Short-chain dehydrogenase, engineering bacterium and application of short-chain dehydrogenase in asymmetric reduction of prochiral carbonyl compound

Abstract

The invention discloses short-chain dehydrogenase, engineering bacteria and application of the short-chain dehydrogenase in asymmetric reduction of prochiral carbonyl compounds, the short-chain dehydrogenase with high activity and strong stereospecificity is obtained through screening, and the short-chain dehydrogenase has the advantages of wide substrate spectrum, mild reaction conditions, simplicity and convenience in operation, high yield and high yield. The produced chiral alcohol has a good application prospect in the creation of chiral drug intermediates and high-selectivity catalysts, and solves the problems of low conversion rate, low yield, low e.e. Value, complex reaction process, many byproducts and the like in the traditional chemical method. The short-chain dehydrogenase provided by the invention has good tolerance to isopropanol, can realize circulation of coenzyme by taking isopropanol as a hydrogen source, and can directly utilize intracellular coenzyme when whole cells are taken as a catalyst, thereby realizing zero addition of coenzyme. In addition, a by-product acetone generated by the enzyme catalytic reaction and isopropanol remaining in the reaction can be separated and recycled through a conventional technical means, so that the production cost is greatly reduced.

Description

(I) Technical field

[0001] The present invention belongs to the field of bioengineering technology, and specifically relates to a short-chain dehydrogenase FRSDR mined from Faecalibacterium errhinopitheci, a coding gene, a recombinant engineering bacterium, and an application of the short-chain dehydrogenase FRSDR as a catalyst in the asymmetric reduction of a potential chiral carbonyl compound to prepare a highly optically active chiral alcohol. (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 intermediate for antiplatelet aggregation drugs; (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. It 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 in the production of penems and carbapenems, among which (R)-(-)-1,3-butanediol is the key intermediate in the production of 4-acetoxyazetidinone, and the reduction of prochiral ketones with short-chain dehydrogenases to obtain (2S,3R)-2-[(benzoylamino)methyl]-3-hydroxybutyric acid methyl ester is the 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] Short-chain dehydrogenase (SDRs) is named because its monomer sequence length is only about 250 amino acid residues. Although some sequences have a low identity of less than 20%, SDRs have a conservative tertiary structure and a typical nucleotide binding domain Rossmann fold. SDRs are a type of oxidoreductase that uses NAD(P)H or NAD(P)+ as a coenzyme to oxidize or synthesize alcohols. They are key enzymes that catalyze the asymmetric reduction of potential chiral ketones to generate chiral alcohols.

[0004] Traditional chiral alcohol synthesis requires expensive chiral noble metal catalysts, harsh reaction conditions, flammable and explosive borohydride reducing agents, and has disadvantages such as low product optical purity, complex separation and purification process, difficult "three wastes" treatment, and high production costs. The carbonyl asymmetric reduction reaction catalyzed by SDRs 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.

[0005] Therefore, it is undoubtedly of great significance to find an economical and readily available SDRs that can catalyze highly stereoselective asymmetric reduction at high substrate concentrations without adding or with very small amounts of coenzymes. (III) Summary of the invention

[0006] The purpose of the present invention is to provide a short-chain dehydrogenase, an encoding gene, an engineered bacterium and the application in asymmetric reduction of potentially chiral carbonyl compounds. The enzyme has the advantages of high activity, strong stereospecificity, mild reaction conditions, simple operation and the like, and can economically and greenly produce a variety of chiral alcohols with high optical selectivity; the produced chiral alcohols have good application prospects in the creation of chiral drug intermediates and highly selective catalysts, and solve the problems of low product optical purity, complex separation and purification process, difficulty in treating "three wastes", high production cost and the like in traditional chiral alcohol production methods.

[0007] The technical solution adopted by the present invention is:

[0008] The invention provides a short-chain dehydrogenase FRSDR derived from Faecalibacter rhinopitheci, and the amino acid sequence is shown in SEQ ID NO.2.

[0009] The present invention also provides a gene encoding the short-chain dehydrogenase FRSDR, and the nucleotide sequence is shown in SEQ ID NO.1.

[0010] The present invention also relates to a recombinant expression vector constructed by the coding gene of the short-chain dehydrogenase FRSDR, and a recombinant genetically engineered bacterium obtained by transforming a host bacterium with the recombinant expression vector. The basic vector of the recombinant vector of the present invention is not limited, as long as it can maintain its replication or autonomous replication in various host cells of prokaryotic and / or eukaryotic cells, and the vector can be various conventional vectors in the art, such as various plasmids, phages or viral vectors, etc., preferably using pET30a (+) plasmid as an expression vector; the host bacterium is preferably Escherichia coli, especially Escherichia coli BL21 cells or Escherichia coli DH5α.

[0011] The present invention also provides an application of the short-chain dehydrogenase FRSDR in the asymmetric reduction of a potential chiral carbonyl compound to prepare a chiral alcohol.

[0012] Furthermore, the prochiral carbonyl compound includes an aromatic ketone compound (1), a single-chain diketone (2), a β-ketoester (3), and 4-hydroxy-2-butanone (4);

[0013]

[0014] In formula (1), R1, R2, R3, R4, R5, and R6 are each independently H, F, Cl, Br, I, NO2, CF3, CN, or C1-C2 haloalkyl;

[0015] In formula (2), n is 0, 1, 2, or 3, and R1 and R2 are each independently a C1-C4 alkane group;

[0016] In formula (3), R1 is a C1-C4 alkyl group or an alkyl group with a substituent, and the substituent is a halogen or a benzene ring; R2 is H or N-methylbenzamide; and R3 is a C1-C2 alkyl group.

[0017] Further, the prochiral carbonyl compound includes acetophenone, 1-(3,5-difluorophenyl)ethanone, 3-chloro-1-phenyl-1-propanone, 1-(2-fluorophenyl)ethanone, 1-(2-chlorophenyl)ethanone, 1-(2-bromophenyl)ethanone, 2-chloro-1-(3,4-difluorophenyl)ethanone, 2-chloro-1-(4-chlorophenyl)ethanone, 1-(3-nitrophenyl)ethanone, 1-(3,5-trifluoromethylphenyl)ethanone, 1-(4-cyanophenyl)ethanone, 1-(2-iodo-5-fluorophenyl)ethanone, 4-chloro-3-hydroxybutyric acid methyl ester, 4-chloro-3-hydroxybutyric acid ethyl ester, 4-phenylacetoacetate, 2-(benzamidomethyl)-3-oxobutyric acid methyl ester, 4-hydroxy-2-butanone, 2,3-butanedione, 2,4-pentanedione, and 2,5-hexanedione.

[0018] Furthermore, the application is: using wet bacteria obtained by fermentation culture of short-chain dehydrogenase recombinant genetically engineered bacteria as a catalyst, using a potentially chiral carbonyl compound as a substrate, and using isopropanol as a reaction medium to form a reaction system, reacting at 100-500rpm (preferably 200rpm) 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 chiral alcohol.

[0019] 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).

[0020] Further, the wet bacteria are prepared as follows: the short-chain dehydrogenase recombinant genetic engineering bacteria are 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 about 1.5-2.5 hours to make the bacterial concentration OD600 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, after inducing expression at 26°C for 12 hours, centrifuged at 4°C and 4000 rpm for 10-20 minutes, and the wet bacteria are collected; LB liquid culture medium: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, the solvent is deionized water, pH 7.0.

[0021] The short-chain dehydrogenase of the present invention can be used to catalyze in the form of whole cells, or in the form of crude enzyme solution obtained by cell crushing or pure enzyme extracted by complete crushing. In addition, the two enzymes can be prepared into immobilized enzymes or enzymes in the form of immobilized cells using specific immobilization technology.

[0022] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0023] The present invention screens out a short-chain dehydrogenase with high activity and strong stereospecificity, and realizes the heterologous expression of the short-chain dehydrogenase in Escherichia coli engineering bacteria.

[0024] The short-chain dehydrogenase of the present invention can greenly and efficiently catalyze the conversion of potentially chiral carbonyl compounds such as aromatic ketone compounds, α-ketoesters, single-chain diketones, etc. into corresponding chiral alcohols, has a wide substrate spectrum, mild reaction conditions, and is easy to operate. The produced chiral alcohols have good application prospects in the creation of chiral drug intermediates and highly selective catalysts, and solve the problems of low conversion rate, low yield, low ee value, complex reaction process and many by-products in traditional chemical methods.

[0025] The short-chain dehydrogenase of the present invention has good tolerance to isopropanol, can use isopropanol as a hydrogen source to realize the circulation of coenzymes, and can directly use the coenzymes in cells when whole cells are used as catalysts, thus realizing zero addition of coenzymes.

[0026] In addition, the by-product acetone produced by the enzyme-catalyzed reaction and the remaining isopropanol can be separated and recovered by conventional technical means, greatly reducing production costs. (IV) Description of the drawings

[0027] Figure 1 , Example 2: Polyacrylamide gel electrophoresis of the crude enzyme solution of short-chain dehydrogenase FRSDR after induced expression and the precipitate.

[0028] Figure 2 , GC graphs of the 1-phenylethanol racemate (a) and (R)-1-phenylethanol (b) in Example 3.

[0029] Figure 3 , GC graphs of the 1-(3,5-difluorophenyl)ethanol racemate (a) and (R)-1-(3,5-difluorophenyl)ethanol (b) in Example 4.

[0030] Figure 4 , GC charts of (R)-3-chloro-1-phenyl-1-propanol racemate (a) and (R)-3-chloro-1-phenyl-1-propanol (b) in Example 5.

[0031] Figure 5 , GC charts of (R)-1-(2-fluorophenyl)ethanol racemate (a) and (R)-1-(2-fluorophenyl)ethanol (b) in Example 6.

[0032] Figure 6 , GC charts of the 1-(2-chlorophenyl)ethanol racemate (a) and (R)-1-(2-chlorophenyl)ethanol (b) in Example 7.

[0033] Figure 7 , GC charts of the 1-(2-bromophenyl)ethanol racemate (a) and (R)-1-(2-bromophenyl)ethanol (b) in Example 8.

[0034] Figure 8 , HPLC charts of the 2-chloro-1-(3,4-difluorophenyl)ethanol racemate (a) and (1S)-2-chloro-1-(3,4-difluorophenyl)ethanol (b) in Example 9.

[0035] Fig. 9 , GC charts of the 2-chloro-1-(4-chlorophenyl)ethanol racemate (a) and (R)-2-chloro-1-(4-chlorophenyl)ethanol (b) in Example 10.

[0036] Fig.10 , GC charts of the 1-(3-nitrophenyl)ethanol racemate (a) and (R)-1-(3-nitrophenyl)ethanol (b) in Example 11.

[0037] Fig.11 , HPLC charts of the 3,5-bis(trifluoromethyl)-α-methylbenzyl alcohol racemate (a) and (R)-3,5-bis(trifluoromethyl)-α-methylbenzyl alcohol (b) in Example 12.

[0038] Fig.12 , GC charts of the 1-(4-cyanophenyl)ethanol racemate (a) and (R)-1-(4-cyanophenyl)ethanol (b) in Example 13.

[0039] Fig.13 , GC charts of the 1-(2-iodo-5-fluorophenyl)ethanol racemate (a) and (R)-1-(2-iodo-5-fluorophenyl)ethanol (b) in Example 14.

[0040] Fig.14 , GC charts of 4-chloro-3-hydroxybutyric acid methyl ester racemate (a) and (S)-4-chloro-3-hydroxybutyric acid methyl ester (b) in Example 15.

[0041] Fig.15 , GC charts of 4-chloro-3-hydroxybutyric acid ethyl ester racemate (a) and (S)-4-chloro-3-hydroxybutyric acid ethyl ester (b) in Example 16.

[0042] Fig.16 , GC charts of 4-phenyl-3-hydroxybutyric acid ethyl ester racemate (a) and (R)-4-phenyl-3-hydroxybutyric acid ethyl ester (b) in Example 17.

[0043] Fig.17 , GC chart of methyl (2S,3R)-2-[(benzoylamino)methyl]-3-hydroxybutyrate in Example 18.

[0044] Fig.18 , GC graphs of the 1,3-butanediol racemate (a) and (R)-(-)-1,3-butanediol (b) in Example 19.

[0045] Fig.19 , GC graphs of the 2,3-butanediol racemate (a) and (2R,3R)-2,3-butanediol (b) in Example 20.

[0046] Fig. 20 , GC graphs of the 2,4-pentanediol racemate (a) and (2R,4R)-2,4-pentanediol (b) in Example 21.

[0047] Fig.21 , GC graphs of 2,5-hexanediol racemate (a) and (2R,5R)-2,5-hexanediol (b) in Example 22. (V) Specific implementation methods

[0048] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0049] LB liquid medium: peptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, solvent is deionized water, pH 7.0. LB plate is LB liquid medium with 18g / L agar added.

[0050] Example 1. Screening of short-chain dehydrogenases, construction of recombinant vectors and preparation of recombinant transformants

[0051] 1. Screening of short-chain dehydrogenase

[0052] The method of genome database mining was adopted, and the amino acid sequence of the short-chain dehydrogenase AXSDR from Algoriella xinjiangensis with good biocatalytic performance was used as a probe. The pBLAST search was performed in the NCBI database to select a batch of predetermined short-chain dehydrogenase gene sequences. Then these candidate genes were cloned and expressed respectively, and recombinant Escherichia coli transformants were constructed. By measuring the activity and stereoselectivity of these short-chain dehydrogenases on the substrates listed in Table 1 (acetophenone, 2,3-butanedione, 4-chloroacetoethyl acetate and their similar compounds), the cloned and expressed short-chain dehydrogenases were repeatedly compared and screened, and finally the short-chain dehydrogenase FRSDR with the best catalytic activity was obtained, which was predicted to be Faecalibacter rhinopitheci in NCBI, and the NCBI accession number is: WP_194182923.1.

[0053] 2. Construction of recombinant vector

[0054] According to the gene sequence of the short-chain dehydrogenase screened in step 1 (NCBI accession number: WP_194182923.1), Beijing Qingke Biotechnology Co., Ltd. was commissioned to directly synthesize the DNA sequence (the nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2) after codon optimization and modification, and constructed it into the vector pET-28a to obtain the recombinant vector pET-28a-FRSDR.

[0055] SEQ ID NO.2

[0056] MGILNEKVAIVTGAGSGIGKAIAKLYAREGAKVIISDIDRKGGNDALFEIQEIGGEAFFVEADTSTPEGNEALVNKAIEIYGKLDIACNNAGIGGAAALTGDYTLEDWKKVIDINFNGVFYGCKY QLKAMENNGGGAIINMASIFGSVAAPYSSAYTSSKHGIVGLTKNIGAEYGPKNIRCNAVGPGGIKTPPLLDSLSDEQLNILTSKHPIGRLGEPEEVAELVLFLSSDKASFITGGYYLVDGGYTAI.

[0057] 3. Construction of recombinant genetically engineered bacteria

[0058] 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-FRSDR prepared in step 2 was added in a clean bench, and the cells were placed in an ice bath at 0°C for 30 min, heat-shocked in a 42°C water bath for 90 s, and placed in an ice bath at 0°C for 2 min. 600 μL of LB medium was added, and the cells were cultured at 37°C and 200 rpm in a shaking incubator for 1 h. The cells were spread on an LB plate containing 50 μg / mL kanamycin resistance and cultured at 37°C for 8-12 h. Clones were randomly selected and plasmids were extracted for sequencing and identification, and recombinant Escherichia coli E. coli BL21 (DE3) / pET-28a-FRSDR containing the expression recombinant plasmid was screened.

[0059] Example 2. Expression of short-chain dehydrogenase FRSDR

[0060] The recombinant E. coli obtained in Example 2 was inoculated into LB liquid culture medium containing 50 μg / mL kanamycin, and cultured at 37°C overnight with shaking. The inoculum was inoculated into a 250mL Erlenmeyer flask containing 100mL of LB liquid culture medium at a rate of 1% (v / v), and cultured on a shaker at 37°C and 180rpm. When the OD600 of the culture reached 0.6, IPTG was added as an inducer at a final concentration of 0.1mM, and induced at 26°C for 14h. The culture was centrifuged, and the cells were resuspended with 3mL of phosphate buffer solution (50mM, pH 6.0), transferred to an EP tube, and the wet cells were stored at -20°C after centrifugation.

[0061] The wet cells were prepared into 50 g / L bacterial solution with phosphate buffer solution (50 mM, pH 6.0), and ultrasonically disrupted at 210 W for 30 min in an ice bath, with a working time of 3 s and a rest time of 1 s. The disrupted mixture was centrifuged at low temperature (4°C), and the supernatant (i.e., crude enzyme solution) and precipitate were collected. The polyacrylamide gel electrophoresis diagram is shown in Figure 1 shown.

[0062] Example 3-22: Asymmetric reduction of carbonyl compounds catalyzed by short-chain dehydrogenase FRSDR

[0063] 1mL reaction system: 50g / L wet bacteria prepared by the method of Example 2, 300mM carbonyl compounds such as ketoesters or halogenated aromatic ketones shown in Table 1, and isopropanol to 1mL. Oscillating reaction at 37°C, 200rpm, sampling 500μL every 1h during the reaction, centrifuging at 12000rpm, adding anhydrous sodium sulfate to the supernatant, drying and diluting the filter membrane, and using GC or HPLC to detect substrate conversion rate and product ee value, stop the reaction when the concentration of the reaction product no longer increases or the concentration of the substrate no longer decreases. The results are shown in Table 1, and the HPLC or GC graph is shown in Figure 2-Figure 21 .

[0064] Table 1. Results of asymmetric reduction of acetophenone and its derivatives catalyzed by FRSDR

[0065]

Claims

1. A short-chain dehydrogenase derived from Faecalibacterium nasopharyngeum, characterized in that The amino acid sequence of the short-chain dehydrogenase is shown in SEQ ID NO.

2.

2. A recombinant genetically engineered bacterium containing a gene encoding the short-chain dehydrogenase according to claim 1.

3. Use of the short-chain dehydrogenase according to claim 1 in the asymmetric reduction of prochiral carbonyl compounds to prepare chiral alcohols.

4. The use according to claim 3, characterized in that The prochiral carbonyl compound includes an aromatic ketone compound (1), a single-chain diketone (2), a β-ketoester (3), and 4-hydroxy-2-butanone (4); In formula (1), R1, R2, R3, R4, R5, and R6 are each independently H, F, Cl, Br, I, NO2, CF3, CN, or C1-C2 haloalkyl; In formula (2), n is 0, 1, 2, or 3, and R1 and R2 are each independently a C1-C4 alkane group; In formula (3), R1 is a C1-C4 alkyl group or an alkyl group with a substituent, and the substituent is a halogen or a benzene ring; R2 is H or N-methylbenzamide; and R3 is a C1-C2 alkyl group.

5. The use according to claim 4, characterized in that The prochiral carbonyl compound includes acetophenone, 1-(3,5-difluorophenyl)ethanone, 3-chloro-1-phenyl-1-propanone, 1-(2-fluorophenyl)ethanone, 1-(2-chlorophenyl)ethanone, 1-(2-bromophenyl)ethanone, 2-chloro-1-(3,4-difluorophenyl)ethanone, 2-chloro-1-(4-chlorophenyl)ethanone, 1-(3-nitrophenyl)ethanone, 1-(3,5-trifluoromethylphenyl)ethanone, 1-(4-cyanophenyl)ethanone, 1-(2-iodo-5-fluorophenyl)ethanone, 4-chloro-3-hydroxybutyric acid methyl ester, 4-chloro-3-hydroxybutyric acid ethyl ester, 4-phenylacetoacetate, 2-(benzamidomethyl)-3-oxobutyric acid methyl ester, 4-hydroxy-2-butanone, 2,3-butanedione, 2,4-pentanedione, and 2,5-hexanedione.

6. The use according to claim 3, characterized in that The application is as follows: using wet bacteria obtained by fermentation culture of short-chain dehydrogenase recombinant genetic engineering bacteria as a catalyst, using a potentially chiral carbonyl compound as a substrate, and using isopropanol as a cosolvent to form a reaction system, reacting at 100-500rpm 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 chiral alcohol.

7. The use according to claim 3, 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 3, characterized in that The wet bacterial cells are prepared as follows: short-chain dehydrogenase recombinant genetic engineering bacteria are inoculated into LB culture solution containing kanamycin with 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 about 1.5-2.5 hours 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 hours, the culture medium was centrifuged at 4°C and 4000 rpm for 10-20 minutes to collect the wet bacteria.

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