Short-chain dehydrogenase mutants and their application in catalyzing the synthesis of symmetrical chiral diols from symmetrical diketones

Abstract

The present invention discloses a short-chain dehydrogenase mutant and its application in catalyzing the synthesis of symmetrical chiral diols from symmetrical diketones. The short-chain dehydrogenase mutant is obtained by single or multiple mutations at positions 94, 145, 153, 188, 196, or 208 of the amino acid sequence of the short-chain dehydrogenase shown in SEQ ID NO.2. The short-chain dehydrogenase mutant of the present invention can convert potential chiral diketones at high substrate concentrations into corresponding chiral diols with high stereoselectivity, solving the problems of low yield, low substrate loading, and low product optical purity in existing methods. The present invention uses pure isopropanol as a cosolvent, effectively preventing problems such as subsequent extraction and emulsification, facilitating the extraction and separation of subsequent products; using wet bacteria as a catalyst, the reaction cycle can be achieved without the addition of additional coenzymes, simplifying production operations and reducing production costs, in line with the concept of green chemistry.

Description

(1) Technical field

[0001] The present invention belongs to the field of biochemical engineering, and particularly relates to a short-chain dehydrogenase mutant, a gene, a recombinant genetic engineering bacterium, and an application in catalyzing the synthesis of a phosphine ligand chiral mother nucleus (symmetrical chiral diol) from a symmetrical diketone. (2) Background technology

[0002] Chiral phosphine ligands are one of the most widely used types of ligands in asymmetric catalysis. Among them, the dihedral angle of the bisphosphine ligand plays an important role in asymmetric catalysis, and the appropriate dihedral angle is crucial to achieving excellent enantioselectivity.

[0003] Utilize the bisphosphine ligand (PNAS2004,16,5815-5820.J.Am.Chem.Soc.2006,128,5955-5965.) of the central chiral compound such as the methyl sulfonate or p-toluenesulfonate of symmetrical chiral diol to synthesize a series of chiral side chains, successfully realized in building-up process by the efficient transmission of central chirality to axial chirality, do not need to split and can obtain the chiral bisphosphine ligand of single configuration, and be successfully applied in asymmetric hydrogenation.Therefore symmetrical chiral diol has very large application prospect in the synthesis of bisphosphine ligand.Currently, the comparatively common process of synthetic symmetrical chiral alcohol process is substantially preceded by chiral ketone and is made through metal catalysis hydrogenation, inevitably increased the complexity and the manufacturing cost of reaction. In addition, due to reaction control issues, a large number of by-products are produced, resulting in low product ee. For example, the (2S,3S)-butanediol and (2R,3S)-butanediol produced during the metal-catalyzed hydrogenation synthesis of (2R,3R)-butanediol cannot be effectively controlled, resulting in a very low product ee value. (3) Summary of the invention

[0004] The present invention aims to provide a short-chain dehydrogenase mutant and its application in catalyzing the asymmetric synthesis of chiral diols from potentially chiral diketones. The reaction can achieve a reaction cycle without the need for the addition of additional coenzymes, thereby simplifying production operations and reducing production costs. This is of certain significance for the green production of highly stereoselective chiral diols and provides a basic guarantee for the synthesis of chiral phosphine ligands.

[0005] The technical solution adopted in the present invention is:

[0006] The present invention provides a short-chain dehydrogenase mutant, which is obtained by performing single mutation or multiple mutation on the 94th, 145th, 153rd, 188th, 196th or 208th position of the short-chain dehydrogenase amino acid sequence shown in SEQ ID NO.2 derived from Faecalibacter rhinopitheci.

[0007] Furthermore, the short-chain dehydrogenase mutant is a mutant in which the amino acid sequence shown in SEQ ID NO. 2 is mutated into one of the following: (1) glycine at position 94 is mutated into alanine (G94A), glutamate (G94Q), or glutamine (G94E); (2) histidine at position 145 is mutated into phenylalanine (H145F); (3) serine at position 153 is mutated into isoleucine (S153I) or leucine (S153L); (4) tyrosine at position 188 is mutated into glycine (Y188G), cysteine ​​(Y188C), or threonine (Y188T); (5) serine at position 196 is mutated into alanine (S196A) or cysteine ​​(S196C); (6) lysine at position 208 is mutated into serine (K208S); and (7) a combined mutation of two or more of the single mutations in steps (1) to (6).

[0008] Further, when the substrate is 2,3-butanedione, the short-chain dehydrogenase mutant is preferably one in which the amino acid sequence shown in SEQ ID NO. 2 is mutated to one of the following: G94Q / H145E, G94Q / H145F, G94Q / H145F / S153I, G94Q / H145F / S153L, G94Q / H145F / S153I / Y188C, G94Q / H145F / S153L / Y188C, G94Q / H145F / S153I / Y188G, G94Q / H145F / S153L / Y188G, G94Q / H145F / S153I / Y188T, or G94Q / H145F / S153L / Y188T.

[0009] Further, when the substrate is 2,4-pentanedione, the short-chain dehydrogenase mutant is preferably one in which the amino acid sequence shown in SEQ ID NO. 2 is mutated to one of the following: G94A / H145F, G94A / H145F / Y188A, G94A / H145F / Y188G, G94A / H145F / Y188C, G94A / H145F / Y188T, G94A / H145F / Y188C / S196A, G94A / H145F / Y188C / S196C, G94A / H145F / Y188T / S196A, G94A / H145F / Y188T / S196C.

[0010] Furthermore, when the substrate is 2,5-hexanedione, it is preferred that the short-chain dehydrogenase mutant is a mutant in which the amino acid sequence shown in SEQ ID NO. 2 is mutated to one of the following: G94A / S196A, G94A / S196C.

[0011] Further, when the substrate is 3,4-hexanedione, the short-chain dehydrogenase mutant is preferably one in which the amino acid sequence shown in SEQ ID NO. 2 is mutated to one of the following: S196A, S196C, G94A / H145F / S153L, G94A / H145F / S153L / Y188G, G94A / H145F / S153L / Y188C, G94A / H145F / S153L / Y188T, G94A / H145F / S153L / Y188T / S196A, G94A / H145F / S153L / Y188T / S196C.

[0012] Furthermore, when the substrate is 3,5-heptanedione, the short-chain dehydrogenase mutant is preferably one in which the amino acid sequence shown in SEQ ID NO. 2 is mutated to one of the following: G94E, G94E / S153I, G94E / S153L, G94E / S153L / Y188A, G94E / S153L / Y188T, or G94E / S153I / 196A.

[0013] Furthermore, when the substrate is 3,6-octanedione, the short-chain dehydrogenase mutant is preferably one in which the amino acid sequence shown in SEQ ID NO. 2 is mutated into one of the following: G94A / H145F / S153I / Y188A / 196C, G94A / H145F / S153I / Y188T / 196A, G94A / H145F / S153I / Y188A / 196C / K208S, or G94A / H145F / S153I / Y188T / 196A / K208S.

[0014] Furthermore, when the substrate is dibenzoyl, the short-chain dehydrogenase mutant is preferably one in which the amino acid sequence shown in SEQ ID NO. 2 is mutated to one of the following: G94A / Y188A, G94A / Y188T, G94A / Y188C, G94A / Y188G, G94A / Y188T / 196A, G94A / Y188T / 196C, G94A / Y188T / 196C / K208S.

[0015] The present invention also relates to the encoding gene of the short-chain dehydrogenase mutant, a recombinant vector constructed from the encoding gene, and a recombinant genetically engineered bacterium prepared by transformation with the recombinant vector. The recombinant vector of the present invention is not limited, as long as it can maintain its replication or autonomous replication in various prokaryotic and / or eukaryotic host cells. The vector can be any conventional vector in the art, such as various plasmids, phages, or viral vectors, preferably using the pET-28a(+) plasmid as an expression vector; the host bacterium is preferably Escherichia coli, such as E. coli BL21 cells or E. coli DH5α.

[0016] The present invention also provides an application of the short-chain dehydrogenase mutant in catalyzing the asymmetric synthesis of chiral diols from potential chiral diketones. The application method comprises the following steps: using wet bacteria obtained by fermentation culture of a recombinant genetically engineered bacterium containing a gene encoding the short-chain dehydrogenase mutant as a catalyst, using the potential chiral diketone as a substrate, and using an organic alcohol as a reaction medium to form a reaction system; carrying out the reaction at 200 rpm and 30-37° C.; obtaining a reaction solution containing the chiral diol after completion of the reaction; and separating and purifying the reaction solution to obtain the chiral diol.

[0017] Furthermore, the organic alcohol includes isopropyl alcohol.

[0018] Furthermore, the prochiral diketone includes 2,3-butanedione, 2,4-pentanedione, 2,5-hexanedione, 3,4-hexanedione, 3,5-heptanedione, 3,6-octanedione, and dibenzoyl.

[0019] Furthermore, in the reaction system, the amount of catalyst used is 10-100 g / L (preferably 50 g / L) based on the weight of the wet bacteria, and the concentration of the substrate added is 0.5-3 M (preferably 1 M).

[0020] Furthermore, the wet cells are prepared as follows: the recombinant engineered bacteria of the short-chain dehydrogenase mutant are inoculated into an LB liquid culture medium containing kanamycin at a final concentration of 50 mg / L, and cultured at 37°C for 8 hours to obtain a seed solution; the seed solution is then inoculated into a sterile LB liquid culture medium containing kanamycin at a final concentration of 50 mg / L at a volume concentration of 2%, and cultured at 37°C for about 1.5-2.5 hours until the cell concentration OD600 is 0.4-0.8, and isopropylthio-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 after inducing expression at 26°C for 12 hours, the wet cells are collected by centrifugation at 4°C and 4000 rpm for 30 minutes; LB liquid culture medium: 10 g / L peptone, 5 g / L yeast powder, 10 g / L sodium chloride, and the solvent is deionized water, pH 7.0.

[0021] The short-chain dehydrogenase mutants described herein can be used for catalysis in whole-cell form, as a crude enzyme solution obtained from disrupted cells, or as a pure enzyme solution extracted from completely disrupted cells. Furthermore, the two enzymes can be prepared as immobilized enzymes or immobilized cells using specific immobilization techniques.

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

[0023] (1) The present invention obtains short-chain dehydrogenase mutants with significantly improved activity and stereoselectivity for potential chiral diketone substrates through mutation screening. It can convert potential chiral diketones at higher substrate concentrations (greater than 2M) into corresponding chiral diols with high stereoselectivity, solving the problems of low yield, low substrate loading, and low product optical purity of existing methods.

[0024] (2) The present invention explores and optimizes the reaction system and uses pure isopropanol as a cosolvent, which effectively prevents problems such as subsequent extraction emulsification and facilitates the extraction and separation of subsequent products. Using wet bacteria as catalysts, the reaction cycle can be achieved without the need for additional coenzymes, simplifying production operations and reducing production costs. The by-products acetone and isopropanol produced by the reaction can be recycled and reused, which is in line with the concept of green chemistry.

[0025] (3) The present invention reduces production costs while avoiding the environmental problems caused by traditional methods. It has certain significance for the green production of high stereoselective chiral diols and provides a basic guarantee for the synthesis of chiral phosphine ligands. (IV) Description of the accompanying drawings

[0026] Figure 1 , GC graphs of the substrate (a) and product (b) of Example 2.

[0027] Figure 2 , GC graphs of the substrate (a) and product (b) of Example 3.

[0028] Figure 3 , GC graphs of the substrate (a) and product (b) of Example 4.

[0029] Figure 4 , GC graphs of the substrate (a) and product (b) of Example 5.

[0030] Figure 5 , GC chart of the product of Example 6.

[0031] Figure 6 , GC graphs of the substrate (a) and product (b) of Example 7.

[0032] Figure 7 , HPLC charts of the substrate (a) and product (b) of Example 8. (V) Specific implementation methods

[0033] The present invention is further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto:

[0034] Example 1: Screening of short-chain dehydrogenase mutants and preparation of recombinant engineered bacteria

[0035] 1. Construction of wild-type short-chain dehydrogenase engineered bacteria

[0036] The oxidoreductase FRSDR of the short-chain dehydrogenase family derived from Faecalibacterrhopitheci in NCBI was artificially synthesized by a gene synthesis company, with the NCBI number WP_194182923.1 (the nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2). It was then integrated into the pET-28a(+) commercial plasmid to directly obtain the recombinant plasmid pET-28a(+)-FRSDRG, which was then transformed into E. coli BL21(DE3) to obtain the wild-type short-chain dehydrogenase engineered bacteria E. coli BL21(DE3) / pET-28a(+)-WT.

[0037] SEQ ID NO.2 protein sequence:

[0038] MGILNEKVAIVTGAGSGIGKAIAKLYAREGAKVIISDIDRKGGNDALFEIQEIGGEAFFVEADTSTPEGNEALVNKAIEIYGKLDIACNNAGIGGAAALTGDYTLEDWKKVIDINFNGVFYGCKY QLKAMENNGGGAIINMASIHGSVAAPYSSAYTSSKHGIVGLTKNIGAEYGPKNIRCNAVGPGYIKTPPLLDSLSDEQLNILTSKHPIGRLGEPEEVAELVLFLSSDKASFITGGYYLVDGGYTAI.

[0039] 2. Selection of short-chain dehydrogenase mutation sites

[0040] The wild-type short-chain dehydrogenase FRSDR in step 1 was subjected to molecular docking and rational analysis to select key sites, which were positions 94, 145, 153, 188, 196, and 208.

[0041] 3. Design of site-directed mutagenesis primers

[0042] According to the mutation site in step 2, design the site-directed primers as shown in Table 1.

[0043] Table 1. Site-directed mutagenesis primers

[0044]

[0045] 4. Mutation

[0046] (1) Single-site site-directed saturation mutagenesis

[0047] Using pET-28a(+)-FRSDR gene as template, PCR amplification was performed using high-fidelity DNA polymerase according to the system in Table 2.

[0048] Table 2. PCR reaction system

[0049]

[0050]

[0051] The PCR reaction program was as follows: pre-denaturation at 95°C for 5 min, followed by the following cycle: denaturation at 95°C for 1 min, annealing at 63°C for 10 s, extension at 72°C for 1 min 30 s, for a total of 30 cycles, and final extension at 72°C for 10 min, followed by storage at 4°C.

[0052] Mix 3 μL of PCR product with 2 μL of Loding Buffer and verify PCR success by gel electrophoresis based on band size. Add 1 μL of Dpn I to the verified PCR product and incubate at 37°C for 1 hour to eliminate the original DNA template left over from PCR. Place the product in a 65°C oven for 10 minutes to inactivate Dpn I. Purify the digested product using a Clean Up kit and store in a refrigerator at 4°C. Obtain the single-site mutants G94A, G94Q, G94E, H145F, S153I, S153L, Y188A, Y188G, Y188T, Y188C, S196A, S196C, and K208S, and express them according to step 5.

[0053] (2) Combined mutation

[0054] The single-site mutants were subjected to double-site or multi-site combination mutation using the primers in Table 1 and the system in Table 2, and expressed according to the method in step 5.

[0055] 5. Construction and induced expression of short-chain dehydrogenase mutant engineered bacteria

[0056] The competent Escherichia coli BL21 (DE3) (Invitrogen) cells stored at -80°C were placed on ice at 0°C for 10 min. 5 μL of the PCR product purified in step 4 was added in a clean bench. The cells were 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 liquid medium was added and the cells were cultured on a shaker at 37°C and 200 rpm for 1 h. Finally, the cells were spread on an LB plate containing 50 μg / mL kanamycin resistance and cultured at 37°C for 8-12 h to obtain mutant transformants.

[0057] After sequencing confirmed that the mutation was successful, the recombinant Escherichia coli carrying the mutant was inoculated into LB liquid culture medium containing 50 μg / mL kanamycin and cultured with shaking at 37°C overnight. The inoculum was inoculated into a 250 mL Erlenmeyer flask containing 100 mL of LB liquid culture medium at a 1% (v / v) inoculum volume and cultured in 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 centrifuged, the wet bacteria were collected, and stored at -20°C.

[0058] 6. Screening of short-chain dehydrogenases and their mutants for 2,3-butanedione

[0059] Reaction system: 0.43 g of 2,3-butanedione, 0.5 g of wet bacteria, isopropanol added to 10 mL, substrate added to a final concentration of 0.5 M, reaction at 30°C, 200 rpm for 30 min, 200 μL of reaction solution was extracted with 200 μL of ethyl acetate, centrifuged, and the supernatant was dried over anhydrous sodium sulfate. The substrate conversion rate and meso (ee)% were detected by GC. The results are shown in Table 3.

[0060] Table 3. Activity of short-chain dehydrogenases and their mutants towards 2,3-butanedione

[0061]

[0062] 7. Screening of short-chain dehydrogenases and their mutants for 2,4-pentanedione

[0063] Reaction system: 0.5 g of 2,4-pentanedione, 0.5 g of wet bacteria, isopropanol added to 10 mL, substrate added to a final concentration of 0.5 M, reaction at 30°C, 200 rpm for 30 min, 200 μL of reaction solution was extracted with 200 μL of ethyl acetate, centrifuged, and the supernatant was dried over anhydrous sodium sulfate. The substrate conversion rate (ee%) was detected by GC. The results are shown in Table 4.

[0064] Table 4. Activity of short-chain dehydrogenases and their mutants towards 2,4-pentanedione

[0065]

[0066] 8. Screening of short-chain dehydrogenases and their mutants for 2,5-hexanedione

[0067] Reaction system: 0.57 g of 2,5-hexanedione, 0.5 g of wet bacteria, isopropanol added to 10 mL, substrate added to a final concentration of 0.5 M, reaction at 30°C, 200 rpm for 30 min, 200 μL of reaction solution was extracted with 200 μL of ethyl acetate, centrifuged, and the supernatant was dried over anhydrous sodium sulfate. The substrate conversion rate (ee%) was detected by GC. The results are shown in Table 5.

[0068] Table 5. Activity of short-chain dehydrogenases and their mutants towards 2,5-hexanedione

[0069]

[0070] 9. Screening of short-chain dehydrogenases and their mutants for 3,4-hexanedione

[0071] Reaction system: 0.57 g of 3,4-hexanedione, 0.5 g of wet bacteria, isopropanol added to 10 mL, substrate added to a final concentration of 0.5 M, reaction at 30°C, 200 rpm for 30 min, 200 μL of reaction solution was extracted with 200 μL of ethyl acetate, centrifuged, and the supernatant was dried over anhydrous sodium sulfate. The substrate conversion rate (ee%) was detected by GC. The results are shown in Table 6.

[0072] Table 6. Activity of short-chain dehydrogenases and their mutants towards 3,4-hexanedione

[0073]

[0074] 10. Screening of short-chain dehydrogenases and their mutants for 3,5-heptanedione

[0075] Reaction system: 0.64 g of 3,5-heptanedione, 0.5 g of wet bacteria, isopropanol was added to 10 mL, the substrate was added to a final concentration of 0.5 M, and the reaction was carried out at 30°C and 200 rpm for 30 min. 200 μL of the reaction solution was extracted with 200 μL of ethyl acetate. After centrifugation, the supernatant was dried over anhydrous sodium sulfate. The substrate conversion rate (ee%) was detected by GC. The results are shown in Table 7.

[0076] Table 7. Activity of short-chain dehydrogenases and their mutants towards 3,5-heptanedione

[0077]

[0078] 11. Screening of short-chain dehydrogenases and their mutants for 3,6-octanedione

[0079] Reaction system: 0.71 g of 3,6-octanedione, 0.5 g of wet bacteria, isopropanol was added to 10 mL, the substrate was added to a final concentration of 0.5 M, and the reaction was carried out at 30°C and 200 rpm for 30 min. 200 μL of the reaction solution was extracted with 200 μL of ethyl acetate. After centrifugation, the supernatant was dried over anhydrous sodium sulfate. The substrate conversion rate (ee%) was detected by GC. The results are shown in Table 8.

[0080] Table 8. Activity of short-chain dehydrogenases and their mutants towards 3,6-octanedione

[0081]

[0082]

[0083] 12. Screening of short-chain dehydrogenases and their mutants for dibenzoylation

[0084] Reaction system: 1.05 g of dibenzoyl, 0.5 g of wet bacteria, add isopropanol to 10 mL, add substrate to a final concentration of 0.5 M, react at 30°C, 200 rpm for 2 h, take 200 μL of the reaction solution and add 200 μL of ethyl acetate for extraction, add anhydrous sodium sulfate for drying and centrifuge, take 10 μL of the supernatant and dry it at 60°C, add 1 mL of chromatographic grade n-hexane for dissolution, filter membrane, and detect the substrate conversion rate (ee%) by HPLC. The results are shown in Table 9.

[0085] Table 9. Activity of short-chain dehydrogenases and their mutants on dibenzoyl

[0086]

[0087] Example 2: Synthesis of (2R,3R)-butanediol from 2,3-butanedione catalyzed by the optimal mutant

[0088] The following reaction was carried out using E. coli BL21(DE3) / pET-28a(+)-FRSDR-G94Q / H145F / S153L / Y188G wet cells as catalyst:

[0089] 10mL reaction system: 2,3-butanedione 2.58g, wet bacteria 0.5g, add isopropanol to 10mL, add substrate to a final concentration of 3M, react at 30℃, 200rpm overnight, GC detection, conversion rate 99.9%, ee 99.8%, GC chart see Figure 1 shown.

[0090] 1 L reaction system: 258 g 2,3-butanedione, 50 g wet bacteria, add isopropanol to 1 L, react at 30°C overnight, GC detection, conversion rate 98%, ee 99.8%.

[0091] Example 3: Catalytic synthesis of (2R,4R)-pentanediol from 2,4-pentanedione

[0092] The following reaction was carried out using E. coli BL21(DE3) / pET-28a(+)-FRSDR-G94A / H145F / Y188T / S196C wet cells as catalyst:

[0093] 10mL reaction system: 2,4-pentanedione 3g, wet bacteria 0.5g, add isopropanol to 10mL, add substrate to a final concentration of 3M, react at 37℃, 200rpm overnight, GC detection, conversion rate 81%, ee 99.9%, GC chart see Figure 2 shown.

[0094] 100 L reaction system: 30 kg of 2,4-pentanedione, 5 kg of wet bacteria, add isopropanol to 100 L, react at 37°C, 200 rpm overnight, GC detection, conversion rate 76%, ee 99.9%.

[0095] Example 4: Catalytic Synthesis of (2R,5R)-Hexanediol from 2,5-Hexanedione

[0096] The following reactions were carried out using E. coli BL21(DE3) / pET-28a(+)-FRSDR-G94A / S196C wet cells as catalyst:

[0097] 10mL reaction system: 2,5-hexanedione 3.42g, wet bacteria 0.5g, add isopropanol to 10mL, add substrate to a final concentration of 3M, react at 37℃, 200rpm overnight, GC detection, conversion rate 80%, ee 99.9%, GC chart see Figure 3 shown.

[0098] 5 L reaction system: 1.7 kg of 2,5-hexanedione, 250 g of wet bacteria, add isopropanol to 5 mL, react at 37°C, 200 rpm overnight, GC detection, conversion rate 83%, ee 99.9%.

[0099] Example 5: Catalytic synthesis of (3R,4R)-hexanediol from 3,4-hexanedione

[0100] The following reaction was carried out using E. coli BL21(DE3)pET-28a(+)-FRSDR-G94A / H145F / S153L / Y188T / S196A wet cells as catalyst:

[0101] 10mL reaction system: 3,4-hexanedione 3.42g, wet bacteria 0.5g, add isopropanol to 10mL, add substrate to a final concentration of 3M, react at 37℃, 200rpm overnight, GC detection, conversion rate 93%, ee 99.8%, GC chart see Figure 4 shown.

[0102] Example 6: Catalytic synthesis of (3R,5R)-heptanediol from 3,5-heptanedione

[0103] The following reactions were carried out using E. coli BL21(DE3) / pET-28a(+)-FRSDR-G94E / S153L wet cells as catalyst:

[0104] 10mL reaction system: 3g 3,5-heptanedione, 0.5g wet bacteria, add isopropanol to 10mL, add substrate to a final concentration of 2.3M, react at 37℃, 200rpm overnight, GC detection, conversion rate 79%, ee 99%, GC chart see Figure 5 shown.

[0105] Example 7: Catalytic Synthesis of (3R,6R)-Octanediol from 3,6-Octanedione

[0106] The following reactions were carried out using E. coli BL21(DE3) / pET-28a(+)-FRSDR-G94A / H145F / S153I / Y188A / S196C / K208S wet cells as catalyst:

[0107] 10mL reaction system: 3,6-octanedione 4g, wet bacteria 0.5g, add isopropanol to 10mL, add substrate to a final concentration of 2.8M, react at 37℃, 200rpm overnight, GC detection, conversion rate 76%, ee 99.3%, GC chart see Figure 6 shown.

[0108] 1L reaction system: 400g of 3,6-octanedione, 50g of wet bacteria, add isopropanol to 10mL, react at 37℃, 200rpm overnight, GC detection conversion rate 72%, ee 99.3%.

[0109] Example 8: Catalytic synthesis of (S,S)-1,2-diphenylethylene glycol from dibenzoyl

[0110] The following reaction was carried out using E. coli BL21(DE3) / pET-28a(+)-FRSDR-G94A / Y188T / S196C wet cells as catalyst:

[0111] 10mL reaction system: 6g of dibenzoyl, 0.5g of wet bacteria, add isopropanol to 10mL, add substrate to a final concentration of 2.85M, react at 37℃, 200rpm overnight, GC detection, conversion rate 99%, ee 99.9%, HPLC chart see Figure 7 shown.

[0112] Table 10. Catalytic results of the best mutants in Examples 2-8

[0113]

Claims

1. A short-chain dehydrogenase mutant, characterized in that The short-chain dehydrogenase mutant is a mutant in which the amino acid sequence shown in SEQ ID NO. 2 is mutated into one of the following: (1) glycine at position 94 is mutated into alanine, and serine at position 196 is mutated into alanine; (2) glycine at position 94 is mutated into alanine, histidine at position 145 is mutated into phenylalanine, serine at position 153 is mutated into leucine, tyrosine at position 188 is mutated into threonine, and serine at position 196 is mutated into alanine; (3) glycine at position 94 is mutated into alanine, tyrosine at position 188 is mutated into threonine, and serine at position 196 is mutated into cysteine; (4) glycine at position 94 is mutated into alanine, histidine at position 145 is mutated into phenylalanine, serine at position 153 is mutated into leucine, tyrosine at position 188 is mutated into threonine, and serine at position 196 is mutated into cysteine. (5) Glycine at position 94 mutated to alanine, histidine at position 145 mutated to phenylalanine, serine at position 153 mutated to isoleucine, tyrosine at position 188 mutated to alanine, serine at position 196 mutated to cysteine, and lysine at position 208 mutated to serine; (6) Glycine at position 94 mutated to glutamine, histidine at position 145 mutated to phenylalanine, serine at position 153 mutated to leucine, and tyrosine at position 188 mutated to glycine.

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

3. Use of the short-chain dehydrogenase mutant according to claim 1 in catalyzing the asymmetric synthesis of chiral diols from prochiral diketones.

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 a recombinant genetically engineered bacterium containing a gene encoding a short-chain dehydrogenase mutant as a catalyst, using a potentially chiral diketone as a substrate, and using an organic alcohol as a reaction medium to form a reaction system; carrying out the reaction at 200 rpm and 30-37° C.; obtaining a reaction solution containing a chiral diol after the reaction is completed; and separating and purifying the reaction solution to obtain the chiral diol.

5. The use according to claim 4, characterized in that The organic alcohol includes isopropyl alcohol.

6. The use according to claim 4, characterized in that The prochiral diketones include 2,3-butanedione, 2,4-pentanedione, 2,5-hexanedione, 3,4-hexanedione, 3,5-heptanedione, and 3,6-octanedione.

7. The use according to claim 4, characterized in that In the reaction system, the amount of catalyst used is 10-100 g / L based on the weight of the wet bacteria, and the concentration of the substrate added is 0.5-3 M.

8. The use according to claim 4, characterized in that The wet bacteria were prepared as follows: recombinant engineered bacteria of the short-chain dehydrogenase mutant were inoculated into LB liquid culture medium containing kanamycin at a final concentration of 50 mg / L, and cultured at 37° C. for 8 h to obtain seed liquid; 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 hours to make the bacterial concentration OD600 of 0.4-0.

8. Isopropylthio-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 hours, the culture was centrifuged at 4°C and 4000 rpm for 30 minutes to collect the wet bacteria.

Images