阅读说明：本技术 具有混合糖同步发酵能力的重组微生物及利用其的二醇的生成方法 (Recombinant microorganism having ability to synchronously ferment mixed sugars and method for producing diol using same ) 是由 朴钟明 切拉杜莱·拉希纳辛格 宋孝鹤 于 2018-11-30 设计创作，主要内容包括：本发明涉及如下的重组微生物,即,对于木质纤维素糖化液中的两种以上的糖具有同步发酵能力,并且,具有二醇生成能力。(The present invention relates to a recombinant microorganism having a capability of simultaneous fermentation of two or more sugars in a lignocellulose saccharification liquid and a capability of producing a diol.)

1. A recombinant microorganism characterized in that it comprises, in a microorganism,

has synchronous fermentation capacity for more than two kinds of sugar in the lignocellulose saccharification liquid,

also, it has a diol generating ability.

2. The recombinant microorganism according to claim 1, wherein the microorganism is of the genus Klebsiella.

3. The recombinant microorganism according to claim 1, wherein the lignocellulose saccharification solution contains pentose and hexose, and the recombinant microorganism has a synchronous fermentation ability of pentose and hexose.

4. The recombinant microorganism according to claim 1, wherein the catabolite repression mechanism is inhibited as compared to a wild-type microorganism.

5. The recombinant microorganism according to claim 1, wherein the glucose-specific phosphotransferase IIA component or the glucose-specific phosphotransferase IIBC component of the phosphotransferase system is inhibited more than in the wild-type microorganism.

6. The recombinant microorganism according to claim 1, wherein the pathway for converting xylose via xylulose to xylulose-5-P or ribulose-5-P or ribose-5-P or fructose-6-P or erythrose-4-P or glyceraldehyde-3-P is more enhanced compared to the wild-type microorganism.

7. The recombinant microorganism according to claim 1, wherein the activity of one or more enzymes selected from the group consisting of xylose isomerase, xylulokinase, D-ribulose-5-phosphate 3-epimerase, ribose 5-phosphate isomerase, transaldolase and transketolase is enhanced.

8. The recombinant microorganism according to claim 1, wherein the activity of a cyclic adenosine monophosphate receptor protein is inhibited.

9. The recombinant microorganism according to claim 1, wherein a gene encoding a cyclic adenosine monophosphate-activated global transcription factor is mutated.

10. A method for producing a diol, comprising:

preparing a medium containing two or more sugars;

a step of inoculating the recombinant microorganism according to any one of claims 1 to 9 to the culture medium; and

culturing the recombinant microorganism.

11. The method of producing a diol according to claim 10, wherein the two or more sugars are one or more sugars selected from the group consisting of xylose, arabinose, and cellobiose, and glucose.

12. The method of producing a diol according to claim 10, wherein the medium comprises a lignocellulose saccharification liquid.

Technical Field

The present invention relates to a recombinant microorganism having the ability to synchronously ferment mixed sugars and a method for producing a diol using the same.

Background

Diols are not only compounds widely used in industry, but also compounds used as various chemical intermediates, and their utility is high. For example, 2, 3-butanediol is a very highly commercially potential chemical substance that can be used as a Precursor (precusor) for 1, 3-Butadiene (1, 3-Butadiene) which is a main raw material of Synthetic rubber (Synthetic rubber) and Methyl Ethyl Ketone (MEK) which is a solvent. In addition, the freezing point of the 2, 3-butanediol is very low, the butanediol can be directly used as an Antifreezing agent only, the octane number (Octanenumber) is high, and the butanediol can be mixed with the existing Gasoline (Gasoline) to be used as an octane number promoter. Further, 1, 3-propanediol is useful as a polymer monomer such as polyester or polyurethane, and is useful as an additive for improving properties of cosmetics, personal hygiene products and the like. In particular, polytrimethylene terephthalate (PTT), which is a linear aromatic polyester produced by the polymerization reaction of 1, 3-propanediol and terephthalic acid (terephthalic acid), has a unique twist called kink (kink) generated in a semi-crystalline molecular structure in a polymer chain, and thus has excellent morphological stability, and is applicable to various fields such as fibers, packaging and films, non-woven fabric structures, engineering plastics, and the like, due to such structural characteristics.

The diol can be produced by a chemical synthesis step or a microbial fermentation step. However, the chemical synthesis process has problems of generating environmental pollutants during the process and high synthesis cost. On the other hand, although the step of producing diols by fermenting microorganisms from renewable resources has an environmental advantage, there are problems such as an increase in the price of grains, a low fermentation yield of strains, and low productivity when used industrially.

For example, when cellulosic biomass (woody and herbaceous plants such as trees, oil palm fruit bunches (EFBs), corn stover, and rice straw (hereinafter, collectively referred to as "lignocelluloses") is used, these biomass are inedible biomass, and therefore, can be produced at low cost without competing with edible biomass (grains and the like), and are advantageous as industrial biomass materials. However, in lignocellulose-derived biomass, pentose and hexose are mixed, and microorganisms use hexose for metabolism first by a catabolite repression (catabolite repression) mechanism and then use pentose for metabolism. Therefore, the sugar consumption rate is slow, the fermentation time is increased, and the productivity is lowered. In addition, when pentose such as xylose remains in the fermentation liquid, it is difficult to separate and purify the diol.

Therefore, in the course of studying microorganisms capable of effectively utilizing lignocellulosic biomass for metabolism, the present inventors have invented a recombinant microorganism having an excellent ability to simultaneously ferment pentoses and hexoses.

Disclosure of Invention

Problems to be solved by the invention

The purpose of the present invention is to provide a recombinant microorganism having excellent ability to simultaneously ferment pentose and hexose.

Another object of the present invention is to produce a diol by using the above recombinant microorganism.

Means for solving the problems

In order to achieve the above object, the present invention provides a recombinant microorganism having a capability of simultaneous fermentation of two or more sugars in a lignocellulose saccharification liquid and a capability of producing a diol.

Also, the present invention provides a method for producing a diol, comprising: preparing a medium containing two or more sugars; inoculating the recombinant microorganism into the culture medium; and culturing the recombinant microorganism.

Effects of the invention

The invention provides a microorganism having the ability to ferment mixed sugars simultaneously and a method for producing a diol using the same.

Drawings

FIG. 1 schematically shows the metabolic pathways of hexose and pentose sugars of Klebsiella oxytoca.

FIG. 2 shows the simultaneous fermentation capacity of glucose and xylose of Klebsiella oxytoca with deposit number KCTC12132 BP.

FIG. 3 shows the simultaneous fermentation capacity of glucose and xylose of the recombinant strain of comparative example 1.

FIG. 4 shows the simultaneous fermentation capacity of glucose and xylose of the recombinant strain of example 1.

FIG. 5 shows the simultaneous fermentation capacity of glucose and xylose of the recombinant strain of example 2.

FIG. 6 shows the simultaneous fermentation capacity of glucose and xylose of the recombinant strain of example 3.

FIG. 7 shows the simultaneous fermentation capacity of glucose and xylose of the recombinant strain of example 4.

FIG. 8 shows the simultaneous fermentation capacity of glucose and xylose of the recombinant strain of example 5.

FIG. 9 shows the simultaneous fermentation capacity of glucose and xylose of the recombinant strain of example 6.

FIG. 10 shows the simultaneous fermentation capacity of glucose and xylose of the recombinant strain of example 7.

FIG. 11 shows the simultaneous fermentation capacity of glucose and xylose of the recombinant strain of example 8.

FIG. 12 shows the simultaneous fermentation capacity of glucose and xylose of the recombinant strain of example 9.

FIG. 13 shows the results of batch fermentation of the recombinant strain of comparative example 1 using a simulant.

FIG. 14 shows the results of batch fermentation of the recombinant strain of example 3 using a simulant.

FIG. 15 shows the results of batch fermentation of the recombinant strain of example 3 using a saccharification liquid derived from Miscanthus sinensis.

FIG. 16 shows the results of batch fermentation of the recombinant strain of example 3 using a tree-derived saccharification liquid.

FIG. 17 shows the results of fed-batch fermentation of the recombinant strain of comparative example 1 using a mock solution.

FIG. 18 shows the results of fed-batch fermentation of the recombinant strain of example 3 using a mock solution.

FIG. 19 is the result of fed-batch fermentation of the recombinant strain of example 3 using a tree-derived saccharification liquid.

Description of reference numerals:

■ glucose

◆ xylose

●: 2, 3-butanediol

▲ lactic acid.

Detailed Description

The present invention relates to a recombinant microorganism having a capability of simultaneous fermentation of two or more sugars in a lignocellulose saccharification liquid and a capability of producing a diol.

Further, the present invention relates to a method for producing a diol, comprising: preparing a medium containing two or more sugars; inoculating the recombinant microorganism into the culture medium; and culturing the recombinant microorganism.

The present invention will be described in detail below.

Lignocellulose saccharification liquid

The recombinant microorganism of the present invention has tolerance to a lignocellulose saccharification liquid and has a simultaneous fermentation ability for two or more sugars in the lignocellulose saccharification liquid. The lignocellulose saccharification liquid is a hydrolysate obtained by hydrolyzing a lignocellulose raw material (for example, a tree, an oil palm empty fruit cluster, a corn stalk, a sugar cane stalk, a reed, a miscanthus sinensis, a straw, or the like), and is preferably a hydrolysate remaining after hydrolyzing the lignocellulose raw material and removing lignin. The lignocellulose saccharification liquid contains mixed sugar, and the mixed sugar contains more than two kinds of sugar. The saccharified solution preferably contains pentoses such as glucose, xylose, mannose, galactose, arabinose, cellobiose, hexoses such as glucose, and disaccharides, and particularly contains high amounts of glucose and xylose.

Lignocellulose saccharification liquid resistance

The recombinant microorganism of the present invention is resistant to lignocellulose saccharification solutions. The fact that the recombinant microorganism is resistant to a lignocellulose saccharification liquid means that the recombinant microorganism can grow in a medium containing the saccharification liquid, and the growth of the microorganism is not inhibited by components in the saccharification liquid.

Synchronous fermentation capacity

The recombinant microorganism of the present invention has a capability of synchronously fermenting two or more sugars in a lignocellulose saccharification liquid. The simultaneous fermentation capacity means that one sugar does not ferment before another sugar. The recombinant microorganism of the present invention has a simultaneous fermentation ability for two or more sugars, and therefore, a phenomenon in which one sugar interferes with the metabolism of the other sugar (i.e., catabolite repression) is prevented between sugars that are targets of the simultaneous fermentation ability.

Synchronous fermentation capacity of recombinant microorganisms

The recombinant microorganism of the present invention has a capability of synchronously fermenting two or more sugars in a lignocellulose saccharification liquid. Preferably, the recombinant microorganism of the present invention has the ability to simultaneously ferment one or more sugars selected from the group consisting of xylose, arabinose, and cellobiose and glucose. More preferably, the recombinant microorganism of the present invention has a xylose simultaneous fermentation rate of 90% or more, and preferably, the recombinant microorganism of the present invention has a xylose simultaneous fermentation rate of 95% or more.

Synchronous fermentation rate (%) [ (total input sugar (g) — total residual sugar (g) after fermentation))/total input sugar (g) ] × 100

Example) xylose synchronous fermentation Rate (%)

Xylose simultaneous fermentation rate [ (total xylose input amount (g) — residual xylose amount (g) after fermentation))/(total xylose input amount (g)) ] × 100

Diols

The diol of the present invention has 5 or less carbon atoms. Preferably, the diol of the present invention is butanediol, more preferably, the diol of the present invention is 2, 3-butanediol.

Recombinant microorganism

The present invention relates to a lignocellulose saccharification liquid having a capability of simultaneous fermentation of two or more sugars and a capability of producing a diol. The recombinant microorganism is resistant to a lignocellulose saccharification liquid, and more preferably resistant to a microbial growth inhibitor in the lignocellulose saccharification liquid. The recombinant microorganism has the ability to ferment hexose and pentose simultaneously, and preferably has the ability to ferment glucose and xylose simultaneously.

Preferably, the recombinant microorganism is a recombinant Klebsiella (Klebsiella). More preferably, the recombinant microorganism of the invention is recombinant Klebsiella oxytoca (Klebsiella oxytoca).

The recombinant microorganism of the present invention can inhibit the mechanism of repression of degradation products (catabolites) compared to a wild-type microorganism. Preferably, the recombinant microorganism of the invention inhibits more the Glucose-specific Phosphotransferase IIA component (PTS) or Glucose-specific Phosphotransferase IIBC component (PTS) of the Phosphotransferase system (PTS, Phosphotransferase system) than the wild-type microorganism.

The recombinant microorganism of the present invention may be a recombinant microorganism that more enhances a pathway of converting xylose to xylulose-5-P or Ribulose (Ribulose) -5-P or Ribose (Ribose) -5-P or Fructose (Fructose) -6-P or erythrose-4-P (erythrose-4-P) or Glyceraldehyde (Glyceraldehyde) -3-P through xylulose (xylulose) than a wild-type microorganism. Preferably, the recombinant microorganism of the present invention is a recombinant microorganism that enhances the activity of one or more enzymes selected from the group consisting of xylose isomerase, xylulokinase, D-ribulose-5-phosphate 3-epimerase, ribose 5-phosphate isomerase, transaldolase and transketolase.

Preferably, the recombinant microorganism of the present invention inhibits cyclic adenosine monophosphate (cAMP) receptor activity of a receptor protein of cyclic adenosine monophosphate. More preferably, in the recombinant microorganism of the present invention, a gene encoding a cyclic adenosine monophosphate-activated global transcription factor is mutated to thereby inhibit the expression of the above gene, or the mutated gene is overexpressed to inhibit the cyclic adenosine monophosphate receptor activity.

Preferably, in the recombinant microorganism of the present invention, the pathway for converting pyruvate into lactate is inhibited. Lactate dehydrogenase (lactate dehydrogenase) controls the conversion of pyruvate to lactate. The pathway for converting pyruvate into lactate can be inhibited by inhibiting the lactate dehydrogenase. The inhibition of the lactate dehydrogenase can be achieved by the inhibition of the expression of lactate dehydrogenase, the inhibition of lactate dehydrogenase activity, or the like. For example, the lactate dehydrogenase can be inhibited by deleting lactate dehydrogenase a (ldha), which is a gene encoding lactate dehydrogenase, or by mutating the gene (mutation such as suppression of normal gene expression by mutation, substitution, or deletion of a part of the base or introduction of a part of the base), or by selecting an appropriate method such as gene expression control in the transcription process or translation process.

Furthermore, it is preferable that the recombinant microorganism of the present invention inhibits a pathway for converting pyruvate into acetyl-CoA and formate. Pyruvate formate lyase (pyruvate-format lyase) catalyzes the conversion of pyruvate to acetyl-CoA and formate under facultative anaerobic conditions (pathway 1).

Route 1

Pyruvic acid → acetyl coenzyme A + formic acid

The above-mentioned pyruvate formate lyase is inhibited to inhibit the conversion pathway from pyruvate to acetyl-CoA and the conversion pathway to formate. The inhibition of the pyruvate formate lyase can be achieved by, for example, inhibition of expression of pyruvate formate lyase or inhibition of the enzymatic activity of pyruvate formate lyase. For example, the pyruvate formate lyase may be inhibited by deleting formate lyase b (pflb) which is a gene encoding pyruvate formate lyase, by mutating, substituting or deleting a part of the base, or by introducing a part of the base to inhibit normal gene expression, or by selecting an appropriate method such as gene expression control in transcription or translation, by a person of ordinary skill.

FIG. 1 shows a pathway in the recombinant Klebsiella oxytoca of the present invention that is more enhanced or inhibited compared to the wild-type strain and genes of enzymes for controlling the pathway. In order to reduce by-products such as lactate (lactate), formate (formate), ethanol (ethanol), the lactate dehydrogenase a, formate lyase B genes were removed. Inhibits pathways (crr, ptsG, crp) involved in the repression mechanism of degradation products (catabolite), and amplifies and expresses pathways (xylA, xylB, rpe, rpiA, tktAB, talB) involved in the extraction (uptake) and metabolism of xylose (pentose).

Culture medium comprising two or more sugars

Preferably, the medium containing two or more kinds of sugars is a medium containing a saccharified solution derived from lignocellulose. The medium may include one or more sugars selected from the group consisting of xylose, arabinose, and cellobiose, and glucose. In this case, 5.5: 4.5 to 9: 1, the ratio of glucose to xylose in the saccharification liquid may preferably be 5.5: 4.5 to 8.0: 2.0 weight ratio of glucose to xylose in the saccharified solution.

Diol-producing ability of recombinant microorganism

The diol-producing ability of the recombinant microorganism of the present invention was calculated in the following manner.

Glycol productivity (g/L/h) of the amount of glycol produced per unit of time and volume

(in this case, the glycol productivity was calculated based on the amount of glycol accumulated in the total phase in the continuous culture in the exponential growth phase in the batch method and fed-batch method.)

Production rate of 2, 3-butanediol (g/L/h) amount of 2, 3-butanediol produced per unit time and unit volume (in this case, in the batch method and fed-batch method, the production rate of 2, 3-butanediol is based on the exponential growth phase, and in the continuous culture, the production rate is calculated based on the amount of accumulated 2, 3-butanediol produced in the total phase)

Yield (%) [ amount of 2, 3-butanediol produced (g)/carbon source (g) ] × 100

Concentration (g/L) the amount of metabolite produced per unit volume

EMBODIMENTS FOR CARRYING OUT THE INVENTION

The advantages and features of the present invention and methods for achieving the advantages and features will become apparent with reference to the embodiments and the accompanying drawings described in detail below. However, the present invention is not limited to the embodiments disclosed below, but may be embodied in various forms different from each other, and the embodiments are only for the purpose of making the disclosure of the present invention more complete, and are provided for informing a person skilled in the art of the scope of the present invention completely, and the present invention is defined only by the scope of the claims.

Materials and methods

The wild-type strain used was a klebsiella oxytoca strain with a deposit number of KCTC12132BP (deposited at 2/8/2012, korea institute of life engineering).

The sugars were analyzed by liquid chromatography. In this case, the mobile phase used was a 0.01N H2SO4 solution, and the column used was Aminex87H from Bio-Rad.

The tree-derived saccharification liquid used in the experimental examples of the present invention was prepared by the following method.

The method includes the steps of cutting waste wood, adding the waste wood to functional groups containing 70% sulfuric acid, and then stirring and reacting the mixture at a temperature of about 100 ℃ for 30 minutes, thereby performing pretreatment, adding an appropriate amount of water to the pretreated slurry to perform hydrolysis, wherein the solution hydrolyzed in the above manner contains glucose and xylose derived from cellulose (cellulose) and hemicellulose (hemicellules), and thus exists in the form of a mixture of a plurality of sugars (hereinafter, the mixture of the sugars is referred to as "mixed sugar").

The saccharification liquid derived from cerbera sinensis used in the experimental examples of the present invention was prepared by the following method.

The present invention relates to a method for producing a biomass-derived saccharified solution, and more particularly, to a method for producing a biomass-derived saccharified solution, which comprises cutting shionogi, adding thereto a functional group containing 70% sulfuric acid, stirring the same at a temperature of about 100 ℃ for 30 minutes, and reacting the same, thereby performing pretreatment, adding an appropriate amount of water to the pretreated slurry to perform hydrolysis, the solution hydrolyzed in the above manner containing glucose and xylose derived from cellulose and hemicellulose, thereby existing as a mixture of a plurality of sugars (hereinafter, the mixture of the sugars is referred to as "mixed sugar"),. separating sulfuric acid from the above hydrolyzed solution (including the mixed sugar) using an anion exchange resin, and producing a saccharified solution derived from shionogi having a mixed sugar concentration of about 100 g/L, concentrating the saccharified solution derived from shionogi produced in the above manner again so that the mixed sugar concentration becomes about 200 g/L, and using the concentrate as a culture solution for continuous.

Experimental example 1: preparation of recombinant Strain

Comparative example 1: preparation of Klebsiella oxytoca (K.oxytoca) Deltalactate dehydrogenase A Deltaformate lyase B

In order to clone lactate dehydrogenase and pyruvate formate lyase of Klebsiella oxytoca, the homologous sites of lactate dehydrogenase A (SEQ ID NO: 1) and formate lyase B (SEQ ID NO: 2) as target genes were amplified using Polymerase Chain Reaction (PCR) (Table 1).

In this case, the amplified deoxyribonucleic acid (DNA) fragment may include an antibiotic resistance gene or the like in order to increase the recombination probability of the target gene, and may further include a levan sucrose transferase (sacB) gene encoding levan sucrase in order to remove the antibiotic resistance gene recombined in the chromosome later.

TABLE 1

The above-mentioned deoxyribonucleic acid fragment was transferred by electroporation (electrophoresis, 25uF, 200. omega., 18kV/cm) to Klebsiella oxytoca deposited as KCTC12132 BP. At this time, recombinant Klebsiella oxytoca from which the lactate dehydrogenase A gene was deleted was prepared by transferring a deoxyribonucleic acid fragment including a homologous site of the lactate dehydrogenase A gene. Then, a deoxyribonucleic acid fragment containing a homologous site of the formate lyase B gene is transferred to the recombinant Klebsiella oxytoca from which the lactate dehydrogenase A gene is deleted.

As a result, a recombinant klebsiella oxytoca (klebsiella oxytoca Δ lactate dehydrogenase a Δ formate lyase B) from which lactate dehydrogenase a and formate lyase B as target genes were removed was prepared.