Method for synthesizing 2,3-dihydroxy ketone compounds by benzaldehyde lyase mutant and application thereof

Abstract

This invention discloses a strain derived from *Pseudomonas fluorescens* (…). Pseudomonas fluorescens ) and Arshan Polymorphobacter ( Polymorphobacter arshaanensis This invention discloses benzaldehyde lyase and its mutants, and their application as catalysts in the catalysis of chiral aromatic 2,3-dihydroxyacetone compounds. The benzaldehyde lyase of this invention exhibits advantages such as high efficiency, excellent stereoselectivity, and broad substrate applicability. It can achieve configurational stereocomplementation and achieve yields up to 93%. ee With a value as high as 99%, it can achieve pilot-scale amplification synthesis, which is conducive to low-cost production and has industrial application prospects.

Description

Technical Field

[0001] This invention belongs to the fields of enzyme engineering and chemical engineering technology, specifically designing benzaldehyde lyase mutants, and in particular designing a method for synthesizing 2,3-dihydroxyketone compounds catalyzed by benzaldehyde lyase mutants and their applications. Background Technology

[0002] Benzaldehyde lyase (BAL) is a thiamine diphosphate (ThDP)-dependent enzyme, originally derived from *Pseudomonas fluorescens*. Pseudomonas fluorescens It was discovered in [the study]. It is a multifunctional enzyme capable of catalyzing the formation and cleavage of C-C bonds between aldehyde substrates. Benzaldehyde lyase is a multifunctional enzyme with broad industrial and scientific research applications. Through enzyme engineering and synthetic biology, its catalytic performance and application range are continuously expanding, providing new tools for green chemistry and the synthesis of chiral compounds.

[0003] The 2,3-dihydroxyketone group is an important component of pharmaceuticals and chemicals, widely found in natural products, drugs, and active molecules. It is also a crucial building block in the synthesis of other carbohydrates and natural products. For example, cytoxazolone (…) was isolated from a Streptomyces bacterium in soil samples collected from Hiroshima Prefecture, Japan. Cytoxazone (IL-4, IL-10) is a novel cytokine modulator that selectively inhibits the production of Th2 cytokines (IL-4, IL-10), exhibiting immunosuppressive activity and showing potential as a therapeutic agent in the field of immunotherapy. (IL-4, IL-10) is derived from natural plants. Cardiobutanolide It exhibits significant biological activity in antitumor and teratogenic effects. (Jiang's A (…)) Jiangrine A Isolated from the fermentation broth of actinomycetes, this is a pyrrole-2-carboxaldehyde derivative with anti-inflammatory activity, exhibiting inhibition of lipopolysaccharide-induced NO production in macrophages. The 2,3-dihydroxyketone unit is an important framework for the preparation of various drugs and bioactive molecules, and may also be a potential anti-tumor drug. However, current synthetic methods are limited, mainly relying on traditional chemical synthesis methods, which suffer from problems such as the use of excessive alkali and organic solvents, CO2 release, complex processes, and difficulties in separation and purification. Furthermore, it has limitations in its applicability and poor stereochemical control. Therefore, there is a need to explore benzaldehyde lyases with superior performance and develop economical, efficient, green, and environmentally friendly low-carbon biocatalytic synthesis processes. Summary of the Invention

[0004] The first aspect of this invention is to provide a method for synthesizing a compound of formula I catalyzed by benzaldehyde lyase or a mutant thereof, the reaction formula of which is as follows: ; The benzaldehyde lyase has an amino acid sequence as shown in SEQ ID NO:1 or SEQ ID NO:2; The amino acid sequence of the benzaldehyde lyase mutant is obtained by mutation of the sequence shown in SEQ ID NO:1 or SEQ ID NO:2; Among them, R1 is selected from: C6-C 10 Aryl groups or C5-C groups containing N, O, or S atoms 10 heteroaryl; the C6-C 10 Aryl groups or C5-C groups containing N, O, or S atoms 10 The heteroaryl group may be optionally replaced by one, two, or three independent R4 groups. The R4 is selected from: hydrogen, hydroxyl, halogen, C1-C6 alkoxy, C1-C6 alkyl, halogen-substituted C1-C6 alkyl, halogen-substituted C1-C6 alkoxy, NH2, NH (C1-C6 alkyl), N (C1-C6 alkyl), or cyano. R2 is selected from N, O or S atoms, and R3 is selected from amino, hydroxyl or thiol groups.

[0005] More preferably, R4 is selected from: hydrogen, hydroxyl, halogen, C1-C6 alkoxy, C1-C6 alkyl, halogen-substituted C1-C6 alkyl. R2 is selected from O; R3 is selected from hydroxyl groups.

[0006] More preferably, R1 is selected from: phenyl, naphthyl, thiophene, or furanyl; the phenyl, naphthyl, thiophene, or furanyl group may optionally be replaced by one, two, or three independent R4 groups. The R4 is selected from: hydrogen, hydroxyl, fluorine, chlorine, bromine, iodine, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, hexyl, -CFH2, -CClH2, -CF2H, -CF3, -CH2CFH2, -CH2CF2H, -CH2CF3, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, or tert-butoxy; More preferably, R1 is selected from... , , , , , , , , , , , , , , , , , , .

[0007] The second aspect of this invention provides two ThDP-dependent enzymes, namely benzaldehyde lyase. Pf BAL or Pa BAL and Pf BAL or Pa This invention utilizes bioinformatics methods to analyze and predict enzyme genes that may exhibit significant catalytic activity against substrates. These genes are then cloned and expressed in suitable expression vectors to construct recombinant *E. coli* cells. By measuring the activity and stereoselectivity of the recombinant ThDP-dependent enzymes, the cloned enzymes are screened to ultimately obtain the mutant enzyme with the best catalytic performance, derived from *Pseudomonas fluorescens*. Pseudomonas fluorescens AAA50176.1, named benzaldehyde lyase Pf BAL, derived from *Alxytrophomyces arshanensis* (… Polymorphobacter arshaanensis )WP_135246357.1, named benzaldehyde lyase Pa BAL. The amino acid sequence of the benzaldehyde lyase is preferably as shown in SEQ ID NO:1 and SEQ ID NO:2 in the sequence listing, and the amino acid sequence has been described in existing literature Gene, 144(1994), 137-138, 10.1016 / 0378-1119(94)90218-6 and Angew. Chem. Int. Ed. It was reported in 2022, 61, e202116344.

[0008] Preferred are derived proteins consisting of novel amino acid sequences formed by substituting one or more amino acids from the amino acid sequences shown in SED ID NO:1 (alanine at position 28, leucine at position 112, glutamic acid at position 113) and SED ID NO:2 (leucine at position 116, glutamic acid at position 117, glycine at position 396, histidine at position 417, glycine at position 421, methionine at position 480, methionine at position 556, and tyrosine at position 571). A further preferred amino acid sequence of the benzaldehyde lyase 1 mutant is as follows: 1. Replace the alanine at position 28 of the amino acid sequence shown in SEQ ID No. 1 with arginine; 2. Replace the alanine at position 28 of the amino acid sequence shown in SEQ ID No. 1 with aspartic acid; 3. Replace the alanine at position 28 of the amino acid sequence shown in SEQ ID No. 1 with glutamic acid; 4. Replace the alanine at position 28 of the amino acid sequence shown in SEQ ID No. 1 with phenylalanine; 5. Replace the alanine at position 28 of the amino acid sequence shown in SEQ ID No. 1 with glycine; 6. Replace the alanine at position 28 of the amino acid sequence shown in SEQ ID No. 1 with histidine; 7. Replace the alanine at position 28 of the amino acid sequence shown in SEQ ID No. 1 with isoleucine; 8. Replace the alanine at position 28 of the amino acid sequence shown in SEQ ID No. 1 with leucine; 9. Replace the alanine at position 28 of the amino acid sequence shown in SEQ ID No. 1 with lysine; 10. Replace the alanine at position 28 of the amino acid sequence shown in SEQ ID No. 1 with methionine; 11 Replace the alanine at position 28 of the amino acid sequence shown in SEQ ID No. 1 with proline; 12. Replace the alanine at position 28 of the amino acid sequence shown in SEQ ID No. 1 with serine; 13 Replace the alanine at position 28 of the amino acid sequence shown in SEQ ID No. 1 with tryptophan; 14. Replace the alanine at position 28 of the amino acid sequence shown in SEQ ID No. 1 with tyrosine; 15. Replace the alanine at position 28 of the amino acid sequence shown in SEQ ID No. 1 with valine; 16. Replace the alanine at position 28 of the amino acid sequence shown in SEQ ID No. 1 with cysteine; 17. Replace the alanine at position 28 of the amino acid sequence shown in SEQ ID No. 1 with asparagine; 18. Replace the alanine at position 28 of the amino acid sequence shown in SEQ ID No. 1 with threonine; 19. Replace the alanine at position 28 of the amino acid sequence shown in SEQ ID No. 1 with glutamine; The amino acids at positions 112 and 113 of the amino acid sequence shown in SED ID NO:1 are also mutated as shown above.

[0009] The amino acid sequence of the further selected benzaldehyde lyase 2 mutant is as follows: 1. Replace glycine at position 421 of the amino acid sequence shown in SEQ ID No. 2 with alanine; 2. Replace glycine at position 421 of the amino acid sequence shown in SEQ ID No. 2 with cysteine; 3. Replace glycine at position 421 of the amino acid sequence shown in SEQ ID No. 2 with aspartic acid; 4. Replace glycine at position 421 of the amino acid sequence shown in SEQ ID No. 2 with glutamic acid; 5. Replace glycine at position 421 of the amino acid sequence shown in SEQ ID No. 2 with phenylalanine; 6. Replace glycine at position 421 of the amino acid sequence shown in SEQ ID No. 2 with histidine; 7. Replace glycine at position 421 of the amino acid sequence shown in SEQ ID No. 2 with isoleucine; 8. Replace glycine at position 421 of the amino acid sequence shown in SEQ ID No. 2 with lysine; 9. Replace glycine at position 421 of the amino acid sequence shown in SEQ ID No. 2 with leucine; 10. Replace glycine at position 421 of the amino acid sequence shown in SEQ ID No. 2 with methionine; 11 Replace glycine at position 421 of the amino acid sequence shown in SEQ ID No. 2 with asparagine; 12 Replace glycine at position 421 of the amino acid sequence shown in SEQ ID No. 2 with proline; 13 Replace glycine at position 421 of the amino acid sequence shown in SEQ ID No. 2 with glutamine; 14. Replace glycine at position 421 of the amino acid sequence shown in SEQ ID No. 2 with arginine; 15. Replace glycine at position 421 of the amino acid sequence shown in SEQ ID No. 2 with serine; 16. Replace glycine at position 421 of the amino acid sequence shown in SEQ ID No. 2 with threonine; 17. Replace glycine at position 421 of the amino acid sequence shown in SEQ ID No. 2 with valine; 18. Replace glycine at position 421 of the amino acid sequence shown in SEQ ID No. 2 with tryptophan; 19. Replace glycine at position 421 of the amino acid sequence shown in SEQ ID No. 2 with tyrosine; The amino acid sequence shown in SED ID NO:2 also has mutations at positions 116, 117, 396, 417, 480, 556, and 571, as shown above.

[0010] Alternatively, the reaction is carried out under the catalytic conditions of benzaldehyde lyase or its mutant, wherein the benzaldehyde lyase is obtained by mutation of the amino acid sequences shown in SED ID NO:1 and SED ID NO:2; and / or the amino acid sequences of the benzaldehyde lyase mutant as shown in SED ID NO:1 and SED ID NO:2, wherein the mutations include: The alanine at position 28 should be replaced with any one of the following: arginine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, tryptophan, tyrosine, valine, cysteine, asparagine, threonine, or glutamine. Alternatively, the leucine at position 112 can be replaced with any one of the following: alanine, arginine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, methionine, proline, serine, tryptophan, tyrosine, valine, cysteine, asparagine, threonine, or glutamine. Alternatively, the glutamic acid at position 113 can be replaced with any one of the following: alanine, arginine, aspartic acid, phenylalanine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, tryptophan, tyrosine, valine, cysteine, asparagine, threonine, or glutamine. The amino acid sequence of the benzaldehyde lyase 2 mutant was obtained by mutation of the sequence shown in SEQ ID NO:2, wherein the mutation includes: The leucine at position 116 is replaced with any one of the following: alanine, arginine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, methionine, proline, serine, tryptophan, tyrosine, valine, cysteine, asparagine, threonine, and glutamine. And / or, the glutamic acid at position 117 may be replaced with any one of the following: alanine, arginine, aspartic acid, phenylalanine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, tryptophan, tyrosine, valine, cysteine, asparagine, threonine, and glutamine. And / or, the glycine at position 396 may be replaced with any one of the following: alanine, arginine, aspartic acid, glutamic acid, phenylalanine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, tryptophan, tyrosine, valine, cysteine, asparagine, threonine, and glutamine. And / or, the histidine at position 417 may be replaced with any one of the following: alanine, arginine, aspartic acid, glutamic acid, phenylalanine, glycine, isoleucine, leucine, lysine, methionine, proline, serine, tryptophan, tyrosine, valine, cysteine, asparagine, threonine, and glutamine. And / or, the glycine at position 421 may be replaced with any one of the following: alanine, arginine, aspartic acid, glutamic acid, phenylalanine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, tryptophan, tyrosine, valine, cysteine, asparagine, threonine, and glutamine. And / or, the methionine at position 480 may be replaced with any one of the following: alanine, arginine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, leucine, lysine, proline, serine, tryptophan, tyrosine, valine, cysteine, asparagine, threonine, or glutamine. And / or, the methionine at position 556 may be replaced with any one of the following: alanine, arginine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, leucine, lysine, proline, serine, tryptophan, tyrosine, valine, cysteine, asparagine, threonine, and glutamine. And / or, the tyrosine at position 571 is replaced with any one of alanine, arginine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, tryptophan, valine, cysteine, asparagine, threonine, and glutamine.

[0011] Furthermore, the catalytic reaction is selected from the benzaldehyde lyase 1 mutant. Pf BAL_A28S and benzaldehyde lyase 2 mutants are Pa BAL_G421S_M556F_Y571H (named as) Pa BAL M3).

[0012] Furthermore, the catalytic reaction is an asymmetric catalytic reaction, and the reaction is carried out in the presence of MgSO4 and ThDP.

[0013] Furthermore, the concentration of compound 1 is 50-100 mM, the concentration of compound 2 is 50-100 mM, the concentration of MgSO4 is 2.0-3.0 mM, the concentration of ThDP is 0.1-0.3 mM, the reaction temperature is 20-30 °C, and the pH value is 7.0-8.0. And / or, preferably, the concentration of the compound of formula 1 is 50 mM or 100 mM, the concentration of the compound of formula 2 is 50 mM or 100 mM, the concentration of MgSO4 is 2.5 mM, the concentration of ThDP is 0.15 mM, the reaction temperature is 30 °C, and the pH value is 8.0.

[0014] For the substrates benzaldehyde and 1,4-dioxane-2,5-diol, the following exemplary method can be used to prepare ( ) in phosphate-buffered saline (PBS) buffer at pH 8.0, in the presence of MgSO4 and ThDP, by the action of the ThDP-dependent enzyme and its mutant strain. R )or( S 2,3-Dihydroxy-1-phenylpropane-1-one. In this reaction, to ensure the reaction occurs and to facilitate structural and electrostatic regulation, MgSO4 and ThDP are additionally added to the reaction system. The amount of MgSO4 is 2.5 mM, and the amount of ThDP is 0.15 mM. The phosphate buffer can be any conventional phosphate buffer in the art, and the concentration of the phosphate buffer can be 50 mmol / L. The temperature of the asymmetric catalytic reaction can be 20-30°C. o C, preferably 30 o C. The reaction time depends on the reactivity of different benzaldehyde lyases or their mutant strains, generally 12-24 h. Yield can be determined by normal-phase high-performance liquid chromatography (HPLC) or nuclear magnetic resonance spectroscopy (NMR). 1 The enantiomeric excess (ee) was determined by ¹H NMR, and the enantiomeric excess value was analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC). Preferably, the yield was determined using an ACE 5 C18-PFP column with a mobile phase of formic acid / water / acetonitrile = 60% : 40% (v / v) and a column temperature of 25°C. o C, flow rate 1 mL / min, UV detection wavelength 250 nm, or use 1 ¹H NMR with 1,3,5-trimethoxybenzene as an internal standard; ee value using IA or OD-H column, mobile phase of n-hexane / isopropanol = 90%:10% (v / v), column temperature 30°C. o C, flow rate 0.8 mL / min, UV detection wavelength 250 nm.

[0015] Reaction treatment method: Add an equal or excess volume of ethyl acetate to the reaction solution to quench the reaction. After centrifuging at 12000 rpm for 1 min, remove the organic phase. Repeat this operation three times. Combine the organic phases, add anhydrous sodium sulfate to dry, and remove the solvent by rotary evaporation.

[0016] In a third aspect, the present invention provides a variety of isolated nucleic acids, said nucleic acids encoding the aforementioned ThDP-dependent enzyme (benzaldehyde lyase).Pf BAL and its mutants and benzaldehyde lyase Pa Nucleic acid molecules (BAL and its mutants).

[0017] In a fourth aspect, this invention provides various recombinant expression vectors containing the aforementioned ThDP-dependent enzyme gene sequences. These recombinant expression vectors can be constructed by cloning the aforementioned enzyme genes into various vectors using conventional methods in the art. Preferably, the expression vectors include various vectors conventional in the art, such as commercially available plasmids, granules, bacteriophages, or viral vectors, and the vectors are preferably copy vectors of pET28a(+) and pET21a(+).

[0018] In a fifth aspect, the present invention provides various recombinant expression cells comprising expression vectors containing the above-mentioned ThDP-dependent enzyme gene or its mutant. The recombinant expression cells are host cells that are compatible with the introduction of the expression vector and capable of inducing the expression of the target gene. The host cells are preferably *Escherichia coli* (E. coli). E.coli BL21(DE3). The aforementioned recombinant expression vector was transformed into... E.coli The preferred genetically engineered strain of this invention can be obtained from BL21 (DE3). For example, the recombinant expression vector pET28a (+)- Pf BAL or pET21a (+)- Pa BAL is converted to Escherichia coli ( E. coli Recombinant Escherichia coli was obtained from BL21 (DE3). E. coli BL21 (DE3) / pET28a (+)- Pf BAL or E. coli BL21 (DE3) / pET21a (+)- Pa BAL.

[0019] In a sixth aspect, this invention provides a preferred expression method for the ThDP-dependent enzyme. The culture medium is preferably Lysogeny Broth (LB) medium: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L. The culture method involves using the recombinant *Escherichia coli* described in this invention, for example... E.coli BL21 (DE3) / pET28a- Pf BAL or E. coli BL21 (DE3) / pET21a (+)- Pa BAL was inoculated into LB medium containing kanamycin or ampicillin and incubated at 37°C. o C. After culturing in a shaker at 220 rpm for 3-4 hours, the bacterial cell concentration in the culture medium was measured using a spectrophotometer (OD). 600When the concentration reaches 0.6–0.8, place the shake flask in an ice-water mixture for 30 min, then add isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.1–1 mmol / L (preferably 1 mmol / L), and incubate at 18 °C. o Induced expression was performed at C for 18-20 h, followed by centrifugation at 9000 rpm for 5 min to obtain bacterial cells still containing the target enzyme. The obtained bacterial cells were then stored at -40°C. o C or -80 o C. Store frozen.

[0020] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0021] As used in this invention, "wild-type benzaldehyde lyase" refers to a naturally occurring, unmodified benzaldehyde lyase whose nucleotides can be obtained through genetic engineering techniques, such as genome sequencing and polymerase chain reaction (PCR), and whose amino acid sequence can be deduced from the nucleotide sequence. The amino acid sequence of the wild-type benzaldehyde lyase is shown in SEQ ID NO:1 and SEQ ID NO:2. SEQ ID NO:1: MAMITGGELVVRTLIKAGVEHLFGLHGAHIDTIFQACLDHDVPIIDTRHEAAAGHAAEGYARAGAKLGVALVTAGGGFTNAVTPIANAWLDRTPVLFLTGSGALRDDETNTLQAGIDQVAMAAPITKWAHRVMATEHIPRLVMQAIRAALSAPRGPVLLDLPWDILMNQIDEDSVIIPDLVLSAHGARPDPADLDQALALLRKAERPVIVLGSEASRTARKTALSAFVAATGVPVFADYEGLSMLSGLPDAMRGGLVQNLYSFAKADAAPDLVLMLGARFGLNTGHGSGQLIPHSAQVIQVDPDACELGRLQGIALGIVADVGGTIEALAQATAQDAAWPDRGDWCAKVTDLAQERYASIAAKSSSEHALHPFHASQVIAKHVDAGVTVVADGALTYLWLSEVMSRVKPGGFLCHGYLGSMGVGFGTALGAQVADLEAGRRTILVTGDGSVGYSIGEFDTLVRKQLPLIVIIMNNQSWGATLHFQQLAVGPNRVTGTRLENGSYHGVAAAFGADGYHVDSVESFSAALAQALAHNRPACINVAVALDPIPPEELILIGMDPFA。

[0022] SEQ ID NO:2: MSSPEARYTGGDLLAQTLHDAGVTKIFALHGGHHEALFKGCIDQGIDLIDFRHEAAAGHAADAYARTTGKLGVCIITAGPGFTNAISAIANAQLDASPVLFLIGAPPLREVETNPLQGGIDQIAMARPAAKWALSIPSTERVRD LTAMAIRKAMTGRKGPVVLEIPIDILHMSVTGAQATPSAGLAVRPQPAPAPEEVAALAELLLRAERPVIVAGLESASAATAVALRALVAKLPLPVFAKPQAYGLLPAGHACDAGAAGNLAVLPIIGAGAPDLVILLGARLGLLMLG GRSGALVPHDAHVVQIYSDASEIGRLRDIDLPIAADCAQTLTALTKALAAVDLPDTSAWTARAAGAKALAASAWPDAEVAGGIHPYHAAKAVANAAGQDAAYVFDGGESSSWGTATVAVDAPARVLSHGYLGCLGIGPGFAIGM QIAHPDRRVVQVTGDGAMGFHIQEFDTMVRHRLPIVTVILNNQVWGMSIHGQQMMYGANYNVITKLGSTQYASIAAAFGCHAERVTAFAEIAPAMARAFASGKPALVEIMTDADVVHPATVAMLGQLAEGSRDIMIPYYENIAAS The mutant protein of this invention, as used herein, is referred to by the terms "mutant," "benzaldehyde lyase mutant protein," "mutant protein," "benzaldehyde lyase mutant protein," "mutant protein of this invention," "benzaldehyde lyase mutant protein of this invention," and "benzaldehyde lyase mutant of this invention," which are used interchangeably and all refer to benzaldehyde lyase that is not naturally occurring. The mutant protein is an artificially modified protein based on the protein shown in SEQ ID NO:1 or SEQ ID NO:2. Furthermore, the mutant protein of this invention has highly efficient catalytic activity for the formation of optically pure amino alcohol compounds.

[0023] The benzaldehyde lyase mutant described in this invention is... Pf BAL_A28S and Pa BAL M3, Pf BAL_A28S is a known mutant disclosed in existing literature (J. Am. Chem. Soc. 2025, 147, 3102). 3109), Pa BAL M3 is a newly invented mutant.

[0024] Pa BAL_G421S_M556F_Y571H (named as) Pa The preferred amino acid sequence of BAL M3 is as shown in SEQ ID NO:3 in the sequence listing, and the amino acid sequence is as follows: SEQ ID NO:3: MSSPEARYTGGDLLAQTLHDAGVTKIFALHGGHHEALFKGCIDQGIDLIDFRHEAAAGHAADAYARTTGKLGVCIITAGPGFTNAISAIANAQLDASPVLFLIGAPPLREVETNPLQGGIDQIAMARPAAKWALSIPSTERVRD LTAMAIRKAMTGRKGPVVLEIPIDILHMSVTGAQATPSAGLAVRPQPAPAPEEVAALAELLLRAERPVIVAGLESASAATAVALRALVAKLPLPVFAKPQAYGLLPAGHACDAGAAGNLAVLPIIGAGAPDLVILLGARLGLLMLG GRSGALVPHDAHVVQIYSDASEIGRLRDIDLPIAADCAQTLTALTKALAAVDLPDTSAWTARAAGAKALAASAWPDAEVAGGIHPYHAAKAVANAAGQDAAYVFDGGESSSWGTATVAVDAPARVLSHGYLSCLGIGPGFAIGM QIAHPDRRVVQVTGDGAMGFHIQEFDTMVRHRLPIVTVILNNQVWGMSIHGQQMMYGANYNVITKLGSTQYASIAAAFGCHAERVTAFAEIAPAMARAFASGKPALVEIMTDADVVHPATVAFLGQLAEGSRDIMIPYHENIAAS In this context, the term “AxxB” indicates that amino acid A at position xx is changed to amino acid B. For example, “A28S” indicates that alanine at position 28 is mutated to serine S, and so on.

[0025] Beneficial technical effects of the present invention: 1. The ThDP-dependent enzyme and its mutant strains provided by this invention can be used for the efficient synthesis of stereocomplementary pure 2,3-dihydroxy aromatic ketones. Using this enzymatic catalytic technology, the yield can reach 93%, the product ee value is higher than 99%, and the atom utilization rate can reach 100%, achieving preparative-grade production of the natural product molecule C-veratrol, up to 5.4 g with a yield of 76%. This invention, with its high product yield and high optical purity, is conducive to the efficient, low-cost, green, and environmentally friendly production of 2,3-dihydroxy aromatic ketones, providing a strong supply route for C-veratrol and possessing great potential for industrial application.

[0026] 2. This invention has advantages such as mild reaction conditions, 100% atom economy, green and environmentally friendly, high yield, three-dimensional complementarity and reaching industrial production level, so it has a good application prospect in industrial production. Attached Figure Description

[0027] Figure 1 Recombinant expression plasmid pET28a (+)- Pf A schematic diagram of the BAL construction.

[0028] Figure 2 : Recombinant expression plasmid pET21a (+)- Pa A schematic diagram of the BAL construction. Detailed Implementation

[0029] The present invention is further illustrated by the following examples, but these are not intended to limit the invention.

[0030] The materials used in the following embodiments are sourced from: Recombinant expression plasmid pET28a- Pf BAL and pET21a (+)- Pa BAL was synthesized at Nanjing GenScript.

[0031] E. coli BL21(DE3) competent cells and agarose gel DNA & PCR product recovery and purification kits were purchased from Beijing Tiangen Biotech Co., Ltd.

[0032] Molecular biology reagents such as the restriction enzyme DpnI, PCR extraction kits, and plasmid mini kits were purchased from ThermoScientific and Omega Biotek.

[0033] Example 1: Benzaldehyde lyase Pf Preparation of BAL recombinant expression cells The recombinant expression plasmid pET28a- Pf BAL transferred in E. coliBL21 (DE3) host cells: Plasmid and E. coli Mix BL21(DE3) competent cells at a volume ratio of 1 / 10, place on ice for 20-25 min, then incubate at 42°C. o Heat shock at C for 90 s, then place in an ice bath for 2-3 min. Next, add 500-600 μL of antibiotic-free LB medium to a clean bench and incubate at 37°C. o Incubate on a shaker for 30 min. Finally, place the cloth on solid LB medium containing Kana resistance (C). E. coli BL21 (DE3) / pET21a (+)- Pa BAL was prepared using amp-resistant solid LB medium and incubated at 37°C. o After overnight culture in a C incubator, single clones were picked and cultured in liquid LB medium containing KANA resistance. E. coli BL21 (DE3) / pET21a(+)- Pa BAL was prepared using amp-resistant liquid LB medium.

[0034] Example 2: Benzaldehyde lyase Pf Construction of BAL_A28S mutant With the aid of molecular docking, two benzaldehyde lyases were subjected to directed evolution to construct a benzaldehyde lyase mutant library: site-directed saturation mutagenesis (SSM) was performed at sites within a 4 Å range of the substrate binding pocket, with pET28a(+)- Pf BAL / pET21a (+)- Pa Using BAL and its mutants as templates, mutant primers were designed, as shown in Tables 1 to 12. PCR was performed using the high-fidelity DNA polymerase KOD One.

[0035] The PCR reaction conditions are as follows: In a PCR reaction system with a total volume of 25 μL, add 0.5~20 ng of template, 12.5 μL of 2×KOD One, 1 μL each of forward and reverse primers, and add deionized water to bring the volume to 25 μL. PCR reaction procedure: (1) Pre-denaturation: 98 o C, 45 sec; (2) Transformation: 98 o C, 15 sec; (3) Annealing: (minimum Tm value - 5) o C, 30 sec; (4) Extension: 68 o C, 150 sec; (5) Post-extension: 68 oC, 150 sec, steps (2) ~ (4) were performed for 30 cycles, and the products were stored at 4 ~ 16 °C. After the PCR products were verified by agarose gel electrophoresis, the restriction endonuclease DpnI was added and digested at 37 °C for 1 ~ 2 h. Immediately afterwards, the PCR products were purified using an agarose gel DNA & PCR product recovery and purification kit, and then the purified products were transferred to E. coli In BL21 (DE3) competent cells, the procedure was performed as described above, and the resulting mutants were then sent to a gene sequencing company for sequencing to confirm the mutation status.

[0036] Different mutant primers were designed based on the same construction process described above. Referring to the examples in Tables 1 to 4, the corresponding genes were obtained and sequenced.

[0037] Table 1 Pf Primer sequence for BAL mutation at position 28 ; Table 2 Pa Primer sequence for the mutation at position 421 of BAL ; Table 3 Pa Primer sequence for the BAL mutation at position 556 ; Table 4 Pa Primer sequence for the BAL mutation at position 571 ; Example 3 Benzaldehyde lyase Pf BAL inducible expression The recombinant expression cells obtained in Example 1 E. coli BL21 (DE3) / pET28a (+)- Pf BAL, inoculated into liquid LB medium (containing kana resistance) to a final concentration of 50 μg / mL. Pa BAL requires the use of AMP resistance), 37 o C. Incubate overnight on a shaker at 220 rpm, then inoculate 1% (v / v) into 100 mL of liquid LB medium containing the corresponding antibiotic and incubate under the same conditions. When the bacterial concentration (OD) in the culture medium... 600 When the concentration reaches 0.6-0.8, place it on ice for 30 minutes, then add IPTG to a final concentration of 1 mmol / L. Let it stand for 16 minutes. o C. Induce expression at 220 rpm for 18-20 h. Finally, centrifuge the culture medium at 9000 rpm for 5 min, collect the bacterial cells, and store at -80°C. oStore at C. Other benzaldehyde lyases and mutants are induced to express in the same way.

[0038] Synthesis and Preparation Example 1 Pf BAL mutant (benzaldehyde lyase) Pf The BAL_A28S mutant catalyzes the synthesis of chiral 2,3-dihydroxy aromatic ketone compounds. Pa BAL (benzaldehyde lyase) Pa The BAL M3 mutant catalyzes the synthesis of chiral 2,3-dihydroxyaromatic ketone compounds. ; Pf BAL mutant reaction system (total 400 μL): Substrate 1 (0.04 mmol, 100 mM), Substrate 2 (0.04 mmol, 100 mM), ThDP (0.15 mM), MgSO4 (2.5 mM), DMSO (5%, v / v), containing 60 mg / mL of [unspecified substance]. Pf BAL mutant whole cells were treated with PBS buffer (0.4 mL, pH 8.0) at 30°C. o C. React at 1000 rpm for 6 hours. After the reaction is complete, add an equal or excess amount of ethyl acetate to quench the reaction, separate the organic phase, repeat three times, and then evaporate the ethyl acetate to dryness using a vacuum concentrator. Obtain the R-configuration product, determine the yield and ee value. R The configuration products were 88% and 97%.

[0039] Pa BAL mutant reaction system (total 500 μL): Substrate 1 (0.02 mmol, 33 mM), Substrate 2 (0.02 mmol, 33 mM), ThDP (0.15 mM), MgSO4 (2.5 mM), DMSO (5%, v / v), containing 0.3% loading Pa BAL mutant purified enzyme in PBS buffer (pH 8.0), 30 o C. React at 1000 rpm for 24 h. After the reaction is complete, add an equal or excess amount of ethyl acetate to quench the reaction, separate the organic phase, repeat three times, and then evaporate the ethyl acetate to dryness using a vacuum concentrator. Determine the yield and ee value. S Configuration products: 32%, 95%. 1 H NMR (400 MHz, CDCl3) δ 7.92 – 7.88 (m,2H), 7.57 (t, J = 7.4 Hz, 1H), 7.45 (t, J = 7.4 Hz, 2H), 5.18 (dd,J = 5.0, 3.1Hz, 1H), 3.99 (dd, J = 11.9, 3.1 Hz, 1H), 3.76 – 3.71 (m, J = 5.0, 11.9 Hz 1H).

[0040] Table 5 Catalytic Synthesis Series ( S )-2,3-dihydroxyaromatic ketone compounds ; Synthesis and Preparation Example 2

[0041] Under the same reaction conditions as in preparation 1, using the benzaldehyde lyase PfBAL_A28S mutant, the structures of substrate 1 and substrate 2 are shown in Table 5, and the product was obtained ( R )-3b, 1 H NMR (400 MHz, CDCl3) δ 7.92 (d, J = 9.0 Hz, 2H), 6.96 (d, J = 9.0 Hz, 2H), 5.14 – 5.09 (m, 1H), 4.02 – 3.97 (m, 1H), 3.87 (s, 3H), 3.71 (m, 1H).

[0042] Synthesis and Preparation Example 3 Under the same reaction conditions as in Synthesis 1, the structures of substrate 1 and substrate 2 are shown in Tables 13 and 14. The product was obtained using the benzaldehyde lyase PfBAL_A28S mutant. R )-3c, using the benzaldehyde lyase PaBAL M3 mutant to obtain ( S )-3c, 1 HNMR (400 MHz, CDCl3) δ 7.48 – 7.42 (m, 2H), 7.37 (t, J = 7.9 Hz, 1H), 7.13 (dd, J = 7.9 Hz, 1H), 5.14 (dd, J = 4.9, 3.2 Hz, 1H), 3.99 (dd, J = 11.8, 3.2 Hz,1H), 3.82 (s, 3H), 3.75 (dd, J = 11.8, 4.9 Hz, 1H).

[0043] Synthesis and Preparation Example 4 Under the same reaction conditions as in Synthesis 1, the structures of substrate 1 and substrate 2 are shown in Tables 13 and 14. The product was obtained using the benzaldehyde lyase PfBAL_A28S mutant. R )-3d, using the benzaldehyde lyase PaBAL M3 mutant to obtain ( S -3d; 1 HNMR (400 MHz, CDCl3) δ 7.83 (d, J = 8.2 Hz, 2H), 7.30 (d, J = 8.2 Hz, 2H), 5.14(dd, J = 5.2, 3.2 Hz, 1H), 4.00 (dd, J = 11.7, 3.2 Hz, 1H), 3.73 (dd, J = 11.7,5.2 Hz, 1H), 2.42 (s, 3H).

[0044] Synthesis and Preparation Example 5 Under the same reaction conditions as in Synthesis 1, the structures of substrate 1 and substrate 2 are shown in Tables 13 and 14. The product was obtained using the benzaldehyde lyase PfBAL_A28S mutant. R )-3e, using the benzaldehyde lyase PaBAL M3 mutant to obtain ( S )-3e, 1 HNMR (400 MHz, CDCl3) δ 7.74 (s, 1H), 7.71 (d, J = 7.6 Hz, 1H), 7.43 (d, J = 8.0Hz, 1H), 7.38 (dd, J = 7.6, 8.0 Hz, 1H), 5.16 (dd, J = 5.0, 3.2 Hz, 1H), 4.01(dd, J = 11.8, 3.2 Hz, 1H), 3.74 (dd, J = 11.8, 5.0 Hz, 1H), 2.41 (s, 3H). Synthesis and Preparation Example 6 Under the same reaction conditions as in Synthesis 1, the structures of substrate 1 and substrate 2 are shown in Tables 13 and 14. The product was obtained using the benzaldehyde lyase PfBAL_A28S mutant. R )-3f, 1 H NMR (400 MHz, DMSO-d 6) δ 9.77 (s, 1H), 7.42(m, 1H), 7.34 – 7.27 (m, 2H), 7.02 (m, 1H), 5.18 (d, J = 6.6 Hz, 1H), 4.96 –4.88 (m, 1H), 4.77 (m, 1H), 3.74 – 3.65 (m, 1H), 3.59 (m, 1H).

[0045] Synthesis and Preparation Example 7 Under the same reaction conditions as in Synthesis 1, the structures of substrate 1 and substrate 2 are shown in Tables 13 and 14. The product was obtained using the benzaldehyde lyase PfBAL_A28S mutant. R )-3g, obtained using the benzaldehyde lyase PaBAL M3 mutant ( S )-3g, 1 HNMR (400 MHz, DMSO- d 6) δ 8.01 (d, J = 8.7 Hz, 2H), 7.59 (d, J = 8.7 Hz, 2H), 5.38 (s, 1H), 4.93 (m, 1H), 4.82 (s, 1H), 3.73 – 3.58 (m, 2H).

[0046] Synthesis and Preparation Example 8 Under the same reaction conditions as in Synthesis 1, the structures of substrate 1 and substrate 2 are shown in Tables 13 and 14. The product was obtained using the benzaldehyde lyase PfBAL_A28S mutant. R )-3h, using the benzaldehyde lyase PaBAL M3 mutant to obtain ( S -3h; 1 HNMR (400 MHz, DMSO- d 6) δ 7.92 (d, J = 8.5 Hz, 2H), 7.73 (d, J = 8.5 Hz, 2H), 5.38 (d, J = 6.5 Hz, 1H), 4.92 (m, 1H), 4.82 (t, J = 5.8 Hz, 1H), 3.66 (m, 2H).

[0047] Synthesis and Preparation Example 9 Under the same reaction conditions as in Synthesis 1, the structures of substrate 1 and substrate 2 are shown in Tables 13 and 14. The product was obtained using the benzaldehyde lyase PfBAL_A28S mutant. R )-3i, obtained using the benzaldehyde lyase PaBAL M3 mutant ( S )-3i, 1 HNMR (400 MHz, DMSO- d 6) δ 8.07 (d, J = 8.8 Hz, 2H), 7.34 (t, J = 8.8 Hz, 2H), 5.33 (d, J = 6.5 Hz, 1H), 4.96 – 4.90 (m, 1H), 4.80 (t, J = 5.8 Hz, 1H), 3.66 (m, 2H).

[0048] Synthesis and Preparation Example 10 Under the same reaction conditions as in Synthesis 1, the structures of substrate 1 and substrate 2 are shown in Tables 13 and 14. The product was obtained using the benzaldehyde lyase PfBAL_A28S mutant. R )-3j, 1 H NMR (400 MHz, DMSO- d 6) δ 8.16 (d, J = 8.2Hz, 2H), 7.89 (d, J = 8.2 Hz, 2H), 5.49 (d, J = 6.4 Hz, 1H), 4.99 – 4.94 (m,1H), 4.86 (t, J = 5.7 Hz, 1H), 3.70 (m, 2H).

[0049] Synthesis and Preparation Example 11 Under the same reaction conditions as in Synthesis 1, the structures of substrate 1 and substrate 2 are shown in Tables 13 and 14. The product was obtained using the benzaldehyde lyase PfBAL_A28S mutant. R -3k, obtained using the benzaldehyde lyase PaBAL M3 mutant ( S -3k, 1 HNMR (400 MHz, CDCl3) δ 7.86 (d, J = 8.4 Hz, 2H), 7.33 (d, J= 8.4 Hz, 2H), 5.15(dd, J = 5.1, 3.2 Hz, 1H), 4.01 (dd, J = 11.8, 3.2 Hz, 1H), 3.73 (dd, J = 11.8, 5.1 Hz, 1H), 2.96 (m, J = 6.9 Hz, 1H), 1.25 (d, J = 6.9 Hz, 6H).

[0050] Synthesis and Preparation Example 12 Under the same reaction conditions as in Synthesis 1, the structures of substrate 1 and substrate 2 are shown in Tables 13 and 14. The product was obtained using the benzaldehyde lyase PfBAL_A28S mutant. R )-3l, 1 H NMR (400 MHz, DMSO- d 6) δ 8.00 (s, 1H), 7.54 (d, J = 3.6 Hz, 1H), 6.73 – 6.70 (m, 1H), 5.40 (d, J = 6.2 Hz, 1H), 4.81 (t, J =5.9 Hz, 1H), 4.63 (m, 1H), 3.65 (m, 2H).

[0051] Synthesis and Preparation Example 13 Under the same reaction conditions as in Synthesis 1, the structures of substrate 1 and substrate 2 are shown in Tables 13 and 14. The product was obtained using the benzaldehyde lyase PfBAL_A28S mutant. R )-3m, obtained using the benzaldehyde lyase PaBAL M3 mutant ( S -3m; 1 HNMR (400 MHz, CDCl3) δ 7.82 (d, J = 3.9 Hz, 1H), 7.77 (d, J = 5.0 Hz, 1H), 7.23– 7.18 (m, J = 3.9, 5.0 Hz 1H), 4.99 – 4.95 (m, 1H), 4.06 (dd, J = 11.7, 3.4Hz, 1H), 3.87 (dd, J = 11.7, 5.1 Hz, 1H).

[0052] Synthesis and Preparation Example 14 Under the same reaction conditions as in Synthesis 1, the structures of substrate 1 and substrate 2 are shown in Tables 13 and 14. The product was obtained using the benzaldehyde lyase PfBAL_A28S mutant. R )-3n, 1 H NMR (400 MHz, CDCl3) δ 8.19 (dd, J = 2.9, 1.3Hz, 1H), 7.56 (dd, J = 5.1, 1.3 Hz, 1H), 7.38 (dd, J = 5.1, 2.9 Hz, 1H), 4.96(m, 1H), 4.02 (dd, J = 11.8, 3.3 Hz, 1H), 3.80 (dd, J = 11.8, 5.0 Hz, 1H).

[0053] Synthesis and Preparation Example 15 Under the same reaction conditions as in Synthesis 1, the structures of substrate 1 and substrate 2 are shown in Tables 13 and 14. The product was obtained using the benzaldehyde lyase PfBAL_A28S mutant. R -3o, 1 H NMR (400 MHz, DMSO- d 6) δ 10.02 (s, 1H), 7.58 (d, J = 8.3 Hz, 1H), 7.51 (s, 1H), 6.89 (d, J = 8.3 Hz, 1H), 5.06 (s, 1H), 4.99(m, J = 4.2, 11.3 Hz,1H), 4.76 (s, 1H), 3.83 (s, 3H), 3.72 (d, J = 11.3 Hz, 1H), 3.64 (d, J = 4.2 Hz, 1H).

[0054] Synthesis and Preparation Example 16 Under the same reaction conditions as in Synthesis 1, the structures of substrate 1 and substrate 2 are shown in Tables 13 and 14. The product was obtained using the benzaldehyde lyase PfBAL_A28S mutant. R )-3p, obtained using the benzaldehyde lyase PaBAL M3 mutant ( S -3p; 1HNMR (400 MHz, CDCl3) δ 7.97 (dd, J = 7.0, 2.2 Hz, 1H), 7.81 (dd, J = 8.7, 2.2Hz, 1H), 7.21 (t, J = 8.5 Hz, 1H), 5.15 – 5.11 (m, 1H), 3.98 (dd, J = 12.0, 3.2Hz, 1H), 3.76 (dd, J = 12.0, 4.9 Hz, 1H).

[0055] Synthesis and Preparation Example 17 Under the same reaction conditions as in Synthesis 1, the structures of substrate 1 and substrate 2 are shown in Tables 13 and 14. The product was obtained using the benzaldehyde lyase PfBAL_A28S mutant. R )-3q, 1 H NMR (400 MHz, CDCl3) δ 8.01 – 7.93 (m, 1H),7.04 – 6.97 (m, 1H), 6.90 (m, 1H), 5.01 (m, J = 1.8, 3.3 Hz, 1H), 3.99 (dd, J =12.1, 1.8 Hz, 1H), 3.80 (m, J = 12.1, 3.3 Hz, 1H).

[0056] Synthesis and Preparation Example 18 Under the same reaction conditions as in Synthesis 1, the structures of substrate 1 and substrate 2 are shown in Tables 13 and 14. The product was obtained using the benzaldehyde lyase PfBAL_A28S mutant. R )-3r, obtained using the benzaldehyde lyase PaBAL M3 mutant ( S )-3r, 1 HNMR (400 MHz, DMSO- d 6) δ 7.58 (s, 2H), 7.26 (s, 1H), 5.15 (d, J = 6.7 Hz, 1H), 4.99 (q, J = 5.7, 4.7 Hz, 1H), 4.76 (t, J = 5.8 Hz, 1H), 3.71 (m, J= 11.1, 4.Hz, 1H), 3.61 (m, J = 11.1, 5.7 Hz, 1H), 2.33 (s, 6H).

[0057] Synthesis and Preparation Example 19 Under the same reaction conditions as in Synthesis 1, the structures of substrate 1 and substrate 2 are shown in Tables 13 and 14. The product was obtained using the benzaldehyde lyase PfBAL_A28S mutant. R -3s, obtained using the benzaldehyde lyase PaBAL M3 mutant ( S -3s 1 HNMR (400 MHz, CDCl3) δ 8.46 (s, 1H), 7.93 (m, 4H), 7.61 m, 2H), 5.34 (d, J =5.2 Hz, 1H), 4.10 (dd, J = 11.8, 3.2 Hz, 1H), 3.82 (dd, J = 11.8, 5.2 Hz, 1H).

[0058] Synthesis and Preparation Example 20 Pf BAL_A28S mutant catalytic synthesis ( R Preparative reactions of 2,3-dihydroxy-1-phenylprop-1-one Reaction system: Substrate 1 (4 mmol), Substrate 2 (4 mmol), DMSO (5%, v / v), ThDP (0.15 mM), MgSO4 (2.5 mM), containing 60 mg / mL of [unspecified substance]. Pf BAL_A28S whole cells in PBS buffer (40 mL, pH 8.0). 30 o C. After reacting in a shaker at 220 rpm for 24 h, the reaction was quenched with an equal volume or excess of ethyl acetate, and the organic phase was extracted and separated. This operation was repeated four times, and the organic phases were combined. Anhydrous sodium sulfate was added to dry the mixture, and the organic solvent was removed by rotary evaporation. The mixture was then purified by silica gel column chromatography, and 0.59 g of pure ( R )-2,3-dihydroxy-1-phenylprop-1-one, with a separation yield of 89%.

[0059] Synthesis and Preparation Example 21 The synthesis was scaled up under the same reaction conditions to prepare 20 samples. R )-2,3-dihydroxy-1-(p-tolyl)propane-1-one was weighed to obtain 0.62 g of pure product, with a separation yield of 87%.

[0060] Synthesis and Preparation Example 22 Pf BAL_A28S mutant catalytic synthesis ( R Preparative reactions of 2,3-dihydroxy-1-(3-methoxyphenyl)propane-1-one Reaction system: Substrate 1 (40 mmol), Substrate 2 (40 mmol), DMSO (5%, v / v), ThDP (0.15 mM), MgSO4 (2.5 mM), containing 60 mg / mL of [unspecified substance]. Pf BAL_A28S whole cells in PBS buffer (400 mL, pH 8.0). 30 o C. After reacting in a shaker at 220 rpm for 24 h, the reaction was quenched with an equal volume or excess of ethyl acetate, and the organic phase was extracted and separated. This operation was repeated four times, and the organic phases were combined. Anhydrous sodium sulfate was added to dry the mixture, and the organic solvent was removed by rotary evaporation. The mixture was then purified by silica gel column chromatography, and 9.72 g of pure ( R )-2,3-dihydroxy-1-(3-methoxyphenyl)propane-1-one, with a separation yield of 89%.

[0061] Synthesis and Preparation Example 23 Pf Preparative-stage reaction of BAL_A28 mutant S-catalyzed synthesis of C-veratrol ethylene glycol Reaction system: Substrate 1 (33 mmol), Substrate 2 (33 mmol), DMSO (5%, v / v), ThDP (0.15 mM), MgSO4 (2.5 mM), containing 60 mg / mL of [unspecified substance]. Pf BAL_A28S whole cells in PBS buffer (400 mL, pH 8.0). 30 o C. After reacting in a shaker at 220 rpm for 24 h, the reaction was quenched with an equal volume or excess of ethyl acetate, and the organic phase was extracted and separated. The same operation was repeated four times, and the organic phases were combined. Anhydrous sodium sulfate was added to dry and remove water. The organic solvent was removed by rotary evaporation and purified by silica gel column chromatography. 5.35 g of pure C-veratrol was obtained, with a separation yield of 76%.

[0062] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for synthesizing compound I by benzaldehyde lyase or its mutant, wherein the reaction formula is as follows: ； The benzaldehyde lyase has an amino acid sequence as shown in SEQ ID NO:1 or SEQ ID NO:2; The amino acid sequence of the benzaldehyde lyase mutant is obtained by mutation of the sequence shown in SEQ ID NO:1 or SEQ ID NO:

2. in, R1 is selected from: C6-C 10 Aryl groups or C5-C groups containing N, O, or S atoms 10 heteroaryl; the C6-C 10 Aryl groups or C5-C groups containing N, O, or S atoms 10 The heteroaryl group may be optionally replaced by one, two, or three independent R4 groups. The R4 is selected from: hydrogen, hydroxyl, halogen, C1-C6 alkoxy, C1-C6 alkyl, halogen-substituted C1-C6 alkyl, halogen-substituted C1-C6 alkoxy, NH2, NH (C1-C6 alkyl), N (C1-C6 alkyl), or cyano. R2 is selected from N, O or S atoms, and R3 is selected from amino, hydroxyl or thiol groups.

2. The method according to claim 1, characterized in that: R4 is selected from: hydrogen, hydroxyl, halogen, C1-C6 alkoxy, C1-C6 alkyl, halogen-substituted C1-C6 alkyl. R2 is selected from O; R3 is selected from hydroxyl groups.

3. The method according to claim 1 or 2, characterized in that: in, R1 is selected from: phenyl, naphthyl, thiophene, or furanyl; the phenyl, naphthyl, thiophene, or furanyl group may be further optionally substituted by one, two, or three independent R4 groups. The R4 is selected from: hydrogen, hydroxyl, fluorine, chlorine, bromine, iodine, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, hexyl, -CFH2, -CClH2, -CF2H, -CF3, -CH2CFH2, -CH2CF2H, -CH2CF3, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, or tert-butoxy; Preferably, R1 is selected from... , , , , , , , , , , , , , , , , , , .

4. The method according to claim 1 or 2, characterized in that: The amino acid sequence of the benzaldehyde lyase 1 mutant was obtained by mutation of the sequence shown in SEQ ID NO:1, wherein the mutation includes: The alanine at position 28 should be replaced with any one of the following: arginine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, tryptophan, tyrosine, valine, cysteine, asparagine, threonine, or glutamine. Alternatively, the leucine at position 112 can be replaced with any one of the following: alanine, arginine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, methionine, proline, serine, tryptophan, tyrosine, valine, cysteine, asparagine, threonine, or glutamine. Alternatively, the glutamic acid at position 113 can be replaced with any one of the following: alanine, arginine, aspartic acid, phenylalanine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, tryptophan, tyrosine, valine, cysteine, asparagine, threonine, or glutamine. Alternatively, the amino acid sequence of the benzaldehyde lyase 2 mutant is obtained by mutation of the sequence shown in SEQ ID NO:2, wherein the mutation includes: The leucine at position 116 is replaced with any one of the following: alanine, arginine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, methionine, proline, serine, tryptophan, tyrosine, valine, cysteine, asparagine, threonine, and glutamine. And / or, the glutamic acid at position 117 may be replaced with any one of the following: alanine, arginine, aspartic acid, phenylalanine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, tryptophan, tyrosine, valine, cysteine, asparagine, threonine, and glutamine. And / or, the glycine at position 396 may be replaced with any one of the following: alanine, arginine, aspartic acid, glutamic acid, phenylalanine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, tryptophan, tyrosine, valine, cysteine, asparagine, threonine, and glutamine. And / or, the histidine at position 417 may be replaced with any one of the following: alanine, arginine, aspartic acid, glutamic acid, phenylalanine, glycine, isoleucine, leucine, lysine, methionine, proline, serine, tryptophan, tyrosine, valine, cysteine, asparagine, threonine, and glutamine. And / or, the glycine at position 421 may be replaced with any one of the following: alanine, arginine, aspartic acid, glutamic acid, phenylalanine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, tryptophan, tyrosine, valine, cysteine, asparagine, threonine, and glutamine. And / or, the methionine at position 480 may be replaced with any one of the following: alanine, arginine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, leucine, lysine, proline, serine, tryptophan, tyrosine, valine, cysteine, asparagine, threonine, or glutamine. And / or, the methionine at position 556 may be replaced with any one of the following: alanine, arginine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, leucine, lysine, proline, serine, tryptophan, tyrosine, valine, cysteine, asparagine, threonine, and glutamine. And / or, the tyrosine at position 571 is replaced with any one of alanine, arginine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, tryptophan, valine, cysteine, asparagine, threonine, and glutamine.

5. The method according to claim 4, wherein the catalytic reaction is selected from a benzaldehyde lyase 1 mutant. Pf BAL_A28S or benzaldehyde lyase 2 mutant is Pa BAL_G421S_M556F_Y571H.

6. The preparation method according to claim 5, characterized in that, The catalytic reaction is an asymmetric catalytic reaction, and the reaction is carried out in the presence of MgSO4 and ThDP; And / or, preferably, the concentration of compound 1 is 50-100 mM, the concentration of compound 2 is 50-100 mM, the concentration of MgSO4 is 2.0-3.0 mM, the concentration of ThDP is 0.1-0.3 mM, the reaction temperature is 20-30 °C, and the pH value is 7.0-8.

0. And / or, preferably, the concentration of the compound of formula 1 is 50 mM or 100 mM, the concentration of the compound of formula 2 is 50 mM or 100 mM, the concentration of MgSO4 is 2.5 mM, the concentration of ThDP is 0.15 mM, the reaction temperature is 30 °C, and the pH value is 8.

0.

7. Benzaldehyde lyase Pa BAL mutant, characterized by: The benzaldehyde lyase Pa The amino acid sequence of the BAL mutant is as shown in SEQ ID NO:

3.

8. An isolated nucleic acid, characterized in that, The nucleic acid described is encoding, for example, a benzaldehyde lyase 2 mutant. Pa The nucleic acid molecule BAL_G421S_M556F_Y571H.

9. Contains a benzaldehyde lyase 2 mutant. Pa Expression vector for the gene encoding BAL_G421S_M556F_Y571H.

10. Contains a benzaldehyde lyase 2 mutant. Pa Recombinant cells encoding the BAL_G421S_M556F_Y571H gene.

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