Ethanol dehydrogenase, recombinant genetically engineered bacteria and their application in the asymmetric reduction of potentially chiral carbonyl compounds
By using a highly active alcohol dehydrogenase derived from Candida linearis, recombinant genetically engineered bacteria were expressed in Escherichia coli, solving the problems of low product purity, complex separation, and high cost in traditional chiral alcohol synthesis, and realizing an efficient and environmentally friendly asymmetric reduction reaction.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- HANGZHOU WENDEJIE BIOTECHNOLOGY CO LTD
- Filing Date
- 2024-12-10
- Publication Date
- 2026-05-05
AI Technical Summary
Traditional chiral alcohol synthesis methods suffer from problems such as low optical purity of products, complex separation and purification processes, difficulty in treating "three wastes" (waste gas, wastewater, and solid waste), and high production costs. In addition, they require expensive chiral precious metal catalysts and flammable and explosive borohydride reducing agents.
A high-activity alcohol dehydrogenase derived from Candida linearis is used, expressed in Escherichia coli through recombinant genetically engineered bacteria, and the whole cell is used as a catalyst to achieve the asymmetric reduction of prochiral carbonyl compounds. The reaction conditions are mild, the coenzyme is recycled, the byproducts are easy to separate, and the production cost is reduced.
This method enables the efficient preparation of highly optically selective chiral alcohols, achieving a substrate conversion rate and stereoselectivity of 99%. The reaction conditions are mild, reducing production costs and environmental pollution.
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Abstract
Description
(I) Technical Field
[0001] This invention belongs to the field of bioengineering technology, specifically relating to an alcohol dehydrogenase, its encoding gene, a recombinant engineered bacterium, and its application in the asymmetric reduction of prochiral carbonyl compounds to prepare highly optically active chiral alcohols. (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 an important class of chiral building blocks widely used in the manufacture of high-value-added products such as pharmaceuticals, fine chemicals, and agrochemicals. For example, (1S)-2-chloro-1-(3,4-difluorophenyl)ethanol is an intermediate for a novel antiplatelet aggregation drug; (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 and is widely used in the prevention and treatment of cardiovascular and cerebrovascular diseases. It is currently the only prescription drug with cumulative sales exceeding US$150 billion; (S)-4-chloro-3-hydroxybutyrate ethyl ester is the first chiral hydroxyl group used to construct atorvastatin calcium. Penicillenes and carbapenems possess broad antibacterial spectra, outstanding antibacterial activity, and stability against most β-lactamases, making them considered "first-line drugs" for treating severe hospital-acquired infections. 4-Acetoxyaziridine [(2R,3R)-3-(R)-1-(tert-butyldimethylsilyloxy)ethyl-4-oxazabut-2-ylacetate] is a key chiral intermediate in the production of penicillenes and carbapenems. Among these, (R)-(-)-1,3-butanediol is a key intermediate in the production of 4-acetoxyaziridine, while methyl (2S,3R)-2-[(benzoylamino)methyl]-3-hydroxybutyrate is a hot topic in the synthesis of 4-acetoxyaziridine. Novel phosphine ligands possess advantages such as high reactivity and enantioselectivity, and can be used in various metal-catalyzed reactions. In their latest synthetic routes, chiral symmetrical diols such as (2R,3R)-2,3-butanediol are widely used as chiral side-chain skeletons.
[0003] Alcohol dehydrogenase (ADH) is abundant in the livers of humans and animals, as well as in plant and microbial cells. As a key enzyme in the metabolism of short-chain alcohols in organisms, it plays an important role in many physiological processes and has broad substrate specificity. It is a key enzyme that catalyzes the asymmetric reduction of prochiral ketones to chiral alcohols.
[0004] Traditional chiral alcohol synthesis requires expensive chiral noble metal catalysts, harsh reaction conditions, and flammable and explosive borohydride reducing agents. It also suffers from drawbacks such as low product optical purity, complex separation and purification processes, difficult waste treatment, and high production costs. ADH-catalyzed carbonyl asymmetric reduction reactions offer significant advantages, including a wide availability of enzymes, outstanding activity and stereoselectivity, mild reaction conditions, high product optical purity, low production costs, and environmental friendliness, making them promising candidates for chiral alcohol synthesis. Therefore, finding an economically available ADH capable of catalyzing highly stereoselective asymmetric reductions at high substrate concentrations, with little or no coenzyme addition, is undoubtedly of paramount importance. (III) Summary of the Invention
[0005] The purpose of this invention is to provide an alcohol dehydrogenase, its encoding gene, a recombinant genetically engineered bacterium, and its application in the asymmetric reduction of prochiral carbonyl compounds to prepare highly optically active chiral alcohols. This enzyme has advantages such as high activity, strong stereospecificity, mild reaction conditions, and simple operation. It can economically and greenly produce a variety of highly optically selective chiral alcohols and has a promising application prospect. It solves the problems of low optical purity of products, complex separation and purification processes, difficulty in treating "three wastes" (waste gas, wastewater, and solid waste), and high production costs in traditional chiral alcohol production methods.
[0006] The technical solution adopted in this invention is:
[0007] This invention provides a highly active alcohol dehydrogenase derived from Candida orthopsilosis Co., the amino acid sequence of which is shown in SEQ ID NO.2.
[0008] The present invention also provides a gene encoding the alcohol dehydrogenase, the nucleotide sequence of which is shown in SEQ ID NO.1.
[0009] This invention also relates to a recombinant vector containing the encoding gene of the alcohol dehydrogenase, and recombinant genetically engineered bacteria prepared by transformation of the recombinant vector. The base vector of the recombinant vector is not limited, as long as it can maintain replication or autonomously replicate in various host cells of prokaryotic and / or eukaryotic cells. The vector can be any conventional vector in the art, such as various plasmids, bacteriophages, or viral vectors, preferably using the pET28a(+) plasmid as the expression vector. The recombinant genetically engineered bacteria use *Escherichia coli* as the expression host (*E. coli* BL21 cells or *E. coli* DH5α).
[0010] This invention provides an application of the alcohol dehydrogenase in the asymmetric reduction of a prochiral carbonyl compound to prepare a chiral alcohol. The method of application is as follows: using wet bacterial cells obtained by fermentation culture of recombinant genetically engineered bacteria containing the 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, the reaction is carried out at 200-700 rpm (preferably 200 rpm) and 37°C. After the reaction is completed, a reaction solution containing a chiral alcohol is obtained. The reaction solution is then separated and purified to obtain the desired chiral alcohol.
[0011] Furthermore, the prochiral carbonyl compounds include single-chain ketones (1), aryl ketones (2) / (4), heterocyclic ketones (such as pyridine, thiazole, etc.) (3), etc.
[0012]
[0013] In Formula 1, R represents C1-C4 alkane groups;
[0014] In Formula 2, R1-R5 are each independently H, F, Cl, Br, I, NO2, CF3, CN, C1-C4 alkyl or haloalkyl.
[0015] Further, 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.
[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 bacterial cells, and the initial concentration of the substrate is 0.1-1 M (preferably 0.3 M).
[0017] Further, the wet bacterial cells are prepared as follows: recombinant engineered bacteria containing the alcohol dehydrogenase encoding gene are inoculated into LB medium containing a final concentration of 50 mg / L kanamycin and cultured at 37°C for 8 h to obtain seed culture; then, the seed culture is inoculated into sterile LB liquid medium containing a final concentration of 50 mg / L kanamycin at a volume concentration of 2% and cultured at 37°C for 1.5-2.5 h to achieve a bacterial cell concentration OD600 of 0.4-0.8; then, isopropyl thio-β-D-galactoside (IPTG) is added to the culture medium at a final concentration of 0.1-1.0 mM (preferably 0.1 mM), and expression is induced at 26°C for 12 h. After centrifugation at 4°C and 4000 rpm for 10-20 min, the wet bacterial cells are collected; LB liquid medium: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, solvent is deionized water, pH 7.0.
[0018] The alcohol dehydrogenase described in this invention can perform catalysis in whole-cell form, or it can be catalyzed using crude enzyme solution obtained from cell disruption or pure enzyme extracted from complete disruption. Furthermore, specific immobilization techniques can be used to prepare the aforementioned crude enzyme solution or pure enzyme into immobilized enzymes, or to prepare wet bacterial cells into immobilized cell form for catalysis.
[0019] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:
[0020] (1) The alcohol dehydrogenase mined by the present invention has good biological properties and has achieved heterologous expression in engineered Escherichia coli. When the whole cell is used as a catalyst, the intracellular coenzyme can be directly utilized, achieving zero addition of coenzyme.
[0021] (2) The alcohol dehydrogenase of the present invention can catalyze the conversion of prochiral ketones such as single-chain ketones, aryl ketones, and heterocyclic ketones into corresponding chiral alcohols in a green and efficient manner. It has high activity, high stereoselectivity and a broad substrate spectrum. 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 coenzyme cycle. Moreover, the by-products of the enzyme-catalyzed reaction—acetone and the remaining isopropanol—can be separated and recovered by technical means, which greatly reduces the production cost. (iv) Description of the attached drawings
[0023] Figure 1 GC chromatograms of (RS)-(+)-3-methyl-2-butanol (a) and (S)-(+)-3-methyl-2-butanol (b) in Example 3.
[0024] Figure 2 GC chromatograms of (RS)-(+)-4-methyl-2-pentanol (a) and (S)-(+)-4-methyl-2-pentanol (b) in Example 4.
[0025] Figure 3 GC chromatograms of (RS)-(+)-2-hexanol (a) and (S)-(+)-2-hexanol (b) in Example 5.
[0026] Figure 4 GC chromatograms of (RS)-1-(2-chlorophenyl)ethanol (a) and (S)-1-(2-chlorophenyl)ethanol (b) in Example 6.
[0027] Figure 5 GC chromatograms of (RS)-1-(3-chlorophenyl)ethanol (a) and (S)-1-(3-chlorophenyl)ethanol (b) in Example 7.
[0028] Figure 6 GC chromatograms of (RS)-1-(2-fluorophenyl)ethanol (a) and (S)-1-(2-fluorophenyl)ethanol (b) in Example 8.
[0029] Figure 7 GC chromatograms of (RS)-1-(2-nitrophenyl)ethanol (a) and (S)-1-(2-nitrophenyl)ethanol (b) in Example 9.
[0030] Figure 8 GC chromatograms of (RS)-1-(4-bromophenyl)ethanol (a) and (S)-1-(4-bromophenyl)ethanol (b) in Example 10.
[0031] Figure 9 GC chromatograms of (RS)-1-(4-iodophenyl)ethanol (a) and (S)-1-(4-iodophenyl)ethanol (b) in Example 11.
[0032] Figure 10 GC chromatograms of (RS)-(-)-4-(1-hydroxyethyl)pyridine (a) and (S)-(-)-4-(1-hydroxyethyl)pyridine (b) in Example 12.
[0033] Figure 11 GC chromatograms of (RS)-1-(2-naphthyl)ethanol (a) and (S)-(-)-1-(2-naphthyl)ethanol (b) in Example 13.
[0034] Figure 12 GC chromatograms of (RS)-1-naphthyl-1-ethanol (a) and (S)-1-naphthyl-1-ethanol (b) in Example 14.
[0035] Figure 13GC chromatograms of (RS)-1-(thiazol-2-yl)ethane-1-ol (a) and (S)-1-(thiazol-2-yl)ethane-1-ol (b) in Example 15.
[0036] Figure 14 GC chromatograms of (RS)-1-(3-bromo-2-pyridyl)ethanol (a) and (S)-1-(3-bromo-2-pyridyl)ethanol (b) in Example 16. (V) Detailed Implementation
[0037] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:
[0038] LB liquid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, solvent is deionized water, pH 7.0.
[0039] LB plates are made 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 transformants
[0041] 1. Screening of alcohol dehydrogenases
[0042] Using the amino acid sequence of the alcohol dehydrogenase CMCR from *Candida metapsilosis*, which exhibits good biocatalytic performance, as a probe, a pBLAST search was performed in the NCBI database to select a batch of predetermined alcohol dehydrogenase gene sequences. These candidate genes were then cloned and expressed to construct recombinant *E. coli* transformants. The conversion rates and stereoselectivities of these alcohol dehydrogenases to substrates 2-hexanone, acetophenone, and their similar compounds were measured, and the cloned and expressed enzymes were repeatedly compared and screened. Some screening results are shown in Table 1. Finally, the alcohol dehydrogenase CoC with the best catalytic activity was obtained, annotated by NCBI as *Candida orthopsilosis Co*, NCBI accession number: XP_003865936.1, and its 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 the recombinant vector
[0048] Based on the sequence of the predicted alcohol dehydrogenase gene of 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 then constructed it into the vector pET-28a to obtain the recombinant vector pET-28a-CoC.
[0049] 3. Construction of recombinant genetically engineered bacteria
[0050] First, competent E. coli BL21(DE3) (Invitrogen) cells stored at -80℃ were incubated on ice at 0℃ for 10 min. Then, 1 μL of the recombinant vector pET-28a-CoC was added to each cell in a clean bench, and the cells were incubated on ice at 0℃ for 30 min, heat-shocked in a water bath at 42℃ for 90 s, and then incubated on ice at 0℃ for 2 min. 600 μL of LB medium was added, and the cells were cultured on a shaker at 37℃ and 200 rpm for 1 h. The cells were then plated on LB plates containing 50 μg / mL kanamycin resistance and cultured at 37℃ for 8-12 h. Clones were randomly picked, plasmids were extracted, and sequenced for identification. Recombinant E. coli BL21(DE3) / pET-28a-CoC containing the recombinant plasmid expression was obtained.
[0051] Example 2: Expression of alcohol dehydrogenase CoC
[0052] The recombinant Escherichia coli constructed in Example 1 was inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C and 180 rpm with shaking. The bacterial culture was then inoculated into a 250 mL Erlenmeyer flask containing 100 mL of LB liquid 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 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 was then centrifuged, and the wet bacterial cells were resuspended in 3 mL of phosphate buffer solution (pH 7.0, 50 mM), transferred to EP tubes, centrifuged, and stored at -20°C.
[0053] The wet bacterial cells were resuspended in phosphate buffer solution (pH 7.0, 50mM) to prepare a bacterial solution of 50g / L. The solution was then sonicated at 210W for 30min under ice bath conditions with a 3s working time followed by a 1s interval. The solution was centrifuged at low temperature (4℃) and the supernatant was collected. This supernatant is the crude enzyme solution of alcohol dehydrogenase CoC.
[0054] Examples 3-16: Asymmetric reduction of carbonyl compounds catalyzed by alcohol dehydrogenase CoC
[0055] Reaction system: 0.05 g of ethanol dehydrogenase CoC wet cells prepared by the method in Example 2, 1 mL of isopropanol, and 300 mM of carbonyl compounds such as ketoesters or haloaryl ketones from Table 1 constitute a 1 mL reaction system.
[0056] The reaction was carried out under shaking conditions at 37℃ and 200 rpm. Samples were taken every 30 minutes during the reaction for real-time GC monitoring or TLC detection. The TLC developing solvent was n-hexane:ethyl acetate = 3:1. The reaction was stopped when the concentration of the product no longer increased or the concentration of the substrate no longer decreased. The reaction solution was centrifuged and the supernatant was collected. A small amount of anhydrous sodium sulfate was added as a stationary phase. The peak areas of the substrate and product were detected by GC. The substrate conversion rate and the ee value of the product were calculated based on the peak area vs. concentration standard curve of the substrate standard. The results are shown in Table 2. Figures 1-14 .
[0057] GC detection method: Rt-βDEXsa chiral column, injection port and detector temperature 230℃, carrier gas N2, flow rate 1m / s. Column oven temperature: 80℃ for 3 min, ramp to 140℃ at 10℃ / min, hold for 3 min (Examples 3, 4); 45℃ for 5 min, ramp to 70℃ at 5℃ / min, hold for 3 min (Example 5); 80℃ for 3 min, ramp to 140℃ at 10℃ / min, hold for 3 min, ramp to 180℃ at 20℃ / min, hold for 5 min (Examples 6, 7, 8, 9, 10, 11); 160℃ for 5 min, ramp to 180℃ at 5℃ / min, hold for 3 min (Examples 13, 14); 140℃ for 5 min, ramp to 180℃ at 30℃ / min, hold for 3 min (Examples 12, 15, 16).
[0058] Table 2. Results of CoC-catalyzed asymmetric reduction reactions of carbonyl compounds
[0059]
Claims
1. The application of an alcohol dehydrogenase in the catalytic asymmetric reduction of a prochiral carbonyl compound to prepare a chiral alcohol, characterized in that, The method of application is as follows: using wet bacterial cells obtained by fermentation culture of recombinant genetically engineered bacteria containing the alcohol dehydrogenase encoding gene as catalyst, using a prochiral ketone compound as substrate, and isopropanol as reaction medium to form a reaction system, the reaction is carried out at 200-700 rpm and 37℃. After the reaction is completed, a reaction solution containing chiral alcohol is obtained. The reaction solution is separated and purified to obtain the desired chiral alcohol. The amino acid sequence of the alcohol dehydrogenase is shown in SEQ ID NO.
2. The prochiral ketone compound is: 3-methyl-2-butanone, 4-methyl-2-pentanone, 2-hexanone, 2'-chloroacetophenone, 3'-chloroacetophenone, 2'-fluoroacetophenone, o-nitroacetophenone, p-bromoacetophenone, 4-iodoacetophenone, 4-acetylpyridine, 2-naphthylacetophenone, 1-naphthylacetophenone, 2-acetylthiazole, 1-(3-bromopyridin-2-yl)acetophenone.
2. The application as described in claim 1, characterized in that, In the reaction system, the amount of catalyst used is 50-300 g / L based on the weight of wet bacterial cells, and the initial concentration of the substrate is 0.1-1 M.
3. The application as described in claim 1, characterized in that, The wet bacterial cells were prepared as follows: recombinant engineered bacteria containing the alcohol dehydrogenase encoding gene were inoculated into LB culture medium containing a final concentration of 50 mg / L kanamycin and cultured at 37°C for 8 h to obtain seed culture. The seed culture was then inoculated into sterile LB liquid medium containing 50 mg / L kanamycin at a volume concentration of 2%. The medium was cultured at 37°C for 1.5-2.5 h until the bacterial cell concentration OD600 was 0.4-0.
8. Isopropyl thio-β-D-galactoside was then added to the culture medium at a final concentration of 0.1-1.0 mM. After inducing expression at 26°C for 12 h, the cells were centrifuged at 4°C and 4000 rpm for 10-20 min and the wet cells were collected.