KR101366763B1 - Methods for Preparing meso-2,3-butanediol - Google Patents

Abstract

The present invention relates to a method for producing meso -2,3-butanediol. More specifically, the method for preparing meso -2,3-butanediol, which comprises the following steps: (a) Escherichia coli (acetoin reductase) nucleotide sequence of Klebsiella spp. codon optimization suitable for expression in coli ) to obtain the nucleotide sequence of SEQ ID NO: 1; (b) inserting the codon optimized nucleotide sequence of the acetoin reductase into an expression vector to produce a recombinant vector; (c) transforming the recombinant vector to Escherichia coli to produce recombinant Escherichia coli; And (d) preparing meso -2,3-butanediol from the recombinant E. coli. The present invention is to improve the codon so that the activity of the enzyme can be highly expressed in E. coli through Codon Optimization of the gene encoding the acetoin reductase of Klebsiella species This enables the production of meso -2,3-butanediol by recombinant E. coli, and shows an increased activity of 3-7 times compared to the existing enzyme activity.

Description

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

The present invention relates to a method for preparing mi-2,3-butanediol.

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, that is, minimize the production and energy consumption of wastes that are harmful to humankind. There is increasing interest in bioethanol, biodiesel, biogas and butanol, represented by bioenergy, 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, interest in hydrocarbon-type compounds, which are new renewable energy resources, is increasing. 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 species are pathogenic bacteria that cause diseases such as pneumonia and enteritis, and are conditional anaerobic strains that undergo lactose fermentation. In general, Klebsiella species biosynthesize a mixture of three isomers of 2,3-butanediol from monosaccharides. Isomers of meso -2,3-butanediol include (R, R) -, meso- and (S, S) -isomers . The biosynthesis ratio of these isomers can be seen to change very rapidly by strain, gene and culture environment.

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

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 using glucose as a carbon source, the skin over the Le meso acid (pyruvate) and an acetonitrile (acetoin) through the meso -2,3- butane diol is produced or butane which passes the circulation diode diacetyl (diacetyl) -2, It has a selective route for 3-butanediol to be produced.

Attempts and efforts to identify strains for the production of meso -2,3-butanediol by microorganisms, to improve the prepared strains, and to efficiently convert them have been made. 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, meso -2,3- meso -2,3- was efficiently isolated from acetoin reductase having excellent efficiency through codon-optimized gene introduction of acetoin reductase nucleotide of Klebsiella into E. coli, which is useful as an industrial strain. By confirming that butanediol can be produced, the present invention has been completed.

Accordingly, it is an object of the present invention to provide a method for producing meso -2,3-butanediol.

Another object of the present invention is to provide meso -2,3-butanediol.

Another object of the present invention is to provide a codon optimized nucleotide sequence of acetoin reductase.

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 a method for preparing meso -2,3-butanediol, comprising the following steps:

(a) Codon optimization of acetoin reductase nucleotide sequence of Klebsiella spp. suitable for expression in Escherichia coli to obtain the nucleotide sequence of SEQ ID NO: 1 step;

(b) inserting the codon optimized nucleotide sequence of the acetoin reductase into an expression vector to produce a recombinant vector;

(c) transforming the recombinant vector to Escherichia coli to produce recombinant Escherichia coli; And

(d) preparing meso -2,3-butanediol from the recombinant E. coli.

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, meso -2,3- meso -2,3- was efficiently isolated from acetoin reductase having excellent efficiency through codon-optimized gene introduction of acetoin reductase nucleotide of Klebsiella into E. coli, which is useful as an industrial strain. It was confirmed that butanediol can be prepared.

The method for preparing meso -2,3-butanediol of the present invention will be described in detail by dividing each step as follows:

Step (a): codon optimization (Codon Optimization )

The present invention is to provide a method for producing all-meso -2,3- dimethyl butane, "meso -2,3- butane diol, manufactured by the present invention are converted from the acetonitrile by acetonitrile of reductase. Acetoin reductase refers to an enzyme that synthesizes acetoin into butanediol through the following scheme.

[Reaction Scheme]

The main feature of the present invention is that the acetoin reductase used in the preparation of meso -2,3-butanediol of the present invention has codon optimized nucleotides.

As used herein, the term 'Codon Optimization' is one of the ways to increase protein production. Nucleic acid sequences encoding protein sequences are composed of codons that are rarely used by the host and are not maximally expressed. Codon optimization basically involves the conversion of codons of the desired nucleic acid sequence (not high in frequency) to the host in favor of the codon expression frequency of the host. The following shows the frequency of codons encoding specific amino acids of E. coli. http://www.kazusa.or.jp/codon/ gives the codon frequency for all living things.

Preferably, the codon optimized acetoin reductase of the present invention is a nucleic acid molecule having an amino acid sequence set forth in SEQ ID NO: 2, more preferably, the nucleic acid molecule has a nucleotide sequence set forth in SEQ ID NO: 1 .

As used herein, the term " nucleic acid molecule " has the meaning inclusive of DNA (gDNA and cDNA) and RNA molecules. In the nucleic acid molecule, the nucleotide which is a basic constituent unit is not only a natural nucleotide, Also included are 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; Phenylalanine, tryptophan and tyrosine have similar shapes. 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). The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol . Biol . 215: 403-10 (1990)) is accessible from NCBI (National Center for Biological Information) 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.

Step (b): Preparation of recombinant vector

Subsequently, a recombinant vector is prepared by inserting the codon optimized nucleotide sequence of the acetoin reductase into an expression vector.

The vector system of the present invention can be constructed through various methods known in the art, and specific methods for this can be found 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 prokaryotic cells as hosts. 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. When E. coli is used as a 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.

Preferably, the codon optimized nucleotide sequence of the acetoin reductase is operatively linked to the lac promoter.

As used herein, the term “operably linked” refers to the functional binding between a nucleic acid expression control sequence (eg, an array of promoters, signal sequences, or transcriptional regulator binding sites) and other nucleic acid sequences, thereby The regulatory sequence will control the transcription and / or translation of said other nucleic acid sequence.

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.

Step (c): Preparation of Recombinant E. Coli

Subsequently, the recombinant vector is transformed into Escherichia coli to prepare recombinant Escherichia coli.

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 Such as Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, and Salmonella typhimurium, such as BL21 (DE3), E. coli RR1, E. coli LE392, E. coli B, E. coli X1776, E. coli W3110, And Staphylococcus aureus and various Pseudomonas species.

Methods for delivering the vector of the present invention into a host cell can be carried out using a CaCl ₂ method (Cohen, SN et al., Proc . Natl . Acac. Sci . USA , 9: 2110-2114 SN et al, Proc Natl Acac Sci USA, 9: 2110-2114 (1973); and Hanahan, D., J. Mol Biol, 166:....... 557-580 (1983)) and electroporation method (Dower, WJ et al., Nucleic. Acids Res . , 16: 6127-6145 (1988)).

According to a preferred embodiment of the present invention, the present invention prepared recombinant E. coli 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 comprises 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.

Step (d): Preparation of meso -2,3- butanediol

Finally, meso -2,3-butanediol is prepared from the recombinant E. coli.

Preferably, the step (d) is carried out at pH 4 to pH 8 conditions and 35 ℃ to 45 ℃ conditions.

Preferably, the method yields a 3-7 fold increase in meso -2,3-butanediol compared to the case of using acetoin reductase nucleotide sequences of non-codon optimized Krebs.

According to a preferred embodiment of the present invention, the codon-optimized acetoin reductase of the present invention has a 3-6-fold increase in enzymatic activity compared to conventional acetoin reductase, from acetoin The conversion to meso -2,3-butanediol also increased more than five times.

According to another aspect of the present invention, the present invention provides meso -2,3-butanediol prepared by the preparation method of the present invention.

According to another aspect of the invention, the invention is codon optimized in Escherichia coli for acetoin reductase of Klebsiella species having the nucleotide sequence of SEQ ID NO: 1 Provide the nucleotide sequence.

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

(a) The present invention uses codon optimization of a gene sequence encoding acetoin reductase of Klebsiella species to codon such that the activity of the enzyme is highly expressed in E. coli. This improved the production of meso -2,3-butanediol by recombinant E. coli.

(b) The codon optimized acetoin reductase of the present invention exhibits 3-6 fold increased activity compared to conventional enzyme activity.

(c) The present invention is expected to have great advantages in terms of eco-friendliness and economics for mass production of meso -2,3-butanediol, which is a platform compound that will be a useful foundation for the chemical industry.

1 is keurep when Ella (Klebsiella) meso-2,3-butane diol from the paper glucose (meso -2,3-butanediol) of acetonitrile in a schematic showing the metabolic pathway to convert meso-2,3-butane The gene encoding the enzyme converting to diol ( budC ) is shown.
Figure 2 Krebsciela expression vector pUC18 And kanamycin resistance for use as an E. coli shuttle vector Schematic representation of the structure in which the gene is inserted. This was named pUC18K. 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.
3 is optimized for E. coli through codon substitution in the shuttle vector pUC18K budC Schematic representation of the structure in which the gene is inserted. This was named pSBCO3 and the codon optimized gene was named CObudC. 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 4 shows the Krebssiella pneumoniae chromosome as a template Electrophoresis picture of pSBCO3 inserted with the gene of acetoin reductase optimized for E. coli through codon substitution to confirm the insertion of the gene by the restriction enzyme treatment. Row 1; Size marker, 2 rows; The result of budC cut off by BamHI and XbaI treatment in E. coli :: pUC18K :: CO budC .
Figure 5 shows the production yield of meso -2,3-butanediol in acetoin-added medium according to pH of the E. coli :: pSBCO3 recombinant strain of the present invention compared with the recombinant containing the gene without codon substitution It is a graph.
Figure 6 shows the meso -2,3-butanediol production yield in acetoin-added medium according to the temperature of the E. coli :: pSBCO3 recombinant strain of the present invention compared with the recombinant containing the codon-free gene It is a graph.

Example

Example 1 Polymerase Chain Reaction (PCR) CObudC Preparation of Recombinant Plasmids Expressing Genes

Based on the chromosome of Klebsiella pneumoniae KCTC 2242, Bionia based on the gene sequence of budC (Genbank, NCBI), which encodes acetoin reductase for meso -2,3-butanediol production CoBudC was produced by requesting codon optimization from Korea. A primer (primer) of the CObudC was prepared and cloned through a polymerase chain reaction (PCR, TaKaRa Korea) method. The genes of Table 1 were amplified using the primers of Table 2 designed to specifically amplify only the target genes. CObudC was amplified to a size of 771 bp.

gene Production enzyme CObudC Acetoin Reductase

gene Primer sequence Restriction enzyme CObudC F 5'-GGATCCATGAAAAAAGTCGCATTAGTTACTGG -3 ' BamHⅠ R 5'-TCTAGATTAATTAAATACCATACCACCGTCG -3 ' XbaⅠ

The polymerase chain reaction was carried out at 95 ° C. / under conventional reaction conditions (10 mM Tris-HCl, pH 9.0), 50 mM KCI, 0.1% Triton X-100, 2 mM MgSO ₄ and Taq DNA polymerase (TaKaRa, Japan). 1 time under conditions of 5 minutes (modification), 66 ° C / 1 minute (annealing) and 72 ° C / 1 minute (extension), followed by 95 ° C / 1 minute (modification), 66 ° C / 30 seconds (annealing), and Thirty replicates were performed under conditions of 72 ° C./1 min (extension). 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. After PCR, the amplified DNA was purified on 0.8% agarose gel, purified and used for T-vector cloning. The recombinant plasmid pGEM-T :: CObudC was produced by ligation to the pGEM-T easy vector (TaKaRa, Japan). The recombinant plasmid was transformed into Escherichia coli ( E. coli DH5α competent cells, RBS) to prepare a strain.

Example 2: Preparation of Recombinant Plasmid pSBCO3

PGEM -T :: the recombinant plasmid of Example 1: CObudC and Restriction enzymes of Table 2 were treated with pUC18K (FIG. 2), a shuttle vector of Escherichia coli and Krebsiella , and reacted for about 2 hours in a 37 ° C water bath. PUC18K used in Example 2 was prepared by cloning a gene having resistance to kanamycin to the existing pET28a vector and inserting it into the pUC18 vector. DNA fragments were digested with the restriction enzymes of Table 2, and then reacted at 16 ° C. using a T4 ligase (Takara, Japan) to a multicloning site of the pUC18K vector to complete a 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, treated with restriction enzymes, and the cut DNA was cut onto 0.8% agarose gel. It was confirmed by electrophoresis. 4 shows the result of cleavage of the recombinant plasmid by restriction enzymes.

The amplified product was treated with the restriction enzymes of Table 2 and introduced into the pUC18K vector to prepare pSBCO3. 3 shows a map of the vector named pSBCO3 (including the gene encoding CObudC ).

Example 3: Heterologous Escherichia Coli Transformant production

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

For the electroshock transformation method, 3 ml of LB (10 g / L tryptone, 10 g / L) in 30 μl (0.1%) of wild E. coli DH5α cultures pre-incubated for 16 hours in a test tube NaCl and 5 g / L yeast extract) medium were inoculated. When the absorbance of the culture reached 0.6 at a wavelength of 600 nm, the supernatant and the cells were separated by centrifugation (12000 rpm, 1 minute) of the 3 ml culture corresponding to the entire culture. The obtained cells were washed once with 1 ml of 10% glycerol, and then centrifuged again (12000 rpm, 1 minute) to separate the supernatant and the cells. The cells were suspended with 80 μl 10% glycerol. 1-3 μl of recombinant plasmid (pSBCO3) was added to the suspended cells.

A mixture of 80 μl of cells and plasmids to which the plasmid was added was placed in a cuvette (BIO-RAD, Gene pulser cuvette) for electroporation, and subjected to electric shock (1800 v, 25 μs, 200 with BIO-RAD, Gene pulser Xcell). Ω) was added. 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 200 rpm and 37 ° C. for 1 hour. Cultured transformants were LB agar medium (10 g / L Triton, 10 g / L NaCl, 5 g / L yeast extract and 20 g agar) with ampicillin (50 μg / ml) and kanamycin (50 μg / ml). / L) was incubated at 37 ℃ until a single cell was produced. This resulted in the development of an E. coli SGJSBCO03 recombinant strain transformed with the recombinant plasmid pSBCO3 to the E. coli DH5α strain (Table 3).

Development strain - E. coli SGJSBCO03 K. pneumoniae KTCT2242 :: pUC18K :: CObudC

Example  4: Recombination In E. coli  Codon-optimized Acetoin  Reducing enzyme codon optimized acetoin  reductase, CObudC A) protein production

E. coli SGJSBCO03 recombinant strain was incubated for 16 hours in LB (containing 50 μg / ml of ampicillin and 50 μg / ml of kanamycin), and the culture medium was inoculated into LB medium containing 50 μg / ml of ampicillin and 50 μg / ml of kanamycin. When the absorbance was 0.6-1.0 at 600 nm, the glycerol concentration was 25%, prepared as a storage solution, and then stored at -80 ° C until the culture experiment.

Incubation of the developed recombinant strain was incubated for 30 hours in 30 μl of storage solution in 3 ml of LB to which 50 μg / ml of ampicillin and 50 μg / ml of kanamycin in 10 ml tubes were added, followed by another 500 ml flask. Acetoin in 200 ml LB (10 g / L tryptone, 10 g / L NaCl and 5 g / L yeast extract) medium containing ampicillin (50 μg / ml) and kanamycin (50 μg / ml) 15 mM was added and 0.5% of the electric culture was inoculated to incubate for 54 hours at 37 ℃, 170 rpm. When protein absorbance reached 0.6 at 600 nm, the inducer was added with IPTG (isopropylthio-β-D-galactoside, sigma, USA) (final concentration 0.1 mM / ml). Was induced.

In order to measure the concentration of meso -2,3-butanediol in the culture medium of the recombinant strain, the sample collected every certain time during the culture was centrifuged (12000 rpm, 10 minutes), and the supernatant and the cells were separated and stored at -40 ° C. It was. Each of the supernatants separated from the culture solution was purified using a 0.2 μm filter using 1 ml each, followed by high performance liquid chromatography (HPLC) quantitative analysis under the conditions of Table 4.

subject Condition HPLC model   (Younglin, Korea) and UV730D (Younglin, Korea) column Aminex HPX-87H (Biorad, USA) Flow rate 0.8 mi / 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 produced a higher amount of acetoin reductase protein than recombinant E. coli containing a codon-unoptimized gene. It was possible.

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

In order to measure the enzyme activity of acetoin reductase expressed in the developed recombinant strains, the culture was carried out with 30 μl of the storage cell solution and 50 μg / ml ampicillin and 50 μg / ml kanamycin added in 10 ml tube. Incubated for 16 hours, then again 200 ml of LB (10 g / L trypsin, 10 g / L NaCl and 5 g /) with 50 μg / ml ampicillin and 50 μg / ml kanamycin in a 500 ml flask L yeast extract) medium was inoculated with 0.5% of the electric culture was incubated for 24 hours at 37 ℃, 170 rpm. When protein absorbance reached 0.6 at 600 nm, the inducer added IPTG (isopropylthio-β-D-galactoside, sigma, USA) (final concentration 1 mM / ml) for protein expression. Was induced. E. coli wild species were cultured in the same manner as above in LB medium without antibiotics, and IPTG was not added.

For enzymatic activity measurement, the culture medium was centrifuged at 4000 rpm for 10 minutes, and then washed twice with PBS buffer. The cells were crushed by sonication for 3 minutes by floating the bacterial solution in 5 ml PBS buffer. 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 at 340 nm to calculate NADH / NAD conversion. In order to measure the change in enzyme activity according to pH, the pH of PBS buffer was prepared under three conditions of pH 5, pH 6, and pH 7, and then added and reacted at 25 ° C. for 60 minutes. In order to measure the change in enzyme activity according to temperature, the cultured cells were washed with a pH 7 PBS buffer and reacted at 25 ° C., 30 ° C., 37 ° C. and 42 ° C. for 60 minutes. Through this, the activity of acetoin reductase according to temperature and pH was measured. In addition, the amount of protein was measured using colorimetric method, and the degree of enzyme activity per protein was calculated. As a result, in the wild E. coli, there was no acetoin reductase, so there was no consumption of NADH, and the enzyme activity was not measured. Are shown (Table 5 and Table 6). This means the enzyme activity according to the pH and temperature of the intracellular acetoin reductase and showed the highest activity at 37 ℃.

In order to measure the extracellular activity of the acetoin reductase of the developed recombinant strain, acetoin (15 mM) was added to the medium under the same conditions as above, followed by pH 5, pH 6 and pH 7 and a temperature of 25 ° C, 30 ° C, 37 After incubation at 24 ° C. and 42 ° C. for 24 hours, 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 yield was about 5 times higher than the original acetoin reductase. In particular, the yield of the original acetoin reductase showed a yield of 0.8-1.0 g / g even at pH 5 and pH 6 conditions of 0.1 g / g or less. In addition, the yield of the original acetoin reductase showed a yield of 0.8 g / g or more even at 37 ℃ and 42 ℃ conditions of 0.2 g / g or less (Figs. 5 and 6). This means the activity of extracellular acetoin reductase and showed high activity at pH 6,7.

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.

Attach an electronic file to a sequence list

Claims (7)

Method for preparing meso -2,3-butanediol, comprising the following steps:
(a) Codon optimization of acetoin reductase nucleotides of Klebsiella spp. suitable for expression in Escherichia coli to obtain nucleotide sequences of SEQ ID NO: 1 ;
(b) inserting the codon optimized nucleotide of the acetoin reductase into an expression vector to produce a recombinant vector;
(c) transforming the recombinant vector to Escherichia coli to produce recombinant Escherichia coli; And
(d) preparing meso -2,3-butanediol from the recombinant E. coli.
The method of claim 1, wherein the codon optimized nucleotide of the acetoin reductase is operatively bound to a lac promoter.
The method according to claim 1, wherein the step (c) of transforming the E. coli is carried out by electroporation.
The method of claim 1, wherein the step (d) is carried out at pH 4 to pH 8 conditions and 35 ℃ to 45 ℃ conditions.
The method according to claim 1, wherein the yield of meso -2,3-butanediol is increased by 3-7 times as compared to the case of using acetoin reductase nucleotides of non-codon optimized Klebsiella spp. How to.
delete Codon optimized nucleotides in Escherichia coli for acetoin reductase of Klebsiella spp. Having the nucleotide sequence of SEQ ID NO: 1.

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