NADP-type formate dehydrogenase mutant and use thereof in enzyme-catalyzed redox reaction

Abstract

Provided are an NADP-type formate dehydrogenase mutant and the use thereof in an enzyme-catalyzed redox reaction. The mutant takes NADP-type formate dehydrogenase XSFDH derived from Xanthobacter sp. 91 with an amino acid sequence as shown in SEQ ID NO: 2 as an original sequence, and has an amino acid sequence with corresponding mutation sites (such as D222Q). Further provided are a coding gene of the NADP-type formate dehydrogenase mutant, a vector, a bioengineered bacterium, and a method for synthesizing Pro-Xylane. The mutant has the advantages of good stereoselectivity, high optical purity of the product, and an improved reaction yield.

Description

A mutant of NADP-type formate dehydrogenase and its application in enzyme-catalyzed redox reactions. Technical Field

[0001] This invention belongs to the fields of enzyme engineering and genetic engineering technology, and in particular relates to an NADP-type formate dehydrogenase mutant and its application in the synthesis of bosine. Background Technology

[0002] Pro-Xylane, chemically known as hydroxypropyltetrahydropyranotriol, is a xylose derivative with anti-aging activity. Pro-Xylane can make the skin stronger and more elastic, improve fine lines on the neck, and prevent aging, and has a wide range of applications in cosmetics. Studies have shown that the activity of S-Pro-Xylane (Ia) is superior to that of R-Pro-Xylane (Ib) (Bioorg Med Chem Lett, 2009, 19(3):845-849; CN100441588).

[0003] Currently, most Bosein products on the market are in mixture form (S / R isomer ratio between 5:5 and 7:3). This is primarily because existing preparation processes use sodium borohydride or sodium triacetyl borohydride for carbonyl reduction, resulting in unsatisfactory stereoselectivity. For example, WO02 / 051828A2 discloses a chemical preparation method for Bosein that uses sodium borohydride to reduce the carbonyl group of β-acetone xyloside (II). This method suffers from low stereoselectivity, difficulty in product separation, and environmental pollution.

[0004] Existing technologies also disclose some methods for biocatalytic synthesis. For example, CN111876452A discloses a one-pot method for preparing Bosein using a bioenzyme, but the information on the carbonyl reductase used in this method is unknown, making it difficult to reproduce. For example, CN113416756A discloses a biocatalytic method using aldehyde-ketone reductase (AKR) and alcohol dehydrogenase, but the content of the S-configuration diastereomer obtained is only 94.6%, and the optical purity of the product is poor. For example, CN114507681A discloses a biocatalytic synthesis method using sorbitase OpCR and its mutants derived from the wild-type structural gene of Ogataea parapolymorpha, but does not disclose the stereoconfiguration and chiral purity of the obtained Bosein product.

[0005] In enzymatic reactions, the NAD(P)H regeneration system offers mild reaction conditions, especially in whole-cell catalysis where no additional cofactors are required. Common coenzyme regeneration systems include glucose dehydrogenase, isopropanol dehydrogenase, and formate dehydrogenase. However, in cofactor-coupled regeneration systems, glucose dehydrogenase (GDH) produces gluconic acid, necessitating acidification and purification. In isopropanol dehydrogenase (ADH) systems, its organic nature reduces enzyme activity and stability (Appl. Environ. Microbiol. 2022, 88, e00341–00322). Formate dehydrogenase (FDH) is a better coenzyme system, with carbon dioxide as its only byproduct, which easily escapes from the reaction system without affecting enzyme activity or product purification. However, wild-type formate dehydrogenase (FDH) is largely NAD-dependent, exhibiting poorer NADP-type catalytic activity compared to other coenzyme regeneration enzymes such as glucose dehydrogenase. Therefore, many studies have focused on the shift in FDH's coenzyme preference. For example, Tishkov et al. reported that coenzyme specificity was altered by NAD+. + Transform into NADP + The mutant PseFDH T5M8 enables it to use NADP. + (Biochem.Biophys.2006,409(1)); Xu et al. derived the coenzyme preference of CdFDH from Candida dubliniensis from NAD + Transform into NADP + Its specific activity was significantly increased to 3.2 U / mg (ChemBioChem 2023, 24, e202300390.). However, the enzyme activity of the currently reported NADP-type formate dehydrogenase is relatively poor, and the hydrogen donation capacity of the coenzyme is insufficient during the reaction, resulting in a large amount of coenzyme required and a low reaction conversion rate. This also leads to low production efficiency and high production costs for high-value chemicals that rely on the NADP coenzyme regeneration cycle system.

[0006] Therefore, there is a need in the field to develop formate dehydrogenases and / or their mutants with better activity, which can provide more options for biocatalytic synthesis methods.

[0007] Summary of the Invention

[0008] To address the problems of relatively poor enzyme activity of NADP-type formate dehydrogenases in biocatalytic synthesis methods, leading to low production efficiency and high production costs, as well as the limited variety of NADP-type formate dehydrogenases available, this invention provides a class of NADP-type formate dehydrogenases with improved enzyme activity. It also provides the corresponding nucleic acid sequence, recombinant expression plasmid, genetically engineered strain, and preparation method of this NADP-type formate dehydrogenase, as well as its application in the stereospecific synthesis of S-Bosoxonine.

[0009] In a first aspect, the present invention provides an NADP-type formate dehydrogenase, wherein the NADP-type formate dehydrogenase is derived from Xanthobacter sp. 91 and has the amino acid sequence shown in SEQ ID NO:2 as the starting sequence, and has the following mutation sites:

[0010] Mutant 2: An amino acid sequence corresponding to SEQ ID NO:2 and having the mutation site D222Q;

[0011] Mutant 6: An amino acid sequence corresponding to SEQ ID NO:2 and having mutation sites A199G and D222Q;

[0012] Mutant 7: An amino acid sequence corresponding to SEQ ID NO:2 and having mutation sites A199G, D222Q, C256A and H380K;

[0013] Mutant 8: An amino acid sequence corresponding to SEQ ID NO:2 and having mutation sites A199G, D222Q, C256A, H380K and S381V;

[0014] Mutant 9: An amino acid sequence corresponding to SEQ ID NO:2 and having mutation sites A199G, D222Q, C256A, H380K, S381V and Y63F;

[0015] Mutant 10: An amino acid sequence corresponding to SEQ ID NO:2 and having mutation sites A199G, D222Q, C256A, H380K, S381V and I280L;

[0016] Mutant 11: An amino acid sequence corresponding to SEQ ID NO:2 and having mutation sites A199G, D222Q, C256A, H380K, S381V and C146S;

[0017] Mutant 12: An amino acid sequence corresponding to SEQ ID NO:2 and having mutation sites A199G, D222Q, C256A, H380K, S381V, Y63F and I280L;

[0018] Mutant 13: an amino acid sequence corresponding to SEQ ID NO:2 and having mutation sites A199G, D222Q, C256A, H380K, S381V, Y63F, C146S and I280L.

[0019] In some specific implementations, the carbonyl reductase is mutant 2.

[0020] In some specific implementations, the carbonyl reductase is mutant 6.

[0021] In some specific embodiments, the carbonyl reductase is mutant 7.

[0022] In some specific embodiments, the carbonyl reductase is mutant 8.

[0023] In some specific implementations, the carbonyl reductase is mutant 9.

[0024] In some specific embodiments, the carbonyl reductase is mutant 10.

[0025] In some specific embodiments, the carbonyl reductase is mutant 11.

[0026] In some specific embodiments, the carbonyl reductase is mutant 12.

[0027] In some specific embodiments, the carbonyl reductase is mutant 13.

[0028] The NADP-type formate dehydrogenase provided in this invention is a molecularly modified NADP-type formate dehydrogenase XSFDH (its amino acid sequence is shown in SEQ ID NO:2) derived from Xanthobacter sp. 91, thereby obtaining a variant of NADP-type formate dehydrogenase with enhanced enzyme activity. This invention uses wild-type carbonyl reductase XSFDH as a template, and performs error-prone PCR using a random point mutagenesis kit, or mutates wild-type formate dehydrogenase XSFDH through directed evolution to obtain a plasmid library containing the evolved formate dehydrogenase gene. High-throughput screening is used to obtain mutation sites that enhance enzyme activity, and mutation libraries are constructed for these mutation sites to screen for mutants with enhanced activity and preferred mutation sites.

[0029] The NADP-type formate dehydrogenase provided by this invention has higher enzyme activity compared with wild-type NADP-type formate dehydrogenase XSFDH, which can reduce the amount of enzyme used in the reaction.

[0030] Secondly, the present invention also provides a coding gene for the NADP-type formate dehydrogenase described in the first aspect. The nucleotide sequence of the coding gene is obtained by taking the sequence shown in SEQ ID NO:1 as the starting sequence and performing corresponding mutations (e.g., base substitutions) on the codons based on the differences between the amino acid sequence of the NADP-type formate dehydrogenase and SEQ ID NO:2.

[0031] The present invention also provides an isolated nucleic acid that encodes the NADP-type formate dehydrogenase described in the first aspect above.

[0032] The preparation method of the nucleic acid can be a conventional preparation method in the art. The preparation method preferably includes: obtaining the nucleic acid molecule encoding the NADP-type formate dehydrogenase by gene cloning technology, or obtaining the nucleic acid molecule encoding the NADP-type formate dehydrogenase by artificial full-sequence synthesis.

[0033] Thirdly, the present invention also provides an expression vector loaded with the encoding gene for NADP-type formate dehydrogenase as described in the second aspect above.

[0034] In a preferred embodiment, the expression vector is a recombinant plasmid.

[0035] In a preferred embodiment, the expression vector is a pET28a(+) vector.

[0036] The recombinant expression vector can be obtained by conventional methods in the art, and is generally constructed by linking the nucleic acid to various expression vectors.

[0037] The present invention also provides a recombinant expression transformant comprising the above-described expression vector. The recombinant expression transformant comprises a host cell and a target gene transferred into the host cell, wherein the target gene comprises the encoding gene for NADP-type formate dehydrogenase as described in the second aspect above.

[0038] The recombinant expression transformant is generally prepared by transforming the expression vector into a host microorganism. The host microorganism is preferably *Escherichia coli* (E. coli), and more preferably *E. coli* BL21(DE3) competent cells. Transforming the recombinant expression vector (e.g., plasmid) into *E. coli* BL21(DE3) competent cells yields the preferred genetically engineered strain of this invention. The transformation method can be any conventional method in the art, such as electroporation or heat shock.

[0039] Fourthly, the present invention also provides the application of the NADP-type formate dehydrogenase described in the first aspect above as a coenzyme regeneration enzyme in enzyme-catalyzed redox reactions.

[0040] Specifically, the present invention provides the application of the NADP-type formate dehydrogenase described in the first aspect above as a coenzyme in the preparation of S-Bosein by carbonyl reductase catalytic reduction of carbonyl groups using β-acetone xyloside as a substrate.

[0041] The specific method is a method for preparing S-Bosorin by carbonyl reductase catalysis of carbonyl reductase reduction of β-pyrone xyloside as a substrate, which includes the following steps:

[0042] (a) In a liquid reaction system, using compound β-acetone xyloside of formula II as a substrate and NADP-type formate dehydrogenase as a coenzyme regenerating enzyme, an asymmetric reduction reaction was carried out in the presence of the coenzyme and catalyzed by carbonyl reductase to obtain compound S-Bosorin of formula Ia.

[0043] (b) Optionally, the compound S-Bosein of formula Ia is isolated from the reaction system following step (a);

[0044] The NADP-type formate dehydrogenase is the NADP-type formate dehydrogenase described in the first aspect above.

[0045] The carbonyl reductase is the carbonyl reductase NgADH gene, accession number KAI8399263.

[0046] In some specific embodiments, the concentration of compound II in the reaction system is 1-1000 g / L.

[0047] In some specific embodiments, the concentration of the compound of formula II in the reaction system is 10 g / L or more, 30 g / L or more, 50 g / L or more, 70 g / L or more, 90 g / L or more, 100 g / L or more, 120 g / L or more, 140 g / L or more, 160 g / L or more, 180 g / L or more, 200 g / L or more, 220 g / L or more, 240 g / L or more, 260 g / L or more, 280 g / L or more, 300 g / L or more, 320 g / L or more, 350 g / L or more, 400 g / L or more, or 450 g / L or more.

[0048] In some specific embodiments, the concentration of the compound of formula II in the reaction system is 50-500 g / L; preferably, the concentration of the compound of formula II is 100-400 g / L; more preferably, it is 150-300 g / L.

[0049] In some specific embodiments, a co-substrate is also present in the reaction system.

[0050] In some specific embodiments, the co-substrate is selected from: ammonium formate, sodium formate, or combinations thereof.

[0051] In some specific embodiments, the cosubstrate is sodium formate.

[0052] In some specific embodiments, the mass-volume concentration of the co-substrate in the reaction system is 5% to 50% (w / v).

[0053] In some specific embodiments, the coenzyme is selected from the group consisting of reducing coenzymes, oxidizing coenzymes, or combinations thereof.

[0054] In some specific embodiments, the reducing coenzyme is selected from the group consisting of NADH, NADPH, or combinations thereof.

[0055] In some specific embodiments, the oxidative coenzyme is selected from the group consisting of NAD+, NADP+, or combinations thereof.

[0056] In some specific embodiments, the ratio of NAD+ dosage to substrate dosage is 0.01% to 2.0% (w / w); preferably, the ratio is 0.1% to 1.0% (w / w).

[0057] In some specific embodiments, the ratio of NADP+ to substrate is 0.01% to 2.0% (w / w); preferably, the ratio is 0.1% to 1.0% (w / w).

[0058] In some specific embodiments, the ratio of the coenzyme regenerating enzyme to the substrate is 0.1% to 10.0% (w / w); preferably, the ratio is 0.1% to 5.0% (w / w); more preferably, the ratio is 0.1% to 2.0% (w / w).

[0059] In some specific embodiments, the reaction temperature in step (a) is 10°C to 45°C. The reaction temperature can also be selected as 20°C to 40°C, or 25°C to 35°C.

[0060] In some specific embodiments, in step (a), the reaction time is 0.1 to 240 hours; preferably, the reaction time is 1 to 72 hours; more preferably, the reaction time is 2 to 48 hours. A suitable reaction endpoint is determined by monitoring the conversion rate of compound I; preferably, when the reaction conversion rate is >98%; more preferably, when the reaction conversion rate is >99%.

[0061] In some specific embodiments, in step (a), the pH of the reaction system is 6.0 to 9.0; preferably, the pH is 6.5 to 8.5; more preferably, the pH is 7.0 to 8.0.

[0062] In some specific embodiments, the reaction system is an aqueous solvent system.

[0063] In some specific embodiments, the reaction system is a phosphate buffer system.

[0064] In some specific embodiments, in step (b), the ee value of the compound of formula II in the reaction system after the reaction is ≥99.5%; preferably, the ee value of the compound of formula II is ≥99.9%.

[0065] The reaction system provided by this invention can perform enzymatic reactions to obtain S-Bosonic with high optical purity through high conversion. Preferably, the optical purity is ee value ≥ 98%; more preferably, the optical purity is ee value ≥ 99%.

[0066] Technical terms

[0067] carbonyl reductase

[0068] In this invention, "carbonyl reductase" is an enzyme that can stereoselectively catalyze the asymmetric reduction of prochiral ketones to chiral alcohols.

[0069] In this invention, the carbonyl reductase includes both wild-type and mutant types. Furthermore, it can be isolated or recombinant. The wild-type carbonyl reductase mentioned in this invention is the NgADH gene of Nakaseomyces glabratus (accession number KAI8399263).

[0070] The carbonyl reductase provided in this invention is a series of mutants obtained through screening based on wild-type carbonyl reductase. They all have higher activity than wild-type carbonyl reductase and can achieve 100% stereoselectivity for S-Bosine.

[0071] The reaction system can use the aforementioned carbonyl reductase in wet cells, crude enzyme solution, crude enzyme powder, or pure enzyme. To obtain higher conversion efficiency, crude enzyme solution is preferred. The ratio of carbonyl reductase to substrate is preferably 1%–6% (w / w) or the ratio of resting cell mass to substrate mass is 10–100%.

[0072] Coenzyme

[0073] In this invention, "coenzyme" refers to a coenzyme that enables electron transfer in redox reactions.

[0074] Typically, the coenzymes of this invention are reducing coenzymes NADH and NADPH or oxidizing coenzymes NAD+ and NADP+. Since reducing coenzymes are relatively expensive, oxidizing coenzymes NAD+ and NADP+ are preferred.

[0075] Coenzyme regeneration enzyme

[0076] Coenzyme regeneration refers to the process of regenerating coenzymes from an oxidized state to a reduced state, or vice versa, in an enzyme-catalyzed reaction, thereby maintaining the coenzyme at a certain catalytic level. The enzymes used to achieve this coenzyme regeneration function are called coenzyme regenerating enzymes. Common coenzyme regenerating enzymes include glucose dehydrogenase systems, isopropanol dehydrogenase systems, and formic acid dehydrogenase systems.

[0077] Stereoisomers

[0078] In this invention, stereoisomers refer to isomers resulting from different spatial arrangements of atoms in a molecule. They can be classified into cis-trans isomers and enantiomers, or further divided into enantiomers and diastereomers. In chemical or enzymatic reactions, the preferential formation of one stereoisomer relative to another is called stereoselectivity. Stereoselectivity can be partial, in which case the formation of one stereoisomer is more favorable than the other, or it can be complete, in which case only one stereoisomer is formed. When the stereoisomers are enantiomers, stereoselectivity refers to enantiomer selectivity, that is, the fraction (usually reported as a percentage) of one enantiomer in the total of the two enantiomers. This (usually a percentage) is typically reported in the art as an enantiomer excess (ee) calculated according to the following formula: [major enantiomer - minor enantiomer] / [major enantiomer + minor enantiomer]. When the stereoisomers are diastereomers, stereoselectivity refers to diastereoselectivity, that is, the fraction (usually reported as a percentage) of one diastereomer in a mixture of two diastereomers, and is typically optionally reported as diastereoselectivity excess (de). Enantiomer excess and diastereoselectivity excess are types of stereoisomer excess. In this invention, the stereoisomers are substantially pure, and the enzyme is capable of converting the substrate into the corresponding product having at least about 95%, 96%, 97%, 98%, or 99% stereoisomer excess; preferably, at least about 98% stereoisomer excess; more preferably, at least about 99% stereoisomer excess.

[0079] Biocatalytic preparation method

[0080] This invention provides a method for preparing the compound S-Bosein (Ia) by catalytic reduction of β-acetone xyloside (II) using a carbonyl reductase. The carbonyl reductase can be NgADH from Nakaseomyces glabratus (accession number KAI8399263), and the reaction formula is shown below:

[0081] The main technical effects of this invention are:

[0082] 1) The NADP-type formate dehydrogenase mutant obtained in this application exhibits significantly increased enzyme activity compared to the wild type, maintaining the coenzyme at a high activity level. This, in turn, reduces the amount of coenzyme regeneration enzyme and / or coenzyme required in the reaction, thus lowering costs. Yield is significantly improved, and costs are reduced.

[0083] 2) The NADP-type formate dehydrogenase mutant obtained in this application is used as a coenzyme regeneration enzyme, which helps to improve the conversion rate and shorten the reaction time in the reaction of catalytic reduction of β-acetone xyloside to prepare S-Bosein.

[0084] 3) This invention provides a new source of NADP-type formate dehydrogenase mutants for enzyme-catalyzed synthesis, which has the advantage of high activity, providing more and better options for industrial production. Attached Figure Description

[0085] Figure 1 shows the chiral HPLC chromatogram of the racemic mixture of Bosein. The retention time of the S-Bosein configuration is approximately 9.151 min and the retention time of the R-Bosein configuration is 9.681 min.

[0086] Figure 2 shows the chiral HPLC chromatogram of the Ib compound obtained by carbonyl reductase catalysis in this application, with the S configuration showing an ee value of 100%. Detailed Implementation

[0087] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0088] Experimental methods in the following examples, unless otherwise specified, were performed under standard conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated. All experimental materials used in this invention are commercially available unless otherwise specified.

[0089] In the following examples, the purity and ee value detection conditions were as follows: chromatographic column: Alphasil XD-Amide 5μm (4.6mm×150mm), column temperature 30℃, using 95% acetonitrile aqueous solution as the mobile phase, flow rate 1mL / min, and detection time 25min.

[0090] Example 1: Construction and screening of engineered formate dehydrogenase bacteria

[0091] Genes of formate dehydrogenases BBFDH (accession number: WP_131856636.1), XSFDH (accession number: WP_029558741.1), and MpFDH (accession number: WP_091675598.1) from Bosea sp. BK604, Xanthobacter sp. 91, and Methylocapsa palsarum were synthesized after codon optimization (performed by Genewiz). These genes were then cloned into pET28a(+) vectors and introduced into host *Escherichia coli* BL21(DE3) competent cells (catalog number: Shanghai Weidi Biotechnology Co., Ltd. EC1002). The cells were cultured on kanamycin-resistant plates, and single colonies were picked and cultured in LB medium to obtain recombinant genetically engineered bacteria containing formate dehydrogenases BBFDH, XSFDH, and MpFDH.

[0092] The formate dehydrogenase-producing bacteria were transferred to 5 mL of LB medium (containing 50 μg / mL kanamycin) and cultured overnight at 37°C and 220 rpm to obtain a seed culture. 10 μL of the formate dehydrogenase seed culture was transferred to a shake flask containing 50 mL of fermentation medium (containing 50 μg / mL kanamycin) and cultured at 37°C and 220 rpm until the OD600 value > 0.8. Formate dehydrogenase expression was induced at 25°C using isopropyl thiogalactoside (IPTG) at a final concentration of 0.1 mM, followed by overnight culture. After fermentation, centrifuge at 12000g for 30 min to collect 1 mL of bacterial cells. The cells were then resuspended in 1 mL of lysis buffer (0.1 M phosphate buffer containing 1000 U lysozyme, pH 7.0). After lysis at 30°C for 1 h, the cells were centrifuged at 4°C, 8000g for 10 min. The clear supernatant was collected to determine the activities of formate dehydrogenases BBFDH, XSFDH, and MpFDH. 190 μL of reaction solution (containing 0.4 mM sodium formate and 1 mM NADP) was added... + or NAD + Add the solution to a new 96-well microplate, then add 10 μL of supernatant, and detect the change in NAD(P)H at an absorbance of 340 nm. The activity of formate dehydrogenase was calculated using the amount of NAD(P)H produced. The relative activities of BBFDH, XSFDH, and MpFDH are shown in Table 1. Finally, XSFDH, which had the best activity, was selected for cofactor preference conversion modification.

[0093] The fermentation medium formula is as follows: yeast extract (2.4%), soybean peptone (1.2%), sodium chloride (0.3%), glycerol (0.5%), dipotassium hydrogen phosphate (0.2%), and magnesium sulfate heptahydrate (0.05%).

[0094] Table 1: Relative activity of formate dehydrogenase NAD(P)

[0095] The catalytic NAD activity of wild-type formate dehydrogenase BBFDH was set to 100%.

[0096] Table 1 shows that the activities of formate dehydrogenases from different sources differ significantly. Among the various formate dehydrogenases, wild-type formate dehydrogenase XSFDH showed better relative activities for both NAD and NADP; however, comparing the relative activities of NAD and NADP revealed that wild-type formate dehydrogenase XSFDH specifically recognizes NAD cofactors, and its utilization rate of NADP is extremely low.

[0097] Example 2: Construction and screening of a formate dehydrogenase mutant library

[0098] Based on the protein structure modeling of wild-type formate dehydrogenase XSFDH, the structures of the coenzyme-binding pockets of NADPH and NADH-dependent enzymes were analyzed, and their primary sequences were aligned to identify key amino acid residues affecting coenzyme preference. A site-directed mutagenesis library was then constructed. Furthermore, using the XSFDH mutant with enhanced activity as a template, random point mutagenesis was performed using a kit… Error-prone PCR can be performed using the Site-Directed Mutagenesis Kit (STMK), or a plasmid library containing the evolved formate dehydrogenase gene can be obtained by mutating the wild-type formate dehydrogenase XSFDH through directed evolution.

[0099] The constructed plasmid library was transferred into *E. coli* BL21(DE3) (catalog number: Shanghai Weidi Biotechnology Co., Ltd. EC1002) and plated onto LB agar containing 50 μg / mL kanamycin. The culture was incubated overnight at 37°C. Single colonies were picked and transferred to 96-well plates containing 400 μL of LB agar (containing 50 μg / mL kanamycin) and incubated overnight at 37°C and 200 rpm to obtain the formate dehydrogenase mutant seed culture. 10 μL of the formate dehydrogenase mutant seed culture was transferred to 96-well plates containing 400 μL of fermentation medium (containing 50 μg / mL kanamycin) and incubated at 37°C and 200 rpm until the OD600 value > 0.8. The mutant expression was induced at 28°C with 1 mM isopropyl thiogalactoside (IPTG), followed by 20 h of further culture. The 96-well plate was centrifuged at 4000g for 30 min to collect bacterial cells. The cells were then resuspended in 200 μL of lysis buffer (0.1 M phosphate buffer containing 1000 U lysozyme, pH 7.0) and lysed at 30°C for 1 h. After lysis, the cells were centrifuged at 4000g for 30 min at 4°C. The clear supernatant was collected to determine mutant activity. 190 μL of reaction solution (containing 0.4 mM sodium formate and 1 mM NADP+) was added to a new 96-well microplate, followed by 10 μL of supernatant. The change in NADPH was detected at 340 nm. The enzyme activity of the mutants was calculated based on the amount of NADPH generated. The relative activities of each mutant are shown in Table 2.

[0100] Table 2: Formate dehydrogenase mutants and their relative activities

[0101] Note: The activity of wild-type formate dehydrogenase XSFDH (SEQ ID NO:1) is set to 100%.

[0102] As shown in Table 2, D222Q is the site of a mutation that enhances the activity of formate dehydrogenase, with a relative activity increase of over 500%. In particular, some mutants containing the D222Q mutation site exhibit even better activity.

[0103] Example 3: Formate dehydrogenase mutant used for the biocatalytic preparation of S-Bosonic.

[0104] Take 100 mL of 0.05 M phosphate buffer and add NADP. +Add 0.01g of formate dehydrogenase mutant 13 (1g), dissolve thoroughly in sodium formate (20g), and add carbonyl reductase NgADH (accession number KAI8399263) (2g). Add substrate β-acetone xyloside (25g), react at 30℃ and 180rpm, and adjust the pH to 6.5-7.5 with sodium bicarbonate. Terminate the reaction when the conversion rate is >98% using differential monitoring. If the conversion rate is still below >98% after 48 hours, terminate the reaction directly. Remove bacterial cells by centrifugation, desalt the supernatant by nanofiltration, concentrate under reduced pressure, and purify by resin column chromatography (solvent: water) to obtain 23.9g of product with a purity of 99.9%, a yield of 95.6%, and an ee value of 100% (as shown in Figure 2). Figure 1 shows the chiral HPLC chromatogram of the racemic mixture.

[0105] Following the same reaction conditions and procedures as described above, only the formate dehydrogenase and its dosage were changed to investigate the reaction effects of the formate dehydrogenase mutant and the wild-type formate dehydrogenase under different feed amounts. Specific data comparisons are shown in Table 3.

[0106] Table 3. Effects of formate dehydrogenase mutant and wild-type formate dehydrogenase on different feed amounts.

[0107] As shown in Table 3, the wild-type formate dehydrogenase exhibits low reactivity and low conversion rate, severely affecting the redox reaction process and reducing product yield. In contrast, the formate dehydrogenase mutant 13 of this invention exhibits high activity, high conversion rate, and fast reaction speed. Furthermore, even at low dosages, it does not affect the redox reaction process and can still achieve a high product yield.

[0108] Note: The CdFDH mutant enzyme, whose coenzyme preference changes from NAD+ to NADP+ (ChemBioChem 2023,24,e202300390.), was prepared according to the method and steps in Example 1.

[0109] Example 4: Formate dehydrogenase mutant used for the biocatalytic preparation of S-Bosonic.

[0110] Take 100 mL of 0.05 M phosphate buffer and add NADP. +Add 0.01g of formate dehydrogenase mutant 12 (1g), dissolve thoroughly in sodium formate (20g), and add carbonyl reductase NgADH (accession number KAI8399263) (3g). Add substrate β-acetone xyloside (25g), react at 30℃ and 250rpm, and adjust the pH to 6.0-8.0 with sodium bicarbonate. Terminate the reaction when the conversion rate is >98%. Remove bacterial cells by centrifugation, desalt the supernatant by nanofiltration, concentrate under reduced pressure, and purify by resin column chromatography (water as solvent) to obtain 23.4g of product with a purity of 99.9%, a yield of 93.6%, and an ee value of 100%.

[0111] Example 5: Formate dehydrogenase mutant used for the biocatalytic preparation of S-Bosonic.

[0112] Take 100 mL of 0.1 M phosphate buffer and add NADP. + Add 0.01g of formate dehydrogenase mutant 11 (1g), dissolve thoroughly in sodium formate (30g), and add carbonyl reductase NgADH (accession number KAI8399263) (3g). Add substrate β-acetone xyloside (30g), react at 30℃ and 250rpm, and adjust the pH to 7.0-8.0 with sodium bicarbonate. Terminate the reaction when the conversion rate is >98%. Remove bacterial cells by centrifugation, desalt the supernatant by nanofiltration, concentrate under reduced pressure, and purify by resin column chromatography (water as solvent) to obtain 28.8g of product with a purity of 99.9%, a yield of 96.0%, and an ee value of 100%.

[0113] Example 6: Formate dehydrogenase mutant used for the biocatalytic preparation of S-Bosonic.

[0114] Take 100 mL of 0.1 M phosphate buffer and add NADP. + Add 0.01g of formate dehydrogenase mutant 13 (0.2g), dissolve thoroughly in sodium formate (30g), and add 2.5g of carbonyl reductase NgADH (accession number KAI8399263). Add 25g of substrate β-acetone xyloside, react at 35℃ and 250rpm, and adjust the pH to 7.0-8.0 with sodium bicarbonate. Terminate the reaction when the conversion rate is >98%. Remove bacterial cells by centrifugation, desalt the supernatant by nanofiltration, concentrate under reduced pressure, and purify by resin column chromatography (water as solvent) to obtain 23.3g of product with a purity of 99.9%, a yield of 93.6%, and an ee value of 100%.

[0115] In summary, the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An NADP-type formate dehydrogenase, characterized in that, The NADP-type formate dehydrogenase is an amino acid sequence derived from Xanthobacter sp. 91 with the amino acid sequence shown in SEQ ID NO:2, and has the following mutation sites: Mutant 2: An amino acid sequence corresponding to SEQ ID NO:2 and having the mutation site D222Q; Mutant 6: An amino acid sequence corresponding to SEQ ID NO:2 and having mutation sites A199G and D222Q; Mutant 7: An amino acid sequence corresponding to SEQ ID NO:2 and having mutation sites A199G, D222Q, C256A and H380K; Mutant 8: An amino acid sequence corresponding to SEQ ID NO:2 and having mutation sites A199G, D222Q, C256A, H380K and S381V; Mutant 9: An amino acid sequence corresponding to SEQ ID NO:2 and having mutation sites A199G, D222Q, C256A, H380K, S381V and Y63F; Mutant 10: An amino acid sequence corresponding to SEQ ID NO:2 and having mutation sites A199G, D222Q, C256A, H380K, S381V and I280L; Mutant 11: An amino acid sequence corresponding to SEQ ID NO:2 and having mutation sites A199G, D222Q, C256A, H380K, S381V and C146S; Mutant 12: An amino acid sequence corresponding to SEQ ID NO:2 and having mutation sites A199G, D222Q, C256A, H380K, S381V, Y63F and I280L; Mutant 13: an amino acid sequence corresponding to SEQ ID NO:2 and having mutation sites A199G, D222Q, C256A, H380K, S381V, Y63F, C146S and I280L.

2. The carbonyl reductase as described in claim 1, characterized in that, The carbonyl reductase is mutant 2.

3. The carbonyl reductase as described in claim 1, characterized in that, The carbonyl reductase is mutant 6.

4. The carbonyl reductase as described in claim 1, characterized in that, The carbonyl reductase is mutant 7.

5. The carbonyl reductase as described in claim 1, characterized in that, The carbonyl reductase is mutant 8.

6. The carbonyl reductase according to claim 1, characterized in that, The carbonyl reductase is mutant 9.

7. The carbonyl reductase according to claim 1, characterized in that, The carbonyl reductase is mutant 10.

8. The carbonyl reductase as described in claim 1, characterized in that, The carbonyl reductase is mutant 11.

9. The carbonyl reductase according to claim 1, characterized in that, The carbonyl reductase is mutant 12.

10. The carbonyl reductase according to claim 1, characterized in that, The carbonyl reductase is mutant 13.

11. A gene encoding a carbonyl reductase as described in any one of claims 1-10.

12. An expression carrier, characterized in that, The expression vector is loaded with the gene encoding the carbonyl reductase as described in claim 11; preferably, the expression vector is a recombinant plasmid; preferably, the expression vector is a pET22b(+) vector.

13. A genetically engineered bacterium, characterized in that, It includes a host cell and a target gene transferred into the host cell, wherein the target gene contains the gene encoding a carbonyl reductase as described in claim 11.

14. The genetically engineered bacteria as described in claim 13, characterized in that, The host cell is Escherichia coli.

15. The use of NADP-type formate dehydrogenase as any one of claims 1-10 as a coenzyme regeneration enzyme in enzyme-catalyzed redox reactions.

16. A method for preparing S-Bosein using NADP-type formate dehydrogenase as a coenzyme regeneration enzyme, characterized in that, Includes the following steps: (a) In a liquid reaction system, using compound β-acetone xyloside of formula II as a substrate and NADP-type formate dehydrogenase as a coenzyme regenerating enzyme, an asymmetric reduction reaction was carried out in the presence of the coenzyme and catalyzed by carbonyl reductase to obtain compound S-Bosorin of formula Ia. (b) Optionally, the compound S-Bosein of formula Ia is isolated from the reaction system following step (a); The NADP-type formate dehydrogenase is the NADP-type formate dehydrogenase as described in any one of claims 1-10.

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