An alpha-hydroxy-beta-keto acid synthase catalyzing the coupling reaction of alpha-keto acids

Abstract

The present application relates to catalytic enzyme technical field, specifically relates to a kind of catalytic alpha-keto acid coupling reaction alpha-hydroxy-beta-keto acid synthase.The alpha-hydroxy-beta-keto acid synthase of a kind of catalytic alpha-keto acid coupling reaction provided in the present application, the amino acid sequence of the alpha-hydroxy-beta-keto acid synthase is as shown in SEQ ID NO:1-SEQ ID NO:4.The alpha-hydroxy-beta-keto acid synthase and its mutant provided in the present application can catalyze the carbon-carbon coupling reaction between multiple alpha-keto acid substrates, expand substrate adaptability, provide more possibilities for diversified synthesis pathway, significantly improve the application prospect of multi-enzyme catalysis in complex molecule synthesis, provide strong catalytic tool for the preparation and development of related natural products and drug lead compounds.

Description

Technical Field

[0001] This invention relates to the field of catalytic enzyme technology, and more specifically to an α-hydroxy-β-keto acid synthase that catalyzes α-keto acid coupling reactions. Background Technology

[0002] α-Hydroxy-β-keto acid synthases are a class of thiamine pyrophosphate (ThDP)-dependent enzymes primarily responsible for catalyzing the coupling reaction between two α-keto acids. Existing research has confirmed that α-hydroxy-β-keto acid synthases are key catalysts for carbon-carbon bond formation in the biosynthesis of various bioactive natural products. For example, in the biosynthetic pathway of furanolide compounds like cyanobacterin, α-hydroxy-β-keto acid synthase CybE catalyzes the coupling of p-hydroxyphenylpyruvate with 3-methyl-2-oxobutyric acid, forming a carbon-carbon bond between the β- and γ-positions of the furanolide core. Nat. Chem. Biol. 2022, 18, 652–658. In the synthesis of carbazole alkaloids neocarazostatin A and carquinostatin A, NzsH and CqsB3 initiate the assembly of the carbazole A ring by catalyzing the coupling of indole-3-pyruvate with pyruvate. J. Org. Chem. 2019, 84, 16323–16328; Angew. Chem. Int. Ed. 2019, 58, 13349–13353). Furthermore, in the biosynthesis of scytonemin, α-hydroxy-β-keto acid synthase ScyA catalyzes the coupling reaction of indole-3-pyruvate with p-hydroxyphenylpyruvate, forming cyclopenta[ b The assembly of the indole skeleton provides a key precursor (J. Am. Chem. Soc. 2008, 130, 15260–15261). Recently, several α-hydroxy-β-keto acid synthases (such as XclA, Thzk0150, Cbei2730, and CbeiHKI805_0381) involved in the synthesis of keto alcohols (such as xenocyloins, sattabacins, sattazolins, and clostrocyloin) have also been reported. ChemBioChem 2014, 15, 369–372; ChemBioChem 2014, 15, 527–532; ACS Chem. Biol. 2019, 14, 1490–1497). Although α-hydroxy-β-keto acid synthases play a crucial role in the biosynthesis of many bioactive natural products, these enzymes are currently known to have the following drawbacks:

[0003] (i) Narrow substrate spectrum: Currently known α-hydroxy-β-keto acid synthases typically only accept a few or specific α-keto acid substrates, limiting the diversity in types and structures of natural products they can catalyze. For example, to date, only a handful of furanolide-like natural products have been reported (…). Nat. Chem. Biol. 2022, 18, 652–658; Angew. Chem. Int. Ed. 2022, 61, e202204545), pseudocladocerans are even rarer natural products ( Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci. 2020, 90, 467–481).

[0004] (II) Insufficient application development: Although many enzymes have been successfully applied to the synthesis of economic or pharmaceutical molecules, the potential of α-hydroxy-β-keto acid synthase as a powerful tool for catalyzing carbon-carbon bond formation has not been fully explored, especially in expanding the structural diversity of natural products and synthesizing novel drug lead compounds, which still needs further exploration and development. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the deficiency of the narrow substrate spectrum of α-keto acids in the prior art, thereby providing an α-hydroxy-β-keto acid synthase that catalyzes the coupling reaction of α-keto acids.

[0006] Another technical problem to be solved by the present invention is to overcome the deficiency of insufficient structural expansion of synthetic drugs in the prior art, thereby providing an α-hydroxy-β-keto acid synthase that catalyzes the α-keto acid coupling reaction.

[0007] On one hand, the present invention provides an α-hydroxy-β-keto acid synthase that catalyzes the α-keto acid coupling reaction, wherein the amino acid sequence of the α-hydroxy-β-keto acid synthase is shown in SEQ ID NO:1 to SEQ ID NO:4.

[0008] In some embodiments, the α-hydroxy-β-keto acid synthase is a homologous protein to the protein with the amino acid sequence shown in SEQ ID NO:1~SEQ ID NO:4, with a homology of 40% or more.

[0009] On the other hand, the present invention also provides a nucleic acid molecular gene encoding the above-mentioned α-hydroxy-β-keto acid synthase, the sequence of which is shown in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, and SEQ ID NO:11.

[0010] Meanwhile, the present invention also provides a method for constructing a recombinant expression vector containing the above-mentioned α-hydroxy-β-keto acid synthase gene, comprising the following steps: amplifying the target gene from the genomic DNA of the strain, or synthesizing the target gene according to the sequence of the target gene; recombining the target gene and plasmid to obtain the expression vector.

[0011] In some embodiments, the primer sequences for amplifying the target gene are shown in SEQ ID NO:13~SEQ ID NO:14 or SEQ ID NO:17~SEQ ID NO:18.

[0012] In some embodiments, before the plasmid is recombinated with the target gene, a step of linearizing the plasmid is included. The primer sequences used for the plasmid linearization step are shown in SEQ ID NO:15~SEQ ID NO:16 or SEQ ID NO:19~SEQ ID NO:20 or SEQ ID NO:21~SEQ ID NO:22, SEQ ID NO:23~SEQ ID NO:24, SEQ ID NO:25~SEQ ID NO:26.

[0013] Furthermore, the present invention provides biological materials comprising any one of the following: (1) a nucleic acid molecule encoding the above-mentioned α-hydroxy-β-keto acid synthase or a nucleic acid molecule containing the gene of the α-hydroxy-β-keto acid synthase as described in claim 3; (2) a recombinant expression vector containing the nucleic acid molecule described in (1); (3) a recombinant microorganism containing the nucleic acid molecule described in (1) or a recombinant microorganism containing the recombinant expression vector described in (2); (4) a recombinant cell line containing the nucleic acid molecule described in (1) or a recombinant cell line containing the recombinant expression vector described in (2).

[0014] On the other hand, the present invention provides an α-hydroxy-β-keto acid, wherein the α-hydroxy-β-keto acid is formed by coupling α-keto acid under the catalysis of α-hydroxy-β-keto acid synthase, wherein the α-hydroxy-β-keto acid synthase is at least one of the above-mentioned α-hydroxy-β-keto acid synthase, an α-hydroxy-β-keto acid synthase encoded by a gene encoding the above-mentioned α-hydroxy-β-keto acid synthase, an α-hydroxy-β-keto acid synthase formed by an expression vector obtained by a method for constructing a recombinant expression vector of the above-mentioned α-hydroxy-β-keto acid synthase gene, or an α-hydroxy-β-keto acid synthase contained in the above-mentioned biological material.

[0015] In some embodiments, the α-keto acid includes at least one of aliphatic α-keto acids or aromatic α-keto acids.

[0016] In some embodiments, the aliphatic α-keto acid includes at least one selected from pyruvate, 3-methyl-2-oxobutyric acid, 2-oxovalerate, 3-methyl-2-oxovalerate, 4-methyl-2-oxovalerate, 2-oxohexanoic acid, 2-cyclopropyl-2-oxoacetic acid, and 2-cyclobutyl-2-oxoacetic acid.

[0017] In some embodiments, the aromatic α-keto acid includes at least one of indole-3-pyruvic acid, p-hydroxyphenylpyruvic acid, phenylpyruvic acid, p-nitrophenylpyruvic acid, 4-hydroxy-3-methoxyphenylpyruvic acid, 2-oxo-4-phenylbutyric acid, and 3-(naphth-1-yl)-2-oxopropionic acid.

[0018] In some embodiments, the α-hydroxy-β-keto acid has the structure shown in formula (I).

[0019]

[0020] Equation (I)

[0021] R1 and R2 are independently selected from -R3 or -CR3.

[0022] R3 is selected from at least one of substituted or unsubstituted C6-C20 aryl, C6-C20 heteroaryl, hydrogen atom, substituted or unsubstituted C1-C20 alkyl, and C3-C20 cycloalkyl; the substituent of the substituted C1-C20 alkyl is selected from C6-C20 aryl or C6-C20 heteroaryl, and the substituent of the substituted C6-C20 aryl is selected from at least one of hydroxyl, C1-C6 alkyl, methoxy or nitro.

[0023] In some of these embodiments, R3 is selected from phenol, phenyl, nitrophenyl, benzyl, naphthyl, indolyl, 3-methoxy-4-hydroxyphenyl, substituted or unsubstituted C1-C6 alkyl and C3-C4 cycloalkyl; wherein the substituent of the substituted C1-C6 alkyl is selected from phenyl.

[0024] On the other hand, the present invention also provides an α,β-hydroxycarboxylic acid compound, wherein the α,β-hydroxycarboxylic acid compound is obtained by reducing the above-mentioned α-hydroxy-β-keto acid.

[0025] In some embodiments, the α,β-hydroxycarboxylic acid compound has the structure shown in formula (II).

[0026]

[0027] Formula (II),

[0028] R1 and R2 are independently selected from -R3 or -CR3.

[0029] R3 is selected from at least one of substituted or unsubstituted C6-C20 aryl, C6-C60 heteroaryl, hydrogen atom, substituted or unsubstituted C1-C20 alkyl, and C3-C20 cycloalkyl; the substituent of the substituted C1-C20 alkyl is selected from C6-C20 aryl or C6-C60 heteroaryl, and the substituent of the substituted C6-C20 aryl is selected from at least one of hydroxyl, C1-C6 alkyl, methoxy or nitro.

[0030] In some of these embodiments, R3 is selected from phenol, phenyl, nitrophenyl, benzyl, naphthyl, indolyl, 3-methoxy-4-hydroxyphenyl, substituted or unsubstituted C1-C6 alkyl and C3-C4 cycloalkyl; wherein the substituent of the substituted C1-C6 alkyl is selected from phenyl.

[0031] In some embodiments, the α,β-hydroxycarboxylic acid compounds have the structures shown in Formulas 1-1 to 1-116.

[0032]

[0033]

[0034]

[0035]

[0036]

[0037]

[0038]

[0039]

[0040]

[0041]

[0042]

[0043]

[0044]

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

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[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070] .

[0071] In some embodiments, a reducing agent is used to reduce α-hydroxy-β-keto acid, said reducing agent including NaBH4.

[0072] In some embodiments, the coupling reaction is carried out at a temperature of 16-55°C for a reaction time of 15-180 min.

[0073] In some embodiments, the molar ratio of the first α-keto acid to the second α-keto acid is 0.8-1.2:0.8-1.2, preferably 1:1.

[0074] In some embodiments, the concentration of the reducing agent in the α-hydroxy-β-keto acid intermediate reaction system is greater than or equal to 10 mM, and the reduction reaction time is greater than or equal to 10 min. For example, the concentration of the reducing agent in the α-hydroxy-β-keto acid intermediate reaction system is 10 mM, 15 mM, 20 mM, 30 mM, 45 mM, 50 mM, 100 mM, or any range of two such values; the reduction reaction time is 10 min, 15 min, 22 min, 25 min, 30 min, 50 min, or any range of two such values.

[0075] In some embodiments, the compound having a furanolactone structure is reacted in the presence of α-hydroxy-β-keto acid synthase with α-keto acid and α,β-unsaturated carboxylic acid as substrates; or, in the presence of α-hydroxy-β-keto acid synthase, it is reacted with α-amino acid, α-keto acid and α,β-unsaturated carboxylic acid as substrates; the α-hydroxy-β-keto acid synthase is at least one of the above-mentioned α-hydroxy-β-keto acid synthase, α-hydroxy-β-keto acid synthase encoded by a gene encoding the above-mentioned α-hydroxy-β-keto acid synthase, α-hydroxy-β-keto acid synthase formed by an expression vector obtained by a method for constructing a recombinant expression vector of the above-mentioned α-hydroxy-β-keto acid synthase gene, or α-hydroxy-β-keto acid synthase contained in the above-mentioned biological material.

[0076] In some embodiments, the compound having a furanolactone structure has the structural formula shown in formula (Ⅲ).

[0077]

[0078] Formula (Ⅲ),

[0079] R4 is selected from substituted or unsubstituted C6-C30 aryl, C6-C30 heteroaryl, hydrogen atom, substituted or unsubstituted C1-C30 alkyl, wherein the substituent of the substituted C6-C30 aryl is selected from at least one of hydroxyl and methoxy, and the substituent of the substituted C1-C30 alkyl is selected from C6-C30 aryl or C6-C30 heteroaryl.

[0080] R5 is selected from -R7 or -CR7, R7 is selected from at least one of substituted or unsubstituted C6-C30 aryl, C6-C30 heteroaryl, hydrogen atom, substituted or unsubstituted C1-C30 alkyl, and C3-C30 cycloalkyl, the substituent of the substituted C6-C30 aryl is selected from at least one of hydroxyl and methoxy, and the substituent of the substituted C1-C30 alkyl is selected from C6-C30 aryl and C6-C30 heteroaryl;

[0081] R6 is selected from substituted or unsubstituted C6-C30 aryl groups, C6-C30 heteroaryl groups, and the substituents of substituted C6-C30 aryl groups are selected from at least one of hydroxyl, halogen, cyano, or C1-C6 alkyl groups.

[0082] In some embodiments, R4 is selected from substituted or unsubstituted C6-C15 aryl, C6-C15 heteroaryl, hydrogen atom, substituted or unsubstituted C1-C15 alkyl, wherein the substituent of the substituted C6-C15 aryl is selected from at least one of hydroxyl and methoxy, and the substituent of the substituted C1-C15 alkyl is selected from C6-C15 aryl and C6-C15 heteroaryl.

[0083] In some embodiments, R5 is selected from -R7 or -CR7, R7 is selected from at least one of substituted or unsubstituted C6-C15 aryl, C6-C30 heteroaryl, hydrogen atom, substituted or unsubstituted C1-C15 alkyl, and C3-C15 cycloalkyl, the substituent of the substituted C6-C15 aryl is selected from at least one of hydroxyl and methoxy, and the substituent of the substituted C1-C15 alkyl is selected from C6-C15 aryl or C6-C15 heteroaryl.

[0084] In some of these embodiments, R6 is selected from substituted or unsubstituted C6-C15 aryl groups, C6-C15 heteroaryl groups, and the substituents of substituted C6-C15 aryl groups are selected from at least one of hydroxyl, halogen, cyano, or C1-C6 alkyl groups.

[0085] In some of these embodiments, R4 is selected from at least one of phenolic, phenyl, o-phenolic, 3-methoxy-4-hydroxyphenyl, 3,5-dimethoxy-4-hydroxyphenyl, and C1-C6 alkyl groups.

[0086] In some embodiments, R5 is selected from at least one of phenolic, phenyl, nitrophenyl, benzyl, naphthyl, indolyl, 3-methoxy-4-hydroxyphenyl, p-cyanophenyl, benzothiophene, substituted or substituted C1-C6 alkyl and C3-C4 cycloalkyl, wherein the substituent of the substituted C1-C6 alkyl is selected from phenyl.

[0087] In some of these embodiments, R7 is selected from at least one of phenolic, phenyl, nitrophenyl, benzyl, naphthyl, indole, 3-methoxy-4-hydroxyphenyl, p-methylphenyl, p-cyanophenyl, benzothiophene, 3,5-dichloro-4-hydroxyphenyl, 3-chloro-4-hydroxyphenyl, and p-fluorophenyl.

[0088] In some of these embodiments, R4 is selected from at least one of isopropyl, n-butyl, or ethyl.

[0089] In some embodiments, R5 is selected from at least one of n-propyl, isopropyl, n-butyl, methyl, or ethyl.

[0090] In some embodiments, the compound having a furanolactone structure has the structures shown in Formulas 2-1 to 2-36.

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102] .

[0103] In some embodiments, when α-keto acids and α,β-unsaturated carboxylic acids are used as substrates and / or α-amino acids, α-keto acids and α,β-unsaturated carboxylic acids are used as substrates, the reaction system further includes coenzyme A, thiamine pyrophosphate, adenine nucleoside triphosphate, a catalytic enzyme, magnesium salt, and buffer solution.

[0104] In some embodiments, the final concentration of α-hydroxy-β-keto acid synthase in the reaction system is 0.01-0.025 mM.

[0105] In some embodiments, the catalytic enzyme includes at least one of 4-coumarate-coenzyme A ligase, furanolactone synthase, and transaminase.

[0106] In some embodiments, the magnesium salt includes at least one of magnesium chloride, magnesium sulfate, or magnesium nitrate.

[0107] In some embodiments, the buffer solution includes at least one of sodium chloride buffer, 4-hydroxyethylpiperazine ethanesulfonic acid buffer, tris(hydroxymethyl)aminomethane hydrochloride buffer, or phosphate buffer.

[0108] The technical solution of this invention has the following advantages:

[0109] This invention provides an α-hydroxy-β-keto acid synthase for catalyzing α-keto acid coupling reactions, the amino acid sequence of which is shown in SEQ ID NO:1 to SEQ ID NO:4. The α-hydroxy-β-keto acid synthase provided by this invention can catalyze carbon-carbon coupling reactions between various α-keto acid substrates, expanding substrate adaptability and providing more possibilities for diverse chemical synthetic pathways. This significantly enhances its application prospects in the synthesis of complex molecules and provides a powerful catalytic tool for the development of related natural products and drug lead compounds. Attached Figure Description

[0110] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0111] Figure 1 This is a schematic diagram of the recombinant plasmid in Embodiment 1 of the present invention;

[0112] Figure 2 This is a schematic diagram of the recombinant plasmid in Embodiment 2 of the present invention;

[0113] Figure 3 This is a schematic diagram of the recombinant plasmid in Example 3 of the present invention;

[0114] Figure 4 This is a schematic diagram of the recombinant plasmid in Example 4 of the present invention;

[0115] Figure 5 This is the nuclear magnetic resonance spectrum of the product obtained in Application Example 1 of this invention;

[0116] Figure 6 This is the nuclear magnetic resonance spectrum of the product obtained in Example 2 of this invention;

[0117] Figure 7 This is the nuclear magnetic resonance spectrum of the product obtained in Example 3 of this invention;

[0118] Figure 8 This is the nuclear magnetic resonance spectrum of the product obtained in Example 4 of this invention;

[0119] Figure 9 This is the nuclear magnetic resonance spectrum of the product obtained in Example 5 of this invention;

[0120] Figure 10 This is the nuclear magnetic resonance spectrum of the product obtained by application example 6 of the present invention;

[0121] Figure 11 This is the nuclear magnetic resonance spectrum of the product obtained in Example 7 of this invention;

[0122] Figure 12 This is the nuclear magnetic resonance spectrum of the product obtained by application example 8 of the present invention. Detailed Implementation

[0123] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0124] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0125] Example 1

[0126] This embodiment provides a method for preparing a gene encoding α-hydroxy-β-keto acid synthase, and the specific steps and parameters are as follows:

[0127] 1. Amplification csmA Gene

[0128] Using PCR to detect *Microbacterium chrysogenum* ( Chryseomicrobium Genomic DNA (genome sequence recorded in Genebank, accession number PP238510.1) of strain sp. PKU-MA01392 was amplified using this as a template. csmA Genes, of which the PCR reaction system is shown in Table 1 and the PCR reaction procedure is shown in Table 2.

[0129] Table 1 PCR Reaction System

[0130]

[0131] The template concentration was 200 ng / μL, the forward primer concentration was 10 μM, and the forward primer in this embodiment was CsmA-Fw, the specific sequence of which is shown in Table 5. The reverse primer concentration was 10 μM, and the reverse primer in this embodiment was CsmA-Fv, the specific sequence of which is shown in Table 5.

[0132] 2× Phanta Max Master Mix were purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0133] Table 2 shows the PCR reaction procedure as follows:

[0134]

[0135] The extraction of genomic DNA followed the method described in the book *Practical Streptomyces Genetics*. The steps were as follows: A strain frozen at -80℃ (isolated from sponge samples collected by our research group from Naozhou Island, Guangdong) was streaked onto solid medium A (1 g / L yeast extract, 5 g / L peptone, 1 g / L beef extract, 0.01 g / L ferric phosphate, 33 g / L sea salt, pH 7.4) and incubated at 28℃ until single colonies appeared. A single colony was picked from the plate and inoculated into 50 mL of liquid medium A and incubated at 28℃ and 250 rpm for 2-3 days. The 50 mL of bacterial culture was centrifuged at 4000 rpm for 15 min, and the supernatant was discarded. The precipitate was resuspended in 15 mL of 10 mM EDTA (pH 8.0), centrifuged at 4000 rpm for 15 min, and the supernatant was discarded. The bacterial pellet was resuspended in 5 mL of SET buffer (5 mM sodium chloride, 25 mM EDTA, 20 mM tris(hydroxymethyl)aminomethane, pH 8.0), and lysozyme was added to a final concentration of 1 mg / mL. The mixture was incubated at 37°C for 60 min. 600 μL of 10% sodium dodecyl sulfate and proteinase K to a final concentration of 0.5 mg / mL were added, and the mixture was incubated at 55°C for 2 h. 2 mL of 5 M sodium chloride was added, and the mixture was thoroughly mixed and allowed to cool to room temperature. 5 mL of chloroform was added, and the mixture was vortexed for 30 min, then centrifuged at 4000 rpm for 30 min. The supernatant was transferred to a new 10 mL centrifuge tube and centrifuged at 4000 rpm for 10 min. The supernatant was then transferred again to a new 10 mL centrifuge tube. Add 0.6 times the volume of isopropanol to a centrifuge tube to precipitate the DNA. Centrifuge at 4000 rpm for 10 min and discard the supernatant. Rinse the DNA precipitate with 5 mL of 70% ethanol, discard the 70% ethanol, and dry the ethanol in a clean bench. Dissolve in 1 mL of enzyme-free sterile water and store at -20°C. Amplification csmA The gene sequence is as shown in SEQ ID NO:5 (see Table 6 for the specific sequence).

[0136] 2. Constructing recombinant plasmids

[0137] pET28a(+)- was constructed using the ClonExpress Ultra One Step Cloning Kit (Novizan). CsmA The expression vector, of which plasmid pET28a(+) was purchased from Sangon Biotech Co., Ltd.

[0138] The specific steps are as follows:

[0139] 2.1 Using pET28a(+) as a template, the linearized pET28a(+) vector fragment was amplified using primers 28a-Fw / Rv (sequence information is shown in Table 5). The amplification reaction system is shown in Table 3, and the reaction procedure is shown in Table 4.

[0140] Table 3 PCR Reaction System II

[0141]

[0142] The template concentration was 200 ng / μL, the forward primer concentration was 10 μM, the reverse primer concentration was 10 μM, and the 2× Phanta Max Master Mix was purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0143] Table 4 PCR Reaction Procedure 2

[0144]

[0145] 2.2. Amplification was performed using the ClonExpress Ultra One Step Cloning Kit (Vazyme). csmA The gene was recombination reaction with the linearized pET28a(+) vector fragment in a 10 μL volume. csmA The gene concentration was 6 ng / μL, the vector fragment concentration was 12 ng / μL, and the reaction conditions were 50 ℃ for 15 min.

[0146] 2.3. Add 5 μL of the recombinant product to 50 μL of Trans1-T1 competent cells (purchased from Beijing TransGen Biotech Co., Ltd.), gently tap the tube wall to mix, and incubate on ice for 30 min. Then, heat shock in a 42°C water bath for 30 s, and immediately place on ice to cool for 3 min.

[0147] 2.4. Add 900 μL of LB liquid medium (without antibiotics) and incubate at 37°C for 1 h. Centrifuge at 4000 rpm (2,500 × g) for 2 min and discard the supernatant. Resuspend the bacterial culture in the remaining medium and gently spread it evenly on a plate containing kanamycin resistance using a sterile spreader.

[0148] 2.5. Incubate upside down in a 37°C incubator for 14 hours, then select single clones for sequencing identification.

[0149] Primer information for this embodiment is shown in Table 1. A schematic diagram of the recombinant plasmid is shown below. Figure 1 The correctness of the recombinant plasmids was confirmed by DNA sequencing. pET28a(+)- CsmAThe sequence of the expression vector is shown in SEQ ID NO:6 (see Table 6 for the specific sequence), and the specific primer information is shown in Table 5.

[0150] Example 2

[0151] This embodiment provides a method for preparing a gene encoding α-hydroxy-β-keto acid synthase, and the specific steps and parameters are as follows:

[0152] 1. Beijing Ruiboxingke Biotechnology Co., Ltd. was commissioned to synthesize... BbmA Gene, BbmA See SEQ ID NO:7 for the gene sequence, using primers. BbmA -Fw / Rv for synthesis BbmA The gene was amplified by PCR to obtain BbmA See Tables 1 and 2 for gene fragments, PCR reaction systems, and reaction procedures.

[0153] 2. Constructing recombinant plasmids

[0154] pET28a(+)- was constructed using the ClonExpress Ultra One Step Cloning Kit (Novizan). BbmA Carrier of expression.

[0155] The specific steps are as follows:

[0156] 2.1 Using pET28a(+) as a template, the linearized pET28a(+) vector fragment was amplified using primers 28a-Fw / Rv. The amplification reaction system and reaction procedure are shown in Tables 3 and 4.

[0157] 2.2. Amplification was performed using the ClonExpress Ultra One Step Cloning Kit (Vazyme). bbm The gene was recombination reaction with the linearized pET28a(+) vector fragment in a 10 μL volume. bbm The gene concentration was 6 ng / μL, the vector fragment concentration was 12 ng / μL, and the reaction conditions were 50 ℃ for 15 min.

[0158] 2.3. Add 5 μL of the recombinant product to 50 μL of Trans1-T1 competent cells, gently tap the tube wall to mix, and incubate on ice for 30 min. Then, heat shock in a 42°C water bath for 30 s, and immediately cool on ice for 3 min.

[0159] 2.4. Add 900 μL of LB liquid medium (without antibiotics) and incubate at 37°C for 1 h. Centrifuge at 4000 rpm (2500 × g) for 2 min and discard the supernatant. Resuspend the bacterial culture in the remaining medium and gently spread it evenly on a plate containing kanamycin resistance using a sterile spreader.

[0160] 2.5. Incubate upside down in a 37°C incubator for 14 hours, then select single clones for sequencing identification.

[0161] See the schematic diagram of the recombinant plasmid. Figure 2 The correctness of the recombinant plasmids was confirmed by DNA sequencing.

[0162] pET28a(+)- BbmA The sequence of the expression vector is shown in SEQ ID NO:8 (see Table 6 for the specific sequence), and the specific primer information is shown in Table 5.

[0163] Example 3

[0164] This embodiment provides a method for preparing a gene encoding α-hydroxy-β-keto acid synthase, and the specific steps and parameters are as follows:

[0165] 1. Mutant CsmA G466F Construction of expression carrier

[0166] 1.1 Using plasmid pET28a(+)- CsmA Using the template, linearized pET28a(+)- was amplified using primers G466F-Fw / Rv. CsmA G466F For the fragments, amplification reaction system and reaction procedure, please refer to Tables 3 and 4.

[0167] 1.2. Using the ClonExpress Ultra One Step Cloning Kit (Vazyme), the above linearized pET28a(+)- CsmA G466F The fragment undergoes self-ligation in a 10 μL reaction system. CsmA G466F The gene concentration was 6 ng / μL (sequence shown in SEQ ID NO:9), the vector fragment concentration was 12 ng / μL, and the reaction conditions were 50 ℃ for 15 min.

[0168] 1.3. Add 5 μL of the recombinant product to 50 μL of Trans1-T1 competent cells, gently tap the tube wall to mix, and incubate on ice for 30 min. Then, heat shock in a 42°C water bath for 30 s, and immediately cool on ice for 3 min.

[0169] 1.4. Add 900 μL of LB liquid medium (without antibiotics) and incubate at 37°C for 1 h. Then, centrifuge at 4000 rpm (2,500 × g) for 2 min and discard the supernatant. Resuspend the bacterial culture in the remaining medium and gently spread it evenly on a plate containing kanamycin resistance using a sterile spreader.

[0170] 1.5. Incubate upside down in a 37°C incubator for 14 h, then select single clones for sequencing identification.

[0171] Constructed pET28a(+)- CsmA G466F See the schematic diagram of the expression carrier. Figure 3 Its sequence is shown in SEQ ID NO:10 (see Table 6 for the specific sequence), and the specific primer information is shown in Table 5.

[0172] Example 4

[0173] This embodiment provides a method for preparing a gene encoding α-hydroxy-β-keto acid synthase, and the specific steps and parameters are as follows:

[0174] 1. Mutant BbmA L112S / W288T / G484F Construction of expression carrier

[0175] 1.1 Using plasmid pET28a(+)- BbmA Using the template, linearized pET28a(+)- was amplified using primers L112S-Fw / Rv. BbmA L112S For the fragments, amplification reaction system and reaction procedure, please refer to Tables 3 and 4.

[0176] 1.2. Using the ClonExpress Ultra One Step Cloning Kit (Vazyme), the above linearized pET28a(+)- BbmA L112S The fragment undergoes self-ligation in a 10 μL reaction system. BbmA L112S / W288T / G484F The gene (sequence shown in SEQ ID NO:11) concentration was 6 ng / μL, the vector fragment concentration was 12 ng / μL, and the reaction conditions were 50 ℃ for 15 min.

[0177] 1.3. Add 5 μL of the recombinant product to 50 μL of Trans1-T1 competent cells, gently tap the tube wall to mix, and incubate on ice for 30 min. Then, 42... o After heat shock in a water bath for 30 seconds, immediately place it on ice to cool for 3 minutes.

[0178] 1.4. Add 900 μL of LB liquid medium (without antibiotics) and incubate at 37°C for 1 h. Then, centrifuge at 4000 rpm (2,500 × g) for 2 min and discard the supernatant. Resuspend the bacterial culture in the remaining medium and gently spread it evenly on a plate containing kanamycin resistance using a sterile spreader.

[0179] 1.5. Incubate upside down in a 37°C incubator for 16 h, then select single clones for sequencing identification.

[0180] 1.6. Using successfully sequenced pET28a(+)- BbmA L112S Starting with the plasmid, linearized pET28a(+)- was amplified using primers W288T-Fw / Rv. BbmA L112S / W288T Repeat steps 1.2-1.5 above to construct plasmid pET28a(+)- BbmA L112S / W288T .

[0181] 1.7 Similarly, using successfully sequenced pET28a(+)- BbmA L112S / W288T Starting with the plasmid, linearized pET28a(+)- was amplified using primers G484F-Fw / Rv. BbmA L112S / W288T / G484F Repeat steps 1.2-1.5 above to construct plasmid pET28a(+)- BbmA L112S / W288T / G484F .

[0182] Constructing plasmid pET28a(+)- BbmA L112S / W288T / G484F See the schematic diagram of the expression carrier. Figure 4 Its sequence is shown in SEQ ID NO:12 (see Table 6 for the specific sequence). Primer information for this experimental example is shown in Table 5.

[0183] Example 5

[0184] Take 3 μL of the expression vector of α-hydroxy-β-keto acid synthase prepared in Examples 1-4 and add it to 50 μL of Escherichia coli BL21(DE3) competent cells. Gently tap the tube wall to mix and let stand on ice for 30 min. Then, heat shock in a 42°C water bath for 30 s and immediately place on ice to cool for 3 min.

[0185] Add 900 μL of LB liquid medium (without antibiotics) and incubate at 37°C for 1 h. Then, centrifuge at 4000 rpm (2,500 × g) for 2 min and discard the supernatant. Resuspend the bacteria in the remaining medium and spread gently on a plate containing kanamycin resistance using a sterile spreader.

[0186] Incubate upside down in a 37°C incubator for 16 h, then pick single colonies and culture them in LB medium containing 30 mg / L kanamycin until they reach OD. 600 When the protein expression reaches 0.8, 200 µM isopropyl-β-D-thiogalactoside (IPTG) is added to induce protein expression at 18 °C, and the incubation continues for 16 hours.

[0187] After collecting cells by centrifugation (4 °C, 4,000 g, 15 min), they were resuspended in lysis buffer (50 mM Tris, 300 mM NaCl, 20 mM imidazole, 10% glycerol, pH 8.0) and sonicated on ice.

[0188] Soluble protein was collected by centrifugation (4 °C, 12,000 g, 60 min) and loaded into a pre-equilibrated HisTrap HP 5 mL column (Buffer A: 50 mM Tris, 300 mM NaCl, 15 mM imidazole, pH 8.0). After washing with 25 mL of Buffer A, the target protein was eluted with 20 mL of Buffer B (50 mM Tris, 300 mM NaCl, 400 mM imidazole, pH 8.0).

[0189] After identification by SDS-PAGE, the protein was replaced with buffer C (50 mM HEPES, 200 mM NaCl, pH 8.0) using a PD-10 desalting column (GE Healthcare) and stored at -80 °C for later use.

[0190] Among them, the expression vector prepared in Example 1 was eluted to obtain α-hydroxy-β-keto acid synthase. CsmA The amino acid sequence is shown in SEQ ID NO:1. The α-hydroxy-β-keto acid synthase obtained by elution of the expression vector prepared in Example 2 BbmA The amino acid sequence is shown in SEQ ID NO:2. The α-hydroxy-β-keto acid synthase obtained by elution of the expression vector prepared in Example 3 is shown in SEQ ID NO:2. CsmA G466F The amino acid sequence is shown in SEQ ID NO:3. The α-hydroxy-β-keto acid synthase obtained by elution of the expression vector prepared in Example 4 BbmA L112S / W288T / G484F The amino acid sequence is shown in SEQ ID NO:4 (see Table 6 for the specific sequence).

[0191] Table 5 Primer information in the examples

[0192]

[0193]

[0194] Table 6 shows the expression vector pET28a(+)- CsmA In the sequence, uppercase letters represent genes. CsmA Sequence, expression vector pET28a(+)- BbmA In the sequence, uppercase letters represent genes. BbmA Sequence, expression vector pET28a(+)- CsmA G466F In the sequence, uppercase letters represent genes. CsmA G466F The sequence, expression vector pET28a(+)- BbmA L112S / W288T / G484F In the sequence, uppercase letters represent genes. BbmA L112S / W288T / G484F sequence.

[0195] Application Example 1

[0196] This application example provides a method for preparing α,β-hydroxycarboxylic acids, with the specific steps and parameters as follows:

[0197] (1) Add 15 mL of reaction buffer (50 mM 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), 200 mM sodium chloride, 2 mM magnesium chloride, pH 8.0) to the reaction flask, and then add the following reagents in sequence: 2 mM indole-3-pyruvate, 2 mM p-hydroxyphenylpyruvate, 0.1 mM ThDP and 0.01 mM α-hydroxy-β-keto acid synthase to a final concentration. CsmA (The α-hydroxy-β-keto acid synthase prepared in Example 5 according to Example 1) was reacted at 37 °C for 1 hour.

[0198] (2) Add NaBH4 with a final concentration of 100 mM to the reaction system obtained after step (1), and continue the reaction at room temperature for 30 minutes. The reaction formula is shown in formula (VI).

[0199] Formula (VI)

[0200] After the reaction was completed, an equal volume of methanol was added to terminate the reaction, and the supernatant was collected by high-speed centrifugation (12,000 g, 30 min).

[0201] The supernatant was concentrated and dried under reduced pressure to obtain the crude product.

[0202] The crude product was then dissolved in 2 mL of 50% methanol-water solution and centrifuged again at high speed (12,000 g, 30 min) to collect the supernatant.

[0203] The product was separated and purified by semi-preparative high performance liquid chromatography (HPLC) using an acetonitrile (B)-water (A) gradient elution (10%-100% B, 40 min). The chromatographic column was YMC-Pack ODS-A (250 × 10 mm, 5 µm, YMC CO., Ltd., Shimogyo-ku, Kyoto, Japan), the flow rate was 2 mL / min, and the detection wavelength was 210 nm.

[0204] The purified product was subjected to NMR scanning, see [link to NMR scan]. Figure 5 As can be seen, the substrate conversion rate obtained in this application example is 63%. 1 H NMR (600 MHz, DMSO-) d 6) δ 3.87 (br d, J = 10.4 Hz, 1H), 2.67 (br d, J = 14.8 Hz, 1H), 2.79 (dd, J = 14.8 Hz, 10.4, 1H), 7.45 (d, J = 7.8 Hz, 1H), 6.94 (t, J = 7.3 Hz,1H), 7.04 (m, 1H), 7.32 (d, J = 8.0 Hz, 1H), 10.72 (br s, 1H), 7.10 (d, J = 1.8Hz, 1H), 2.85 (d, J = 13.7 Hz, 1H), 2.96 (d, J = 13.7 Hz, 1H), 7.02 (d,J = 8.4Hz, 2H), 6.64 (d, J = 8.4 Hz, 2H).

[0205] Application Example 2

[0206] This application example provides a method for preparing α,β-hydroxycarboxylic acid. The specific steps and parameters are the same as in application example 1. The difference is that equimolar amounts of 2-oxo-4-phenylbutyric acid are used to replace indole-3-pyruvic acid in step (1), and equimolar amounts of 4-hydroxy-3-methoxyphenylpyruvic acid are used to replace p-hydroxyphenylpyruvic acid in step (1).

[0207] The purified product was subjected to NMR scanning, see [link to NMR scan]. Figure 6 As can be seen, the substrate conversion rate obtained in this application example is 67%. 1 H NMR (600 MHz, DMSO-) d 6) δ 3.54 (br d, J = 10.6 Hz, 1H), 1.49 (m, 1H), 1.78 (m, 1H), 2.53 (m, 1H), 2.82 (m, 1H), 7.17 (d, J = 7.1 Hz, 2H), 7.26 (t, J = 7.7 Hz, 2H), 7.16 (t, J = 7.1 Hz, 1H), 2.78 (d, J = 13.7 Hz, 1H), 2.90 (d, J = 13.7 Hz, 1H), 6.75 (d, J = 1.7 Hz, 1H), 8.62 (s, 1H), 6.59 (d, J = 8.0 Hz, 1H), 6.54 (br d, J =8.0 Hz, 1.7, 1H), 3.70 (s, 3H).

[0208] Application Example 3

[0209] This application example provides a method for preparing α,β-hydroxycarboxylic acids. The specific steps and parameters are the same as in Application Example 1, except that an equimolar amount of pyruvate is used to replace p-hydroxyphenylpyruvate in step (1), and an equimolar amount of α-hydroxy-β-keto acid synthase is used. BbmA (The α-hydroxy-β-keto acid synthase obtained in Example 5 according to Example 2) replaces the α-hydroxy-β-keto acid synthase. CsmA(α-hydroxy-β-keto acid synthase prepared in Example 5 according to Example 1).

[0210] The purified product was subjected to NMR scanning, see [link to NMR scan]. Figure 7 As can be seen, the substrate conversion rate obtained in this application example is 84%. 1 H NMR (600 MHz, DMSO-) d 6) δ 3.89 (q, J = 6.3 Hz, 1H), 1.18 (d, J = 6.3 Hz, 3H), 2.78 (d, J =14.4 Hz, 1H), 3.02 (d, J = 14.4 Hz, 1H), 7.55 (d, J = 7.7 Hz, 1H), 6.92 (br t, J =7.5 Hz, 1H), 7.01 (br t, J = 7.5 Hz, 1H), 7.28 (d, J = 8.1 Hz, 1H), 10.77 (br s,1H), 7.10 (d, J = 2.2 Hz, 1H).

[0211] Application Example 4

[0212] This application example provides a method for preparing α,β-hydroxycarboxylic acids. The specific steps and parameters are the same as in application example 1, except that an equimolar amount of 4-methyl-2-oxopentanoic acid is used to replace indole-3-pyruvic acid in step (1), an equimolar amount of 3-(naphthyl-1-yl)-2-oxopropionic acid is used to replace p-hydroxyphenylpyruvic acid in step (1), and an equimolar amount of α-hydroxy-β-keto acid synthase is used. BbmA (The α-hydroxy-β-keto acid synthase obtained in Example 5 according to Example 2) replaces the α-hydroxy-β-keto acid synthase. CsmA (α-hydroxy-β-keto acid synthase prepared in Example 5 according to Example 1).

[0213] The purified product was subjected to NMR scanning, see [link to NMR scan]. Figure 8 As can be seen, the substrate conversion rate obtained in this application example is 77%. 1 H NMR (600 MHz, DMSO-) d 6) δ 3.76 (br d, J = 10.7 Hz, 1H), 0.98 (t, J= 11.9 Hz, 1H), 1.58(m, 1H), 1.83 (m, 1H), 0.86 (d, J = 6.7 Hz, 3H), 0.91 (d, J = 6.7 Hz, 3H), 3.31(d, J = 14.2 Hz, 1H), 3.68 (d, J = 14.2 Hz, 1H), 8.24 (m, 1H), 7.47 (m, 2H), 7.84(m, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.38 (t, J = 7.6 Hz, 1H), 7.43 (br d, J = 6.9Hz, 1H).

[0214] Application Example 5

[0215] This application example provides a method for preparing α,β-hydroxycarboxylic acids. The specific steps and parameters are the same as in application example 1, except that an equimolar amount of phenylpyruvic acid is used to replace indole-3-pyruvic acid in step (1), an equimolar amount of 2-oxopentanoic acid is used to replace p-hydroxyphenylpyruvic acid in step (1), and an equimolar amount of α-hydroxy-β-keto acid synthase is used. CsmA G466F (The α-hydroxy-β-keto acid synthase obtained in Example 5 from Example 3) replaces the α-hydroxy-β-keto acid synthase. CsmA (α-hydroxy-β-keto acid synthase prepared in Example 5 according to Example 1).

[0216] The purified product was subjected to NMR scanning, see [link to NMR scan]. Figure 9 As can be seen, the substrate conversion rate obtained in this application example is 61%. 1 H NMR (600 MHz, DMSO-) d 6) δ 3.65 (dd, J = 10.5, 1.9 Hz, 1H), 2.46 (dd, J = 14.0, 10.5 Hz,1H), 2.85 (br d, J = 14.0 Hz, 1H), 7.21 (d, J = 7.6 Hz, 2H), 7.25 (t, J = 7.5 Hz, 2H), 7.16 (t, J= 7.2 Hz, 1H), 1.47 (m, 2H), 1.66 (td, J = 12.9, 4.1 Hz, 1H),1.09 (m, 1H), 0.87 (t, J = 7.3 Hz, 3H).

[0217] Application Example 6

[0218] This application example provides a method for preparing α,β-hydroxycarboxylic acids. The specific steps and parameters are the same as in application example 1, except that an equimolar amount of p-nitrophenylpyruvic acid is used to replace indole-3-pyruvic acid in step (1), an equimolar amount of 2-oxopentanoic acid is used to replace p-hydroxyphenylpyruvic acid in step (1), and an equimolar amount of α-hydroxy-β-keto acid synthase is used. BbmA L112S / W288T / G484F (The α-hydroxy-β-keto acid synthase obtained in Example 5 from Example 4) replaces the α-hydroxy-β-keto acid synthase. CsmA (α-hydroxy-β-keto acid synthase prepared in Example 5 according to Example 1).

[0219] The purified product was subjected to NMR scanning, see [link to NMR scan]. Figure 10 As can be seen, the substrate conversion rate obtained in this application example is 54%. 1 HNMR (600 MHz, DMSO- d 6) δ 3.72 (dd, J = 10.6, 2.1 Hz, 1H), 2.65 (dd, J = 13.9 Hz,10.6 Hz, 1H), 2.99 (br d, J = 13.9 Hz, 1H), 7.52 (d, J = 8.7 Hz, 2H), 8.14 (d, J =8.7 Hz, 2H), 1.49 (m, 2H), 1.70 (td, J = 12.5, 4.6 Hz, 1H), 1.10 (m, 1H), 0.88(t, J = 7.3 Hz, 3H).

[0220] Application Example 7

[0221] This application example provides a method for preparing furan lactone compounds, with the specific steps and parameters as follows:

[0222] (1) Add 20 mL of reaction buffer (50 mM HEPES, 200 mM sodium chloride, 5 mM magnesium chloride, pH 8.0) to the reaction flask, and then add the following reagents in sequence: 2 mM p-hydroxyphenylpyruvic acid, 2 mM 4-methyl-2-oxovaleric acid, 2 mM p-coumaric acid, 2 mM coenzyme A (CoA), 0.1 mM ThDP, 5 mM adenine nucleoside triphosphate (ATP), and 0.025 mM α-hydroxy-β-keto acid synthase. BbmA The reaction was carried out at 25 °C for 12 hours with 0.05 mM 4-coumaroyl-CoA ligase 1 (Gm4CL1) and 0.05 mM furanolactone synthase (CybF). The reaction formula is shown in formula (VII).

[0223]

[0224] Formula (VII)

[0225] (2) The reaction system obtained after step (1) was extracted three times with an equal volume of ethyl acetate. The ethyl acetate layers were combined, concentrated under reduced pressure and dried to obtain the crude product.

[0226] The crude product was reconstituted with acetonitrile and then centrifuged at high speed (12,000g, 30 minutes) to obtain the supernatant.

[0227] The product was separated and purified by semi-preparative high-performance liquid chromatography (HPLC) using an acetonitrile (B)-water (A) gradient elution (10%-100% B, 40 min). The chromatographic column was a YMC-Pack ODS-A (250 × 10 mm, 5 µm, YMC CO., Ltd., Kyoto, Japan), the flow rate was 2 mL / min, and the detection wavelength was 340 nm.

[0228] The purified product was subjected to NMR scanning, see [link to NMR scan]. Figure 11 As can be seen, the substrate conversion rate obtained in this application example is 55%. 1 HNMR (600 MHz, DMSO- d 6) δ 3.56 (s, 2H), 7.01 (d, J = 8.5 Hz, 2H), 6.67 (d, J = 8.5Hz, 2H), 6.33 (s, 1H), 7.64 (d, J = 8.8 Hz, 2H), 6.82 (d, J = 8.8 Hz, 2H), 2.48(d, J= 7.5 Hz, 2H), 1.84 (hept, J = 6.6 Hz, 1H), 0.90 (d, J = 6.6, 6H).

[0229] Application Example 8

[0230] This application example provides a method for preparing furanolide compounds, with the specific steps and parameters as follows:

[0231] (1) Add 20 mL of reaction buffer (50 mM HEPES, 200 mM sodium chloride, 5 mM magnesium chloride, pH 8.0) to the reaction flask, and then add the following reagents in sequence: 2 mM 2-oxo-4-phenylbutyric acid, 2 mM 3,5-dichloro-L-tyrosine, 2 mM p-coumaric acid, 1 mM pyridoxal phosphate (PLP), 4 mM α-ketoglutarate (α-KG), 2 mM CoA, 0.1 mM ThDP, 5 mM ATP, 0.05 mM AroH, and 0.01 mM α-hydroxy-β-keto acid synthase. CsmA 0.05 mM Gm4Cl1 and 0.05 mM CybF were reacted at 25 °C for 12 hours. The reaction formula is shown in equation (VIII).

[0232]

[0233] Formula (VIII)

[0234] (2) The reaction system obtained after step (1) was extracted three times with an equal volume of ethyl acetate. The ethyl acetate layers were combined, concentrated under reduced pressure and dried to obtain the crude product.

[0235] The crude product was reconstituted with acetonitrile and then centrifuged at high speed (12,000g, 30 minutes) to obtain the supernatant.

[0236] The product was separated and purified by semi-preparative high-performance liquid chromatography (HPLC) using an acetonitrile (B)-water (A) gradient elution (10%-100% B, 40 min). The chromatographic column was a YMC-Pack ODS-A (250 × 10 mm, 5 µm, YMC CO., Ltd., Kyoto, Japan), the flow rate was 2 mL / min, and the detection wavelength was 340 nm.

[0237] The purified product was subjected to NMR scanning, see [link to NMR scan]. Figure 12 It can be seen that the substrate conversion rate is 36%. 1 H NMR (600 MHz, DMSO- d 6) δ3.43 (s, 2H), 6.99 (d, J = 8.5 Hz, 2H), 6.68 (d, J = 8.5 Hz, 2H), 6.40(s, 1H), 7.79 (s, 2H), 2.83 (dd, J = 6.5, 9.5 Hz, 2H), 2.70 (dd, J = 6.5, 9.5 Hz, 2H), 7.25 (d, J = 8.2 Hz, 2H), 7.30 (t, J = 7.5 Hz, 2H), 7.21 (t, J = 7.5 Hz, 1H).

[0238] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. The application of α-hydroxy-β-keto acid synthase in catalyzing α-keto acid coupling reactions, characterized in that, The amino acid sequence of the α-hydroxy-β-keto acid synthase is shown in SEQ ID NO:1 to SEQ ID NO:

4.

2. The application according to claim 1, characterized in that, The gene sequences encoding α-hydroxy-β-keto acid synthase are shown in SEQ ID NO:5, SEQ ID NO:7, and SEQ ID NO:

9.

3. The application according to claim 2, characterized in that, A method for constructing a recombinant expression vector encoding the gene for α-hydroxy-β-keto acid synthase includes the following steps: The target gene was amplified from the genomic DNA of the strain. or Synthesize the target gene according to its sequence; The target gene and plasmid are recombined to obtain an expression vector.

4. The application according to claim 3, characterized in that, The primer sequences for amplifying the target gene are shown in SEQ ID NO:13~SEQ ID NO:14 or SEQ ID NO:17~SEQ ID NO:18; and / or, Before the plasmid is recombinated with the target gene, a step of linearizing the plasmid is also included. The primer sequences used for the plasmid linearization step are shown in SEQ ID NO:15~SEQ ID NO:

16.

5. The application according to any one of claims 1-4, characterized in that, The catalytic α-keto acid coupling reaction includes the step of synthesizing α-hydroxy-β-keto acid, which includes the coupling of α-keto acid under the catalysis of α-hydroxy-β-keto acid synthase.

6. The application according to claim 5, characterized in that, The α-keto acid includes at least one of aliphatic α-keto acids or aromatic α-keto acids.

7. The application according to claim 6, characterized in that, The aliphatic α-keto acid includes at least one selected from pyruvate, 3-methyl-2-oxobutyric acid, 2-oxovalerate, 3-methyl-2-oxovalerate, 4-methyl-2-oxovalerate, 2-oxohexanoic acid, 2-cyclopropyl-2-oxoacetic acid, and 2-cyclobutyl-2-oxoacetic acid; and / or, The aromatic α-keto acids include at least one of indole-3-pyruvic acid, p-hydroxyphenylpyruvic acid, phenylpyruvic acid, p-nitrophenylpyruvic acid, 4-hydroxy-3-methoxyphenylpyruvic acid, 2-oxo-4-phenylbutyric acid and 3-(naphth-1-yl)-2-oxopropionic acid.

8. The application according to claim 5, characterized in that, The α-hydroxy-β-keto acid has the structure shown in formula (Ⅰ). Equation (Ⅰ), R1 and R2 are independently selected from -R3 or -CR3. R3 is selected from at least one of substituted or unsubstituted C6-C20 aryl, C6-C20 heteroaryl, hydrogen atom, substituted or unsubstituted C1-C20 alkyl, and C3-C20 cycloalkyl; The substituents of the substituted C1-C20 alkyl groups are selected from C6-C20 aryl or C6-C20 heteroaryl groups. The substituents of the substituted C6-C20 aryl group are selected from at least one of hydroxyl, C1-C6 alkyl, methoxy or nitro groups.

9. The application according to claim 8, characterized in that, R3 is selected from at least one of phenolic, phenyl, nitrophenyl, benzyl, naphthyl, indole, 3-methoxy-4-hydroxyphenyl, substituted or unsubstituted C1-C6 alkyl and C3-C4 cycloalkyl; The substituents of the substituted C1-C6 alkyl groups are selected from phenyl groups.

10. The application according to claim 4, characterized in that, The catalytic α-keto acid coupling reaction includes a method for synthesizing a compound having a furanolactone structure, wherein the steps of synthesizing the compound having a furanolactone structure include reacting an α-keto acid and an α,β-unsaturated carboxylic acid as substrates in the presence of α-hydroxy-β-keto acid synthase. or, The reaction takes α-amino acids, α-keto acids and α,β-unsaturated carboxylic acids as substrates in the presence of α-hydroxy-β-keto acid synthase.

11. The application according to claim 10, characterized in that, The compound having a furanolactone structure has the structural formula shown in formula (Ⅲ). Formula (Ⅲ), R4 is selected from substituted or unsubstituted C6-C30 aryl groups, C6-C30 heteroaryl groups, hydrogen atoms, and substituted or unsubstituted C1-C30 alkyl groups. The substituents of the substituted C6-C30 aryl group are selected from at least one of hydroxyl and methoxy groups, and the substituents of the substituted C1-C30 alkyl group are selected from C6-C30 aryl or C6-C30 heteroaryl groups. R5 is selected from -R7 or -CR7, and R7 is selected from at least one of substituted or unsubstituted C6-C30 aryl, C6-C30 heteroaryl, hydrogen atom, substituted or unsubstituted C1-C30 alkyl, and C3-C30 cycloalkyl. The substituents of the substituted C6-C30 aryl group are selected from at least one of hydroxyl and methoxy groups, and the substituents of the substituted C1-C30 alkyl group are selected from C6-C30 aryl or C6-C30 heteroaryl groups. R6 is selected from substituted or unsubstituted C6-C30 aryl groups, or C6-C30 heteroaryl groups. The substituents of the substituted C6-C30 aryl group are selected from at least one of hydroxyl, halogen atom, cyano, or C1-C6 alkyl.

12. The application according to claim 11, characterized in that, R4 is selected from at least one of substituted or unsubstituted C6-C15 aryl, C6-C15 heteroaryl, hydrogen atom, and substituted or unsubstituted C1-C15 alkyl. The substituents of the substituted C6-C15 aryl group are selected from at least one of hydroxyl and methoxy groups, and the substituents of the substituted C1-C15 alkyl group are selected from C6-C15 aryl and C6-C15 heteroaryl groups. R5 is selected from -R7 or -CR7, and R7 is selected from at least one of substituted or unsubstituted C6-C15 aryl, C6-C30 heteroaryl, hydrogen atom, substituted or unsubstituted C1-C15 alkyl, and C3-C15 cycloalkyl. The substituents of the substituted C6-C15 aryl group are selected from at least one of hydroxyl and methoxy groups, and the substituents of the substituted C1-C15 alkyl group are selected from C6-C15 aryl or C6-C15 heteroaryl groups. R6 is selected from substituted or unsubstituted C6-C15 aryl groups, or C6-C15 heteroaryl groups. The substituents of the substituted C6-C15 aryl group are selected from at least one of hydroxyl, halogen atom, cyano, or C1-C6 alkyl.

13. The application according to claim 12, characterized in that, R4 is selected from at least one of phenolic, phenyl, o-phenolic, 3-methoxy-4-hydroxyphenyl, 3,5-dimethoxy-4-hydroxyphenyl, and C1-C6 alkyl groups; and / or, R5 is selected from at least one of phenolic, phenyl, nitrophenyl, benzyl, naphthyl, indole, 3-methoxy-4-hydroxyphenyl, p-cyanophenyl, benzothiophene, substituted or substituted C1-C6 alkyl and C3-C4 cycloalkyl groups. The substituents of the substituted C1-C6 alkyl groups are selected from phenyl groups; and / or, R7 is selected from at least one of phenol, phenyl, nitrophenyl, benzyl, naphthyl, indole, 3-methoxy-4-hydroxyphenyl, p-methylphenyl, p-cyanophenyl, benzothiophene, 3,5-dichloro-4-hydroxyphenyl, 3-chloro-4-hydroxyphenyl, and p-fluorophenyl.

14. The application according to claim 13, characterized in that, R4 is selected from at least one of isopropyl, n-butyl, or ethyl; and / or, R5 is selected from at least one of n-propyl, isopropyl, n-butyl, methyl, or ethyl.

15. The application according to any one of claims 10-14, characterized in that, The compounds having a furanolactone structure have structures shown in Formulas 2-1 to 2-36. 。 16. The application according to claim 15, characterized in that, When α-keto acids and α,β-unsaturated carboxylic acids are used as substrates and / or α-amino acids, α-keto acids and α,β-unsaturated carboxylic acids are used as substrates, the reaction system also includes coenzyme A, thiamine pyrophosphate, adenine nucleoside triphosphate, catalytic enzyme, magnesium salt, and buffer solution.

17. The application according to claim 16, characterized in that, The final concentration of α-hydroxy-β-keto acid synthase in the reaction system is 0.01-0.025 mM; and / or, The catalytic enzyme includes at least one of 4-coumarate-coenzyme A ligase, furanolactone synthase, and transaminase; and / or, The magnesium salt includes at least one of magnesium chloride, magnesium sulfate, or magnesium nitrate; and / or, The buffer solution includes at least one of sodium chloride buffer, 4-hydroxyethylpiperazine ethanesulfonic acid buffer, tris(hydroxymethyl)aminomethane hydrochloride buffer, or phosphate buffer.

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