KR102642882B1 - Recombinant microorganisms enhanced ability of producing acetoin and method for preparing acetoin using the same - Google Patents

Abstract

The present invention relates to a recombinant microorganism with enhanced acetoin production ability and a method for producing acetoin using the same. It was confirmed that the recombinant microorganism of the present invention can maintain a high production yield of acetoin even under continuous aerobic conditions through metabolic engineering methods, and that the production cost of acetoin can be effectively reduced by culturing it in minimal medium. It can be used as a core technology for

Description

Recombinant microorganisms with enhanced ability to produce acetoin and method for producing acetoin using the same {Recombinant microorganisms enhanced ability of producing acetoin and method for preparing acetoin using the same}

The present invention relates to a recombinant microorganism with enhanced acetoin production ability and a method for producing acetoin using the same.

Global warming gases and waste resulting from the use of fossil fuels are causing a serious environmental crisis for humanity. Accordingly, there is a need to develop a new environmentally friendly biological process using biomass as a raw material that can replace chemical industry, that is, minimize the production of waste and energy consumption harmful to humanity. Interest in bioethanol, biodiesel, and biogas, widely known as bioenergy, is increasing, and all of the mentioned types of bioenergy are used for power generation or transportation fuel, but due to some shortcomings in the production method, new renewable energy Interest in hydrocarbon-type compounds as energy resources is increasing. Accordingly, interest in recombinant strains that can produce acetoin, a platform compound with various industrial values, as a metabolite is increasing in many industries such as food, pharmaceuticals, and cosmetics.

First, the recombinant strains studied so far have been cultured in complex media to produce acetoin. However, the above complex medium contains Yeast extract. It contains ingredients such as corn steep liquor, and these ingredients account for more than 50% of the total cost of the culture medium, resulting in an increase in production costs. In addition, the components of various complex media cause additional problems in the process of purifying acetoin after culturing microorganisms, which is an obstacle to actual commercialization.

Meanwhile, a strain that can produce acetoin as a metabolite Enterobacteriaceae has two metabolic pathways that decompose glucose as a carbon source: the PTS metabolic pathway, which converts it to 6-Phospho-glucose through the Phosphotransferase system (PTS), and the Non-PTS metabolic pathway, which converts it to gluconate using glucose dehydrogenase. There is (see Figure 1). The strain allows glucose to enter the cell through the PTS metabolic pathway and undergoes glycolysis to produce acetoin by Acetolactate synthase and Acetolactate decarboxylase. The acetoin produced is further metabolized by Acetoin reductase. Because 2,3-butanediol is produced, the production yield of acetoin is greatly reduced. In addition, in the process of cultivating the above strain, aerobic conditions must be maintained to increase acetoin production efficiency, but in Enterobacteriaceae , if aerobic conditions continue, glucose is decomposed through the non-PTS metabolic pathway, not the PTS cycle. As 2-Ketogluconate accumulates, acetoin production efficiency drastically decreases. Therefore, for mass production of acetoin, it is necessary to develop a recombinant strain that blocks additional metabolism in the PTS metabolic pathway and maintains high production efficiency of acetoin even under continuous aerobic conditions.

Against this background, research on new recombinant strains that can improve the production efficiency of acetoin and reduce production costs is being actively conducted, but is still insufficient.

Korean Patent Publication No. 2015-0076410

The present invention was created to solve the above problems, and the present inventors not only suppressed the additional metabolism of acetoin in the PTS (Phosphotransferase system) metabolic pathway, but also prevented the Non-PTS metabolic pathway from being activated even under continuous aerobic conditions. For the first time, a recombinant microorganism was identified that did not contain any recombinant microorganisms, and through this, it was confirmed that the production yield of acetoin, a metabolite, could be significantly improved, and based on this, the present invention was completed.

Accordingly, the object of the present invention is a microorganism having 2,3-butandiol and lactate biosynthetic pathways, including lactate dehydrogenase and acetoin reductase. The aim is to provide a recombinant microorganism, a bacterium of the Enterobacteriaceae family, whose activity is weakened or inactivated and whose acetoin production ability is enhanced.

In addition, another object of the present invention is to provide a recombinant microorganism with an enhanced acetoin production ability in which the activity of glucose dehydrogenase is additionally weakened or inactivated.

In addition, another object of the present invention is, (a) culturing the recombinant microorganism in a medium containing a carbon source; and (b) recovering acetoin from the medium in which the recombinant microorganism was cultured.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

In order to achieve the object of the present invention as described above, the present invention is a microorganism having a 2,3-butandiol and lactate biosynthetic pathway, lactate dehydrogenase and acetoin. Provided is a recombinant microorganism, a bacterium of the Enterobacteriaceae family, in which the activity of acetoin reductase is weakened or inactivated and the acetoin production ability is enhanced.

In one embodiment of the present invention, the microorganism has ldhA encoding lactate dehydrogenase. The budC gene, which encodes acetoin reductase, may be deleted.

In another embodiment of the present invention, the activity of glucose dehydrogenase may be further weakened or inactivated in the microorganism, and the gcd gene encoding glucose dehydrogenase may be additionally deleted. there is.

As another embodiment of the present invention, the bacteria may be of the genus Enterobacter or Klebsiella , preferably Enterobacter aerogenes. aerogenes ).

The present invention includes the steps of (a) culturing the recombinant microorganism in a medium containing a carbon source; and (b) recovering acetoin from the medium in which the recombinant microorganism was cultured.

In one embodiment of the present invention, the bacterium is Enterobacter It may be the genus or Krebsiella, and preferably, it may be Enterobacter aerogenes.

In another embodiment of the present invention, the culture medium contains Na ₂ HPO ₄ , KH ₂ PO ₄ , NaCl, NH ₄ Cl, MgSO ₄ , and CaCl ₂ It may be the minimum badge.

As another embodiment of the present invention, the culture may be carried out under aerobic conditions.

The recombinant microorganism according to the present invention can not only significantly improve the production yield of acetoin, a metabolite, even under continuous aerobic conditions through metabolic engineering methods, but also can be cultured in a minimal medium, unlike the conventional technology in which it is cultured in a complex medium. By doing so, the production cost of acetoin can be effectively reduced, and it is expected that it can be used as a core technology for acetoin production.

Figure 1 is a diagram showing the metabolic pathways blocked in the PTS glucose decomposition cycle and the non-PTS glucose decomposition cycle in the recombinant strain of the present invention.
Figure 2 is a diagram briefly showing the lambda red recombination technology (λ-red-recombination) used in the present invention.
Figure 3 is a comparison of the production yield of acetoin in the acetoin production method using the recombinant strain of the present invention, where A is the control strain, B is the recombinant strain I, and C is acetoin produced from the recombinant strain II. This is a result showing the production yield of.
Figure 4, (a) Serratia marcescens H32 Complex medium for culture, (b) for culturing Paenibacillus polymyxa CS107J This is a result showing the composition and production cost of the complex medium and (c) the M9 minimal medium of the present invention.
Figure 5 compares the concentration of 2,3-butanediol produced in the acetoin production method using the recombinant strain of the present invention, where A is the control strain, B is the recombinant strain I, and C is the recombinant strain II. This result shows the concentration of 2,3-butanediol produced.
Figure 6 compares the concentration of 2-ketogluconate produced in the acetoin production method using the recombinant strain of the present invention, where A is the control strain, B is the recombinant strain I, and C is the recombinant strain II. This result shows the concentration of 2-ketogluconate produced.

Hereinafter, the present invention will be described in detail.

The present invention is a microorganism having a 2,3-butandiol and lactate biosynthetic pathway, and the activities of lactate dehydrogenase and acetoin reductase are weakened or Provided is a recombinant microorganism, a bacterium of the Enterobacteriaceae family , which is inactivated and has an enhanced acetoin production ability.

In addition, the present invention provides a recombinant microorganism with an enhanced acetoin production ability in which the activity of glucose dehydrogenase is additionally weakened or inactivated.

In the present invention, “Acetoin” is a precursor of 2,3-butanediol, also called 3-Hydroxybutanone or acetyl methyl carbinol, and is a phosphotransferase system (PTS). ) is one of the metabolites of the metabolic pathway. Recently, various recombinant strains have been developed for mass production of acetoin, but they are experiencing difficulties due to the uneconomical use of the culture medium and the non-PTS metabolic pathway activated under aerobic conditions.

The terms “lactic acid dehydrogenase” and “acetoin reductase” used in the present invention are enzymes involved in the Phosphotransferase system (PTS) metabolic pathway. The lactate dehydrogenase has the amino acid sequence of SEQ ID NO: 1 or the base of SEQ ID NO: 2. By sequence, the acetoin reductase may consist of the amino acid sequence of SEQ ID NO: 3 or the base sequence of SEQ ID NO: 4, and each contains at least 70%, preferably at least 80%, of the amino acid or base sequence of SEQ ID NOs: 1 to 4. , more preferably 90% or more, and most preferably 95% or more sequence homology. As shown in Figure 1, they catalyze the reaction of converting pyruvic acid to lactic acid and converting acetoin to 2,3-butanediol, respectively. That is, during the metabolism of glucose, by-products lactic acid and 2,3-butanediol are produced by lactate dehydrogenase and acetoin reductase, which acts as a factor in reducing the production yield of acetoin. There is a need to weaken or inactivate their activity.

The term “glucose dehydrogenase” used in the present invention is an enzyme involved in the Non-PTS metabolic pathway that is activated under aerobic conditions. The glucose dehydrogenase may consist of the amino acid sequence of SEQ ID NO: 5 or the base sequence of SEQ ID NO: 6. and a base sequence having sequence homology of at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% with the amino acid of SEQ ID NO: 5 and the base sequence of SEQ ID NO: 6, respectively. may include. As shown in Figure 1, it catalyzes the reaction that converts glucose to gluco-1,5-lactone. In other words, the production yield of acetoin can be further improved under aerobic conditions, but if these aerobic conditions continue, glucose is broken down through the non-PTS metabolic pathway, not the PTS cycle, and 2-Ketogluconate accumulates, leading to the production of acetoin. Efficiency decreases sharply. Therefore, in order to improve the production yield of acetoin, it is necessary to block the Non-PTS metabolic pathway by weakening or inactivating the activity of the enzyme.

In the present invention, the “weakening of enzyme activity” means a decrease in the activity of the enzyme compared to the activity of the enzyme in the original state or before modification of the microorganism. The weakening occurs when the activity of the enzyme itself is reduced compared to the activity of the enzyme originally possessed by the microorganism due to mutation of the gene encoding the enzyme, or when the activity of the enzyme encoding the enzyme is inhibited or translation is inhibited within the cell. This concept includes cases where the overall degree of enzyme activity is lower than that of the natural type or strain before modification.

In addition, “inactivation” refers to a case where the expression of the gene encoding the enzyme is not expressed at all compared to the natural strain or the strain before modification, and even when expressed, there is no activity.

Weakening or inactivating this enzyme activity can be achieved by applying various methods well known in the art. Examples of the method include a method of replacing a gene encoding the enzyme on a chromosome with a gene mutated to reduce the activity of the enzyme, including when the activity of the enzyme is removed; A method of introducing a mutation into the expression control sequence of a gene on a chromosome encoding the enzyme; A method of replacing the expression control sequence of the gene encoding the enzyme with a sequence with weak or no activity; A method of deleting all or part of the gene on the chromosome encoding the enzyme; A method of introducing an antisense oligonucleotide (eg, antisense RNA) that binds complementary to the transcript of the gene on the chromosome and inhibits translation from the mRNA to the enzyme; A method of artificially adding a sequence complementary to the SD sequence in front of the SD sequence of the gene encoding the enzyme to form a secondary structure to make attachment of ribosomes impossible, and a method of making the attachment of ribosomes impossible, and the method of making the attachment of the ribosome impossible There is a reverse transcription engineering (RTE) method that adds a promoter to the 3' end to enable reverse transcription.

Specifically, ldhA, encoding lactate dehydrogenase. The activity of each enzyme can be weakened or inactivated by deleting all or part of the gene, the budC gene encoding acetoin reductase, or the gcd gene encoding glucose dehydrogenase, but is not limited thereto.

In the present invention, the bacteria may be of the Enterobacter genus or Klebsiella genus, preferably Enterobacter aerogenes , but 2,3-butanediol (2, Any microorganism having a 3-butandiol) and lactate biosynthetic pathway may be included without limitation.

In one embodiment of the present invention, ldhA and budC are formed through Lambda Red recombination technology. Recombinant strains Ⅰ and ldhA , budC , and gcd with genes deleted Recombinant strain II with the gene removed was prepared, and it was confirmed that the production yield of acetoin can be significantly increased through this, and the production cost of acetoin can be effectively reduced by culturing it in minimal medium (see Example 1) ).

In addition, it was confirmed that by producing acetoin using the above recombinant strain, the production of 2,3-butanediol, a by-product of the PTS metabolic pathway, and 2-ketogluconate, a by-product of the Non-PTS metabolic pathway, could be reduced ( See Example 2).

Accordingly, the present invention includes the steps of (a) culturing the recombinant microorganism in a medium containing a carbon source; and (b) recovering acetoin from the medium in which the recombinant microorganism was cultured.

In the present invention, the bacteria may be of the Enterobacter genus or Klebsiella genus, preferably Enterobacter aerogenes , but 2,3-butanediol (2, Any microorganism having a 3-butandiol) and lactate biosynthetic pathway may be included without limitation.

The medium used in the method of the present invention contains Na ₂ HPO ₄ , KH ₂ PO ₄ , NaCl, NH ₄ Cl, MgSO ₄ , and CaCl ₂ As a minimal medium, preferably 5-7 g/l of Na ₂ HPO ₄ , 2-4 g/l of KH ₂ PO ₄ , 0.3-0.7 g/l of NaCl, 0.5-1.5 g/l of NH ₄ Cl, 0.39- It may consist of 0.59 g/l of MgSO ₄ and 0.004 to 0.0024 g/l of CaCl ₂ , but is not limited thereto.

Carbon sources used in the medium used in the method of the present invention include common carbohydrates that can be added to the medium as carbon sources, preferably sucrose, lactose, maltose, trehalose, turanose, cellobiose, and raffinose. , melechytose, malothriose, acarbose, stachyose, glucose, amylose, cellulose, fructose, anose, or galactose, but is not limited thereto.

In addition, during the culture of the present invention, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid can be added to the culture in an appropriate manner to adjust the pH of the culture, and to maintain the aerobic state of the culture. Oxygen or oxygen-containing gas can also be injected into the water.

Below, preferred embodiments are presented to aid understanding of the present invention. However, the following examples are provided only to make the present invention easier to understand, and the content of the present invention is not limited by the following examples.

Manufacturing example One. Acetoin production capacity Preparation of recombinant strains

In this example, Enterobacter aerogenes (KCTC 2190), in which the lactate dehydrogenase gene ( ldhA ) has been deleted, was used as the parent strain, and a recombinant strain with improved acetoin production ability was obtained through lambda red recombination technology (λ-red-recombination), a metabolic engineering method. A strain was prepared. That is, from the parent strain, recombinant strain I (△ ldhA , △ budC ) in which the pathway for converting acetoin to 2,3-butandiol is blocked by removing the Acetoin reductase gene (budC), Recombinant strain II (△ldhA, △budC, △gcd) was prepared by deleting not only the acetoin reductase gene but also the glucose dehydrogenase gene ( gcd ) , thereby blocking the Non-PTS glucose decomposition pathway activated under aerobic conditions.

For the expression of λ-red-recombinase enzyme and Flippase enzyme, pRedET plasmid and 707FLP plasmid from Gene bridge GmbH were used, respectively, and cassette DNA containing kanamycin sequence was prepared using pKD4 plasmid as a template.

Specifically, the pRedET plasmid was introduced into the parent strain through electroporation (1800V), and then cultured in a medium supplemented with tetracycline at 30°C. In addition, using the pKD4 plasmid containing the FRT-kanamycin-FRT cassette as a template, primers containing base pairs (homologous region, 50bp) homologous to the surrounding regions of the target gene to be removed were prepared, and then primers were prepared with a Tm value of 45. Cassette DNA was prepared by performing PCR according to the GS taq polymerase protocol, and sequence information for the primers is shown in Table 1 below.

The parent strain into which pRedET was introduced was inoculated into LB medium supplemented with tetracycline, and when the OD600 reached about 0.2, 20% L-arabinose (0.9ml) was added to express Lambda Red recombinase. Then, the cells were cultured again until the OD600 reached 0.5 to prepare cells in a competent state. Afterwards, the cells were diluted with a 10% glycerol solution, transformed by introducing the prepared cassette DNA (100ng) through electroporation (1800V), cultured in LB medium for 1 hour, and then plated with kanamycin antibiotics. was screened.

Afterwards, colony PCR was performed to confirm the introduction of the cassette DNA in the transformed strain. First, the colonies screened with the kanamycin antibiotic plate were diluted with 10 μl of DW and then heated at 100°C for 5 minutes to obtain genomic DNA. Afterwards, PCR was performed according to the GS taq polymerase protocol using colony PCR primers using the diluted colonies (1 ㎕) as a template, and the PCR result was electrophoresed to confirm the introduction of cassette DNA. Sequence information for the primers is shown in Table 2 below.

Afterwards, the 707FLP plasmid was introduced again through electroporation (1800V), cultured in LB medium at 30°C, and then cultured O/N at 37°C to remove all cassette DNA and plasmid, thereby extracting the target gene from the parent strain. budC or gcd were removed, respectively.

Example One. Acetoin production capacity Check the enhancement effect

In the process of producing acetoin using recombinant strains I and II prepared in Preparation Example 1, the production yield of acetoin, the target product, was evaluated.

To this end, glucose, the sole carbon source, was added to the M9 minimal medium, and then each of the above recombinant strains was cultured in flask batches for 24 hours under aerobic conditions. As a control, a recombinant strain in which only the lactate dehydrogenase gene ( ldhA ) was deleted was used.

As a result, as shown in Figure 3, while the production yield of acetoin in the control group was very low at 0.02 g/g, it was confirmed that the production yield of acetoin was greatly increased in both methods using recombinant strains I and II, especially , recombinant strain II showed the highest production yield at 0.38 g/g.

Meanwhile, in the production method of acetoin using existing microorganisms, Serratia marcescens CS107J , or Paenibacillus polymyxa H32 is known as the producing strain, As these culture media, complex media are used. However, as shown in FIGS. 4A and 4B, the complex media contain ingredients such as corn steep powder or yeast extract, and these ingredients account for more than 50% of the total in terms of cost. In addition, these have the potential to incur additional costs during the purification process of acetoin, which is a major factor in increasing the unit cost of acetoin production.

On the other hand, the M9 minimal medium in which the recombinant strain of the present invention is cultured is a medium that does not contain any ingredients such as corn steep powder or yeast extract, as shown in Figure 4c, and is cheaper than the complex medium in terms of cost. It is about 10 times cheaper, or about 25 times cheaper, and acetoin can be easily separated and purified from the culture medium, making it possible to lower the production cost of acetoin.

Considering the above results, the method for producing acetoin using the recombinant strain of the present invention can reduce production costs by culturing it in minimal medium, and can block metabolic pathways, especially Non-PTS, which is activated under aerobic conditions. It can be seen that the production yield of acetoin can be maximized by blocking the target path.

Example 2. Confirm the effect of reducing by-product production

In the same manner as Example 1, in the process of producing acetoin using recombinant strains I and II prepared in Preparation Example 1, 2,3-butandiol by the PTS glucose decomposition pathway, and The concentration of 2-ketogluconate by the non-PTS glucose decomposition pathway activated under aerobic conditions was measured. As a control, a recombinant strain in which only the lactate dehydrogenase gene ( ldhA ) was deleted was used.

As a result, as shown in Figure 5, the concentration of 2,3-butanediol produced in the control group was about 8.87 g/l, while the concentration of 2,3-butanediol was significantly reduced in both methods using recombinant strains I and II. was able to confirm. Similar to the above results, as shown in Figure 6, the concentration of 2-ketogluconate produced in the control group was about 4 g/l, while the concentration of 2-ketogluconate was significantly reduced in recombinant strains I and II. In particular, in recombinant strain II, in which not only the acetoin reductase gene but also the glucose dehydrogenase gene ( gcd ) was deleted, 2-ketogluconate was not detected at all.

From the above results, it can be seen that the acetoin production method using the recombinant strain of the present invention can improve the production yield of the target product, acetoin, by minimizing the production of by-products during the production process.

The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive.

<110> KOREA UNIVERSITY RESEARCH AND BUSINESS FOUNDATION <120> Recombinant microorganisms enhanced ability of producing acetoin and method for preparing acetoin using the same <130> DP-2016-0024_P16U13C0037 <160> 15 <170> KoPatentIn 3.0 <210> 1 <211 > 329 <212> PRT <213> Artificial Sequence <220> <223> lactate dehydrogenase_amino acid <400> 1 Met Lys Ile Ala Val Tyr Ser Thr Lys Gln Tyr Asp Lys Lys Tyr Leu 1 5 10 15 Gln His Val Asn Asp Ala Tyr Gly Phe Glu Leu Glu Phe Phe Asp Phe 20 25 30 Leu Leu Thr Glu Lys Thr Ala Lys Thr Ala Asn Gly Cys Glu Ala Val 35 40 45 Cys Ile Phe Val Asn Asp Asp Gly Ser Arg Pro Val Leu Glu Glu Leu 50 55 60 Lys Ala His Gly Val Lys Tyr Ile Ala Leu Arg Cys Ala Gly Phe Asn 65 70 75 80 Asn Val Asp Leu Glu Ala Ala Lys Glu Leu Gly Leu Arg Val Val Arg 85 90 95 Val Pro Ala Tyr Ser Pro Glu Ala Val Ala Glu His Ala Ile Gly Met 100 105 110 Met Met Ser Leu Asn Arg Arg Ile His Arg Ala Tyr Gln Arg Thr Arg 115 120 125 Asp Ala Asn Phe Ser Leu Glu Gly Leu Thr Gly Phe Thr Met Tyr Gly 130 135 140 Lys Thr Ala Gly Val Ile Gly Thr Gly Lys Ile Gly Val Ala Thr Leu 145 150 155 160 Arg Ile Leu Lys Gly Phe Gly Met Arg Leu Leu Ala Phe Asp Pro Tyr 165 170 175 Pro Ser Ala Ala Ala Leu Asp Leu Gly Val Glu Tyr Val Asp Leu Pro 180 185 190 Thr Leu Tyr Ala Gln Ser Asp Val Ile Ser Leu His Cys Pro Leu Thr 195 200 205 Asn Glu Asn Tyr His Leu Leu Asn Gln Ala Ala Phe Asp Gln Met Lys 210 215 220 Asp Gly Val Met Val Ile Asn Thr Ser Arg Gly Ala Leu Ile Asp Ser 225 230 235 240 Gln Ala Ala Ile Asp Ala Leu Lys His Gln Lys Ile Gly Ala Leu Gly 245 250 255 Met Asp Val Tyr Glu Asn Glu Arg Asp Leu Phe Phe Glu Asp Lys Ser 260 265 270 Asn Asp Val Ile Gln Asp Asp Val Phe Arg Arg Leu Ser Ala Cys His 275 280 285 Asn Val Leu Phe Thr Gly His Gln Ala Phe Leu Thr Ala Glu Ala Leu 290 295 300 Ile Gly Ile Ser Glu Thr Thr Leu Gly Asn Leu Gln Gln Val Ala Lys 305 310 315 320 Gly Glu Thr Cys Pro Asn Ala Leu Val 325 <210> 2 <211> 990 <212> DNA <213> Artificial Sequence <220> <223> lactate dehydrogenase_nucleotide <400> 2 atgaaaatcg ctgtttatag taccaagcag tacgataaaa agtatctgca gcatgttaac 60 gacgcatacg gctttgaact ggaatttttc gatttcctgc tgaccgaaaa gaccgcgaaa 120 acggccaacg gctgtgaagc ggtatgtatc ttcgttaatg acgacggtag ccgcccggtg 180 ctggaagagc ta aaagccca cggcgtgaaa tatatcgcgc tgcgctgcgc cggctttaac 240 aacgtcgatc ttgaggcggc taaagagctg ggcctgcgcg tcgtgcgcgt cccggcctac 300 tcgccggaag ccgttgctga acacgccatc ggtatgatga tgtcgttgaa ccgtcgcatt 360 catcgcgcct atcagcgtac ccgcgatgcc aacttctcgc tggaagggct gaccggcttc 420 acgatgtacg gtaaaaccgc aggggtgatc ggcaccggta aaatcggcgt tgcgacgctg 480 cggatcctca aaggtttcgg tatgcgcctg ctggcgtttg atccctatcc gagcgcggcg 540 gcgctggatc tcggcgttga atatgtcgac ctgccgacgc tgtacgcgca gtccgacgtc 600 at ctccctgc actgcccgct taccaacgaa aactatcacc tgctcaacca ggcggcattc 660 gatcagatga aagacggcgt gatggtcatt aataccagcc gcggcgcgct tatcgattca 720 caagcggcta tcgacgcgct gaagcatcag aaaatcggcg cgctgggaat ggacgtgtat 780 gaaaatgaac gcgatctgtt ctttgaagat aaatcgaatg atgtcatcca ggatgacgtg 840 ttccgccggc tctccgcctg ccacaacgtc ctgtttaccg ggcaccaggc attcctgacg 900 gctgaggcgc tgatcggtat ttccgagaca acgcttggca atctgcagca ggtagctaag 960 ggcgaaacct gcccgaacgc gctggtctaa 990 <210> 3 <211> 256 <212> PRT <213> Artificial Sequence <220> < 223> acetoin reductase_amino acid <400> 3 Met Lys Lys Val Ala Leu Val Thr Gly Ala Gly Gln Gly Ile Gly Lys 1 5 10 15 Ala Ile Ala Leu Arg Leu Val Lys Asp Gly Phe Ala Val Ala Ile Ala 20 25 30 Asp Tyr Asn Asp Val Thr Ala Lys Ala Val Ala Asp Glu Ile Asn Gln 35 40 45 His Gly Gly Arg Ala Ile Ala Val Lys Val Asp Val Ser Asp Arg Glu 50 55 60 Gln Val Phe Ala Ala Val Glu Gln Ala Arg Lys Thr Leu Gly Gly Phe 65 70 75 80 Asn Val Ile Val Asn Asn Ala Gly Val Ala Pro Ser Thr Pro Ile Glu 85 90 95 Ser Ile Thr Pro Glu Ile Val Asp Lys Val Tyr Asn Ile Asn Val Lys 100 105 110 Gly Val Ile Trp Gly Ile Gln Ala Ala Val Glu Ala Phe Lys Lys Glu 115 120 125 Gly His Gly Gly Lys Ile Ile Asn Ala Cys Ser Gln Ala Gly His Val 130 135 140 Gly Asn Pro Glu Leu Ala Val Tyr Ser Ser Ser Lys Phe Ala Val Arg 145 150 155 160 Gly Leu Thr Gln Thr Ala Ala Arg Asp Leu Ala Pro Leu Gly Ile Thr 165 170 175 Val Asn Gly Tyr Cys Pro Gly Ile Val Lys Thr Pro Met Trp Ala Glu 180 185 190 Ile Asp Arg Gln Val Ser Glu Ala Ala Gly Lys Pro Leu Gly Tyr Gly 195 200 205 Thr Ala Glu Phe Ala Lys Arg Ile Thr Leu Gly Arg Leu Ser Glu Pro 210 215 220 Glu Asp Val Ala Ala Cys Val Ser Tyr Leu Ala Ser Pro Asp Ser Asp 225 230 235 240 Tyr Met Thr Gly Gln Ser Leu Leu Ile Asp Gly Gly Met Val Phe Asn 245 250 255 <210> 4 <211> 711 <212> DNA <213> Artificial Sequence <220> <223> acetoin reductase_nucleotide <400> 4 cgcctcgtga aggacggttt tgccgtggcg atcgccgatt acaatgacgt cacagcgaaa 60 gccgtggcgg atgaaatca a ccagcacggc ggccgggcaa tcgcggtcaa agtcgatgtt 120 tccgaccgtg agcaggtgtt tgccgccgtc gaacaggcgc gaaaaacgct gggcggattc 180 aacgtcatcg tcaataacgc cggggtcgcg ccatcaacgc ctatcgaatc cattacgccg 240 gagattgtcg acaaggtcta caacatcaac gttaaagggg tgatctgggg gattcaggcg 300 gcagtcgagg cctttaaaaa agaggggcac ggcggcaaaa tcatcaacgc ctgttcgcag 360 gccggacacg tcggcaaccc ggaactggcg gtctacagct cgagcaaatt cgccgtacgc 420 ggtttaacgc aaaccgccgc tcgcgacctg gcgccgctgg gtattaccgt taacggctac 480 tgcccgggga ttgtgaaaac gccgatgtgg gccgagat cg atcgtcaggt atccgaagcg 540 gcgggtaaac ctctgggcta cgggacagcc gaattcgcca aacgcatcac cctcggccgc 600 ctgtctgagc cagaagatgt cgccgcctgc gtctcttatc tcgccagccc ggattccgat 660 tatatgaccg gtcaatcgct gctgatcgat ggcgggatgg tattcaatta a 711 <210> 5 <211> 796 <212> PRT <213> Artificial Sequence <220 > <223> glucose dehydrogenase_amino acid <400> 5 Met Ala Glu Thr Lys Ser Gln Gln Ser Arg Leu Leu Val Thr Leu Thr 1 5 10 15 Ala Leu Phe Ala Ala Phe Cys Gly Leu Tyr Leu Leu Ile Gly Gly Val 20 25 30 Trp Leu Ala Ala Ile Gly Gly Ser Trp Tyr Tyr Pro Ile Ala Gly Leu 35 40 45 Val Met Leu Ala Val Thr Val Met Leu Phe Arg Gly Lys Arg Ala Ala 50 55 60 Leu Trp Leu Tyr Ala Ala Leu Leu Leu Ala Thr Met Ile Trp Gly Val 65 70 75 80 Trp Glu Val Gly Phe Asp Phe Trp Ala Leu Thr Pro Arg Ser Asp Ile 85 90 95 Leu Val Phe Phe Gly Ile Trp Leu Ile Leu Pro Phe Val Trp Arg Arg 100 105 110 Leu Pro Val Pro Ser Ala Gly Ala Val Gly Gly Leu Val Ile Ala Leu 115 120 125 Leu Ile Ser Gly Gly Ile Leu Thr Trp Ala Gly Phe Asn Asp Pro Gln 130 135 140 Glu Val Asn Gly Thr Leu Ser Ala Asp Ala Thr Pro Ala Ala Pro Ile 145 150 155 160 Ser Thr Val Ala Asp Ser Asp Trp Pro Ala Tyr Gly Arg Asn Gln Glu 165 170 175 Gly Gln Arg Tyr Ser Pro Leu Lys Gln Ile Asn Thr Asp Asn Val Lys 180 185 190 Asn Leu Lys Glu Ala Trp Val Phe Arg Thr Gly Asp Leu Lys Gln Pro 195 200 205 Asn Asp Pro Gly Glu Ile Thr Asn Glu Val Thr Pro Ile Lys Val Gly 210 215 220 Asp Met Leu Tyr Leu Cys Thr Ala His Gln Arg Leu Phe Ala Leu Asp 225 230 235 240 Ala Ala Thr Gly Lys Glu Lys Trp His Phe Asp Pro Gln Leu Asn Ala 245 250 255 Asp Pro Ser Phe Gln His Val Thr Cys Arg Gly Val Ser Tyr His Glu 260 265 270 Ala Lys Ala Asp Asn Ala Pro Ala Asp Val Val Ala Asp Cys Pro Arg 275 280 285 Arg Ile Ile Leu Pro Val Asn Asp Gly Arg Leu Phe Ala Val Asn Ala 290 295 300 Asp Asn Gly Lys Leu Cys Glu Thr Phe Ala Asn Lys Gly Ile Leu Asn 305 310 315 320 Leu Gln Thr Asn Met Pro Val Thr Thr Pro Gly Met Tyr Glu Pro Thr 325 330 335 Ser Pro Pro Ile Ile Thr Asp Lys Thr Ile Val Ile Ala Gly Ala Val 340 345 350 Thr Asp Asn Phe Ser Thr Arg Glu Pro Ser Gly Val Ile Arg Gly Phe 355 360 365 Asp Val Asn Thr Gly Lys Leu Leu Trp Ala Phe Asp Pro Gly Ala Lys 370 375 380 Asp Pro Asn Ala Ile Pro Ser Asp Glu His His Phe Thr Leu Asn Ser 385 390 395 400 Pro Asn Ser Trp Ala Pro Ala Ala Tyr Asp Ala Lys Leu Asp Leu Val 405 410 415 Tyr Leu Pro Met Gly Val Thr Thr Pro Asp Ile Trp Gly Gly Asn Arg 420 425 430 Thr Pro Glu Gln Glu Arg Tyr Ala Ser Ser Ile Val Ala Leu Asn Ala 435 440 445 Thr Thr Gly Lys Leu Ala Trp Ser Tyr Gln Thr Val His His Asp Leu 450 455 460 Trp Asp Met Asp Met Pro Ser Gln Pro Thr Leu Ala Asp Ile Asp Val 465 470 475 480 Asn Gly Lys Thr Val Pro Val Ile Tyr Ala Pro Ala Lys Thr Gly Asn 485 490 495 Ile Phe Val Leu Asp Arg Arg Asn Gly Glu Leu Val Val Pro Ala Pro 500 505 510 Glu Lys Pro Val Pro Gln Gly Ala Ala Lys Gly Asp Tyr Val Ala Lys 515 520 525 Thr Gln Pro Phe Ser Glu Leu Ser Phe Arg Pro Lys Lys Asp Leu Thr 530 535 540 Gly Ala Asp Met Trp Gly Ala Thr Met Phe Asp Gln Leu Val Cys Arg 545 550 555 560 Val Ile Phe His Gln Met Arg Tyr Glu Gly Ile Phe Thr Pro Pro Ser 565 570 575 Glu Gln Gly Thr Leu Val Phe Pro Gly Asn Leu Gly Met Phe Glu Trp 580 585 590 Gly Gly Ile Ser Val Asp Pro Asn Arg Gln Val Ala Ile Ala Asn Pro 595 600 605 Met Ala Leu Pro Phe Val Ser Lys Leu Ile Pro Arg Gly Pro Gly Asn 610 615 620 Pro Met Glu Pro Pro Lys Asp Ala Lys Gly Ser Gly Thr Glu Ser Gly 625 630 635 640 Val Gln Pro Gln Tyr Gly Val Pro Phe Gly Val Thr Leu Asn Pro Phe 645 650 655 Leu Ser Pro Phe Gly Leu Pro Cys Lys Gln Pro Ala Trp Gly Tyr Ile 660 665 670 Ser Ala Leu Asp Leu Lys Thr Asn Glu Val Val Trp Lys Lys Arg Ile 675 680 685 Gly Thr Pro Gln Asp Ser Leu Pro Phe Pro Leu Pro Val Pro Leu Pro 690 695 700 Phe Asn Met Gly Met Pro Met Leu Gly Gly Pro Ile Ser Thr Ala Gly 705 710 715 720 Asn Val Leu Phe Ile Ala Ala Thr Ala Asp Asn Tyr Leu Arg Ala Tyr 725 730 735 Asn Met Ser Asn Gly Glu Lys Leu Trp Gln Ala Arg Leu Pro Ala Gly 740 745 750 Gly Gln Ala Thr Pro Met Thr Tyr Glu Val Asn Gly Lys Gln Tyr Val 755 760 765 Val Ile Ser Ala Gly Gly His Gly Ser Phe Gly Thr Lys Met Gly Asp 770 775 780 Tyr Ile Val Ala Tyr Ala Leu Pro Asp Asp Ala Lys 785 790 795 <210> 6 <211> 2391 <212> DNA <213> Artificial Sequence <220> <223> glucose dehydrogenase_nucleotide <400> 6 atggcagaaa caaaatctca acaatcacgg ttacttgtca cgctgacggc gctgtttgcc 60 gccttctgtg gcctgtatct gttaatcggt ggggtatggc tggccgctat tggcggttcc 120 tggtactacc ctatcgcagg cctggtgatg ctggccgtca ccgttatgct gttccgcggc 180 aagcgcgctg cgctgtggct gtacgccgcc ctgctgctgg caaccatgat ttggggggta 240 tgggaagtcg gtttcgactt ctgggcgctg acgccgcgta gcgacatcct ggtcttcttc 300 ggcatctggc tgattctgcc atttgtctgg cgtcgtctg c cggtcccttc cgccggtgcc 360 gttggcggtc tggttatcgc cctgctgatt agcggcggga tcctgacctg ggccggtttc 420 aacgatccgc aggaagtgaa cggtacgctg agcgctgacg cgacgccggc tgcgccgatt 480 tctaccgtcg ccgatag cga ctggccggct tatggccgca accaggaagg ccagcgttat 540 tcaccgctga agcaaattaa taccgataac gtgaagaacc tgaaggaagc ctgggtattc 600 cgcaccggcg acctgaagca gccgaacgat ccgggtgaaa tcactaacga agtgacgccg 660 attaaagttg gcgacatgct gtatctgtgt accgcgcacc agcgtctgtt cgcgcttgac 720 gcggccaccg gtaaagagaa gtggcatttt gacccgcagc tgaacg ccga tccgtcgttc 780 cagcacgtga cctgccgtgg cgtctcctat catgaagcca aagcagataa cgcgcctgcc 840 gacgtcgtcg ccgactgccc gcgccgtatt attctgccgg tcaacgatgg ccgtctgttc 900 gcggtgaacg ccgacaacgg taagctgt gc gaaacctttg ccaacaaagg cattctcaac 960 ctgcaaacca atatgccggt aaccacgccg ggtatgtatg aaccgacgtc gccgccgatt 1020 atcaccgata aaactatcgt cattgccggc gcggtaaccg ataacttctc aacccgcgag 1080 ccatcaggcg tgatccgcgg ctttgatgtg aataccggta aactgctgtg ggccttcgac 1140 ccgggtgcga aagaccctaa tgcgatcccg agcgatgagc atcacttcac actcaattca 1200 cctaactcct gggcgcctgc cgcctatgac gcgaagctgg atttagtcta tctgccgatg 1260 ggcgttacca cgccggatat ctggggcggc aatcgcacgc cggaacagga acgttacgcc 1320 agctctatcg tagcgctgaa cgcaaccacc gggaaactgg catggagcta ccaaaccgta 1380 caccacgatc tgtgggatat ggatatgccg tcgcagccga cgctggccga cattgatgtg 1440 aacggtaaaa ccgtaccggt gatttacgct ccggccaaaa ccggcaacat cttcgtgctg 1500 gatcgccgta atggcgagct ggtggtcccg gcgccggaaa aaccggttcc gcaaggtgcg 1560 gcgaaaggcg attatgttgc taaaacccag ccgttctccg agctgagctt ccgtccgaag 1 620 aaagacctga ccggcgcaga tatgtggggc gccaccatgt ttgaccaact ggtgtgccgc 1680 gttatcttcc atcagatgcg ctatgaaggt atcttcactc cgccgtctga acagggcacg 1740 ctggtcttcc cggggaacct ggggatgttt gaatggggcg gtatct ccgt cgatccaaac 1800 cgtcaggtgg ctatcgccaa cccgatggcg ctgccgttcg tctctaaact gatcccacgc 1860 ggcccgggca acccgatgga accgccaaaa gacgcgaaag gctctggtac cgagtccggc 1920 gttcagccgc agtatggcgt cccgttcggc gtcacgctga acccgttcct gtcgccgttt 1980 ggcctgccgt gtaaacaacc ggcatggggt tatatctcgg cgctggatct gaagaccaac 2040 gaagt ggtgt ggaaaaaacg catcggtacg ccgcaggata gtctgccgtt cccgctgcct 2100 gttccactgc cattcaatat gggtatgccg atgctgggcg ggccgatctc aaccgccggt 2160 aacgtgctgt ttatcgccgc caccgccgat aactacctgc gcgcttacaa catgag taat 2220 ggtgaaaaac tgtggcaggc gcgtctgcct gcaggtggcc aggcgacgcc gatgacctat 2280 gaagtgaatg gcaagcagta cgttgtcatc tctgccggcg gtcatggctc gttcggtact 2340 aaaatgggcg actacatcgt tgcctatgcg ttaccggatg acgcgaaata a 2391 <210> 7 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> budC_Forward <400> 7 ccagctacac ctgagt caaa ttctttaagt catcacaaaa ggaaatggaa gtgtaggctg 60 gagctgcttc 70 <210> 8 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> budC_Reverse <400> 8 aaagcccctg cgaacaggca ggggcaaacc atgtcagagc ttatttttta gtccatatga 60 atatcctcct 70 <210> 9 <211> 70 <212> DNA <21 3> Artificial Sequence <220> <223> gcd_Forward <400> 9 tcctataatt aattgcagtt aaaaaattaa caactgaaga gtgtctttct gtgtaggctg 60 gagctgcttc 70 <210> 10 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> gcd_Reverse < 400> 10 aggggaggggg gcaaacaaaa aaacggcaac tttcgttgcc gttttgcgtt gtccatatga 60 atatcctcct 70 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> budC_Colony_Forward <400> 11 aaagcctatg ccgaatcctt 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> budC_Colony_Reverse <400> 12 cttctccggg ctaccaaatc 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> gcd_Colony_Forward <400> 13 atttcactgc cg ctgtcttt 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> gcd_Colony_Reverse <400> 14 ggtgagtgaa gtgctggtga 20 <210> 15 <211> 1580 <212> DNA <213> Artificial Sequence < 220> <223> cassette_nucleotide <400> 15 tcctataatt aattgcagtt aaaaaattaa caactgaaga gtgtctttct gtgtaggctg 60 gagctgcttc gaagttccta tactttctag agaataggaa cttcggaata ggaacttcaa 120 gatcccctca cgctgccgca agcactcagg g cgcaagggc tgctaaagga agcggaacac 180 gtagaaagcc agtccgcaga aacggtgctg accccggatg aatgtcagct actgggctat 240 ctggacaagg gaaaacgcaa gcgcaaagag aaagcaggta gcttgcagtg ggcttacatg 300 gcgatagcta gactgggcgg ttttatggac agcaagcgaa ccggaattgc cagctggggc 360 gccctctggt aaggttggga agccctgcaa agtaaactgg atggctttct tgccgccaag 420 gatctgatgg cgcaggggat caagatctga tcaagagaca ggatgaggat cgtttcgcat 48 0 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg 540 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc 600 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaact gca 660 ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct 720 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga 780 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg 840 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat 900 cgagc gagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga 960 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg 1020 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaaat gg 1080 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat 1140 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct 1200 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga 1260 cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg 13 20 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt 1380 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc 1440 caccccagct tcaaaagcgc tctgaagttc ctatactttc tagagaatag ga acttcgga 1500 ataggaacta aggaggatat tcatatggac aacgcaaaac ggcaacgaaa gttgccgttt 1560ttttgtttgc cccctcccct 1580

Claims (11)

In microorganisms with 2,3-butandiol and lactate biosynthetic pathways,
The microorganism has the ldhA gene encoding lactate dehydrogenase, the budC gene encoding acetoin reductase, and the gcd gene encoding glucose dehydrogenase deleted, or
Characterized in that it is a bacterium of the Enterobacteriaceae family in which the activity of lactate dehydrogenase, acetoin reductase, and glucose dehydrogenase expressed from each of the above genes is weakened or inactivated and the acetoin production ability is enhanced. Recombinant microorganisms.
delete delete delete According to paragraph 1,
The bacterium is a recombinant microorganism, characterized in that it is a bacterium of the genus Enterobacter or Klebsiella .
According to paragraph 1,
The bacteria are Enterobacter aerogenes ( Enterobacter aerogenes ), a recombinant microorganism.
(a) culturing the recombinant microorganism of any one of claims 1 and 5 to 6 in a medium containing a carbon source; and
(b) A method for producing acetoin, comprising the step of recovering acetoin from the medium in which the recombinant microorganism was cultured.
In clause 7,
A manufacturing method, characterized in that the bacteria are bacteria of the genus Enterobacter or Krebsiella.
In clause 7,
A manufacturing method, characterized in that the bacteria are Enterobacter aerogenes.
In clause 7,
The culture medium contains Na ₂ HPO ₄ , KH ₂ PO ₄ , NaCl, NH ₄ Cl, MgSO ₄ , and CaCl ₂ A manufacturing method, characterized in that it is a minimal medium.
In clause 7,
A production method, characterized in that the culture is carried out under aerobic conditions.