Short-chain dehydrogenase mutant and application of short-chain dehydrogenase mutant in catalytic synthesis of symmetric chiral diol from symmetric diketone

Abstract

The invention discloses a short-chain dehydrogenase mutant and application of the short-chain dehydrogenase mutant in catalyzing symmetric diketone to synthesize symmetric chiral diol. The short-chain dehydrogenase mutant is obtained by performing single mutation or multiple mutation on the 94th site, the 145th site, the 153rd site, the 188th site, the 196th site or the 208th site of a short-chain dehydrogenase amino acid sequence as shown in SEQ ID NO.2. Through short-chain dehydrogenase mutation, prochiral diketone with high substrate concentration can be converted into corresponding chiral diol with high stereoselectivity, and the problems that an existing method is low in yield, low in substrate loading capacity, low in product optical purity and the like are solved. Pure isopropanol is adopted as a cosolvent, so that the problems of subsequent extraction emulsification and the like are effectively prevented, and subsequent extraction and separation of products are facilitated; the wet thallus is used as a catalyst, so that reaction circulation can be realized without additionally adding coenzyme, the production operation is simplified, the production cost is reduced, and the method conforms to the concept of green chemistry.

Description

(I) Technical field

[0001] The 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 symmetrical diketones. (II) 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-toluene sulfonate of symmetrical chiral diol to synthesize a series of chiral side chains, successfully realize the efficient transfer from central chirality to axial chirality in the building-up process, do not need to split and 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 process that is relatively common in the process of synthesizing symmetrical chiral alcohol is basically obtained by metal catalyzed hydrogenation with chiral ketone before, which inevitably increases the complexity and 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 product ee value that is too low. (III) Summary of the invention

[0004] The purpose of the present invention is to provide a short-chain dehydrogenase mutant and its application in catalyzing the asymmetric synthesis of chiral diols from potential chiral diketones. The reaction can realize the reaction cycle without the need for additional addition of coenzymes, thereby simplifying the production operation and reducing the production cost. It is of certain significance for the green production of chiral diols with high stereoselectivity and provides a basic guarantee for the synthesis of chiral phosphine ligands.

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

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

[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) the glycine at position 94 is mutated into alanine (G94A), glutamic acid (G94Q), or glutamine (G94E); (2) the histidine at position 145 is mutated into phenylalanine (H145F); (3) the serine at position 153 is mutated into isoleucine (S153I) or leucine (S153L); (4) the tyrosine at position 188 is mutated into glycine (Y188G), cysteine ​​(Y188C), or threonine (Y188T); (5) the serine at position 196 is mutated into alanine (S196A) or cysteine ​​(S196C); (6) the lysine at position 208 is mutated into serine (K208S); (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, 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 into 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, G94Q / H145F / S153L / Y188T.

[0009] Further, when the substrate is 2,4-pentanedione, the short-chain dehydrogenase mutant is preferably a mutant in which the amino acid sequence shown in SEQ ID NO.2 is mutated into 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, 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: 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] Further, when the substrate is 3,5-heptanedione, 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: G94E, G94E / S153I, G94E / S153L, G94E / S153L / Y188A, G94E / S153L / Y188T, G94E / S153I / 196A.

[0013] Further, when the substrate is 3,6-octanedione, the short-chain dehydrogenase mutant is preferably a mutant 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, G94A / H145F / S153I / Y188T / 196A / K208S.

[0014] Further, 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 into 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 coding gene of the short-chain dehydrogenase mutant, the recombinant vector constructed by the coding gene, and the recombinant genetic engineering bacteria prepared by transformation of 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 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 virus vectors, etc., preferably pET-28a (+) plasmid is used as an expression vector; the host bacteria is preferably Escherichia coli, such as Escherichia coli BL21 cells or Escherichia 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 is: using wet bacteria obtained by fermentation culture of recombinant genetic engineering bacteria containing a gene encoding the short-chain dehydrogenase mutant as a catalyst, using potential chiral diketones as substrates, and using organic alcohols as reaction media to form a reaction system, reacting at 200 rpm and 30-37° C. After the reaction is completed, a reaction solution containing the chiral diol is obtained, and the reaction solution is separated and purified to obtain the chiral diol.

[0017] Furthermore, the organic alcohol includes isopropanol.

[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] Further, the wet bacteria are prepared as follows: the recombinant engineering bacteria of the short-chain dehydrogenase mutant are inoculated into an LB liquid culture medium containing kanamycin with a final concentration of 50 mg / L, 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 kanamycin with a final concentration of 50 mg / L at an inoculum amount of 2% by volume, and cultured at 37°C for about 1.5-2.5 hours so that the bacterial concentration OD600 is 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 30 minutes, and the wet bacteria are collected; LB liquid culture medium: peptone 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L, the solvent is deionized water, pH 7.0.

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

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

[0023] (1) The present invention obtains a short-chain dehydrogenase mutant with significantly improved activity and stereoselectivity for a potential chiral diketone substrate through mutation screening, and can convert a potential chiral diketone with a high substrate concentration (greater than 2M) into a corresponding chiral diol with high stereoselectivity, thereby solving the problems of low yield, low substrate loading, and low product optical purity of the existing method.

[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. The wet bacteria are used as catalysts, and the reaction cycle can be achieved without the need for additional coenzymes, which simplifies the production operation and reduces the production cost. 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 the production cost while avoiding the environmental problems caused by the traditional method, which is of 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 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 in conjunction with 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 engineering 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 being 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), and was 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 QLKAMENNGGGAIINMASIFGSVAAPYSSAYTSSKHGIVGLTKNIGAEYGPKNIRCNAVGPGGIKTPPLLDSLSDEQLNILTSKHPIGRLGEPEEVAELVLFLSSDKASFITGGYYLVDGGYTAI.

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

[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 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] Take 3 μL of PCR product and mix it with 2 μL of Loding buffer, and use gel electrophoresis to verify whether PCR is successful according to the band size. Add 1 μL of Dpn I to the successfully verified PCR product, place it at 37°C for 1 hour to eliminate the original DNA template left during PCR, and place it in a 65°C oven for 10 minutes to inactivate Dpn I. Purify the digested product according to the Clean up kit and store the purified product in a 4°C refrigerator. Obtain the single-site mutants G94A, G94Q, G94E, H145F, S153I, S153L, Y188A, Y188G, Y188T, Y188C, S196A, S196C, 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 engineering bacteria

[0056] 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 5 μL of the PCR product purified in step 4 was added in a clean bench, followed by an ice bath at 0°C for 30 min, heat shock in a 42°C water bath for 90 s, and an ice bath at 0°C for 2 min. 600 μL of LB liquid culture medium was added, and the cells were cultured at 37°C and 200 rpm in a shaking incubator 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 at 37°C with shaking overnight. The inoculum was inoculated into a 250mL Erlenmeyer flask containing 100mL of LB liquid culture medium at a 1% (v / v) inoculation amount, and cultured in a shaker at 37°C and 180rpm. When the OD600 of the culture reached 0.6, IPTG with a final concentration of 0.1mM was added as an inducer, and the culture was induced at 26°C for 14h. The culture was centrifuged, the wet bacteria were collected, and stored at -20°C.

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

[0059] Reaction system: 0.43 g of 2,3-butanedione, 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 30 min, take 200 μL of the reaction solution and add 200 μL of ethyl acetate for extraction, add anhydrous sodium sulfate to the supernatant after centrifugation and dry, use GC to detect the substrate conversion rate, meso (meso) ee%, the results are shown in Table 3.

[0060] Table 3. Activity of short-chain dehydrogenase and its mutants on 2,3-butanedione

[0061]

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

[0063] Reaction system: 0.5 g of 2,4-pentanedione, 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 30 min, take 200 μL of reaction solution and add 200 μL of ethyl acetate for extraction, add anhydrous sodium sulfate to the supernatant after centrifugation and dry, use GC to detect substrate conversion rate, ee%, the results are shown in Table 4.

[0064] Table 4. Activity of short-chain dehydrogenase and its mutants on 2,4-pentanedione

[0065]

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

[0067] Reaction system: 0.57 g of 2,5-hexanedione, 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 30 min, take 200 μL of reaction solution and add 200 μL of ethyl acetate for extraction, add anhydrous sodium sulfate to the supernatant after centrifugation and dry, use GC to detect substrate conversion rate, ee%, the results are shown in Table 5.

[0068] Table 5. Activity of short-chain dehydrogenase and its mutants on 2,5-hexanedione

[0069]

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

[0071] Reaction system: 0.57 g of 3,4-hexanedione, 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 30 min, take 200 μL of reaction solution and add 200 μL of ethyl acetate for extraction, add anhydrous sodium sulfate to the supernatant after centrifugation and dry, use GC to detect substrate conversion rate, ee%, the results are shown in Table 6.

[0072] Table 6. Activity of short-chain dehydrogenase and its mutants on 3,4-hexanedione

[0073]

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

[0075] Reaction system: 0.64 g of 3,5-heptanedione, 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 30 min, take 200 μL of reaction solution and add 200 μL of ethyl acetate for extraction, add anhydrous sodium sulfate to the supernatant after centrifugation and dry, use GC to detect substrate conversion rate, ee%, the results are shown in Table 7.

[0076] Table 7. Activity of short-chain dehydrogenase and its mutants on 3,5-heptanedione

[0077]

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

[0079] Reaction system: 0.71 g of 3,6-octanedione, 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 30 min, take 200 μL of reaction solution and add 200 μL of ethyl acetate for extraction, add anhydrous sodium sulfate to the supernatant after centrifugation and dry, use GC to detect substrate conversion rate, ee%, the results are shown in Table 8.

[0080] Table 8. Activity of short-chain dehydrogenase and its mutants on 3,6-octanedione

[0081]

[0082]

[0083] 12. Screening of short-chain dehydrogenase and its mutants against dibenzoyl

[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 reaction solution and add 200 μL of ethyl acetate for extraction, add anhydrous sodium sulfate to dry and centrifuge, take 10 μL of supernatant and dry at 60°C, add 1 mL of chromatographic grade n-hexane to dissolve, filter membrane, and detect substrate conversion rate (ee%) by HPLC. The results are shown in Table 9.

[0085] Table 9. Bis(2-aminobenzoyl)activity of short-chain dehydrogenase and its mutants

[0086]

[0087] Example 2: The optimal mutant catalyzes the synthesis of (2R,3R)-butanediol from 2,3-butanedione

[0088] The following reactions were 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 overnight at 30℃, 200rpm, GC detection, conversion rate 99.9%, ee 99.8%, GC chart see Figure 1 shown.

[0090] 1L reaction system: 258g 2,3-butanedione, 50g wet bacteria, add isopropanol to 1L, react at 30℃ 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 reactions were 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 overnight at 37℃, 200rpm, GC detection, conversion rate 81%, ee 99.9%, GC chart see Figure 2 shown.

[0094] 100L reaction system: 2,4-pentanedione 30kg, wet bacteria 5kg, add isopropanol to 100L, react at 37℃, 200rpm 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 overnight at 37℃, 200rpm, GC detection, conversion rate 80%, ee 99.9%, GC chart see Figure 3 shown.

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

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

[0100] The following reactions were 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 overnight at 37℃, 200rpm, 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: 3,5-heptanedione 3g, wet bacteria 0.5g, add isopropanol to 10mL, add substrate to a final concentration of 2.3M, react overnight at 37℃, 200rpm, 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 overnight at 37℃, 200rpm, 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 overnight at 37℃ and 200rpm. GC detection conversion rate is 72%, ee99.3%.

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

[0110] The following reactions were 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 overnight at 37℃, 200rpm, 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 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.

2. The short-chain dehydrogenase mutant according to claim 1, 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) the glycine at position 94 is mutated into alanine, glutamic acid, and glutamine respectively; (2) the histidine at position 145 is mutated into phenylalanine; (3) the serine at position 153 is mutated into isoleucine or leucine; (4) the tyrosine at position 188 is mutated into glycine, cysteine, or threonine; (5) the serine at position 196 is mutated into alanine or cysteine; (6) the lysine at position 208 is mutated into serine; (7) a combined mutation of two or more of the single mutations in steps (1) to (6).

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

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

5. The use according to claim 4, characterized in that The application method comprises the following steps: using wet bacteria obtained by fermentation culture of recombinant genetically engineered bacteria 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, reacting at 200 rpm and 30-37° C. After the reaction is completed, a reaction liquid containing a chiral diol is obtained, and the reaction liquid is separated and purified to obtain the chiral diol.

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

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

8. The use according to claim 5, characterized in that In the reaction system, the amount of catalyst used is 10-100 g / L based on the weight of wet bacteria, and the concentration of substrate added is 0.5-3 M.

9. The use according to claim 5, characterized in that The wet bacterial cells are prepared as follows: the recombinant engineering bacteria of the short-chain dehydrogenase mutant are inoculated into LB liquid culture medium containing kanamycin with a final concentration of 50 mg / L, and cultured at 37° C. for 8 hours 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 h to make the bacterial concentration OD600 of 0.4-0.

8. Isopropylthio-β-D-galactoside with a final concentration of 0.1-1.0 mM was then added to the culture medium. After inducing expression at 26°C for 12 h, the culture medium was centrifuged at 4°C and 4000 rpm for 30 min to collect the wet bacteria.

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