Alcohol dehydrogenase, recombinant genetically engineered bacterium and application of recombinant genetically engineered bacterium in asymmetric reduction of prochiral carbonyl compounds

Abstract

The invention discloses ethanol dehydrogenase, recombinant genetically engineered bacteria and application of the ethanol dehydrogenase in asymmetric reduction of prochiral carbonyl compounds, the ethanol dehydrogenase has good biological performance, heterologous expression in Escherichia coli engineering bacteria is achieved, intracellular coenzyme can be directly utilized when whole cells are adopted as a catalyst, and the catalyst has a good application prospect. Zero addition of the coenzyme is realized, the enzyme has the advantages of high activity, high stereoselectivity, wider substrate spectrum and the like, prochiral ketone compounds can be greenly and efficiently catalyzed to be converted into corresponding chiral alcohols, and the problems of low conversion rate, low yield, low e.e. Value, complex reaction process, more byproducts and the like in a traditional chemical method are solved. The ethanol dehydrogenase has good tolerance to isopropanol, and the isopropanol can be used as a hydrogen source to realize the circulation of coenzyme. And the byproduct acetone generated by the enzyme catalytic reaction and the isopropanol remaining in the reaction can be separated and recovered by a technical means, so that the production cost is greatly reduced.

Description

(I) Technical field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to an alcohol dehydrogenase, a coding gene, a recombinant engineering bacterium and an application thereof in preparing highly optically active chiral alcohols by asymmetric reduction of potentially chiral carbonyl compounds. (II) Background technology

[0002] Chiral alcohols are important intermediates for many best-selling drugs and chiral chemicals. Among them, chiral α-phenylethanol and its derivatives are important chiral building blocks and are widely used in the manufacture of high value-added products such as medicines, fine chemicals and agricultural chemicals. For example, (1S)-2-chloro-1-(3,4-difluorophenyl)ethanol is a new antiplatelet drug intermediate, (R)-3,5-bis(trifluoromethyl)-phenylethanol is a traditional Chinese medicine intermediate for the chemotherapy antiemetic drug aprepitant; Atorvastatin calcium is a third-generation fully synthetic statin drug with outstanding lipid-lowering effects. It is widely used in the prevention and treatment of cardiovascular and cerebrovascular diseases and is currently the only prescription drug with cumulative sales exceeding US$150 billion. (S)-4-chloro-3-hydroxybutyric acid ethyl ester is the first chiral hydroxyl group in the construction of atorvastatin calcium; Penems and carbapenems have a broad antibacterial spectrum, outstanding antibacterial activity and stability against most β-lactamases, and are considered to be the "first-line drugs" for the treatment of severe nosocomial infections. 4-Acetoxyazetidinone [(2R,3R)-3-(R)-1-(tert-butyldimethylsilyloxy)ethyl-4-oxazetidin-2-yl acetate] is a key chiral intermediate for the production of penems and carbapenems, among which (R)-(-)-1,3-butanediol is a key intermediate for the production of 4-acetoxyazetidinone, and (2S,3R)-2-[(benzoylamino)methyl]-3-hydroxybutyric acid methyl ester is a hot spot for the synthesis of 4-acetoxyazetidinone. The new phosphine ligand has the advantages of high reactivity and enantioselectivity, and can be used in a variety of metal-catalyzed reactions. In its latest synthetic route, chiral symmetrical diols such as (2R,3R)-2,3-butanediol are widely used as chiral side chain skeletons.

[0003] Alcohol dehydrogenase (ADH) is present in large quantities in human and animal liver, plant and microbial cells. As a key enzyme in the metabolism of major short-chain alcohols in organisms, it plays an important role in many physiological processes. It has a wide range of substrate specificity and is a key enzyme that catalyzes the asymmetric reduction of prochiral ketones to generate chiral alcohols.

[0004] Traditional chiral alcohol synthesis requires expensive chiral noble metal catalysts, harsh reaction conditions, and flammable and explosive borohydride reducing agents, and has the disadvantages of low product optical purity, complex separation and purification process, difficult treatment of "three wastes", and high production costs. The ADH-catalyzed carbonyl asymmetric reduction reaction has outstanding advantages such as a wide range of enzyme sources, outstanding activity and stereoselectivity, mild reaction conditions, high product optical purity, low production cost, and environmental friendliness. It has broad application prospects in the synthesis of chiral alcohols. Therefore, it is undoubtedly of great significance to find an economical and readily available ADH that can catalyze asymmetric reduction with high stereoselectivity at high substrate concentrations without adding or adding very small amounts of coenzymes. (III) Summary of the invention

[0005] The present invention aims to provide an alcohol dehydrogenase, an encoding gene, a recombinant genetic engineering bacterium and the use thereof in the asymmetric reduction of a potential chiral carbonyl compound to prepare a chiral alcohol with high optical activity. The enzyme has the advantages of high activity, strong stereospecificity, mild reaction conditions, simple operation and the like, can economically and greenly produce a variety of chiral alcohols with high optical selectivity, has a good application prospect, and solves the problems of low optical purity of products, complex separation and purification process, difficulty in treating "three wastes", high production cost and the like in traditional chiral alcohol production methods.

[0006] The technical solution adopted by the present invention is:

[0007] The invention provides a high-activity alcohol dehydrogenase derived from Candida orthopsilosis Co. The amino acid sequence of the alcohol dehydrogenase is shown in SEQ ID NO.2.

[0008] The present invention also provides a gene encoding the alcohol dehydrogenase, and the nucleotide sequence is shown in SEQ ID NO.1.

[0009] The present invention also relates to a recombinant vector containing the gene encoding the alcohol dehydrogenase, and a recombinant genetically engineered bacterium prepared by transforming the recombinant vector. The basic vector of the recombinant vector is not limited, as long as it can maintain its replication or autonomous replication in various host cells of prokaryotic and / or eukaryotic cells; the vector can be various conventional vectors in the art, such as various plasmids, phages or virus vectors, etc., preferably using pET28a (+) plasmid as an expression vector; the recombinant genetically engineered bacterium uses Escherichia coli as an expression host (Escherichia coli BL21 cells or Escherichia coli DH5α).

[0010] The invention provides an application of the alcohol dehydrogenase in catalyzing the asymmetric reduction of a prochiral carbonyl compound to prepare a chiral alcohol. The application method comprises the following steps: using wet bacteria obtained by fermentation culture of a recombinant genetic engineering bacterium containing an alcohol dehydrogenase encoding gene as a catalyst, using a prochiral carbonyl compound as a substrate, and using pure isopropanol as a reaction medium to form a reaction system, reacting at 200-700 rpm (preferably 200 rpm) and 37° C., obtaining a reaction solution containing the chiral alcohol after the reaction is completed, and separating and purifying the reaction solution to obtain the desired chiral alcohol.

[0011] Furthermore, the prochiral carbonyl compound includes single-chain ketone compounds (1), aromatic ketone compounds (2) / (4), heterocyclic ketone compounds (such as pyridine, thiazole, etc.) (3), etc.:

[0012]

[0013] In Formula 1, R is a C1-C4 alkane group;

[0014] In formula 2, R1-R5 are each independently H, F, Cl, Br, I, NO2, CF3, CN, C1-C4 alkyl or halogenated alkyl.

[0015] Further, the prochiral carbonyl compound includes: (RS)-(+)-3-methyl-2-butanol, (RS)-(+)-4-methyl-2-pentanol, (RS)-(+)-2-hexanol, (RS)-1-(2-chlorophenyl)ethanol, (RS)-1-(3-chlorophenyl)ethanol, (RS)-1-(2-fluorophenyl)ethanol, (RS)-1-(2-nitrophenyl)ethanol, (RS)-1-(4-bromophenyl)ethanol, (RS)-1-(4-iodophenyl)ethanol, (RS)-(-)-4-(1-hydroxyethyl)pyridine, (RS)-1-(2-naphthyl)ethanol, (RS)-1-naphthyl-1-ethanol, (RS)-1-(thiazol-2-yl)ethane-1-ol, and (RS)-1-(3-bromo-2-pyridyl)ethanol.

[0016] Furthermore, in the reaction system, the amount of catalyst used is 50-300 g / L (preferably 50 g / L) based on the weight of the wet bacteria, and the initial concentration of the substrate added is 0.1-1 M (preferably 0.3 M).

[0017] Further, the wet bacterial cells are prepared as follows: a recombinant engineered bacterium containing an alcohol dehydrogenase encoding gene is inoculated into an LB culture medium containing a final concentration of 50 mg / L kanamycin, and cultured at 37°C for 8 hours to obtain a seed solution; then the seed solution is inoculated into a sterile LB liquid culture medium containing a final concentration of 50 mg / L kanamycin at an inoculum amount of 2% by volume, and cultured at 37°C for 1.5-2.5 hours to make the bacterial cell concentration OD600 be 0.4-0.8, and then isopropylthio-β-D-galactoside (IPTG) with a final concentration of 0.1-1.0 mM (preferably 0.1 mM) is added to the culture solution, and after inducing expression at 26°C for 12 hours, the wet bacterial cells are collected by centrifugation at 4°C and 4000 rpm for 10-20 minutes; LB liquid culture medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, and the solvent is deionized water, pH 7.0.

[0018] The alcohol dehydrogenase of the present invention can be used to perform catalysis in the form of whole cells, or in the form of crude enzyme liquid obtained by cell crushing or pure enzyme extracted by complete crushing. In addition, the crude enzyme liquid or pure enzyme can be prepared into an immobilized enzyme or wet bacteria can be prepared into an immobilized cell form for catalysis by using a specific immobilization technology.

[0019] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0020] (1) The alcohol dehydrogenase mined from the genome database of the present invention has good biological properties and achieves heterologous expression in Escherichia coli engineered bacteria. When the whole cell is used as a catalyst, the intracellular coenzyme can be directly used, achieving zero addition of the coenzyme.

[0021] (2) The alcohol dehydrogenase of the present invention can greenly and efficiently catalyze the conversion of potential chiral ketone compounds such as single-chain ketone compounds, aromatic ketone compounds, heterocyclic ketone compounds into corresponding chiral alcohols, has high activity, high stereoselectivity and a wide substrate spectrum, and the substrate conversion rate and stereoselectivity are both as high as 99%, which solves the problems of low conversion rate, low yield, low ee value, complex reaction process, and many by-products in traditional chemical methods.

[0022] (3) The alcohol dehydrogenase of the present invention has good tolerance to isopropanol and can use isopropanol as a hydrogen source to realize the circulation of the coenzyme. Moreover, the byproduct acetone produced by the enzyme catalytic reaction and the isopropanol remaining in the reaction can be separated and recovered by technical means, which greatly reduces the production cost. (IV) Description of the drawings

[0023] Figure 1 , GC graphs of (RS)-(+)-3-methyl-2-butanol (a) and (S)-(+)-3-methyl-2-butanol (b) in Example 3.

[0024] Figure 2 , GC graphs of (RS)-(+)-4-methyl-2-pentanol (a) and (S)-(+)-4-methyl-2-pentanol (b) in Example 4.

[0025] Figure 3 , GC graphs of (RS)-(+)-2-hexanol (a) and (S)-(+)-2-hexanol (b) in Example 5.

[0026] Figure 4 , GC graphs of (RS)-1-(2-chlorophenyl)ethanol (a) and (S)-1-(2-chlorophenyl)ethanol (b) in Example 6.

[0027] Figure 5 , GC graphs of (RS)-1-(3-chlorophenyl)ethanol (a) and (S)-1-(3-chlorophenyl)ethanol (b) in Example 7.

[0028] Figure 6 , GC graphs of (RS)-1-(2-fluorophenyl)ethanol (a) and (S)-1-(2-fluorophenyl)ethanol (b) in Example 8.

[0029] Figure 7 , GC graphs of (RS)-1-(2-nitrophenyl)ethanol (a) and (S)-1-(2-nitrophenyl)ethanol (b) in Example 9.

[0030] Figure 8 , GC graphs of (RS)-1-(4-bromophenyl)ethanol (a) and (S)-1-(4-bromophenyl)ethanol (b) in Example 10.

[0031] Fig. 9 , GC charts of (RS)-1-(4-iodophenyl)ethanol (a) and (S)-1-(4-iodophenyl)ethanol (b) in Example 11.

[0032] Fig.10 , GC graphs of (RS)-(-)-4-(1-hydroxyethyl)pyridine (a) and (S)-(-)-4-(1-hydroxyethyl)pyridine (b) in Example 12.

[0033] Fig.11 , GC graphs of (RS)-1-(2-naphthyl)ethanol (a) and (S)-(-)-1-(2-naphthyl)ethanol (b) in Example 13.

[0034] Fig.12 , GC charts of (RS)-1-naphthyl-1-ethanol (a) and (S)-1-naphthyl-1-ethanol (b) in Example 14.

[0035] Fig.13, GC charts of (RS)-1-(thiazol-2-yl)ethane-1-ol (a) and (S)-1-(thiazol-2-yl)ethane-1-ol (b) in Example 15.

[0036] Fig.14 , GC charts of (RS)-1-(3-bromo-2-pyridinyl)ethanol (a) and (S)-1-(3-bromo-2-pyridinyl)ethanol (b) in Example 16. (V) Specific implementation methods

[0037] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0038] LB liquid culture medium: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, solvent is deionized water, pH 7.0.

[0039] LB plates were prepared by adding 20 g / L agar to LB liquid medium.

[0040] Example 1. Screening of alcohol dehydrogenase, construction of recombinant vector and preparation of recombinant transformant

[0041] 1. Screening of alcohol dehydrogenase

[0042] The amino acid sequence of alcohol dehydrogenase CMCR from Candida metapsilosis with good biocatalytic performance was used as a probe, and a pBLAST search was performed in the NCBI database to select a batch of predetermined alcohol dehydrogenase gene sequences. Then, these candidate genes were cloned and expressed respectively to construct recombinant Escherichia coli transformants. By measuring the conversion rate and stereoselectivity of these alcohol dehydrogenases to substrates 2-hexanone, acetophenone and their similar compounds, the cloned and expressed enzymes were repeatedly compared and screened. Some screening results are shown in Table 1, and finally the alcohol dehydrogenase CoC with the best catalytic activity was obtained, which was annotated by NCBI as Candida orthopsilosis Co, NCBI accession number: XP_003865936.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0043] SEQ ID NO.2

[0044] MFKSISILKSIKPTNSRIPHILSIPLIRNMSIPSTQYGFVYTKQSGLNLQSDLPVHKPKAGQLLLKIDAVGLCHSDLHVIYEGLDCGDNYVMGHEIAGTVAAVGDDVNSYKVGDRVACVGPNGCGGCKYCRGSIDNVCKRAFGDWFGLGYDGGYQQYLLVTRPRNLALIPDNVSSDVAAASTD AVLTPYHAIKMAKVSPTSNLLLIGAGGLGGNAIQVAKSFGAKVTVLDKKKEARDQAKKLGADEVYESLPGSISPGSFSACFDFVSVQATFDLCQKYVEPKGVIVPVGLGAPKLSFDLGDLALREIQVLGSFWGTTNDLDEVLQLVSEGKVKPVVQSAKLKELPEYIEKLRKNAYEGRVVFNP.

[0045] Table 1. Partial screening results of alcohol dehydrogenase

[0046]

[0047] 2. Construction of recombinant vector

[0048] Based on the sequence of the alcohol dehydrogenase gene predicted to be from Candida orthopsilosis Co (NCBI accession number: XP_003865936.1) included in NCBI, Beijing Qingke Biotechnology Co., Ltd. was commissioned to directly synthesize the DNA sequence (SEQ ID NO.1) after codon optimization and modification, and construct it into the vector pET-28a to obtain the recombinant vector pET-28a-CoC.

[0049] 3. Construction of recombinant genetically engineered bacteria

[0050] First, the competent cells of Escherichia coli BL21 (DE3) (Invitrogen) stored at -80°C were placed in an ice bath at 0°C for 10 min, and then 1 μL of the recombinant vector pET-28a-CoC was added in a clean bench, ice bathed at 0°C for 30 min, heat shocked in a 42°C water bath for 90 s, and ice bathed at 0°C for 2 min. 600 μL of LB medium was added and cultured at 37°C, 200 rpm in a shaking incubator for 1 h; spread on an LB plate containing 50 μg / mL kanamycin resistance and cultured at 37°C for 8-12 h. Clones were randomly picked to extract plasmids for sequencing and identification, and recombinant E. coli BL21 (DE3) / pET-28a-CoC containing the expression recombinant plasmid was screened.

[0051] Example 2, expression of alcohol dehydrogenase CoC

[0052] The recombinant Escherichia coli constructed in Example 1 was inoculated into LB liquid culture medium containing 50 μg / mL kanamycin, and cultured overnight at 37°C and 180 rpm. The bacterial solution was inoculated into a 250 mL Erlenmeyer flask containing 100 mL of LB liquid culture medium at a 1% (v / v) inoculation rate, and cultured on a shaker at 37°C and 180 rpm. When the OD600 of the culture solution reached 0.6, IPTG with a final concentration of 0.1 mM was added as an inducer, and induced at 26°C for 14 h. The culture solution was centrifuged, and the wet bacteria were resuspended with 3 mL of phosphate buffer solution (pH 7.0, 50 mM), transferred to an EP tube, and stored at -20°C after centrifugation.

[0053] The wet bacteria were resuspended in phosphate buffer solution (pH 7.0, 50 mM) to form a 50 g / L bacterial solution, and ultrasonically disrupted at 210 W for 30 min in an ice bath, with a working time of 3 s and a 1 s interval. The supernatant was collected at low temperature (4°C) to obtain the crude enzyme solution of alcohol dehydrogenase CoC.

[0054] Example 3-16, Asymmetric reduction of carbonyl compounds catalyzed by alcohol dehydrogenase CoC

[0055] Reaction system: 0.05 g of alcohol dehydrogenase CoC wet bacteria prepared by the method of Example 2, 1 mL of isopropanol, and 300 mM of substrate ketoester or halogenated aromatic ketone or other carbonyl compounds in Table 1 constitute 1 mL of the reaction system.

[0056] The reaction was shaken at 37°C and 200 rpm. Samples were taken every 30 minutes during the reaction, and real-time GC monitoring or TLC detection was performed. The TLC developing solvent was n-hexane: ethyl acetate = 3:1. The reaction was stopped when the concentration of the reaction product no longer increased or the concentration of the substrate no longer decreased. The reaction solution was centrifuged to obtain the supernatant, and a small amount of anhydrous sodium sulfate was added as the stationary phase. The peak areas of the substrate and product were detected by GC. The substrate conversion rate and the product ee value were calculated based on the peak area and concentration standard curve of the substrate standard. The results are shown in Table 2. Figure 1-Figure 14 .

[0057] GC detection method: Rt-βDEXsa chiral column, injection port and detector temperature 230°C, carrier gas N2, flow rate 1m / s. Column oven temperature, 80°C for 3min, 10°C / min temperature rise to 140°C, and 3min for 3min (Examples 3 and 4); 45°C for 5min, 5°C / min temperature rise to 70°C, and 3min for 3min (Example 5); 80°C for 3min, 10°C / min temperature rise to 140°C, and 3min for 3min, 20°C / min temperature rise to 180°C, and 5min for 5min (Examples 6, 7, 8, 9, 10, 11); 160°C for 5min, 5°C / min temperature rise to 180°C, and 3min for 3min (Examples 13 and 14); 140°C for 5min, 30°C / min temperature rise to 180°C, and 3min for 3min (Examples 12, 15, and 16).

[0058] Table 2. Results of asymmetric reduction of carbonyl compounds catalyzed by CoC

[0059]

Claims

1. A highly active alcohol dehydrogenase, characterized in that: The amino acid sequence of the alcohol dehydrogenase is shown in SEQ ID NO.

2.

2. A recombinant genetically engineered bacterium containing the gene encoding the alcohol dehydrogenase according to claim 1.

3. Use of the alcohol dehydrogenase according to claim 1 in catalyzing the asymmetric reduction of prochiral carbonyl compounds to prepare chiral alcohols.

4. The use according to claim 3, characterized in that The application method comprises the following steps: using wet bacteria obtained by fermentation culture of recombinant genetically engineered bacteria containing an alcohol dehydrogenase encoding gene as a catalyst, using a potentially chiral carbonyl compound as a substrate, and using isopropanol as a reaction medium to form a reaction system, and reacting at 200-700 rpm and 37° C. After the reaction is completed, a reaction liquid containing chiral alcohol is obtained, and the reaction liquid is separated and purified to obtain the desired chiral alcohol.

5. The use according to claim 4, characterized in that The potential chiral carbonyl compounds include single-chain ketone compounds, aromatic ketone compounds, and heterocyclic ketone compounds.

6. The use according to claim 5, characterized in that The prochiral carbonyl compounds include: (RS)-(+)-3-methyl-2-butanol, (RS)-(+)-4-methyl-2-pentanol, (RS)-(+)-2-hexanol, (RS)-1-(2-chlorophenyl)ethanol, (RS)-1-(3-chlorophenyl)ethanol, (RS)-1-(2-fluorophenyl)ethanol, (RS)-1-(2-nitrophenyl)ethanol, (RS)-1-(4-bromophenyl)ethanol, (RS)-1-(4-iodophenyl)ethanol, (RS)-(-)-4-(1-hydroxyethyl)pyridine, (RS)-1-(2-naphthyl)ethanol, (RS)-1-naphthyl-1-ethanol, (RS)-1-(thiazol-2-yl)ethane-1-ol, and (RS)-1-(3-bromo-2-pyridyl)ethanol.

7. The use according to claim 4, characterized in that In the reaction system, the amount of catalyst used is 50-300 g / L based on the weight of wet bacteria, and the initial concentration of substrate added is 0.1-1 M.

8. The use according to claim 4, characterized in that The wet bacterial cells are prepared as follows: the recombinant engineered bacteria containing the alcohol dehydrogenase encoding gene are inoculated into LB culture solution containing kanamycin at a final concentration of 50 mg / L, and cultured at 37° C. for 8 hours to obtain seed solution; The seed liquid was then inoculated into a sterile LB liquid culture medium containing a final concentration of 50 mg / L kanamycin at an inoculum concentration of 2% by volume, and cultured at 37°C for 1.5-2.5 h to make the bacterial concentration OD600 of 0.4-0.

8. Isopropylthio-β-D-galactoside with a final concentration of 0.1-1.0 mM was then added to the culture medium. After inducing expression at 26°C for 12 h, the culture medium was centrifuged at 4°C and 4000 rpm for 10-20 min to collect the wet bacteria.

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