阅读说明：本技术 类胡萝卜素和脱辅基类胡萝卜素的生产 (Production of carotenoids and apocarotenoids ) 是由 张聪强 陈茜娴 朱兴奋 于 2018-02-26 设计创作，主要内容包括：公开生产类胡萝卜素和脱辅基类胡萝卜素的方法。方法包括以下步骤：在宿主细胞中表达表达模块,该表达模块包含表达载体,该表达载体具有编码至少一种优化的类胡萝卜素或脱辅基类胡萝卜素生成酶的编码区,该编码区与启动子可操作地连接。还提供宿主细胞和试剂盒,所述宿主细胞包含表达载体,所述表达载体具有编码至少一种优化的类胡萝卜素或脱辅基类胡萝卜素生成酶的编码区,所述编码区与启动子可操作地连接。特别地,类胡萝卜素或脱辅基类胡萝卜素生成酶形成选自以下的操纵子：a.用于生产α-紫罗酮的ΔN50-LsLcyE和TrxA-桂花CCD1；或b.用于生产β-紫罗酮的crtY和phCCD1；或c.用于生产ε-胡萝卜素的ΔN50-LsLcyE；或d.用于生产视黄醛的crtY和blh；或e.用于生产视黄醇的crtY、blh和ybbO。(Disclosed are methods for producing carotenoids and apoprotenoids. The method comprises the following steps: expressing an expression module in a host cell, the expression module comprising an expression vector having a coding region encoding at least one optimized carotenoid or apophotocarotenoid producing enzyme, the coding region being operably linked to a promoter. Also provided are host cells and kits comprising an expression vector having a coding region encoding at least one optimized carotenoid or apoproteolytic carotenoid producing enzyme, the coding region being operably linked to a promoter. In particular, the carotenoid or apoproteolytic carotenoid-producing enzyme forms an operon selected from the group consisting of: a. delta N50-LsLcyE and TrxA-sweet osmanthus CCD1 for producing alpha-ionone; crtY and phCCD1 for beta-ionone production; Δ N50-LsLcyE e for the production of epsilon-carotene; crtY and blh for the production of retinal; crtY, blh and ybbO for retinol production.)

1. a process for producing a carotenoid or an apoprotecarotenoid, the process comprising the steps of: expressing in a host cell an expression module comprising an expression vector having a coding region encoding at least one optimized apoprotecarotenoid or carotenoid-producing enzyme, said coding region being operably linked to a promoter.

2. The method of claim 1, wherein the carotenoid is selected from among phytoene, lycopene, alpha-carotene, gamma-carotene, delta-carotene, epsilon-carotene, or beta-carotene, and wherein the apo-carotenoid is selected from among alpha-ionone, beta-ionone, pseudoionone (or psi-ionone), hydroxyionone, beta-cyclocitral, trans-geranylacetone, 6-methyl-5-hepten-2-one (MHO), retinal, retinol, 8 ', 10-diphocarotene-8', 10-dialdehyde (C17), or 10 ', 6-diphocarotene-10', 6-dialdehyde (C19).

3. The method according to claim 1 or 2, wherein the at least one optimized carotenoid or apoproteolytic carotenoid is selected from the group consisting of crtY, CCD1, CCD2, CCD4, BCDO, LcyE, blh or ybbO.

4. The method of claim 3, wherein the CCD2 is selected from CaCCD2 or CsCCD 2.

5. The method of claim 3, wherein the CCD4 is selected from the group consisting of: AtCCD4, BoCCD4b, CmCCD4, CsCCD4a, MaCCD4, MdCCD4, OfCCD4, PpCCD4, RdCCD4 and VvCCD4 a.

6. The process according to claim 2 or claim 3, wherein the apo-carotenoid is alpha-ionone, and wherein the at least one optimized apo-carotenoid production enzyme is selected from LcyE and CCD1, and forms an operon having the following structure: LcyE-CCD 1.

7. the method according to claim 6, wherein the LcyE is derived from lettuce (Lactuca sativa) and is N-terminally truncated (ranging from 1 to 100 amino acids of LsLcyE, in particular Δ N50-LsLcyE).

8. The method of claim 6, wherein the CCD1 is expressed as a fusion protein selected from TrxA-CCD1, SUMO-CCD1, or MBP-CCD 1.

9. The method of claim 8, wherein the fusion protein is TrxA-CCD 1.

10. The method of claim 9, wherein the fusion protein is selected from TrxA-Osmanthus fragrans (Osmanthus fragrans) CCD1 or TrxA-Petunia (Petunia hybrid) CCD 1.

11. The method of claim 10, wherein the CCD1 is derived from or is osmanthus fragrans CCD1(OfCCD1) and comprises one or more of the following mutations: N154Y, M152T, L151F.

12. The method according to any one of claims 6 to 11, comprising screening for alpha-ionone expression levels in amounts of up to 100 and 1000mg/L over a 24 hour period.

13. The method according to claim 2 or claim 3, wherein the apoprotenoid is β -ionone, and wherein the at least one optimized apoproteolytic enzyme is selected from the group consisting of crtY and CCD1, and forms an operon having the structure crtY-CCD 1.

14. The method of claim 13, wherein the CCD1 is derived from petunia (PhCCD 1).

15. The method of claim 13, wherein the crtY is derived from Pantoea ananatis (Pantoea ananatis).

16. The method of any one of claims 13 to 15, further comprising screening for the expression level of β -ionone in an amount of up to 10-1000mg/L over a 24 hour period.

17. The method according to claim 2 or claim 3, wherein the apo-carotenoid is retinal, and wherein the at least one optimized apo-carotenoid producing enzyme is selected from the group consisting of crtY and blh, and forms an operon with the structure crtY-blh.

18. The method of claim 17, wherein the crtY is derived from pantoea ananatis.

19. The method of claim 17, wherein the blh is derived from uncultured marine bacterium HF10_19P 19.

20. The method of claims 17-19, further comprising screening for retinal expression levels in an amount of 10-1000mg/L over a 24 hour period.

21. The method according to claim 2 or claim 3, wherein the carotenoid is epsilon-carotene, and wherein the at least one optimized carotenoid producing enzyme is LcyE.

22. The method of claim 21, wherein the LcyE is derived from lettuce and is N-terminally truncated (Δ N50-LsLcyE).

23. The method of any one of claims 21 to 22, further comprising screening for epsilon-carotene expression levels in amounts of 10-1000mg/L over a 24 hour period.

24. The method of claim 2, wherein the carotenoid is phytoene.

25. The method of claim 24, further comprising screening for phytoene expression levels in amounts of 10-1000mg/L over a 24 hour period.

26. The method of any one of claims 1 to 25, further comprising expressing in the host cell: a first expression module comprising an expression vector having a first coding region encoding one or more optimized first gene products selected from the group consisting of atoB, hmgS, thmgR, and optionally crtY, the first coding region being operably linked to a promoter; a second expression module comprising an expression vector having a second coding region encoding one or more optimized second gene products selected from mevK, pmk, pmd, or idi, the second coding region operably linked to a promoter; a third expression module comprising an expression vector having a third coding region encoding one or more optimized third gene products selected from ispA, crtE, crtB, or crtI, the third coding region operably linked to a promoter.

27. The method of claim 26, wherein the one or more first gene products form an operon having the following structure: atoB-hmgS-thmgR.

28. The method of claim 26, wherein the one or more first gene products form an operon having the following structure: crtY-atoB-hmgS-thmgR.

29. The method of claim 26, wherein the one or more second gene products form an operon having the following structure: mevK-pmk-pmd-idi.

30. The method of claim 26, wherein the one or more third gene products form an operon having the following structure: crtE-crtB-ispA.

31. The method of claim 26, wherein the one or more third gene products form an operon having the following structure: crtE-crtB-crtI-ispA.

32. The method of any one of claims 1 to 31, wherein the host cell is Escherichia coli (Escherichia coli) selected from BL21DE3 or MG1655DE 3.

33. The method of any one of claims 1 to 32, wherein the promoter is selected from one or more of a TM1, TM2 or TM3, a T7RNA polymerase promoter, a T5RNA polymerase promoter, a T3RNA polymerase promoter, a SP6RNA polymerase promoter, or an inducible promoter.

34. The method according to any one of claims 1 to 33, wherein the optimization of the apophotocarotenoid producing enzyme and the optimized gene product is achieved by codon optimization or site-directed mutagenesis.

35. A host cell comprising an expression module comprising an expression vector having a coding region encoding at least one optimized apoprotecarotenoid or carotenoid-producing enzyme, said coding region being operably linked to a promoter.

36. The host cell of claim 35, wherein the at least one optimized apoproteolytic carotenoid produces an enzyme selected from the group consisting of crtY, CCD1, CCD2, CCD4, BCDO, LcyE, blh, and ybbO.

37. The host cell of claim 36, wherein the optimized gene product is selected from the group consisting of: delta N50-LsLcyE and TrxA-sweet osmanthus CCD 1; crtY and phCCD 1; Δ N50-LsLcLyE; crtY and blh; and crtY, blh and ybbO.

38. The host cell of claim 37, wherein the Osmanthus CCD1 comprises one or more of the following mutations: N154Y, M152T, L151F.

39. The host cell of any one of claims 26-38, further comprising:

A first expression module comprising an expression vector having a first coding region encoding one or more optimized first gene products selected from the group consisting of atoB, hmgS, thmgR, and optionally crtY, the first coding region being operably linked to a promoter;

A second expression module comprising an expression vector having a second coding region encoding one or more optimized second gene products selected from mevK, pmk, pmd, or idi, the second coding region operably linked to a promoter;

A third expression module comprising an expression vector having a third coding region encoding one or more optimized third gene products selected from ispA, crtE, crtB, or crtI, the third coding region operably linked to a promoter.

40. The host cell of any one of claims 26-39, wherein the host cell is E.coli selected from BL21DE3 or MG1655DE 3.

41. A vector encoding one or more optimized gene products selected from the group consisting of atoB, hmgS, thmgR, and optionally crtY, operably linked to a promoter.

42. A vector encoding one or more optimized gene products selected from mevK, pmk, pmd or idi operably linked to a promoter.

43. A vector encoding one or more optimized gene products selected from ispA, crtE, crtB or crtI operably linked to a promoter.

44. A vector encoding one or more optimized gene products selected from the group consisting of crtY, CCD1, BCDO, LcyE, blh, or ybbO operably linked to a promoter.

45. The vector of claim 44, wherein the optimized gene product is selected from the group consisting of: delta N50-LsLcyE and TrxA-sweet osmanthus CCD 1; crtY and phCCD 1; Δ N50-LsLcLyE; crtY and blh; and crtY, blh and ybbO.

46. The vector of claim 45, wherein the Osmanthus CCD1 comprises one or more of the following mutations: N154Y, M152T, L151F.

47. A system for producing a carotenoid or an apophotocarotenoid, the system comprising an expression module comprising an expression vector having a coding region encoding at least one optimized carotenoid or apophotocarotenoid-producing enzyme, the coding region being operably linked to a promoter, wherein the at least one optimized carotenoid or apophotocarotenoid-producing enzyme is selected from the group consisting of:

a. Delta N50-LsLcyE and TrxA-sweet osmanthus CCD1 for producing alpha-ionone; or

b. crtY and phCCD1 for beta-ionone production; or

c. Δ N50-LsLcyE e for production of epsilon-carotene; or

d. crtY and blh for the production of retinal; or

e. crtY, blh and ybbO for retinol production.

48. The system of claim 47, further comprising:

A first expression module comprising an expression vector having a first coding region encoding one or more optimized first gene products selected from the group consisting of atoB, hmgS, thmgR, and optionally crtY, the first coding region being operably linked to a promoter;

A second expression module comprising an expression vector having a second coding region encoding one or more optimized second gene products selected from mevK, pmk, pmd, or idi, the second coding region operably linked to a promoter;

A third expression module comprising an expression vector having a third coding region encoding one or more optimized third gene products selected from ispA, crtE, crtB, or crtI, the third coding region operably linked to a promoter.

49. A kit for the production of an apo-carotenoid or carotenoid when used in a method according to any one of claims 1 to 34, the kit comprising one or more of:

A first vector encoding one or more optimized first gene products selected from the group consisting of atoB, hmgS, thmgR, and optionally crtY, operably linked to a promoter;

A second vector encoding one or more optimized second gene products selected from mevK, pmk, pmd, or idi operably linked to a promoter;

A third vector encoding one or more optimized third gene products selected from ispA, crtE, crtB or crtI operably linked to a promoter; and

A fourth vector encoding one or more optimized gene products selected from the group consisting of:

a. Delta N50-LsLcyE and TrxA-sweet osmanthus CCD1 which are used for producing alpha-ionone and are operably connected with a promoter; or

b. crtY and phCCD1 operably linked to a promoter for the production of beta-ionone;

Or

c. Δ N50-LsLcyE e operably linked to a promoter for the production of epsilon-carotene;

Or

d. crtY and blh operably linked to a promoter for production of retinal; or

e. crtY, blh and ybbO for retinol production.

50. A process for producing a carotenoid or an apoprotecarotenoid, the process comprising the steps of:

a. Contacting a host cell according to any one of claims 35 to 40 with a substrate for carotenoid or apoprotecarotenoid production in a chemically-defined medium;

b. Incubating the host cell in the chemically-defined medium to produce one or more preselected carotenoids or apocarotenoids; and

c. Extracting the one or more preselected carotenoids or apocarotenoids from the chemically-defined medium using an organic layer.

51. The method of claim 50, wherein the organic layer is coconut oil or soybean oil.

52. the method of claim 50 or 51, wherein the apocarotenoid is selected from a-ionone, β -ionone, pseudoionone (or ψ -ionone), hydroxy ionone, β -cyclocitral, trans-geranylacetone, 6-methyl-5-hepten-2-one (MHO), retinal, retinol, 8 ', 10-diapocarotene-8', 10-dialdehyde (C17), or 10 ', 6-diapocarotene-10', 6-dialdehyde (C19), and wherein the carotenoid is selected from phytoene, lycopene, α -carotene, γ -carotene, δ -carotene, ε -carotene, or β -carotene.

53. A kit for the production of carotenoids when used in the method according to any one of claims 1 to 34, comprising one or more of: a first vector encoding one or more optimized first gene products selected from the group consisting of atoB, hmgS, thmgR; a second vector encoding one or more optimized second gene products selected from mevK, pmk, pmd, or idi operably linked to a promoter; a third vector encoding one or more optimized third gene products selected from ispA, crtE or crtB, optionally crtI, operably linked to a promoter for the production of phytoene or lycopene.

Technical Field

The present invention is in the field of carotenoids and apocarotenoids (apocarotenoids), and in particular, the present invention relates to an improved process for producing apocarotenoids and carotenoids.

Background

Carotenoids are a class of natural products synthesized in many plants, algae, and certain bacteria and fungi. Carotenoids have many unsaturated carbon combinations that are conjugated, which contribute to different characteristics, bright colors (ranging from light yellow to orange to red), and effective UV and antioxidant resistance. Due to these characteristics, carotenoids have been widely used as natural pigments and nutraceuticals (nutraceuticals). In particular, phytoene (phytoene) has an effective UVB absorption ability and reduces melanin synthesis in human skin, and thus has an increasing market in cosmetics. Lycopene, beta-carotene and alpha-carotene are well known for their antioxidant action and have been widely used in food, cosmetic, nutraceutical and animal feed products.

Apoprotenoids are a class of compounds derived from carotenoids by carotenoid-cleaving oxygenases. Apocarotenoids are widely distributed in bacteria, fungi, plants and animals, acting as flavour (aroma) and odour (gent) compounds (alpha-ionone and beta-ionone), photopigments (bixin, crocin), hormones (abscisic acid) and signalling compounds (strigolactone). Among the various apocarotenoids, alpha-ionone and beta-ionone are two important aromatic compounds. Alpha-ionone has a sweet and violet-like aroma with an odor threshold of 0.4 ppb. Its isomer, beta-ionone, has a warm, woody and violet scent and an even lower odor threshold, of 0.007ppb in air and 1ppb in water. They are widely used in the cosmetics and perfumery industries due to their significantly low odor threshold and pleasant odor. Besides ionone, retinol (or vitamin a) is another commercially important apoprotenoid. Retinol plays a vital role as an antioxidant in vision, bone development and skin health. The market size of retinol, an effective drug for active cosmetic ingredients and skin disorders, is estimated to be about $ 16 billion.

Despite their high commercial value, the supply of natural ionones and retinoids (retinoids) is severely limited by their extremely low abundance in nature. Ionones are present at sub-ppm levels in many flowers and fruits such as roses, sweet osmanthus, iris roots and raspberry. For example, an agricultural area of 100 tons of raspberry or 20 hectares is required to produce only 1 gram of alpha ionone. For retinoids, no natural sources from plants exist. Very low amounts of retinoids are present in some foods of animal origin, such as eggs and butter. Thus, the increasing demand for natural ionones and retinoids cannot currently be met by supplies extracted from natural sources. While these compounds can be chemically synthesized, apocarotenoids such as alpha-ionone have chiral centers, and the synthesized compounds are usually mixtures of different enantiomers. It is known that different isomers of many perfume (fragrance) compounds have different odours, and it is therefore important to synthesise a single isomer rather than a mixture of isomers. More importantly, consumers tend to prefer natural perfumes over synthetic perfumes, and thus natural ingredients have significantly higher prices. The production of alpha-ionone was previously demonstrated in engineered E.coli (Escherichia coli), but the yields were very low.

Thus, there is a need to provide naturally produced compounds in increased yields.

SUMMARY

In one aspect, a method of producing a carotenoid or an apoprotecarotenoid is provided, the method comprising the step of expressing in a host cell an expression module comprising an expression vector having a coding region encoding at least one optimized carotenoid or apoproteolytic carotenoid-producing enzyme, the coding region being operably linked to a promoter.

In one aspect, a host cell is provided comprising an expression vector having a coding region encoding at least one optimized carotenoid or apoprotecarotenoid-producing enzyme, said coding region being operably linked to a promoter.

In one aspect, there is provided a kit for the production of carotenoids when used in a method as described herein, the kit comprising one or more of: a first vector encoding one or more optimized first gene products selected from the group consisting of atoB, hmgS, thmgR; a second vector encoding one or more optimized second gene products selected from mevK, pmk, pmd, or idi operably linked to a promoter; a third vector encoding one or more optimized third gene products selected from ispA, crtE or crtB, optionally crtI, operably linked to a promoter for the production of phytoene or lycopene.

In one aspect, there is provided a system for producing a carotenoid or an apoprotecarotenoid, the system comprising an expression vector having a coding region encoding at least one optimized carotenoid or apoproteolytic carotenoid-producing enzyme, the coding region being operably linked to a promoter, wherein the at least one optimized carotenoid or apoproteolytic carotenoid-producing enzyme is selected from the group consisting of: a. Δ N50-LsLcyE and TrxA-CCD1 (preferably TrxA-Osmanthus fragrans (Osmanthus fragrans) CCD1) for producing alpha-ionone; crtY and CCD1 (preferably Petunia hybrid CCD1) for the production of beta-ionone; Δ N50-LsLcyE e for the production of epsilon-carotene; crtY and blh for production of retinal or retinol.

In one aspect, there is provided a kit for the production of a carotenoid or an apoprotecarotenoid when used in a method as described herein, the kit comprising one or more of: a first vector encoding one or more optimized first gene products selected from the group consisting of atoB, hmgS, thmgR, and optionally crtY, operably linked to a promoter; a second vector encoding one or more optimized second gene products selected from mevK, pmk, pmd, or idi operably linked to a promoter; a third vector encoding one or more optimized third gene products selected from ispA, crtE, crtB or crtI operably linked to a promoter; and a fourth vector encoding one or more optimized gene products selected from the group consisting of: a. delta N50-LsLcyE and TrxA-sweet osmanthus CCD1 which are used for producing alpha-ionone and are operably connected with a promoter; crtY and phCCD1 operably linked to a promoter for the production of beta-ionone; Δ N50-LsLcyE e operably linked to a promoter for the production of epsilon-carotene; crtY and blh operably linked to a promoter for production of retinal or retinol.

In one aspect, a process for producing a carotenoid or an apoprotecarotenoid, said process comprising the steps of: a. contacting a host cell as described herein with a substrate for the production of an apoptopic carotenoid or carotenoid in a chemically-defined medium; b. incubating the host cell in the chemically-defined medium to produce one or more preselected carotenoids or apocarotenoids; extracting one or more pre-selected carotenoids or apocarotenoids from the chemically-defined medium using an organic layer.

Definition of

As used herein, the term "coding region," also referred to as a coding sequence or CDS (from a coding DNA sequence), is a portion of DNA or RNA that is comprised of exons that encodes a protein.

As used herein, "operon" (operon) refers to a group of genes or DNA segments that function as a single transcription unit. It may comprise an operator (operator), a promoter and one or more structural genes that are transcribed into a polycistronic mRNA.

Brief Description of Drawings

The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:

FIG. 1 is a diagram of a "plug-and-play" platform for the biosynthesis of carotenes and apoprotenoids. The starting material is an inexpensive sugar (e.g., glucose) or glycerol. The platform can produce a variety of carotenes (phytoene, lycopene, alpha-carotene, beta-carotene, delta-carotene, and epsilon-carotene) and apoprotenoids (alpha-ionone, beta-ionone, psi-ionone, retinol, dialdehydes (C17 and C19), geranylacetone, beta-cyclocitral, and 6-methyl-5-hepten-2-one (MHO)).

FIG. 2 is a schematic of an expression module for carotene synthesis. The upstream MVA pathway (expression module 1), the downstream MVA pathway (module 2), the phytoene or lycopene pathway (expression module 3), the carotene pathway (expression module 4). The expressed genes encode the following enzymes: atoB, acetoacetyl-CoA thiolase; hmgS, HMG-CoA synthetase; thmgR, a truncated HMG-CoA reductase; mevk, mevalonate kinase; pmk, phosphomevalonate kinase; pmd, mevalonate pyrophosphate decarboxylase; idi, IPP isomerase; ispA, FPP synthase; crtE, GGPP synthetase; crtB, phytoene synthase; crtI, phytoene desaturase; crtY, lycopene- β -cyclase; LCYe, lycopene-epsilon-cyclase. Abbreviations for the compounds: G3P, glyceraldehyde D-3-phosphate; DHAP, dihydroxyacetone phosphate; HMG-CoA, 3-hydroxy-3-methyl-glutaryl-coenzyme a; MVA, mevalonic acid; MVAP, mevalonate phosphate; MVAPP, mevalonate diphosphate; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; HMBPP, (E) -4-hydroxy-3-methyl-but-2-enyl pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate. The dashed arrows indicate the various enzymatic steps.

fig. 3 shows the UV absorption spectra of various carotenoids. UV-B and UV-A wavelengths are shown.

FIG. 4 shows optimization of phytoene production by controlling SAR, MPPI and EBIA modules with T7 promoter variants using an experimental design-aided systematic path optimization (EDASPO) approach. Numbers represent different strengths of T7 promoter variants, with 1, 2, and 3 representing approximately 92%, 37%, and 16% of the strength of the native T7 promoter, respectively. Expression module 1 or SAR module, hmgrs-atoB-hmgR; expression module 2 or MPPI module, mevK, pmk, pmd and idi; expression module 3 or EBA module, phytoene biosynthetic pathway (crtEB and ispA).

Fig. 5 shows the optimization of phytoene production using the EDASPO method by controlling SAR, MPPI and EBIA modules with the T7 promoter variant. Numbers represent different strengths of T7 promoter variants, with 1, 2, and 3 representing approximately 92%, 37%, and 16% of the strength of the native T7 promoter, respectively. Expression module 1 or SAR module, hmgrs-atoB-hmgR; expression module 2 or MPPI module, mevK, pmk, pmd and idi; expression module 3 or EBIA module, lycopene biosynthetic pathway (crtEBI and ispA).

Fig. 6 shows the production of epsilon-carotene by the lycopene accumulating e.coli (e. Carotenoid production by strains expressing different forms of the LCYe enzyme. Schematic representation of promoters and regulated genes of different strains. Different N-terminally truncated LCYes from lettuce (Lactuca sativa) were tested. Abbreviations are as follows. LL, wild-type LCYe enzyme from lettuce; l25, LCYe enzyme with the first 25 amino acids removed; l34, LCYe enzyme with the first 34 amino acids removed; l50, LCYe enzyme with the first 50 amino acids removed; l75, LCYe enzyme with the first 75 amino acids removed; l92, LCYe enzyme with the first 92 amino acids removed; l100, LCYe enzyme with the first 100 amino acids removed. See strain description in table 3. All measurements are the mean of three replicates, with standard error bars shown in the figure. The percentage of epsilon-carotene in the final total carotenoids (lycopene + delta-carotene + epsilon-carotene) is also shown.

Figure 7 shows protein and mRNA expression analysis of LsLcyE and OfCCD1 with N-terminal modifications. (A) SDS-PAGE gel images of whole cell protein expression with various N-terminal modifications of LsLcyE and OfCCD 1. The protein of interest is indicated by blue arrows. Lane 1: e.coli cells with overexpressed LsLCYe. Lane 2: e.coli cells with over-expressed Δ 50-LsLCYe (L50). Lane 3: e.coli cells with overexpressed Δ 100-LsLCYe (L100). Lane 4: e.coli cells with over-expressed OfCCD 1. Lane 5: e.coli cells with over-expressed SUMO-OfCCD1 (SUMO-O). Lane 6: e.coli cells with over-expressed MBP-OfCCD1 (MBP-O). Lane 7: e.coli cells with over-expressed TrxA-OfCCD1 (TrxA-O). Lane 8: e.coli cells without any over-expression (control). Quantitative analysis of the yield of overexpressed protein was based on the intensity of the bands on the SDS PAGE gel. (B) LsLCYe expression with various N-terminal truncations. (C) mRNA expression levels of LsLCYe and truncated forms thereof. (D) OfCCD1 expression with different fusion partners (partner). (E) mRNA expression levels of OfCCD1 with different fusion proteins. Relative fold changes in protein expression were normalized by expression of LsLCYe (B and C) and SUMO-OfCCD1(D and E). The results are the average of three replicates, with standard error bars shown.

FIG. 8 is a schematic representation of the biosynthetic pathway of apocarotenoids (e.g., alpha, beta-ionone and retinoids). The biosynthetic pathway is grouped into four major modules: the upstream MVA pathway (expression module 1), the downstream MVA pathway (expression module 2), the lycopene pathway (expression module 3) and the apoprotecarotenoid pathway (expression module 4). The expressed genes encode the following enzymes: atoB, acetoacetyl-CoA thiolase; hmgS, HMG-CoA synthetase; thmgR, a truncated HMG-CoA reductase; mevk, mevalonate kinase; pmk, phosphomevalonate kinase; pmd, mevalonate pyrophosphate decarboxylase; idi, IPP isomerase; ispA, FPP synthase; crtE, GGPP synthetase; crtB, phytoene synthase; crtI, phytoene desaturase; LCYe, lycopene epsilon-cyclase; crtY, lycopene beta-cyclase; CCD1, carotenoid-cleaving dioxygenase; BCDO (or blh), beta-carotene dioxygenase; ybbO, NADP + dependent aldehyde reductase. Abbreviations for the compounds: G3P, glyceraldehyde D-3-phosphate; DHAP, dihydroxyacetone phosphate; HMG-CoA, 3-hydroxy-3-methyl-glutaryl-coenzyme a; MVA, mevalonic acid; MVAP, mevalonate phosphate; MVAPP, mevalonate diphosphate; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; HMBPP, (E) -4-hydroxy-3-methyl-but-2-enyl pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate. The dashed arrows indicate the various enzymatic steps.

FIG. 9 shows the production of alpha-ionone in E.coli strains that accumulate epsilon-carotene. (A) Optimization of CCD1 with N-terminal Fusion Partner (FP), supplemented with inset pictures with different y-axis ratios to compare ionone titers for strains 121-LO and 121-L50-O. (B) Intracellular carotenoids that remain uncleaved. (C) Schematic representation of promoters and regulated genes of different strains. Abbreviations are as follows. LO, LsLCYe and OfCCD1 are expressed on the same plasmid in a polycistronic manner; L50-O (LsLCYe enzyme (L50) with the first 50 amino acids removed) and OfCCD1 were expressed in a polycistronic manner. L50-SUMO-O, L50 was co-expressed with SUMO fused OfCCD 1. L50-MBP-O, L50 was co-expressed with MBP fused OfCCD 1. L50-TrxA-O, L50 and TrxA fused OfCCD1 were co-expressed.

fig. 10 shows the protein solubility of OfCCD1 with N-terminal modification. The protein of interest is shown by the arrow. Lane 1: insoluble OfCCD 1. Lane 2: soluble OfCCD 1. Lane 3: insoluble SUMO-OfCCD 1. Lane 4: soluble SUMO-OfCCD 1. Lane 5: insoluble MBP-OfCCD 1. Lane 6: soluble MBP-OfCCD 1. Lane 7: insoluble TrxA-OfCCD 1. Lane 6: soluble TrxA-OfCCD 1.

FIG. 11 shows the homology modeling of OfCCD1 based on the crystal structure of vivopaous 14 from maize (Zea mays) (PDB: 3 NPE). Two membrane interaction helices are labeled. The membrane is indicated by the shaded area. Cofactors Fe2+ and oxygen are labeled.

fig. 12 shows the engineering of the second membrane interaction helix of OfCCD 1. (A) Comparison of titers of α -ionone and ψ -ionone between wild-type ofCCD1 and engineered ofCCD1 with mutations (L151F, M152T and N154Y). (B) Biomass of the engineered strain. (C) Fold change in solubility and total expression of TrxA fused wild-type OfCCD1 and TrxA fused mutant OfCCD 1. (D) SDS PAGE gel images of TrxA fused wild-type OfCCD1 and TrxA fused mutant OfCCD 1. Lane M, molecular marker. Lane 1, total protein of e.coli cells expressing trxA fused wild-type OfCCD 1. Lane 2, soluble protein of E.coli cells expressing trxA fused wild-type OfCCD 1. Lane 3, total protein of e.coli cells expressing trxA-fused mutant OfCCD 1. Lane 4, soluble protein of e.coli cells expressing trxA-fused mutant OfCCD 1.

FIG. 13 shows fed-batch fermentation of an apocarotenoid. (A) Time course curves for ionone and biomass. (B) Schematic representation of the promoters and regulated genes for strains used in fed-batch fermentations. The process consists of two main stages (by induced separation), a biomass stage where only biomass is accumulated and a production stage where ionone and biomass are produced simultaneously. Isopropyl myristate was used as the organic layer to extract alpha-ionone, beta-ionone and psi-ionone. (C) Schematic representation of the in situ isolation of apocarotenoids in a bioreactor. Food grade coconut oil (or soybean oil, etc.) as an organic layer to extract alpha-ionone, beta-ionone, and psi-ionone. The use of chemically defined media without any amino acids and vitamins reduces the cost of the media.

Figure 14 shows chiral analysis of alpha-ionone. The product is the 100% R-enantiomer of alpha-ionone, identical to the native alpha-ionone. In contrast, the chemically synthesized ionone is a mixture of 50% of the R-enantiomer and 50% of the S-enantiomer.

Figure 15 shows a metabolic engineering strategy for the production of beta-ionone. Wild type OfCCD1, N-terminally modified OfCCD1 and the complement crtY were compared. In the Of, TOf and crtY strains, the operon/genes crtY-OfCCD1, crtY-TrxA-OfCCD1 and crtY were expressed, respectively. In the TOf + Y strain, the p15A-spec vector was supplemented with an additional copy of the crtY gene. (A) Ionone production in engineered strains. Strain crtY is a control strain that does not express the CCD1 gene, and therefore produces only beta-carotene. (B) Carotenoids accumulated in the engineered strain. (C) Biomass of the engineered strain. Although thioredoxin fusion did not improve the production of β -ionone, it resulted in a 37-fold increase in the production of the byproduct ψ -ionone, which was obtained from the conversion of lycopene by TOfCCD 1. Supplementation with additional crtY effectively converts more lycopene to beta-carotene, so that beta-ionone production is significantly increased and psi-ionone is significantly reduced.

FIG. 16 shows GC-chromatograms Of the products in the Of, TOF and TOf + Y strains.

Figure 17 shows mass spectra of alpha-ionone, beta-ionone, and psi-ionone.

figure 18 shows an alignment of different CCD1 variants in this study. Alignment was performed by the Clustal Omega multiple sequence alignment tool. Information about CCD1 is summarized in table 4.

Figure 19 shows screening of CCDs with higher selectivity. (A) Ionone production using different CCDs. (B) Carotenoids accumulated in E.coli cells with different CCDs. In the strain TOF, At, Vv, Ph, TPh, operon/gene crtY-TrxA-OfCCD1, crtY-AtCCD1, crtY-VvCCD1, crtY-PhCCD1 and crtY-TrxA-PhCCD1 are expressed, respectively. In the Ph + Y and TPh + Y strains, the p15A-spec vector was supplemented with an additional copy of the crtY gene. PhCCD1 was found to be more selective for beta-carotene than VvCCD1 and OfCCD1, thus producing higher titers of ionone and lower amounts of ψ -ionone. (C) Fed-batch fermentation with beta-ionone of strain TPh + Y. (D) Schematic representation of promoters and regulated genes of different strains.

FIG. 20 shows the mass spectra of the dialdehyde and diol produced in the fermentor. C17 and C19 for dialdehyde and diol were detected, but C14 was not detected.

Figure 21 shows screening for CCD2 and CCD4 with higher activity and selectivity. The upper panel shows the use of beta-carotene as a substrate for beta-ionone production. The lower panel shows the use of carotene as a substrate for alpha-ionone production.

Figure 22 shows the production of retinoids in a modular system. (A) Retinoid production using a different blh gene and increased gene copy of crtY. (B) Carotenoids accumulated in different E.coli cells. To produce other apocarotenoids we can simply replace the CCD1 enzyme with the other corresponding carotenoid-cleaving oxygenase. Here, we use bcdo (blh) to produce retinoids. (C) Schematic representation of a modular system for the production of different apocarotenoids.

Detailed description of the invention

In a first aspect, the present invention relates to a process for the production of a carotenoid or an apoprotecarotenoid. The method comprises the following steps: expressing an expression module in a host cell, said expression module comprising an expression vector having a coding region encoding at least one optimized carotenoid or apophotocarotenoid producing enzyme, said coding region being operably linked to a promoter.

The carotenoid may be selected from among phytoene, lycopene, alpha-carotene, gamma-carotene, delta-carotene, epsilon-carotene or beta-carotene, and wherein the apocarotenoid may be selected from among alpha-ionone, beta-ionone, pseudoionone (or psi-ionone), hydroxyionone, beta-cyclocitral, trans-geranylacetone, 6-methyl-5-hepten-2-one (MHO), retinal, retinol, 8 ', 10-diapocarotene (diapocarene) -8', 10-dialdehyde (C17) or 10 ', 6-diapocarotene-10', 6-dialdehyde (C19).

The lycopene cyclase may be selected from lycE or crtY or truncated forms thereof.

as described herein, the at least one optimized apocarotenoid-producing enzyme may be selected from crtY, CCD1, CCD2, CCD4, BCDO, LcyE, blh or ybbO.

The CCD2 may be selected from the group consisting of CaCCD2 or CsCCD 2. The CCD4 may be selected from the group consisting of: AtCCD4, BoCCD4b, CmCCD4, CsCCD4a, MaCCD4, MdCCD4, OfCCD4, PpCCD4, RdCCD4 and VvCCD4 a.

In one embodiment, the apoprotenoid may be alpha-ionone and the at least one optimized apoprotenoid-producing enzyme may be selected from the group consisting of LcyE and CCD1, and may form an operon having the structure: LcyE-CCD 1.

In some embodiments, the LcyE may be derived from lettuce and is N-terminally truncated (ranging from 1 to 100 amino acids of LsLcyE, particularly Δ N50-LsLcyE).

The CCD1 may be expressed as a fusion protein selected from TrxA-CCD1, SUMO-CCD1 or MBP-CCD 1.

In some embodiments, the fusion protein is TrxA-CCD 1. The fusion protein can be selected from TrxA-sweet osmanthus CCD1 or TrxA-petunia CCD 1.

In some embodiments, CCD1 may be derived from osmanthus fragrans or may be osmanthus fragrans CCD1(OfCCD 1). In some embodiments, OfCCD1 as described herein can be optimized between amino acid positions F148 to I167. In particular, OfCCD1 may contain one or more of the following mutations: N154Y, M152T, L151F.

In some embodiments, the method may comprise screening for alpha-ionone expression levels in an amount of from 10 to 1000mg/L, from 200 to 800mg/L, from 300 to 700mg/L, from 400 to 600mg/L over a 24 hour period. In some embodiments, the amount of alpha-ionone is about 500mg/L over a 24 hour period.

in some embodiments, the apoprotenoid is beta-ionone. The at least one optimized apoproteolytic carotenoid producing enzyme may be selected from the group consisting of crtY and CCD1, and may form an operon having the structure crtY-CCD 1. In some embodiments, crtY as described herein may be derived from Pantoea ananatis (Pantoea ananatis).

In some embodiments, CCD1 as described herein may be derived from petunia (PhCCD 1).

In some embodiments, the method may further comprise screening for the level of beta-ionone expression in an amount from 10 to 1000mg/L, from 200 to 800mg/L, from 300 to 700mg/L, from 400 to 600mg/L over a 24 hour period. In some embodiments, the amount of alpha-ionone is about 500mg/L over a 24 hour period.

the apocarotenoids may be retinal or retinol. In one embodiment, the apoprotectane may be retinal. The at least one optimized apoproteolytic carotenoid producing enzyme may be selected from the group consisting of crtY and blh, and may form an operon having the structure crtY-blh. In some embodiments, the apo-carotenoid can be retinol. The at least one optimized apoproteolytic carotenoid producing enzyme may be selected from the group consisting of crtY, blh or ybbO, and may form an operon having the structure crtY-blh-ybbO. In some embodiments, crtY as described herein may be derived from pantoea ananatis. In some embodiments, blh as described herein may be derived from Uncultured marine bacteria (unculized marine bacteria) HF10_19P 19. In some embodiments, the method may further comprise screening for retinal expression levels in an amount of from 10 to 1000mg/L, from 200 to 800mg/L, from 300 to 700mg/L, from 400 to 600mg/L over a 24 hour period. In some embodiments, the amount of alpha-ionone is about 500mg/L over a 24 hour period.

The carotenoid may be epsilon-carotene. The at least one optimized carotenoid producing enzyme may be LcyE. In some embodiments, the LcyE may be derived from lettuce (LsLcyE) and may be N-terminally truncated. In some embodiments, LsLcyE may comprise an N-terminal truncation of 1-100 amino acids, 30-70 amino acids, or 40-60 amino acids. In some embodiments, the LsLcyE comprises an N-terminal truncation of 50 amino acids (Δ N50-LsLcyE).

In some embodiments, the method may further comprise expressing in the host cell: a first expression module comprising an expression vector having a first coding region encoding one or more optimized first gene products selected from the group consisting of atoB, hmgS, thmgR, and optionally crtY, the first coding region being operably linked to a promoter; a second expression module comprising an expression vector having a second coding region encoding one or more optimized second gene products selected from mevK, pmk, pmd, or idi, the second coding region operably linked to a promoter; a third expression module comprising an expression vector having a third coding region encoding one or more optimized third gene products selected from ispA, crtE, crtB, or crtI, the third coding region operably linked to a promoter.

in some embodiments, the one or more first gene products may form an operon having the following structure: atoB-hmgS-thmgR.

In some embodiments, the one or more first gene products may form an operon having the following structure: crtY-atoB-hmgS-thmgR.

In some embodiments, the one or more second gene products may form an operon having the following structure: mevK-pmk-pmd-idi.

In some embodiments, the one or more third gene products may form an operon having the following structure: crtE-crtB-ispA.

in some embodiments, the one or more third gene products may form an operon having the following structure: crtE-crtB-crtI-ispA.

The host cell as described herein may be an Escherichia coli (Escherichia coli) selected from BL21DE3 or MG1655DE 3.

The promoter may be selected from one or more of TM1, TM2 or TM3, a T7RNA polymerase promoter, a T5RNA polymerase promoter, a T3RNA polymerase promoter, an SP6RNA polymerase promoter, or an inducible promoter.

In some embodiments, the optimization of the apophotocarotenoid producing enzyme and the optimized gene product is achieved by codon optimization or site-directed mutagenesis.

In another aspect, a host cell is provided comprising an expression module comprising an expression vector having a coding region encoding at least one optimized apoprotecarotenoid or carotenoid producing enzyme, said coding region being operably linked to a promoter.

in some embodiments, the at least one optimized apoproteolytic carotenoid can be selected from the group consisting of crtY, CCD1, CCD2, CCD4, BCDO, LcyE, blh, or ybbO.

The host cell may further comprise a first expression module comprising an expression vector having a first coding region encoding one or more optimized first gene products selected from the group consisting of atoB, hmgS, thmgR, and optionally crtY, the first coding region being operably linked to a promoter; a second expression module comprising an expression vector having a second coding region encoding one or more optimized second gene products selected from mevK, pmk, pmd, or idi, the second coding region operably linked to a promoter; a third expression module comprising an expression vector having a third coding region encoding one or more optimized third gene products selected from ispA, crtE, crtB, or crtI, the third coding region operably linked to a promoter.

in another aspect, vectors are provided that encode one or more optimized gene products selected from the group consisting of atoB, hmgS, thmgR, and optionally crtY, operably linked to a promoter.

In another aspect, vectors are provided that encode one or more optimized gene products selected from mevK, pmk, pmd, or idi operably linked to a promoter.

In another aspect, vectors are provided that encode one or more optimized gene products selected from ispA, crtE, crtB or crtI operably linked to a promoter.

In another aspect, vectors are provided that encode one or more optimized gene products selected from the group consisting of crtY, CCD1, BCDO, LcyE, blh, or ybbO operably linked to a promoter.

In some embodiments, the optimized gene product may be selected from the group consisting of: delta N50-LsLcyE and TrxA-sweet osmanthus CCD 1; crtY and phCCD 1; Δ N50-LsLcLyE; crtY and blh; and crtY, blh and ybbO.

In another aspect, there is provided a system for producing a carotenoid or an apoprotecarotenoid, the system comprising an expression module comprising an expression vector having a coding region encoding at least one optimized carotenoid or apoprotecarotenoid-producing enzyme, the coding region being operably linked to a promoter, wherein the at least one optimized carotenoid or apoprotecarotenoid-producing enzyme is selected from the group consisting of: a. delta N50-LsLcyE and TrxA-sweet osmanthus CCD1 for producing alpha-ionone; crtY and phCCD1 for beta-ionone production; Δ N50-LsLcyE e for the production of epsilon-carotene; crtY and blh for production of retinal or retinol; crtY, blh and ybbO for retinol production.

In some embodiments, the system may further comprise: a first expression module comprising an expression vector having a first coding region encoding one or more optimized first gene products selected from the group consisting of atoB, hmgS, thmgR, and optionally crtY, the first coding region being operably linked to a promoter; a second expression module comprising an expression vector having a second coding region encoding one or more optimized second gene products selected from mevK, pmk, pmd, or idi, the second coding region operably linked to a promoter; a third expression module comprising an expression vector having a third coding region encoding one or more optimized third gene products selected from ispA, crtE, crtB, or crtI, the third coding region operably linked to a promoter.

In another aspect, there is provided a kit for the production of a carotenoid or an apoprotecarotenoid when used in a method as described herein, the kit comprising one or more of: a first vector encoding one or more optimized first gene products selected from the group consisting of atoB, hmgS, thmgR; a second vector encoding one or more optimized second gene products selected from mevK, pmk, pmd, or idi operably linked to a promoter; a third vector encoding one or more optimized third gene products selected from ispA, crtE or crtB, optionally crtI, operably linked to a promoter for the production of phytoene or lycopene.

In another aspect, there is provided a kit for the production of an apo-carotenoid or carotenoid when used in a method as described herein, the kit comprising one or more of: a first vector encoding one or more optimized first gene products selected from the group consisting of atoB, hmgS, thmgR, and optionally crtY, operably linked to a promoter; a second vector encoding one or more optimized second gene products selected from mevK, pmk, pmd, or idi operably linked to a promoter; a third vector encoding one or more optimized third gene products selected from ispA, crtE, crtB or crtI operably linked to a promoter; and a fourth vector encoding one or more optimized gene products selected from the group consisting of: a. delta N50-LsLcyE and TrxA-sweet osmanthus CCD1 which are used for producing alpha-ionone and are operably connected with a promoter; crtY and phCCD1 operably linked to a promoter for the production of beta-ionone; Δ N50-LsLcyE e operably linked to a promoter for the production of epsilon-carotene; crtY and blh operably linked to a promoter for production of retinal; crtY, blh and ybbO for retinol production.

In another aspect, there is provided a process for producing an apo-carotenoid or carotenoid, the process comprising the steps of: a. contacting a host cell as described herein in a chemically-defined medium with a substrate for apo-carotenoid or carotenoid production; b. incubating the host cell in the chemically-defined medium to produce one or more preselected apocarotenoids or carotenoids; extracting one or more pre-selected apocarotenoids or carotenoids from the chemically-defined medium using an organic layer.

In some embodiments, the organic layer may be coconut oil or soybean oil or other edible oil.

In some embodiments, the apoprotenoid may be selected from alpha-ionone, beta-ionone, pseudoionone (or psi-ionone), hydroxy ionone, trans-geranylacetone, 6-methyl-5-hepten-2-one (MHO), retinal, retinol, 8 ', 10-diphocarotene-8', 10-dialdehyde (C17) or 10 ', 6-diphocarotene-10', 6-dialdehyde (C19), and wherein the carotenoid may be selected from phytoene, lycopene, alpha-carotene, gamma-carotene, delta-carotene, epsilon-carotene or beta-carotene.

The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing" and the like are to be construed broadly and not as limiting. Additionally, the terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention, in the use of such terms and expressions, of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

The present invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

Other embodiments are within the following claims and non-limiting examples. Further, where features or aspects of the invention are described in terms of markush groups, those skilled in the art will recognize that the invention thus also describes any individual member or subgroup of members of the markush group.

experimental part

Non-limiting examples of the present invention and comparative examples will be further described in more detail by reference to specific examples, which should not be construed as limiting the scope of the present invention in any way.

Materials and methods

In this study, the E.coli strain Bl21-Gold DE3 (Stratagene) was used. The genes hmgS, hmgR, mevK, pmk and pmd (or MVD1) were amplified by PCR using Saccharomyces cerevisiae (Saccharomyces cerevisiae) chromosomal DNA. The genes atoB and idi were amplified from E.coli genomic DNA. All genes were cloned into two plasmids, p15A-spec-hmgS-atoB-hmgR (L2-8) and p15A-cam-mevK-pmk-pmd-idi (L2-5). The gene in the lycopene biosynthetic pathway (crtEBI) amplified from the pAC-LYC plasmid was introduced into the p15A-kan-crtEBI-ispA plasmid. The LCYe gene from lettuce (LsLCYe enzyme), the crtY gene from pantoea ananatis, the CCD1 gene from Arabidopsis thaliana (Arabidopsis thaliana), osmanthus fragrans, Vitis vinifera (Vitis vinifera) and petunia, the blh gene (blh1) from the uncultured marine bacterium HF10_19P19 and the blh gene (blh2) from the uncultured marine bacterium 66a03 were codon optimized and synthesized by Integrated DNA Technologies. The LsLCYe gene and the crtY gene were first cloned into plasmids p15A-amp-LsLCYe (L2-9) and p15A-amp-crtY (L2-9), respectively. The OfCCD1 gene was inserted into the p15A-amp-LsLCYe (L2-9) plasmid. A different CCD1 or blh gene was subsequently inserted into the p15A-amp-crtY (L2-9) strain. Site-directed mutagenesis was introduced from primers synthesized by Integrated DNA Technologies. An additional copy of crtY was inserted into the p15A-spec-hmgS-atoB-hmgR (L2-8) plasmid. All p15A plasmids were derived from pAC-LYC plasmid. The stem-loop structure of RNA I in the origin of the p15A plasmid was mutated to make the plasmids compatible with each other. The T7 promoter variants TM1, TM2 and TM3 are summarized in table 1. Information on the plasmids and the strains is summarized in tables 2 and 3.

the promoter sequences used are summarized in table 1.

Table 2 summarizes the strains and plasmids used.

The strains used in this study are described in table 3.

Culture medium and culture conditions

all cells were grown in 2XPY medium (20g/L peptone, 10g/L yeast extract and 10g/L NaCl) supplemented with 10g/L glycerol, 50mM 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES). For prolonged incubation (48h), 5mg/L Tween80 was also added to the medium to prevent cell aggregation. Briefly, 10. mu.L of fresh cell culture was inoculated into 1mL of fresh medium in 14mL BD falcon tubes. Cells were initially grown at 37 ℃ with an oscillation rate of 300rpm and induced by a series of IPTG concentrations (as shown herein) when the OD600 reached about 0.6. After induction, 200 μ L of dodecane was supplemented to the culture to extract ionone or retinoid, and the cells were incubated at 28 ℃ for additional 20h or 48h, followed by harvesting. The medium was supplemented with appropriate antibiotics (100mg/L ampicillin, 34mg/L chloramphenicol, 50mg/L kanamycin, and 50mg/L spectinomycin) to maintain the respective plasmids.

Quantification of carotenoids and retinoids

Intracellular carotenoids were extracted from the cell pellet (pellet) according to the acetone extraction method. Briefly, 10-50. mu.L of bacterial culture (depending on the carotenoid content of the cells) was collected and centrifuged. The cell pellet was washed with PBS and resuspended in 20. mu.L of water, followed by addition of 180. mu.L of acetone. The HPLC method employed an Agilent 1260Infinity LC system equipped with ZORBAX, Eclipse Plus C18, 4.6mm X250 mm, 5 μm column and Diode Array Detector (DAD). Isocratic conditions (50% methanol, 48% ethyl acetate and 2% water) were maintained at 1.5mL/min for 5 min. Carotenoids were detected at a wavelength of 450 nm. A standard curve was generated using commercial standards (containing epsilon-carotene, beta-carotene, lycopene, retinol, and retinal). Extracellular retinoid samples were prepared by diluting 20-50 μ L of the organic layer into 1000 μ L of hexane and analyzed by the same HPLC system. The isocratic conditions were used as follows. The mobile phase was 95% methanol and 5% acetonitrile and the flow rate was 1.5 ml/min. The retinoid is detected at a wavelength of 340 nm.

Quantification of alpha-ionone, beta-ionone and psi-ionone

Samples of alpha-ionone or beta-ionone were prepared by diluting 20-50. mu.L of the organic layer into 1000. mu.L of hexane. Samples were analyzed on an Agilent 7980B gas chromatograph (GC/MS) equipped with an Agilent VF-WAXms column and an Agilent 7200 precision mass quadrupole time of flight. Sample injections were performed in a non-split mode at 240 ℃. The temperature program (open program) starts at 100 ℃ for 2min, then the temperature is raised to 240 ℃ at 30 ℃/min and held at 240 ℃ for another 2 min. Ionone concentrations were calculated by interpolation from standard curves prepared with commercial standards. The mass spectrometer was run in EI mode by full scan analysis (m/z 30-300,1 spetra/s).

Chiral analysis

The chirality of alpha-ionones from the samples was analyzed by Agilent 7980B gas chromatography (GC/MS) equipped with an Agilent Cyclosil-B GC column and Agilent 7200 accurate mass quadrupole time of flight. The temperature ramp program starts at 80 ℃ for 2min, then the temperature is raised to 210 ℃ at 5 ℃/min and to 250 ℃ at 20 ℃/min, and finally held at 250 ℃ for another 2 min. Ionone concentrations were calculated by interpolation from standard curves prepared with commercial standards. The mass spectrometer was run in EI mode by full scan analysis (m/z 30-300,2 spetra/s).

Fed-batch fermentation

The starting medium was a chemically defined modified medium containing 15g/L glucose, 2g/L (NH4)2SO4, 4.2g/L KH2PO4 and 11.24g/L K2HPO4, 1.7g/L citric acid, 0.5g/L MgSO4 and 10mL/L trace element solution. The trace element solution (100X) contained 0.25g/L CoCl2 & 6H2O, 1.5g/L MnSO4 & 4H2O, 0.15g/L CuSO4 & 2H2O, 0.3g/L H3BO3, 0.25g/L Na2MoO4 & 2H2O, 0.8g/L Zn (CH3COO)2, 5g/L ferric citrate (III) and 0.84g/L EDTA, pH 8.0. Feed medium (500g/L glucose and 5g/L MgSO4) was pumped into a 250mL Mini bioreactor (Applikon Biotechnology) at an initial rate of 0.6mL/h and increased to 1.8mL/h within 12h and held at 1.5mL/h for an additional 24h in an approximately exponential manner. When the OD reached about 40, the cells were induced by 0.03mM IPTG. After induction, 30mL of isopropyl myristate was replenished into the bioreactor to extract ionone.

Examples

Production of carotene by' plug and play