Biocatalysis method for synthesizing material precursor alpha-hydroxy ketone

Abstract

The invention discloses a biocatalysis method for synthesizing a material precursor alpha-hydroxy ketone, and belongs to the technical field of biocatalysis synthesis. According to the invention, a coenzyme circulating enzyme lactic dehydrogenase LcLDH1 in an alpha-hydroxy ketone synthesis system is replaced by NADH oxidase EfNox from enterococcus faecalis, so that the yield of 2-hydroxy-4-chloroacetophenone reaches 84.7 or above, and the highest yield reaches 90%. According to the method, the coenzyme regeneration cost is effectively reduced, the overall economical efficiency of a reaction system is improved, and a more favorable technical support is provided for large-scale synthesis and industrial application of the alpha-hydroxy ketone compound.

Description

Technical Field

[0001] The invention relates to a biocatalytic method for synthesizing a material precursor alpha-hydroxyketone, and belongs to the technical field of biocatalytic synthesis. Background Art

[0002] α-Hydroxy ketones are an important class of organic compounds with a hydroxyl group located α-to the reactive carbonyl group. Due to their unique functional group arrangement and remarkable chemical reactivity, α-hydroxy ketones hold significant research value and broad application prospects in drug synthesis, functional material development, and fine chemical manufacturing.

[0003] In recent years, with the increasing demand for the application of α-hydroxyketone compounds in the fields of medicine and materials, the research on their synthesis methods has also received significant attention. The main synthetic routes currently include chemical catalysis and biocatalysis. Typical strategies of chemical catalysis include: (1) using substituted methyl ketone as raw material, generating α-bromoketone intermediates through bromination reagents (such as Br2, HBr or N-bromosuccinimide), and then hydrolyzing to obtain the corresponding α-hydroxyketone; (2) using olefins as substrates, selectively oxidizing double bonds through halogen reagents (such as I2, N-bromosuccinimide) or transition metal catalysts (such as palladium catalysts); (3) using aldehyde compounds as starting materials, through N-heterocyclic carbene catalysts and polyformaldehyde coupling reaction to prepare α-hydroxyketone. However, these chemical synthesis methods generally have the disadvantages of harsh reaction conditions, a large number of by-products, and heavy environmental load, which limit their widespread promotion in practical applications.

[0004] Multienzyme cascade catalysis utilizes multiple enzymes working together in a single reaction system to efficiently synthesize complex target compounds from simple starting materials. In comparison, in vitro multienzyme cascade catalysis systems have attracted increasing attention in the biomanufacturing field in recent years and show significant development prospects due to their advantages, including fewer side reactions, high product yields, rapid reaction rates, easy product separation and purification, high tolerance to toxic environments, and easy and precise control of reaction conditions. In 2024, our research group developed a three-enzyme cascade catalytic reaction system, using the epoxide hydrolase mutant RpEHL360V from Rhodotorula paludigena, the (2,3)-butanediol dehydrogenase mutant BsBDHAI49L / V266L / G292A from Bacillus subtilis, and the lactate dehydrogenase LcLDH1 from Lactobacillus casei. Under mild reaction conditions, 50 mmol / L racemic epoxide was successfully converted into 2-hydroxyacetophenone with a final product yield of 73.1% (Synthesis of α-hydroxy ketones through novel one-pot multi-enzyme cascade system from racemic epoxides).

[0005] Compared to chemical methods, multi-enzyme cascade catalysis integrates the synergistic action of multiple enzymes within a single reaction system, efficiently converting simple substrates into complex target products in a single step. In vitro multi-enzyme cascade catalysis systems, in particular, offer advantages such as high reaction specificity, minimal side reactions, high product purity, rapid reaction speed, mild conditions, environmental friendliness, and excellent process economics. In recent years, they have become a key development direction in the field of biocatalysis and synthesis.

[0006] Therefore, developing a new method for synthesizing α-hydroxyketones by multi-enzyme cascade catalysis with better economy and industrial adaptability has extremely high practical and economic value. Summary of the Invention

[0007] In 2024, the inventors constructed a three-enzyme cascade catalytic system, specifically including the epoxide hydrolase mutant RpEH from marine red yeast (Rhodotorula paludigena) L360V , (2,3)-Butanediol dehydrogenase mutant BsBDHA from Bacillus subtilis I49L / V266L / G292A, and the lactate dehydrogenase LcLDH1 from Lactobacillus casei. Under relatively mild reaction conditions, they successfully achieved the efficient conversion of 50 mmol / L racemic epoxide to 2-hydroxyacetophenone, with a final product yield of 73.1% (disclosed in the literature "Synthesis of α-hydroxy ketones through a novel one-pot multi-enzyme cascade system from racemic epoxides").

[0008] To further improve the yield of α-hydroxyketones, the present invention replaces the coenzyme recycling enzyme lactate dehydrogenase LcLDH1 in the original system with NADH oxidase EfNox from Enterococcus faecium. This optimization strategy effectively reduces the cost of coenzyme regeneration and improves the overall economic efficiency of the reaction system, providing more favorable technical support for the large-scale synthesis and industrial application of α-hydroxyketone compounds.

[0009] The first object of the present invention is to provide a genetically engineered Escherichia coli bacterium for producing α-hydroxyketone, wherein the genetically engineered Escherichia coli bacterium simultaneously overexpresses (2,3)-butanediol dehydrogenase and NADH oxidase.

[0010] In one embodiment, the amino acid sequence of (2,3)-butanediol dehydrogenase is shown as SEQ ID NO.5, and the amino acid sequence of NADH oxidase is shown as SEQ ID NO.7.

[0011] In one embodiment, the amino acid sequence of the (2,3)-butanediol dehydrogenase is shown in SEQ ID NO. 5, that is, the (2,3)-butanediol dehydrogenase mutant BsBDHA. I49L / V266L / G292A , the nucleotide sequence is shown in SEQ ID NO.6.

[0012] In one embodiment, the NADH oxidase is from Enterococcus faecium, and the nucleotide sequence of the NADH oxidase is shown in SEQ ID NO.8.

[0013] In one embodiment, (2,3)-butanediol dehydrogenase and NADH oxidase are overexpressed by a plasmid vector comprising any one of pETDuet-1, pCDFDuet-1, or pRSFDuet-1.

[0014] In one embodiment, the plasmid vector is pCDFDuet-1.

[0015] The second object of the present invention is to provide the use of any of the above-mentioned genetically engineered Escherichia coli bacteria in the preparation of α-hydroxyketones.

[0016] The third object of the present invention is to provide a method for synthesizing α-hydroxyketone using a multi-enzyme cascade catalysis, comprising the steps of:

[0017] (1) Using racemic styrene oxide and its derivatives as starting substrates, an epoxy ring-opening reaction is carried out in a reaction system containing epoxy hydrolase to generate the corresponding chiral 1,2-diol intermediate;

[0018] (2) adding any of the above-mentioned genetically engineered Escherichia coli (E. coli / pET-BN) and catalyzing the reaction in the presence of oxygen and coenzyme NADH to oxidatively convert the chiral 1,2-diol intermediate into α-hydroxyketone.

[0019] In one embodiment, the racemic epoxystyrene and its derivatives in step (1) include any one or more of rac-p-chloroepoxystyrene, rac-epoxystyrene and rac-m-chloroepoxystyrene.

[0020] In one embodiment, the amino acid sequence of the epoxide hydrolase in step (1) is shown as SEQ ID NO.1 or SEQ ID NO.3.

[0021] In one embodiment, the nucleotide sequence of the epoxy hydrolase in step (1) is as shown in SEQ ID NO. 2 or SEQ ID NO. 4.

[0022] In one embodiment, in step (2), the concentration of the genetically engineered E. coli / pET-BN is 10-25 mg / mL, and the NADH concentration is 4-10 mmol / L;

[0023] In step (2), the presence of oxygen is conditional on sufficient stirring and ventilation, that is, the volume ratio of the reaction liquid to the volume of air is 1-1.5:1-3.

[0024] In one embodiment, in step (1) and step (2), the reaction temperature is 25-40°C.

[0025] In one embodiment, in step (1) and step (2), the reaction temperature is 30°C.

[0026] In one embodiment, the concentration of E. coli / pET-BN cells is 15-25 mg / mL.

[0027] In one embodiment, the NADH concentration is 8-10 mmol / L.

[0028] In one embodiment, the ratio of the reaction liquid volume to the air volume is 1:1 to 3, preferably, the ratio of the reaction liquid volume to the air volume is 1:3.

[0029] The fourth object of the present invention is to provide the use of any of the above methods in the synthesis of α-hydroxyketones.

[0030] Beneficial effects of the present invention

[0031] The efficient conversion of epoxides can be effectively achieved through a multi-enzyme cascade system, which has the advantages of mild reaction conditions, high selectivity, few by-products, and environmental friendliness. It greatly reduces production costs, improves the yield and purity of reaction products, is suitable for industrial-scale applications, and has significant economic and social benefits.

[0032] Specifically:

[0033] The genetically engineered bacteria E.coli / pCDF-BN of the present invention has a yield of 2-hydroxy-4-chloroacetophenone of more than 84.7% under different substrates at a concentration of 50 mmol / L, with the highest reaching 90%, which is significantly better than 73.1% under the same conditions in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 bsbdha obtained by PCR amplification I49L / V266L / G292A gene (lane 1).

[0035] Figure 2 Three linearized vectors (1-3 are schematic diagrams of nucleic acid gels after double enzyme digestion of pETDuet-1, pCDFDuet-1 and pRSFDuet-1).

[0036] Figure 3 The efnox gene was amplified by PCR (lane 1).

[0037] Figure 4 These are three linearized vectors (1-3 are schematic diagrams of nucleic acid gels after double enzyme digestion of pETDuet-bdh, pCDFDuet-bdh and pRSFDuet-bdh respectively).

[0038] Figure 5 Schematic diagram of the structures of three recombinant expression vectors: pETDuet-bdh-nox, pCDFDuet-bdh-nox and pRSFDuet-bdh-nox.

[0039] Figure 6 Schematic diagram of cascade catalytic reaction. DETAILED DESCRIPTION

[0040] The following describes preferred embodiments of the present invention. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0041] The raw materials used in the embodiment are:

[0042] Mutant RpEH L360V , recombinant strain E. coli / rpeh L360V , mutant BsBDHA I49L / V266L / G292A , recombinant plasmid pET-28a-bsbdha I49L / V266L / G292A This research has been published in the literature “Synthesis of α-hydroxy ketones through novel one-pot multi-enzyme cascade system from racemic epoxides; doi.org / 10.1016 / j.mcat.2024.114464”.

[0043] The EfNox gene was inserted between Nde I and Xho I of pET-28a and synthesized by Sangon Biotech (Shanghai) Co., Ltd. to obtain the plasmid pET-28a-efnox.

[0044] pETDuet-1 was purchased from Fenghui Biotechnology Co., Ltd.;

[0045] pCDFDuet-1 was purchased from Fenghui Biotechnology Co., Ltd.;

[0046] pRSFDuet-1 was purchased from Fenghui Biotechnology Co., Ltd.

[0047] Example 1: Construction of a recombinant plasmid for co-expression of (2,3)-butanediol dehydrogenase and NADH oxidase genes

[0048] 1. Construction of pETDuet-bdh, pCDFDuet-bdh and pRSFDuet-bdh recombinant plasmids

[0049] Plasmid pET-28a-bsbdha I49L / V266L / G292A As a PCR amplification template, specific primers (BDH-F, BDH-R) were used to amplify BsBDHA I49L / V266L / G292A Gene (amino acid sequence as shown in SEQ ID NO.5, nucleotide sequence as shown in SEQ ID NO.6), amplification results as shown in Figure 1 The specific primer sequences are shown in Table 1

[0050] Table 1 Primers

[0051] name sequence BDH-F: 5'-ATCACCACAGCCAGGATCCGATGAAGGCAGCAAGATGG-3' BDH-R: 5'-TCGACTTAAGCATTATGCGGCCGCTTAGTTAGGTCTAACAAG-3' NOx-F: 5'-TAAGAAGGAGATATACATATGAAAGTAGTAGTAATTGG-3' NOx-R: 5'-TTCTTTACCAGACTCGAGTTATTGTTTTGTTTCCATTT-3'

[0052] The vectors pETDuet-1, pCDFDuet-1, and pRSFDuet-1 were linearized using restriction enzymes SwiftCut Nde I and SwiftCut Xho I (purchased from Nanjing Novozymes Biotech Co., Ltd.). Figure 2 shown.

[0053] Exnase II-mediated BsBDHA I49L / V266L / G292A The gene was homologously recombined with three linearized vectors (pETDuet-1, pCDFDuet-1 and pRSFDuet-1) to construct the recombinant plasmids pETDuet-bdh, pCDFDuet-bdh and pRSFDuet-bdh.

[0054] 2. Construction of pETDuet-bdh-nox, pCDFDuet-bdh-nox and pRSFDuet-bdh-nox recombinant plasmids

[0055] Using plasmid pET-28a-efnox as a template, primers (NOx-F, NOx-R, as shown in Table 1) were used to amplify the EfNox gene (amino acid sequence shown in SEQ ID NO.7, nucleotide sequence shown in SEQ ID NO.8). The amplification results are shown in Figure 1. Figure 3 shown.

[0056] The recombinant plasmids pETDuet-bdh, pCDFDuet-bdh, and pRSFDuet-bdh constructed in step 1 were linearized using restriction enzymes SwiftCut Nde I and SwiftCut Xho I, respectively. Figure 4 shown.

[0057] Exnase II was used to mediate homologous recombination of EfNox gene with three linearized vectors (pETDuet-bdh, pCDFDuet-bdh and pRSFDuet-bdh) to construct recombinant plasmids pETDuet-bdh-nox, pCDFDuet-bdh-nox and pRSFDuet-bdh-nox. The results are shown in Figure 2. Figure 5 shown.

[0058] The constructed recombinant plasmids (recombinant plasmids pETDuet-bdh-nox, pCDFDuet-bdh-nox and pRSFDuet-bdh-nox) were transformed into E. coli BL21 (DE3) to obtain recombinant strains, which were named E. coli / pET-BN, E. coli / pCDF-BN and E. coli / pRSF-BN according to the different plasmids.

[0059] Example 2: Induced expression of recombinant bacteria and preparation of lyophilized powder

[0060] Preparation of freeze-dried bacterial powder, comprising the steps of:

[0061] Take the recombinant strain constructed in Example 1 and the one expressing RpEH L360V (amino acid sequence as SEQ ID NO.1, nucleotide sequence as SEQ ID NO.2) recombinant strain E. coli / rpeh L360V The single colonies were inoculated into LB medium (2 mL) containing the corresponding antibiotics and cultured overnight at 37 ° C and 220 rpm; 2 mL of the overnight culture was inoculated into 100 mL of fresh LB medium (containing the corresponding antibiotics) and continued to culture. 600 When the pH value was about 0.6-0.8, IPTG was added to a final concentration of 0.1 mmol / L and the expression was induced at 16°C for 20 h.

[0062] After expression, the bacteria were collected and prepared into a bacterial suspension with a concentration of 200 mg / mL using 10 mmol / L, pH 7.0 sodium phosphate buffer (Na2HPO4-NaH2PO4), and freeze-dried for 8 h to obtain dry bacterial freeze-dried powder, which was accurately weighed and sealed for storage.

[0063] Example 3: Optimization of cascade catalytic reaction conditions

[0064] Take the recombinant E. coli / rpeh prepared in Example 2 L360V and E. coli / pET-BN lyophilized powder were prepared into bacterial suspensions with a concentration of 20 mg / mL using sodium phosphate buffer (10 mmol / L, pH 7.0) for reaction system optimization. The reaction diagram is shown in the figure. Figure 6 shown.

[0065] 1. Optimization of E. coli / pET-BN cell concentration

[0066] Using rac-p-chloroepoxyphenylethane (rac-1a, 20mmol / L) as substrate, 5mg / mL of E. coli / rpeh L360VThe bacterial suspension was reacted at 30°C for 1 h to convert rac-1a to (R)-1-(4-chlorophenyl)-1,2-ethanediol (1b); then 5-25 mg / mL E. coli / pET-BN lyophilized powder and 4 mmol / L NADH were added and the reaction was continued for 12 h;

[0067] After the reaction was completed, an equal volume of ethyl acetate was used for extraction, and the content of 2-hydroxy-4-chloroacetophenone (1c) in the reaction system was analyzed by gas chromatography (GC).

[0068] The results are shown in Table 2. When the concentration of E. coli / pET-BN cells was 15 mg / mL, the yield of 1c reached the maximum, which was 28.4%.

[0069] Table 2 Effect of bacterial concentration on 1c yield

[0070]

[0071] 2. Optimization of NADH concentration

[0072] Under the determined optimal E. coli / pET-BN concentration (15 mg / mL), the NADH concentration (2-10 mmol / L) was further optimized and the reaction was carried out for 12 h.

[0073] The results are shown in Table 3. The results show that when the NADH concentration is 10 mmol / L, the yield of 1c is the highest, which is 41.6%.

[0074] Table 3 Effect of NADH concentration on 1c yield

[0075]

[0076] 3. Optimization of oxygen supply ratio

[0077] Under the previously optimized E. coli / pET-BN and NADH concentration conditions, the ratio of the reaction liquid volume to the air volume was changed (1:3 to 3:1) and the reaction was carried out for 12 hours.

[0078] The results are shown in Table 4. The results show that when the volume ratio of reaction liquid to air is 1:3, the yield of 1c reaches the highest, which is 56.7%.

[0079] Table 4 Effect of oxygen supply ratio on 1c yield

[0080]

[0081] The above results show that the optimal reaction conditions for cascade catalysis are: bacterial concentration 15 mg / mL, NADH concentration 10 mmol / L, and a volume ratio of reaction liquid to air 1:3.

[0082] Example 4: Comparison of catalytic effects of recombinant bacteria expressed in different vectors

[0083] Under the optimal reaction conditions of Example 3, 20 mmol / L rac-1a was used as the substrate and 5 mg / mL E. coli / rpeh L360V The reaction was catalyzed by lyophilized buffer for 1 h, and then different plasmid co-expression recombinant bacteria—E. coli / pET-BN, E. coli / pCDF-BN, and E. coli / pRSF-BN lyophilized powders at a concentration of 15 mg / mL were added and catalysis was continued for 12 h. The NADH concentration was 10 mmol / L, and the liquid volume to air volume ratio was 1:3.

[0084] After the reaction, the optimal recombinant strain was determined based on the highest yield of 1c. The results are shown in Table 5. The results show that under the same reaction conditions, the highest yield of 1c obtained by E. coli / pCDF-BN was 62.9%.

[0085] Table 5 Effect of different carriers on 1c yield

[0086] Recombinant bacteria species Yield (%) E.coli / pET-BN 56.7 E.coli / pCDF-BN 62.9 E.coli / pRSF-BN 21.6

[0087] Example 5: Application of a multi-enzyme cascade catalytic system to synthesize α-hydroxyketones

[0088] According to the method of Example 4, 50 mmol / L of different substrates (rac-p-chloroepoxyphenylethane, rac-epoxyphenylethane and rac-m-chloroepoxyphenylethane, i.e., rac-1a, rac-2a and rac-3a) were used, and the optimal reaction conditions in Example 3 (30°C at E. coli / rpeh L360V 1c was synthesized by catalyzing the reaction for 1 h with a 5 mg / mL bacterial suspension, followed by the addition of 15 mg / mL E. coli / pCDF-BN lyophilized powder and 10 mmol / L NADH, and continued for 12 h under sufficient stirring and aeration conditions. The reaction was continued for 12 h, and the 1c concentration was determined. The results are shown in Table 6.

[0089] Table 6 Effect of different substrates on the yield of 1c

[0090]

[0091] The results showed that at the same substrate concentration and different substrates, the yield of 1c by E. coli / pCDF-BN was high, significantly better than the 73.1% in the existing technology (Synthesis of α-hydroxy ketones through novel one-pot multi-enzyme cascade system from racemic epoxides).

[0092] RhE L360V Amino acid sequence SEQ ID NO.1:

[0093] MAAHSFTAPPAPYNIDFAPQVDDLHRRLDAARWPGQDVVPDDVDYGEHGAFGLGAGPSLALMKELAQEWRGQDQKQLQDHLNSYKNYRVEIEGLDIHFLHYP SARADAFPLILCHGWPGGYHEFLHVLERLTEPKDQGSRAFHVVVPSMPGYAFSSPPKTAKWGMEDTARVFDKLMTGLGYFRYAAQGGDWGSITARCLGSLHKE NCVAVHLNFCPVPPPPFPLNMFNPRTLLDWMPRFVLPDERRAKLERGVAYIERGSSYYAMQNLTPRTPAYGLNDSPIGLAWIGEKMIPGIDKAVKHPNATLNREALFTTLSIYWFTGSIGSSFLPYALNPHFGTFLVSPRHHLPNFALSNFPDEVFTPEERDARRTGNLRWYKDAEDGGHFAALEKPEVFAEHVREAMGVLLSNQA

[0094] RhE L360V Nucleotide sequence SEQ ID NO.2:

[0095]

[0096] RpEH L360C Amino acid sequence SEQ ID NO.3:

[0097] MAAHSFTAPPAPYNIDFAPQVDDLHRRLDAARWPGQDVVPDDVDYGEHGAFGLGAGPSLALMKELAQEWRGQDQKQLQDHLNSYKNYRVEIEGLDIHFLHYPSARADAFPLILCHGWPGGYHEFLHVLERLTEPKDQGSRAFHVVVPSMPGYAFSSPPKTAKWGMEDTARVFDKLMTGLGYFRYAAQGGDWGSITARCLGSLHKENCVAVHLNFCPVPPPFPLNMFNPRTLLDWMPRFVLPDERRAKLERGVAYIERGSSYYAMQNLTPRTPAYGLNDSPIGLLAWIGEKMIPGIDKAVKHPNATLNREALFTTLSIYWFTGSIGSSFLPYALNPHFGTFLVSPRHHLPNFALSNFPDECFTPEERDARRTGNLRWYKDAEDGGHFAALEKPEVFAEHVREAMGVLLSNQA

[0098] RpEH L360C Nucleotide sequence SEQ ID NO.4:

[0099]

[0100] BsBDHA I49L / V266L / G292A Amino acid sequence SEQ ID NO.5:

[0101] MKAARWHNQKDIRIEHIEEPKTEPGKVKIKVKWCGICGSDLHEYLGGPLFIPVDKPHPLTNETAPVTMGHEFSGEVVEVGEGVENYKVGDRVVVEPIFATHGHQGAYNLDEQMGFLGLAGGGGGFSEYVSVDEELLFKLPDELSYEQGALVEPSAVALYAVRSSKLKAGDKAAVFGCGPIGLLVIEALKAAGATDIYAVELSPERQQKAEELGAIIVDPSKTDDVVAEIAERTGGGVDVAFEVTGVPVVLRQAIQSTTIAGETVILSIWEKGAEIHPNDIVIKERTVKGIIAYRDIFPAVLSLMKEGYFSADKLVTKKIVLDDLIEEGFGALIKEKSQVKILVRPN

[0102] BsBDHA I49L / V266L / G292A Nucleotide sequence SEQ ID NO.6:

[0103]

[0104] EfNox amino acid sequence SEQ ID NO.7:

[0105] MKVVVIGCTHAGTAAVKSILKNHPEAEVTVYERNDNISFLSCGIALYVGGVVKDPAGLFYSNPEELSSLGATVKMEHDVTDVDTENKTVTAKDLKTGETETVSYDKLVMTTGSWPIIPPISGIDAKNVLLCKNYNQANEIIAQAKEAKRLVIVGGGYIGIELVEAFVESGKQVTLIDGLDRILNKYLDKPFTDILEKELTDRGVTLALGENVTEFITDEEGKVKQVATPTDTFDTDMVILCVGFRPNTKLIEGKVETLPNGAIKVNEYMQTSDPDIFAAGDSAVVNYNPSGTQNYIPLATNAVRQGLLVGNNLTEHKMAYRGTQGTSGLYLFGWTIGSTGVTKESATLNDLEVQATVFEDNYRPEFMPTTEKVMMELVYEKGTNRIVGAQFMSKYDITQSANTMSLAVQNKMTVEDLALSDFFFQPHFDRPWNYLNLLAQAALGEIETAQ

[0106] EfNox nucleotide sequence SEQ ID NO.8:

[0107]

[0108] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A genetically engineered Escherichia coli for producing α-hydroxyketone, characterized in that: The genetically engineered Escherichia coli bacteria simultaneously overexpress (2,3)-butanediol dehydrogenase and NADH oxidase.

2. The genetically engineered Escherichia coli according to claim 1, characterized in that The amino acid sequence of (2,3)-butanediol dehydrogenase is shown in SEQ ID NO.5, and the amino acid sequence of NADH oxidase is shown in SEQ ID NO.

7.

3. The genetically engineered Escherichia coli according to claim 1, characterized in that (2,3)-butanediol dehydrogenase and NADH oxidase are overexpressed by a plasmid vector, wherein the plasmid vector includes any one of pETDuet-1, pCDFDuet-1 or pRSFDuet-1.

4. Use of the genetically engineered Escherichia coli according to any one of claims 1 to 3 in the preparation of α-hydroxyketone.

5. A method for synthesizing α-hydroxyketone using multi-enzyme cascade catalysis, characterized in that: Including steps: (1) Using racemic styrene oxide and its derivatives as starting substrates, an epoxy ring-opening reaction is carried out in a reaction system containing epoxy hydrolase to generate the corresponding chiral 1,2-diol intermediate; (2) Adding the genetically engineered Escherichia coli according to any one of claims 1 to 3, catalyzing the reaction in the presence of oxygen and coenzyme NADH, and oxidatively converting the chiral 1,2-diol intermediate into α-hydroxyketone.

6. The method according to claim 5, characterized in that In step (1), racemic epoxystyrene and its derivatives include any one or more of rac-p-chloroepoxystyrene, rac-epoxystyrene and rac-m-chloroepoxystyrene.

7. The method according to claim 5, characterized in that The amino acid sequence of the epoxide hydrolase in step (1) is shown in SEQ ID NO.1 or SEQ ID NO.

3.

8. The method according to claim 5, characterized in that In step (2), the concentration of the genetically engineered Escherichia coli cells is 10-25 mg / mL, and the NADH concentration is 4-10 mmol / L; In step (2), the presence of oxygen is such that the volume ratio of the reaction liquid to the volume of air is 1-1.5:1-3.

9. The method according to claim 5, characterized in that In step (1) and step (2), the reaction temperature is 25-40°C.

10. Use of the method according to any one of claims 5 to 9 in the synthesis of α-hydroxyketone.

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