KR20150121789A - Recombinant microorganism having enhanced butanediol producing ability and method for producing butanediol using the same - Google Patents

Abstract

The present invention relates to recombinant microorganisms for producing 2,3-butanediol, wherein a pathway for converting pyruvate into alpha-acetolactate, a pathway for converting the alpha-acetolactate into acetoin or a pathway for converting the acetoin into 2,3-butanediol are promoted.

Description

TECHNICAL FIELD The present invention relates to a recombinant microorganism having enhanced 2,3-butanediol production ability and a method for producing 2,3-butanediol using the recombinant microorganism. [0002]

The present invention relates to a recombinant microorganism having enhanced 2,3-butanediol production ability and a method for producing 2,3-butanediol using the recombinant microorganism.

2,3-butanediol, which is one of the alcohols having four carbon atoms and two hydroxyl groups (-OH) (CH3CHOHCHOHCH3), can be produced by reacting 1,3-butadiene and 1,3- (Mie et al., Biotechnol. Adv., 29: 351, 2011), which can be chemically catalyzed by using methyl ethyl ketone (MEK) as an additive and solvent. In addition, 2,3-butanediol is an industrially important intermediate because it can be mixed with gasoline as an octane booster (Celinska et al., Biotechnol. Adv., 27: 715, 2009).

2,3-butanediol can be produced through a chemical synthesis process and a microbial fermentation process. However, since 2,3-butanediol production cost is very high, 2,3-butanediol production on a commercial scale has not been achieved. In recent years, along with the rapid development of 2,3-butanediol production technology through the microbial fermentation process, the rapid increase of the price of raw materials of fossil materials and the regulation of international environmental pollution have been strengthened, Interest in production and the importance of research and development are increasing.

The method of producing bio-based 2,3-butanediol is to convert a renewable biomass material into 2,3-butanediol through fermentation of a microorganism having 2,3-butanediol producing ability. 2,3-butanediol is Klebsiella, Enterobacter, Bacillus. Serratia species (Species) (Maddox IS, Biotechnol., 6: 269, 1996). Particularly, K. pneumoniae, K. oxytoca and Paenibacillus polymyxa produce a relatively large amount of 2,3-butanediol, and particularly Kleb CIELA NUMONIERE and KLEPSYELLA OXITOKA have the advantage of being able to produce 2,3-butanediol from various kinds of bio-raw materials including 5-carbon sugars derived from wood-based materials, which are easy to cultivate and have a high growth rate (Ji Jansen et al., Adv., 29: 351, 2011; Chandel et al., Sustainable Biotechnol., 63, 2010; Jansen et al., Biotechnol. Bioeng., 26: Biochem. Eng., 27: 85, 1983).

Studies on the production of bio-based 2,3-butanediol by microbial fermentation process are divided into optimization of fermentation process (temperature, pH, dissolved oxygen, etc.) and microbial development (microbial discovery, physiological characterization, mutation, genetic engineering etc.) have. In terms of optimization of the fermentation process, various conditions such as temperature, pH, and dissolved oxygen concentration capable of efficiently producing 2,3-butanediol have been identified (Ji et al., Bioresource Technol., 100: 3410, 2009; Nakashimada et al , J. Biosci. Bioeng., 90: 661, 2000; Nakashimada et al., Biotechnol. Lett., 20: 1133, 1998). However, the production of 2,3-butanediol through the microbial fermentation process under the above conditions still has a problem in that it is difficult to apply directly to a commercial process due to low productivity and yield. In addition, in the fermentation process, various by-products such as organic acids including lactic acid and alcohols including ethanol are formed together with 2,3-butanediol. The production of by-products not only lowers the yield of 2,3-butanediol for the bio-raw material but also requires a great deal of separation and purification cost in the 2,3-butanediol recovery process from the culture broth.

Therefore, the development of microorganisms related to the production of 2,3-butanediol has mainly proceeded to reduce by-products. Typically, Ji et al. Have succeeded in partially inhibiting the production of by-products of organic acids by exposing UV, a kind of physical / chemical mutagenesis method, to the wild-type Klebsiella oxytoca strain (Ji et al., Biotechnol. Lett., 30: 731, 2008). In addition, there have also been attempts to improve 2,3-butanediol production by applying an ion beam method to a strain of Klebsiella nymonii to increase the consumption rate of biomass (Ma et al., Appl. Microbiol Biotechnol., 82: 49, 2009). However, the above-described strains still have a problem of being directly applied to commercial processes in terms of 2,3-butanediol productivity, final concentration (concentration) and yield.

Therefore, the inventors of the present invention found that when recombinant microorganisms having high productivity, concentration and yield of 2,3-butanediol were studied, the recombinant microorganism into which specific genes were introduced had high selectivity and productivity of 2,3-butanediol and completed the present invention Respectively.

An object of the present invention is to provide a recombinant microorganism having enhanced 2,3-butanediol production ability and a method for producing 2,3-butanediol using the recombinant microorganism.

According to an aspect of the present invention,

In the 2,3-butanediol producing microorganism,

A pathway for converting pyruvate to alpha-acetolactate, a pathway for converting alpha-acetolactate to acetone, or a pathway for converting acetone to 2,3-butanediol,

A recombinant microorganism for producing 2,3-butanediol is provided.

Further, according to the present invention,

Inoculating the recombinant microorganism of the present invention; and

And culturing the recombinant microorganism. The present invention also provides a method for producing 2,3-butanediol.

The recombinant microorganism of the present invention has high productivity of 2,3-butanediol.

Figure 1 shows the biosynthetic pathway of 2,3-butanediol in the 2,3-butanediol producing strain.
Figure 2 shows the pGSC-budA plasmid.
Figure 3 shows the pGSC-budAB plasmid.
Figure 4 shows the pGSC-budABC plasmid.
Fig. 5 shows the fermentation results of the wild-type K. oxytoca wild type strain of Comparative Example 1. Fig.
6 is a result of fermentation of the recombinant Klebsiella oxytoca strain (K. oxytoca? LdhA? PflB) of Comparative Example 2. Fig.
7 is a result of fermentation of the recombinant Klebsiella oxytoca strain (K. oxytoca wild type + pGSC-budRABC) of Example 1. Fig.
8 is a result of fermentation of the recombinant Klebsiella oxytoca strain K. oxytoca [Delta] ldhA [Delta] pflB + pGSC-budRA of Example 2].
Fig. 9 shows the results of fermentation of the recombinant Klebsiella oxytoca strain (K. oxytoca [Delta] dhA [Delta] pflB + pGSC-budRAB)
10 is a result of fermentation of the recombinant Klebsiella oxytoca strain of Example 4 (K. oxytocaΔldhAΔpflB + pGSC-budRABC).

According to the present invention,

In the 2,3-butanediol producing microorganism,

A pathway for converting pyruvate to alpha-acetolactate, a pathway for converting alpha-acetolactate to acetone, or a pathway for converting acetone to 2,3-butanediol,

The present invention relates to a recombinant microorganism for producing 2,3-butanediol.

Further, according to the present invention,

Inoculating the recombinant microorganism of the present invention; and

And culturing the recombinant microorganism. The present invention also provides a method for producing 2,3-butanediol.

Hereinafter, the present invention will be described in detail.

2,3- Butanediol Productive Increased  Recombinant microorganism

The recombinant microorganism of the present invention

In the 2,3-butanediol producing microorganism,

A pathway for converting pyruvate to alpha-acetolactate, a pathway for converting alpha-acetolactate to acetone, or a pathway for converting acetone to 2,3-butanediol,

Is a recombinant microorganism for producing 2,3-butanediol.

In addition, the recombinant microorganism of the present invention,

The 2,3-butanediol producing microorganism is a recombinant microorganism having increased activity of at least one enzyme selected from the group consisting of acetolactate synthase, acetolactate dicarboxylase and acetyl reductase.

In addition, the recombinant microorganism of the present invention,

In the 2,3-butanediol producing microorganism, it is a recombinant microorganism having increased activity of two or more enzymes selected from the group consisting of acetolactate synthase, acetolactate dicarboxylase and acetyl reductase.

In addition, the recombinant microorganism of the present invention,

In the 2,3-butanediol producing microorganism, the activity of acetolactate synthase, acetolactate dicarboxylase, and acetyl reductase is increased.

In addition, the recombinant microorganism of the present invention

In the 2,3-butanediol producing microorganism,

The pathway for converting pyruvate to acetyl koide, the pathway for converting pyruvate to formic acid, or the pathway for converting pyruvate to lactate is inhibited,

A pathway for converting pyruvate to alpha-acetolactate, a pathway for converting alpha-acetolactate to acetone, or a pathway for converting acetone to 2,3-butanediol,

May be a recombinant microorganism for producing 2,3-butanediol.

In this case, the recombinant microorganism of the present invention is a 2,3-butanediol producing microorganism,

The pathway for converting pyruvate to acetylcohexyl, the pathway for converting pyruvate to formic acid, and the pathway for converting pyruvate to lactate are inhibited,

The pathway for converting pyruvate to alpha-acetolactate may be promoted

In this case, the recombinant microorganism of the present invention is a microorganism producing 2,3-butanediol,

The pathway for converting pyruvate to acetylcohexyl, the pathway for converting pyruvate to formic acid, and the pathway for converting pyruvate to lactate are inhibited,

The pathway for converting pyruvate to alpha-acetolactate and the pathway for converting alpha-acetolactate to acetone may be facilitated

In this case, the recombinant microorganism of the present invention is a microorganism producing 2,3-butanediol,

The pathway for converting pyruvate to acetylcohexyl, the pathway for converting pyruvate to formic acid, and the pathway for converting pyruvate to lactate are inhibited,

The pathway for converting pyruvate to alpha-acetolactate, the pathway for converting alpha-acetolactate to acetone, and the pathway for converting acetone to 2,3-butanediol may be facilitated

In addition, the recombinant microorganism of the present invention is a recombinant microorganism that inhibits the activity of pyruvate-formate lyase and lactate dehydrogenase in the 2,3-butanediol producing microorganism and increases the activity of acetolactate synthase have

In addition, the recombinant microorganism of the present invention is characterized in that the activity of pyruvate-formate lyase and lactate dehydrogenase is inhibited in the 2,3-butanediol producing microorganism, and the activity of acetolactate synthase and acetolactate decarboxylase May be an increased recombinant microorganism

In addition, the recombinant microorganism of the present invention is characterized in that the activity of pyruvate-formate lyase and lactate dehydrogenase is inhibited in the 2,3-butanediol producing microorganism, and the activity of acetolactate synthase, acetolactate decarboxylase, Lt; RTI ID = 0.0 > reductase < / RTI > activity may be an increased recombinant microorganism

Also, the recombinant microorganism of the present invention has high selectivity, yield, concentration and productivity of 2,3-butanediol. In addition, the recombinant microorganism of the present invention may be such that the production ability of by-products such as formic acid, lactate, etc. is suppressed more than that of the wild-type microorganism due to the recombination.

Acetyl Koei  Biosynthetic pathway

The recombinant microorganism of the present invention may be a recombinant microorganism produced using a 2,3-butanediol producing microorganism having acetyl-CoA and lactate biosynthesis pathway. The acetyl-CoA biosynthetic pathway of the present invention refers to the pathway through which acetylcoe is synthesized from a specific metabolite in a microorganism. The acetyl-CoA biosynthesis pathway of the present invention may be a pathway in which acetyl CoA is synthesized from pyruvate.

Lactate  Biosynthetic pathway

The recombinant microorganism of the present invention may be a recombinant microorganism produced using a 2,3-butanediol producing microorganism having acetyl-CoA and lactate biosynthesis pathway. The lactate biosynthetic pathway of the present invention refers to the route through which lactate is synthesized from a particular metabolite in a microorganism. The lactate biosynthesis pathway of the present invention may be a pathway in which lactate is synthesized from pyruvate.

2,3 Butanediol  Production microorganism

The recombinant microorganism of the present invention is a recombinant microorganism produced by genetically recombining 2,3-butanediol producing microorganisms. The 2,3-butanediol producing microorganism may be, for example, a microorganism belonging to the genus of Klebsiella, Bacillus, Serratia or Enterobacter, preferably Klebsiella oxytoca K. oxytoca, K. pneumoniae, and the like, and most preferably K. oxytoca. Preparation of the recombinant microorganism of the present invention using Klebsiella oxytoca is advantageous for the industrial scale production of 2,3 butanediol rather than using Klebsiella pneumoniae.

Pyruvate  Acetyl Koeiro  Suppression of switching paths

Pyruvate-formate lyase regulates the conversion of pyruvate to acetylcopherol. By inhibiting the pyruvate-formate lyase, the pathway for converting pyruvate to acetyl koide can be inhibited. Inhibition of the pyruvate-formate lyase may be accomplished by inhibiting the expression of pyruvate-formate lyase, inhibiting the enzyme activity of pyruvate-formate lyase, and the like. For example, it is possible to delete pflB, which is a gene encoding pyruvate-formate lyase, or to mutate (mutation such as mutation, substitution, deletion, deletion of some bases or introduction of some bases to suppress expression of normal gene) Or regulating gene expression in transcription or translation processes, the skilled artisan can select the appropriate method to inhibit pyruvate-formate lyase.

Pyruvate  Inhibition of pathway to formic acid

Pyruvate-formate lyase regulates the conversion of pyruvate to formic acid. By inhibiting the pyruvate-formate lyase, the pathway for converting pyruvate to formic acid can be inhibited. Inhibition of the pyruvate-formate lyase may be accomplished by inhibiting the expression of pyruvate-formate lyase, inhibiting the enzyme activity of pyruvate-formate lyase, and the like. For example, it is possible to delete pflB, which is a gene encoding pyruvate-formate lyase, or to mutate (mutation such as mutation, substitution, deletion, deletion of some bases or introduction of some bases to suppress expression of normal gene) Or regulating gene expression in transcription or translation processes, the skilled artisan can select the appropriate method to inhibit pyruvate-formate lyase.

Pyruvate With lactate  Suppression of switching paths

Lactate dehydrogenase regulates the conversion of pyruvate to lactate. By inhibiting the lactate dehydrogenase, the pathway for converting pyruvate to lactate can be inhibited. The inhibition of the lactate dehydrogenase can be achieved by inhibiting the expression of lactate dehydrogenase, inhibiting the enzyme activity of lactate dehydrogenase, and the like. For example, ldhA, which is a gene encoding lactate dehydrogenase, is deleted or a mutation (mutation such as mutation, substitution, deletion, deletion of some bases, introduction of some bases to suppress the expression of normal gene) Or by controlling the gene expression in transcription or translation processes, the skilled artisan can select an appropriate method to inhibit lactate dehydrogenase.

Pyruvate  Alpha- Acetolactate  Facilitating the transition path

Acetolactate synthase regulates the conversion of pyruvate to alpha-acetolactate, which is involved in the production of alpha-acetolactate. By increasing the activity of the acetolactate synthase, the pathway to convert pyruvate to alpha-acetolactate can be promoted. The increase in the activity of the acetolactate synthase can be achieved by amplification and expression of a gene of acetolactate synthase, an increase in enzyme activity of acetolactate synthase, and the like. For example, it is possible to introduce budB, which is a gene encoding acetolactate synthase, into a microorganism or cause mutation (mutation such as amplification of gene expression by mutating, substituting or deleting some bases or introducing some bases) Or controlling the gene expression in the transcription or translation process, the skilled artisan can select the appropriate method to increase the activity of the acetolactate synthase.

Alpha- Acetolactate With acetone  Facilitating the transition path

Acetolactate decarboxylase regulates the conversion of alpha-acetolactate to acetone and is involved in the production of acetone. By increasing the activity of the acetolactate dicarboxylic silage, the pathway for converting alpha-acetolactate to acetone can be promoted. The increase in the activity of the acetolactate dicarboxylic silage can be achieved by amplifying and expressing the gene of acetolactate dicarboxylic silage and increasing the enzyme activity of the acetolactate dicarboxylic silage. For example, it is possible to introduce budA, which is a gene encoding acetolactate dicarboxylic silage, into a microorganism, or to mutate (mutation such as amplification of gene expression by introducing some bases, mutation, substitution or deletion of some bases, , Or by controlling the gene expression in the transcription or translation process, the skilled artisan can select an appropriate method to increase the activity of the acetolactate dicarboxylic silage.

Acetone  2,3- With butanediol  Facilitating the transition path

Acetoin reductase modulates the conversion of acetone to 2,3-butanediol and is involved in the production of 2,3-butanediol. By increasing the activity of the acetyl reductase, the pathway for converting the acetyl reductase to 2,3-butanediol can be promoted. The increase in the activity of the acetyl reductase can be achieved by amplification and expression of the gene of the acetyl reductase and an increase in the enzyme activity of the acetyl reductase. For example, budC, which is a gene encoding acetyl reductase, is introduced into a microorganism, or a mutation (mutation such as amplification of gene expression by introducing some bases, mutation, substitution or deletion of some bases, , The transcription process or the regulation of gene expression in the translation process, the skilled artisan can select an appropriate method to increase the activity of the acetylin reductase.

2,3- Butanediol  Production method

The method for producing 2,3-butanediol of the present invention comprises culturing the recombinant microorganism of the present invention and recovering 2,3-butanediol from the culture.

culture

The culture of the recombinant microorganism of the present invention is carried out under aerobic conditions, preferably under microaerobic conditions. For example, the culture is carried out while supplying oxygen, i.e., air, during the culture, and as a specific example, it may be performed through stirring, but is not limited thereto. The recombinant microorganism of the present invention can be cultured in a complex culture medium. The type of the complex culture medium is not particularly limited, and a person skilled in the art will generally be able to appropriately select and use commercially available complex culture media.

Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described in detail below. It should be understood, however, that the invention is not limited to the disclosed embodiments, but is capable of many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, To fully disclose the scope of the invention to those skilled in the art, and the invention is only defined by the scope of the claims.

≪ Materials and methods >

The wild-type strain Klebsiella oxytoca KCTC 12132BP was prepared and used as a strain of Comparative Example 1. The recombinant microorganisms were prepared in the following <1-1> to <1-3>.

-2,3-butanediol concentration (g / L): Amount of 2,3-butanediol produced per unit volume

-2,3-butanediol yield (g / g): 2,3-butanediol yield (g) / carbon source (g) × 100

-2,3-butanediol Productivity (g / L / h): Amount of 2,3-butanediol produced per unit time, unit volume

&Lt; 1-1 > Preparation of recombinant K. oxytocin in which ldhA and pflB were deleted

Production of DNA fragment containing homologous region of target gene

In order to inactivate the target gene of Klebsiella oxytoca, a recombinant mechanism of bacteria was used and the homologous region of the gene to be removed was amplified by PCR. After transferring the corresponding DNA fragment containing the homologous portion to the bacteria, a recombination mechanism between the homologous region of the gene in the DNA fragment and the gene in the chromosome of Klebsiella oxytoca recombine to produce the target gene .

First, the homologous part 1 (SEQ ID NO: 2) of the target gene ldhA (SEQ ID NO: 1) was amplified by PCR using the primers of SEQ ID NOS: 3 and 4 to clone the lactate dehydrogenase of Klebsiella oxytoca . Homologous site 2 (SEQ ID NO: 5) was also amplified by PCR using primers of SEQ ID NOS: 6 and 7. Thereafter, the homologous sites 1 and 2 were used as a template and amplified by PCR to complete a DNA fragment (SEQ ID NO: 8) containing homologous sites 1 and 2.

On the other hand, in order to clone the homology region of the pyruvate formate lyase of Klebsiella oxytoca, the homology region 1 (SEQ ID NO: 10) of the target gene pflB (SEQ ID NO: 9) was amplified by PCR using the primers of SEQ ID NOS: 11 and 12 . Homologous site 2 (SEQ ID NO: 13) was amplified by PCR using the primers of SEQ ID NOS: 14 and 15. Thereafter, homologous sites 1 and 2 were amplified by PCR using the template as a template to complete homologous sites 1 and 2 (SEQ ID NO: 16) (Table 1). In order to increase the recombination probability of the target gene, the completed DNA fragment may include an antibiotic resistance gene and the like, and may contain a sacB gene encoding Levansucracase enzyme to remove the antibiotic resistance gene recombined in the chromosome .

ldhA  And pflB Recombinant K. &lt; / RTI &gt; Oxytocin  Produce

The prepared DNA fragments were transferred to Klebsiella oxytoca KCTC 12132BP using electroporation (25 uF, 200 OMEGA, 18 kV / cm) and the target gene was removed using the recombinant mechanism of the microorganism there was.

 A recombinant Klebsiella oxytoca was prepared by transferring a DNA fragment containing the homologous region of the ldhA gene to the wild-type Klebsiella k. After removal of the ldhA gene from the wild-type Klebsiella oxytoca, a DNA fragment containing the homologous region of the pflB gene was transferred to prepare a Klebsiella oxytoca in which the pflB gene was removed in addition to the ldhA gene.

The following recombinant process and fermentation experiments were carried out using the K. oxytoca KCTC 12132BP recombinant microorganism (hereinafter referred to as &quot; K. oxytoca DELTA ldhA DELTA pflB &quot;) in which ldhA and pflB thus prepared were deleted .

<1-2> Preparation of pGSC-budA plasmid, pGSC-budAB plasmid and pGSC-budABC plasmid

A recombinant plasmid to be used for the expression amplification of a gene encoding acetolactate dicarboxylase (budA), acetolactate synthase enzyme (budB) and acetylin reductase enzyme (budC) derived from Klebsiella oxytocin was prepared Respectively.

In order to construct a recombinant vector that amplifies the target gene expression of Klebsiella lucidum, pBBR1MCS (Kovach et al., Biotechniques, 800-802, 1994) containing a restriction enzyme site, multiple cloning site and a chloramphenicol resistance gene ) The gene to be amplified should be cloned into the plasmid. After cloning the plasmid into the bacteria, the gene expression is amplified by the replication mechanism of the plasmid in the cell.

(BudA, SEQ ID NO: 17) encoding the acetolactate dicarboxylase enzyme of Klebsiella oxytoca and the gene (budB, SEQ ID NO: 18) encoding the acetolactate synthase enzyme and the acetyl reductase enzyme The target gene was amplified by PCR to clone the coding gene (budC, SEQ ID NO: 19) (Table 2). At this time, the regulator gene budR was also introduced (SEQ ID NO: 20). For amplification, primers containing restriction sites (XbaI, ApaI, etc.) in the multiple cloning site of the plasmid were amplified. The DNA fragment containing each gene and the plasmid were treated in the same manner as the restriction enzyme in the multiple cloning site, and the two fragments were ligated with T4 DNA ligase to complete pGSC-budA, pGSC-budAB and pGSC-budABC plasmids (Figs. 2 to 4).

&Lt; 1-3 > Expression of a gene encoding acetolactate dicarboxylase, acetolactate synthase, and acetyl reductase

Expression of a gene encoding acetolactate dicarboxylase (budA), acetolactate synthase enzyme (budB) and acetylin reductase enzyme (budC) derived from chlorhexilaoxitocarn was amplified.

The pGSC-budA plasmid, the pGSC-budAB plasmid and the pGSC-budABC plasmid prepared in <1-2> were subjected to electrophoresis (25 uF, 200 Ω, 18 kV / cm) 2 &lt; / RTI &gt; recombinant Klebsiella lysozyme. More specifically, the pGSC-budABC plasmid was cloned into wild-type Klebsiella koktaoka KCTC 12132BP (Comparative Example 1) to complete the recombinant Klebsiella oxytoca amplified expression of the genes (hereinafter referred to as "K oxytoca wild type + pGSC-budABC &quot;). The pGSC-budA plasmid, the pGSC-budAB plasmid and the pGSC-budABC plasmid were cloned into the recombinant microorganism of Comparative Example 2, which had both ldhA and pflB removed simultaneously, and the recombinant complementase Ellaoxytocar was completed (Examples 2 to 4). At this time, the regulator gene budR was also introduced. After the electroporation, the recombinant Klebsiella oxytocars were incubated at 30 DEG C for 1 hour and stabilized. The cells were then spread in an LB complex solid medium containing chloramphenicol at 37 DEG C, respectively. After that, colonies grown in a solid medium containing chloramphenicol were picked out. The plasmids contained in the selected colonies were minipreped to confirm the cloning of the genes by electrophoresis. As a result, the following recombinant microorganisms were prepared (Table 3).

<Experimental Example 1> 2,3-butanediol production ability of wild-type microorganism and recombinant microorganism in which ldhA and pflB were deleted

The production and efficacy of 2,3-butanediol was evaluated using the recombinant Klebsiella strain of Comparative Example 2 in which wild-type Klebsiella of Comparative Example 1 and ldhA and pflB were simultaneously removed.

Fermentation was performed on the recombinant Klebsiella oxytocar of Comparative Example 1 and the recombinant Klebsiella oxytocar of Comparative Example 2 in which ldhA and pflB were simultaneously removed. The cultivation of the microorganisms was carried out as follows.

First, the microorganisms were inoculated into 250 ml of a complex medium containing 9 g / L glucose (50 mM glucose), cultured at 37 ° C for 16 hours, and the culture was inoculated into a 3 L complex medium. Was set at a micro-aerobic condition (aerobic rate 1 vvm, stirring speed 150 rpm), 90 g / L initial glucose concentration, pH 6.5, and incubation temperature 37 ° C. In the medium, 25 mg / L of chloramphenicol was added. 5N NaOH was used to adjust pH during fermentation. Samples were collected during fermentation against the wild type and recombinant Clevisiella, and the growth rate was measured by measuring the OD600 (optical density) of the collected samples. The collected samples were centrifuged at 13,000 rpm for 10 minutes, The metabolite of the liquid and the concentration of 2,3-butanediol were analyzed by liquid chromatography (HPLC)

As a result, when lactic acid dehydrogenase (ldhA) and pyruvic acid-formate lyase (pflB) were removed from Klebsiella oxytocar, 2,3-butanediol production was 39.49 g / L, 2,3- (2,3-butanediol g / glucose g) was 0.45. The productivity (g / L / h) of the recombinant microorganism of Comparative Example 2 was 0.58. Compared with the wild-type strain of Comparative Example 1, the recombinant microorganism of Comparative Example 2 showed a remarkable decrease in the production of lactic acid, formic acid, and ethanol, and the productivity of 2,3-butanediol was improved and 2,3-butanediol production concentration, production yield, , And the selectivity was improved (Table 4) (Figs. 5 and 6).

<Experimental Example 2> 2,3-butanediol production ability of recombinant microorganisms selectively amplified and expressed by budA, budAB and budABC genes

The recombinant Klebsiella oxytoca of Examples 1 to 4 prepared in the above <1-3> was fermented.

The specific fermentation process is as follows. The recombinant microorganisms to be tested were inoculated into 250 ml of complex medium containing 9 g / L glucose (50 mM, glucose) and cultured at 37 ° C for 16 hours. The culture was inoculated in a 3 L complex medium, Was set at a micro-aerobic condition (aerobic rate 1 vvm, stirring speed 150 rpm), 90 g / L initial glucose concentration, pH 6.5, and incubation temperature 37 ° C. In the medium, 25 mg / L of chloramphenicol was added. 5N NaOH was used to adjust pH during fermentation. The samples were collected during the fermentation of the recombinant microorganisms. The OD600 (optical density) of the collected samples was measured to determine the growth rate. The collected samples were centrifuged at 13,000 rpm for 10 minutes, The product and 2,3-butanediol concentrations were analyzed by liquid chromatography (HPLC)

As a result, it was found that when the budABC was amplified and expressed in the wild type (Example 1), the yield and concentration of 2,3-butanediol were increased as compared with Comparative Example 1 (wild type). However, it was found that the yield of by-products such as lactate was still higher than that of 2,3-butanediol.

On the other hand, Example 1, in which genes pflB and ldhA were not removed, was superior to Example 4 in which genes pflB and ldhA were removed, and Example 4 was excellent in the effect of reducing byproducts such as lactic acid and the like, The yield and the concentration of the polymer were better than those of the polymer (see Table 4, Fig. 7 and Fig. 10)

In addition, the recombinant microorganism of Example 2 showed a significant increase in the yield and productivity of 2,3-butanediol as compared with the recombinant microorganism of Comparative Example 2 (K. oxytoca? LdhA? PflB). Also, comparing the recombinant microorganisms of Example 2 and Example 3, it was confirmed that the production performance of 2,3-butanediol was improved even when budA as well as budB were amplified. In addition, comparing the recombinant microorganisms of Example 3 and Example 4, it was confirmed that the production performance of 2,3-butanediol was improved even when budC was amplified. As a result, it was found that 2,3-butanediol production performance was most improved when the recombinant microorganism of Example 4, which amplified all of budABC, was compared with the recombinant microorganism of Comparative Example 2.

Therefore, in the production of 2,3-butanediol, not only the removal of the genes pflB and ldhA for the by-product reduction but also the removal of the genes encoding acetolactate dicarboxylic acidase of Klebsiella, the gene encoding acetolactate synthase, It was confirmed that it is very important to amplify the expression of the gene encoding the reductase gene (Table 5, FIGS. 7 to 10, and 7: Example 1, FIG. 8: Example 2, 10: Example 4).

<110> gs caltex <120> RECOMBINANT MICROORGANISM HAVING ENHANCED BUTANEDIOL PRODUCING          ABILITY AND METHOD FOR PRODUCING BUTANEDIOL USING THE SAME <130> GSP130003 <160> 20 <170> Kopatentin 2.0 <210> 1 <211> 990 <212> DNA <213> Klebsiella oxytoca <400> 1 atgaaaatcg ctgtgtatag tacaaaacag tacgacaaga agtatctgca gcatgttaat 60 gatgcatatg gctttgaact ggagtttttt gacttcctgc taaccgaaaa aaccgccaaa 120 accgccaacg gctgtgaagc ggtgtgtatc ttcgtaaacg atgacggtag ccgcccggta 180 cttgaagaac tgaaagccca cggcgtgcag tacatcgcgc tgcgctgcgc ggggttcaac 240 aacgttgacc tcgatgccgc caaagagctg ggcctgcggg tggtgcgcgt cccggcctac 300 tcgccggaag cggtcgctga gcacgcgatc ggcatgatga tgtcgctgaa ccgccgcatt 360 caccgtgcct atcagcgcac ccgcgacgcg aacttctctc tggaagggct gaccggtttc 420 accatgcacg gtaaaaccgc cggcgttatt ggcaccggta aaatcggcgt cgccgcgctg 480 cgcattctta aaggcttcgg tatgcgtctg ctggcgtttg atccctaccc aagcgccgcc 540 gcgctggata tgggcgtgga gtatgtcgat cttgaaaccc tgtaccggga gtccgatgtt 600 atctcactgc actgcccact gaccgatgaa aactaccatt tgctgaacca tgccgcgttc 660 gatcgcatga aagacggggt gatgatcatc aacaccagcc gcggcgcgct catcgattcg 720 caggcagcga tcgacgccct gaagcatcag aaaattggcg cgctggggat ggacgtgtat 780 gagaacgaac gcgatctgtt ctttgaagat aagtctaatg acgtgattca ggatgatgtg 840 ttccgccgtc tctccgcctg ccataacgtc ctgtttaccg gtcaccaggc gtttctgacc 900 gcggaagcgt tgatcagcat ttcgcaaacc accctcgaca acctgcgtca agtggatgca 960 ggcgaaacct gtcctaacgc actggtctga 990 <210> 2 <211> 595 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 2 atgacgttcg ctaaatcctg cgccgtcatc tcgctgctga tcccgggcac ctccgggcta 60 ctgctgttcg gcaccctggc atcggccagc ccgggacatt tcctgttaat gtggatgagc 120 gccagcctcg gcgctatcgg cggattctgg ctctcgtggc tgacgggcta ccgctaccgg 180 taccatctgc atcgtatccg ctggcttaat gccgaacgcc tcgctcgcgg ccagttgttc 240 ctgcgccgcc acggcgcgtg ggcagtcttt tttagccgct ttctctctcc gcttcgcgcc 300 accgtgccgc tggtaaccgg cgccagcggc acctctctct ggcagtttca gctcgccaac 360 gtcagctccg ggctgctctg gccgctgatc ctgctggcgc caggcgcgtt aagcctcagc 420 ttttgatgaa aggtattgtc ttttaaagag atttcttaac accgcgatat gctctagaat 480 tattactata acctgctgat taaactagtt tttaacattt gtaagattat tttaattatg 540 ctaccgtgac ggtattatca ctggagaaaa gtcttttttc cttgcccttt tgtgc 595 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 3 cacggatcca tgacgttcgc taaatcctgc 30 <210> 4 <211> 40 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 4 gcacaaaagg gcaaggaaaa aagacttttc tccagtgata 40 <210> 5 <211> 591 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 5 tatcactgga gaaaagtctt ttttccttgc ccttttgtgc tcccccttcg cggggggcac 60 attcagataa tccccacaga aattgcctgc gataaagtta caatcccttc atttattaat 120 acgataaata tttatggaga ttaaatgaac aagtatgctg cgctgctggc ggtgggaatg 180 ttgctatcgg gctgcgttta taacagcaag gtgtcgacca gagcggaaca gcttcagcac 240 caccgttttg tgctgaccag cgttaacggg cagccgctga atgccgcgga taagccgcag 300 gagctgagct tcggcgaaaa gatgcccatt acgggcaaga tgtctgtttc aggtaatatg 360 tgcaaccgct tcagcggcac gggcaaagtc tctgacggcg agctgaaggt tgaagagctg 420 gcaatgaccc gcatgctctg cacggactcg cagcttaacg ccctggacgc cacgctgagc 480 aaaatgctgc gcgaaggcgc gcaggtcgac ctgacggaaa cgcagctaac gctggcgacc 540 gccgaccaga cgctggtgta taagctcgcc gacctgatga attaataatt a 591 <210> 6 <211> 40 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 6 tatcactgga gaaaagtctt ttttccttgc ccttttgtgc 40 <210> 7 <211> 32 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 7 cctgcggccg ctaattatta attcatcagg tc 32 <210> 8 <211> 1146 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 8 atgacgttcg ctaaatcctg cgccgtcatc tcgctgctga tcccgggcac ctccgggcta 60 ctgctgttcg gcaccctggc atcggccagc ccgggacatt tcctgttaat gtggatgagc 120 gccagcctcg gcgctatcgg cggattctgg ctctcgtggc tgacgggcta ccgctaccgg 180 taccatctgc atcgtatccg ctggcttaat gccgaacgcc tcgctcgcgg ccagttgttc 240 ctgcgccgcc acggcgcgtg ggcagtcttt tttagccgct ttctctctcc gcttcgcgcc 300 accgtgccgc tggtaaccgg cgccagcggc acctctctct ggcagtttca gctcgccaac 360 gtcagctccg ggctgctctg gccgctgatc ctgctggcgc caggcgcgtt aagcctcagc 420 ttttgatgaa aggtattgtc ttttaaagag atttcttaac accgcgatat gctctagaat 480 tattactata acctgctgat taaactagtt tttaacattt gtaagattat tttaattatg 540 ctaccgtgac ggtattatca ctggagaaaa gtcttttttc cttgcccttt tgtgctcccc 600 cttcgcgggg ggcacattca gataatcccc acagaaattg cctgcgataa agttacaatc 660 ccttcattta ttaatacgat aaatatttat ggagattaaa tgaacaagta tgctgcgctg 720 ctggcggtgg gaatgttgct atcgggctgc gtttataaca gcaaggtgtc gaccagagcg 780 gaacagcttc agcaccaccg ttttgtgctg accagcgtta acgggcagcc gctgaatgcc 840 gcggataagc cgcaggagct gagcttcggc gaaaagatgc ccattacggg caagatgtct 900 gtttcaggta atatgtgcaa ccgcttcagc ggcacgggca aagtctctga cggcgagctg 960 aaggttgaag agctggcaat gacccgcatg ctctgcacgg actcgcagct taacgccctg 1020 gacgccacgc tgagcaaaat gctgcgcgaa ggcgcgcagg tcgacctgac ggaaacgcag 1080 ctaacgctgg cgaccgccga ccagacgctg gtgtataagc tcgccgacct gatgaattaa 1140 taatta 1146 <210> 9 <211> 2283 <212> DNA <213> Klebsiella oxytoca <400> 9 atgtccgagc ttaatgaaaa gttagccaca gcctgggaag gttttgcgaa aggtgactgg 60 cagaacgaag tcaacgtccg cgacttcatc cagaaaaact ataccccgta cgaaggtgac 120 gagtccttcc tggctggcgc aactgacgcg accaccaagc tgtgggacac cgtaatggaa 180 ggcgttaaac aggaaaaccg cactcacgcg cctgttgatt ttgatacttc ccttgcatcc 240 accatcactt ctcatgacgc tggctacatc gagaaaggtc tcgagaaaat cgttggtctg 300 cagactgaag ctccgctgaa acgcgcgatt atcccgttcg gcggcatcaa aatggtcgaa 360 ggttcctgca aagcgtacga tcgcgagctg gacccgatgc tgaagaaaat cttcactgaa 420 taccgtaaaa ctcacaacca gggcgtgttt gacgtttaca ccaaagacat cctgaactgc 480 cgtaaatctg gtgttctgac cggtctgccg gatgcctatg gccgtggtcg tatcatcggt 540 gactaccgtc gcgttgcgct gtacggtatc gacttcctga tgaaagacaa atacgctcag 600 ttcgtttctc tgcaagagaa actggaaaac ggcgaagatc tggaagcaac catccgtctg 660 cgcgaagaaa tctctgaaca gcaccgcgcg ctgggtcaga tcaaagaaat ggcggctaaa 720 tatggctgcg atatctctgg tcctgctacc accgctcagg aagctatcca gtggacctac 780 ttcggttacc tggctgccgt aaaatctcag aacggcgcgg caatgtcctt cggtcgtacc 840 tccagcttcc tggacatctt catcgaacgt gacctgaaag ccggtaaaat caccgagcaa 900 gacgcacagg aaatgattga ccacctggtc atgaaactgc gtatggttcg tttcctgcgt 960 acccctgaat atgatgaact gttctctggc gacccgatct gggcaacaga atctatcggc 1020 ggtatgggcg ttgacggccg tactctggtc accaaaaaca gcttccgttt cctgaacacc 1080 ctgtacacca tggggccgtc tccggagccg aacatcacca ttctgtggtc tgaaaaactg 1140 ccgctgagct tcaaaaaata cgccgcgaaa gtgtccatcg atacctcttc tctgcagtac 1200 gagaacgatg acctgatgcg tcctgacttc aacaacgatg actacgctat cgcttgctgc 1260 gtaagcccga tggttgttgg taagcaaatg cagttcttcg gcgcgcgtgc taacctggcg 1320 aaaaccatgc tgtacgcaat caacggcggc gttgatgaaa aactgaaaat gcaggttggt 1380 cctaaatctg aaccgatcaa aggcgacgtt ctgaacttcg acgaagtgat ggaccgcatg 1440 gatcacttca tggactggct ggctaaacag tacgtcactg cgctgaacat catccactac 1500 atgcacgaca agtacagcta cgaagcttcc ctgatggcgc tgcacgaccg tgatgttatc 1560 cgcaccatgg catgtggtat cgcaggtctt tccgttgcgg ctgactccct gtctgcaatc 1620 aaatatgcga aagttaaacc gattcgtgac gaaaacggtc tggctgtcga cttcgaaatc 1680 gaaggcgaat acccgcagtt tggtaacaac gactctcgcg tcgatgatat ggccgttgac 1740 ctggttgaac gtttcatgaa gaaaattcag aaactgcaca cctaccgcaa cgctatcccg 1800 actcagtccg ttctgaccat cacctctaac gttgtgtatg gtaagaaaac cggcaacacc 1860 cctgacggtc gtcgcgctgg cgctccgttc ggaccaggtg ctaacccgat gcacggccgt 1920 gaccagaaag gcgctgttgc ctctctgacc tccgttgcaa aactgccgtt tgcttacgcg 1980 aaagatggta tttcttacac cttctctatc gtgccgaacg cgctgggtaa agacgacgaa 2040 gttcgtaaaa ctaacctcgc cggcctgatg gatggttact tccaccacga agcgtccatc 2100 gaaggcggtc agcatctgaa cgtcaacgtt atgaaccgcg aaatgctgct cgacgcgatg 2160 gaaaacccgg aaaaatatcc gcagctgacc atccgcgtat ccggctacgc agtacgtttt 2220 aactccctga ctaaagaaca gcagcaggac gttattactc gtaccttcac tcagaccatg 2280 taa 2283 <210> 10 <211> 635 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 10 gggtcaactg gcgaaaaact ggctcaacgt ctatgttggt aacctgattg gttgcttact 60 gtttgtattg ctgatgtggc tttcaggcga atatatgact gccaacggtc aatggggact 120 taacgttctg caaaccgccg accacaaaat gcaccatact tttgttgaag ccgtgtgcct 180 gggtatcctg gcaaacctga tggtctgcct tgcggtatgg atgagttact ccggccgtag 240 cctgatggat aaagccatga ttatggtttt accggtggca atgtttgttg ccagcgggtt 300 tgagcacagt atcgcgaaca tgtttatgat cccgctgggt atcgttatcc gcgactttgc 360 aagcccggaa ttctggaccg cagttggttc aactccggaa agtttctctc acctgaccgt 420 catgaacttc atcactgata acctgattcc ggtaactatc gggaacatca tcggcggtgg 480 tctgctggtt gggttgacat actgggtcat ttacctgcgt ggcgacgacc atcactaagg 540 gttgtttcag gcagtaaata aaaaatccac ttaagaaggt aggtgttaca tgtccgagct 600 taatgaaaag ttacagcagc aggacgttat tactc 635 <210> 11 <211> 39 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 11 atcggatccg ggtcaactgg cgaaaaactg gctcaacgt 39 <210> 12 <211> 46 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 12 ggtaataac gtcctgctgc tgtaactttt cattaagctc ggacat 46 <210> 13 <211> 669 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 13 atgtccgagc ttaatgaaaa gttacagcag caggacgtta ttactcgtac cttcactcag 60 accatgtaat ggtattgact gaaatcgtac agtaaaaagc gtacaataaa ggctccacgc 120 aagtggggcc tttttagcaa tatcatcctg ccccagtctc ttttgtctgc tgtctatact 180 ttatggataa cagccaaaac agactcgaca tagcctttga gctgtgcatc tacataggcc 240 ccggatgggc caaattcgga gatatcaccg caatgtcaac aattggtcgc attcactcct 300 ttgaatcctg tggcaccgtc gatggcccgg ggattcgctt tatcaccttc ttccagggct 360 gcctgatgcg ctgcctctat tgccacaacc gcgatacctg ggatacccac ggcggcaaag 420 agattaccgt tgaagagctg atgaaagagg tggtgaccta tcgccacttt atgaacgctt 480 ccggcggcgg cgtgacggca tccggcggcg aggctatcct gcaggccgaa tttgttcgcg 540 actggttccg cgcctgtaag aaagaaggta ttcatacctg tctcgatacc aacggctttg 600 tgcgccgcta cgatccggtt attgatgaac tgctggaggt caccgacctg gtgatgctcg 660 atctcaagc 669 <210> 14 <211> 46 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 14 atgtccgagc ttaatgaaaa gttacagcag caggacgtta ttactc 46 <210> 15 <211> 41 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 15 actgcggccg cgcttgagat cgagcatcac caggtcggtg a 41 <210> 16 <211> 1258 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 16 gggtcaactg gcgaaaaact ggctcaacgt ctatgttggt aacctgattg gttgcttact 60 gtttgtattg ctgatgtggc tttcaggcga atatatgact gccaacggtc aatggggact 120 taacgttctg caaaccgccg accacaaaat gcaccatact tttgttgaag ccgtgtgcct 180 gggtatcctg gcaaacctga tggtctgcct tgcggtatgg atgagttact ccggccgtag 240 cctgatggat aaagccatga ttatggtttt accggtggca atgtttgttg ccagcgggtt 300 tgagcacagt atcgcgaaca tgtttatgat cccgctgggt atcgttatcc gcgactttgc 360 aagcccggaa ttctggaccg cagttggttc aactccggaa agtttctctc acctgaccgt 420 catgaacttc atcactgata acctgattcc ggtaactatc gggaacatca tcggcggtgg 480 tctgctggtt gggttgacat actgggtcat ttacctgcgt ggcgacgacc atcactaagg 540 gttgtttcag gcagtaaata aaaaatccac ttaagaaggt aggtgttaca tgtccgagct 600 taatgaaaag ttacagcagc aggacgttat tactcgtacc ttcactcaga ccatgtaatg 660 gtattgactg aaatcgtaca gtaaaaagcg tacaataaag gctccacgca agtggggcct 720 ttttagcaat atcatcctgc cccagtctct tttgtctgct gtctatactt tatggataac 780 agccaaaaca gactcgacat agcctttgag ctgtgcatct acataggccc cggatgggcc 840 aaattcggag atatcaccgc aatgtcaaca attggtcgca ttcactcctt tgaatcctgt 900 ggcaccgtcg atggcccggg gattcgcttt atcaccttct tccagggctg cctgatgcgc 960 tgcctctatt gccacaaccg cgatacctgg gatacccacg gcggcaaaga gattaccgtt 1020 gaagagctga tgaaagaggt ggtgacctat cgccacttta tgaacgcttc cggcggcggc 1080 gtgacggcat ccggcggcga ggctatcctg caggccgaat ttgttcgcga ctggttccgc 1140 gcctgtaaga aagaaggtat tcatacctgt ctcgatacca acggctttgt gcgccgctac 1200 gatccggtta ttgatgaact gctggaggtc accgacctgg tgatgctcga tctcaagc 1258 <210> 17 <211> 780 <212> DNA <213> Klebsiella oxytoca <400> 17 atgaaccatt ctgctgaatg ctcttgtgaa gagagcctgt gtgaaactct acgaggattt 60 tccgcgcaac atcccgatag cgtcatctac cagacctctc tgatgagcgc gctgttaagc 120 ggcgtttatg aaggtaatac gaccatcgcc gatttgctca cccacggcga tttcggcctc 180 gggaccttta atgaactgga cggcgagctg atcgcgttta gcagcgaagt ttaccagctg 240 cgcgccgacg gcagcgcccg caaagcccgt atggaacagc gtacgccgtt cgcggtgatg 300 acctggtttc agccgcagta tcgcaaaacg ttcgataaac cggtcagccg tgaacaactg 360 cacgacatca tcgaccagca aattccgtcg gacaatctgt tctgcgccct gcgtattaac 420 ggccattttc gccacgccca tacccgcacg gtaccgcgcc agacgccgcc ataccgggcg 480 atgaccgacg tactcgacga ccagccggtt tttcgcttca accagcgcga gggtgtcctg 540 gttgggttca gaacgccgca gcatatgcag ggcattaacg tggccggcta ccatgaacac 600 ttcatcaccg atgaccgcca gggcggcggc catctgctcg attatcagct cgaccacggc 660 gttctgacct ttggcgagat ccacaaattg atgattgacc ttcctgccga tagcgccttc 720 ctgcaggcgg atctgcatcc agacaatctt gatgccgcca ttcgctcagt cgaaaactaa 780                                                                          780 <210> 18 <211> 1680 <212> DNA <213> Klebsiella oxytoca <400> 18 gtggataatc aacatcaacc gcgccagtgg gcgcacggcg ccgacctcat cgtcagccag 60 cttgaggccc agggggtacg ccaggtgttc ggcattccgg gggccaaaat cgataaagtc 120 ttcgattcgc tgctcgactc ctccattcgc attatcccgg tgcgccacga agccaacgcc 180 gcctttatgg ccgccgcggt tggccgcatc accggcaaag cgggcgtcgc gctggtcacg 240 tccggaccgg gctgctctaa cctgattacc gggatggcaa cggccaatag cgaaggcgac 300 ccggtggtgg cgctgggcgg cgcggtaaaa cgcgccgaca aggccaaaca ggtgcaccag 360 agtatggata cggtgacgat gtttagcccg gtgaccaaat actcggtgga agtcaccgcg 420 ccggaagcgc tggcggaagt tgtctccaac gcgtttcgtg cagccgagca gggacgcccc 480 ggcagcgcct tcgtcagcct gccgcaggac gtggtcgacg ggccggtcca cgccagggtt 540 ctgcccgccg gcgatgcgcc gcagaccggc gcggcgccgg acgacgccat tgcgcgagtc 600 gcgaagatga ttgccggcgc gaaaaatccg atatttctgc tcggcctgat ggccagccag 660 acggaaaaca gcgcggcgct gcgcgaattg ctgaaaaaaa gtcatatccc ggtgaccagc 720 acctatcagg ccgccggcgc agtcaatcag gatcacttta cccgcttcgc cggacgggtt 780 ggtctgttca acaaccaggc aggggatcga ctcctgcatc tcgccgacct ggtcatctgc 840 atcggctata gtccggtgga gtacgagccg gccatgtgga ataacggtaa cgccacgctg 900 gtacatatcg acgtgctgcc cgcttacgaa gagcgtaatt ataccccgga cgtcgagctg 960 gtcggcaata tcgccgccac cctgaacaaa ctgtctcaac gcatcgacca ccagctggtg 1020 ctctcgccgc aggccgccga gatccttgtc gaccgccagc atcagcggga gctcctcgac 1080 cgccgcggtg cgcacctgaa ccagttcgcg cttcatccgc tgcgcatcgt tcgcgccatg 1140 caggacatcg tcaatagcga tgtcaccctg accgtcgata tggggagctt tcatatctgg 1200 atcgcccgct atctctacag ctttcgcgcc cgtcaggtca tgatttccaa cggtcaacag 1260 accatgggcg tggcgctgcc gtgggcgatt ggcgcctggc tggtcaatcc gcagcgcaaa 1320 gtggtttccg tttccggcga cggcggtttc ctgcagtcca gcatggagct ggagaccgct 1380 gtacggctga aagcgaacgt cctgcatatc atctgggtcg ataacggcta caacatggtg 1440 gcgattcagg aggagaaaaa ataccagcgg ctctccggcg ttgagttcgg cccggtggat 1500 tttaaagtct acgccgaagc cttcggcgcc aaagggtttg cggtagagag cgccgaagcc 1560 cttgagccga cgctgcgggc ggcgatggac gtcgacggcc ccgccgtcgt agccatcccc 1620 gtggattacc gcgataaccc gctgctgatg ggccagctcc atctcagtca actactttga 1680                                                                         1680 <210> 19 <211> 771 <212> DNA <213> Klebsiella oxytoca <400> 19 atgaaaaaag tcgcactcgt caccggcgcg ggccagggta tcggtaaagc tatcgccctt 60 cgtctggtga aagatggttt tgccgtggct atcgccgatt ataacgacgc caccgcgcag 120 gcggtcgctg atgaaattaa ccgcagcggc ggccgggcgc tagcggtgaa ggtggatgtg 180 tctcaacgcg atcaggtttt tgccgccgtc gaacaggcgc gcaagggtct cggcggtttt 240 gacgtgatcg tcaacaacgc cggggttgcg ccctccacac caatcgaaga gattcgcgag 300 gaggtgatcg ataaagtcta caatatcaac gttaaaggcg ttatctgggg catccaggcc 360 gcggtagagg cgtttaaaaa agagggccac ggcggcaaaa ttatcaacgc ctgctcccag 420 gcgggccatg taggtaaccc ggagctggcg gtctatagct ccagtaaatt tgccgtgcgc 480 ggcctgacgc aaaccgccgc ccgcgatctg gcgcatctgg ggattaccgt aaacggctac 540 tgcccgggga tcgtcaaaac cccaatgtgg gcggaaattg accgccaggt ttccgaagcg 600 gcgggtaaac cgctgggcta cggaacccag gagttcgcca aacgcattac ccttgggcgg 660 ctatccgagc cggaagacgt cgcagcctgc gtctcttatc tcgccggtcc ggactccaat 720 tatatgaccg gccaatcgct gctgatcgat ggcggcatgg tatttaacta a 771 <210> 20 <211> 873 <212> DNA <213> Klebsiella oxytoca <400> 20 atggaacttc gttatctgcg ctacttcgtc gccgtcgccg aggcgcggca cttcactaaa 60 gcggcaaaag aacttggtat ctcccagcca ccgctaagtc agcaaattca acggcttgaa 120 agagaaattg gcacaccgct tttccgccga ttaacgcggg gcgtcgagct gacggaagcg 180 ggggagtcgt tttatgagga tgcttgccag attctggcgt taagcgacgc ggcgctggaa 240 aaaaccaagg gaattgcccg cgggctgaac ggtcggctct cgctgggcat taccagctct 300 gatgcttttc atccgcaaat ctttagcctg ttacagcagt ttcagcagag ttatccgggg 360 gtgacgttgc atcaggtcga aggcaatatg gcgacgctga tgggggcgct tggcgaagcg 420 gagctggata ttgcttttgt acgcctgccc tgcgagagca gtaaagcgtt caatctgcgg 480 attatcgatc aagaaccgat ggtgattgcc ctggcgcgtt cgcacccgct atcgtcccac 540 cgcgagctga cgctggagca actgacggac gtggcgccga ttcttttccc gcgtgaagtg 600 gcccctggcc tgtatgagct ggtgttcaac agctatctgc gcgccggtgt tgacgtgcag 660 cgagcctatc agtcatcgca aatatcatcg tcgctcagca tggtggaggc tggcttcggc 720 tttgcgctgg tgccgcagtc gatgacctgc atcacgctcc ccaacgtgag ctatcacccg 780 ctgatgggta caccgctgaa aaccgatatc gctatcgcct ggcggcgcta cgagcgctcg 840 cgcacggtga agcgttttct ggccatgttt taa 873

Claims (16)

In the 2,3-butanediol producing microorganism,
A pathway for converting pyruvate to alpha-acetolactate, a pathway for converting alpha-acetolactate to acetone, or a pathway for converting acetone to 2,3-butanediol,
Recombinant microorganism for producing 2,3-butanediol.
The method according to claim 1,
Wherein the activity of acetolactate synthase is increased to promote the pathway for converting pyruvate to alpha-acetolactate.
The method according to claim 1,
Wherein the pathway for converting pyruvate to alpha-acetolactate is promoted by increasing the expression of budB, a gene encoding acetolactate synthase.
The method according to claim 1,
Wherein the activity of acetolactate dicarboxylic silage is increased so that a pathway for converting alpha-acetolactate to acetone is promoted.
The method according to claim 1,
Wherein the pathway for converting acetolactate to acetone is promoted by increasing the expression of budA, a gene encoding acetolactate dicarboxylic silage.
The method according to claim 1,
Wherein the pathway for converting acetone to 2,3-butanediol is promoted by increasing the activity of the acetyl reductase.
The method according to claim 1,
Wherein the expression of budC which is a gene encoding an acetylin reductase is increased so that a pathway for converting acetone to 2,3-butanediol is promoted.
The method according to claim 1,
Wherein the pathway for converting pyruvate to acetyl koide, the pathway for converting pyruvate to formic acid, or the pathway for converting pyruvate to lactate is inhibited.
9. The method of claim 8,
Wherein the pathway for converting pyruvate into acetyl koi or the pathway for converting pyruvate to formic acid is inhibited by suppressing pyruvate-formate lyase.
9. The method of claim 8,
Wherein the pathway for converting pyruvate to lactate is inhibited by inhibiting lactate dehydrogenase.
9. The method of claim 8,
Wherein at least one of pflB, which is a gene encoding pyruvate-formate lyase, and ldhA, which is a gene encoding lactate dehydrogenase, is deleted or suppressed.
The method according to claim 1,
Wherein the recombinant microorganism is Klebsielline.
The method according to claim 1,
Wherein the recombinant microorganism is Klebsiella oxytocina.
The method according to claim 1,
Wherein the recombinant microorganism is characterized in that the productivity of 2,3-butanediol is higher than that of lactate.
The method according to claim 1,
The expression of two or more genes selected from the group consisting of budB, which is a gene encoding acetolactate synthase, budA, which is a gene encoding acetolactate dicarboxylic silage, and budC, which is a gene that codes acetone reductase, Wherein the recombinant microorganism is a recombinant microorganism.
15. A method for producing a recombinant microorganism, which comprises: inoculating the recombinant microorganism of any one of claims 1 to 15;
And culturing the recombinant microorganism.

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