Short-chain dehydrogenase, engineered bacteria and application thereof in asymmetric reduction of latent chiral carbonyl compounds

Abstract

The application discloses a short-chain dehydrogenase, an engineering bacterium and application of the short-chain dehydrogenase in asymmetric reduction of a latent chiral carbonyl compound. The application screens a short-chain dehydrogenase with high activity and strong stereospecificity, has a wide substrate spectrum, is easy to operate and has mild reaction conditions. The produced chiral alcohol has a good application prospect in chiral drug intermediates and creation of a high-selectivity catalyst, and solves the problems of low conversion rate, low yield, low e.e. value, complex reaction process and many by-products in traditional chemical methods. The short-chain dehydrogenase has good tolerance to isopropyl alcohol, can realize coenzyme circulation by taking isopropyl alcohol as a hydrogen source, and can directly utilize intracellular coenzyme when adopting whole cells as a catalyst, so that zero addition of coenzyme is realized. In addition, the by-product acetone produced in the enzyme catalysis reaction and the residual isopropyl alcohol can be separated and recovered through conventional technical means, so that the production cost is greatly reduced.

Description

(I) Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a short-chain dehydrogenase FRSDR, its encoding gene, recombinant engineered bacteria, and its application as a catalyst in the asymmetric reduction of prochiral carbonyl compounds to prepare highly optically active chiral alcohols, which was discovered from Faecalibacterium praecox. (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 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 novel 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 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. Penicillin and carbapenems possess a broad antibacterial spectrum, outstanding antibacterial activity, and stability against most β-lactamases, making them considered "first-line drugs" for treating severe hospital-acquired infections. 4-Acetoxyazacyclobutanone [(2R,3R)-3-(R)-1-(tert-butyldimethylsilyloxy)ethyl-4-oxazabut-2-ylacetate] is a key chiral intermediate in the production of penicillin and carbapenems. Among these, (R)-(-)-1,3-butanediol is a crucial intermediate in the production of 4-acetoxyazacyclobutanone, while the reduction of the prochiral ketone by short-chain dehydrogenases to obtain methyl (2S,3R)-2-[(benzoylamino)methyl]-3-hydroxybutyrate is a hot topic in the synthesis of 4-acetoxyazacyclobutanone. Novel phosphine ligands have advantages such as high reactivity and enantioselectivity, and can be used in a variety of 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] Short-chain dehydrogenases (SDRs) are named for their monomeric sequences, which are only about 250 amino acid residues long. Although some have sequence identity as low as 20%, SDRs possess conserved tertiary structures and all exhibit typical Rossmann folds with nucleotide-binding domains. SDRs are a class of oxidoreductases that use NAD(P)H or NAD(P)+ as coenzymes to oxidize or synthesize alcohols. They are key enzymes catalyzing 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. SDR-catalyzed carbonyl asymmetric reduction reactions offer significant advantages, including a wide range of enzyme sources, 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.

[0005] Therefore, finding an economically available SDR that can catalyze highly stereoselective asymmetric reduction at high substrate concentrations, with little or no added coenzyme, is undoubtedly of great significance. (III) Summary of the Invention

[0006] The purpose of this invention is to provide a short-chain dehydrogenase, its encoding gene, an engineered bacterium, and its application in the asymmetric reduction of potentially chiral carbonyl compounds. This enzyme has advantages such as high activity, strong stereospecificity, mild reaction conditions, and simple operation, enabling the economical and green production of a variety of chiral alcohols with high optical selectivity. The produced chiral alcohols have excellent application prospects in the creation of chiral drug intermediates and highly selective catalysts, solving the problems of low optical purity of products, complex separation and purification processes, difficult treatment of "three wastes," and high production costs associated with traditional chiral alcohol production methods.

[0007] The technical solution adopted in this invention is:

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

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

[0010] This invention also relates to a recombinant expression vector constructed from the encoding gene of the short-chain dehydrogenase FRSDR, and recombinant genetically engineered bacteria obtained by transforming the recombinant expression vector into host bacteria. The base vector of the recombinant vector of this 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. The vector can be any conventional vector in the art, such as various plasmids, bacteriophages, or viral vectors, preferably using the pET30a(+) plasmid as the expression vector; the host bacteria are preferably *Escherichia coli*, especially *E. coli* BL21 cells or *E. coli* DH5α.

[0011] The present invention also provides the application of the short-chain dehydrogenase FRSDR in the asymmetric reduction of prochiral carbonyl compounds to prepare chiral alcohols.

[0012] Furthermore, the prochiral carbonyl compounds include aryl ketones (1), single-chain diketones (2), β-keto esters (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, the substituent being a halogen or a benzene ring; R2 is H or N-methylbenzamide; and R3 is a C1-C2 alkyl group.

[0017] Furthermore, the prochiral carbonyl compounds include acetophenone, 1-(3,5-difluorophenyl) acetophenone, 3-chloro-1-phenyl-1-propanone, 1-(2-fluorophenyl) acetophenone, 1-(2-chlorophenyl) acetophenone, 1-(2-bromophenyl) acetophenone, 2-chloro-1-(3,4-difluorophenyl) acetophenone, 2-chloro-1-(4-chlorophenyl) acetophenone, 1-(3-nitrophenyl) acetophenone, 1-(3,5-trifluoromethylphenyl) acetophenone, 1-(4-cyanophenyl) acetophenone, 1-(2-iodo-5-fluorophenyl) acetophenone, methyl 4-chloro-3-hydroxybutyrate, ethyl 4-chloro-3-hydroxybutyrate, ethyl 4-phenylacetoacetate, methyl 2-(benzoylaminomethyl)-3-oxobutyrate, 4-hydroxy-2-butanone, 2,3-butanedione, 2,4-pentanedione, and 2,5-hexanedione.

[0018] Furthermore, the application is as follows: using wet bacterial cells obtained by fermentation culture of short-chain dehydrogenase recombinant genetically engineered bacteria as catalyst, using a prochiral carbonyl compound as substrate, and using isopropanol as reaction medium to form a reaction system, the reaction is carried out at 100-500 rpm (preferably 200 rpm) and 37°C. After the reaction is completed, a reaction solution containing chiral alcohol is obtained, and the reaction solution 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 bacterial cells, and the initial concentration of the substrate is 0.1-1 M (preferably 0.3 M).

[0020] Further, the wet bacterial cells are prepared as follows: Recombinant genetically engineered short-chain dehydrogenase bacteria are inoculated into LB broth containing a final concentration of 50 mg / L kanamycin and cultured at 37°C for 8 hours to obtain a seed culture. Then, the seed culture is inoculated at a volume concentration of 2% into sterile LB liquid medium containing a final concentration of 50 mg / L kanamycin and cultured at 37°C for approximately 1.5-2.5 hours until the bacterial cell concentration (OD600) is 0.4-0.8. Isopropyl thio-β-D-galactoside (IPTG) is then added to the culture medium at a final concentration of 0.1-1.0 mM (preferably 0.1 mM). Expression is induced at 26°C for 12 hours, followed by centrifugation at 4°C and 4000 rpm for 10-20 minutes to collect the wet bacterial cells. LB liquid medium consists of 10 g / L peptone, 5 g / L yeast extract, and 10 g / L sodium chloride, with deionized water as the solvent and a pH of 7.0.

[0021] The short-chain dehydrogenases described in this invention can perform catalysis in whole-cell form, or they can be catalyzed using crude enzyme solutions obtained from cell disruption or pure enzymes extracted from complete disruption. Furthermore, specific immobilization techniques can be used to prepare the above two enzymes into immobilized enzymes or enzymes in immobilized cell form.

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

[0023] This invention screened and obtained a short-chain dehydrogenase with high activity and strong stereospecificity, and achieved heterologous expression of the short-chain dehydrogenase in engineered Escherichia coli.

[0024] The short-chain dehydrogenase of this invention can catalyze the conversion of aryl ketones, α-keto esters, single-chain diketones, and other prochiral carbonyl compounds into corresponding chiral alcohols in a green and efficient manner. It has a broad substrate spectrum, mild reaction conditions, and simple operation. The produced chiral alcohols have great application prospects in the creation of chiral drug intermediates and highly selective catalysts. It solves 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 this invention has good tolerance to isopropanol, can use isopropanol as a hydrogen source to achieve coenzyme cycling, and can directly utilize intracellular coenzymes when using whole cells as catalysts, thus achieving zero coenzyme addition.

[0026] In addition, the byproducts of the enzyme-catalyzed reaction—acetone and the remaining isopropanol—can be separated and recovered using conventional techniques, greatly reducing production costs. (iv) Description of the attached drawings

[0027] Figure 1 Example 2: The crude enzyme solution of the short-chain dehydrogenase FRSDR after induction expression was subjected to polyacrylamide gel electrophoresis along with the precipitate.

[0028] Figure 2 GC chromatograms of racemic 1-phenylethanol (a) and (R)-1-phenylethanol (b) in Example 3.

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

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

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

[0032] Figure 6 GC diagrams of racemic 1-(2-chlorophenyl)ethanol (a) and (R)-1-(2-chlorophenyl)ethanol (b) in Example 7.

[0033] Figure 7 GC diagrams of racemic mixture (a) and (R)-1-(2-bromophenyl)ethanol (b) in Example 8.

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

[0035] Figure 9 GC chromatograms of racemic 2-chloro-1-(4-chlorophenyl)ethanol (a) and (R)-2-chloro-1-(4-chlorophenyl)ethanol (b) in Example 10.

[0036] Figure 10 GC diagrams of racemic 1-(3-nitrophenyl)ethanol (a) and (R)-1-(3-nitrophenyl)ethanol (b) in Example 11.

[0037] Figure 11 HPLC chromatograms of racemic mixture (a) and (R)-3,5-di(trifluoromethyl)-α-methylbenzyl alcohol (b) in Example 12.

[0038] Figure 12 GC diagrams of racemic 1-(4-cyanophenyl)ethanol (a) and (R)-1-(4-cyanophenyl)ethanol (b) in Example 13.

[0039] Figure 13 GC diagrams of racemic mixture (a) and (R)-1-(2-iodo-5-fluorophenyl)ethanol (b) in Example 14.

[0040] Figure 14 GC chromatograms of racemic methyl 4-chloro-3-hydroxybutyrate (a) and (S)-4-chloro-3-hydroxybutyrate (b) in Example 15.

[0041] Figure 15 GC chromatograms of racemic 4-chloro-3-hydroxybutyrate ethyl ester (a) and (S)-4-chloro-3-hydroxybutyrate ethyl ester (b) in Example 16.

[0042] Figure 16 GC chromatograms of racemic 4-phenyl-3-hydroxybutyrate ethyl ester (a) and (R)-4-phenyl-3-hydroxybutyrate ethyl ester (b) in Example 17.

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

[0044] Figure 18 GC chromatograms of racemic 1,3-butanediol (a) and (R)-(-)-1,3-butanediol (b) in Example 19.

[0045] Figure 19 GC chromatograms of racemic 2,3-butanediol (a) and (2R,3R)-2,3-butanediol (b) in Example 20.

[0046] Figure 20 GC chromatograms of racemic 2,4-pentanediol (a) and (2R,4R)-2,4-pentanediol (b) in Example 21.

[0047] Figure 21 GC chromatograms of racemic 2,5-hexanediol (a) and (2R,5R)-2,5-hexanediol (b) in Example 22. (V) Detailed Implementation

[0048] 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:

[0049] LB liquid medium: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, solvent: deionized water, pH 7.0. LB plates are made by adding 18 g / L agar to LB liquid medium.

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

[0051] 1. Screening of short-chain dehydrogenases

[0052] Using genomic database mining, the amino acid sequence of the short-chain dehydrogenase AXSDR from *Algoriella xinjiangensis*, exhibiting good biocatalytic performance, was used as a probe in the NCBI database for pBLAST searching, selecting a batch of predetermined short-chain dehydrogenase gene sequences. These candidate genes were then cloned and expressed to construct recombinant *E. coli* transformants. The activities and stereoselectivity of these short-chain dehydrogenases to the substrates listed in Table 1 (acetophenone, 2,3-butanedione, ethyl 4-chloroacetoacetate, and their similar compounds) were repeatedly compared and screened. Ultimately, the short-chain dehydrogenase FRSDR, with the best catalytic activity, was obtained. NCBI predicts its inclusion in *Faecalibacter rhinopitheci*, NCBI accession number: WP_194182923.1.

[0053] 2. Construction of the recombinant vector

[0054] Based on 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 (nucleotide sequence as shown in SEQ ID NO.1, amino acid sequence as 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] E. coli BL21(DE3) (Invitrogen) competent cells stored at -80℃ were incubated on ice at 0℃ for 10 min. Then, 1 μL of the recombinant vector pET-28a-FRSDR prepared in step 2 was added to each cell in a clean bench. 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 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-FRSDR containing the recombinant plasmid was obtained.

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

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

[0061] Wet bacterial cells were prepared into a 50 g / L bacterial suspension using phosphate buffer (50 mM, pH 6.0). The suspension was then sonicated at 210 W for 30 min under ice bath conditions, with a 3-second sonication interval of 1 second. The disrupted mixture was centrifuged at low temperature (4 °C), and the supernatant (crude enzyme solution) and precipitate were collected. The polyacrylamide gel electrophoresis image is shown below. Figure 1 As shown.

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

[0063] A 1 mL reaction system consisted of 50 g / L of wet bacterial cells prepared according to the method in Example 2, 300 mM of carbonyl compounds such as ketoesters or haloacetones as shown in Table 1, and isopropanol to a final volume of 1 mL. The reaction was carried out at 37°C and 200 rpm with shaking. During the reaction, 500 μL samples were taken every 1 hour. After centrifugation at 12000 rpm, the supernatant was dried and diluted with anhydrous sodium sulfate, filtered through a filter membrane, and the substrate conversion and product ee value were detected by GC or HPLC. The reaction was stopped when the product concentration no longer increased or the substrate concentration no longer decreased. The results are shown in Table 1, and the HPLC or GC chromatograms are shown in [Table 1]. Figures 2-21 .

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

[0065]

Claims

1. A type of bacteria derived from *Heliotropium indicum* (… Faecalibacter rhinopitheci The short-chain dehydrogenase of ) is characterized by, The amino acid sequence of the short-chain dehydrogenase is shown in SEQ ID NO.

2.

2. A recombinant genetically engineered bacterium containing the encoding gene of the short-chain dehydrogenase as described in claim 1.

3. The application of the short-chain dehydrogenase of claim 1 in the asymmetric reduction of prochiral carbonyl compounds to prepare chiral alcohols, characterized in that, The prochiral carbonyl compound is an aryl ketone (1), a single-chain diketone (2), a β-keto ester (3), or a 4-hydroxy-2-butanone (4). （1） （2） （3） （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, 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, the substituent being a halogen or a benzene ring; R2 is H or N-methylbenzamide; and R3 is a C1-C2 alkyl group.

4. The application as described in claim 3, characterized in that, The proximal carbonyl compounds include acetophenone, 1-(3,5-difluorophenyl) acetophenone, 3-chloro-1-phenyl-1-propanone, 1-(2-fluorophenyl) acetophenone, 1-(2-chlorophenyl) acetophenone, 1-(2-bromophenyl) acetophenone, 2-chloro-1-(3,4-difluorophenyl) acetophenone, 2-chloro-1-(4-chlorophenyl) acetophenone, 1-(3-nitrophenyl) acetophenone, 1-(3,5-trifluoromethylphenyl) acetophenone, 1-(4-cyanophenyl) acetophenone, 1-(2-iodo-5-fluorophenyl) acetophenone, methyl 4-chloro-3-hydroxybutyrate, ethyl 4-chloro-3-hydroxybutyrate, ethyl 4-phenylacetoacetate, methyl 2-(benzoylaminomethyl)-3-oxobutyrate, 4-hydroxy-2-butanone, 2,3-butanedione, 2,4-pentanedione, and 2,5-hexanedione.

5. The application as described in claim 3, characterized in that, The application is as follows: using wet bacterial cells obtained by fermentation culture of short-chain dehydrogenase recombinant genetically engineered bacteria as catalyst, using a prochiral carbonyl compound as substrate, and using isopropanol as a co-solvent to form a reaction system, the reaction is carried out at 100-500 rpm and 37°C. After the reaction is completed, a reaction solution containing chiral alcohol is obtained. The reaction solution is then separated and purified to obtain the chiral alcohol.

6. The application as described in claim 5, 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.

7. The application as described in claim 5, characterized in that, The wet bacterial cells were prepared as follows: short-chain dehydrogenase recombinant genetically engineered bacteria 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 at a volume concentration of 2% into sterile LB liquid medium containing a final concentration of 50 mg / L kanamycin. 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. Expression was induced at 26°C for 12 h, and the cells were collected by centrifugation at 4°C and 4000 rpm for 10-20 min.

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