, using antibodies that recognize and bind to the protein. See Sambrook et al, 1989, supra.

The term "reducing" or "reducing" the expression level or enzymatic activity of a gene refers to partially or completely inhibiting the expression or enzymatic activity of the gene. Such inhibition of expression or activity may be inhibition of gene expression, deletion of all or part of the promoter region required for gene expression, deletion of the gene coding region, or replacement of the wild-type promoter by a weaker natural or synthetic promoter. For example, the genes may be deleted completely and may be replaced with selection marker genes to facilitate identification, isolation and purification of the strains according to the invention. Alternatively, endogenous genes may be knocked out or deleted to favor new metabolic pathways. In yet another embodiment, the expression of the gene may be reduced or diminished by using a weak promoter or by introducing certain mutations.

As used herein, the term "non-naturally occurring" when used in reference to a microorganism or enzyme activity of the present disclosure is intended to mean that the microorganism or enzyme has at least one genetic alteration that is not commonly found in strains that naturally occur in the reference species (including wild-type strains of the reference species). Genetic alterations include, for example, modifications to introduce expressible nucleic acids encoding metabolic polypeptides, addition of other nucleic acids, deletion of nucleic acids, and/or other functional disruption of the genetic material of the microorganism. Such modifications include, for example, coding regions for heterologous, homologous, or heterologous and homologous polypeptides and functional fragments thereof for reference species. Other modifications include, for example, non-coding regulatory regions, wherein the modification alters expression of a gene or operon. Exemplary non-naturally occurring microbial or enzymatic activities include the hydroxylation activities described above.

The term "exogenous" as used herein with respect to various molecules (e.g., polynucleotides, polypeptides, enzymes, etc.) refers to molecules and/or molecules produced therefrom that are not normally or naturally occurring in a given yeast, bacterium, organism, microorganism, or cell in nature.

On the other hand, the terms "endogenous" or "native" as used herein with respect to various molecules, e.g., polynucleotides, polypeptides, enzymes, etc., refer to molecules that are normally or naturally occurring in and/or produced by a given yeast, bacterium, organism, microorganism, or cell in nature.

The term "heterologous" as used herein in the context of modifying a host cell refers to various molecules, such as polynucleotides, polypeptides, enzymes, and the like, wherein at least one of the following is true: (a) the molecule is foreign ("exogenous") to the host cell (i.e., does not naturally occur in the host cell); (b) a molecule occurs naturally (e.g., "endogenous") in a given host microorganism or host cell, but is produced at a non-natural location in the cell or in a non-natural amount in the cell; and/or (c) the molecule differs in nucleotide or amino acid sequence from the endogenous nucleotide or amino acid sequence such that the molecule that differs in nucleotide or amino acid sequence from the endogenous nucleotide or amino acid is produced in the cell in a non-native (e.g., greater than naturally occurring) amount. Heterologous expression of the polynucleotide can be achieved by: one or more vectors (e.g., plasmids, cosmids, viral vectors, etc.) comprising a gene of interest are introduced into a host microorganism or by integrating a construct comprising the gene of interest into the genome of the host microorganism.

As used herein, a "homolog" with respect to an original enzyme or gene of a first family or species refers to a different enzyme or gene of a second family or species, which is determined by functional, structural or genomic analysis to be the enzyme or gene of the second family or species corresponding to the original enzyme or gene of the first family or species. Homologs typically have functional, structural, or genomic similarities. Techniques are known in which enzymes or homologues of genes can be easily cloned using gene probes and PCR. Functional assays and/or genomic mapping of genes can be used to confirm identity of cloned sequences to homologs.

A protein is "homologous" to or is "homologous" to a second protein if the amino acid sequence encoded by the gene has a similar amino acid sequence to the amino acid sequence encoded by the second gene. Alternatively, if two proteins have "similar" amino acid sequences, the proteins have homology to the second protein. Thus, the term "homologous protein" is intended to mean that two proteins have similar amino acid sequences. In some cases, homology between two proteins indicates that they have a common ancestor associated with evolution. The terms "homologous sequence" or "homologue" are considered, believed or known to be functionally related. The functional relationships may be represented in any of a number of ways, including but not limited to: (a) the degree of sequence identity and/or (b) the same or similar biological function. Preferably, refer to both (a) and (b). The degree of sequence identity may vary, but in one embodiment (when using standard sequence alignment programs known in the art) is at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least 98.5%, or at least about 99%, or at least 99.5%, or at least 99.8%, or at least 99.9% sequence identity. Homology can be determined using software programs readily available in the art, such as those discussed in Current Protocols in Molecular Biology (F.M. Ausubel et al, eds., 1987) suppl.30, section 7.718, Table 7.71. Some alignment programs are MacVector (Oxford Molecular Ltd, Oxford, UK) and ALIGN Plus (Scientific and economic Software, Pa.). Other non-limiting alignment programs include Sequencher (Gene Codes, Annelberg, Mich.), AlignX, and Vector NTI (Invitrogen, Calsbarda). Similar biological functions may include, but are not limited to: catalyze the same or similar enzymatic reactions; the same or similar selectivity for substrate or cofactor; have the same or similar stability; equal or similar tolerance to various fermentation conditions (temperature, pH, etc.); and/or have the same or similar tolerance to various metabolic substrates, products, byproducts, intermediates, and the like. The degree of similarity of biological functions can vary, but in one embodiment is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least 98.5%, at least about 99%, or at least 99.5%, or at least 99.8%, or at least 99.9%, according to one or more assays known to those skilled in the art for determining a given biological function.

The term "variant" refers to any polypeptide or enzyme described herein. Variants also include one or more components of a multimer, a multimer comprising an individual component, a multimer comprising multiple individual components (e.g., a multimer of a reference molecule), a chemical breakdown product, and a biological breakdown product. In particular non-limiting embodiments, an enzyme may be a "variant" relative to a reference enzyme due to a change in any portion of the polypeptide sequence encoding the reference enzyme. A variant of a reference enzyme can have at least 10%, at least 30%, at least 50%, at least 80%, at least 90%, at least 100%, at least 105%, at least 110%, at least 120%, at least 130% or more of the enzyme activity in a standard assay used to measure the enzyme activity of a reference enzyme preparation. In some embodiments, a variant may also refer to at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a full-length, or unprocessed, enzyme of the present disclosure. In some embodiments, a variant may also refer to at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a mature, or processed, enzyme of the present disclosure.

As used herein, the term "yield potential" refers to the yield of a product from a biosynthetic pathway. In one embodiment, the yield potential may be expressed as a percentage of the weight of the final product per weight of starting compound.

As used herein, the term "thermodynamic maximum yield" refers to the maximum yield of a product obtained from fermentation of a given feedstock (e.g., glucose) based on the amount of energy of the product compared to the feedstock. For example, in normal fermentation, the product will not contain more energy than the feedstock if no additional energy source (e.g., light, hydrogen, or methane, or electricity) is used. The thermodynamic maximum yield represents the product yield from all energy and mass from the feedstock converted to product. The yield can be calculated and is independent of the particular route. If the yield of a particular pathway to a product is below the thermodynamic maximum yield, that particular pathway will lose quality and it is likely that particular pathway will be improved or replaced with a more efficient pathway to the product.

The term "redox balance" refers to the total amount of redox cofactor in a given set of reactions. At a shortage of redox cofactors, the redox balance is negative because of the need to burn the feedstock to meet the cofactor requirements, and the yield of this approach would be impractical. When the redox cofactor is in excess, the redox balance is considered positive and the yield of this pathway is below the maximum yield (Dugar et al, "Relative potential of biological pathways for biological and bio-based products" Nature biotechnology 29.12(2011): 1074). Furthermore, when the pathway produces the same amount of redox cofactor that it consumes, the redox balance is zero, and the pathway may be referred to as a "redox balance". Designing metabolic pathways and engineered organisms such that redox cofactors are balanced or nearly balanced typically results in more efficient, higher yield production of the desired compound than unbalanced pathways. Since the two half-reactions (one oxidation and the other reduction) occur simultaneously, the redox reactions always occur simultaneously. In a redox process, a reducing agent transfers electrons to an oxidizing agent. Therefore, in the reaction, the reducing agent or the reducing agent loses electrons and is oxidized, and the oxidizing agent or the oxidizing agent gains electrons and is reduced. In one embodiment, the redox reaction occurs in a biological system. The term redox state is generally used to describe the balance of NAD +/NADH and NADP +/NADPH of natural or unnatural metabolic pathways in biological systems (e.g., microbial cells). The redox state is reflected in the equilibrium of several groups of metabolites (e.g., lactate and pyruvate, β -hydroxybutyrate and acetoacetate), the interconversion of which depends on these ratios. In one embodiment, the use of an external source of hydrogen or electrons, in combination with or without a hydrogenase enzyme capable of converting hydrogen to nad (p) H, may be beneficial in increasing product yield in metabolic pathways with negative redox balance, i.e., when redox cofactors (e.g., nad (p) H) are in short supply.

Background introduction

The Glyoxylate Shunt (GS), also known as the glyoxylate cycle, is a variation of the tricarboxylic acid cycle (TCA cycle) and is an anabolic pathway occurring in plants, bacteria, protists and fungi. The TCA cycle differs from the glyoxylate shunt in that in the glyoxylate shunt, isocitrate is cleaved (clean) by isocitrate lyase into glyoxylate and succinate rather than decarboxylation and dehydrogenation into α -ketoglutarate. This bypasses the two decarboxylation steps that occur in the TCA cycle, allowing conversion of acetyl-coa to TCA cycle intermediates without carbon loss. Glyoxylic acid is converted to malic acid by incorporation of an acetyl-coa molecule.

The use of the Glyoxylate Shunt (GS) pathway for the production of glycolic acid is described in U.S. patent No. 9,034,615, which is incorporated herein by reference in its entirety. This patent discloses GA production by attenuating the glyoxylate consuming pathway and increasing the activity of NAD (P) H dependent glyoxylate reductase. U.S. patent No. 8,945,888; U.S. pre-authorization publication No. 2014/0295510; and PCT publication No. WO 2016/193540, which is incorporated herein by reference in its entirety, also discloses the use of the glyoxylate shunt pathway for the production of glycolic acid. However, the glyoxylate shunt pathway has a reduced overall yield potential of 0.84g _ GA/g _ glucose, while the thermodynamic maximum yield of glucose → GA conversion is 1.70 g/g. This pathway is also not redox balanced and has a high excess of 4mol NADH and 2mol quinol per mole glucose consumed, all of which require re-oxidation to render cells viable. The overall stoichiometry and yield potential of this route can be summarized as follows: glucose->2GA+2CO₂+4NADH +2 quinol +2 ATP; y is 0.84g/g, y (max) is 1.70g/g, y is 49% of y (max).

The production of GA by a pentose derivative-based pathway to glycolaldehyde is described in PCT publication nos. WO2017/059236 and WO 2016/79440, and U.S. pre-grant publication nos. US2016/0076061 and US2015/0147794, the entire contents of which are incorporated herein by reference. However, these approaches also have the potential for reduced overall yields. For example, GA production using xylose as a source has a reduced yield potential of 1.01g _ GA/g _ xylose, while the thermodynamic maximum yield of xylose → GA conversion is 1.71 g/g. The overall stoichiometry and yield potential of the xylose-based pathway can be summarized as follows: xylose->2GA+1CO₂+3NADH +1 quinol +0 ATP; y is 1.01g/g, Y (max) is 1.71g/g, and Y is 59% of Y (max). As can be seen from this equation, the xylose-based pathway also produces excess NADH and CO₂。

PCT publication No. WO 2015/181074, incorporated herein by reference in its entirety, discloses a method for producing D-erythrose and the subsequent conversion of D-erythrose to glycolaldehyde. Glycolaldehyde may be further converted to glycolic acid and/or glycine. This pathway has a reduced yield potential of 1.27g _ GA/g _ glucose, while the thermodynamic maximum yield is 1.70 g/g. The overall stoichiometry and yield potential based on the erythrose pathway can be summarized as follows: glucose- >3GA +2NADH +1 quinol-1 ATP, Y1.27 g/g, Y (max) 1.70g/g, Y75% of Y (max). This pathway is also not redox balanced and has a high excess of 2mol NADH and 1mol quinol per mole glucose consumed, all of which require re-oxidation to render cells viable.

The serine/hydroxypyruvate pathway is described for GA production in U.S. patent No. 8,911,978, which is incorporated herein by reference in its entirety.

All these pathways produce excess NADH and release excess CO₂I.e. these routes do not reach the maximum thermodynamically possible yield. They usually oxidize more sugar carbons than desired to CO₂Thereby reducing product yield.

The present application relates to recombinant glyoxylate producing microorganisms having one or more biosynthetic pathways for the production of Glycolic Acid (GA) and/or glycine. In one embodiment, the recombinant glyoxylate producing microorganism of the invention comprises a reverse glyoxylate shunt based route (route) which increases the yield of GA and glycine. In another embodiment, the recombinant glyoxylate producing microorganism of the invention comprises the previously described metabolic pathways and modifications for the production of GA and/or glycine and a reverse glyoxylate shunt based route to further increase the yield of GA and glycine. The terms "glycolic acid" and "glycolic acid" are used interchangeably throughout this disclosure.

Certain patent documents disclose the reverse glyoxylate shunt pathway. For example, U.S. patent No. 9,410,131 discloses a reverse glyoxylate shunt pathway for the production of oxaloacetate and malonyl-coa. U.S. pre-grant publication No. 2016/369292 discloses the use of the retro-glyoxylate shunt for producing isocitrate and acetyl-coa. EP patent No. 2738247B1 discloses the use of the retro-glyoxylate shunt for the production of acetyl-coa. However, none of these patent documents discloses a reverse glyoxylate shunt for increasing glyoxylate production and subsequently increasing glycolic acid and/or glycine production. Furthermore, none of these patent documents discloses a reverse glyoxylate shunt wherein acetyl-coa generated by the activity of malyl-coa lyase is re-incorporated into the metabolic pathway (e.g., citric acid is produced by combining oxaloacetate in the glyoxylate shunt) for increased production of glycolic acid and/or glycine.

The present disclosure provides for the first time a carbon fixation route for GA or glycine production making it suitable for most current CO-bearing applications₂And the GA and glycine pathways with excess NADH. By providing a suitable common pathway, the present disclosure addresses the problem of NADH excess in all Glycolic Acid (GA) and glycine pathways described so far, and enables higher GA and glycine yields than previously described individual pathways, including the recently published high-yield pathway using xylose.

The present disclosure provides for the first time a retro-glyoxylate shunt route that utilizes carboxylation reactions for the production of GA and glycine. None of the GA or glycine production pathways described to date utilize carboxylation reactions to synthesize GA or glycine.

In certain embodiments, the carboxylation-based retro-glyoxylate shunt pathway of the present disclosure can be used synergistically with known GA or glycine production pathways.

The present disclosure includes the use of the genes described herein, and/or homologues of the enzymes encoded by these genes, as well as natural variants, or engineered variants.

Microorganisms, pathways and methods of the invention

In one embodiment, the present invention provides a recombinant glyoxylate producing microorganism that produces glycolate and/or glycine from glyoxylate using a reverse glyoxylate shunt pathway. Expression of the reverse glyoxylate shunt pathway increases glyoxylate production as an intermediate and increases the production of the final products glycolic acid and glycine. In some embodiments, the glyoxylate-producing recombinant microorganism of the invention co-produces glycolic acid and glycine. In another embodiment, the glyoxylate-producing recombinant microorganism of the invention co-produces glycolic acid and another byproduct such as, but not limited to, succinic acid or lactic acid. In yet another embodiment, the glyoxylate-producing recombinant microorganism of the invention co-produces glycine and another byproduct, such as, but not limited to, succinic acid or lactic acid.

In some embodiments, the present disclosure provides a recombinant glyoxylate-producing microorganism for the synthesis of glycolate and/or glycin, wherein the microorganism comprises: (a) a gene encoding pyruvate carboxylase which catalyzes the conversion of pyruvate to Oxaloacetate (OAA), and/or a gene encoding phosphoenolpyruvate carboxylase which catalyzes the conversion of phosphoenolpyruvate to OAA, and/or a gene encoding phosphoenolpyruvate carboxykinase which catalyzes the conversion of phosphoenolpyruvate to OAA; (b) a gene encoding malate thiokinase catalyzing the conversion of malate into malyl-coa; (c) a gene encoding a malyl-coa lyase that catalyzes the conversion of malyl-coa to glyoxylate and acetyl-coa, wherein acetyl-coa produced by the malyl-coa lyase is combined with OAA to increase the biosynthesis of GA and/or glycine. In these embodiments, the recombinant microorganism has reduced phosphoglucose isomerase activity, or more preferably does not catalyze the conversion of glucose-6-phosphate to fructose-6-phosphate by the enzyme phosphoglucose isomerase. In addition, the recombinant microorganism may or may not comprise an overexpressed endogenous or exogenous enzyme: citrate synthase, isozymeCitrate lyase and/or glyoxylate reductase. By reducing the activity of the phosphoglucose isomerase, or more preferably by deleting the gene encoding the phosphoglucose isomerase (e.g. the gene pgi in E.coli), which catalyzes the conversion of glucose-6-phosphate to fructose-6-phosphate, the carbon source can be diverted at least in part to the pentose-phosphate pathway (PPP) to provide the additional NADPH that may be required for optimal conversion of glyoxylate to glycolate. In some embodiments, by using the carboxylase and carboxykinase suggested herein, it is possible to reincorporate the CO generated by the PPP route₂。

In one embodiment, the retro glyoxylate shunt-based pathway of the present disclosure comprises carboxylating pyruvate to malate; conversion of malate to malyl-coa (coa) and malyl-coa to glyoxylate and acetyl-coa. Accordingly, in one embodiment, provided herein is a recombinant microorganism comprising a gene encoding malate dehydrogenase converting pyruvate to malate, a gene encoding malate thiokinase converting malate to malyl-coa, and a gene encoding malyl-coa lyase converting malyl-coa to glyoxylate and acetyl-coa. In one embodiment, the gene encoding malate dehydrogenase encodes a modified malate dehydrogenase that catalyzes the conversion of pyruvate to malate but does not catalyze the reverse reaction of malate to pyruvate or exhibits reduced conversion of malate to pyruvate. In some embodiments, the gene encoding malate dehydrogenase may comprise a deletion or loss of function mutation. The modified malate dehydrogenase may be a naturally occurring variant, or an engineered variant.

In another embodiment, the reverse glyoxylate shunt-based pathway of the present disclosure comprises carboxylation of phosphoenolpyruvate (PEP) and/or pyruvate to Oxaloacetate (OAA); conversion of OAA to malic acid; malic acid to malyl-coa (coa), and malyl-coa to glyoxylic acid and acetyl-coa. Thus, in one embodiment, provided herein is a recombinant microorganism comprising the following genes: a gene encoding pyruvate carboxylase which converts pyruvate into OAA, and/or a gene encoding phosphoenolpyruvate carboxylase which converts phosphoenolpyruvate into OAA, and/or a gene encoding phosphoenolpyruvate carboxykinase which converts phosphoenolpyruvate into OAA in combination with a gene encoding malate dehydrogenase which catalyzes the conversion of OAA into malate, a gene encoding malate thiokinase which converts malate into malyl-CoA, and a gene encoding malyl-CoA lyase which converts malyl-CoA into glyoxylate and acetyl-CoA.

In another embodiment, the retrograde glyoxylate shunt-based pathway of the present disclosure comprises carboxylation of phosphoenolpyruvate (PEP) or pyruvate to Oxaloacetate (OAA), and/or pyruvate to malate; conversion of OAA to malic acid; malic acid to malyl-coa (coa), and malyl-coa to glyoxylic acid and acetyl-coa. Thus, in one embodiment, provided herein is a recombinant microorganism comprising the following genes: a gene encoding pyruvate carboxylase which converts pyruvate into OAA, and/or a gene encoding phosphoenolpyruvate carboxylase which converts phosphoenolpyruvate into OAA, and/or a gene encoding phosphoenolpyruvate carboxykinase which converts phosphoenolpyruvate into OAA; and/or a gene encoding a malate dehydrogenase that catalyzes the conversion of pyruvate to malate; and/or a gene encoding malate dehydrogenase that catalyzes the conversion of OAA to malate; a gene encoding malate thiokinase converting malate into malyl-coa; and a gene encoding malyl-coa lyase for converting malyl-coa to glyoxylate and acetyl-coa. In one embodiment, the gene encoding malate dehydrogenase encodes a modified malate dehydrogenase that catalyzes the conversion of pyruvate to malate, or OAA to malate, but does not catalyze the reverse reaction of malate to pyruvate, or malate to OAA, or exhibits reduced conversion of malate to pyruvate or malate to OAA. The modified malate dehydrogenase may be a naturally occurring variant, or an engineered variant.

In another embodiment, the reverse glyoxylate shunt-based pathway of the present disclosure comprises carboxylation of phosphoenolpyruvate (PEP) and/or pyruvate to Oxaloacetate (OAA); converting malic acid to malyl-coa (coa) and malyl-coa to glyoxylic acid and acetyl-coa; wherein the reverse glyoxylate shunt pathway does not involve conversion of OAA to malate. Thus, in one embodiment, provided herein is a recombinant microorganism comprising the following genes: a gene encoding pyruvate carboxylase which converts pyruvate into OAA, and/or a gene encoding phosphoenolpyruvate carboxylase which converts phosphoenolpyruvate into OAA, and/or a gene encoding phosphoenolpyruvate carboxykinase which converts phosphoenolpyruvate into OAA; a gene encoding malate thiokinase that converts malate to malyl-coa, and a gene encoding malyl-coa lyase that converts malyl-coa to glyoxylate and acetyl-coa, wherein the microorganism does not comprise a gene encoding malate dehydrogenase that catalyzes the conversion of OAA to malate, or comprises a loss of function mutation in a gene encoding malate dehydrogenase that catalyzes the conversion of OAA to malate.

In another embodiment, the reverse glyoxylate shunt-based pathway of the present disclosure comprises carboxylation of phosphoenolpyruvate (PEP) and/or pyruvate to Oxaloacetate (OAA); carboxylating pyruvic acid to malic acid; converting malic acid to malyl-coa (coa) and malyl-coa to glyoxylic acid and acetyl-coa; wherein the reverse glyoxylate shunt pathway does not comprise conversion of OAA to malate. Thus, in one embodiment, provided herein is a recombinant microorganism comprising the following genes: a gene encoding pyruvate carboxylase which converts pyruvate into OAA, and/or a gene encoding phosphoenolpyruvate carboxylase which converts phosphoenolpyruvate into OAA, and/or a gene encoding phosphoenolpyruvate carboxykinase which converts phosphoenolpyruvate into OAA; a gene encoding malate dehydrogenase that catalyzes the conversion of pyruvate to malate; a gene encoding malate thiokinase that converts malate to malyl-coa, and a gene encoding malyl-coa lyase that converts malyl-coa to glyoxylate and acetyl-coa, wherein the microorganism does not comprise a gene encoding malate dehydrogenase that catalyzes the conversion of OAA to malate, or comprises a loss of function mutation in a gene encoding malate dehydrogenase that catalyzes the conversion of OAA to malate.

Glyoxylate produced by the retro-glyoxylate shunt and other pathways can be converted to glycolate and/or glycine. To increase GA production, the recombinant microorganism of any of the embodiments disclosed herein can comprise a gene encoding an nad (p) H-dependent glyoxylate reductase that catalyzes the conversion of glyoxylate to glycolate. In one embodiment, the recombinant microorganism can overexpress nad (p) H-dependent glyoxylate reductase to increase GA yield. In another embodiment, the recombinant microorganism of any of the embodiments disclosed herein can comprise an gain-of-function mutation in a gene encoding an nad (p) H-dependent glyoxylate reductase, such that the nad (p) H-dependent glyoxylate reductase activity is increased as compared to a microorganism lacking the mutation.

The term "NAD (P) H-dependent" as used herein includes NADH-dependent as well as NADPH-dependent enzyme activities.

To increase glycine production, the recombinant microorganism of any of the embodiments disclosed herein may comprise one or more genes encoding enzymes that catalyze the conversion of glyoxylate to glycine. For example, the recombinant microorganism of any of the embodiments disclosed herein can comprise the following genes: a gene encoding alanine-glyoxylate transaminase, a gene encoding glycine dehydrogenase, a gene encoding glycine transaminase, a gene encoding serine-glyoxylate transaminase, and/or a gene encoding glycine oxidase. In one embodiment, the recombinant microorganism of any of the embodiments disclosed herein may overexpress one or more of these genes to increase the yield of glycine. In another embodiment, the recombinant microorganism of any of the embodiments disclosed herein can comprise gain-of-function mutations in one or more of the genes encoding glycine-producing enzymes described above, such that the activity of these genes is increased as compared to a microorganism lacking the mutation.

In one embodiment, the recombinant microorganism of the present disclosure does not produce malonyl-coa via the rGS pathway.

The recombinant microorganisms of the invention comprise a gene encoding malate dehydrogenase, which catalyzes the carboxylation of pyruvate to malate and/or catalyzes the reduction of OAA to malate. In one embodiment, the malate dehydrogenase catalyzing the conversion of OAA to malate is from, but not limited to, enzyme class (E.C.) 1.1.1.37. In another embodiment, the malate dehydrogenase catalyzing the conversion of malate to OAA is from EC 1.1.5.4. Malate dehydrogenase, which catalyzes the conversion of malate to OAA, is also called malate: a quinol oxidoreductase. In certain embodiments, the malate dehydrogenase that catalyzes the carboxylation of pyruvate to malate is from, but is not limited to, enzyme class (E.C.)1.1.1.38, e.c.1.1.1.39, or e.c. 1.1.1.40.

In one embodiment, a recombinant microorganism of the present disclosure may comprise a gene encoding malate dehydrogenase, wherein the malate dehydrogenase may catalyze the conversion of pyruvate to malate and/or the conversion of OAA to malate, but does not catalyze the reverse reaction of malate to pyruvate or malate to OAA, or catalyzes the reverse reaction but has a reduced effect. In one embodiment, a recombinant microorganism of the present disclosure comprises a gene encoding malate dehydrogenase, wherein the malate dehydrogenase can catalyze the conversion of oxaloacetate to malate, but not pyruvate to malate, or malate to pyruvate. In one embodiment, the gene encoding malate dehydrogenase may comprise a mutation resulting in partial or complete inhibition of malate dehydrogenase activity catalyzing the conversion of oxaloacetate to malate, or malate to oxaloacetate, or pyruvate to malate, or malate to pyruvate.

In an exemplary embodiment, the gene encoding malate dehydrogenase that catalyzes the carboxylation of pyruvate to malate is derived from, but is not limited to, a bacterium such as Escherichia (e.g., a gene maeA or maeB from Escherichia coli), Pseudomonas, Bacillus (e.g., a gene maeA from Bacillus subtilis), Rhizobium (e.g., a gene dme from Rhizobium melilote (R.melilote)), Mycobacterium (e.g., a gene mez from Mycobacterium tuberculosis), Salmonella (e.g., a gene maeB derived from; or from a yeast such as a gene mae1 from Saccharomyces cerevisiae), or from a plant (e.g., a gene nad-me1, or nad-me2, or nad-me3, or nadp-me1, or nadp-me2 from Arabidopsis thaliana).

In an exemplary embodiment, the gene encoding malate dehydrogenase that catalyzes the conversion of oxaloacetate to malate is derived from, but is not limited to, bacteria such as escherichia (e.g., gene mdh from escherichia coli), corynebacterium, streptomyces (e.g., gene mdh from streptomyces coelicolor); or from a yeast, such as yeast (e.g., gene mdh1/2/3 from Saccharomyces cerevisiae); or from a plant, such as Arabidopsis thaliana. In another embodiment, the gene encoding malate dehydrogenase that catalyzes the conversion of malate to oxaloacetate (also referred to as malate: quinol oxidoreductase) is from, but not limited to, Escherichia (e.g., gene mqo from E.coli), Pseudomonas (e.g., gene mqo from Pseudomonas putida), or Bacillus.

Malic acid is converted to malyl-coa by malate thiokinase activity (also known as malate-coa ligase or malyl-coa synthetase), or by succinyl-coa ligase activity (also known as succinyl-coa synthetase). In one embodiment, the malate thiokinase is from, but not limited to EC6.2.1.4, EC 6.2.1.5, EC 6.2.1.9, or EC6.2.1. In an exemplary embodiment, the gene encoding sulfomalate kinase or succinyl-coa ligase is from a bacterium such as escherichia (e.g., sucCD-2 gene from escherichia coli), Thermus thermophilus (Thermus thermophilus), Clostridium kluyveri (Clostridium kluyveri), Bacillus subtilis (Bacillus subtilis), methanococcus (methanococcus) (e.g., mtkAB or sucCD gene from methanococcus jannaschii), Staphylococcus aureus (Staphylococcus aureus), methanotrophus thermoautotrophicum (methanotrophus), pseudomonas, Methylococcus (methanococcus sp.), methylobacterium (e.g., mtkabab or pseudomonas), methylobacterium (e.g., mtneb or pseudomonas nitrobacter) gene from methylobacterium (m. exquerystorens), pseudomonas aeruginosa (pseudomonas aeruginosa), pseudomonas aeruginosa (pseudomonas), pseudomonas aeruginosa (Bacillus subtilis), pseudomonas denitrificans (Bacillus nitrobacter), pseudomonas (pseudomonas), pseudomonas aeruginosa (Bacillus subtilis), pseudomonas (pseudomonas aeruginosa (pseudomonas), pseudomonas (pseudomonas), pseudomonas (e, pseudomonas), pseudomonas (e, pseudomonas), pseudomonas (pseudomonas), pseudomonas (pseudomonas), pseudomonas (pseudomonas), pseudomonas (pseudomonas), pseudomonas (pseudomonas), pseudomonas (pseudomonas), pseudomonas (pseudomonas), pseudomonas (pseudomonas), pseudomonas (pseudomonas), pseudomonas (pseudomonas), pseudomonas (pseudomonas), pseudomonas (pseudomonas), pseudomonas (pseudomonas), pseudomonas (pseudomonas), pseudomonas (pseudomonas), pseudomonas (pseudomonas), pseudomonas (pseudomonas), pseudomonas (pseudomonas), pseudomonas (pseudomonas), pseudomonas (pseudomonas), pseudomonas (pseudomonas), pseudomonas (pseudomonas), pseudomonas (pseudomonas), pseudomonas (pseudomonas), pseudomonas) or pseudomonas (pseudomonas), pseudomonas (pseudomonas), pseudomonas (pseudomonas) or pseudomonas (pseudomonas), pseudomonas (pseudomonas), methylococcus capsulatus, Rhodobacteraceae (Rhodobacteraceae) or Rhizobium (Rhizobium). In one embodiment, the malate thiokinase or succinyl-coa ligase has high activity and/or high specificity for malate and low activity and/or low specificity for other compounds such as succinate. This can be achieved by enzymatic engineering.

Maloyl-CoA is converted by maloyl-CoA lyase to glyoxylate and acetyl-CoA. In one embodiment, the malyl-coa lyase is from, but not limited to, EC 4.1.3.24 or EC 4.1.3.25. In an exemplary embodiment, the gene encoding malyl-coa lyase is from methylobacterium (e.g., gene mclA from demethylobacterium), demethylobacterium, Thalassobius activus, rhodobacter (e.g., gene mcl1 from rhodobacter sphaeroides (r. sphaeroides)), roseobacterium maritima (Roseobacter litoralis), streptomycetes, streptococcus, mycobacterium ((e.g., gene mcl1 from mycobacterium smegmatis), humicola fulva (hyphomium methylvorum), rosa (Roseobacter) (e.g., gene mcl1 from roseobacterium maritima), Nitrosomonas (nitromonas europaea), cuprum pesticidum (cuppriavium monocator), pseudomonas aeruginosa (e.g., chlorella), gene mcl from phomophilus pyrococcus (c.aurantiacus), Nereida (e.g. gene mcl1 from n.ignawava), hyphomycete denitrificans (rhodobacter denitificans), Rhodococcus fascians (Rhodococcus fascians).

PEP is carboxylated to oxaloacetate by phosphoenolpyruvate carboxylase or phosphoenolpyruvate carboxykinase. In one embodiment, the PEP carboxylase is from, but not limited to, EC 4.1.1.31. In an exemplary embodiment, the gene encoding the PEP carboxylase is from, but is not limited to, bacteria, such as escherichia (e.g., the gene ppc from escherichia coli), Rhodothermus (Rhodothermus) (e.g., the gene ppc or pepC from Rhodothermus marinus (r. marinus)), corynebacterium, salmonella, Hyphomicrobium (Hyphomicrobium), streptococcus, streptomyces, Pantoea (Pantoea), bacillus, clostridium, pseudomonas, Rhodopseudomonas (Rhodopseudomonas), methanobacter methanophilus (methanobacteria) (e.g., the gene ppcA from methanothermus thermoautotrophicum); plants, such as hybrid sugarcane (Saccharum hybrid), soybean (glycine) (e.g. the gene ppc from soybean (g.max)), Nicotiana tabacum (Nicotiana tabacum), Amaranthus (Amaranthus hypochondriacus), Triticum aestivum (Triticum aestivum), alfalfa (Medicago sativa), maize (Zea mays) (e.g. the gene pep1) or arabidopsis thaliana (e.g. the gene ppc1 or ppc2 or ppc3 from arabidopsis thaliana (a.thaliana)); archaea or yeast. In one embodiment, the phosphoenolpyruvate carboxykinase is from, but not limited to, EC 4.1.1.32 or EC 4.1.1.49. In an exemplary embodiment, the encoded PEP carboxykinase is from, but is not limited to, bacteria such as escherichia (e.g., gene pck or pckA from escherichia coli), Selenomonas (e.g., gene pckA from Selenomonas ruminata), salmonella (e.g., gene pckA from salmonella typhimurium), mycobacteria, pseudomonas, rhodopseudomonas, clostridium, thermus (Thermococcus), streptococcus (e.g., gene pck or pckA from streptococcus bovis (s.bovis), Ruminococcus (e.g., gene pck or pckA from Ruminococcus albus) and Ruminococcus flavus (r.flavus), Actinobacillus (e.g., gene pck or pckA from Actinobacillus succinogenes), bacillus (r.flavus), bacillus subtilis), bacillus (r.thermus), bacillus (reicola), and clostridium (reichersoniana), and clostridium (reimbucillus (reissulus) producing bacteria (e.g., bacteria), bacillus (reissus) Thermus (Thermus); yeasts, such as yeasts (Saccharomyces) (e.g.genes pck1 or pepc or ppc1 from Saccharomyces cerevisiae); or Trypanosoma (Trypanosoma) (e.g., a gene from Trypanosoma brucei (t.

Pyruvate carboxylation to oxaloacetate is catalyzed by pyruvate carboxylase. In one embodiment, the pyruvate carboxylase is from, but not limited to, EC 6.4.1.1. In an exemplary embodiment, the gene encoding pyruvate carboxylase is from a bacterium, such as bacillus (e.g., gene pyc from bacillus subtilis), candida (e.g., gene pyc1 from candida glabrata (c.glabrata)), cuprioides (cuprioidus) (e.g., gene pyc1 from copper insecticidal (c.necator)), mycobacteria (e.g., gene pyc from mycobacterium smegmatis), Corynebacterium (Corynebacterium) (e.g., gene pyc from Corynebacterium glycinatum (c.glycithiilum)), nocardia (e.g., gene pyc1 from nocardia neostella (n.nova)); or yeasts such as yeasts (Saccharomyces) (e.g., genes pyc1 and pyc2 from Saccharomyces cerevisiae), Pichia (Pichia) (e.g., pyc from Pichia pastoris); or Caenorhabditis (e.g., pyc from Caenorhabditis elegans); or from Homo sapiens (Homo sapiens).

NADH glyoxylate reductase or NADPH glyoxylate reductase reduces glyoxylate to produce glycolic acid. In one embodiment, the NADH glyoxylate reductase is from EC 1.1.1.26. In one embodiment, the NADPH glyoxylate reductase is from EC 1.1.1.79. In an exemplary embodiment, the genes encoding NADH-or NADPH-dependent glyoxylate reductase activity are the genes "ycdW/ghrA" and/or "yiAE" in E.coli, the gene "GLYR 1" from Arabidopsis thaliana, the gene "GOR 1" from Saccharomyces cerevisiae, and the "gyAR" from Thermococcus litoralis. In some embodiments, the cofactor preference (NADH or NADPH) of an enzyme can be altered by enzyme engineering. In some embodiments, the enzyme NADPH-dependent glyoxylate reductase encoded by the gene "ycdW/ghrA" or "yiaE" from e.coli, or "GLYR 1" from arabidopsis thaliana, is engineered to be an NADH-dependent glyoxylate reductase that accepts NADH and the cofactor NADPH accepted by the native enzyme, and which still exhibits the same glyoxylate to glycolate conversion properties (i.e., converts the cofactor without compromising its kinetic parameters in the desired reaction).

In one embodiment, glyoxylate production, and ultimately glycolic acid and/or glycine production, can be increased by co-utilizing the rGS pathway with the Glyoxylate Shunt (GS) pathway. For example, acetyl-coa produced in the rGS pathway (i.e., acetyl-coa produced by malyl-coa lyase activity on malyl-coa) may be re-incorporated into the metabolic pathway to further increase glyoxylate production: that is, citric acid is converted to isocitric acid, which is converted to succinic acid and glyoxylic acid by entering the GS pathway to combine with OAA to produce citric acid. Succinate produced by the GS pathway can be converted to malate via fumarate, malate produced by this pathway can enter the rGS pathway where it is converted to malyl-coa, which is further converted to glyoxylate and acetyl-coa.

In the recombinant microorganism of the present invention, the rGS pathway may be operated first followed by the GS pathway, or the GS pathway may be operated first followed by the rGS pathway.

In one embodiment where rGS and the GS pathway are utilized together, the PEP may be converted to OAA (PEP carboxylase or PEP carboxykinase) and/or pyruvate may be converted to OAA (pyruvate carboxylase) or to malate (malate dehydrogenase); OAA can be converted to malate (malate dehydrogenase); malic acid can be converted to malyl-coa (malate thiokinase); malyl-coa can be converted to glyoxylate and acetyl-coa (malyl-coa lyase); acetyl-coa can combine with OAA to form citrate (citrate synthase); citric acid can be converted into aconitic acid (citrate hydrolase); aconitic acid can be converted into isocitric acid (D-threo isocitric acid hydrolase or aconitase); isocitrate can be converted to succinate and glyoxylate (isocitrate lyase); succinic acid can be converted to fumaric acid (succinate dehydrogenase); and fumaric acid can be converted into malic acid (fumarase). Malic acid can re-enter the rGS pathway and can be converted to malyl-coa.

In another embodiment that utilizes both rGS and the GS pathway, PEP may be converted to OAA (PEP carboxylase or PEP carboxykinase) and/or pyruvate may be converted to OAA (pyruvate carboxylase) or to malate (malate dehydrogenase); OAA can be converted to citrate (citrate synthase) by combination with acetyl-coa; citric acid can be converted into aconitic acid (citrate hydrolase); aconitic acid can be converted into isocitric acid (D-threo isocitric acid hydrolase or aconitase); isocitrate can be converted to succinate and glyoxylate (isocitrate lyase); succinic acid can be converted to fumaric acid (succinate dehydrogenase); fumaric acid can be converted into malic acid (fumarase); and malate may be converted to malyl-coa (malate thiokinase), and malyl-coa may be converted to glyoxylate and acetyl-coa. In this embodiment, OAA may be combined exclusively with acetyl-coa to form citric acid (i.e., by blocking conversion of OAA to malate, e.g., inactivating a malate dehydrogenase that catalyzes the conversion of OAA to malate), or a portion thereof, may be converted to malate.

The recombinant microorganism in any of the embodiments disclosed herein may comprise a gene encoding an enzyme involved in the GS pathway. In one embodiment, the recombinant microorganism comprises (a) a gene encoding citrate synthase that converts acetyl-coa and OAA to citrate; (b) a gene encoding citrate hydrolase which converts citric acid into aconitic acid; (c) a gene encoding D-threo isocitrate hydrolase or aconitase converting cis-aconitic acid into isocitric acid; (d) a gene encoding an isocitrate lyase that converts isocitrate to succinate and glyoxylate; (e) a gene encoding succinate dehydrogenase converting succinate into fumarate; and (f) a gene encoding a fumarase for converting fumarate into malate.

Glyoxylate produced by the GS and rGS pathway can be converted to malate by malate synthase. However, this reaction reduces the yield of glyoxylic acid and thus the production of GA and glycine. Thus, in one embodiment, a loss-of-function mutation in a gene encoding malate synthase may be comprised in a recombinant microorganism described herein. Loss of function mutations as referred to herein can result in complete or partial loss of function. Loss-of-function mutations may also include complete deletions of the gene of interest. In an exemplary embodiment, genes encoding malate synthase that can be inactivated according to the present disclosure include aceB and/or glcB in E.coli or DAL7 and MLS1 in Saccharomyces cerevisiae.

In one embodiment, the flux ratio (flux ratio) of co-utilized rGS and GS is adjusted to obtain the lowest possible net NADH production corresponding to the optimal yield, based on the amount of excess NADH in a given pathway.

The one or more genes encoding the enzymes of interest disclosed herein can be endogenously present, can be inserted into the genome of the microorganism and/or can be expressed by one or more vectors (e.g., plasmids, cosmids, viral vectors, etc.) introduced into the microorganism. High levels of enzymatic activity can be obtained by using or inserting copies of one or several genes on the genome, which can be introduced on the genome by recombinant methods known to those of ordinary skill in the art. For expression by means of vectors, different types of vectors can be used, for example plasmids which differ in their origin of replication and thus in their copy number in the cell. Exemplary plasmids for expressing a gene of interest include, but are not limited to, pSK bluescript II, pSC101, RK2, pACYC, pRSF1010, etc.). The gene encoding the enzymatic polypeptide may be expressed using promoters of different strengths that may or may not be inducible by the inducing molecule. Examples of promoters include Ptrc, Ptac, Plac, lambda promoter cI, or other promoters known to those of ordinary skill in the art. Expression of a gene can also be enhanced by elements (Carrier and Keasling (1998) biotechnol. prog.15,58-64) or proteins (e.g., GST tags, Amersham Biosciences) that stabilize the corresponding messenger RNAs.

In one embodiment, the endogenous Glyoxylate Shunt (GS) pathway and/or other central metabolic pathways in the recombinant microorganism can be modified, for example, by avoiding competitive routes or byproduct formation, and bypassing carbon loss reactions to maximize carbon influx to biosynthesis via the reverse glyoxylate shunt to glycolate and/or glycine. For example, in one embodiment, a recombinant microorganism comprising the rGS pathway may be modified to delete or attenuate expression of at least one gene encoding an enzyme selected from the group consisting of:

(a) malate synthase (e.g., gene aceB and/or glcB in E.coli or gene DAL7 and MLS1 in Saccharomyces cerevisiae);

(b) isocitrate dehydrogenases (e.g., gene icd in E.coli or genes IDP2 and IDH1/2 in Saccharomyces cerevisiae);

(c) pyruvate dehydrogenase (e.g., pdhc and/or lpd gene in E.coli), pyruvate oxidase (e.g., poxB gene in E.coli), and/or pyruvate formate lyase (e.g., pfl gene in E.coli); and

(d) pyruvate kinase (e.g., the gene pykA and/or pykF in E.coli).

In some embodiments, the endogenous glyoxylate-consuming pathway in a recombinant microorganism comprising the rGS pathway can be deleted or attenuated to further increase the yield of glycolate and/or glycine. For example, in one embodiment, a recombinant microorganism comprising the rGS pathway is modified to delete or attenuate the expression, or inhibit the activity, of at least one gene selected from the group consisting of:

(a) a gene encoding glyoxylate aldehyde ligase (carboligase) (for example, gene gcl in E.coli);

(b) a gene encoding 2-oxo-4-hydroxyglutarate aldolase (e.g., edA in E.coli);

(d) genes encoding glycolate oxidase (for example, genes glcD, glcE, glcF and glcG in escherichia coli);

(e) a gene encoding the repressor of isocitrate lyase (for example, the gene iclR in E.coli); and

(f) the gene encoding glucose-6-phosphate isomerase (e.g., the gene pgi in Escherichia coli).

The expression of a gene or the inhibition of the activity of an enzyme encoded by the gene can be attenuated by introducing mutations into the gene which reduce the activity of the corresponding enzyme, or by replacing the native promoter by a low-strength promoter, or by using agents which destabilize the corresponding messenger RNA or protein. The expression of a gene, or the activity of an enzyme encoded by the gene, can be attenuated by deleting the corresponding gene from the microorganism using techniques known in the art.

In one embodiment, a recombinant microorganism of the present disclosure expresses a set of genes encoding:

(a) malate dehydrogenase catalyzing conversion of pyruvate to malate;

(b) malate thiokinase catalyzing the conversion of malate into malyl-coa; and

(d) optionally a phosphoenolpyruvate carboxylase which catalyses the conversion of PEP to oxaloacetate, and/or a phosphoenolpyruvate carboxykinase which catalyses the conversion of PEP to oxaloacetate, and/or a pyruvate carboxylase which catalyses the conversion of pyruvate to oxaloacetate;

and comprising at least one modification selected from the following:

(a) deleting or attenuating the gene encoding malate synthase;

(b) deleting or attenuating a gene encoding isocitrate dehydrogenase;

(d) deleting or attenuating a gene encoding malate dehydrogenase which catalyzes the conversion of oxaloacetate to malate, or malate to oxaloacetate (malate: quinol oxidoreductase); and

(e) deleting or attenuating the gene encoding pyruvate kinase.

In another embodiment, a recombinant microorganism of the present disclosure expresses a set of genes encoding:

(a) a phosphoenolpyruvate carboxylase which catalyzes the conversion of PEP to oxaloacetate, and/or a phosphoenolpyruvate carboxykinase which catalyzes the conversion of PEP to oxaloacetate, and/or a pyruvate carboxylase which catalyzes the conversion of pyruvate to oxaloacetate, and/or a malate dehydrogenase which catalyzes the conversion of pyruvate to malate;

(b) malic acid thiokinase;

(d) optionally, a malate dehydrogenase that catalyzes the conversion of oxaloacetate to malate;

and comprising at least one modification selected from the following:

(a) deleting or attenuating the gene encoding malate synthase;

(b) deleting or attenuating a gene encoding isocitrate dehydrogenase;

(d) deleting or attenuating a gene encoding a malate dehydrogenase that catalyzes the conversion of malate to pyruvate; and

(e) deleting or attenuating the gene encoding pyruvate kinase.

In one embodiment, a recombinant microorganism comprising the rGS pathway expresses a set of genes encoding:

(a) malate dehydrogenase catalyzing conversion of pyruvate to malate;

(b) malate thiokinase catalyzing the conversion of malate into malyl-coa; and

and comprising at least one modification selected from the following:

(a) deleting or attenuating the gene encoding malate synthase;

(b) deleting or attenuating a gene encoding isocitrate dehydrogenase;

(d) deleting or attenuating a gene encoding malate dehydrogenase catalyzing the conversion of oxaloacetate to malate, or malate to oxaloacetate;

(e) deleting or attenuating a gene encoding pyruvate kinase;

(f) deleting or attenuating a gene encoding glyoxylate aldehyde ligase;

(g) deleting or attenuating a gene encoding 2-oxo-4-hydroxyglutarate aldolase;

(h) deleting or attenuating the gene encoding glycolaldehyde reductase;

(i) deleting or attenuating a gene encoding glycolate oxidase; and

(j) deleting or attenuating a gene encoding a repressor of isocitrate lyase.

In one embodiment, the total stoichiometry of glycolic acid produced using the rGS pathway and/or the modifications in one or more of the genes described above is: glucose +2CO₂->2GA +2 acetyl-coenzyme A-2NAD (P) H-2 ATP. In another embodiment, the total stoichiometry of glycolic acid produced using the rGS pathway and/or the modifications in one or more of the genes described above is: glucose +2CO₂+2NAD(P)H+2ATP->4GA +2 quinol.

Redox balance is important to fine-tune metabolic pathways to achieve maximum product yield potential. An imbalance in redox status (e.g., an imbalance in intracellular pool (pool) of NADPH and NADH co-factors, an imbalance in net ATP, and/or a lack of reducing agents) can result in lower product yields and the production of unwanted by-products. The present disclosure includes recombinant microorganisms with fine-tuned redox balance and methods of use thereof. For example, a recombinant microorganism in any of the embodiments disclosed herein can comprise a gene encoding a transhydrogenase and/or an NAD kinase to increase NADH and/or NADPH concentration within the cell, thereby achieving increased product yield. In exemplary embodiments, the recombinant microorganism in any of the embodiments disclosed herein can comprise a gene encoding a transhydrogenase (e.g., "pntAB" and/or "udhA" from e.coli), and/or a gene encoding an NAD kinase (e.g., "yfjB" from e.coli). Expression of these genes can, for example, increase intracellular NADPH concentrations to increase NADPH-dependent glyoxylate reductase activity to facilitate conversion of glyoxylate to glycolate.

The use of transhydrogenases (e.g., the genes pntAB and/or udhA in e.coli), and/or NAD kinases (e.g., the gene yfjB in e.coli) to increase intracellular NADH or NADPH concentrations has been described in US20140335578, Cui et al, Microbial Cell industries 2014,13:21, and Shi et al, Metabolic Engineering16(2013) 1-10; the entire contents of which are incorporated herein by reference.

In one embodiment, reducing agents such as sulfur-containing compounds (e.g., sulfites, sulfur dioxide, and cysteine) and/or hydrogen may be added to the media as an additional source of reducing power to adjust the redox balance in the metabolic pathway to increase the yield of the product. In another embodiment, an exogenous source of hydrogen or other additional electron/NAD (P) H source may be added to the medium for metabolic pathways with negative balance of NADH or NADPH.

In certain embodiments, the malic acid-encoding protein is inactivated by deletion or attenuation in a recombinant microorganism comprising a reverse GS pathway: the gene for quinol oxidoreductase (also known as malate dehydrogenase).

The rGS pathway of the present disclosure can be combined with known GA and glycine production pathways. Currently known GA and/or glycine production pathways include the serine/hydroxypyruvate pathway, described in U.S. patent nos. 8,911,978; the Glyoxylate Shunt (GS) pathway, described in U.S. patent nos. 9,034,615 and 8,945,888, PCT publication No. WO 2016/193540, and U.S. pre-grant publication No. 2014/0295510; the D-erythrose-based pathway is described in PCT publication No. WO 2015/181074; routes based on pentose derivatives to glycolaldehyde are described in PCT publication nos. WO2017/059236 and WO 2016/79440 and U.S. pre-grant publication nos. US2016/0076061 and US 2015/0147794. All these pathways produce excess NADH and release excess CO2, i.e. they do not reach the maximum thermodynamically possible yield. By combining these known GA and glycine producing pathways with the rGS pathway of the present disclosure, the yield of GA and/or glycine can be significantly increased.

GA yields using some of the previously published routes:

the serine/hydroxypyruvate pathway:

1 glucose->->2GA+2CO₂+6NADH+0ATP；y＝0.84g/g，Y(max)1.70g/g, y is 49% of Y (max)

The GS pathway:

glucose->2GA+2CO₂+4NADH +2 quinol +2 ATP; y is 0.84g/g, Y (max) is 1.70g/g, y is 49% of Y (max)

The pentose derivative pathway using GS:

xylose->2GA+1CO₂+3NADH +1 quinol +0 ATP; y 1.01g/g, Y (max) 1.71g/g, y 59% of Y (max)

The erythrose pathway:

glucose- >3GA +2NADH +1 quinol-1 ATP, y ═ 1.27g/g, y (max) ═ 1.70g/g, y is 75% of y (max).

By combining or co-utilizing the above pathway with the rGS pathway of the present invention, the yield of GA can be significantly increased. For example, in certain embodiments, co-using a known GA production pathway with the rGS pathway of the invention can provide increased GA yields as follows:

the GS and rGS pathway:

glucose +2/3CO2+2/3ATP- >10/3GA +2 quinol; y 1.41g/g, Y (max) 1.69g/g, y 83% of Y (max)

The GS and rGS pathway (no flux on malate dehydrogenase):

glucose +2CO2+2NAD (P) H- >4GA +2 quinol; y 1.69g/g, Y (max) 1.69g/g, y is 100% of Y (max)

The pentose derivative pathway using GS and rGS:

xylose + CO2+1ATP- >3GA +1 quinol; y 1.52g/g, Y (max) 1.69g/g, y 90% of Y (max)

The serine, GS and rGS pathways:

glucose + CO2+1.5ATP- >3.5GA +1.5 quinol; y is 1.48g/g, y (max) is 1.69g/g, y is 88% of y (max).

In one embodiment, the reverse glyoxylate shunt pathway of the invention utilizes NADH and CO produced by other glycolate glycine production pathways and/or glycolaldehyde production pathways₂And/or CO supplied from an external source₂And/or HCO3^-And/or other carbon sources, thereby increasing yield potential. For example, in one embodiment, the present inventionThe reverse glyoxylate shunt pathway disclosed utilizes NADH and CO produced by the serine/hydroxypyruvate-based pathway described in U.S. Pat. No. 8,911,978₂. In another embodiment, the reverse glyoxylate shunt pathway utilizes NADH and CO produced by the glyoxylate shunt pathway₂The glyoxylate shunt pathway is described in U.S. patent nos. 9,034,615 and 8,945,888, PCT publication No. WO 2016/193540, and U.S. pre-grant publication No. 2014/0295510. In yet another embodiment, the retro glyoxylate shunt pathway utilizes NADH and CO generated by the D-erythrose and pentose derivative to glycolaldehyde based pathway₂Described in PCT publication nos. WO 2015/181074, WO2017/059236, and WO 2016/79440 and U.S. pre-grant publication nos. US2016/0076061 and US 2015/0147794.

Recombinant microorganisms of the present disclosure include bacteria, yeast, or fungi. In certain embodiments, the microorganism is selected from, but not limited to, enterobacteriaceae, clostridiaceae, bacillaceae, streptomycetaceae, corynebacteriaceae, and saccharomyces. In one embodiment, the microorganism is a species of Escherichia, Clostridium, Bacillus, Klebsiella, Pantoea, Salmonella, Lactobacillus, Corynebacterium, or Saccharomyces. In one embodiment, the microorganism is Escherichia coli, or Corynebacterium glutamicum, or Clostridium acetobutylicum, or Bacillus subtilis, or Saccharomyces cerevisiae.

Glycine production

Glyoxylate produced using any of the above pathways can be converted to glycine using a variety of enzymes. For example, glycine may be produced from glyoxylic acid by transamination of alanine, for example by alanine-glyoxylate aminotransferase (EC 2.6.1.44). Typically, the native pathway utilizes glutamate as an amino donor in another transamination reaction, catalyzing pyruvate by alanine transaminase (EC 2.6.1.2) to complement alanine. Can be prepared by adding a common nitrogen source NH₃Immobilization in the resulting 2-oxoglutarate to complement glutamate itself requires NAD (P) H glutamate synthase (EC 1.4.1.13, EC 1.4.1.14). The total stoichiometry is glyoxylic acid + NH3+1NAD (P) H->Glycine. Others may promote acetaldehydeEnzymes that convert acid to glycine include glycine dehydrogenase (e.c.1.4.1.10), glycine transaminase (e.c.2.6.1.4), serine-glyoxylate transaminase (e.c.2.6.1.45), and glycine oxidase (e.c. 1.4.3.19). Thus, a recombinant microorganism in any of the embodiments disclosed herein may comprise one or more genes selected from the group consisting of: a gene encoding alanine-glyoxylate aminotransferase (EC 2.6.1.44), a gene encoding glycine dehydrogenase (e.c.1.4.1.10), a gene encoding glycine aminotransferase (e.c.2.6.1.4), a gene encoding serine-glyoxylate aminotransferase (e.c.2.6.1.45) and/or a gene encoding glycine oxidase (e.c. 1.4.3.19).

In an exemplary embodiment, the gene encoding glycine dehydrogenase (e.c.1.4.1.10) may be from mycobacterium (e.g., mycobacterium tuberculosis, mycobacterium smegmatis), pseudomonas, flavobacterium (Xanthobacter sp), or bacillus.

In an exemplary embodiment, the gene encoding glycine transaminase (EC 2.6.1.4) may be from Rhodopseudomonas palustris (Rhodopseudomonas palustris), Lactobacillus (Lactobacillus sp.), Hydrogenobacter (Hydrogenobacter sp.), rat (Rattus sp.) or Rhodopseudomonas sp.

Yield of glycine using previously published or natural route:

glyoxylic acid (glyyxylic acid) using the glyoxylic acid branch:

glucose- >2 glyoxylic acid +6NADH +2 quinol +2 ATP; y is 0.82g/g, Y (max) is 2.51g/g, y is 33% of Y (max)

Glycine by transcarbamation of glyoxylic acid:

glucose +2NH₃->2 glycine +4NADH +2 quinol +2 ATP; y is 0.701g/g (glucose +2 NH)₃) Y (max) is 1.20g/g, y is 58% of Y (max)

Glycine decarboxylation by serine:

glucose +2THF +2NH₃->2 glycine +2M-THF +2NADH +0 ATP; y is 0.701g/g (glucose +2 NH)₃)。

Glycine yield by co-using the known pathway with the rGS pathway of the present invention:

pentose derivative pathway, GS and rGS, using glyoxylic acid transamination:

xylose +3NH₃+CO₂+1ATP->3 glycine +1 quinol; y is 1.12g/g (xylose +3 NH)₃) Y (max) is 1.25g/g, y is 90% of Y (max)

GS and rGS, using glyoxylic acid transamination:

glucose +10/3NH₃+2/3CO₂+2/3ATP->10/3 glycine +2 quinol; y is 1.06g/g (glucose +10/3 NH)₃) Y (max) is 1.24g/g, y is 85% of Y (max)

The GS and rGS pathway, using glyoxylate transamination (no flux on malate dehydrogenase):

glucose +4NH₃+2CO2+2NAD(P)H->4 glycine +2 quinol; y 1.24g/g, Y (max) 1.24g/g, y is 100% of Y (max)

The serine, GS and rGS pathways:

glucose +3.5NH₃+CO₂+1.5ATP->3.5 Glycine +1.5 Quinonol; y is 1.10g/g (glucose +3.5 NH)₃) Y (max) is 1.24g, and y is 89% of y (max).

In one embodiment, the expression level of at least one gene in a recombinant microorganism comprising the rGS pathway is increased to increase glycine production, said gene being selected from the group consisting of:

(a) a gene encoding alanine-glyoxylate aminotransferase;

(b) a gene encoding glycine dehydrogenase;

(d) a gene encoding serine-glyoxylate transaminase;

(e) a gene encoding glycine oxidase;

(f) a gene encoding alanine aminotransferase; and

(g) a gene encoding NAD (P) H-dependent glutamate synthase. In another embodiment, one or more of these genes may comprise an gain-of-function mutation that increases the activity of the enzyme encoded by these genes.

The recombinant glyoxylate-producing microorganism of the invention can also co-produce glycolic acid and glycine.

In some embodiments, the microorganisms of the present invention do not produce isopropanol. In one embodiment, the microorganism of the invention does not produce serine and/or glutamate by the reverse glyoxylate shunt pathway. In some embodiments, the microorganism of the invention may not comprise one or more enzymes that convert glycine to serine. For example, in one embodiment, a microorganism of the invention may comprise a loss-of-function mutation in a gene encoding serine hydroxymethyltransferase. In another embodiment, the microorganism may not comprise a glycine consuming pathway.

Microorganisms comprising the reverse glyoxylate pathway exhibit increased production of glycolate and glycine. In one embodiment, the microorganisms of the present disclosure lack a pathway for converting glycolic acid and/or glycine to other products or intermediates.

rGS pathway and other glycolic acid production pathways

In certain embodiments, recombinant microorganisms comprising a reverse glyoxylate shunt pathway utilize other carbon sources (CO) provided by other glycolate and/or glycine production pathways and/or exogenously₂And/or HCO₃ ^-And/or other carbonic acid) produced NADH and CO₂. For example, in one embodiment, the rGS pathway of the invention can be used in conjunction with the pentose derivative to glycolaldehyde based pathway described in WO2017/059236, US2016/0076061, US2015/0147794 and WO 2016/079440.

Thus, in one embodiment, a recombinant microorganism comprising the rGS pathway may further comprise the pathways and/or modifications described in these documents. For example, a recombinant microorganism comprising the rGS pathway may have reduced or eliminated xylulokinase activity, or reduced or eliminated expression of xylulokinase, may recombinantly express an enzyme that interconverts xylulose into ribulose, may recombinantly express a D-ribulose-phosphate aldolase (e.g., a fucA gene from e.coli), may recombinantly express a D-ribulose kinase (e.g., a gene fucok from e.coli), and/or may recombinantly express a glycolaldehyde dehydrogenase, such as aldehyde dehydrogenase a (e.g., a gene aldA from e.coli).

Recombinant microorganisms comprising the rGS pathway may have reduced or eliminated xylulokinase activity, or reduced or eliminated expression of xylulokinase, recombinant expression of an enzyme that converts D-xylulose to D-xylulose 1P (e.g., khk-C from homo sapiens), recombinant expression of a D-xylulose 1-phosphate aldolase (e.g., gene aldoB from homo sapiens), and recombinant expression of a glycolaldehyde dehydrogenase, such as aldehyde dehydrogenase A (e.g., gene aldA from E.coli). A recombinant microorganism comprising the rGS pathway may have reduced or eliminated activity of an enzyme that interconverts xylose to D-xylulose (e.g., the gene xylA from E.coli), or reduced or eliminated expression of said enzyme, recombinantly expressing an enzyme that converts xylose to D-xylonic acid, recombinantly expressing an enzyme that converts D-xylonic acid to 2-dehydro-3-deoxy-D-pentanoate (2-dehydro-3-deoxy-D-pentanate, DPP) (e.g., gene yagF from E.coli), recombinantly expressing a 2-keto-3-deoxy-D-pentanoate aldolase (e.g., gene yagE from E.coli), and recombinantly expressing a glycolaldehyde dehydrogenase, e.g., aldehyde dehydrogenase A (e.g., gene aldA from E.coli).

In some embodiments, a recombinant microorganism comprising the rGS pathway may comprise a deletion of a gene encoding xylulokinase (e.g., the gene xylB from e. In some embodiments, the enzyme that interconverts xylulose and ribulose is a D-tagatose 3-epimerase (e.g., gene dte from Pseudomonas cichorii). In certain embodiments, the D-tagatose 3-epimerase is encoded by the dte gene from pseudomonas chicory (p.cichorii), which is codon optimized for escherichia coli or saccharomyces cerevisiae. In some embodiments, a recombinant microorganism comprising the rGS pathway may have reduced or eliminated glycolaldehyde reductase activity, or reduced or eliminated expression of the enzyme. For example, the recombinant microorganism can comprise a deletion of a gene encoding glycolaldehyde reductase (e.g., gene fucO).

In another embodiment, the rGS pathway of the invention can be utilized in conjunction with the serine/hydroxypyruvate pathway described in U.S. patent No. 8,911,978. Thus, in one embodiment, a recombinant microorganism comprising the rGS pathway may exhibit increased expression levels of pyruvate decarboxylase (e.g., pyruvate decarboxylase encoded by genes Pdc1, Pdc5, Pdc6 from yeast), aldehyde dehydrogenase (e.g., aldehyde dehydrogenase encoded by genes aldA, aldB, aldH, and gabD), serine transaminase, and/or serine oxidase.

In another embodiment, the recombinant microorganism comprising the rGS pathway may further comprise genetic modifications described below: U.S. patent nos. 9,034,615, and 8,945,888; PCT publication nos. WO 2016/193540 and WO 2015/181074; and U.S. pre-grant publication number 2014/0295510.

Method

The present invention provides methods for producing glycolic acid and glycine using any of the recombinant microorganisms described herein.

In one embodiment, the method comprises culturing in a suitable culture medium a recombinant microorganism expressing a gene encoding malate dehydrogenase catalyzing the conversion of pyruvate to malate, a gene encoding malate thiokinase catalyzing the conversion of malate to malyl-coa, and a gene encoding malyl-coa lyase catalyzing the conversion of malyl-coa to glyoxylate and acetyl-coa. The gene encoding malate dehydrogenase may encode a malate dehydrogenase which catalyses the conversion of pyruvate to malate and/or the conversion of OAA to malate, but preferably does not catalyse (or catalyses less efficiently) the reverse reaction from malate to pyruvate or from malate to OAA.

In one embodiment, the method comprises culturing in a suitable culture medium a recombinant microorganism expressing a gene encoding a pyruvate carboxylase which catalyzes the conversion of pyruvate to OAA, and/or a gene encoding a phosphoenolpyruvate carboxylase which catalyzes the conversion of phosphoenolpyruvate to OAA, and/or a gene encoding a phosphoenolpyruvate carboxykinase which catalyzes the conversion of phosphoenolpyruvate to OAA, in combination with a gene encoding a malate thiokinase which catalyzes the conversion of malate to malyl-coa, and a gene encoding a malyl-coa lyase which catalyzes the conversion of malyl-coa to glyoxylate and acetyl-coa, wherein the acetyl-coa produced by the malyl-coa lyase is combined with OAA to increase the biosynthesis of GA and/or glycine. In the same embodiment, the recombinant microorganism may or may not have a downregulation or deletion of glucose-6-phosphate isomerase, pyruvate kinase, pyruvate dehydrogenase and/or malate dehydrogenase.

In one embodiment, the method comprises culturing in a suitable culture medium a recombinant microorganism expressing a gene encoding a pyruvate carboxylase which catalyzes the conversion of pyruvate to OAA, and/or a gene encoding a phosphoenolpyruvate carboxylase which catalyzes the conversion of phosphoenolpyruvate to OAA, and/or a gene encoding a phosphoenolpyruvate carboxykinase which catalyzes the conversion of phosphoenolpyruvate to OAA, in combination with a gene encoding a malate dehydrogenase which catalyzes the conversion of OAA to malate, a gene encoding a malate thiokinase which catalyzes the conversion of malate to malyl-CoA, and a gene encoding a malyl-CoA lyase which catalyzes the conversion of malyl-CoA to glyoxylate and acetyl-CoA. The gene encoding malate dehydrogenase may encode a malate dehydrogenase which catalyses the conversion of pyruvate to malate and/or the conversion of OAA to malate, but preferably does not catalyse (or catalyses less efficiently) the reverse reaction from malate to pyruvate or from malate to OAA.

In another embodiment, the method comprises culturing in a suitable culture medium a recombinant microorganism expressing a gene encoding a pyruvate carboxylase catalyzing the conversion of pyruvate to OAA, and/or a gene encoding a phosphoenolpyruvate carboxylase catalyzing the conversion of phosphoenolpyruvate to OAA, and/or a gene encoding a phosphoenolpyruvate carboxykinase catalyzing the conversion of phosphoenolpyruvate to OAA; a gene encoding malate dehydrogenase catalyzing the conversion of pyruvate to malate; a gene encoding malate dehydrogenase catalyzing the conversion of OAA to malate; a gene encoding malate thiokinase catalyzing the conversion of malate into malyl-coa; and a gene encoding malyl-coa lyase which catalyzes the conversion of malyl-coa to glyoxylate and acetyl-coa. In one embodiment, the gene encoding malate dehydrogenase encodes a modified malate dehydrogenase that catalyzes the conversion of pyruvate to malate or OAA to malate but does not catalyze the reverse reaction from malate to pyruvate, or from malate to OAA, or exhibits a reduced conversion from malate to pyruvate, or from malate to OAA.

In another embodiment, the method comprises culturing in a suitable culture medium a recombinant microorganism expressing a gene encoding a pyruvate carboxylase catalyzing the conversion of pyruvate to OAA, and/or a gene encoding a phosphoenolpyruvate carboxylase catalyzing the conversion of phosphoenolpyruvate to OAA, and/or a gene encoding a phosphoenolpyruvate carboxykinase catalyzing the conversion of phosphoenolpyruvate to OAA; a gene encoding malate thiokinase which catalyzes the conversion of malate into malyl-coa, and a gene encoding malyl-coa lyase which catalyzes the conversion of malyl-coa into glyoxylate and acetyl-coa, wherein the microorganism does not comprise a gene encoding malate dehydrogenase which catalyzes the conversion of OAA into malate, or comprises a deletion or loss-of-function mutation in a gene encoding malate dehydrogenase which catalyzes the conversion of OAA into malate.

In another embodiment, the method comprises culturing in a suitable culture medium a recombinant microorganism expressing a gene encoding a pyruvate carboxylase catalyzing the conversion of pyruvate to OAA, and/or a gene encoding a phosphoenolpyruvate carboxylase catalyzing the conversion of phosphoenolpyruvate to OAA, and/or a gene encoding a phosphoenolpyruvate carboxykinase catalyzing the conversion of phosphoenolpyruvate to OAA; a gene encoding malate dehydrogenase catalyzing the conversion of pyruvate to malate; a gene encoding malate thiokinase which catalyzes the conversion of malate into malyl-coa, and a gene encoding malyl-coa lyase which catalyzes the conversion of malyl-coa into glyoxylate and acetyl-coa, wherein the microorganism does not comprise a gene encoding malate dehydrogenase which catalyzes the conversion of OAA into malate, or comprises a loss of function mutation in a gene encoding malate dehydrogenase which catalyzes the conversion of OAA into malate.

In one embodiment, glyoxylate is reduced to glycolate by an NAD (P) H-dependent glyoxylate reductase expressed by the recombinant microorganism.

In one embodiment, glyoxylate is converted to glycine using an alanine-glyoxylate aminotransferase, a glycine dehydrogenase, a glycine aminotransferase, a serine-glyoxylate aminotransferase, and/or a glycine oxidase expressed by a recombinant microorganism.

Suitable media for use in the methods of the present disclosure comprise a fermentable carbon source. In one embodiment, the carbon source is selected from the group consisting of sugars, glycerol, alcohols, organic acids, alkanes, fatty acids, lignocelluloses, proteins, carbon dioxide, and carbon monoxide. In exemplary embodiments, the carbon source is a sugar. In another exemplary embodiment, the carbon source is a hexose and/or pentose sugar. In another embodiment, the carbon source is glucose or an oligomer of glucose, or the carbon source comprises a biomass hydrolysate comprising hemicellulose. In another embodiment, the carbon source is a monosaccharide (e.g., glucose, xylose, arabinose, fructose, and mannose), a disaccharide (e.g., sucrose, lactose, and maltose), an oligosaccharide (e.g., galactose), or a polysaccharide (e.g., cellulose).

In another embodiment, the method for producing GA and/or glycine comprises culturing a recombinant microorganism expressing a set of genes encoding:

(a) malate dehydrogenase catalyzing carboxylation of pyruvate to malate;

(b) malate thiokinase catalyzing the conversion of malate into malyl-coa; and

(d) optionally phosphoenolpyruvate carboxylase, and/or phosphoenolpyruvate carboxykinase, and/or pyruvate carboxylase;

and comprising at least one modification selected from the following:

(a) deleting or attenuating the gene encoding malate synthase;

(b) deleting or attenuating a gene encoding isocitrate dehydrogenase;

(d) deleting or attenuating a gene encoding a malate dehydrogenase that catalyzes the conversion of oxaloacetate to malate, or the conversion of malate to oxaloacetate; and

(e) deleting or attenuating the gene encoding pyruvate kinase.

In another embodiment, the method for producing GA and/or glycine comprises culturing a recombinant microorganism expressing a set of genes encoding:

(a) malate dehydrogenase catalyzing carboxylation of pyruvate to malate;

(b) malate thiokinase catalyzing the conversion of malate into malyl-coa; and

and comprises:

(a) attenuating a gene encoding malate dehydrogenase catalyzing the conversion of oxaloacetate to malate, or a malate dehydrogenase catalyzing the conversion of malate to oxaloacetate, or attenuating/mutating a gene encoding malate dehydrogenase catalyzing the carboxylation of pyruvate to malate, such that it shows a reduced conversion of malate to pyruvate; and

(b) the genes encoding malate synthase (e.g., aceB and/or glcB genes in E.coli, or DAL7 and MLS1 genes in s.cerevisiae) are deleted or attenuated.

In another embodiment, the method for producing GA and/or glycine comprises culturing a recombinant microorganism expressing a set of genes encoding:

(a) a phosphoenolpyruvate carboxylase which catalyzes the carboxylation of PEP to oxaloacetate, and/or a phosphoenolpyruvate carboxykinase which catalyzes the carboxylation of PEP to oxaloacetate, and/or a pyruvate carboxylase which catalyzes the carboxylation of pyruvate to oxaloacetate, and/or a malate dehydrogenase which catalyzes the carboxylation of pyruvate to malate;

(b) malate thiokinase catalyzing the conversion of malate into malyl-coa; and

(d) and optionally a malate dehydrogenase catalyzing the conversion of oxaloacetate to malate,

and;

wherein the microorganism comprises at least one modification selected from the group consisting of:

(a) deleting or attenuating the gene encoding malate synthase;

(b) deleting or attenuating a gene encoding isocitrate dehydrogenase;

(d) deleting or attenuating a gene encoding a malate dehydrogenase that catalyzes the conversion of malate into pyruvate, or deleting or attenuating a gene encoding a malate dehydrogenase that catalyzes the conversion of malate into oxaloacetate;

(e) deleting or attenuating a gene encoding pyruvate kinase;

(f) deleting or attenuating a gene encoding glyoxylate aldehyde ligase;

(g) deleting or attenuating a gene encoding 2-oxo-4-hydroxyglutarate aldolase;

(h) deleting or attenuating the gene encoding glycolaldehyde reductase;

(i) deleting or attenuating a gene encoding glycolate oxidase; and

(j) deleting or attenuating a gene encoding a repressor of isocitrate lyase.

In yet another embodiment, the method for producing GA and/or glycine comprises culturing in a suitable culture medium a recombinant microorganism that exhibits increased expression levels, or increased activity (i.e., enhanced kinetic parameters for the desired reaction), or greater specificity (i.e., specificity of the engineered enzyme for the target substrate as compared to the wild-type enzyme) of one or more enzymes>5x、>10¹x、>10²x、>10³x、10⁴x, or is preferred>10⁵(ii) a Or a new homologous enzyme) selected from: pyruvate carboxylase, phosphoenolpyruvate carboxykinase, malate dehydrogenase, malate thiokinase, malyl-coa lyase, NADH-dependent glyoxylate reductase, and NADPH-dependent glyoxylate reductase.

In other embodiments, the methods for producing GA and/or glycine comprise culturing a recombinant microorganism in a suitable culture medium, the recombinant microorganism exhibiting a reduced expression level of at least one enzyme selected from the group consisting of: malate synthase, isocitrate dehydrogenase, pyruvate oxidase and/or pyruvate formate lyase, pyruvate kinase, glyoxylate carboligase, 2-oxo-4-hydroxyglutarate aldolase, glucose-6-phosphate isomerase, glycolaldehyde reductase, and glycolate oxidase.

In another embodiment, the method for the production of GA and/or glycine may comprise deletions or modifications which reduce the activity of pyruvate dehydrogenase, prevent or at least reduce the loss of the predominant carbon in the conversion from pyruvate to acetyl-coa, and facilitate the rerouting of carbon from pyruvate or phosphoenolpyruvate to oxaloacetate by the carboxylation activity of the enzyme candidates presented herein.

In another embodiment, the method for the production of GA and/or glycine may comprise deletions or modifications which reduce the activity of pyruvate kinase and facilitate carbon fixation from phosphoenolpyruvate into oxaloacetate by carboxylation activity of the enzyme candidates presented herein.

The methods of the present disclosure can provide yields of glycolic acid ranging from about 1.1g glycolic acid per gram carbon source to about 2.0g/g glycolic acid, including values and ranges therebetween. For example, the yield of glycolic acid can be about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or about 2.0 g/g. The yield of glycolic acid can be from about 1.1 to about 1.8g/g, about 1.2 to about 1.8g/g, about 1.3 to about 1.8g/g, about 1.4 to 1.7g/g, or about 1.4 to 1.6 g/g.

The methods of the present disclosure can provide yields of glycine ranging from about 1.0g glycine to about 1.5g/g glycine per gram of carbon source, including values and ranges therebetween. For example, the yield of glycine may be about 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 g/g. The yield of glycine can range from about 1.0 to about 1.4g/g, about 1.0 to about 1.3g/g, about 1.0 to about 1.2g/g, about 1.1 to 1.5g/g, about 1.1 to 1.4g/g, or about 1.1 to 1.3g/g, or about 1.2 to 1.4 g/g.

Production of polyglycolic acid (PGA)

The present disclosure also includes methods of producing polyglycolic acid (PGA). Glycolic acid produced by the recombinant microorganisms of the present disclosure can be used to produce PGA. PGA can be produced from GA by in vivo polymerization or chemical polymerization.

In one embodiment, the production of PGA using the in vivo polymerization route is described in U.S. pre-grant publication No. 2011/0118434a1, which is incorporated herein by reference in its entirety. In this route, once GA is produced, two classes of enzymes-coa transferase/synthase and PHA synthase can be used to produce PGA in cells. Thus, in one embodiment, a recombinant microorganism in any of the embodiments disclosed herein can comprise a gene encoding Polyhydroxyalkanoate (PHA) synthase.

Four major classes of PHA synthases are known (Rhem, b., 2003). Class I and class II PHA synthases comprise enzymes consisting of only one type of subunit (PhaC). Based on their in vivo and in vitro specificity, class I PHA synthases (e.g., in alcaligenes eutropha) preferentially utilize coa-thioesters of various hydroxy fatty acids containing 3 to 5 carbon atoms, while class II PHA synthases (e.g., in Pseudomonas aeruginosa) preferentially utilize coa-thioesters of various hydroxy fatty acids containing 6 to 14 carbon atoms. Class III synthases (e.g. in allochromyces vinosus) comprise a complex composed of two different types of subunits: PhaC and PhaE subunit. These PHA synthases preferably comprise coenzyme A-thioesters of hydroxy fatty acids of 3 to 5 carbon atoms. Class IV PHA synthases (e.g., in Bacillus megaterium) resemble class III PHA synthases, but PhaE is replaced by PhaR.

In one embodiment, the gene encoding PHA synthase is phaC, phaEC and/or phaCR.

In one embodiment, glycolic acid is converted to glycolyl-coa by one or more enzymes selected from the group consisting of: acyl-coa synthetase, acyl-coa transferase and butyrate kinase related phosphotransbutyrylase (phosphotransbutyrylase).

In an exemplary embodiment, the enzyme that converts glycolate into glycolyl-coa is from a species of the family enterobacteriaceae. In an exemplary embodiment, the recombinant microorganism of any of the embodiments described herein may comprise a gene prpE encoding a propionyl-coa synthetase from escherichia coli or Salmonella typhimurium (Salmonella typhimurium), a gene acs encoding an acetyl-coa transferase from escherichia coli, a gene ptb encoding a phosphotransbutyrylase, and/or a gene buk encoding a butyrate kinase.

If not by in vivo polymerization, chemical polymerization methods are known in the art: ring-opening polymerization from Kureha with high molecular weight PGA (literature attached), and direct polycondensation to achieve low molecular weight PGA (Singh & Tiwari, 2010).

Alternatively, PGA may be prepared by a chemical polymerization route, such as ring-opening polymerization of cyclic diesters or polycondensation of 2-hydroxycarboxylic acids. In an exemplary embodiment, PGA can be produced using a ring-opening polymerization method described by Yamane et al (Polymer Journal, 8 months 2014, pages 1-7) to obtain high molecular weight PGA. In another exemplary embodiment, PGA may be produced by direct polycondensation to obtain low molecular weight PGA (Singh & Tiwari, International Journal of Polymer Science, Vol. 2010, 652719, pp. 23, doi: 10.1155/2010/652719).

The present disclosure also provides methods of producing recombinant microorganisms capable of producing glycolate and/or glycine from glyoxylate using a reverse glyoxylate shunt. In one embodiment, the method for producing a recombinant microorganism comprises introducing into a microorganism one or more genes selected from the group consisting of:

(a) a gene encoding pyruvate carboxylase which converts pyruvate to OAA;

(b) a gene encoding phosphoenolpyruvate carboxylase which converts phosphoenolpyruvate to OAA;

(d) a gene encoding a malate dehydrogenase that converts OAA to malate and/or pyruvate to malate;

(e) a gene encoding malate thiokinase converting malate into malyl-coa;

(f) a gene encoding malyl-coa lyase for converting malyl-coa to glyoxylate and acetyl-coa;

(g) a gene encoding an NADH-dependent glyoxylate reductase that converts glyoxylate to glycolate; and

(h) a gene encoding an NADPH-dependent glyoxylate reductase that converts glyoxylate to glycolate.

The nucleotide sequences of genes encoding the above polypeptides are known in the art and are publicly available (www.ncbi.nlm.nih.gov/genbank /). Methods for incorporating the desired nucleic acid sequence into the genome of a microorganism or into an expression vector are also known. For example, U.S. patent No. 9,034,615, incorporated herein by reference, discloses a method of incorporating the gene ycdW (encoding an NADPH-dependent glyoxylate reductase) into an expression vector.

In certain embodiments, the recombinant microorganism comprises a deletion or modification that attenuates the expression of an endogenous gene. Exemplary methods for producing these microorganisms include deleting a gene, or attenuating expression of a gene by replacing a native promoter with a low-strength promoter, or by introducing a mutation that results in a decrease in enzyme activity into a gene.

In some embodiments, a method for producing a recombinant microorganism comprises introducing into the microorganism a deletion or modification that attenuates the expression, or inhibits the activity, of an enzyme encoded by at least one endogenous gene selected from the group consisting of:

(a) a gene encoding malate synthase;

(b) a gene encoding isocitrate dehydrogenase;

(d) a gene encoding pyruvate kinase;

(e) a gene encoding malate dehydrogenase;

(f) a gene encoding glyoxylate aldehyde ligase;

(g) a gene encoding 2-oxo-4-hydroxyglutarate aldolase;

(h) a gene encoding glycolaldehyde reductase;

(i) a gene encoding glycolate oxidase;

(j) a gene encoding a repressor of isocitrate lyase; and

(l) A gene encoding glucose-6-phosphate isomerase.

The method for producing a recombinant microorganism may further comprise (a) attenuating nucleic acid encoding malic acid: a deletion or modification of the gene expression of the quinol oxidoreductase and/or (b) the introduction of a gain of function mutation in a gene encoding malate dehydrogenase, a gene encoding pyruvate carboxylase, a gene encoding phosphoenolpyruvate carboxykinase, a gene encoding malate thiokinase, a gene encoding malyl-coa lyase, a gene encoding alanine-glyoxylate aminotransferase; a gene for glycine dehydrogenase; a gene encoding glycine transaminase; a gene encoding serine-glyoxylate transaminase; a gene encoding glycine oxidase; a gene encoding alanine aminotransferase and/or a gene encoding NADPH-dependent glutamate synthase.

The foregoing detailed description has been given for clearness of understanding only, and no unnecessary limitations should be understood therefrom, as modifications will be obvious to those skilled in the art.

While the disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the disclosure following, in general, the principles of the disclosure and including such departures from the present disclosure as come within known or customary practice within the art to which the disclosure pertains and as may be applied to the essential features hereinbefore set forth and as follows within the scope of the appended claims.

Examples

Example 1:

in silico analysis of ethanol biosynthesis and improvement by reverse glyoxylate shunt Activity in E.coli Acid production

Flux Balance Analysis (FBA) was performed to mimic the effect of the genetic modifications described herein on glycolic acid production yield in each case (fig. 3 and 4). To this end, a genome-scale metabolic model iJO1366(Orth JD. et al (2011) A complex genome-scale architecture of Escherichia coli metabolism-2011. Mol Syst biol.7:535), which contains all known E.coli metabolic reactions, was modified to mimic Glycolic Acid (GA) production using a Glyoxylate (GS) and retro-glyoxylate (rGS) arm combination. The model was modified to include other reactions and corresponding metabolites, including the malate thiokinase reaction (EC 6.2.1.9), the malyl-coa ligase reaction (EC 4.1.3.24) and the pyruvate carboxylase reaction (EC6.4.1.1).

Simulations were performed using OptFlux software (Rocha L. (2010) OptFlux: an open-source software platform for in silico metabolic engineering. BMC Syst biol.4: 45). In an exemplary embodiment, a brief flux balance analysis was performed to evaluate the maximum theoretical production yield of GA by GS/rGS engineering. According to exemplary embodiments, the transport system used is hexokinase HXK (e.c.2.7.1.1), or phosphotransferase system (PTS), and the carboxylase used to enter the TCA cycle is phosphoenolpyruvate carboxykinase (PEPCK) (e.c.4.1.1.32), phosphoenolpyruvate carboxylase (PPC) (e.c.4.1.1.31), or pyruvate carboxylase PPC (EC 6.4.1.1).

The simulation was performed by applying a set of constraints (constraints) that are easily reproducible under in vivo culture conditions of E.coli strains, where glucose is the carbon substrate and under aerobic conditions. Glucose substrate flux was arbitrarily set at 10. mu. mole. g CDW-1. h-1. No restrictions are set on minimum biomass yield or cell maintenance costs. The simulation results describing the maximum theoretical production of GA are shown in table 1.

TABLE 1 simulation results describing maximum theoretical production of GA

The simulated flux plots are depicted in fig. 3 and 4. Simulations show that the theoretical production yield of GA from glucose by GS/rGS can reach 0.83 to 1.43g, depending on the glucose transport system and carboxylase used_GA/g_GlucoseIn the meantime. Strains that rely on PEPCK or PPC as carboxylase perform poorly in PTS + strains. This may be due to competition between the PTS system and PEPCK/PPC for its common substrate, phosphoenolpyruvate. However, in PTS-deficient strains (where glucose is transported mostly through hexokinase hxhk), the performance of the strain can be improved. As depicted by the flux plots (fig. 3 and 4), the maximum yield can only be achieved if 86% to 100% of the carbon flux from glucose is diverted to the pentose phosphate pathway for providing the redox cofactor for the final glyoxylate reductase reaction (e.c. 1.1.1.26). This carbon flux towards the pentose phosphate pathway is seen as an alternative to provide the NADPH co-factor required by NADPH-dependent glyoxylate reductase.

Example 2:

by the reaction of the retro-glyoxylate shunt activity in E.coli with carboxylation by pyruvate carboxylase activity In combination, in vivo biosynthesis and improvement of glycolic acid production

Production of Glycolic Acid (GA) in E.coli can be enhanced by inactivating all of the glyoxylate-consuming annotation reactions, i.e., malate synthase encoded by aceB (GenBank Gene ID: 948512) and glcB (GenBank Gene ID: 948857), glyoxylate carboligase encoded by gcl (GenBank Gene ID: 945394), and 2-oxo-4-hydroxyglutarate aldolase encoded by eda (GenBank Gene ID:946367), as previously described (Alkim C. (2016) the synthetic xylose-1 phosphate pathway production of glyconic acid from sugar mixtures, biotechnol Biofuels,9: 201). Reoxidation of GA can be further prevented by deletion of the glcDEFG operon (GenBank Gene ID: 947353, 2847718, 2847717, 947473) encoding glycolate oxidase.

Thus, the following experiments were performed in E.coli K12 strain MG 1655. delta. aceB. delta. glcDEFGB. delta. gcl. delta. edd-eda. This strain, known as SGK rGS-00, is a gift from Alkim et al (Alkim C. (2016) The synthetic xylose-1 phosphate pathway innovations of glycolic acid from rich sugar mixtures Biotechnol Biofuels,9: 201).

Deletion of the pgi locus encoding phosphoglucose isomerase

To construct strains with enhanced pentose phosphate activity and NADPH pooling, pgi encoding glucose-6-phosphate isomerase (GenBank Gene ID:948535) was deleted by CRISPR-Cas9(Jiang Y. et al (2015) Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. apple Environ Microbiol,81: 2506-Astro 2514) according to standard procedures. Plasmids pTargetF containing guide RNA (pMB1 aadA sgRNA-cadA), and pCas containing cas9 gene and lambda-Red recombinase (repA101-Ts kan Pcas-cas9 ParaB-Red lacIq Ptrc-sgRNA-pMB1) were obtained from AddGene (Addgene plasmids #62226 and #62225, respectively; Addgene, Cambridge, USA).

pTargetF pMB1 aadA sgRNA-Pgi, the expression guide RNA of which has an N20 sequence targeting the Pgi locus, was obtained by overlap PCR using the primers Pgi _ N20_ FW and Pgi _ N20_ RV described in table 2. PCR fragments were obtained by amplifying upstream 500bp and downstream 500bp of the Pgi locus by overlap PCR using primers Pgi _ H1_ FW, Pgi _ H1_ RV, Pgi _ H2_ FW and Pgi _ H2_ RV (see table 2) and combining them to provide donor DNA/disruption cassettes as PCR fragments.

TABLE 2 oligonucleotides for disruption of pgi by CRISPR-Cas 9. The binding region is underlined. The N20 sequence specific for pgi is shown in italics.

Genome editing was performed by adjusting the protocol from (Jiang Y. et al (2015) Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. apple Environ Microbiol,81: 2506-. Strain SGK _ rGS _00 was first transformed with pCAS Plasmid by electroporation using standard procedures (wood CA. (2003) Plasmid vectors. Competent cells of strain SGK-rGS-00 containing pCAS were prepared while the lambda-Red recombinase was induced with arabinose (10mM final concentration) as described previously (Jiang Y. et al (2015) Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. apple Environ Microbiol,81: 2506-2514). Then, 50. mu.l of competent cells were mixed with 100ng of pTargetF plasmid and 400ng of donor DNA. Electroporation was carried out in a 2-mm electroporation cuvette (VWR) at 2.5kV, and the product was immediately suspended in 1ml of LB medium (pre-heated at 30 ℃). The cells were recovered overnight at 30 ℃ and then inoculated onto LB agar containing kanamycin (50. mu.g/ml) and spectinomycin (50. mu.g/ml) and incubated overnight at 30 ℃. For the identification of transformants by colony PCR and sequencing. The resulting strain was called SGK _ rGS _ 01: MG 1655. DELTA. aceB. DELTA. glcDEFGB. DELTA. gcl. delta. edd-eda. delta. pgi.

Deletion of the aceE locus of subunit E1 encoding pyruvate dehydrogenase

A pyruvate-accumulating strain was constructed using MG 1655. delta. i:. KanR strain JW0110, which deletes pyruvate dehydrogenase subunit E1 aceE in strain SGK _ rGS _01, according to standard procedures (Thomason LC. (2007) E.coli Genome management by P1transduction. curr protocol Mol biol.79:1.17) using the MG 1655. delta. i obtained from the Keio single gene deletion collection (Baba T. et al (2006) Construction of Escherichia coli K-12in-frame, single-gene deletion variants: the Keio collection. Mol Syst biol.2:2006.0008) to enhance the use of carboxylase (e.g., pyruvate carboxylase) into the Krebs cycle. Transformants were selected on LB agar supplemented with 100. mu.g/ml kanamycin and identified by colony PCR and sequencing. Antibiotic markers were further removed by specific recombination of the FTR region using Flp recombination as described previously in the literature (Datsenko KA. et al (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12using PCR products Proc Natl Acad Sci USA.97(12): 6640-5). The resulting strain was named SGK-rGS-02 MG 1655. delta. aceB. delta. glcDEFGB. delta. gcl. delta. edd. eda. delta. pgi. delta. aceE.

Expressing pyruvate carboxylase, citrate synthase, isocitrate lyase and glyoxylate reductase to enhance carbon fixation, Glyoxylate shunt activity and glycolic acid synthesis

By passing(Leiborin, Germany) pyruvate carboxylase (SEQ ID NO:20) from the Rhizobium phaseoloides (Rhizobium etli) strain CFN42 was synthesized (Uniprot accession No.: Q2K 340). The genes for the native isocitrate lyase aceA (SEQ ID NO:21) (GeneBank Gene ID:948517) and glyoxylate reductase ghRA (SEQ ID NO:23) (GeneBank Gene ID:946431) were amplified by PCR from the genome of E.coli MG1655 using the primers in Table 3. PCR was performed from plasmid pACT3w-ppc by PCR as described by Trichez et al (Trichez D. (2018) Engineering of Escherichia coli for Krebs cycle-dependent production of Mass acid. Microb Cell fact.17:113)_K620S-gltA_R163LMiddle recovery of NADH insensitive citrate synthase mutant gltA_R163L(SEQ ID NO:22)。

To express thisThese genes, which are synthetic operons, first replaced P with a J23119 constitutive promoter (SEQ ID NO:19) (http:// parts. item. org/Promoters/Catalog/Anderson)_A1lacO-1The pZS13-Luc plasmid (Expressys) was modified by the promoter and introduced into the multiple cloning site. Is obtained byThe J23119 promoter (Leibtin, Germany) was synthesized as a synthetic gene fragment. Subsequently, it was cloned into the pZS13-Luc plasmid by restriction cloning between the restriction sites AatII and KpnI. The multiple cloning site was recovered from pZA21-MCS plasmid (Expressys) by digestion with KpnI and AvrII and incorporated into the plasmid by restriction cloning between the restriction sites KpnI and AvrII. The resulting plasmid was named pZS 1-J23119-MCS.

All genes were amplified by PCR using the primers described in table 3. The PCR fragment was purified on a Gel using the EZ-10Spin Column DNA Gel Extraction Kit (Biobasic) according to the manufacturer's protocol. Then, according to the manufacturer's operating scheme, by usingHiFi DNA Assembly Cloning Kit (New England Biolabs), the purified fragment was cloned into pZS1-J23119-MCS plasmid linearized by restriction digestion with KpnI and HindIII. Construction was confirmed by PCR and sequencing. The resulting synthetic operon was designated J23119-pyc-aceA-gltA_R163LghRA (SEQ ID NO:24), and the plasmid was named pZS1-pyc (see Table 4).

TABLE 3 oligonucleotides used to construct the synthetic operon J23119-pyc-aceA-gltAR 164L-ghRA. The binding region is underlined. Use ofThe HiFi DNA Assembly Cloning Kit (New England Biolabs) uses overhangs to assemble clones.

TABLE 4 oligonucleotides used to construct the synthetic operon PTAc-sucCD-mcl. The binding region is underlined. Use ofThe HiFi DNA Assembly Cloning Kit (New England Biolabs) uses overhangs to assemble clones.

Expression of Malate Sulfokinase and Maloyl-CoA ligase to introduce reverse glyoxylate shunt Activity

The sucC2-sucD2 operon (SEQ ID NO:26) (Uniprot Q607L9 and Q607L8) encoding malate thiokinase from the Methylococcus capsulatus strain Bath, and the genes encoding malyl-CoA lyase and mcl (SEQ ID NO:27) (Uniprot C5B113) from Methylobacterium extorquens AM1 as synthetic genes from Methylococcus capsulatus(leybin, germany) order. To express these genes as synthetic operons, P recovered from the standard pACT3 plasmid was used first_TacSubstitution of P by inducible promoter (SEQ ID NO:25)_LtetO-1The promoter thus modifies the pZS13-MCS plasmid (Expressys) and is then cloned by restriction cloning between the restriction sites AatII and KpnI. The resulting plasmid was designated pZA3-P_Tac-MCS。

All genes were amplified by PCR using the primers described in table 4. The PCR fragment was purified on a Gel using the EZ-10Spin Column DNA Gel Extraction Kit (Biobasic) according to the manufacturer's protocol. Then, according to the manufacturer's operating scheme, by usingHiFi DNA Assembly Cloning Kit (New England Biolabs), fragment purifiedCloned as a synthetic operon into pZA3-P linearized by restriction digestion with EcoRI and MluI_Tac-MCS plasmid. Construction was confirmed by PCR and sequencing. The resulting synthetic operon is designated P_TacsucCD-mcl (SEQ ID NO:28), and the plasmid designated pZA3-rGS (see Table 5).

Determination of glycolic acid production

Three E.coli strains were tested for GA production. Tests (i) contained no plasmid as negative control, (ii) contained only plasmid pZS1-pyc, (ii) contained plasmid pZA3-rGS, (iv) wild strain MG1655 containing both plasmids, and engineered strains SGK _ rGS _01 and SGK _ rGS _ 02. All strains were transformed with the corresponding plasmids by electroporation using standard procedures (wood CA. (2003) Plasmid vectors. The genotypes of the plasmids and strains are shown in Table 5.

TABLE 5 genotypes of plasmids and strains used for glycolic acid production assays.

The strain was grown in M9 glucose medium (20g/L glucose) supplemented with 15mM acetic acid and 1g/L casamino acid for about 50 hours. Ampicillin and chloramphenicol were added to give final concentrations of 100. mu.g/mL and 25. mu.g/mL, respectively (i.e., ampicillin for the strain carrying pZS1-pyc and chloramphenicol for the strain carrying pZA 3-rGS). When the OD600 of the culture reached about 0.6-0.8, the culture was induced with IPTG (final concentration 0.5 mM). By OD₆₀₀Growth was monitored. Samples were taken during the growth to stationary phase. Then, by HPLC-UV/RI (Dionex Ultimate 3000, Thermo Fisher Scientific), using a Rezex ROA-Organic Acid column (Phenomenex) at 80 ℃ using H₂SO₄Glucose consumption and metabolite production were analyzed at 0.5mM as mobile phase (0.5 mL/min). The GA titer and GA yield after 24 hours are shown in Table 6.

As shown in Table 6, no significant glycolic acid production was detected in the MG1655 wild-type control strain without plasmid or with plasmid pZS1-pyc or pZA3-RGS alone. This was expected because all major competing pathways (i.e., glyoxylate and glycolate degradation pathways) were still active in the wild-type strain. Interestingly, a limited amount of GA was detected when the pZS1-pyc plasmid and pZA3-rGS were expressed in the MG1655 wild-type strain. The titer of GA reached 0.11g/L, which is the first indication that the combination of the GS/rGS pathway has a positive effect on GA production even in wild-type strains.

For the engineered strain SGK _ rGS _01, no significant GA production was detected in the blank control strain or the strain with only pZA 3-rGS. However, GA production up to titers of about 0.18g/L could be detected when the glyoxylate shunt activity and glyoxylate reductase activity were enhanced using plasmid pZS 1-pyc. After pZA3-rGS plasmid was added to the strain, the GA titer was improved by 910% to 1.91 g/L. The production yield after 22.5 hours reaches 0.24g_GA/g_GlucoseAn improvement of 2400% was shown compared to the production yield without the rGS engineering.

For the engineered strain SGK _ rGS _02, no significant GA production was detected in the control strain with only a single plasmid. GA production was detected only in the combination of the two plasmids, up to a titer of about 0.28 g/L.

TABLE 6 evaluation of GA Titers and yield during glycolic acid production assay 24 hours after growth

Example 3:

by combining the retro-glyoxylate shunt activity in E.coli with carboxylation by pep carboxylase activity Synbiotic synthesis and improved production of glycolic acid

The following experiments were carried out in E.coli K12 strain MG 1655. delta. aceB. delta. glcDEFGB. delta. gcl. delta. edd-eda. This strain, known as SGK rGS-00, is a gift from Alkim et al (Alkim C. et al (2016) The Synthetic xylolose-1 phosphate pathway innovations of glycolic acid from rich catalysts. Biotechnol Biofuels,9: 201).

Deletion of the pgi locus encoding phosphoglucose isomerase

To construct strains with enhanced pentose phosphate activity and NADPH sink, pgi encoding glucose-6-phosphate isomerase was deleted by CRISPR-Cas9 (GenBank Gene ID:948535) according to standard procedures (Jiang Y. et al (2015) Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. apple Environ Microbiol,81: 2506-2514). The strain was disrupted as previously described in example 2 and designated SGK _ rGS _01 (table 5).

Deletion of the pykF locus encoding pyruvate kinase I

MG 1655. delta. pgi obtained from the Keio monogenic deletion pool (Baba T. et al (2006) Construction of Escherichia coli K-12in-frame, single-Gene knock out variants: the Keio collection. Mol Syst biol.2:2006.0008) KanR strain JW1666, was used for transduction according to standard procedures (Thomason LC. (2007) E.coli Genome Manipulation by P1transduction. Curr Protoc Mol biol.79:1.17), and pyruvate kinase I pykF (GenBank Gene ID:946179) was deleted in strain SGK-rGS-01, to construct strains that accumulate phosphoenolpyruvate to enhance the use of carboxylase (e.g., pep carboxylase) into the Krebs cycle. Transformants were selected on LB agar supplemented with 100. mu.g/ml kanamycin and identified by colony PCR and sequencing. Antibiotic markers were further removed by specific recombination of the FTR region using Flp recombination as described previously in the literature (Datsenko KA. et al (2000) One-step inactivation of chromosogenes in Escherichia coli K-12using PCR products. Proc Natl Acad Sci USA.97(12): 6640-5). The resulting strain was designated SGK-rGS-03 MG 1655. delta. aceB. delta. glcDEFGB. delta. gcl. delta. edd-eda. delta. pgi. delta. pykF.

Expression of pep carboxylase, citrate synthase, isocitrate lyase and glyoxylate reductase to enhance carbon fixation, glyoxylate shunt activity and glycolate synthesis

Plasmid pACT3w-ppc_K620S-gltA_R163LObtained from Trichez et al (Trichez D. (2018) Engineering of Escherichia coli for Krebs cycle-dependent production of male acid. Microb Cell fact.17: 113). The plasmid is contained in inducible P_TacMalic acid-insensitive pep carboxylase mutant ppc under control of promoter_K620SNADH insensitive citrate synthase mutant gltA_R163L. It was further modified as described below and used as a scaffold for the construction of synthetic operons. The Gene encoding the natural isocitrate lyase aceA (GeneBank Gene ID:948517) and the Gene encoding the glyoxylate reductase ghrA (GeneBank Gene ID:946431) were amplified by PCR from the genome of E.coli MG1655 using the primers aceA _ FW/aceA _ RV and ghrA _ FW/ghrA _ RV described in Table 7, respectively. The plasmid pACT3w-ppc was used_K620S-gltA_R163LAs a template, and the primers pACT3_ FW/ppc _ RV described in Table 7, the gene ppc_K620S(SEQ ID NO:29) was amplified by PCR together with the plasmid backbone pACT 3. Finally, the plasmid pACT3w-ppc was used_K620S-gltA_R163LAs a template, and the primers gltA _ FW/gltA _ RV described in Table 7, the gene gltA was amplified by PCR_R163L。

TABLE 7 oligonucleotides used to construct the synthetic operon J23119-pyc-aceA-gltAR 164L-ghRA. The binding region is underlined. Use ofThe HiFi DNA Assembly Cloning Kit (New England Biolabs) uses overhangs to assemble clones.

The PCR fragment was purified on a Gel using the EZ-10Spin Column DNA Gel Extraction Kit (Biobasic) according to the manufacturer's protocol. Then, according to the manufacturer's operating scheme, by usingHiFi DNA Assembly Cloning Kit (New England Biolabs), the purified fragments were assembled. Construction was confirmed by PCR and sequencing. The resulting synthetic operon is designated P_tac-ppc_K620S-aceA-gltA_R163LghRA (SEQ ID NO:30), and the plasmid named pACT 3-ppc.

Expression of malate thiokinase and Maloyl-CoA ligase for introduction of reverse glyoxylate shunt Activity

As described in example 2, the sucC2-sucD2 operon encoding malate thiokinase (SEQ ID NO:26) (Uniprot Q607L9 and Q607L8) from Methylococcus capsulatus strain Bath, and the mcl gene encoding malyl-CoA lyase (Uniprot C5B113) from Methylobacterium extorquens AM1 were synthesized from(leybin, germany) order. The operon P containing the synthesis was obtained as described in example 2_TacPlasmid pZA3-rGS of sucCD-mcl (SEQ ID NO: 28). For expression of P in a background compatible with plasmid pACT3-ppc_TacsucCD-mcl, P by restriction cloning between the restriction sites AvrII and BglII_Tac-sucCD-mcl was further transferred into pZE23-MCS plasmid (Expressys). The resulting plasmid was designated pZE 2-rGS.

Determination of glycolic acid production

Two strains were tested for GA production assays. Tests (i) contained plasmid pACT3-ppc only, (ii) plasmid pZE2-rGS only, and (iii) wild-type MG1655 containing both plasmids and engineered strain SGK _ rGS _ 03. All strains were transformed with the corresponding plasmids by electroporation using standard procedures (wood CA. (2003) Plasmid vectors. The genotypes of the plasmids and strains are shown in Table 8.

TABLE 8 genotypes of plasmids and strains used for glycolic acid production assays.

The strain was grown in M9 glucose medium (20g/L glucose) supplemented with 15mM acetic acid and 1g/L casamino acid for about 50 hours. Adding chloramphenicol and kanamycinSo that the final concentrations were 25. mu.g/mL and 50. mu.g/mL, respectively (i.e., chloramphenicol for the strain carrying pACT3-ppc and kanamycin for the strain carrying pZE 2-rGS). When the OD600 of the culture reached about 0.6-0.8, the culture was induced with IPTG (final 0.5 mM). By OD₆₀₀Growth was monitored. Samples were taken during the growth to stationary phase. Then, by HPLC-UV/RI (Dionex Ultimate 3000, Thermo Fisher Scientific), using a Rezex ROA-Organic Acid column (Phenomenex) at 80 ℃ using H₂SO₄Glucose consumption and metabolite production were analyzed at 0.5mM as mobile phase (0.5 mL/min). Glucose titer, GA titer and GA yield are shown in table 9.

As shown in Table 9, when pACT3-ppc and pZE2-rGS were expressed, and pACT3-ppc did not have pZE2-rGS, GA production could be detected in the wild-type control, but the maximum yield was only 0.02g_GA/g_Glucose(ii) a While no production of glycolic acid was detected in the SGK _ rGS _03 strain with only plasmid pZE 2-rGS. However, GA production up to a titer of about 0.8g/L could be detected in this strain when carbon fixation, glyoxylate shunt activity and glyoxylate reductase activity were enhanced using the plasmid pACT 3-ppc. Addition of pZE2-rGS plasmid to SGK _ rGS _03 did not improve GA titer. After 46 hours, the production yield reaches 0.21g_GA/g_GlucoseShows 525% improvement compared to the strain expressing pACT3-ppc and 1050% improvement compared to the wild type strain expressing both plasmids.

TABLE 9 evaluation of GA Titers and yield during glycolic acid production assay after 46 hours

Illustrative embodiments

1. A recombinant glyoxylate producing microorganism for the synthesis of Glycolic Acid (GA) and/or glycine comprising:

(a) a gene encoding malate dehydrogenase catalyzing the conversion of pyruvate to malate;

(b) a gene encoding malate thiokinase catalyzing the conversion of malate into malyl-coa; and

2. A recombinant glyoxylate producing microorganism for the synthesis of Glycolic Acid (GA) and/or glycine comprising:

(b) a gene encoding malate dehydrogenase catalyzing the conversion of OAA to malate;

3. A recombinant glyoxylate producing microorganism for the synthesis of Glycolic Acid (GA) and/or glycine comprising:

(b) a gene encoding malate thiokinase catalyzing the conversion of malate into malyl-coa; and

4. The recombinant microorganism of any preceding embodiment, wherein the recombinant microorganism does not produce isopropanol, ethanol, acetone, citric acid, itaconic acid, acetic acid, butyric acid, (poly) 3-hydroxybutyric acid, 3-hydroxyisobutyric acid, 3-aminoisobutyric acid, 2-hydroxyisobutyric acid, methacrylic acid, (poly) glutamic acid, arginine, ornithine, citrulline, leucine, isoleucine, or proline from acetyl-coa produced by malyl-coa lyase.

5. The recombinant microorganism of any preceding embodiment, wherein acetyl-coa produced by malyl-coa lyase is combined with OAA to increase biosynthesis of GA and/or glycine.

6. A recombinant microorganism in any preceding embodiment, wherein the microorganism comprises a mutation in a gene encoding malate dehydrogenase, wherein the mutation results in partial or complete inhibition of malate dehydrogenase activity that catalyzes the conversion of oxaloacetate to malate, malate to pyruvate, and/or malate to oxaloacetate.

7. The recombinant microorganism of any one of the preceding embodiments, wherein the microorganism comprises a gene encoding an NADH-dependent glyoxylate reductase that catalyzes the conversion of glyoxylate to glycolate or a gene encoding an NADPH-dependent glyoxylate reductase that catalyzes the conversion of glyoxylate to glycolate.

8. The recombinant microorganism of any one of the preceding embodiments, wherein the microorganism comprises a gene encoding alanine-glyoxylate aminotransferase, a gene encoding glycine dehydrogenase, a gene encoding glycine aminotransferase, a gene encoding serine-glyoxylate aminotransferase, and/or a gene encoding glycine oxidase that catalyzes the conversion of glyoxylate to glycine.

9. The recombinant microorganism of any preceding embodiment, wherein the malate dehydrogenase catalyzing the carboxylation of pyruvate to malate is from enzyme class (E.C.)1.1.1.38, e.c.1.1.1.39, or e.c. 1.1.1.40.

10. The recombinant microorganism of any preceding embodiment, wherein the malate dehydrogenase catalyzing the conversion of oxaloacetate to malate is from enzyme class (E.C.) 1.1.1.37.

11. The recombinant microorganism of any preceding embodiment, wherein the gene encoding malate dehydrogenase catalyzing the carboxylation of pyruvate to malate is selected from the group consisting of: maeA, maeB, dme, mez, mae1, and name-me 1 and name-me 2, or homologues thereof.

12. The recombinant microorganism of any preceding embodiment, wherein the gene maeA is from escherichia coli, pseudomonas, or bacillus; the gene maeB is from escherichia coli or salmonella; the gene dme is from rhizobia; the gene mez is from a mycobacterium; the gene mae1 is from saccharomyces cerevisiae; and the gene nad-me1 or nad-me2 is from Arabidopsis thaliana.

13. The recombinant microorganism of any preceding embodiment, wherein the gene maeA is from bacillus subtilis; the gene dme is from Rhizobium meliloti; or the gene mez is from mycobacterium tuberculosis.

14. The recombinant microorganism of any preceding embodiment, wherein the gene encoding malate dehydrogenase catalyzing the conversion of oxaloacetate to malate is selected from the group consisting of: the gene mdh from Escherichia coli, Corynebacterium, Streptomyces, Yeast and Arabidopsis or homologues thereof.

15. The recombinant microorganism of any preceding embodiment, wherein the gene mdh is from streptomyces coelicolor(s) or the gene mdh1/2/3 is from saccharomyces cerevisiae.

16. The recombinant microorganism of any preceding embodiment, wherein the gene encoding malate thiokinase is sucCD and/or sucCD-2 and/or mtkAB from methylobacterium, demethylobacterium, escherichia coli, thermus thermophilus, rhizogenes (Hyphomicrobium sp.), methanococcus jannaschii, methanopyrus thermoautotrophicum, rhizobium, methylococcus capsulatus, or Pseudomonas (Pseudomonas), or a homolog thereof.

17. The recombinant microorganism of any one of the preceding embodiments, wherein the gene encoding malyl-coa lyase is from Methylobacterium extorquens (Methylobacterium extorquens), Rhodobacter sphaeroides (Rhodobacter sphaeroides), streptomyces, Rhodobacter thermophilus (Chloroflexus aurantiacaus), Nitrosomonas eurotia (nitromonas europaea), Methylococcus capsulatus (methylcoccus capsulatus), Nereida, pythium aphanidermatum (Hyphomicrobium methylvorum), Rhodobacter caldarius (thalassobium vulgare), Rhodobacter sphaeroides, Mycobacterium smegmatis (mycobacteroidis) or Rhodococcus Rhodobacter zonatum (Rhodococcus Rhodobacter sphaeroides), and/or a homolog thereof 1 or a homolog thereof.

18. The recombinant microorganism of any preceding embodiment, wherein the gene encoding pyruvate carboxylase is PYC from Rhizobium phaseolus (Rhizobium etli), PYC1 or PYC2 from yeast, or PYC from bacillus subtilis (b.subtilis) or a homologue thereof.

19. The recombinant microorganism of any one of the preceding embodiments, wherein the gene encoding phosphoenolpyruvate carboxylase is ppc from escherichia coli, ppc or pepC from Rhodothermus marinus (r. marinus), ppcA from methanothermus thermoautotrophus (m. thermothermophilus), pep1 from maize (z. mays), ppc1/2/3 from arabidopsis thaliana (a. thaliana), ppc from soybean (g.max), or from Rhodothermus rubrum (Rhodothermus), Corynebacterium (Corynebacterium), Salmonella (Salmonella), Hyphomicrobium (Hyphomicrobium), Streptococcus (Streptococcus) and Streptomyces (Streptomyces), Pantoea (Pantoea), Bacillus (Bacillus), Clostridium (Clostridium), Pseudomonas pseudomonads (Pseudomonas), rhodobacter sphaeroides (rhodobacter sphaeroides), or lucerne. Or a homologue thereof.

20. The recombinant microorganism of any one of the preceding embodiments, wherein the gene encoding phosphoenolpyruvate carboxykinase is pck or pckA from escherichia coli, pckA from Selenomonas ruminata (selenia ruminants), pckA from Salmonella typhimurium (Salmonella typhimurium), pck or pckA from Klebsiella (Klebsiella sp.), pckA from Thermus (Thermus sp), pckA from enterococcus ruminis (Ruminococcus albus) and Ruminococcus flaviviens (Ruminococcus flavefaciens), pckA from Actinobacillus succinogenes (Actinobacillus succinogenes), pck or pckA from Streptococcus bovis (Streptococcus bovis), or pck or pckA from bacillus, clostridium thermocatenum (clostridium thermocellum), Mycobacterium tuberculosis (Mycobacterium tuberculosis), Mycobacterium tuberculosis; or a homologue thereof.

21. The recombinant microorganism of any preceding embodiment, wherein the microorganism comprises:

(a) a gene encoding citrate synthase that converts OAA and acetyl-coa produced by malyl-coa lyase to citrate;

(b) a gene encoding citrate hydrolase which converts citric acid into aconitic acid;

(d) a gene encoding an isocitrate lyase that converts isocitrate to succinate and glyoxylate;

(e) a gene encoding succinate dehydrogenase converting succinate into fumarate; and

(f) a gene encoding a fumarase enzyme which converts fumaric acid into malic acid.

22. The recombinant microorganism of any preceding embodiment, wherein the microorganism comprises a loss of function mutation in a gene encoding malate synthase, or a deletion of said gene.

23. The recombinant microorganism of any preceding embodiment, wherein the gene encoding glyoxylate reductase activity is selected from the group consisting of: ycdW and/or yiaE from escherichia coli, GOR1 from saccharomyces cerevisiae (s. cerevisiae), gyaR from thermus maritima (Thermococcus litoralis) and/or GLYR1 from arabidopsis thaliana (a. thaliana).

24. The recombinant microorganism of any preceding embodiment, wherein the pyruvate carboxylase to convert pyruvate to OAA is from enzyme classification system number e.c. 6.4.1.1; phosphoenolpyruvate carboxylase to convert phosphoenolpyruvate to OAA is from e.c. 4.1.1.31; phosphoenolpyruvate carboxykinases that convert phosphoenolpyruvate to OAA are from e.c.4.1.1.32 and e.c. 4.1.1.49.

25. The recombinant microorganism of any preceding embodiment, wherein the malate thiokinase that converts malate to malyl-coa is from the enzyme classification system number e.c.6.2.1.4, e.c.6.2.1.5, e.c.6.2.1.9, or e.c. 6.2.1-; and/or a malyl-coa lyase for converting malyl-coa to glyoxylate and acetyl-coa from e.c.4.3.1.24 or e.c. 4.3.1.25.

26. The recombinant microorganism of any preceding embodiment, wherein one or more genes are heterologously expressed.

27. The recombinant microorganism of any preceding embodiment, wherein the microorganism comprises a deletion or modification that reduces the activity of one or more endogenous genes selected from the group consisting of:

(a) a gene encoding isocitrate dehydrogenase;

(b) a gene encoding pyruvate dehydrogenase, pyruvate oxidase, and/or pyruvate formate lyase;

(d) a gene encoding glycolate oxidase.

28. The recombinant microorganism of any preceding embodiment, wherein the gene encoding malate synthase is aceB and/or glcB from E.coli, or DAL7 and/or MLS1 from yeast.

29. The recombinant microorganism of any preceding embodiment, wherein the gene encoding isocitrate dehydrogenase is icd from escherichia coli, or IDP2 and/or IDH1/2 from yeast.

30. The recombinant microorganism of any preceding embodiment, wherein the gene encoding pyruvate dehydrogenase is aceE and/or aceF from E.coli.

31. The recombinant microorganism of any preceding embodiment, wherein the gene encoding pyruvate kinase is pykA and/or pykF from escherichia coli.

32. The recombinant microorganism of any preceding embodiment, wherein the gene encoding glycolate oxidase is glcD, glcE, glcF and/or glcG from e.

33. The recombinant microorganism of any preceding embodiment, wherein said yeast is Saccharomyces cerevisiae.

34. The recombinant microorganism of any preceding embodiment, wherein the microorganism comprises a deletion or modification that reduces the activity of one or more endogenous genes selected from the group consisting of:

(a) a gene encoding glyoxylate aldehyde ligase;

(b) a gene encoding 2-oxo-4-hydroxyglutarate aldolase;

(d) a gene encoding a repressor of isocitrate lyase.

35. The recombinant microorganism of any preceding embodiment, wherein the gene encoding glyoxylate aldehyde ligase is gcl; the gene encoding 2-oxo-4-hydroxyglutarate aldolase is edA; the gene encoding glycolaldehyde reductase is fucO and/or gldA; and the gene encoding the repressor of isocitrate lyase is iclR.

36. The recombinant microorganism of any one of the preceding embodiments, wherein the expression level of a gene encoding alanine-glyoxylate aminotransferase, a gene encoding glycine dehydrogenase, a gene encoding glycine aminotransferase, a gene encoding serine-glyoxylate aminotransferase, and/or a gene encoding glycine oxidase is increased.

37. The recombinant microorganism of any one of the preceding embodiments, wherein the expression level of a gene encoding alanine aminotransferase, and/or a gene encoding NADPH-dependent glutamate synthase is increased.

38. The recombinant microorganism of any preceding embodiment, wherein said microorganism can utilize NADH and CO produced by other glycolate and/or glycine production pathways in a reaction catalyzed by malate dehydrogenase, pyruvate carboxylase, phosphoenolpyruvate carboxykinase, malate thiokinase, and malyl-coa lyase₂。

39. The recombinant microorganism of any preceding embodiment, wherein the microorganism can utilize exogenously added CO in a reaction catalyzed by malate dehydrogenase, pyruvate carboxylase, phosphoenolpyruvate carboxykinase, malate thiokinase, and malyl-CoA lyase₂Carbonic acid, and/or a reducing agent.

40. The recombinant microorganism of any preceding embodiment, wherein the reducing agent is hydrogen, electrons, and/or nad (p) H.

41. The recombinant microorganism of any preceding embodiment, wherein said reducing agent is from an external source.

42. The recombinant microorganism of any preceding embodiment, wherein the microorganism utilizes NADH and CO produced based on the serine/hydroxypyruvate pathway in a reaction catalyzed by malate dehydrogenase, pyruvate carboxylase, phosphoenolpyruvate carboxykinase, malate thiokinase, and malyl-coa lyase₂。

43. The recombinant microorganism of any preceding embodiment, wherein the microorganism utilizes NADH and CO produced by the glyoxylate shunt pathway in a reaction catalyzed by malate dehydrogenase, pyruvate carboxylase, phosphoenolpyruvate carboxykinase, malate thiokinase, and malyl-coa lyase₂。

44. The recombinant microorganism of any preceding embodiment, wherein the microorganism utilizes NADH and CO produced by the D-erythrose to glycolaldehyde based pathway in a reaction catalyzed by malate dehydrogenase, pyruvate carboxylase, phosphoenolpyruvate carboxykinase, malate thiokinase, and malyl-coa lyase₂。

45. The recombinant microorganism of any preceding embodiment, wherein the microorganism utilizes NADH and CO produced by the pentose derivative to glycolaldehyde based pathway in a reaction catalyzed by malate dehydrogenase, pyruvate carboxylase, phosphoenolpyruvate carboxykinase, malate thiokinase, and malyl-coa lyase₂。

46. The recombinant microorganism of any preceding embodiment, wherein the microorganism is selected from the group consisting of bacteria, yeast, and fungi.

47. The recombinant microorganism of any one of the preceding embodiments, wherein the microorganism is a bacterium selected from the group consisting of enterobacteriaceae, clostridiaceae, bacillaceae, streptomycetaceae, and corynebacteriaceae.

48. The recombinant microorganism of any one of the preceding embodiments, wherein the microorganism is a species of escherichia, clostridium, bacillus, klebsiella, pantoea, salmonella, lactobacillus, or corynebacterium.

49. The recombinant microorganism of any one of the preceding embodiments, wherein the microorganism is escherichia coli, or corynebacterium glutamicum, or clostridium acetobutylicum, or bacillus subtilis.

50. The recombinant microorganism of any preceding embodiment, wherein the microorganism is a yeast selected from the family saccharomyces.

51. The recombinant microorganism of any preceding embodiment, wherein the microorganism is a yeast species.

52. The recombinant microorganism of any preceding embodiment, wherein said microorganism is Saccharomyces cerevisiae.

53. The recombinant microorganism of any preceding embodiment, wherein the synthesis of glycolate and/or glycine is increased by increasing the expression level, or activity, or specificity of at least one enzyme selected from the group consisting of: pyruvate carboxylase, phosphoenolpyruvate carboxykinase, malate dehydrogenase, malate thiokinase, malyl-coa lyase, alanine-glyoxylate aminotransferase, glycine dehydrogenase, glycine aminotransferase, serine-glyoxylate aminotransferase, glycine oxidase, NADH-dependent glyoxylate reductase and NADPH-dependent glyoxylate reductase.

54. The recombinant microorganism of any preceding embodiment, wherein the synthesis of glycolate and/or glycine is increased by decreasing the expression level, or activity, or specificity of at least one enzyme selected from the group consisting of: malate synthase, isocitrate dehydrogenase, pyruvate oxidase and/or pyruvate formate lyase, pyruvate kinase, glucose-6-phosphate isomerase, glyoxylate carboligase, 2-oxo-4-hydroxyglutarate aldolase, glycolaldehyde reductase and glycolate oxidase.

55. The recombinant microorganism of any one of the preceding embodiments, wherein the synthesis of glycolate and/or glycine is increased by decreasing the expression level of a gene encoding a repressor of isocitrate lyase.

56. A method of producing glycolic acid and/or glycine using a recombinant microorganism in any preceding embodiment, wherein the method comprises culturing the recombinant microorganism in a culture medium containing a feedstock providing a carbon source until glycolic acid and/or glycine is produced.

57. The method of any one of the preceding embodiments, wherein the carbon source is selected from the group consisting of: sugars, glycerol, alcohols, organic acids, alkanes, fatty acids, hemicellulose, lignocellulose, proteins, carbon dioxide and carbon monoxide.

58. The method of any one of the preceding embodiments, wherein the carbon source is a hexose and/or pentose sugar.

59. The method of any one of the preceding embodiments, wherein the carbon source is glucose.

60. The method of any one of the preceding embodiments, wherein the carbon source is sucrose.

61. The method of any preceding embodiment, wherein the carbon source comprises a biomass hydrolysate comprising hemicellulose.

62. The method of any one of the preceding embodiments, wherein the carbon source is CO₂Or carbonic acid.

63. The method of any preceding embodiment, wherein the carbonic acid is HCO₃ ^-。

64. A method of producing a recombinant microorganism that produces glycolate and/or glycin from glyoxylate, comprising introducing into said microorganism:

(a) a gene encoding malate dehydrogenase catalyzing the conversion of pyruvate to malate;

(b) a gene encoding malate thiokinase catalyzing the conversion of malate into malyl-coa; and

65. A method of producing a recombinant microorganism that produces glycolate and/or glycin from glyoxylate, comprising introducing into said microorganism:

A gene encoding phosphoenolpyruvate carboxykinase which catalyzes the conversion of phosphoenolpyruvate to OAA;

(b) a gene encoding malate dehydrogenase catalyzing the conversion of OAA to malate;

66. A method of producing a recombinant microorganism that produces glycolate and/or glycin from glyoxylate, comprising introducing into said microorganism:

(b) a gene encoding malate thiokinase catalyzing the conversion of malate into malyl-coa; and

67. A method as in any preceding embodiment, wherein the gene encoding malate dehydrogenase comprises a mutation that results in partial or complete inhibition of malate dehydrogenase activity that catalyzes the conversion of oxaloacetate to malate, malate to pyruvate, or malate to oxaloacetate.

68. A method as in any preceding embodiment, comprising introducing into a microorganism:

(a) a gene encoding an NADH-dependent glyoxylate reductase that catalyzes the conversion of glyoxylate to glycolate;

(b) a gene encoding an NADPH-dependent glyoxylate reductase that catalyzes the conversion of glyoxylate to glycolate; or

(i) A gene encoding alanine-glyoxylate aminotransferase, a gene encoding glycine dehydrogenase, a gene encoding glycine aminotransferase, a gene encoding serine-glyoxylate aminotransferase, and/or a gene encoding glycine oxidase which catalyzes the conversion of glyoxylate to glycine.

69. A method according to any one of the preceding embodiments, comprising introducing into the microorganism a loss of function mutation in a gene encoding malate synthase, or a deletion of said gene.

70. A method according to any one of the preceding embodiments, comprising introducing into the microorganism a deletion or modification that reduces the activity of one or more enzymes encoded by a gene selected from the group consisting of:

(a) a gene encoding isocitrate dehydrogenase;

(b) a gene encoding pyruvate dehydrogenase, pyruvate oxidase, and/or pyruvate formate lyase;

(d) a gene encoding glycolate oxidase; and

(e) a gene encoding glucose-6-phosphate isomerase.

71. A method according to any one of the preceding embodiments, comprising introducing into said microorganism a deletion or modification that reduces the activity of one or more enzymes encoded by a gene selected from the group consisting of:

(a) a gene encoding glyoxylate aldehyde ligase;

(b) a gene encoding 2-oxo-4-hydroxyglutarate aldolase;

(d) a gene encoding a repressor of isocitrate lyase.

72. A method according to any one of the preceding embodiments, comprising introducing a gain-of-function mutation into a gene encoding alanine-glyoxylate aminotransferase, a gene encoding alanine-glyoxylate aminotransferase which converts glyoxylate to glycine, a gene encoding glycine dehydrogenase, a gene encoding glycine aminotransferase, a gene encoding serine-glyoxylate aminotransferase, and/or a gene encoding glycine oxidase which catalyzes the conversion of glyoxylate to glycine.

73. A method in any preceding embodiment, comprising introducing a gain-of-function mutation into a gene encoding alanine aminotransferase and/or a gene encoding NADPH-dependent glutamate synthase.

74. The method of any preceding embodiment, wherein the recombinant microorganism is selected from the group consisting of bacteria, yeast and fungi.

75. The method of any one of the preceding embodiments, wherein the recombinant microorganism is a bacterium selected from the group consisting of enterobacteriaceae, clostridiaceae, bacillaceae, streptomycetaceae, and corynebacteriaceae.

76. The method of any one of the preceding embodiments, wherein the recombinant microorganism is a species of escherichia, clostridium, bacillus, klebsiella, pantoea, salmonella, lactobacillus, or corynebacterium.

77. The method of any one of the preceding embodiments, wherein the recombinant microorganism is escherichia coli, or corynebacterium glutamicum, or clostridium acetobutylicum, or bacillus subtilis.

78. The method of any preceding embodiment, wherein the recombinant microorganism is a yeast selected from the family saccharomyces.

79. The method of any preceding embodiment, wherein said recombinant microorganism is a yeast species.

80. The method of any preceding embodiment, wherein said recombinant microorganism is saccharomyces cerevisiae.

Incorporation by reference

All references, articles, publications, patents, patent publications and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

However, a reference to any reference, article, publication, patent publication or patent application cited herein is not, and should not be taken as, an acknowledgment or any form of suggestion that they form part of the common general knowledge in any country in the world.

Sequence listing

<110> Brassico

<120> microorganisms and methods for the production of glycolic acid and glycine by the reverse glyoxylate shunt

<130> BRSK-010/02WO (331051-2041)

<150> 62/806,195

<151> 2019-02-15

<160> 38

<170> PatentIn version 3.5

<210> 1

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> primer Pgi _ N20_ FW

<400> 1

gtcctaggta taatactagt ccgattatct ggggtgaacc gttttagagc tagaaatagc 60

<210> 2

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> primer Pgi _ N20_ RV

<400> 2

actagtatta tacctaggac tgag 24

<210> 3

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Pgi_H1_FW

<400> 3

atgaaaaaca tcaatccaac gc 22

<210> 4

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> primer Pgi _ H1_ RV

<400> 4

ggtggatcag tcggtcacca tgtatgggc 29

<210> 5

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> Pgi_H2_FW

<400> 5

tggtgaccga ctgatccacc agggaacca 29

<210> 6

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> primer Pgi _ H2_ RV

<400> 6

catatcgacg atgattaacc gc 22

<210> 7

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<223> primer pvc _ FW

<400> 7

ttgtttaact ttaaggaggt ttggaggtac catgcccata tccaag 46

<210> 8

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> primer pvc _ RV

<400> 8

ttttcatacg gttcctcctt ctagatcatc cgccgtaaac cg 42

<210> 9

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223> primer aceA _ FW

<400> 9

cggatgatct agaaggagga accgtatgaa aacccgtaca caacaaat 48

<210> 10

<211> 47

<212> DNA

<213> Artificial sequence

<220>

<223> primer aceA _ RV

<400> 10

ttgtatcagc catcgtgtgc ctcctttaga actgcgattc ttcagtg 47

<210> 11

<211> 49

<212> DNA

<213> Artificial sequence

<220>

<223> primer gltA _ FW

<400> 11

atcgcagttc taaaggaggc acacgatggc tgatacaaaa gcaaaactc 49

<210> 12

<211> 52

<212> DNA

<213> Artificial sequence

<220>

<223> primer gltA _ RV

<400> 12

agatgatatc catcgtgtgc ctcctttaac gcttgatatc gcttttaaag tc 52

<210> 13

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> primer ghrA _ FW

<400> 13

tatcaagcgt taaaggaggc acacgatgga tatcatcttt tatcacccaa c 51

<210> 14

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<223> primer ghrA _ RV

<400> 14

ggctgcagga attcgatatc atagattagt agccgcgtgc gcg 43

<210> 15

<211> 49

<212> DNA

<213> Artificial sequence

<220>

<223> primer sucCD _ FW

<400> 15

acaatttcac acaggaaaca gaattcctat aattttgttt aactttaag 49

<210> 16

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> primer sucCD _ RV

<400> 16

tatagtctag atcagaatct gattccgtg 29

<210> 17

<211> 49

<212> DNA

<213> Artificial sequence

<220>

<223> primer mcl _ FW

<400> 17

gaatcagatt ctgatctaga ctataatttt gtttaacttt aaggaggtt 49

<210> 18

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> primer mcl _ RV

<400> 18

tagcacgcgt ttactttccg cccatcgcg 29

<210> 19

<211> 148

<212> DNA

<213> Artificial sequence

<220>

<223> J23119 promoter

<400> 19

gacgtccaca gctaacacca cgtcgtccct atctgctgcc ctaggtctat gagtggttgc 60

tggataactt gacagctagc tcagtcctag gtataatgct agctaataga aataattttg 120

tttaacttta aggaggtttg gaggtacc 148

<210> 20

<211> 3465

<212> DNA

<213> Rhizobium phaseoloides pyc

<400> 20

atgcccatat ccaagatact cgttgccaat cgctctgaaa tagccatccg cgtgttccgc 60

gcggccaacg agcttggaat aaaaacggtg gcgatctggg cggaagagga caagctggcg 120

ctgcaccgct tcaaggcgga cgagagttat caggtcggcc gcggaccgca tcttgcccgc 180

gacctcgggc cgatcgaaag ctatctgtcg atcgacgagg tgatccgcgt cgccaagctt 240

tccggtgccg acgccatcca tccgggctac ggcctcttgt cggaaagccc cgaattcgtc 300

gatgcctgca acaaggccgg catcatcttc atcggcccga aggccgatac gatgcgccag 360

cttggcaaca aggtcgcagc gcgcaacctg gcgatctcgg tcggcgtacc ggtcgtgccg 420

gcgaccgagc cactgccgga cgatatggcc gaagtggcga agatggcggc ggcgatcggc 480

tatcccgtca tgctgaaggc atcctggggc ggcggcggtc gcggcatgcg cgtcattcgt 540

tccgaggccg acctcgccaa ggaagtgacg gaagccaagc gcgaggcgat ggcggccttc 600

ggcaaggacg aggtctatct cgaaaaactg gtcgagcgcg cccgccacgt cgaaagccag 660

atcctcggcg acacccacgg caatgtcgtg catctcttcg agcgcgactg ttccgttcag 720

cgccgcaatc agaaggtcgt cgagcgcgcg cccgcaccct atctttcgga agcgcagcgc 780

caggaactcg ccgcctattc gctgaagatc gcaggggcga ccaactatat cggcgccggc 840

accgtcgaat atctgatgga tgccgatacc ggcaaatttt acttcatcga agtcaatccg 900

cgcatccagg tcgagcacac ggtgaccgaa gtcgtcaccg gcatcgatat cgtcaaggcg 960

cagatccaca tcctggacgg cgccgcgatc ggcacgccgc aatccggcgt gccgaaccag 1020

gaagacatcc gtctcaacgg tcacgccctg cagtgccgcg tgacgacgga agatccggag 1080

cacaacttca ttccggatta cggccgcatc accgcctatc gctcggcttc cggcttcggc 1140

atccggcttg acggcggcac ctcttattcc ggcgccatca tcacccgcta ttacgatccg 1200

ctgctcgtca aggtcacggc ctgggcgccg aacccgctgg aagccatttc ccgcatggac 1260

cgggcgctgc gcgaattccg catccgtggc gtcgccacca acctgacctt cctcgaagcg 1320

atcatcggcc atccgaaatt ccgcgacaac agctacacca cccgcttcat cgacacgacg 1380

ccggagctct tccagcaggt caagcgccag gaccgcgcga cgaagcttct gacctatctc 1440

gccgacgtca ccgtcaatgg ccatcccgag gccaaggaca ggccgaagcc cctcgagaat 1500

gccgccaggc cggtggtgcc ctatgccaat ggcaacgggg tgaaggacgg caccaagcag 1560

ctgctcgata cgctcggccc gaaaaaattc ggcgaatgga tgcgcaatga gaagcgcgtg 1620

cttctgaccg acaccacgat gcgcgacggc caccagtcgc tgctcgcaac ccgcatgcgt 1680

acctatgaca tcgccaggat cgccggcacc tattcgcatg cgctgccgaa cctcttgtcg 1740

ctcgaatgct ggggcggcgc caccttcgac gtctcgatgc gcttcctcac cgaagatccg 1800

tgggagcggc tggcgctgat ccgagagggg gcgccgaacc tgctcctgca gatgctgctg 1860

cgcggcgcca atggcgtcgg ttacaccaac tatcccgaca atgtcgtcaa atacttcgtc 1920

cgccaggcgg ccaaaggcgg catcgatctc ttccgcgtct tcgactgcct gaactgggtc 1980

gagaatatgc gggtgtcgat ggatgcgatt gccgaggaga acaagctctg cgaggcggcg 2040

atctgctaca ccggcgatat cctcaattcc gcccgcccga aatacgactt gaaatattac 2100

accaaccttg ccgtcgagct tgagaaggcc ggcgcccata tcattgcggt caaggatatg 2160

gcgggccttc tgaagccggc tgctgccaag gttctgttca aggcgctgcg tgaagcaacc 2220

ggcctgccga tccatttcca cacgcatgac acctcgggca ttgcggcggc aacggttctt 2280

gccgccgtcg aagccggtgt cgatgccgtc gatgcggcga tggatgcgct ctccggcaac 2340

acctcgcaac cctgtctcgg ctcgatcgtc gaggcgctct ccggctccga gcgcgatccc 2400

ggcctcgatc cggcatggat ccgccgcatc tccttctatt gggaagcggt gcgcaaccag 2460

tatgccgcct tcgaaagcga cctcaaggga ccggcatcgg aagtctatct gcatgaaatg 2520

ccgggcggcc agttcaccaa cctcaaggag caggcccgct cgctggggct ggaaacccgc 2580

tggcaccagg tggcgcaggc ctatgccgac gccaaccaga tgttcggcga tatcgtcaag 2640

gtgacgccat cctccaaggt cgtcggcgac atggcgctga tgatggtctc ccaggacctg 2700

accgtcgccg atgtcgtcag ccccgaccgc gaagtctcct tcccggaatc ggtcgtctcg 2760

atgctgaagg gcgatctcgg ccagcctccg tctggatggc cggaagcgct gcagaagaaa 2820

gcattgaagg gcgaaaagcc ctatacggtg cgccccggct cgctgctcaa ggaagccgat 2880

ctcgatgcgg aacgcaaagt catcgagaag aagcttgagc gcgaggtcag cgacttcgaa 2940

ttcgcttcct atctgatgta tccgaaggtc ttcaccgact ttgcgcttgc ctccgatacc 3000

tacggtccgg tttcggtgct gccgacgccc gcctattttt acgggttggc ggacggcgag 3060

gagctgttcg ccgacatcga gaagggcaag acgctcgtca tcgtcaatca ggcggtgagc 3120

gccaccgaca gccagggcat ggtcactgtc ttcttcgagc tcaacggcca gccgcgccgt 3180

atcaaggtgc ccgatcgggc ccacggggcg acgggagccg ccgtgcgccg caaggccgaa 3240

cccggcaatg ccgcccatgt cggtgcgccg atgccgggcg tcatcagccg tgtctttgtc 3300

tcttcaggcc aggccgtcaa tgccggcgac gtgctcgtct ccatcgaggc catgaagatg 3360

gaaaccgcga tccatgcgga aaaggacggc accattgccg aagtgctggt caaggccggc 3420

gatcagatcg atgccaagga cctgctggcg gtttacggcg gatga 3465

<210> 21

<211> 1305

<212> DNA

<213> Escherichia coli aceA

<400> 21

atgaaaaccc gtacacaaca aattgaagaa ttacagaaag agtggactca accgcgttgg 60

gaaggcatta ctcgcccata cagtgcggaa gatgtggtga aattacgcgg ttcagtcaat 120

cctgaatgca cgctggcgca actgggcgca gcgaaaatgt ggcgtctgct gcacggtgag 180

tcgaaaaaag gctacatcaa cagcctcggc gcactgactg gcggtcaggc gctgcaacag 240

gcgaaagcgg gtattgaagc agtctatctg tcgggatggc aggtagcggc ggacgctaac 300

ctggcggcca gcatgtatcc ggatcagtcg ctctatccgg caaactcggt gccagctgtg 360

gtggagcgga tcaacaacac cttccgtcgt gccgatcaga tccaatggtc cgcgggcatt 420

gagccgggcg atccgcgcta tgtcgattac ttcctgccga tcgttgccga tgcggaagcc 480

ggttttggcg gtgtcctgaa tgcctttgaa ctgatgaaag cgatgattga agccggtgca 540

gcggcagttc acttcgaaga tcagctggcg tcagtgaaga aatgcggtca catgggcggc 600

aaagttttag tgccaactca ggaagctatt cagaaactgg tcgcggcgcg tctggcagct 660

gacgtgacgg gcgttccaac cctgctggtt gcccgtaccg atgctgatgc ggcggatctg 720

atcacctccg attgcgaccc gtatgacagc gaatttatta ccggcgagcg taccagtgaa 780

ggcttcttcc gtactcatgc gggcattgag caagcgatca gccgtggcct ggcgtatgcg 840

ccatatgctg acctggtctg gtgtgaaacc tccacgccgg atctggaact ggcgcgtcgc 900

tttgcacaag ctatccacgc gaaatatccg ggcaaactgc tggcttataa ctgctcgccg 960

tcgttcaact ggcagaaaaa cctcgacgac aaaactattg ccagcttcca gcagcagctg 1020

tcggatatgg gctacaagtt ccagttcatc accctggcag gtatccacag catgtggttc 1080

aacatgtttg acctggcaaa cgcctatgcc cagggcgagg gtatgaagca ctacgttgag 1140

aaagtgcagc agccggaatt tgccgccgcg aaagatggct ataccttcgt atctcaccag 1200

caggaagtgg gtacaggtta cttcgataaa gtgacgacta ttattcaggg cggcacgtct 1260

tcagtcaccg cgctgaccgg ctccactgaa gaatcgcagt tctaa 1305

<210> 22

<211> 1284

<212> DNA

<213> E.coli gltAR163L

<400> 22

atggctgata caaaagcaaa actcaccctc aacggggata cagctgttga actggatgtg 60

ctgaaaggca cgctgggtca agatgttatt gatatccgta ctctcggttc aaaaggtgtg 120

ttcacctttg acccaggctt cacttcaacc gcatcctgcg aatctaaaat tacttttatt 180

gatggtgatg aaggtatttt gctgcaccgc ggtttcccga tcgatcagct ggcgaccgat 240

tctaactacc tggaagtttg ttacatcctg ctgaatggtg aaaaaccgac tcaggaacag 300

tatgacgaat ttaaaactac ggtgacccgt cataccatga tccacgagca gattacccgt 360

ctgttccatg ctttccgtcg cgactcgcat ccaatggcag tcatgtgtgg tattaccggc 420

gcgctggcgg cgttctatca cgactcgctg gatgttaaca atcctcgtca ccgtgaaatt 480

gccgcgttcc tcctgctgtc gaaaatgccg actatggccg cgatgtgtta caagtattcc 540

attggtcagc catttgttta cccgcgcaac gatctctcct acgccggtaa cttcctgaat 600

atgatgttct ccacgccgtg cgaaccgtat gaagttaatc cgattctgga acgtgctatg 660

gaccgtattc tgatcctgca cgctgaccat gaacagaacg cctctacctc caccgtgcgt 720

accgctggct cttcgggtgc gaacccgttt gcctgtatcg cagcaggtat tgcttcactg 780

tggggacctg cgcacggcgg tgctaacgaa gcggcgctga aaatgctgga agaaatcagc 840

tccgttaaac acattccgga atttgttcgt cgtgcgaaag acaaaaatga ttctttccgc 900

ctgatgggct tcggtcaccg cgtgtacaaa aattacgacc cgcgcgccac cgtaatgcgt 960

gaaacctgcc atgaagtgct gaaagagctg ggcacgaagg atgacctgct ggaagtggct 1020

atggagctgg aaaacatcgc gctgaacgac ccgtacttta tcgagaagaa actgtacccg 1080

aacgtcgatt tctactctgg tatcatcctg aaagcgatgg gtattccgtc ttccatgttc 1140

accgtcattt tcgcaatggc acgtaccgtt ggctggatcg cccactggag cgaaatgcac 1200

agtgacggta tgaagattgc ccgtccgcgt cagctgtata caggatatga aaaacgcgac 1260

tttaaaagcg atatcaagcg ttaa 1284

<210> 23

<211> 939

<212> DNA

<213> Escherichia coli ghrA

<400> 23

atggatatca tcttttatca cccaacgttc gatacccaat ggtggattga ggcactgcgc 60

aaagctattc ctcaggcaag agtcagagca tggaaaagcg gagataatga ctctgctgat 120

tatgctttag tctggcatcc tcctgttgaa atgctggcag ggcgcgatct taaagcggtg 180

ttcgcactcg gggccggtgt tgattctatt ttgagcaagc tacaggcaca ccctgaaatg 240

ctgaaccctt ctgttccact ttttcgcctg gaagataccg gtatgggcga gcaaatgcag 300

gaatatgctg tcagtcaggt gctgcattgg tttcgacgtt ttgacgatta tcgcatccag 360

caaaatagtt cgcattggca accgctgcct gaatatcatc gggaagattt taccatcggc 420

attttgggcg caggcgtact gggcagtaaa gttgctcaga gtctgcaaac ctggcgcttt 480

ccgctgcgtt gctggagtcg aacccgtaaa tcgtggcctg gcgtgcaaag ctttgccgga 540

cgggaagaac tgtctgcatt tctgagccaa tgtcgggtat tgattaattt gttaccgaat 600

acccctgaaa ccgtcggcat tattaatcaa caattactcg aaaaattacc ggatggcgcg 660

tatctcctca acctggcgcg tggtgttcat gttgtggaag atgacctgct cgcggcgctg 720

gatagcggca aagttaaagg cgcaatgttg gatgttttta atcgtgaacc cttaccgcct 780

gaaagtccgc tctggcaaca tccacgcgtg acgataacac cacatgtcgc cgcgattacc 840

cgtcccgctg aagctgtgga gtacatttct cgcaccattg cccagctcga aaaaggggag 900

agggtctgcg ggcaagtcga ccgcgcacgc ggctactaa 939

<210> 24

<211> 7260

<212> DNA

<213> Artificial sequence

<220>

<223> J23119-pyc-aceA-gltAR163L-ghrA synthetic operon

<400> 24

ttgacagcta gctcagtcct aggtataatg ctagctaata gaaataattt tgtttaactt 60

taaggaggtt tggaggtacc atgcccatat ccaagatact cgttgccaat cgctctgaaa 120

tagccatccg cgtgttccgc gcggccaacg agcttggaat aaaaacggtg gcgatctggg 180

cggaagagga caagctggcg ctgcaccgct tcaaggcgga cgagagttat caggtcggcc 240

gcggaccgca tcttgcccgc gacctcgggc cgatcgaaag ctatctgtcg atcgacgagg 300

tgatccgcgt cgccaagctt tccggtgccg acgccatcca tccgggctac ggcctcttgt 360

cggaaagccc cgaattcgtc gatgcctgca acaaggccgg catcatcttc atcggcccga 420

aggccgatac gatgcgccag cttggcaaca aggtcgcagc gcgcaacctg gcgatctcgg 480

tcggcgtacc ggtcgtgccg gcgaccgagc cactgccgga cgatatggcc gaagtggcga 540

agatggcggc ggcgatcggc tatcccgtca tgctgaaggc atcctggggc ggcggcggtc 600

gcggcatgcg cgtcattcgt tccgaggccg acctcgccaa ggaagtgacg gaagccaagc 660

gcgaggcgat ggcggccttc ggcaaggacg aggtctatct cgaaaaactg gtcgagcgcg 720

cccgccacgt cgaaagccag atcctcggcg acacccacgg caatgtcgtg catctcttcg 780

agcgcgactg ttccgttcag cgccgcaatc agaaggtcgt cgagcgcgcg cccgcaccct 840

atctttcgga agcgcagcgc caggaactcg ccgcctattc gctgaagatc gcaggggcga 900

ccaactatat cggcgccggc accgtcgaat atctgatgga tgccgatacc ggcaaatttt 960

acttcatcga agtcaatccg cgcatccagg tcgagcacac ggtgaccgaa gtcgtcaccg 1020

gcatcgatat cgtcaaggcg cagatccaca tcctggacgg cgccgcgatc ggcacgccgc 1080

aatccggcgt gccgaaccag gaagacatcc gtctcaacgg tcacgccctg cagtgccgcg 1140

tgacgacgga agatccggag cacaacttca ttccggatta cggccgcatc accgcctatc 1200

gctcggcttc cggcttcggc atccggcttg acggcggcac ctcttattcc ggcgccatca 1260

tcacccgcta ttacgatccg ctgctcgtca aggtcacggc ctgggcgccg aacccgctgg 1320

aagccatttc ccgcatggac cgggcgctgc gcgaattccg catccgtggc gtcgccacca 1380

acctgacctt cctcgaagcg atcatcggcc atccgaaatt ccgcgacaac agctacacca 1440

cccgcttcat cgacacgacg ccggagctct tccagcaggt caagcgccag gaccgcgcga 1500

cgaagcttct gacctatctc gccgacgtca ccgtcaatgg ccatcccgag gccaaggaca 1560

ggccgaagcc cctcgagaat gccgccaggc cggtggtgcc ctatgccaat ggcaacgggg 1620

tgaaggacgg caccaagcag ctgctcgata cgctcggccc gaaaaaattc ggcgaatgga 1680

tgcgcaatga gaagcgcgtg cttctgaccg acaccacgat gcgcgacggc caccagtcgc 1740

tgctcgcaac ccgcatgcgt acctatgaca tcgccaggat cgccggcacc tattcgcatg 1800

cgctgccgaa cctcttgtcg ctcgaatgct ggggcggcgc caccttcgac gtctcgatgc 1860

gcttcctcac cgaagatccg tgggagcggc tggcgctgat ccgagagggg gcgccgaacc 1920

tgctcctgca gatgctgctg cgcggcgcca atggcgtcgg ttacaccaac tatcccgaca 1980

atgtcgtcaa atacttcgtc cgccaggcgg ccaaaggcgg catcgatctc ttccgcgtct 2040

tcgactgcct gaactgggtc gagaatatgc gggtgtcgat ggatgcgatt gccgaggaga 2100

acaagctctg cgaggcggcg atctgctaca ccggcgatat cctcaattcc gcccgcccga 2160

aatacgactt gaaatattac accaaccttg ccgtcgagct tgagaaggcc ggcgcccata 2220

tcattgcggt caaggatatg gcgggccttc tgaagccggc tgctgccaag gttctgttca 2280

aggcgctgcg tgaagcaacc ggcctgccga tccatttcca cacgcatgac acctcgggca 2340

ttgcggcggc aacggttctt gccgccgtcg aagccggtgt cgatgccgtc gatgcggcga 2400

tggatgcgct ctccggcaac acctcgcaac cctgtctcgg ctcgatcgtc gaggcgctct 2460

ccggctccga gcgcgatccc ggcctcgatc cggcatggat ccgccgcatc tccttctatt 2520

gggaagcggt gcgcaaccag tatgccgcct tcgaaagcga cctcaaggga ccggcatcgg 2580

aagtctatct gcatgaaatg ccgggcggcc agttcaccaa cctcaaggag caggcccgct 2640

cgctggggct ggaaacccgc tggcaccagg tggcgcaggc ctatgccgac gccaaccaga 2700

tgttcggcga tatcgtcaag gtgacgccat cctccaaggt cgtcggcgac atggcgctga 2760

tgatggtctc ccaggacctg accgtcgccg atgtcgtcag ccccgaccgc gaagtctcct 2820

tcccggaatc ggtcgtctcg atgctgaagg gcgatctcgg ccagcctccg tctggatggc 2880

cggaagcgct gcagaagaaa gcattgaagg gcgaaaagcc ctatacggtg cgccccggct 2940

cgctgctcaa ggaagccgat ctcgatgcgg aacgcaaagt catcgagaag aagcttgagc 3000

gcgaggtcag cgacttcgaa ttcgcttcct atctgatgta tccgaaggtc ttcaccgact 3060

ttgcgcttgc ctccgatacc tacggtccgg tttcggtgct gccgacgccc gcctattttt 3120

acgggttggc ggacggcgag gagctgttcg ccgacatcga gaagggcaag acgctcgtca 3180

tcgtcaatca ggcggtgagc gccaccgaca gccagggcat ggtcactgtc ttcttcgagc 3240

tcaacggcca gccgcgccgt atcaaggtgc ccgatcgggc ccacggggcg acgggagccg 3300

ccgtgcgccg caaggccgaa cccggcaatg ccgcccatgt cggtgcgccg atgccgggcg 3360

tcatcagccg tgtctttgtc tcttcaggcc aggccgtcaa tgccggcgac gtgctcgtct 3420

ccatcgaggc catgaagatg gaaaccgcga tccatgcgga aaaggacggc accattgccg 3480

aagtgctggt caaggccggc gatcagatcg atgccaagga cctgctggcg gtttacggcg 3540

gatgatctag aaggaggaac cgtatgaaaa cccgtacaca acaaattgaa gaattacaga 3600

aagagtggac tcaaccgcgt tgggaaggca ttactcgccc atacagtgcg gaagatgtgg 3660

tgaaattacg cggttcagtc aatcctgaat gcacgctggc gcaactgggc gcagcgaaaa 3720

tgtggcgtct gctgcacggt gagtcgaaaa aaggctacat caacagcctc ggcgcactga 3780

ctggcggtca ggcgctgcaa caggcgaaag cgggtattga agcagtctat ctgtcgggat 3840

ggcaggtagc ggcggacgct aacctggcgg ccagcatgta tccggatcag tcgctctatc 3900

cggcaaactc ggtgccagct gtggtggagc ggatcaacaa caccttccgt cgtgccgatc 3960

agatccaatg gtccgcgggc attgagccgg gcgatccgcg ctatgtcgat tacttcctgc 4020

cgatcgttgc cgatgcggaa gccggttttg gcggtgtcct gaatgccttt gaactgatga 4080

aagcgatgat tgaagccggt gcagcggcag ttcacttcga agatcagctg gcgtcagtga 4140

agaaatgcgg tcacatgggc ggcaaagttt tagtgccaac tcaggaagct attcagaaac 4200

tggtcgcggc gcgtctggca gctgacgtga cgggcgttcc aaccctgctg gttgcccgta 4260

ccgatgctga tgcggcggat ctgatcacct ccgattgcga cccgtatgac agcgaattta 4320

ttaccggcga gcgtaccagt gaaggcttct tccgtactca tgcgggcatt gagcaagcga 4380

tcagccgtgg cctggcgtat gcgccatatg ctgacctggt ctggtgtgaa acctccacgc 4440

cggatctgga actggcgcgt cgctttgcac aagctatcca cgcgaaatat ccgggcaaac 4500

tgctggctta taactgctcg ccgtcgttca actggcagaa aaacctcgac gacaaaacta 4560

ttgccagctt ccagcagcag ctgtcggata tgggctacaa gttccagttc atcaccctgg 4620

caggtatcca cagcatgtgg ttcaacatgt ttgacctggc aaacgcctat gcccagggcg 4680

agggtatgaa gcactacgtt gagaaagtgc agcagccgga atttgccgcc gcgaaagatg 4740

gctatacctt cgtatctcac cagcaggaag tgggtacagg ttacttcgat aaagtgacga 4800

ctattattca gggcggcacg tcttcagtca ccgcgctgac cggctccact gaagaatcgc 4860

agttctaaag gaggcacacg atggctgata caaaagcaaa actcaccctc aacggggata 4920

cagctgttga actggatgtg ctgaaaggca cgctgggtca agatgttatt gatatccgta 4980

ctctcggttc aaaaggtgtg ttcacctttg acccaggctt cacttcaacc gcatcctgcg 5040

aatctaaaat tacttttatt gatggtgatg aaggtatttt gctgcaccgc ggtttcccga 5100

tcgatcagct ggcgaccgat tctaactacc tggaagtttg ttacatcctg ctgaatggtg 5160

aaaaaccgac tcaggaacag tatgacgaat ttaaaactac ggtgacccgt cataccatga 5220

tccacgagca gattacccgt ctgttccatg ctttccgtcg cgactcgcat ccaatggcag 5280

tcatgtgtgg tattaccggc gcgctggcgg cgttctatca cgactcgctg gatgttaaca 5340

atcctcgtca ccgtgaaatt gccgcgttcc tcctgctgtc gaaaatgccg actatggccg 5400

cgatgtgtta caagtattcc attggtcagc catttgttta cccgcgcaac gatctctcct 5460

acgccggtaa cttcctgaat atgatgttct ccacgccgtg cgaaccgtat gaagttaatc 5520

cgattctgga acgtgctatg gaccgtattc tgatcctgca cgctgaccat gaacagaacg 5580

cctctacctc caccgtgcgt accgctggct cttcgggtgc gaacccgttt gcctgtatcg 5640

cagcaggtat tgcttcactg tggggacctg cgcacggcgg tgctaacgaa gcggcgctga 5700

aaatgctgga agaaatcagc tccgttaaac acattccgga atttgttcgt cgtgcgaaag 5760

acaaaaatga ttctttccgc ctgatgggct tcggtcaccg cgtgtacaaa aattacgacc 5820

cgcgcgccac cgtaatgcgt gaaacctgcc atgaagtgct gaaagagctg ggcacgaagg 5880

atgacctgct ggaagtggct atggagctgg aaaacatcgc gctgaacgac ccgtacttta 5940

tcgagaagaa actgtacccg aacgtcgatt tctactctgg tatcatcctg aaagcgatgg 6000

gtattccgtc ttccatgttc accgtcattt tcgcaatggc acgtaccgtt ggctggatcg 6060

cccactggag cgaaatgcac agtgacggta tgaagattgc ccgtccgcgt cagctgtata 6120

caggatatga aaaacgcgac tttaaaagcg atatcaagcg ttaaaggagg cacacgatgg 6180

atatcatctt ttatcaccca acgttcgata cccaatggtg gattgaggca ctgcgcaaag 6240

ctattcctca ggcaagagtc agagcatgga aaagcggaga taatgactct gctgattatg 6300

ctttagtctg gcatcctcct gttgaaatgc tggcagggcg cgatcttaaa gcggtgttcg 6360

cactcggggc cggtgttgat tctattttga gcaagctaca ggcacaccct gaaatgctga 6420

acccttctgt tccacttttt cgcctggaag ataccggtat gggcgagcaa atgcaggaat 6480

atgctgtcag tcaggtgctg cattggtttc gacgttttga cgattatcgc atccagcaaa 6540

atagttcgca ttggcaaccg ctgcctgaat atcatcggga agattttacc atcggcattt 6600

tgggcgcagg cgtactgggc agtaaagttg ctcagagtct gcaaacctgg cgctttccgc 6660

tgcgttgctg gagtcgaacc cgtaaatcgt ggcctggcgt gcaaagcttt gccggacggg 6720

aagaactgtc tgcatttctg agccaatgtc gggtattgat taatttgtta ccgaataccc 6780

ctgaaaccgt cggcattatt aatcaacaat tactcgaaaa attaccggat ggcgcgtatc 6840

tcctcaacct ggcgcgtggt gttcatgttg tggaagatga cctgctcgcg gcgctggata 6900

gcggcaaagt taaaggcgca atgttggatg tttttaatcg tgaaccctta ccgcctgaaa 6960

gtccgctctg gcaacatcca cgcgtgacga taacaccaca tgtcgccgcg attacccgtc 7020

ccgctgaagc tgtggagtac atttctcgca ccattgccca gctcgaaaaa ggggagaggg 7080

tctgcgggca agtcgaccgc gcacgcggct actaatctat gatatcgaat tcctgcagcc 7140

cgggggatcc catggtacgc gtgctagagg catcaaataa aacgaaaggc tcagtcgaaa 7200

gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat 7260

<210> 25

<211> 260

<212> DNA

<213> Artificial sequence

<220>

<223> Ptac promoter from pACT3

<400> 25

cggagcttat cgactgcacg gtgcaccaat gcttctggcg tcaggcagcc atcggaagct 60

gtggtatggc tgtgcaggtc gtaaatcact gcataattcg tgtcgctcaa ggcgcactcc 120

cgttctggat aatgtttttt gcgccgacat cataacggtt ctggcaaata ttctgaaatg 180

agctgttgac aattaatcat cggctcgtat aatgtgtgga attgtgagcg gataacaatt 240

tcacacagga aacagaattc 260

<210> 26

<211> 2123

<212> DNA

<213> Artificial sequence

<220>

<223> sucCD of Methylococcus capsulatus strain Bath

<400> 26

gaattcctat aattttgttt aactttaagg aggggtacca tgaatatcca tgagtaccag 60

gccaaggagc tgctcaagac ctatggcgtg cccgtgcccg acggcgccgt tgcctattcc 120

gacgcgcagg ccgccagcgt cgccgaggag atcggcggca gccgctgggt ggtcaaggcg 180

cagatccatg ccggcggtcg cggcaaggcc gggggcgtaa aggtcgccca ctccatcgag 240

gaagtccgcc aatacgccga cgccatgctc ggcagccacc tcgtcaccca tcagaccggc 300

ccgggaggct cgctggttca gcgtctgtgg gtggaacagg ccagccatat caaaaaggaa 360

tactacctgg gcttcgtgat cgatcgcggc aatcaacgca tcaccctgat cgcctccagc 420

gagggcggca tggaaatcga ggaagtcgca aaggaaaccc cggagaaaat cgtcaaggaa 480

gtcgtcgatc cggccatagg cctgctggac ttccagtgcc gcaaggtcgc cacggcgatc 540

ggcctgaaag gcaaactgat gccccaggcc gtcaggctga tgaaggccat ctaccgctgc 600

atgcgcgaca aagatgccct gcaggccgaa atcaatcctc tggccatcgt gggcgaaagc 660

gacgaatcgc tcatggtcct ggatgccaag ttcaacttcg acgacaacgc cctgtaccgg 720

cagcgcacca tcaccgagat gcgcgacctg gccgaggaag acccgaaaga ggtcgaagcc 780

tccggccacg gtctcaatta catcgccctc gacggcaaca tcggctgcat cgtcaatggc 840

gccggcctcg ccatggcttc gctcgacgcc atcaccctgc atggcggccg tccggccaac 900

ttcctcgacg tgggcggcgg cgcctccccc gagaaggtca ccaatgcctg ccgcatcgta 960

ctggaagatc ccaacgtccg ctgcatcctg gtcaacatct ttgccggcat caaccgctgt 1020

gactggatcg ccaagggcct gatccaggcc tgcgacagcc tgcagatcaa ggtgccgctg 1080

atcgtgcgcc tggccgggac gaacgtcgac gagggccgca agatcctggc cgaatccggc 1140

ctctccttca tcaccgcgga aaatctggac gacgcggccg ccaaggccgt cgccatcgtc 1200

aagggataac agtcatgagc gtattcgtta acaagcactc caaggtcatc ttccagggct 1260

tcaccggcga gcacgccacc ttccacgcca aggacgccat gcggatgggc acccgggtgg 1320

tcggcggtgt cacccctggc aaaggcggca cccgccatcc cgatcccgaa ctcgctcatc 1380

tgccggtgtt cgacaccgtg gctgaagccg tggccgccac cggcgccgac gtctccgccg 1440

tgttcgtgcc gccgcccttc aatgcggacg cgttgatgga agccatagac gccggcatcc 1500

gggtcgccgt gaccatcgcc gacggcatcc cggtacacga catgatccga ctgcagcgct 1560

accgggtggg taaggattcc atcgtgatcg gaccgaacac ccccggcatc atcacgccgg 1620

gcgagtgcaa ggtgggcatc atgccttcgc acatttacaa gaagggcaac gtcggcatcg 1680

tgtcgcgctc cggcaccctc aattacgagg cgacggaaca gatggccgcg cttgggctgg 1740

gcatcaccac ctcggtcggt atcggcggtg accccatcaa cggaaccgat ttcgtcactg 1800

tcctgcgcgc cttcgaagcc gacccggaaa ccgagatcgt ggtgatgatc ggcgaaatcg 1860

gcggccccca ggaagtcgcc gccgcccgct gggccaagga aaacatgaca aagccggtca 1920

tcggcttcgt cgcaggcctt gccgcaccga ccggccgacg catgggccat gccggcgcca 1980

tcatctccag cgaggccgac accgccggag ccaagatgga cgccatggaa gccttggggc 2040

tgtatgtcgc ccgcaacccg gcacagatcg gccagaccgt gctacgcgcc gcgcaggaac 2100

acggaatcag attctgatct aga 2123

<210> 27

<211> 975

<212> DNA

<213> Artificial sequence

<220>

<223> mcl of Methylobacterium Proteus AM1

<400> 27

atgagcttca ccctgatcca gcaggccacc ccgcgcctgc accgctcgga actcgcggtt 60

cccggctcca acccgacctt catggagaag tcggccgcct cgaaggccga cgtgatcttc 120

ctcgacctcg aggacgcggt tgcgcccgac gacaaggagc aggcccgcaa gaacatcatc 180

caggccctca acgacctgga ttggggcaac aagaccatga tgatccgcat caacggtctc 240

gacacccact acatgtaccg cgacgtggtg gacatcgtgg aggcctgccc gcgcctcgac 300

atgatcctga tccccaaggt cggcgtgccg gccgacgtct acgccatcga cgtgctgacg 360

acgcagatcg agcaggccaa gaagcgcgag aagaagatcg gcttcgaggt gctgatcgag 420

accgcgctcg gcatggccaa tgtcgaggcg atcgcgacct cgtctaagcg ccttgaggcg 480

atgtccttcg gtgtcgccga ctacgccgct tccacccgcg cccgctccac cgtgatcggc 540

ggcgtcaacg ccgattacag cgtgctcacc gacaaggacg aggccggcaa ccgccagacc 600

cactggcagg atccgtggct gttcgcccag aaccgcatgc tggtcgcctg ccgcgcctac 660

ggcctgcgcc cgatcgacgg tcccttcggc gacttctccg atccggacgg ctacacctcg 720

gccgctcgcc gctgcgccgc gctcggcttc gagggcaagt gggcgatcca cccctcgcag 780

atcgatctcg ccaacgaggt cttcaccccc tccgaggccg aggtcaccaa ggcccgccgc 840

atcctggaag ccatggaaga ggccgccaag gccggccgcg gcgccgtctc gctcgacggc 900

cgtctcatcg acatcgcctc gatccgcatg gccgaggcgc tgatccagaa ggccgacgcg 960

atgggcggaa agtaa 975

<210> 28

<211> 3489

<212> DNA

<213> Artificial sequence

<220>

<223> Ptac _ sucC _ sucD _ mcl synthesized operon

<400> 28

cggagcttat cgactgcacg gtgcaccaat gcttctggcg tcaggcagcc atcggaagct 60

gtggtatggc tgtgcaggtc gtaaatcact gcataattcg tgtcgctcaa ggcgcactcc 120

cgttctggat aatgtttttt gcgccgacat cataacggtt ctggcaaata ttctgaaatg 180

agctgttgac aattaatcat cggctcgtat aatgtgtgga attgtgagcg gataacaatt 240

tcacacagga aacagaattc ctataatttt gtttaacttt aaggaggggt accatgaata 300

tccatgagta ccaggccaag gagctgctca agacctatgg cgtgcccgtg cccgacggcg 360

ccgttgccta ttccgacgcg caggccgcca gcgtcgccga ggagatcggc ggcagccgct 420

gggtggtcaa ggcgcagatc catgccggcg gtcgcggcaa ggccgggggc gtaaaggtcg 480

cccactccat cgaggaagtc cgccaatacg ccgacgccat gctcggcagc cacctcgtca 540

cccatcagac cggcccggga ggctcgctgg ttcagcgtct gtgggtggaa caggccagcc 600

atatcaaaaa ggaatactac ctgggcttcg tgatcgatcg cggcaatcaa cgcatcaccc 660

tgatcgcctc cagcgagggc ggcatggaaa tcgaggaagt cgcaaaggaa accccggaga 720

aaatcgtcaa ggaagtcgtc gatccggcca taggcctgct ggacttccag tgccgcaagg 780

tcgccacggc gatcggcctg aaaggcaaac tgatgcccca ggccgtcagg ctgatgaagg 840

ccatctaccg ctgcatgcgc gacaaagatg ccctgcaggc cgaaatcaat cctctggcca 900

tcgtgggcga aagcgacgaa tcgctcatgg tcctggatgc caagttcaac ttcgacgaca 960

acgccctgta ccggcagcgc accatcaccg agatgcgcga cctggccgag gaagacccga 1020

aagaggtcga agcctccggc cacggtctca attacatcgc cctcgacggc aacatcggct 1080

gcatcgtcaa tggcgccggc ctcgccatgg cttcgctcga cgccatcacc ctgcatggcg 1140

gccgtccggc caacttcctc gacgtgggcg gcggcgcctc ccccgagaag gtcaccaatg 1200

cctgccgcat cgtactggaa gatcccaacg tccgctgcat cctggtcaac atctttgccg 1260

gcatcaaccg ctgtgactgg atcgccaagg gcctgatcca ggcctgcgac agcctgcaga 1320

tcaaggtgcc gctgatcgtg cgcctggccg ggacgaacgt cgacgagggc cgcaagatcc 1380

tggccgaatc cggcctctcc ttcatcaccg cggaaaatct ggacgacgcg gccgccaagg 1440

ccgtcgccat cgtcaaggga taacagtcat gagcgtattc gttaacaagc actccaaggt 1500

catcttccag ggcttcaccg gcgagcacgc caccttccac gccaaggacg ccatgcggat 1560

gggcacccgg gtggtcggcg gtgtcacccc tggcaaaggc ggcacccgcc atcccgatcc 1620

cgaactcgct catctgccgg tgttcgacac cgtggctgaa gccgtggccg ccaccggcgc 1680

cgacgtctcc gccgtgttcg tgccgccgcc cttcaatgcg gacgcgttga tggaagccat 1740

agacgccggc atccgggtcg ccgtgaccat cgccgacggc atcccggtac acgacatgat 1800

ccgactgcag cgctaccggg tgggtaagga ttccatcgtg atcggaccga acacccccgg 1860

catcatcacg ccgggcgagt gcaaggtggg catcatgcct tcgcacattt acaagaaggg 1920

caacgtcggc atcgtgtcgc gctccggcac cctcaattac gaggcgacgg aacagatggc 1980

cgcgcttggg ctgggcatca ccacctcggt cggtatcggc ggtgacccca tcaacggaac 2040

cgatttcgtc actgtcctgc gcgccttcga agccgacccg gaaaccgaga tcgtggtgat 2100

gatcggcgaa atcggcggcc cccaggaagt cgccgccgcc cgctgggcca aggaaaacat 2160

gacaaagccg gtcatcggct tcgtcgcagg ccttgccgca ccgaccggcc gacgcatggg 2220

ccatgccggc gccatcatct ccagcgaggc cgacaccgcc ggagccaaga tggacgccat 2280

ggaagccttg gggctgtatg tcgcccgcaa cccggcacag atcggccaga ccgtgctacg 2340

cgccgcgcag gaacacggaa tcagattctg atctagacta taattttgtt taactttaag 2400

gaggtttgga atgagcttca ccctgatcca gcaggccacc ccgcgcctgc accgctcgga 2460

actcgcggtt cccggctcca acccgacctt catggagaag tcggccgcct cgaaggccga 2520

cgtgatcttc ctcgacctcg aggacgcggt tgcgcccgac gacaaggagc aggcccgcaa 2580

gaacatcatc caggccctca acgacctgga ttggggcaac aagaccatga tgatccgcat 2640

caacggtctc gacacccact acatgtaccg cgacgtggtg gacatcgtgg aggcctgccc 2700

gcgcctcgac atgatcctga tccccaaggt cggcgtgccg gccgacgtct acgccatcga 2760

cgtgctgacg acgcagatcg agcaggccaa gaagcgcgag aagaagatcg gcttcgaggt 2820

gctgatcgag accgcgctcg gcatggccaa tgtcgaggcg atcgcgacct cgtctaagcg 2880

ccttgaggcg atgtccttcg gtgtcgccga ctacgccgct tccacccgcg cccgctccac 2940

cgtgatcggc ggcgtcaacg ccgattacag cgtgctcacc gacaaggacg aggccggcaa 3000

ccgccagacc cactggcagg atccgtggct gttcgcccag aaccgcatgc tggtcgcctg 3060

ccgcgcctac ggcctgcgcc cgatcgacgg tcccttcggc gacttctccg atccggacgg 3120

ctacacctcg gccgctcgcc gctgcgccgc gctcggcttc gagggcaagt gggcgatcca 3180

cccctcgcag atcgatctcg ccaacgaggt cttcaccccc tccgaggccg aggtcaccaa 3240

ggcccgccgc atcctggaag ccatggaaga ggccgccaag gccggccgcg gcgccgtctc 3300

gctcgacggc cgtctcatcg acatcgcctc gatccgcatg gccgaggcgc tgatccagaa 3360

ggccgacgcg atgggcggaa agtaaacgcg tgctagaggc atcaaataaa acgaaaggct 3420

cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt 3480

aggacaaat 3489

<210> 29

<211> 2652

<212> DNA

<213> Escherichia coli ppcK620S

<400> 29

atgaacgaac aatattccgc attgcgtagt aatgtcagta tgctcggcaa agtgctggga 60

gaaaccatca aggatgcgtt gggagaacac attcttgaac gcgtagaaac tatccgtaag 120

ttgtcgaaat cttcacgcgc tggcaatgat gctaaccgcc aggagttgct caccacctta 180

caaaatttgt cgaacgacga gctgctgccc gttgcgcgtg cgtttagtca gttcctgaac 240

ctggccaaca ccgccgagca ataccacagc atttcgccga aaggcgaagc tgccagcaac 300

ccggaagtga tcgcccgcac cctgcgtaaa ctgaaaaacc agccggaact gagcgaagac 360

accatcaaaa aagcagtgga atcgctgtcg ctggaactgg tcctcacggc tcacccaacc 420

gaaattaccc gtcgtacact gatccacaaa atggtggaag tgaacgcctg tttaaaacag 480

ctcgataaca aagatatcgc tgactacgaa cacaaccagc tgatgcgtcg cctgcgccag 540

ttgatcgccc agtcatggca taccgatgaa atccgtaagc tgcgtccaag cccggtagat 600

gaagccaaat ggggctttgc cgtagtggaa aacagcctgt ggcaaggcgt accaaattac 660

ctgcgcgaac tgaacgaaca actggaagag aacctcggct acaaactgcc cgtcgaattt 720

gttccggtcc gttttacttc gtggatgggc ggcgaccgcg acggcaaccc gaacgtcact 780

gccgatatca cccgccacgt cctgctactc agccgctgga aagccaccga tttgttcctg 840

aaagatattc aggtgctggt ttctgaactg tcgatggttg aagcgacccc tgaactgctg 900

gcgctggttg gcgaagaagg tgccgcagaa ccgtatcgct atctgatgaa aaacctgcgt 960

tctcgcctga tggcgacaca ggcatggctg gaagcgcgcc tgaaaggcga agaactgcca 1020

aaaccagaag gcctgctgac acaaaacgaa gaactgtggg aaccgctcta cgcttgctac 1080

cagtcacttc aggcgtgtgg catgggtatt atcgccaacg gcgatctgct cgacaccctg 1140

cgccgcgtga aatgtttcgg cgtaccgctg gtccgtattg atatccgtca ggagagcacg 1200

cgtcataccg aagcgctggg cgagctgacc cgctacctcg gtatcggcga ctacgaaagc 1260

tggtcagagg ccgacaaaca ggcgttcctg atccgcgaac tgaactccaa acgtccgctt 1320

ctgccgcgca actggcaacc aagcgccgaa acgcgcgaag tgctcgatac ctgccaggtg 1380

attgccgaag caccgcaagg ctccattgcc gcctacgtga tctcgatggc gaaaacgccg 1440

tccgacgtac tggctgtcca cctgctgctg aaagaagcgg gtatcgggtt tgcgatgccg 1500

gttgctccgc tgtttgaaac cctcgatgat ctgaacaacg ccaacgatgt catgacccag 1560

ctgctcaata ttgactggta tcgtggcctg attcagggca aacagatggt gatgattggc 1620

tattccgact cagcaaaaga tgcgggagtg atggcagctt cctgggcgca atatcaggca 1680

caggatgcat taatcaaaac ctgcgaaaaa gcgggtattg agctgacgtt gttccacggt 1740

cgcggcggtt ccattggtcg cggcggcgca cctgctcatg cggcgctgct gtcacaaccg 1800

ccaggaagcc tgaaaggcgg cctgcgcgta accgaacagg gcgagatgat ccgctttagc 1860

tatggtctgc cagaaatcac cgtcagcagc ctgtcgcttt ataccggggc gattctggaa 1920

gccaacctgc tgccaccgcc ggagccgaaa gagagctggc gtcgcattat ggatgaactg 1980

tcagtcatct cctgcgatgt ctaccgcggc tacgtacgtg aaaacaaaga ttttgtgcct 2040

tacttccgct ccgctacgcc ggaacaagaa ctgggcaaac tgccgttggg ttcacgtccg 2100

gcgaaacgtc gcccaaccgg cggcgtcgag tcactacgcg ccattccgtg gatcttcgcc 2160

tggacgcaaa accgtctgat gctccccgcc tggctgggtg caggtacggc gctgcaaaaa 2220

gtggtcgaag acggcaaaca gagcgagctg gaggctatgt gccgcgattg gccattcttc 2280

tcgacgcgtc tcggcatgct ggagatggtc ttcgccaaag cagacctgtg gctggcggaa 2340

tactatgacc aacgcctggt agacaaagca ctgtggccgt taggtaaaga gttacgcaac 2400

ctgcaagaag aagacatcaa agtggtgctg gcgattgcca acgattccca tctgatggcc 2460

gatctgccgt ggattgcaga gtctattcag ctacggaata tttacaccga cccgctgaac 2520

gtattgcagg ccgagttgct gcaccgctcc cgccaggcag aaaaagaagg ccaggaaccg 2580

gatcctcgcg tcgaacaagc gttaatggtc actattgccg ggattgcggc aggtatgcgt 2640

aataccggct aa 2652

<210> 30

<211> 6602

<212> DNA

<213> Artificial sequence

<220>

<223> Ptac-ppcK 620S-aceA-gltAR163L-ghrA synthetic operon

<400> 30

cggagcttat cgactgcacg gtgcaccaat gcttctggcg tcaggcagcc atcggaagct 60

gtggtatggc tgtgcaggtc gtaaatcact gcataattcg tgtcgctcaa ggcgcactcc 120

cgttctggat aatgtttttt gcgccgacat cataacggtt ctggcaaata ttctgaaatg 180

agctgttgac aattaatcat cggctcgtat aatgtgtgga attgtgagcg gataacaatt 240

tcacacagga aacagaattc gagctcggta cccgggatga acgaacaata ttccgcattg 300

cgtagtaatg tcagtatgct cggcaaagtg ctgggagaaa ccatcaagga tgcgttggga 360

gaacacattc ttgaacgcgt agaaactatc cgtaagttgt cgaaatcttc acgcgctggc 420

aatgatgcta accgccagga gttgctcacc accttacaaa atttgtcgaa cgacgagctg 480

ctgcccgttg cgcgtgcgtt tagtcagttc ctgaacctgg ccaacaccgc cgagcaatac 540

cacagcattt cgccgaaagg cgaagctgcc agcaacccgg aagtgatcgc ccgcaccctg 600

cgtaaactga aaaaccagcc ggaactgagc gaagacacca tcaaaaaagc agtggaatcg 660

ctgtcgctgg aactggtcct cacggctcac ccaaccgaaa ttacccgtcg tacactgatc 720

cacaaaatgg tggaagtgaa cgcctgttta aaacagctcg ataacaaaga tatcgctgac 780

tacgaacaca accagctgat gcgtcgcctg cgccagttga tcgcccagtc atggcatacc 840

gatgaaatcc gtaagctgcg tccaagcccg gtagatgaag ccaaatgggg ctttgccgta 900

gtggaaaaca gcctgtggca aggcgtacca aattacctgc gcgaactgaa cgaacaactg 960

gaagagaacc tcggctacaa actgcccgtc gaatttgttc cggtccgttt tacttcgtgg 1020

atgggcggcg accgcgacgg caacccgaac gtcactgccg atatcacccg ccacgtcctg 1080

ctactcagcc gctggaaagc caccgatttg ttcctgaaag atattcaggt gctggtttct 1140

gaactgtcga tggttgaagc gacccctgaa ctgctggcgc tggttggcga agaaggtgcc 1200

gcagaaccgt atcgctatct gatgaaaaac ctgcgttctc gcctgatggc gacacaggca 1260

tggctggaag cgcgcctgaa aggcgaagaa ctgccaaaac cagaaggcct gctgacacaa 1320

aacgaagaac tgtgggaacc gctctacgct tgctaccagt cacttcaggc gtgtggcatg 1380

ggtattatcg ccaacggcga tctgctcgac accctgcgcc gcgtgaaatg tttcggcgta 1440

ccgctggtcc gtattgatat ccgtcaggag agcacgcgtc ataccgaagc gctgggcgag 1500

ctgacccgct acctcggtat cggcgactac gaaagctggt cagaggccga caaacaggcg 1560

ttcctgatcc gcgaactgaa ctccaaacgt ccgcttctgc cgcgcaactg gcaaccaagc 1620

gccgaaacgc gcgaagtgct cgatacctgc caggtgattg ccgaagcacc gcaaggctcc 1680

attgccgcct acgtgatctc gatggcgaaa acgccgtccg acgtactggc tgtccacctg 1740

ctgctgaaag aagcgggtat cgggtttgcg atgccggttg ctccgctgtt tgaaaccctc 1800

gatgatctga acaacgccaa cgatgtcatg acccagctgc tcaatattga ctggtatcgt 1860

ggcctgattc agggcaaaca gatggtgatg attggctatt ccgactcagc aaaagatgcg 1920

ggagtgatgg cagcttcctg ggcgcaatat caggcacagg atgcattaat caaaacctgc 1980

gaaaaagcgg gtattgagct gacgttgttc cacggtcgcg gcggttccat tggtcgcggc 2040

ggcgcacctg ctcatgcggc gctgctgtca caaccgccag gaagcctgaa aggcggcctg 2100

cgcgtaaccg aacagggcga gatgatccgc tttagctatg gtctgccaga aatcaccgtc 2160

agcagcctgt cgctttatac cggggcgatt ctggaagcca acctgctgcc accgccggag 2220

ccgaaagaga gctggcgtcg cattatggat gaactgtcag tcatctcctg cgatgtctac 2280

cgcggctacg tacgtgaaaa caaagatttt gtgccttact tccgctccgc tacgccggaa 2340

caagaactgg gcaaactgcc gttgggttca cgtccggcga aacgtcgccc aaccggcggc 2400

gtcgagtcac tacgcgccat tccgtggatc ttcgcctgga cgcaaaaccg tctgatgctc 2460

cccgcctggc tgggtgcagg tacggcgctg caaaaagtgg tcgaagacgg caaacagagc 2520

gagctggagg ctatgtgccg cgattggcca ttcttctcga cgcgtctcgg catgctggag 2580

atggtcttcg ccaaagcaga cctgtggctg gcggaatact atgaccaacg cctggtagac 2640

aaagcactgt ggccgttagg taaagagtta cgcaacctgc aagaagaaga catcaaagtg 2700

gtgctggcga ttgccaacga ttcccatctg atggccgatc tgccgtggat tgcagagtct 2760

attcagctac ggaatattta caccgacccg ctgaacgtat tgcaggccga gttgctgcac 2820

cgctcccgcc aggcagaaaa agaaggccag gaaccggatc ctcgcgtcga acaagcgtta 2880

atggtcacta ttgccgggat tgcggcaggt atgcgtaata ccggctaatc tagaaggagg 2940

aaccgtatga aaacccgtac acaacaaatt gaagaattac agaaagagtg gactcaaccg 3000

cgttgggaag gcattactcg cccatacagt gcggaagatg tggtgaaatt acgcggttca 3060

gtcaatcctg aatgcacgct ggcgcaactg ggcgcagcga aaatgtggcg tctgctgcac 3120

ggtgagtcga aaaaaggcta catcaacagc ctcggcgcac tgactggcgg tcaggcgctg 3180

caacaggcga aagcgggtat tgaagcagtc tatctgtcgg gatggcaggt agcggcggac 3240

gctaacctgg cggccagcat gtatccggat cagtcgctct atccggcaaa ctcggtgcca 3300

gctgtggtgg agcggatcaa caacaccttc cgtcgtgccg atcagatcca atggtccgcg 3360

ggcattgagc cgggcgatcc gcgctatgtc gattacttcc tgccgatcgt tgccgatgcg 3420

gaagccggtt ttggcggtgt cctgaatgcc tttgaactga tgaaagcgat gattgaagcc 3480

ggtgcagcgg cagttcactt cgaagatcag ctggcgtcag tgaagaaatg cggtcacatg 3540

ggcggcaaag ttttagtgcc aactcaggaa gctattcaga aactggtcgc ggcgcgtctg 3600

gcagctgacg tgacgggcgt tccaaccctg ctggttgccc gtaccgatgc tgatgcggcg 3660

gatctgatca cctccgattg cgacccgtat gacagcgaat ttattaccgg cgagcgtacc 3720

agtgaaggct tcttccgtac tcatgcgggc attgagcaag cgatcagccg tggcctggcg 3780

tatgcgccat atgctgacct ggtctggtgt gaaacctcca cgccggatct ggaactggcg 3840

cgtcgctttg cacaagctat ccacgcgaaa tatccgggca aactgctggc ttataactgc 3900

tcgccgtcgt tcaactggca gaaaaacctc gacgacaaaa ctattgccag cttccagcag 3960

cagctgtcgg atatgggcta caagttccag ttcatcaccc tggcaggtat ccacagcatg 4020

tggttcaaca tgtttgacct ggcaaacgcc tatgcccagg gcgagggtat gaagcactac 4080

gttgagaaag tgcagcagcc ggaatttgcc gccgcgaaag atggctatac cttcgtatct 4140

caccagcagg aagtgggtac aggttacttc gataaagtga cgactattat tcagggcggc 4200

acgtcttcag tcaccgcgct gaccggctcc actgaagaat cgcagttcta aaggaggcac 4260

acgatggctg atacaaaagc aaaactcacc ctcaacgggg atacagctgt tgaactggat 4320

gtgctgaaag gcacgctggg tcaagatgtt attgatatcc gtactctcgg ttcaaaaggt 4380

gtgttcacct ttgacccagg cttcacttca accgcatcct gcgaatctaa aattactttt 4440

attgatggtg atgaaggtat tttgctgcac cgcggtttcc cgatcgatca gctggcgacc 4500

gattctaact acctggaagt ttgttacatc ctgctgaatg gtgaaaaacc gactcaggaa 4560

cagtatgacg aatttaaaac tacggtgacc cgtcatacca tgatccacga gcagattacc 4620

cgtctgttcc atgctttccg tcgcgactcg catccaatgg cagtcatgtg tggtattacc 4680

ggcgcgctgg cggcgttcta tcacgactcg ctggatgtta acaatcctcg tcaccgtgaa 4740

attgccgcgt tcctcctgct gtcgaaaatg ccgactatgg ccgcgatgtg ttacaagtat 4800

tccattggtc agccatttgt ttacccgcgc aacgatctct cctacgccgg taacttcctg 4860

aatatgatgt tctccacgcc gtgcgaaccg tatgaagtta atccgattct ggaacgtgct 4920

atggaccgta ttctgatcct gcacgctgac catgaacaga acgcctctac ctccaccgtg 4980

cgtaccgctg gctcttcggg tgcgaacccg tttgcctgta tcgcagcagg tattgcttca 5040

ctgtggggac ctgcgcacgg cggtgctaac gaagcggcgc tgaaaatgct ggaagaaatc 5100

agctccgtta aacacattcc ggaatttgtt cgtcgtgcga aagacaaaaa tgattctttc 5160

cgcctgatgg gcttcggtca ccgcgtgtac aaaaattacg acccgcgcgc caccgtaatg 5220

cgtgaaacct gccatgaagt gctgaaagag ctgggcacga aggatgacct gctggaagtg 5280

gctatggagc tggaaaacat cgcgctgaac gacccgtact ttatcgagaa gaaactgtac 5340

ccgaacgtcg atttctactc tggtatcatc ctgaaagcga tgggtattcc gtcttccatg 5400

ttcaccgtca ttttcgcaat ggcacgtacc gttggctgga tcgcccactg gagcgaaatg 5460

cacagtgacg gtatgaagat tgcccgtccg cgtcagctgt atacaggata tgaaaaacgc 5520

gactttaaaa gcgatatcaa gcgttaaagg aggcacacga tggatatcat cttttatcac 5580

ccaacgttcg atacccaatg gtggattgag gcactgcgca aagctattcc tcaggcaaga 5640

gtcagagcat ggaaaagcgg agataatgac tctgctgatt atgctttagt ctggcatcct 5700

cctgttgaaa tgctggcagg gcgcgatctt aaagcggtgt tcgcactcgg ggccggtgtt 5760

gattctattt tgagcaagct acaggcacac cctgaaatgc tgaacccttc tgttccactt 5820

tttcgcctgg aagataccgg tatgggcgag caaatgcagg aatatgctgt cagtcaggtg 5880

ctgcattggt ttcgacgttt tgacgattat cgcatccagc aaaatagttc gcattggcaa 5940

ccgctgcctg aatatcatcg ggaagatttt accatcggca ttttgggcgc aggcgtactg 6000

ggcagtaaag ttgctcagag tctgcaaacc tggcgctttc cgctgcgttg ctggagtcga 6060

acccgtaaat cgtggcctgg cgtgcaaagc tttgccggac gggaagaact gtctgcattt 6120

ctgagccaat gtcgggtatt gattaatttg ttaccgaata cccctgaaac cgtcggcatt 6180

attaatcaac aattactcga aaaattaccg gatggcgcgt atctcctcaa cctggcgcgt 6240

ggtgttcatg ttgtggaaga tgacctgctc gcggcgctgg atagcggcaa agttaaaggc 6300

gcaatgttgg atgtttttaa tcgtgaaccc ttaccgcctg aaagtccgct ctggcaacat 6360

ccacgcgtga cgataacacc acatgtcgcc gcgattaccc gtcccgctga agctgtggag 6420

tacatttctc gcaccattgc ccagctcgaa aaaggggaga gggtctgcgg gcaagtcgac 6480

cgcgcacgcg gctactaatc tagaaagctt ctgttttggc ggatgagaga agaaattcgt 6540

cgcccgccat aaactgccag gcatcaaatt aagcagaagg ccatcctgac ggatggcctt 6600

tt 6602

<210> 31

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> primer pACT _ FW

<400> 31

tctagaaagc ttctgttttg gc 22

<210> 32

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> ppc_RV

<400> 32

gttcctcctt ctagattagc cg 22

<210> 33

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223> primer aceA _ FW

<400> 33

cggctaatct agaaggagga accgtatgaa aacccgtaca caacaaat 48

<210> 34

<211> 47

<212> DNA

<213> Artificial sequence

<220>

<223> primer aceA _ RV

<400> 34

ttgtatcagc catcgtgtgc ctcctttaga actgcgattc ttcagtg 47

<210> 35

<211> 49

<212> DNA

<213> Artificial sequence

<220>

<223> primer gltA _ FW

<400> 35

atcgcagttc taaaggaggc acacgatggc tgatacaaaa gcaaaactc 49

<210> 36

<211> 52

<212> DNA

<213> Artificial sequence

<220>

<223> primer gltA _ RV

<400> 36

agatgatatc catcgtgtgc ctcctttaac gcttgatatc gcttttaaag tc 52

<210> 37

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> primer ghrA _ FW

<400> 37

tatcaagcgt taaaggaggc acacgatgga tatcatcttt tatcacccaa c 51

<210> 38

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<223> primer ghrA _ RV

<400> 38

tccgccaaaa cagaagcttt ctagattagt agccgcgtgc gcg 43

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Microorganisms and methods for the production of glycolic acid and glycine through a reverse glyoxylate shunt

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