CN112143688B

CN112143688B - Construction and application of recombinant escherichia coli

Info

Publication number: CN112143688B
Application number: CN201910567022.9A
Authority: CN
Inventors: 张贵民; 全艳彩; 黄文波
Original assignee: Lunan Pharmaceutical Group Corp
Current assignee: Lunan Pharmaceutical Group Corp
Priority date: 2019-06-27
Filing date: 2019-06-27
Publication date: 2024-05-07
Anticipated expiration: 2039-06-27
Also published as: CN112143688A

Abstract

The invention belongs to the field of enzyme catalysis, and particularly discloses construction and application of recombinant escherichia coli for preparing (S) -4-chloro-3-hydroxybutyrate by biocatalysis. The recombinant escherichia coli provided by the invention can express ketoreductase and glucose dehydrogenase simultaneously and in a periplasmic space of cells, the expressed ketoreductase and glucose dehydrogenase have high enzyme activities, and the constructed recombinant escherichia coli is used for preparing the (S) -4-chloro-3-hydroxybutyrate by whole cell catalysis without adding coenzyme or coenzyme factors, so that the problem of high cost for preparing the (S) -4-chloro-3-hydroxybutyrate by the traditional microbial catalysis method is solved, the conversion rate is up to 99.9%, and the ee value is up to 100%.

Description

Construction and application of recombinant escherichia coli

Technical Field

The invention belongs to the field of enzyme catalysis, and particularly relates to construction and application of recombinant escherichia coli, in particular to construction and application of the recombinant escherichia coli for preparing (S) -4-chloro-3-hydroxybutyrate by biocatalysis.

Background

Rosuvastatin calcium was originally developed by the japanese wild-type company, 4 th month of 2007 as approved by the national food and drug administration for rosuvastatin, an alliance pharmaceutical company. Rosuvastatin calcium is a selective HMG-CoA reductase inhibitor, and has the main functions of treating three diseases such as hypercholesterolemia, simple hyperlipidemia and triglyceride and mixed blood lipid disorder, and particularly has outstanding effect on cholesterol and oil index reduction by using the medicament. Optically pure Ethyl (S) -4-chloro-3-hydroxybutyrate (Ethyl 4-chloro-3-hydroxyrate, (S) -CHBE) is an important organic intermediate, the most important application of which is as a precursor compound for synthesizing the cholesterol-lowering drug atorvastatin. The chiral single enantiomer (S) -4-chloro-3-hydroxy ethyl butyrate ((S) -CHBE) can be also used for synthesizing other active medicaments, such as a hydroxymethylglutaryl CoA (HMG-CoA) reductase inhibitor, a1, 4-dihydropyridine beta-blocker and the like.

The potential chiral substance 4-chloroacetoacetic acid Ethyl ester (Ethyl 4-chloro-3-oxobutanoate, COBE) has the advantages of easy synthesis and low price, and the potential chiral substance is used as a reaction substrate to carry out asymmetric reduction reaction to obtain (S) -CHBE, thus being a very economic and effective preparation way.

Currently known methods for reduction from COBE to chiral alcohols have three main routes:

1. asymmetric reduction of chemical catalysts: and (3) performing asymmetric reduction on the COBE by using rhodium, ruthenium and other metal catalysts. The method has the defects that the stereoselectivity of the product is not high enough, the catalytic reduction reaction needs high hydrogen pressure and the energy consumption is high;

2. enzyme-catalyzed asymmetric reduction using an enzyme (commercial enzyme) such as acetaldehyde reductase or the like after extraction and purification has the disadvantage of requiring the addition of expensive coenzymes;

3. The microbial catalytic asymmetric reduction process is achieved by stereoselective biocatalysis of intact microbial cells (e.g. baker's yeast).

In the three methods, the chemical catalyst is asymmetric reduction method, the adopted metal catalyst is expensive, in addition, the stereoselectivity of the product is not high enough, the catalytic reduction reaction needs high hydrogen pressure, the energy consumption is high, and the waste heavy metal catalyst has great pollution to the environment. The enzyme catalysis asymmetric reduction method has the defects of complicated process operation and easy enzyme inactivation, and requires the addition of expensive coenzyme (typically NADH or NADPH), thereby having higher cost. The microbial catalysis method has the advantages of mild reaction conditions, rapid reaction, less byproduct generation, simple product treatment and the like, and therefore, the microbial catalysis method is attracting more attention. However, it is difficult to screen for excellent microorganism strains with high stereoselectivity; meanwhile, coenzyme is also required to be added, energy substances are continuously supplied, and the toxicity of a substrate to thalli and the instability problem in an aqueous solution are also caused; because of the complex enzyme system present in microbial cells, the optical purity of the catalytic product is often not high.

The catalytic reaction is carried out directly with recombinant reductase, and a large amount of expensive coenzyme (NADH, NADPH) is required to be added according to the stoichiometric amount. Therefore, in order to obtain (S) -CHBE in high yield and high purity, it is necessary to clone the dehydrogenase gene to achieve efficient in situ regeneration of the coenzyme, so as to provide a high-efficiency coenzyme regeneration cycle system.

CN103173503a discloses a recombinant escherichia coli for asymmetric preparation of (S) -CHBE, which is escherichia coli into which a ketoreductase gene KRED is introduced. For providing the E.coli expressing Glucose Dehydrogenase (GDH) with the reducing power NAD (P) H necessary for the catalytic reaction. According to the method, ketoreductase and glucose dehydrogenase are respectively introduced into different escherichia coli, then the two escherichia coli with different enzymes are selected and added into a reaction system, a small amount of NAD (P) H is needed to be added in the reaction process, the escherichia coli BL21 (DE 3)/pET 28a-KRED has low activity and a reaction time of 12 hours, and the reaction is influenced by a plurality of factors such as COBE substrate concentration, dual-bacteria mass ratio, glucose concentration, product concentration and the like, so that the product conversion rate is low, and the highest conversion rate reaches 92% after the reaction is carried out for 12 hours.

CN104651292a discloses a recombinant escherichia coli for preparing (S) -4-chloro-3-hydroxybutyrate through asymmetric transformation and application thereof, wherein the recombinant escherichia coli simultaneously has carbonyl reductase gene and glucose dehydrogenase gene, and can simultaneously express carbonyl reductase and glucose dehydrogenase. However, the carbonyl reductase and glucose dehydrogenase which are recombined and expressed by the recombined escherichia coli belong to intracellular expression of the escherichia coli, the expressed enzymes exist in an inclusion form, the protein cannot be folded correctly to form an inactive or low-activity state, the recombined protein can exert normal biological functions after being crushed, dissolved and renatured, and the protease activity is very low or basically inactive by directly using whole cell catalysis. In addition, in the application of (S) CHBE, freeze-dried thalli is needed to be used as a catalyst, the cost is high, and the survival rate of the thalli after freeze-drying is low.

In summary, the existing microbial catalysis asymmetric reduction of COBE has the problems that expensive coenzyme NADH or NADPH needs to be added, the expressed enzyme activity is low, or the expressed enzyme is an intracellular product, and the recombinant protease can exert normal biological functions only after wall breaking and renaturation are needed, so that the operation is complex, the process is complicated, the production cost is high, and the like. Therefore, there is an urgent need to construct a recombinant E.coli for biocatalytically preparing (S) -4-chloro-3-hydroxybutyrate.

Disclosure of Invention

The invention aims to provide recombinant escherichia coli for preparing (S) -4-chloro-3-hydroxybutyrate through asymmetric transformation and application thereof, so as to solve the problems that in the prior art, the yield of a chemical method catalytic product is low, the energy consumption is high, the pollution is large, the yield of a microbial method catalytic product is low, the optical purity is not high, coenzyme is required to be additionally added, the expressed enzyme activity is low, and ketoreductase and glucose dehydrogenase cannot be co-expressed in a periplasmic space at the same time.

In a first aspect, the invention provides a recombinant E.coli co-expressing a Ketoreductase (KRD) of Saccharomyces rouxii and a Glucose Dehydrogenase (GDH) of Bacillus subtilis. The Ketoreductase (KRD) and Glucose Dehydrogenase (GDH) are encoded by the optimized gene sequences of the present invention.

The ketoreductase (ketoreductase, KRD) gene of the Russell yeast (Zygosaccharomyces rouxii) has 1014bp basic group, the recording number of which in GenBank is AF178079.1, and the gene sequence of which is shown as SEQ ID NO. 1. The ketoreductase coded by the gene comprises 338 amino acids, the accession number of the ketoreductase in GenBank is AAF22287.1, and the amino acid sequence of the ketoreductase is shown as SEQ ID NO. 2.

The glucose dehydrogenase (glucose dehydrogenase, GDH) gene of bacillus subtilis (Bacillus subtilis) contains 783bp base, the accession number of which in Genbank is M12276.1, and the gene sequence of which is shown in SEQ ID NO. 6. The glucose dehydrogenase encoded by the gene comprises 260 amino acids, the accession number of which in Genbank is AAA22463.1, and the amino acid sequence of which is shown in SEQ ID NO. 7.

The ketoreductase KRD gene sequence of the optimized Saccharomyces rouxii is shown as SEQ ID NO. 4; the optimized glucose dehydrogenase GDH gene of the bacillus subtilis has a sequence shown in SEQ ID NO. 9.

The recombinant escherichia coli disclosed by the invention can express ketoreductase KRD of the saccharomyces rouxii and glucose dehydrogenase GDH of the bacillus subtilis in a periplasmic space of cells.

The second object of the present invention is to provide a method for constructing recombinant E.coli co-expressing the above two genes. Firstly, the expression vector is connected with a ketoreductase KRD gene in vitro, and then is connected with a glucose dehydrogenase GDH gene after verification of correctness, the correctness of the vector connection is verified through double digestion and sequencing, and the construction of recombinant escherichia coli is completed by transforming host bacteria through a chemical method by the correct expression vector.

In one test protocol, the expression vector pET-26b.

The following details the construction method of the recombinant E.coli of the present invention:

(1) Optimization and synthesis of Ketoreductase (KRD) and Glucose Dehydrogenase (GDH) genes

The optimized ketoreductase (ketoreductase, KRD) gene of the pre-roux yeast (Zygosaccharomyces rouxii) has 1014bp basic group, the recording number of the ketoreductase gene in GenBank is AF178079.1, and the gene sequence of the ketoreductase gene is shown as SEQ ID NO. 1.

Firstly, bamHI is added to the 5 'of KRD gene and SacI enzyme cutting site is added to the 3' of KRD gene, the designed gene is entrusted to Shanghai biological engineering (Shanghai) stock company to carry out total gene synthesis sequencing, and the synthesized gene sequence is shown as SEQ ID NO. 3. And BamHI is added to the 5 'and SacI cleavage sites are added to the 3' of the optimized nucleotide sequence, and the synthesized gene sequences are shown in SEQ ID NO.4 and SEQ ID NO. 5.

The glucose dehydrogenase (glucose dehydrogenase, GDH) gene of the optimized bacillus subtilis (Bacillus subtilis) contains 783bp base, the recording number of the gene in Genbank is M12276.1, and the gene sequence of the gene is shown as SEQ ID NO. 6.

The SD sequence and pelB leader signal peptide sequence of colibacillus are added in front of GDH gene, and the enzyme cutting sites SalI and NotI are added at two ends respectively, named SGDH, and the designed gene is entrusted to complete gene synthesis sequencing by Shanghai biological engineering (Shanghai) stock company, and the synthesized gene sequence is shown in SEQ ID NO. 8. And the SD sequence and pelB leader signal peptide sequence of the escherichia coli are added in front of the optimized gene sequence, and the two ends of the SD sequence and the pelB leader signal peptide sequence are respectively added with enzyme cutting sites SalI and NotI which are named SGDH, and the synthesized gene sequences are shown as SEQ ID NO.9 and SEQ ID NO. 10.

(2) Construction of Co-expression vectors and transformation of hosts

Firstly, carrying out in vitro connection on an expression vector pET-26b and a Ketoreductase (KRD) gene with a gene sequence of SEQ ID NO.3, verifying that the correct pET-26b-KRD is connected with SGDH gene with a gene sequence of SEQ ID NO.8 by the same method, and verifying the connection accuracy of pET-26b-KRD-SGDH by double enzyme digestion and sequencing. The correct expression vector was transformed into host BL21 (DE 3) by chemical method, and shake flask test confirmed that neither the protease nor glucose dehydrogenase was expressed.

The expression vector pET-26b is connected with a Ketoreductase (KRD) gene with a gene sequence of SEQ ID NO.3 in vitro, the correct pET-26b-KRD is verified to be connected with SGDH gene with a gene sequence of SEQ ID NO.9 by the same method, and the connection accuracy of pET-26b-KRD-SGDH is verified by double enzyme digestion and sequencing. The correct expression vector is transformed into host bacterium BL21 (DE 3) by a chemical method, and shake flask tests prove that the protein ketone reductase is not expressed and the glucose dehydrogenase is correctly expressed.

The expression vector pET-26b is connected with a Ketoreductase (KRD) gene with a gene sequence of SEQ ID NO.3 in vitro, the correct pET-26b-KRD is verified to be connected with SGDH gene with a gene sequence of SEQ ID NO.10 by the same method, and the connection accuracy of pET-26b-KRD-SGDH is verified by double enzyme digestion and sequencing. The correct expression vector is transformed into host bacterium BL21 (DE 3) by a chemical method, and shake flask tests prove that the proteinase is not expressed, the glucose dehydrogenase is correctly expressed, but the electrophoresis band is not clear.

The expression vector pET-26b is connected with a Ketoreductase (KRD) gene with a gene sequence of SEQ ID NO.4 in vitro, the correct pET-26b-KRD is verified to be connected with SGDH gene with a gene sequence of SEQ ID NO.8 by the same method, and the connection accuracy of pET-26b-KRD-SGDH is verified by double enzyme digestion and sequencing. The correct expression vector is transformed into host bacterium BL21 (DE 3) by a chemical method, and a shake flask test proves that the proteinase is correctly expressed and the glucose dehydrogenase is not expressed.

The expression vector pET-26b is connected with a Ketoreductase (KRD) gene with a gene sequence of SEQ ID NO.4 in vitro, the correct pET-26b-KRD is verified to be connected with SGDH gene with a gene sequence of SEQ ID NO.9 by the same method, and the connection accuracy of pET-26b-KRD-SGDH is verified by double enzyme digestion and sequencing. The correct expression vector is transformed into host bacterium BL21 (DE 3) by a chemical method, shake flask tests prove that the proteinase and glucose dehydrogenase can be correctly expressed, and an electrophoresis result shows that the proteinase has obvious protein expression bands.

The expression vector pET-26b is connected with a Ketoreductase (KRD) gene with a gene sequence of SEQ ID NO.4 in vitro, the correct pET-26b-KRD is verified to be connected with SGDH gene with a gene sequence of SEQ ID NO.10 by the same method, and the connection accuracy of pET-26b-KRD-SGDH is verified by double enzyme digestion and sequencing. The correct expression vector was transformed into host BL21 (DE 3) by chemical means, and shake flask experiments confirmed that the proteolytic ketoreductase and glucose dehydrogenase were able to be expressed correctly, but the electrophoretic bands were not clear.

The expression vector pET-26b is connected with a Ketoreductase (KRD) gene with a gene sequence of SEQ ID NO.5 in vitro, the correct pET-26b-KRD is verified to be connected with SGDH gene with a gene sequence of SEQ ID NO.8 by the same method, and the connection accuracy of pET-26b-KRD-SGDH is verified by double enzyme digestion and sequencing. The correct expression vector is transformed into host bacterium BL21 (DE 3) by a chemical method, and a shake flask test proves that the proteinase is correctly expressed, but the electrophoresis band is not clear, and the glucose dehydrogenase is not expressed.

The expression vector pET-26b is connected with a Ketoreductase (KRD) gene with a gene sequence of SEQ ID NO.5 in vitro, the correct pET-26b-KRD is verified to be connected with SGDH gene with a gene sequence of SEQ ID NO.9 by the same method, and the connection accuracy of pET-26b-KRD-SGDH is verified by double enzyme digestion and sequencing. The correct expression vector was transformed into host BL21 (DE 3) by chemical means, and shake flask experiments confirmed that the proteolytic enzyme ketoreductase and glucose dehydrogenase were correctly expressed, but the electrophoretic bands were not clear.

The expression vector pET-26b is connected with a Ketoreductase (KRD) gene with a gene sequence of SEQ ID NO.5 in vitro, the correct pET-26b-KRD is verified to be connected with SGDH gene with a gene sequence of SEQ ID NO.10 by the same method, and the connection accuracy of pET-26b-KRD-SGDH is verified by double enzyme digestion and sequencing. The correct expression vector was transformed into host BL21 (DE 3) by chemical means, and shake flask experiments confirmed that the proteolytic enzyme ketoreductase and glucose dehydrogenase were correctly expressed, but the electrophoretic bands were not clear.

Thus, the Ketoreductase (KRD) gene sequence of the invention is preferably SEQ ID NO.4 and the Glucose Dehydrogenase (GDH) gene sequence is preferably SEQ ID NO.9.

The recombinant escherichia coli BL21 (DE 3) (pET-26 b-KRD-SGDH) co-expresses two proteases of Ketoreductase (KRD) and Glucose Dehydrogenase (GDH), and the two enzymes have different SD sequences and signal peptide sequences in one expression system, and the expression of the two enzymes does not interfere with each other, so that the uniformity of two enzyme systems can be achieved in time and space.

The periplasm of the E.coli contains a series of enzymes, which can promote the correct folding of the target protein and improve the yield of the active enzyme protein. In addition, the content of the hetero protein in the periplasm space is low, so that the degradation in the cell of the recombinant protein can be avoided, and the stable existence is facilitated, thereby being beneficial to the concentration of a target product. The periplasmic space is located between the two cell membranes and more readily releases the protein of interest from the cell to the outside without affecting the integrity of the cell.

The third object of the invention is to provide the application of recombinant escherichia coli in the preparation of (S) -4-chloro-3-hydroxybutyric acid ethyl ester by asymmetric reduction of 4-chloroacetoacetic acid ethyl ester.

The asymmetric reduction reaction adopts a water phase system conversion method. In the prior art, the two-phase method and the single-water phase catalysis are common, and compared with the single-water phase catalysis, the method avoids the toxic action of a large amount of organic reagents on the catalyst, and is beneficial to improving the reaction efficiency.

The invention relates to a preparation method of recombinant escherichia coli BL21 (DE 3) (pET-26 b-KRD-SGDH) for catalyzing COBE to be completely converted into (S) -4-chloro-3-ethylhydroxybutyrate in an aqueous phase system under the condition of no additional coenzyme.

In a water phase reaction system, 4-chloroacetoacetic acid ethyl ester is taken as a raw material, isopropanol is taken as a hydrogen donor, and the recombinant escherichia coli is added for conversion reaction to prepare the (S) -4-chloro-3-hydroxybutyric acid ethyl ester.

The preparation method comprises the following steps: culturing recombinant escherichia coli BL21 (DE 3) (pET-26 b-KRD-SGDH), adding whole cells into a reaction system, taking 4-chloroacetoacetic acid ethyl ester as a raw material, taking isopropanol as a hydrogen donor, and synthesizing (S) -4-chloro-3-hydroxybutyric acid ethyl ester with high enantioselectivity under the condition of not adding exogenous coenzyme by utilizing a coenzyme system of the cells. The reaction system contains aqueous phase buffer solution, and the reaction temperature is controlled to be 25-35 ℃.

Specifically, a water phase buffer solution, isopropanol, 4-chloroacetoacetic acid ethyl ester and recombinant escherichia coli thallus are added into a reaction system, the reaction temperature is controlled to be 25-35 ℃, stirring reaction is carried out, GC detection is carried out, substrate conversion is complete, stirring is stopped, and standing and liquid separation are carried out. The organic phase is added with anhydrous sodium sulfate, filtered and concentrated to obtain (S) -4-chloro-3-hydroxybutyric acid ethyl ester.

Preferably, the aqueous phase buffer solution is phosphate buffer solution, the concentration of the aqueous phase buffer solution is 50-200 mM, and the pH value is 6.0-7.0; the mass volume ratio of the aqueous phase buffer solution to the substrate 4-chloroacetoacetic acid ethyl ester is 3-7:1, ml/g.

Preferably, the mass of the recombinant escherichia coli thallus is 0.5-1.5% of the mass of the substrate 4-chloroacetoacetic acid ethyl ester.

Preferably, the mass ratio of the isopropanol to the substrate 4-chloroacetoacetic acid ethyl ester is 0.3-0.8:1.

Compared with the prior art, the invention has the following advantages:

(1) The Ketoreductase (KRD) gene and the Glucose Dehydrogenase (GDH) gene are optimized, so that the Ketoreductase (KRD) gene and the Glucose Dehydrogenase (GDH) gene are simultaneously expressed in escherichia coli, and the Ketoreductase (KRD) gene and the Glucose Dehydrogenase (GDH) maintain space-time consistency in a system, thereby being more beneficial to the catalytic preparation of (S) -4-chloro-3-hydroxy ethyl butyrate.

(2) The Ketoreductase (KRD) and the Glucose Dehydrogenase (GDH) are expressed in the periplasm space of the escherichia coli, the content of the hetero-protein in the periplasm space is low, and the activity of the hetero-protease is lower than that in cytoplasm, so that the expressed protease can avoid intracellular degradation and exist stably, and the expression of the target protein is facilitated.

(3) The recombinant escherichia coli constructed by the invention has high expressed Ketoreductase (KRD) activity, and can be used for catalyzing the ethyl 4-chloroacetoacetate by directly adding the whole cell of the recombinant escherichia coli into a reaction system without a purification preparation process.

(4) The recombinant escherichia coli BL21 (DE 3) (pET-26 b-KRD-SGDH) constructed by the invention is used for preparing the (S) -4-chloro-3-hydroxybutyric acid ethyl ester by whole cell catalysis, no coenzyme or coenzyme factor is required to be added, the problem of high cost for preparing the (S) -4-chloro-3-hydroxybutyric acid ethyl ester by the traditional microbiological catalysis is solved, the conversion rate is up to 99.9%, and the ee value is up to 100%.

Drawings

FIG. 1 is a physical map of the recombinant co-expression vector pET-26b-KRD-GDH of the present invention;

FIG. 2 is an SDS-PAGE electropherogram, M: the low molecular weight protein marker,1 is a negative control band, and 2 is a band of electrophoresis of co-expression of ketoreductase and glucose dehydrogenase in periplasmic space of cells.

Detailed Description

The invention will be further illustrated with reference to specific examples. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The materials used in the present invention are commercially available without any particular explanation.

Example 1: construction of recombinant plasmid pET-26b-KRD-SGDH

1. Gene design

The Ketoreductase (KRD) gene of the Russell yeast (Zygosaccharomyces rouxii) has 1014bp basic group, the recording number of the KRD gene in GenBank is AF178079.1, and the gene sequence of the KRD gene is shown as SEQ ID NO. 1. The ketoreductase coded by the gene comprises 338 amino acids, the accession number of the ketoreductase in GenBank is AAF22287.1, and the amino acid sequence of the ketoreductase is shown as SEQ ID NO. 2.

According to the sequence of GenBank, bamHI is added to the 5 'and SacI cleavage site is added to the 3' of KRD gene, the designed gene is entrusted to Shanghai biological engineering (Shanghai) Co., ltd for total gene synthesis sequencing, and the synthesized gene sequence is shown as SEQ ID NO. 3.

Meanwhile, the method combines with the preferred codon of the escherichia coli, performs synonymous mutation according to the degeneracy of the codon on the premise of not changing amino acid, adds BamHI at the 5 'end and SacI enzyme cutting site at the 3' end of a designed gene, and entrusts Shanghai biological engineering (Shanghai) stock company to perform total gene synthesis sequencing, wherein the synthesized gene sequences are shown as SEQ ID NO.4 and SEQ ID NO. 5.

The Glucose Dehydrogenase (GDH) gene of bacillus subtilis (Bacillus subtilis) contains 783bp base, the recording number of which in GenBank is M12276.1, and the gene sequence of which is shown in SEQ ID NO. 6. The glucose dehydrogenase coded by the gene comprises 260 amino acids, the accession number of which in GenBank is AAA22463.1, and the amino acid sequence of which is shown in SEQ ID NO. 7.

According to the sequence of GenBank, the SD sequence of E.coli and the pelB leader signal peptide sequence are added in front of GDH gene, and the cleavage sites SalI and NotI are respectively added at two ends, and are named SGDH, the designed gene entrusted with Shanghai biological engineering (Shanghai) stock company to complete gene synthesis sequencing, and the synthesized gene sequence is shown as SEQ ID NO. 8. The base is optimized according to the preference of the escherichia coli codon, an escherichia coli SD sequence and a pelB leader signal peptide sequence are added in front of the genes, enzyme cutting sites SalI and NotI are respectively added at two ends of the genes, and the synthesized gene sequences are shown as SEQ ID NO.9 and SEQ ID NO. 10.

2. Extraction of plasmids

PUC57-KRD-DH5 alpha with KRD gene is respectively entrusted to the synthesis and identification of Shanghai engineering biological engineering (Shanghai) Co., ltd, glycerinum streak is cultivated overnight at 37 ℃, single colony is picked up from a flat plate and inoculated to LB culture medium containing 100 mug/ml ampicillin, cultivated overnight at 37 ℃ at 150rpm, 4ml bacterial solution is taken for centrifugation to collect bacterial body, plasmids are extracted, 60 mug of sterilized water is added for elution, and the bacterial body is preserved at-20 ℃.

PUC57-GDH-DH5 alpha with GDH gene is put into Shanghai engineering and biological engineering (Shanghai) Co., ltd for synthesis and identification, glycerinum is streaked, cultured overnight at 37 ℃, single colony is picked up from a flat plate and inoculated into LB culture medium containing 100 mug/ml ampicillin, cultured overnight at 37 ℃ at 150rpm, 4ml bacterial liquid is collected by centrifugation, plasmid is extracted, 60 mug of sterilized water is added for eluting, and the culture medium is preserved at-20 ℃.

3. Construction of vector pET-26b-KRD-GDH

PET-26b and pUCk57-KRD in the extracted plasmid are subjected to double digestion with BamHI and SacI, and the digestion system is as follows: pET-26b or pUC57-KRD 30.0. Mu.L, 10 XK buffer 2.0. Mu.L, enzyme BamHI 0.5. Mu.L, enzyme SacI 3.5. Mu. L, ddH ₂ O4. Mu.L. 1% agarose electrophoresis, recovering double enzyme fragments of the vector and the gene, eluting with sterile water, wherein the recovered large fragment of the pET-26b vector is 5.35kb, and the recovered small fragment of pUC57-KRD is 1032bp. After recovery of the glue, the two are linked into a loop in the system in which T4 ligase is present. And transforming the connected product into a cloning host DH5 alpha, picking up a transformant, performing enzyme digestion and sequencing, and performing the next experiment on the verified plasmid pET-26 b-KRD.

The correctly ligated plasmids pET-26b-KRD and pUC57-GDH were digested with SalI and Not I in the following manner: pUC57-GDH or pET-26b-KRD 30.0. Mu.L, 10 XH buffer 4.0. Mu.L, enzyme SalI 1.5. Mu.L, enzyme NotI 0.5. Mu. L, ddH ₂ O4. Mu.L. The double digested fragments were recovered, the pET-26b-KRD vector recovered large fragment was 6.38kb, and the pUC57-GDH recovered small fragment was 955bp. After in vitro connection, the cloning host DH5 alpha is transformed, double enzyme digestion verification and gene sequencing are carried out, and the pET-26b-KRD-GDH plasmid is obtained after verification.

EXAMPLE 2 construction of recombinant E.coli

1. Preparation of competent cells of E.coli BL21 (DE 3):

1. a single clone of E.coli BL21 (DE 3) was picked from LB plates and cultured overnight at 37℃for about 16 h.

2. The bacterial liquid is transferred into fresh 50ml LB liquid culture medium according to the proportion of 1:50, and is cultured for 2.5-3 h at 37 ℃ in a shaking way, and when the OD ₆₀₀ is 0.3-0.6, the culture is stopped.

3. Transferring the bacterial liquid into a centrifugal tube precooled on ice, carrying out ice bath for 30min, and centrifuging at 4000r/min for 10min under the condition of 4 ℃.

4. The supernatant was discarded, and the cells were gently suspended in a pre-chilled 0.1MCaCl ₂ solution and centrifuged at 4000r/min for 10min at 4 ℃.

5. The step 4 is repeated once.

6. The supernatant was discarded, 100. Mu.l of pre-chilled CaCl ₂ solution was added, and the cells were carefully suspended to make a competent cell suspension.

2. Plasmid pET-26b-KRD-GDH transformed E.coli BL21 (DE 3)

1. Mu.l of pET-26b-KRD-GDH and 100. Mu.l of BL21 (DE 3) competent cells were gently mixed, respectively, and ice-bathed for 30min.

2. And rapidly placing in ice bath for 5min at 42 ℃ by heat shock for 90S.

3. Respectively adding 800 mu lSOC culture solution, culturing at 37deg.C for 45min

4. Mu.l of each of the bacterial solutions was plated on a resistance plate containing 50. Mu.g/ml kanamycin, and cultured upside down at 37 ℃.

3. Recombinant BL21 (DE 3) (pET-26 b-KRD-SGDH) expression verification

The transformants obtained in the above experiment were inoculated into LB liquid medium containing 50. Mu.g/ml kanamycin resistance, cultured at 37℃until the cell concentration OD600 was about 0.6-1.0, isopropyl-beta-D-thiogalactoside (IPTG) was added at a final concentration of 0.2mM, and after induction culture at 25℃for 20 hours, the cells were collected by centrifugation at 6000rpm, respectively, to obtain recombinant E.coli BL21 (DE 3) (pET-26 b-KRD-SGDH) wet cells containing intracellular expression recombinant plasmid (pET-26 b-KRD-SGDH). 1g of wet cells were collected, and suspended with 10ml of 10mmol/L Tris-HCl solution (pH 8.0) containing 20% sucrose, the temperature was maintained at 4℃for 30 minutes, the cells were collected by centrifugation, suspended with 10ml of 10mmol/L Tris-HCl solution (pH 8.0), the temperature was maintained at 4℃for 30 minutes, and the supernatant was collected by centrifugation at 12000rpm, and SDS-PAGE was performed, and the results are shown in Table 1.

TABLE 1 Ketone reductase and glucose reductase target protein expression

As can be seen from the results in Table 1, when the target gene sequences contained in the plasmids are SEQ ID NO.4 and SEQ ID NO.9, the ketoreductase and glucose dehydrogenase can be correctly expressed in the periplasmic space of the E.coli cells, the construction pattern of the recombinant plasmids is shown in FIG. 1, and the SDS-PAGE electrophoresis pattern is shown in FIG. 2.

Example 3 measurement of enzyme Activity

Recombinant E.coli BL21 (DE 3) (pET-26 b-KRD-SGDH) containing the ketoreductase gene SEQ ID No.4 sequence and glucose dehydrogenase gene SEQ ID No.9 with best expression was picked up into LB liquid medium containing kana resistance, and shake-cultured overnight at 37 ℃. Then, the cells were inoculated into fresh culture medium at an inoculum size of 2%, respectively, and cultured at 37℃until OD600 was about 0.8, IPTG was added to a final concentration of 0.2mmol/L, after induction of expression at 25℃and 200rpm for 20 hours, the cells were centrifuged (4℃and 4000rpm,30 min), the bacterial sludge was resuspended in 50mM Tris-HCl buffer (pH 8.0), the cells were sonicated (power: 300W, sonicated for 5s, intermittent for 5s, 5min total), and the protein content in the cell-free crude enzyme solution was measured by the Bradford method by centrifugation (4℃and 12000rpm,10 min).

Reductase Ketoreductase (KRD) activity assay: the reaction system was 50mM Tris-HCl buffer (pH 8.0) containing 0.3mM ADH,5mM substrate ethyl 4-chloroacetoacetate and an appropriate amount of enzyme crude liquid. The reaction system was scanned for changes in absorbance at 340nm immediately after the enzyme activity was added.

Glucose Dehydrogenase (GDH) activity assay system: activity was measured by the Sadoff method. The assay system contained 200mM substrate glucose, 4uM NAD+,20nM MnSO ₄, and an appropriate amount of crude enzyme solution in 50mM Tris-HCl buffer (pH 8.0) to a final volume of 2.0mL. The reaction system was scanned for changes in absorbance at 340nm immediately after the enzyme activity was added.

The results showed that the specific enzyme activity of ketoreductase of recombinant E.coli BL21 (DE 3) (pET-26 b-KRD-SGDH) was 45.8U/mg and the specific enzyme activity of glucose dehydrogenase of recombinant E.coli BL21 (DE 3) (pET-26 b-KRD-SGDH) was 50.6U/mg.

EXAMPLE 4 preparation of recombinant E.coli BL21 (DE 3) (pET-26 b-KRD-SGDH) Whole-cell

The recombinant E.coli BL21 (DE 3) (pET-26 b-KRD-SGDH) single colony obtained in example 2 is selected from a kana resistance plate, inoculated into a liquid LB culture medium containing kanamycin resistance, cultured in a shaking way at 200rpm at 37 ℃ until the OD600 value reaches 0.6-1.0, IPTG is added until the concentration reaches 0.2mM, the culture is continued for 20 hours at 25 ℃, the thalli is collected by centrifugation at 4 ℃, physiological saline is added for cleaning twice, and the thalli is collected by centrifugation again to obtain the E.coli BL21 (DE 3) (pET-26 b-KRD-SGDH) whole cell containing recombinant ketoreductase and glucose dehydrogenase.

EXAMPLE 5 application of recombinant E.coli in the preparation of (S) -CHBE by catalyzing COBE

The whole recombinant cell prepared in example 4 was used as a catalyst. To the reaction flask, 150ml of a phosphate buffer solution (200 mM) having a pH of 6.0, 9g of isopropyl alcohol, 30g of ethyl 4-chloroacetoacetate and 0.15g of recombinant E.coli wet cells were added. The temperature was controlled at 25℃and the reaction was stirred for 1h. GC detection shows that the substrate conversion rate reaches 99.9%, stirring is stopped, and the mixture is kept stand and separated. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to give ethyl (S) -4-chloro-3-hydroxybutyrate in 97.8% yield and 100% optical purity.

The GC detection analysis conditions were: the column was a 0.3mm capillary column 30m (dimma), FID detector. The chromatographic column temperature is 208 ℃, the gasification and detection temperatures are 230 ℃, the carrier gas flow is 20-30ml/min, the sample injection amount is 0.3-0.5 mu L, and the carrier gas is N ₂.

The optical purity detection conditions of the product are as follows: the chromatographic column is a CP-Chirasil-DEX CB chiral capillary column, and the rest conditions are the same as above.

EXAMPLE 6 application of recombinant E.coli in catalyzing preparation of (S) -CHBE from COBE

The whole recombinant cell prepared in example 4 was used as a catalyst. To the reaction flask, 150ml of a phosphate buffer solution (100 mM) having a pH of 6.5, 40g of isopropyl alcohol, 50g of ethyl 4-chloroacetoacetate and 0.5g of recombinant E.coli wet cells were added. The temperature is controlled at 30 ℃, and the reaction is stirred for 0.5h. GC detection shows that the substrate conversion rate reaches 99.9%, stirring is stopped, and the mixture is kept stand and separated. The organic phase is added with anhydrous sodium sulfate, filtered and concentrated to obtain (S) -4-chloro-3-hydroxybutyric acid ethyl ester, the yield is 98.2 percent, and the optical purity is 100 percent.

EXAMPLE 7 application of recombinant E.coli in the preparation of (S) -CHBE by catalyzing COBE

The whole recombinant cell prepared in example 4 was used as a catalyst. To the reaction flask, 150ml of Tris-HCl buffer (50 mM) having a pH of 7.5, 11g of isopropanol, 21.5g of ethyl 4-chloroacetoacetate and 0.32g of recombinant E.coli wet cells were added. The temperature is controlled at 35 ℃, and the reaction is stirred for 0.5h. GC detection shows that the substrate conversion rate reaches 98.3%, stirring is stopped, and the mixture is kept stand and separated. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to give ethyl (S) -4-chloro-3-hydroxybutyrate in 92.6% yield with 100% optical purity.

EXAMPLE 8 application of recombinant E.coli in the preparation of (S) -CHBE by catalyzing COBE

The whole recombinant cell prepared in example 4 was used as a catalyst. To the reaction flask, 150ml of a phosphate buffer solution (100 mM) having a pH of 6.5, 15g of isopropyl alcohol, 30g of ethyl 4-chloroacetoacetate and 0.3g of recombinant E.coli wet cells were added. The temperature is controlled to be 30 ℃, and the reaction is stirred for 10min. GC detection shows that the substrate conversion rate reaches 99.9%, stirring is stopped, and the mixture is kept stand and separated. The organic phase is added with anhydrous sodium sulfate, filtered and concentrated to obtain (S) -4-chloro-3-hydroxybutyric acid ethyl ester, the yield is 98.4 percent, and the optical purity is 100 percent.

EXAMPLE 9 application of recombinant E.coli in the preparation of (S) -CHBE by catalyzing COBE

The whole recombinant cell prepared in example 4 was used as a catalyst. To the reaction flask, 150ml of a phosphate buffer solution (100 mM) having a pH of 6.5, 40g of isopropyl alcohol, 50g of ethyl 4-chloroacetoacetate and 0.2g of recombinant E.coli wet cells were added. The temperature is controlled at 30 ℃, and the reaction is stirred for 1h. GC detection shows that the substrate conversion rate reaches 99.9%, stirring is stopped, and the mixture is kept stand and separated. The organic phase is added with anhydrous sodium sulfate, dried, filtered and concentrated to obtain (S) -4-chloro-3-hydroxybutyric acid ethyl ester, the yield is 99.2 percent, and the optical purity is 100 percent.

Sequence listing

<110> Lunan pharmaceutical group Co., ltd

<120> Construction and use of recombinant E.coli

<130> 2019

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1017

<212> DNA

<213> Russell Yeast (Zygosaccharomyces rouxii)

<400> 1

atgacaaaag tcttcgtaac aggtgccaac ggattcgttg ctcaacacgt cgttcatcaa 60

ctattagaaa agaactatac agtggttgga tctgtccgtt caactgagaa aggtgataaa 120

ttagctaaat tgctaaacaa tccaaaattt tcatatgaga ttattaaaga tatggtcaat 180

tcgagagatg aattcgataa ggctttacaa aaacattcag atgttgaaat tgtcttacat 240

actgcttcac cagtcttccc aggtggtatt aaagatgttg aaaaagaaat gatccaacca 300

gctgttaatg gtactagaaa tgtcttgtta tcaatcaagg ataacttacc aaatgtcaag 360

agatttgttt acacttcttc attagctgct gtccgtactg aaggtgctgg ttatagtgca 420

gacgaagttg tcaccgaaga ttcttggaac aatattgcat tgaaagatgc caccaaggat 480

gaaggtacag cttatgaggc ttccaagaca tatggtgaaa aagaagtttg gaatttcttc 540

gaaaaaacta aaaatgttaa tttcgatttt gccatcatca acccagttta tgtctttggt 600

cctcaattat ttgaagaata cgttactgat aaattgaact tttccagtga aatcattaat 660

agtataataa aaggtgaaaa gaaggaaatt gaaggttatg aaattgatgt tagagatatt 720

gcaagagctc atatctctgc tgttgaaaat ccagcaacta cacgtcaaag attaattcca 780

gcagttgcac catacaatca acaaactatc ttggatgttt tgaatgaaaa cttcccagaa 840

ttgaaaggta aaatcgatgt tgggaaacca ggttctcaaa atgaatttat taaaaaatat 900

tataaattag ataactcaaa gaccaaaaaa gttttaggtt ttgaattcat ttcccaagag 960

caaacaatca aagatgctgc tgctcaaatc ttgtccgtta aaaatggaaa aaaataa 1017

<210> 2

<211> 338

<212> PRT

<213> Russell Yeast Ketone reductase (Zygosaccharomyces rouxii Ketoreductase)

<400> 2

Met Thr Lys Val Phe Val Thr Gly Ala Asn Gly Phe Val Ala Gln His

1 5 10 15

Val Val His Gln Leu Leu Glu Lys Asn Tyr Thr Val Val Gly Ser Val

20 25 30

Arg Ser Thr Glu Lys Gly Asp Lys Leu Ala Lys Leu Leu Asn Asn Pro

35 40 45

Lys Phe Ser Tyr Glu Ile Ile Lys Asp Met Val Asn Ser Arg Asp Glu

50 55 60

Phe Asp Lys Ala Leu Gln Lys His Ser Asp Val Glu Ile Val Leu His

65 70 75 80

Thr Ala Ser Pro Val Phe Pro Gly Gly Ile Lys Asp Val Glu Lys Glu

85 90 95

Met Ile Gln Pro Ala Val Asn Gly Thr Arg Asn Val Leu Leu Ser Ile

100 105 110

Lys Asp Asn Leu Pro Asn Val Lys Arg Phe Val Tyr Thr Ser Ser Leu

115 120 125

Ala Ala Val Arg Thr Glu Gly Ala Gly Tyr Ser Ala Asp Glu Val Val

130 135 140

Thr Glu Asp Ser Trp Asn Asn Ile Ala Leu Lys Asp Ala Thr Lys Asp

145 150 155 160

Glu Gly Thr Ala Tyr Glu Ala Ser Lys Thr Tyr Gly Glu Lys Glu Val

165 170 175

Trp Asn Phe Phe Glu Lys Thr Lys Asn Val Asn Phe Asp Phe Ala Ile

180 185 190

Ile Asn Pro Val Tyr Val Phe Gly Pro Gln Leu Phe Glu Glu Tyr Val

195 200 205

Thr Asp Lys Leu Asn Phe Ser Ser Glu Ile Ile Asn Ser Ile Ile Lys

210 215 220

Gly Glu Lys Lys Glu Ile Glu Gly Tyr Glu Ile Asp Val Arg Asp Ile

225 230 235 240

Ala Arg Ala His Ile Ser Ala Val Glu Asn Pro Ala Thr Thr Arg Gln

245 250 255

Arg Leu Ile Pro Ala Val Ala Pro Tyr Asn Gln Gln Thr Ile Leu Asp

260 265 270

Val Leu Asn Glu Asn Phe Pro Glu Leu Lys Gly Lys Ile Asp Val Gly Lys

275 280 285

Pro Gly Ser Gln Asn Glu Phe Ile Lys Lys Tyr Tyr Lys Leu Asp Asn

290 295 300

Ser Lys Thr Lys Lys Val Leu Gly Phe Glu Phe Ile Ser Gln Glu Gln

305 310 315 320

Thr Ile Lys Asp Ala Ala Ala Gln Ile Leu Ser Val Lys Asn Gly

325 330 335

Lys Lys

<210> 3

<211> 1032

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 3

ggatccatga caaaagtctt cgtaacaggt gccaacggat tcgttgctca acacgtcgtt 60

catcaactat tagaaaagaa ctatacagtg gttggatctg tccgttcaac tgagaaaggt 120

gataaattag ctaaattgct aaacaatcca aaattttcat atgagattat taaagatatg 180

gtcaattcga gagatgaatt cgataaggct ttacaaaaac attcagatgt tgaaattgtc 240

ttacatactg cttcaccagt cttcccaggt ggtattaaag atgttgaaaa agaaatgatc 300

caaccagctg ttaatggtac tagaaatgtc ttgttatcaa tcaaggataa cttaccaaat 360

gtcaagagat ttgtttacac ttcttcatta gctgctgtcc gtactgaagg tgctggttat 420

agtgcagacg aagttgtcac cgaagattct tggaacaata ttgcattgaa agatgccacc 480

aaggatgaag gtacagctta tgaggcttcc aagacatatg gtgaaaaaga agtttggaat 540

ttcttcgaaa aaactaaaaa tgttaatttc gattttgcca tcatcaaccc agtttatgtc 600

tttggtcctc aattatttga agaatacgtt actgataaat tgaacttttc cagtgaaatc 660

attaatagta taataaaagg tgaaaagaag gaaattgaag gttatgaaat tgatgttaga 720

gatattgcaa gagctcatat ctctgctgtt gaaaatccag caactacacg tcaaagatta 780

attccagcag ttgcaccata caatcaacaa actatcttgg atgttttgaa tgaaaacttc 840

ccagaattga aaggtaaaat cgatgttggg aaaccaggtt ctcaaaatga atttattaaa 900

aaatattata aattagataa ctcaaagacc aaaaaagttt taggttttga attcatttcc 960

caagagcaaa caatcaaaga tgctgctgct caaatcttgt ccgttaaaaa tggaaaaaaa 1020

taataagagc tc 1032

<210> 4

<211> 1032

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 4

ggatccatga ccaaagtttt cgttaccggt gctaacggtt tcgttgctca gcacgttgtt 60

caccagctgc tggaaaaaaa ctacaccgtt gttggttctg ttcgttctac cgaaaaaggt 120

gacaaactgg ctaaactgct gaacaacccg aaattctctt acgaaatcat caaagacatg 180

gttaactctc gtgacgaatt cgacaaagct ctgcagaaac actctgacgt tgaaatcgtt 240

ctgcacaccg cttctccggt tttcccgggt ggtatcaaag acgttgaaaa agaaatgatc 300

cagccggctg ttaacggtac ccgtaacgtt ctgctgtcta tcaaagacaa cctgccgaac 360

gttaaacgtt tcgtttacac ctcttctctg gctgctgttc gtaccgaagg tgctggttac 420

tctgctgacg aagttgttac cgaagactct tggaacaaca tcgctctgaa agacgctacc 480

aaagacgaag gtaccgctta cgaagcttct aaaacctacg gtgaaaaaga agtttggaac 540

ttcttcgaaa aaaccaaaaa cgttaacttc gacttcgcta tcatcaaccc ggtttacgtt 600

ttcggtccgc agctgttcga agaatacgtt accgacaaac tgaacttctc ttctgaaatc 660

atcaactcta tcatcaaagg tgaaaaaaaa gaaatcgaag gttacgaaat cgacgttcgt 720

gacatcgctc gtgctcacat ctctgctgtt gaaaacccgg ctaccacccg tcagcgtctg 780

atcccggctg ttgctccgta caaccagcag accatcctgg acgttctgaa cgaaaacttc 840

ccggaactga aaggtaaaat cgacgttggt aaaccgggtt ctcagaacga attcatcaaa 900

aaatactaca aactggacaa ctctaaaacc aaaaaagttc tgggtttcga attcatctct 960

caggaacaga ccatcaaaga cgctgctgct cagatcctgt ctgttaaaaa cggtaaaaaa 1020

taataagagc tc 1032

<210> 5

<211> 1032

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 5

ggatccatga ccaaagtctt cgtaacaggc gcaaacggtt tcgtggcgca gcacgtagtg 60

caccagctgt tagagaagaa ttatactgtt gtaggcagtg tccgctcgac cgagaaaggg 120

gacaaattgg caaagctgtt aaataaccca aagtttagct acgagattat taaagatatg 180

gtaaactcgc gcgatgagtt tgacaaggct ctgcagaagc actcagatgt cgagatcgta 240

ttgcacaccg ctagtcccgt gtttcccgga gggatcaagg acgtggaaaa agaaatgatt 300

caaccagcgg tcaatggaac tcgcaacgtt cttttgagta ttaaggacaa cttacccaac 360

gtcaaacgtt ttgtatatac gtcttcctta gccgccgtgc gcaccgaggg ggccggttac 420

tcggctgatg aagtagttac ggaagactcg tggaataaca tcgccttaaa ggatgcaact 480

aaagacgaag ggaccgccta tgaagcgtcc aaaacatatg gcgaaaagga agtttggaat 540

ttctttgaga agactaagaa cgttaacttc gatttcgcta tcatcaatcc agtgtatgtt 600

ttcggccctc aactgttcga ggaatacgtc accgataagc tgaatttttc ttctgaaatt 660

attaactcga ttatcaaagg cgaaaaaaag gaaattgaag ggtacgagat cgacgttcgt 720

gatatcgcac gcgcacacat ctctgcagtt gagaacccgg ctaccacacg tcagcgcctt 780

atcccggctg tggcaccgta taatcaacag acgatcttag atgttttaaa cgagaatttc 840

cctgaattga aaggaaagat cgatgtagga aagccaggat cacagaatga atttatcaag 900

aagtattata aactggacaa ttccaaaact aagaaagttt taggatttga gttcattagt 960

caggaacaaa caatcaagga tgctgctgca cagatcctgt ccgttaaaaa cggtaagaag 1020

taataagagc tc 1032

<210> 6

<211> 783

<212> DNA

<213> Bacillus subtilis (Bacillus subtilis)

<400> 6

atgtatccgg atttaaaagg aaaagtcgtc gctattacag gagctgcttc agggctcgga 60

aaggcgatgg ccattcgctt cggcaaggag caggcaaaag tggttatcaa ctattatagt 120

aataaacaag atccgaacga ggtaaaagaa gaggtcatca aggcgggcgg tgaagctgtt 180

gtcgtccaag gagatgtcac gaaagaggaa gatgtaaaaa atatcgtgca aacggcaatt 240

aaggagttcg gcacactcga tattatgatt aataatgccg gtcttgaaaa tcctgtgcca 300

tctcacgaaa tgccgctcaa ggattgggat aaagtcatcg gcacgaactt aacgggtgcc 360

tttttaggaa gccgtgaagc gattaaatat ttcgtagaaa acgatatcaa gggaaatgtc 420

attaacatgt ccagtgtgca cgcgtttcct tggccgttat ttgtccacta tgcggcaagt 480

aaaggcggga taaagctgat gacagaaaca ttagcgttgg aatacgcgcc gaagggcatt 540

cgcgtcaata atattgggcc aggtgcgatc aacacgccaa tcaatgctga aaaattcgct 600

gaccctaaac agaaagctga tgtagaaagc atgattccaa tgggatatat cggcgaaccg 660

gaggagatcg ccgcagtagc agcctggctt gcttcgaagg aagccagcta cgtcacaggc 720

atcacgttat tcgcggacgg cggtatgaca caatatcctt cattccaggc aggccgcggt 780

taa 783

<210> 7

<211> 260

<212> PRT

<213> Bacillus subtilis glucose dehydrogenase (Bacillus subtilis glucose dehydrogenase)

<400> 7

Met Tyr Pro Asp Leu Lys Gly Lys Val Val Ala Ile Thr Gly Ala Ala

1 5 10 15

Ser Gly Leu Gly Lys Ala Met Ala Ile Arg Phe Gly Lys Glu Gln Ala

20 25 30

Lys Val Val Ile Asn Tyr Tyr Ser Asn Lys Gln Asp Pro Asn Glu Val

35 40 45

Lys Glu Glu Val Ile Lys Ala Gly Gly Glu Ala Val Val Val Gln Gly

50 55 60

Asp Val Thr Lys Glu Glu Asp Val Lys Asn Ile Val Gln Thr Ala Ile

65 70 75 80

Lys Glu Phe Gly Thr Leu Asp Ile Met Ile Asn Asn Ala Gly Leu Glu

85 90 95

Asn Pro Val Pro Ser His Glu Met Pro Leu Lys Asp Trp Asp Lys Val

100 105 110

Ile Gly Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala Ile

115 120 125

Lys Tyr Phe Val Glu Asn Asp Ile Lys Gly Asn Val Ile Asn Met Ser

130 135 140

Ser Val His Ala Phe Pro Trp Pro Leu Phe Val His Tyr Ala Ala Ser

145 150 155 160

Lys Gly Gly Ile Lys Leu Met Thr Glu Thr Leu Ala Leu Glu Tyr Ala

165 170 175

Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Ile Asn Thr

180 185 190

Pro Ile Asn Ala Glu Lys Phe Ala Asp Pro Lys Gln Lys Ala Asp Val

195 200 205

Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Glu Pro Glu Glu Ile Ala

210 215 220

Ala Val Ala Ala Trp Leu Ala Ser Lys Glu Ala Ser Tyr Val Thr Gly

225 230 235 240

Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Gln Tyr Pro Ser Phe Gln

245 250 255

Ala Gly Arg Gly

260

<210> 8

<211> 955

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 8

gtcgacttaa tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa 60

ataattttgt ttaactttaa gaaggagata tacatatgaa atacctgctg ccgaccgctg 120

ctgctggtct gctgctcctc gctgcccagc cggcgatggc catgtatccg gatttaaaag 180

gaaaagtcgt cgctattaca ggagctgctt cagggctcgg aaaggcgatg gccattcgct 240

tcggcaagga gcaggcaaaa gtggttatca actattatag taataaacaa gatccgaacg 300

aggtaaaaga agaggtcatc aaggcgggcg gtgaagctgt tgtcgtccaa ggagatgtca 360

cgaaagagga agatgtaaaa aatatcgtgc aaacggcaat taaggagttc ggcacactcg 420

atattatgat taataatgcc ggtcttgaaa atcctgtgcc atctcacgaa atgccgctca 480

aggattggga taaagtcatc ggcacgaact taacgggtgc ctttttagga agccgtgaag 540

cgattaaata tttcgtagaa aacgatatca agggaaatgt cattaacatg tccagtgtgc 600

acgcgtttcc ttggccgtta tttgtccact atgcggcaag taaaggcggg ataaagctga 660

tgacagaaac attagcgttg gaatacgcgc cgaagggcat tcgcgtcaat aatattgggc 720

caggtgcgat caacacgcca atcaatgctg aaaaattcgc tgaccctaaa cagaaagctg 780

atgtagaaag catgattcca atgggatata tcggcgaacc ggaggagatc gccgcagtag 840

cagcctggct tgcttcgaag gaagccagct acgtcacagg catcacgtta ttcgcggacg 900

gcggtatgac acaatatcct tcattccagg caggccgcgg ttaataagcg gccgc 955

<210> 9

<211> 955

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 9

gtcgacttaa tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa 60

ataattttgt ttaactttaa gaaggagata tacatatgaa atacctgctg ccgaccgctg 120

ctgctggtct gctgctcctc gctgcccagc cggcgatggc catgtaccct gacttgaaag 180

gtaaggtcgt ggcgattacg ggcgccgcct cgggtcttgg gaaggccatg gccattcgct 240

tcggtaaaga acaagccaag gtcgtgatta actattattc taacaaacag gacccgaatg 300

aggtaaaaga agaagtcatt aaggcgggag gggaggccgt cgtggtacag ggagatgtaa 360

ctaaggaaga agatgtaaaa aacatcgtcc aaacggccat taaagagttt ggaacactgg 420

acattatgat taacaacgct ggattggaaa atcccgtgcc ttcgcatgag atgcctctga 480

aagattggga caaggttatc gggacgaact tgacaggagc gttcttgggc tctcgtgagg 540

cgatcaaata cttcgtggaa aacgatatta aaggtaacgt catcaatatg tcctctgtac 600

acgcttttcc atggcccctg ttcgttcact atgcggctag taaaggaggg attaagttga 660

tgactgagac gcttgcctta gaatatgccc ctaaagggat tcgtgttaat aatattggcc 720

ccggtgcaat taacacccct atcaatgccg agaaatttgc ggacccgaag caaaaggcgg 780

atgtggagag catgattccg atgggataca tcggcgagcc agaggagatc gccgccgtag 840

ctgcttggct tgcatcgaag gaggcctctt atgtcacggg catcacattg tttgcggacg 900

ggggaatgac acagtacccg agttttcaag cgggacgtgg ataataagcg gccgc 955

<210> 10

<211> 955

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 10

gtcgacttaa tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa 60

ataattttgt ttaactttaa gaaggagata tacatatgaa atacctgctg ccgaccgctg 120

ctgctggtct gctgctcctc gctgcccagc cggcgatggc catgtacccg gacctgaaag 180

gtaaagttgt tgctatcacc ggtgctgctt ctggtctggg taaagctatg gctatccgtt 240

tcggtaaaga acaggctaaa gttgttatca actactactc taacaaacag gacccgaacg 300

aagttaaaga agaagttatc aaagctggtg gtgaagctgt tgttgttcag ggtgacgtta 360

ccaaagaaga agacgttaaa aacatcgttc agaccgctat caaagaattc ggtaccctgg 420

acatcatgat caacaacgct ggtctggaaa acccggttcc gtctcacgaa atgccgctga 480

aagactggga caaagttatc ggtaccaacc tgaccggtgc tttcctgggt tctcgtgaag 540

ctatcaaata cttcgttgaa aacgacatca aaggtaacgt tatcaacatg tcttctgttc 600

acgctttccc gtggccgctg ttcgttcact acgctgcttc taaaggtggt atcaaactga 660

tgaccgaaac cctggctctg gaatacgctc cgaaaggtat ccgtgttaac aacatcggtc 720

cgggtgctat caacaccccg atcaacgctg aaaaattcgc tgacccgaaa cagaaagctg 780

acgttgaatc tatgatcccg atgggttaca tcggtgaacc ggaagaaatc gctgctgttg 840

ctgcttggct ggcttctaaa gaagcttctt acgttaccgg tatcaccctg ttcgctgacg 900

gtggtatgac ccagtacccg tctttccagg ctggtcgtgg ttaataagcg gccgc 955

Claims

1. A recombinant escherichia coli, which is characterized in that a ketoreductase KRD gene of a saccharomyces rouxii and a glucose dehydrogenase GDH gene of bacillus subtilis are expressed simultaneously; the ketoreductase KRD gene sequence of the Russell yeast is shown as SEQ ID NO. 4; the GDH gene sequence of the glucose dehydrogenase of the bacillus subtilis is shown as SEQ ID NO. 9; the ketoreductase KRD gene of the Russell yeast and the glucose dehydrogenase GDH gene of the bacillus subtilis are simultaneously expressed in the periplasmic space of the recombinant escherichia coli cells.

2. A method for constructing recombinant escherichia coli as set forth in claim 1, wherein the recombinant escherichia coli is constructed by firstly connecting an expression vector with a ketoreductase KRD gene in vitro, connecting the expression vector with a glucose dehydrogenase GDH gene after verification of correctness, verifying the correctness of the connection of the vector by double digestion and sequencing, and transforming a host bacterium by a chemical method by the correct expression vector.

3. The use of the recombinant escherichia coli according to claim 1 in the preparation of (S) -4-chloro-3-hydroxybutyric acid ethyl ester by asymmetric reduction of 4-chloroacetoacetic acid ethyl ester.

4. A method for producing (S) -4-chloro-3-hydroxybutyric acid ethyl ester is characterized in that 4-chloroacetoacetic acid ethyl ester is taken as a raw material, isopropanol is taken as a hydrogen donor, and the recombinant escherichia coli of claim 1 is added for conversion reaction to prepare the (S) -4-chloro-3-hydroxybutyric acid ethyl ester.

5. The method for producing (S) -4-chloro-3-hydroxybutyric acid ethyl ester according to claim 4, wherein the aqueous reaction system is provided with phosphate buffer solution, the concentration of the phosphate buffer solution is 50-200 mM, and the pH is 6.0-7.0; the mass volume ratio of the aqueous phase buffer solution to the substrate 4-chloroacetoacetic acid ethyl ester is 3-7:1, ml/g.

6. The method for producing ethyl (S) -4-chloro-3-hydroxybutyrate according to claim 4, wherein the mass ratio of isopropyl alcohol to ethyl 4-chloroacetoacetate is 0.3 to 0.8:1.

7. The method for producing ethyl (S) -4-chloro-3-hydroxybutyrate according to claim 4, wherein the mass of the recombinant E.coli cells is 0.5 to 1.5% of the mass of ethyl 4-chloroacetoacetate.

8. The process for producing ethyl (S) -4-chloro-3-hydroxybutyrate as claimed in claim 4, wherein the reaction temperature is 25 to 35 ℃.