KR20130058236A - Methods for preparing meso-2,3-butanediol - Google Patents

Abstract

PURPOSE: A method for preparing meso-2,3-butandiol from acetoin using a recombinant microorganism containing a novel acetoin reductase is provided to obtain a large amount of compounds for a platform. CONSTITUTION: Acetoin reductase has an amino acid sequence of sequence number 2. A nucleic acid molecule encodes acetoin reductase and has a nucleotide sequence of sequence number 1. A vector contains the nucleic acid molecule. A microorganism is transformed by the nucleic acid molecule. The transformed microorganism is transformed E.coli. A method for preparing 2,3-butanediol comprises a step of culturing the transformed microorganism.

Description

Method for Preparing meso-2,3-butanediol {Methods for Preparing meso-2,3-butanediol}

The present invention relates to a meso-2,3-butanediol-producing heterologous E. coli and a preparation method.

The use of fossil fuels has led to the massive production of global warming gases and wastes, causing a serious environmental crisis for humanity. There is a need to develop new environmentally friendly biological processes that use biomass as a raw material that can replace the chemical industry produced from fossil fuels, that is, minimize the production and energy consumption of wastes that are harmful to mankind. Interest in bioethanol, biodiesel, biogas and butanol, represented by bioenergy, is increasing, but all of the mentioned types of bioenergy can be used as fuels for power generation or transportation, but some disadvantages of performance and production methods As a result, there is a growing interest in compounds in the form of hydrocarbons, a new renewable energy source. Accordingly, interest in recombinant strains that can produce meso -2,3-butanediol, a compound for platforms with various industrial values, as a metabolite is also increasing.

Klebsiella oxytoca is a pneumococcal bacterium that is gram-negative, motility, and lactose fermented conditionally anaerobic strain. Oxytoca is a Klebsiella genus of Enterobacteriaceae similar to Klebsiella pneumonia , but with indole-negative, melezitose and 3-hydroxy It is distinguished by its inability to grow in hydroxybutyrate media. It is mainly found in soil and about 30% has nitrogen fixing ability under anaerobic conditions. Klebsiella oxytoca causes disease.

Also, in general, Klebsiella oxytoca ) biosynthesizes a mixture of three isomers of meso -2,3-butanediol from monosaccharides. Isomers of meso -2,3-butanediol include three isomers of the form (R, R) -, meso- , and (S, S) -. The biosynthesis ratio of these isomers can be seen to change very rapidly by the strain, gene, and culture environment of the strain.

meso -2,3-butanediol is a chemical used in the synthesis of solvents, anti-freeze solutions and plasticizers. Chemical conversion, butadiene (1,3-butadiene) used for the manufacture of synthetic rubber, methyl ethyl ketone (MEK), a liquid fuel additive, acetoin, diacetyl, used as a food additive fragrance (diacetyl) and precursors of polyurethanes are possible.

Meso -2,3-butanediol can be produced by biological methods, developed on a commercial scale during World War II, and has recently gained attention with the development of industrial biotechnology. Meso -2,3-butanediol is produced through mixed acid fermentation metabolic pathways and shows various production quantities depending on strains and carbon sources. When glucose is used as a carbon source, meso -2,3-butanediol is produced through pyruvate and acetoin or through a butanediol cycle through diacetyl. Meso -2,3-butanediol has a selective pathway to produce.

Attempts and efforts have been made to identify strains for the production of meso -2,3-butanediol by microorganisms, to improve the prepared strains and to efficiently convert them. In particular, Krebsiella spp. Is known to have high production of meso -2,3-butanediol.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors endeavored to develop a recombinant microorganism capable of producing meso-2,3-butanediol to produce a hydrocarbon type compound which is a renewable energy resource that can minimize the production of hazardous waste and energy consumption. As a result, it was confirmed that meso-2,3-butanediol can be produced by introducing a novel acetoin reductase gene of Klebsiella oxytoca into E. coli, which is useful as an industrial strain. The present invention has been completed.

An object of the present invention is to provide a novel acetoin reductase (acetoin reductase).

Another object of the present invention is to provide a nucleic acid molecule encoding acetoin reductase.

Still another object of the present invention is to provide a vector containing a nucleic acid molecule.

Another object of the present invention is to provide a transforming microorganism.

Another object of the present invention to provide a method for producing 2,3-butanediol comprising culturing the transformed microorganism.

Another object of the present invention to provide a method for producing 2,3-butanediol comprising culturing transformed Escherichia coli.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides an acetoin reductase having an amino acid sequence set forth in SEQ ID NO: 2 sequence.

The present inventors endeavored to develop a recombinant microorganism capable of producing meso-2,3-butanediol to produce a hydrocarbon type compound which is a renewable energy resource that can minimize the production of hazardous waste and energy consumption. As a result, it was confirmed that meso-2,3-butanediol can be produced by introducing a novel acetoin reductase gene of Klebsiella oxytoca into E. coli, which is useful as an industrial strain.

As used herein, the term “acetoin reductase” refers to an enzyme that synthesizes acetoin into butanediol through the following scheme.

[Reaction Scheme]

Preferably, it is a nucleic acid molecule encoding acetoin reductase having an amino acid sequence described in SEQ ID NO: 2, more preferably, the nucleic acid molecule has a nucleotide sequence described in SEQ ID NO: 1 .

As used herein, the term “nucleic acid molecule” is meant to encompass DNA (gDNA and cDNA) and RNA molecules inclusively, and the nucleotides, which are the basic structural units in nucleic acid molecules, are modified from sugar or base sites as well as natural nucleotides. It also includes analogues (Scheit, Nucleotide Analogs , John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90: 543-584 (1990).

Variations in nucleotides do not cause changes in the protein. Such nucleic acids include functionally equivalent codons or codons that encode the same amino acid (eg, by degeneracy of the codon, there are six codons for arginine or serine), or codons that encode biologically equivalent amino acids. And nucleic acid molecules.

In addition, mutations in nucleotides may result in changes in acetoin reductase itself. Even in the case of a mutation that causes a change in the amino acid of acetoin reductase, it can be obtained that exhibits almost the same activity as the acetoin reductase of the present invention.

It will be apparent to those skilled in the art that biological equivalents that may be included in the acetoin reductase of the present invention will be limited to variations in amino acid sequences that exert biological activity equivalent to the acetoin reductase of the present invention.

Such amino acid variations are made based on the relative similarity of the amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, and the like. By analysis of the size, shape and type of amino acid side chain substituents, arginine, lysine and histidine are both positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have a similar shape. Thus, based on these considerations, arginine, lysine and histidine; Alanine, glycine and serine; And phenylalanine, tryptophan and tyrosine are biologically functional equivalents.

In introducing mutations, hydropathic idex of amino acids may be considered. Each amino acid is assigned a hydrophobicity index according to its hydrophobicity and charge: isoleucine (+4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine / cysteine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); Histidine (-3.2); Glutamate (-3.5); Glutamine (-3.5); Aspartate (-3.5); Asparagine (-3.5); Lysine (-3.9); And arginine (-4.5).

The hydrophobic amino acid index is very important in imparting the interactive biological function of proteins. It is known that substitution with an amino acid having a similar hydrophobicity index can retain similar biological activities. When a mutation is introduced with reference to a hydrophobic index, substitution is made between amino acids showing a hydrophobic index difference preferably within ± 2, more preferably within ± 1, even more preferably within ± 0.5.

On the other hand, it is also well known that the substitution between amino acids having similar hydrophilicity values leads to proteins with homogeneous biological activity. As disclosed in US Pat. No. 4,554,101, the following hydrophilicity values are assigned to each amino acid residue: arginine (+3.0); Lysine (+3.0); Aspartate (+ 3.0 ± 1); Glutamate (+ 3.0 ± 1); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (-0.4); Proline (-0.5 ± 1); Alanine (-0.5); Histidine (-0.5); Cysteine (-1.0); Methionine (-1.3); Valine (-1.5); Leucine (-1.8); Isoleucine (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5); Tryptophan (-3.4). When a mutation is introduced with reference to the hydrophilicity value, the amino acid is substituted preferably within ± 2, more preferably within ± 1, even more preferably within ± 0.5.

Amino acid exchange in proteins that do not globally alter the activity of the molecule is known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges are amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thr / Phe, Ala / Exchange between Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, Asp / Gly.

Considering the above-described variations with biologically equivalent activity, the acetoin reductase or nucleic acid molecule encoding the same of the present invention is also interpreted to include sequences that exhibit substantial identity with the sequences listed in the Sequence Listing. Such substantial identity may, for example, be at least 99% when the sequences of the invention are aligned with the maximal correspondence of any of the sequences of the present invention, and the aligned sequences are analyzed using algorithms commonly used in the art. It means a sequence showing homology of. Alignment methods for sequence comparison are well known in the art. Various methods and algorithms for alignment are described by Smith and Waterman, Adv . Appl . Math . 2: 482 (1981) ; Needleman and Wunsch, J. Mol . Bio . 48: 443 (1970); Pearson and Lipman, Methods in Mol . Biol . 24: 307-31 (1988); Higgins and Sharp, Gene 73: 237-44 (1988); Higgins and Sharp, CABIOS 5: 151-3 (1989); Corpet et al., Nuc . Acids Res. 16: 10881-90 (1988); Huang et al., Comp . Appl . BioSci . 8: 155-65 (1992) and Pearson et al., Meth. Mol . Biol . 24: 307-31 (1994). NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol . Biol . 215: 403-10 (1990)) is accessible from the National Center for Biological Information (NCBI) and the like, and is available on the Internet in blastp, blastm, It can be used in conjunction with sequencing programs such as blastx, tblastn and tblastx. BLSAT is available at http://www.ncbi.nlm.nih.gov/BLAST/. A method for comparing sequence homology using this program can be found at http://www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

According to another aspect of the invention, the invention is a vector comprising acetoin reductase having an amino acid sequence described in SEQ ID NO: 2 or a nucleic acid molecule having a nucleotide sequence described in SEQ ID NO: 1.

The vector system of the present invention can be constructed through various methods known in the art, and specific methods thereof are described in Sambrook et al., Molecular . Cloning , A Laboratory Manual , Cold Spring Harbor Laboratory Press (2001), which is incorporated herein by reference.

The vector of the present invention can typically be constructed as a vector for cloning or as a vector for expression. In addition, the vector of the present invention can be constructed using a prokaryotic cell as a host. The vector of the present invention can typically be constructed as a vector for cloning or as a vector for expression.

For example, when the vector of the present invention is an expression vector and the prokaryotic cell is used as a host, a strong promoter (for example, a tac promoter, lac) capable of promoting transcription can be obtained. Promoter, lac UV5 promoter, lpp promoter, p _L ^λ promoter, p _R ^λ promoter, rac 5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.), ribosomal binding site for initiation of detoxification And transcription / detox termination sequences. If E. coli is used as the host cell, E. coli Promoter and operator sites of the tryptophan biosynthetic pathway (Yanofsky, C., J. Bacteriol . , 158: 1018-1024 (1984)) and the leftward promoter of phage λ (p _L ^λ promoter, Herskowitz, I. and Hagen, D., . Ann Rev Genet, 14:. . 399-445 (1980)) may be used as a control region.

On the other hand, vectors that can be used in the present invention are plasmids (eg, pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pUC19, pET, etc.), phages (eg, λgt4λB, λ-Charon, λΔz1 which are often used in the art). And M13, etc.) or viruses (eg, SV40, etc.).

On the other hand, the vector of the present invention includes, as a selection marker, an antibiotic resistance gene commonly used in the art, for example, ampicillin, gentamicin, carbinicillin, chloramphenicol, streptomycin, kanamycin, And resistance genes for tetracycline.

In the vector of the present invention, the acetoin reductase gene has a minimum length including an enzyme overexpression function and inserts only the base sequence including the necessary portion for RBS (Ribosomal binding site) and the enzyme expression as a sequence and inserts the host cell. In terms of reducing the metabolic burden of the present invention, and according to a preferred embodiment of the present invention, the present invention was used to improve the pUC18 vector.

According to another aspect of the invention, the invention is a microorganism transformed by acetoin reductase having an amino acid sequence described in SEQ ID NO: 2 or a nucleic acid molecule having a nucleotide sequence described in SEQ ID NO: 1.

A host cell capable of continuously cloning and expressing the vector of the present invention in a stable manner can be any host cell known in the art, and examples include E. coli JM109, E. coli Bacillus sp. Strains such as BL21 (DE3), E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, and Salmonella typhimurium Enterobacteria and strains such as Um, Serratia Marcesons and various Pseudomonas species.

The method of carrying the vector of the present invention into a host cell is performed by the CaCl ₂ method (Cohen, SN et al., Proc . Natl . Acac . Sci . USA , 9: 2110-2114 (1973)), one method (Cohen, SN et al., Proc . Natl . Acac . Sci . USA , 9: 2110-2114 (1973); and Hanahan, D., J. Mol. Biol . , 166: 557-580 (1983)) and electroporation methods (Dower, WJ et al., Nucleic . Acids Res . , 16: 6127-6145 (1988)) and the like.

According to a preferred embodiment of the present invention, the present invention produced a transformed microorganism using the electroporation method.

Vectors injected into a host cell can be expressed in the host cell, in which case a large amount of acetoin reductase is obtained. For example, when the expression vector contains a lac promoter, the host cell may be treated with IPTG to induce gene expression.

As a microorganism which can be transformed by an acetoin reductase having an amino acid sequence described in SEQ ID NO: 2 of the present invention or a nucleic acid molecule having a nucleotide sequence described in SEQ ID NO: 1 of the present invention, E. coli DH5α , E. coli JM109, E. coli Bacillus sp. Strains such as BL21 (DE3), E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, and Salmonella typhimurium Enterobacteria and strains, such as Umm, Serratia Marcesons and various Pseudomonas species, are available, but are not limited to these.

Preferably, the transformed microorganism is Escherichia coli and the transformed Escherichia coli is meso -2,3-butanediol enantioselective overexpression Escherichia coli.

As used herein, the term 'enantiomer selective' refers to the selective expression of certain enantiomers of the enantiomers. According to a preferred embodiment of the invention, the transformed microorganism of the invention produces the meso -2,3-butanediol isomer.

According to another aspect of the invention, 2 comprising culturing the transformed microorganism by acetoin reductase having an amino acid sequence described in SEQ ID NO: 2 or a nucleic acid molecule having a nucleotide sequence described in SEQ ID NO: 1 Provided is a method for preparing 3-butanediol.

According to another aspect of the present invention, the method comprises culturing the transformed Escherichia coli by acetoin reductase having an amino acid sequence as set forth in SEQ ID NO: 2 or a nucleic acid molecule having a nucleotide sequence as set forth in SEQ ID NO: 1 Provided is a method for preparing 2,3-butanediol.

Preferably, the 2,3-butanediol is meso -2,3-butanediol.

Preferably, the transforming microorganism exhibits an optimal yield of 2,3-butanediol at pH 5 to pH 7 conditions, more preferably pH 5.5 to pH 6.5.

Preferably, the transforming microorganism exhibits an optimum yield of 2,3-butanediol at 35 ° C. to 45 ° C., more preferably 36 ° C. to 43 ° C.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a method for producing meso -2,3-butanediol from acetoin using a recombinant microorganism incorporating a novel acetoin reductase.

(b) Through the present invention, it was confirmed that the acetoin reductase of Krebssiella oxytoca converts acetoin into meso -2,3-butanediol and confirmed that the protein can be expressed in E. coli.

(c) The method for producing meso -2,3-butanediol according to the present invention can mass-produce a compound for a platform, which is useful for chemical industry, and is environmentally friendly and economical.

1 Bacteria Gram-negative bacteria in E. coli Krebsiela If the introduction of a gene coding for the enzyme involved in the metabolic conversion of glucose to the oxy cytokine meso -2,3- butane diol (meso -2,3-butanediol), intermediate and meso -2,3- butane A schematic showing the biosynthetic pathway of diols ( meso- 2,3-butanediol) is shown.
2 shows the structure of pUC18K, an expression vector. pUC18K Klebsiella And kanamycin resistance to the pUC18 vector, an E. coli shuttle vector It is a structure where a gene is inserted. The arrowhead (>) indicates the directionality of 5 'to 3' of the gene sequence, and the horizontal section () indicates the portion where the restriction site is located.
Figure 3 when keurep in the shuttle vector pUC18K Ella Oxytoca budC Schematic representation of the structure in which the gene is inserted. This was named pSB3. The arrowhead (>) indicates the directionality of 5 'to 3' of the gene sequence, and the horizontal section () indicates the portion where the restriction site is located.
4 Klebsiella Using restriction enzymes to insert the pSB3 oxy one cytokine as a template chromosome of acetonitrile reductase (acetoin reductase, budC) gene, shows the electrophoresis photograph confirming whether the insertion of the gene. Column 1 is the size marker, and column 2 is the result of restriction enzyme treatment of BamHI and XbaI on pSB3. The size markers (Takara, Japan) used a 500 bp DNA ladder, and when they came back from the marker shown at the bottom, the size was 500 bp, 1000 bp, 1500 bp and 2000 bp, respectively.
Figure 5 shows the SDS-PAGE gel electrophoresis confirming the expression of the protein when the pSB3 is introduced into E. coli and cultured when overexpression is induced by IPTG 0.1 mM. Column 1 is the size marker, column 2 is the wild type E. coli, and column 3 shows the expression results of the budC protein of Escherichia coli with pSB3.
6 is E. coli of the present invention :: PSB3 recombinant strain is a graph showing the production of meso -2,3-butanediol over time in acetoin-added medium.
7 is E. coli of the present invention It is a graph showing the yield of meso -2,3-butanediol in acetoin-added medium according to pH of :: pSB3 recombinant strain.
8 is E. coli of the present invention The graph shows the yield of meso -2,3-butanediol in acetoin-added medium according to the temperature of :: pSB3 recombinant strain.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example 1: budC Preparation of Expression Recombinant Plasmids

Krebsiela Oxytoka ( Klebsiella) oxytoca ) encoding the enzyme for meso -2,3-butanediol production, using the chromosome of ATCC 43863 as a template budC of cloned via gene sequence (Genbank non-register sequences, full-genome sequence analysis (Macrogen, Republic of Korea) with reference to the polymerase chain reaction using a designed primers (primer) (PCR, TAKARA, Japan) method. Table 1 budC Genes are shown and Table 2 shows primers and restriction enzymes for amplification of the budC gene.

gene Production enzyme budC Acetoin reductase

gene Primer sequence Restriction enzyme budC Forward 5'-GGA TCC ATG AAA AAA GTC GCA CTT GTT ACC-3 ' BamHⅠ Reverse 5'-TCT AGA TTA GTT AAA CAC CAT GCC GC-3 ' XbaⅠ

In order to amplify the budC gene, amplification was carried out using a primer designed specifically for the budC gene (Table 2). This budC gene was amplified to a size of 780 bp.

The polymerase chain reaction was carried out at 95 ° C./5 under conventional reaction conditions (10 mM pH 9.0 Tris-HCl, 50 mM KCI, 0.1% Trypton X-100, 2 mM MgSO ₄ and Taq DNA polymerase (TAKARA, Japan)). Once performed at min (modification), 66 ° C./1 min (annealing) and 72 ° C./1 min (extension), then 95 ° C./1 min (modification), 66 ° C./30 sec (annealing) and 72 ° C. / Thirty replicates were performed under conditions of 1 minute (kidney). As a final step, the cells were reacted at 95 ° C / 1 min (denaturation), 66 ° C / 1 min (annealing) and 72 ° C / 5 min (elongation) for stable elongation. The DNA amplified after the polymerase chain reaction was identified and purified on 0.8% agarose gel and used for T-vector cloning. Ligation was carried out on the pGEM-T easy vector (TAKARA, Japan) to prepare a recombinant plasmid pGEM-T :: budC . The recombinant plasmid was transformed in E. coli DH5α competent cells, RBS bioscience to prepare strains.

Example 2: Preparation of Recombinant Plasmid pSB3

PGEM -T :: budC which is the recombinant plasmid of Example 1 The shuttle vector of pUC18K (Fig. 2) of the E. coli and keurep when Ella at 37 ℃ constant temperature water bath (water bath) with the restriction enzymes listed in Table 2 was reacted for about 2 hours. The pUC18K used here was prepared by cloning a gene (814 bp) having kanamycin resistance to the existing pET28a vector at the NdeI restriction enzyme site of the pUC18 vector and inserting it into the pUC18 vector (Takara, Japan). Each DNA fragment was digested with the above listed restriction enzymes and then ligated at 16 ° C. using a T4 ligase (TAKARA) as shown in FIG. 3 at the multi cloning site of the pUC18K vector. Gated to complete the recombinant plasmid. In order to confirm the insertion of the inserted gene at each step, E. coli DH5α competent cells (RBS) were transformed, and recombinant plasmids were extracted and treated with restriction enzymes at the original insertion sites. A method of comparing the sizes by electrophoresis on 0.8% agarose gel was used. 4 shows the result of cleavage of the recombinant plasmid by restriction enzyme.

The amplified product was introduced into the pUC18K vector using the restriction enzymes listed in Table 2 to prepare pSB3. 3 is a diagram showing a map of the vector named pSB3 (including the gene encoding budC ).

Example  3: heterologous E. coli Transformant production

In general, strains commonly used for cloning are used to make competent cells using CaCl ₂ buffer, and then introduce plasmids into host cells by heat shock (42 ° C.). In order to increase the transformation method by electroporation (electroporation) was used.

For the electric shock transformation method, 3 ml of LB (10 g / L tryptone, 10 g / L) in 30 μl 0.1% wild type Escherichia coli ( E. coli DH5α) culture solution pre-incubated for 16 hours in a test tube When inoculated with NaCl and 5 g / L yeast extract) and the absorbance of the culture reached 0.6 at a wavelength of 600 nm, 3 ml of the medium corresponding to the whole culture was centrifuged at 12000 rpm for 1 minute to obtain a supernatant and The cells were isolated. The collected cells were washed once with 1 ml of 10% glycerol, and then centrifuged at 12000 rpm for 1 minute, and the supernatant and cells were divided. The cells were suspended in 80 μl of 10% glycerol. 1-3 μl of pSB3 was added to the suspended cells.

80 μl of the plasmid-added cells were placed in a cuvette (BIO-RAD, Gene pulser cuvette) for electroporation, and subjected to electric shock (1800) with a Gene pulser Xcell (BIO-RAD, USA). v, 25 uF, 200 Ω). 1 ml LB (10 g / L tryptone, 10 g / L NaCl and 5 g / L yeast extract) prepared in advance was added thereto, followed by shaking culture at 37 ° C. for 1 hour at 200 rpm. Cultured transformants were single in LB agar (50 μg / ml ampicillin, 50 μg / ml kanamycin, 10 g / L tryptone, 10 g / L NaCl, 5 g / L yeast extract and 20 g / L agar). The cells were incubated at 37 ° C until production. This recombinant plasmid pSB3 via developed a transformed E. coli SGJSB03 a recombinant strain in E. coli DH5α strain (Table 3).

Development strain - E. coli SGJSB03 K. oxytoca ATCC43863 :: pUC18K :: budC

Example  4: Recombination In E. coli Acetoin  Reductase ( acetoin reductase , budC A) Gene expression and protein production

E. coli SGJSB03 recombinant strains were incubated for 16 hours in LB medium containing 50 μg / ml Ampicillin and 50 μg / ml Kanamycin, and the culture was again incubated with 50 μg / ml Ampicillin and 50 μg / ml Kanamycin. Inoculated into the LB medium containing the absorbance was made to the storage solution so that the glycerol concentration is 25% when the absorbance is 0.6-1.0 at 600 nm and then stored at -80 ℃ until the culture experiment.

In order to confirm the expression of budC gene by the vector inserted into the recombinant strain developed above, SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel) to confirm the expression of budC gene of recombinant plasmid (pSB3) in Proteometec Electrophoresis) was commissioned. The experiment was divided into sample preparation and SDS, and the sample preparation process was carried out for 16 hours in 3 ml LB containing 50 µg / ml ampicillin and 50 µg / ml kanamycin in 30 ml storage solution in a 10 ml tube. Incubated for 200 ml of LB (50 μg / ml ampicillin, 50 μg / ml kanamycin, 10 g) with 1 mM IPTG (Iisopropylthio-β-D-galactoside, sigma, USA) added to a 500 ml flask. / L tryptone, 10 g / L NaCl and 5 g / L yeast extract) medium was inoculated with 0.5% of the electric culture was incubated for 12 hours at 37 ℃, 170 rpm. After incubation, the cells were separated by centrifugation, washed with PBS (Phosphate Buffered Saline) buffer, and mixed with the cell lysate in a volume of 1: 1 and sonicated. Thereafter, the supernatant was collected by centrifugation, a protease inhibition factor was added thereto, and the protein was quantified by Bradford method, and electrophoresed on SDS gel by 20 μl. The gel kit used mini PROTEAN 3 cells (BIO-RAD, USA), mobile phase was 10% SDS buffer, reaction time 30-40 minutes at 50 volts, and Coomassie Blue dye G25 was used for staining. The glass plate was washed with soap and water, then washed twice with ethanol and reacted with a spacer to run the running gel reactant (4.295 ml of distilled water, 3 ml of 40% bis / acrylamide, 2.5 ml of 0.5 M pH 6.8 tris, 10%). 0.1 ml of SDS, 0.1 ml of ammonium persulfate (APS), and 0.005 ml of Tetramethylethylenediamine (TEMED) with a final volume of 10 ml) were polymerized for 30 minutes. After inserting the comb at right angles, a small amount of stacking gel was poured at both ends of the comb to seal the gel, and the gel glass pedestal and chambers were placed by removing the comb after polymerization for 10-20 minutes. . Samples were separated by filling the SDS mobile phase above and below the chamber and applying an electrode to the gel. As a result, budC confirmed that 27 KDa protein was expressed through recombinant plasmid (FIG. 5).

Cultivation of the developed recombinant strain was incubated for 16 hours in 30 ㎖ of storage bacteria solution in 3 ㎖ LB added 50 ㎍ / ㎖ ampicillin and 50 ㎍ / ㎖ kanamycin in a 10 ㎖ tube, and again 500 ml flask 15 mM acetoin was added to 200 ml LB (50 μg / ml ampicillin, 50 μg / ml kanamycin, 10 g / L tryptone, 10 g / L NaCl and 5 g / L yeast extract) medium and 0.5% The whole culture was inoculated and incubated at 37 ° C. and 170 rpm for 54 hours. When protein absorbance reached 0.6 at 600 nm, the inducer IPTG (isopropylthio-β-D-galactoside, sigma, USA) was added to a final concentration of 0.1 mM / ml. Expression of the protein was induced.

In order to measure the concentration of meso -2,3-butanediol in the culture medium of the recombinant strain, the samples collected every hour during the culture were centrifuged at 12000 rpm for 10 minutes and the supernatant and cells were separated and stored at -40 ℃. . Each of the supernatant separated from the culture solution was purified by using a 0.2 μm filter each, and then subjected to high performance liquid chromatography (HPLC) quantitative analysis under the following conditions. Table 4 shows the HPLC conditions for meso -2,3-butanediol analysis.

subject Condition HPLC Model   LC-10AT unit (Younglin, Korea), UV730D (210 nm, Younglin, Korea) column Aminex HPX-87H (Biorad, USA) Flow rate 0.8 ml / min Injection volume 30 μl Mobile phase 0.001 N sulfuric acid Oven temperature 60 ° C Operating time 20 minutes

The recombinant strain developed in the present invention produced meso -2,3-butanediol in acetoin -added medium, which confirmed that acetoin reductase protein can be produced in recombinant E. coli at a conversion rate of 53% or more (Fig. 6).

Example  5: Recombination In E. coli  Expressed Acetoin  Reductase ( acetoin reductase , budC ) Enzyme activity and Conversion rate  Measure

In order to measure the enzymatic activity of acetoin reductase expressed in the recombinant strains developed, a 30 μl stock solution was stored in 3 ml of LB (50 μg / ml ampicillin and 50 μg / ml kanamycin) in a 10 ml tube. After incubation for a period of time, it was again contained in 200 ml of LB (50 μg / ml ampicillin, 50 μg / ml kanamycin, 10 g / L tryptone, 10 g / L NaCl and 5 g / L yeast in a 500 ml flask). Extract) was inoculated with 0.5% of the electric culture medium and incubated for 24 hours at 37 ℃, 170 rpm. For protein expression, when the absorbance of the culture medium reached 0.6 at 600 nm, the inducer was added with isopropylthio-β-D-galactoside (sigma, USA) to induce the recombination of the recombinant protein at a final concentration of 1 mM / ml. Expression was induced. For wild-type E. coli, the cells were cultured in the same manner as above in LB medium without antibiotics and IPTG was not added.

For enzyme activity measurement, the culture medium was centrifuged at 4000 rpm for 10 minutes, washed twice with PBS buffer, and then suspended in 5 ml of PBS buffer. The cells were crushed by sonication for 3 minutes. After centrifugation, 0.1 ml of 1 mM NADH and 0.1 ml of 50 mM acetoin were added to 0.7 ml of the supernatant. The solution was reacted for 60 minutes and then measured by UV (340 nm) to calculate NADH (Nicotinamide Adenine Dinucleotide Hydrogenase) / NAD (Nicotinamide Adenine Dinucleotide) conversion. In order to measure the change in enzyme activity according to pH, the pH of the PBS buffer was prepared under the three conditions of pH 5, pH 6 or pH 7 and added thereto, followed by reaction at 25 ° C. for 60 minutes. On the contrary, in order to measure the change in enzyme activity according to the temperature, the cultured cells were washed with pH 7 PBS buffer and reacted for 60 minutes at a temperature between 25 ° C, 30 ° C, 37 ° C and 42 ° C in the last step. Through this, the activity of acetoin reductase according to temperature and pH was measured. In addition, the amount of protein was measured using a colorimetric method, and the enzyme activity per protein was calculated through the calculation. As a result, in the wild type E. coli, there was no acetoin reductase, so there was no consumption of NADH, so the enzyme activity was not measured. The recombinant showed high enzyme activity at pH 6 and at temperatures of 37 ° C and 42 ° C (Tables 5 and 6). . This means enzyme activity according to pH and temperature of acetoin reductase in cells. Table 5 is a table showing the measured enzyme activity according to the temperature of acetoin reductase (acetoin reductase, budC ) overexpressed in the E. coli :: pSB3 recombinant strain of the present invention, Table 6 is E. coli :: pSB3 recombinant It is a table showing the enzyme activity measurement value according to the pH of acetoin reductase ( budC ) overexpressed in the strain.

Strain Temperature
(℃) Specific enzyme activity
(/ Min / mg protein) Wild type Escherichia coli 25 ND 30 ND 37 ND 42 ND E. coli DH5α :: pUC18K :: K. oxy 43863 budC 25 27.20 ± 0.001 30 37.12 ± 0.003 37 55.42 ± 0.003 42 63.93 ± 0.004

Strain pH Specific enzyme activity
(/ Min / mg protein) Wild type Escherichia coli 5 ND 6 ND 7 ND E. coli DH5α :: pUC18K :: K. oxy 43863 budC 5 ND 6 28.51 ± 0.002 7 37.12 ± 0.003

In order to measure the extracellular activity of the acetoin reductase of the recombinant strain, 15 mM acetoin was added in the medium under the same conditions, followed by pH 5, pH 6 and pH 7 or 25 ° C, 30 ° C, 37 ° C and After incubation for 24 hours at 42 ° C., meso -2,3-butanediol was measured by HPLC. The yield was calculated in units of g / g by dividing the amount of butanediol by the amount of acetoin added. As a result, the activity of the extracellular enzyme was found to be high at pH 6 and 42 ° C. (FIG. 7 and FIG. 8).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> Industry-University Cooperation Foundation Sogang University Methods for Preparing meso-2,3-butanediol <130> PN110650 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 771 <212> DNA <213> Klebsiella oxytoca 43863 budC <400> 1 atgaaaaaag tcgcactcgt gaccggcgcg ggccagggca tcgggaaagc tatcgccctt 60 cgcctggtta aagatggttt tgccgtggct atcgccgatt acaatgacgc caccgcacag 120 gcggtcgccg atgaaattaa ccgcggcggc ggtcaggcgc tggcggtgaa ggtggatgtc 180 tctaaacgcg accaggtttt tgccgccgta gaacaggcgc ggaagggcct gggcggtttt 240 gacgtgattg tgaacaacgc cggggtggcg ccctccacgc ctatcgaaga gattcgcgag 300 gacgtgatcg ataaagtcta caatatcaac gtcaaaggcg ttatctgggg tatccaggcc 360 gcggtagagg cgtttaaaaa agagggccac ggcggcaaaa tcatcaacgc ctgctcccag 420 gcgggccacg tgggtaaccc ggaactggcg gtctatagct caagtaagtt tgccgtgcgc 480 ggcctgacgc aaaccgccgc ccgcgatctg gcgcatctgg gaattaccgt taacggctac 540 tgcccgggga tcgttaaaac cccaatgtgg gtggaaatcg accgtcaggt ttccgaagcg 600 gcgggtaaac cgctgggcta cgggacccag gagttcgcca aacgcattac ccttggacgg 660 ctttcagagc cggaagacgt cgcggcctgc gtctcttatc tcgccggtcc ggactccagc 720 tatatgaccg gccaatcgct gctgatcgat ggcggcatgg tatttagcta a 771 <210> 2 <211> 256 <212> PRT <213> Klebsiella oxytoca 43863 budC <400> 2 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 Ala Thr Ala Gln Ala Val Ala Asp Glu Ile Asn Arg          35 40 45 Gly Gly Gly Gln Ala Leu Ala Val Lys Val Asp Val Ser Lys Arg Asp      50 55 60 Gln Val Phe Ala Ala Val Glu Gln Ala Arg Lys Gly Leu Gly Gly Phe  65 70 75 80 Asp Val Ile Val Asn Asn Ala Gly Val Ala Pro Ser Thr Pro Ile Glu                  85 90 95 Glu Ile Arg Glu Asp Val Ile 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 His Leu Gly Ile Thr                 165 170 175 Val Asn Gly Tyr Cys Pro Gly Ile Val Lys Thr Pro Met Trp Val Glu             180 185 190 Ile Asp Arg Gln Val Ser Glu Ala Ala Gly Lys Pro Leu Gly Tyr Gly         195 200 205 Thr Gln 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 Gly Pro Asp Ser Ser 225 230 235 240 Tyr Met Thr Gly Gln Ser Leu Leu Ile Asp Gly Gly Met Val Phe Ser                 245 250 255

Claims (11)

Acetoin reductase having an amino acid sequence set forth in SEQ ID NO: 2 sequence.
A nucleic acid molecule encoding acetoin reductase having an amino acid sequence set forth in SEQ ID NO: 2 sequence.
The nucleic acid molecule of claim 2, wherein the nucleic acid molecule has a nucleotide sequence as set forth in SEQ ID NO: 1.
A vector comprising the nucleic acid molecule of claim 2 or 3.
Microorganisms transformed by the nucleic acid molecule of claim 2 or 3.
The microorganism of claim 5, wherein the transformed microorganism is Escherichia coli and the transformed Escherichia coli is meso -2,3-butanediol enantioselective overexpression Escherichia coli.
A method for producing 2,3-butanediol comprising culturing the transformed microorganism of claim 5.
Method for producing 2,3-butanediol comprising culturing the transformed E. coli of claim 6.
8. The method of claim 7, wherein said 2,3-butanediol is meso -2,3-butanediol.
8. The method of claim 7, wherein the transforming microorganism exhibits an optimum yield of 2,3-butanediol at pH 5 to pH 7 conditions.

The method of claim 7, wherein the transforming microorganism is characterized in that the optimum yield of 2,3-butanediol at 35 ℃ to 45 ℃ conditions.

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