Method for synthesizing heterocyclic acid through whole-cell catalysis

Abstract

The invention provides a method for expressing succinic acid-semialdehyde dehydrogenase from klebsiella pneumoniae (Klebsiella pneumoniae) in escherichia coli (Escherichia coli) BL21 (DE3), which is characterized in that the succinic acid-semialdehyde dehydrogenase can be used for expressing the succinic acid-semialdehyde dehydrogenase from the klebsiella pneumoniae (Klebsiella pneumoniae). And in a buffer solution system, carrying out whole-cell catalysis on the heterocyclic aldehyde to synthesize the heterocyclic acid. On the basis, 5-methyl-2-formyl pyrazine is used as a substrate, so that the production of the 5-methyl-2-pyrazine carboxylic acid is realized. The method is simple to operate and mild in reaction condition, does not need to additionally provide cofactors, and is beneficial to large-scale industrial production.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a method for catalytically synthesizing 5-methyl-2-pyrazinecarboxylic acid by expressing proteins using Escherichia coli. Technical Background

[0002] Heterocyclic acid compounds are important intermediates for many drugs. For example, 5-methyl-2-pyrazinecarboxylic acid is mainly used in the synthesis of the second-generation hypoglycemic drug glipizide and the lipid-lowering drug acipimox. In addition, 5-methyl-2-pyrazinecarboxylic acid is also an effective catalyst for synthesizing metal complexes.

[0003] Currently, 5-methyl-2-pyrazinecarboxylic acid produced industrially is mainly synthesized by intermolecular cyclization, or can be synthesized by multi-step oxidation with strong oxidants or electrochemical oxidation using 2,5-dimethylpyrazine as a substrate. CN1155581C discloses a preparation method of 5-methyl-2-pyrazinecarboxylic acid, in which glyoxal and o-phenylenediamine are catalytically cyclized at high temperature with sodium pyrosulfite, and the product is obtained through oxidation, sulfuric acid acidification, decarboxylation, extraction and separation with methyl ethyl ketone, and crystallization. CN116217499A discloses a continuous preparation method of 5-methylpyrazine-2-carboxylic acid, in which 2,5-dimethylpyrazine is catalyzed by potassium permanganate solution, and 5-methylpyrazine-2-carboxylic acid is obtained through acid neutralization, extraction with methyl ethyl ketone, and vacuum distillation. CN109369544A discloses a method for catalytic oxidation to prepare 5-methylpyrazine-2-carboxylic acid, using Mn-W-Co / diatomite as a catalyst, and synthesizing 5-methylpyrazine-2-carboxylic acid by oxidizing 2,5-dimethylpyrazine with oxygen. The reaction process of traditional chemical synthesis methods is complex, the operation is cumbersome and time-consuming, which will increase the reaction cost. Multiple steps involve strong oxidants and organic solvents, making the operation safety and environmental protection not guaranteed. The biosynthetic method has the advantages of catalytic selectivity and less environmental pollution, and has good development prospects. CN107312806B discloses an enzymatic method for producing 5-methylpyrazine-2-carboxylic acid, providing an aldehyde dehydrogenase from Pseudomonas putida, and connecting a tag to its N-terminus or / and C-terminus to obtain a fusion protein, using 5-methyl-2-formylpyrazine as a substrate, and obtaining 17.02 mM of 5-methylpyrazine-2-carboxylic acid, but it requires an additional cofactor NAD+.

[0004] In recent years, with the continuous development of downstream products of heterocyclic acids, their demand has been increasing year by year. It is particularly important to develop a simpler, high-yield, sustainable and suitable for large-scale industrial production method for preparing heterocyclic acids. Summary of the Invention

[0005] The object of the present invention is to provide a method for catalytically producing heterocyclic acids using whole microbial cells.

[0006] A method for producing heterocyclic acids by whole-cell catalysis according to the present invention comprises the following steps:

[0007]

[0008] Using the heterocyclic aldehyde shown in Formula I as a reaction substrate, in a buffer reaction system, in the presence of Escherichia coli cells expressing aldehyde dehydrogenase, the heterocyclic acid product shown in Formula II is obtained.

[0009] Specifically, the aldehyde dehydrogenase can be selected from the following different species sources:

[0010] (1) Succinic semialdehyde dehydrogenase KpSSADH from Klebsiella pneumoniae;

[0011] (2) Aldehyde dehydrogenase SlaADH from Streptomyces laurentii;

[0012] (3) Benzaldehyde dehydrogenase AviBADH from Acinetobacter vivianii;

[0013] (4) Benzaldehyde dehydrogenase EhaBADH from Egicoccus halophilus;

[0014] (5) Benzaldehyde dehydrogenase RopBADH from Rhodococcus opacus.

[0015] Preferably, the aldehyde dehydrogenase can be selected from any one of the following:

[0016] (1) Succinic semialdehyde dehydrogenase KpSSADH from Klebsiella pneumoniae, whose amino acid sequence is shown in SEQ ID NO.1;

[0017] (2) Aldehyde dehydrogenase SlaADH from Streptomyces laurentii, whose Genbank number is BAU84253.1;

[0018] (3) Benzaldehyde dehydrogenase AviBADH from Acinetobacter vivianii, whose Genbank number is GGI58546.1;

[0019] (4) Benzaldehyde dehydrogenase EhaBADH from Egicoccus halophilus, whose Genbank number is GGI08474.1;

[0020] (5) Benzaldehyde dehydrogenase RopBADH derived from Rhodococcus opacus, with Genbank accession number CAG7585578.1).

[0021] More preferably, the nucleotide sequence of the succinic semialdehyde dehydrogenase is as shown in SEQ ID NO.2.

[0022] In a specific embodiment, the substrate concentration is 30 - 125 mM.

[0023] Specifically, the temperature of the reaction is 10 - 60 °C; the pH of the reaction system is 4.0 - 10.0; the reaction time is 6 - 24 h.

[0024] In a specific embodiment, the buffer solution is any one of phosphate buffered saline, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, bis(2-hydroxyethyl)amino(trimethyl)methane, Tris-HCl buffer, citrate-sodium citrate buffer, glycine-sodium hydroxide buffer.

[0025] Preferably, Escherichia coli cells expressing aldehyde dehydrogenase are used as whole-cell catalysts to perform whole-cell catalysis of heterocyclic aldehyde substrates in a buffer solution to obtain heterocyclic acid products.

[0026] The Escherichia coli cells expressing aldehyde dehydrogenase are obtained by constructing the gene of the aldehyde dehydrogenase on a gene expression vector such as pET22b and then introducing it into Escherichia coli, more specifically Escherichia coli E. coli BL21(DE3).

[0027] More specifically, 5-methyl-2-pyrazinecarboxylic acid, 6-methyl-2-pyrazinecarboxylic acid, 5-isopropylpyrazine-2-carboxylic acid, 5-ethylpyrazine-2-carboxylic acid, 5-chloropyrazine-2-carboxylic acid, pyrrole-2-carboxylic acid, pyrrole-3-carboxylic acid, 2-furoic acid, 3-furoic acid and 5H-pyrrolo[2,3-b]pyrazine-7-carboxylic acid are synthesized using 5-methyl-2-pyrazinecarboxaldehyde, 6-methyl-2-pyrazinecarboxaldehyde, 5-isopropylpyrazine-2-carboxaldehyde, 5-ethylpyrazine-2-carboxaldehyde, 5-chloropyrazine-2-carboxaldehyde, pyrrole-2-carboxaldehyde, pyrrole-3-carboxaldehyde, 2-furaldehyde, 3-furaldehyde and 5H-pyrrolo[2,3-b]pyrazine-7-carboxaldehyde as substrates respectively.

[0028] The beneficial effects of the present invention are as follows: 1. By expressing a specific succinic semialdehyde dehydrogenase using recombinant Escherichia coli, heterocyclic aldehydes can be catalytically synthesized into heterocyclic acids. 2. Applying this whole-cell enzyme to the synthesis of 5-methyl-2-pyrazinecarboxylic acid from 5-methyl-2-formylpyrazine, the culture conditions and the optimal conditions for the catalytic reaction are optimized. 3. Expand the substrate spectrum of KpSSADH. The recombinant construction method of the recombinant Escherichia coli of the present invention is simple. The method of whole-cell catalysis is green and environmentally friendly, easy to use, and easy to scale up, with good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 . It is the plasmid map of the expression vector pET22b-KpSSADH.

[0030] Figure 2 . It is the liquid chromatography diagram of the reaction process for synthesizing 5-methyl-2-pyrazinecarboxylic acid.

[0031] Figure 3 . It is the diagram of the influence of different pH values on the substrate conversion rate during catalysis.

[0032] Figure 4 . It is the diagram of the influence of different buffers on the substrate conversion rate.

[0033] Figure 5 . It is the diagram of the influence of different temperatures on the substrate conversion rate during catalysis. DETAILED DESCRIPTION OF THE INVENTION

[0034] The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all obtained from regular commercial suppliers unless otherwise specified. In the following quantitative tests, more than three repeated experiments are set, and the results are averaged.

[0035] The protein sequence of succinic semialdehyde dehydrogenase KpSSADH (Genbank No.: SVJ49636.1) derived from Klebsiella pneumoniae is shown in SEQ ID NO.1. The gabD sequence of the succinic semialdehyde dehydrogenase KpSSADH gene derived from Klebsiella pneumoniae is shown in SEQ ID NO.2. The DNA described in SEQ ID NO.2 encodes the amino acid sequence of SEQ ID NO.1.

[0036] Example 1: Construction of the expression vector and recombinant Escherichia coli

[0037] According to the DNA sequence published by NCBI (Genbank No.: SVJ49636.1), the codon-optimized gabD gene (as shown in SEQ ID NO.2) was artificially synthesized by Suzhou Genewiz Biotechnology Co., Ltd. The fragment between the NcoI and HindIII restriction sites of the pET22b(+) plasmid was replaced with the double-stranded DNA molecule shown in SEQ ID NO.2 to obtain the recombinant expression vector pET22b-KpSSADH. The plasmid map is shown in Figure 1 . Similarly, the expression vectors pET22b-SlaADH, pET22b-AviBADH, pET22b-EhaBADH, and pET22b-RopBADH were constructed. Expression was induced by isopropyl-β-D-thiogalactopyranoside (IPTG). E. coli BL21(DE3) used in the present invention was preserved in this laboratory. The recombinant expression vector was transformed into E. coli BL21(DE3) to obtain recombinant bacteria.

[0038] The recombinant Escherichia coli was streaked on a solid Luria-Bertani (LB) plate containing 50 μg / mL carbenicillin and cultured overnight at 37°C. A single colony was picked and inoculated into an LB liquid medium containing 50 μg / mL carbenicillin and cultured at 37°C and 220 rpm for 16 h to obtain the primary seed culture. The primary seed culture was inoculated into a Terrific Broth (TB) liquid medium containing 50 μg / mL carbenicillin at 1% inoculum and cultured at 37°C and 220 rpm until the OD 600 of the bacterial solution reached 0.65. Then, an inducer IPTG with a final concentration of 0.1 mM was added to the culture system, and induction was carried out at 16°C and 220 rpm for 14 h. The culture system was centrifuged at 6000 rpm for 10 min to collect the cell pellet. The cells were washed twice with PBS buffer at pH 7.5. The weight was measured and resuspended to obtain a whole-cell catalyst at 500 g / L.

[0039] In a 1 mL reaction system, 796.34 μL of PBS buffer (pH = 8, 100 mmol / L) was added, followed by 200 μL of whole cells (final concentration 100 g / L) and 3.66 μL of 5-methyl-2-formylpyrazine (final concentration 30 mM). The reaction was carried out at 20°C on a shaker at 220 rpm for 10 h. 200 μL of the reaction solution was taken, centrifuged at 12000 rpm for 1 min, and the supernatant was taken for HPLC detection of the product concentration.

[0040] The detection method uses HPLC chromatography for detection. Instrument model: SHIMADZU LC-2030Plus; chromatographic column: 5μm C18-AQ, 4.6×250mm (HSS); column temperature: 30°C; ultraviolet detection wavelength: 270nm; injection volume: 10μL; flow rate: 1.0mL / min; mobile phase ratio: 0.5% (volume percentage) trifluoroacetic acid aqueous solution: acetonitrile = 96:4. The standard substance used is 5-methyl-2-pyrazinecarboxylic acid (Shanghai Haohong Biopharmaceutical Technology Co., Ltd., CAS: 5521-55-1), and the peak emergence time is 17.3±0.2min; within ±0.2min of the peak emergence position under the same conditions, they are all identified as the same substance. After screening five enzymes, the whole-cell catalytic effect of KpSSADH is the best.

[0041] Example 2: Optimal induction temperature for enzyme expression

[0042] Pick a single colony of E. coli pET22b-KpSSADH revived by streaking in Example 1 and inoculate it into LB liquid medium containing 50μg / mL carbenicillin, and culture the primary seed liquid at 37°C and 220rpm for 16h. Inoculate the primary seed liquid into Terrific Broth (TB) liquid medium containing 50μg / mL carbenicillin at 1%, and culture it at 37°C and 220rpm until the OD 600 of the bacterial solution = 0.65. Add IPTG with a final concentration of 0.1mM to the culture system, and induce at 16°C, 20°C, 25°C, 30°C, and 220rpm for 14h respectively. Centrifuge the culture system at 6000rpm for 10min to collect the bacterial cell precipitate. Wash the bacterial cells twice with PBS buffer at pH 7.5. Weigh and resuspend to a whole-cell catalyst of 500g / L.

[0043] In a 1mL reaction system, add 796.34μL of PBS buffer (pH = 8, 100mmol / L), 200μL of whole cells (final concentration 100g / L), and 3.66μL of 5-methyl-2-formylpyrazine (final concentration 30mM), and react at 20°C in a shaker at 220rpm for 10h. Take 200μL of the reaction solution, centrifuge at 12000rpm for 1min, and take the supernatant for HPLC detection of the product concentration. The detection method is the same as in Example 1. When the induction temperature is 20°C, the product production is the highest.

[0044] Example 3: Optimal inducer addition amount for enzyme expression

[0045] Pick the E. coli pET22b-KpSSADH monoclonal that was revived by drawing a line in Example 1 and inoculate it into LB liquid medium containing 50 μg / mL carbenicillin. Culture the primary seed solution at 37 °C and 220 rpm for 16 h. Inoculate the primary seed solution into TB liquid medium containing 50 μg / mL carbenicillin at 1%, and culture it at 37 °C and 220 rpm until the OD 600 of the bacterial solution 600 = 0.65. Add IPTG with final concentrations of 0.1, 0.25, 0.5, 0.75, and 1.0 mM to the culture system respectively, and induce at 20 and 220 rpm for 14 h. Centrifuge the culture system at 6000 rpm for 10 min to collect the bacterial cell precipitate. Wash the bacterial cells twice with PBS buffer at pH 7.5. Weigh and resuspend to obtain a whole-cell catalyst at 500 g / L.

[0046] In a 1 mL reaction system, add 796.34 μL of PBS buffer (pH = 7.5, 100 mmol / L), 200 μL of whole cells (final concentration 100 g / L), and 3.66 μL of 5-methyl-2-formylpyrazine (final concentration 30 mM). React at 220 rpm in a shaker at 20 °C for 10 h. Take 200 μL of the reaction solution, centrifuge at 12000 rpm for 1 min, and take the supernatant for HPLC detection of the product concentration. The detection method is the same as that in Example 1. When the addition amount of the inducer is 0.25 mM, the product production amount is the highest.

[0047] Example 4: Optimal pH for enzyme expression

[0048] Using the whole cells cultured with an IPTG final concentration of 0.25 mM in Example 3, in a 1 mL reaction system, add 796.34 μL of different pH buffers (Sodium citrate buffers with pH 4.0, pH 5.0, pH 6.0, PBS buffers with pH 6.0, pH 7.0, pH 8.0, Tris-HCl with pH 8.0, pH 9.0, and Glycine-NaOH buffers with pH 9.0, pH 10.0), 200 μL of whole cells (final concentration 100 g / L), and 3.66 μL of 5-methyl-2-formylpyrazine (final concentration 30 mM). React at 220 rpm in a shaker at 20 °C for 5 h. Take 200 μL of the reaction solution, centrifuge at 12000 rpm for 1 min, and take the supernatant for HPLC detection of the product concentration. The detection method is the same as that in Example 1. The results are as Figure 3 shown. When the reaction pH is 8.0, the product production amount is the highest.

[0049] Example 5: Optimal buffer for whole-cell catalyzed synthesis of 5-methyl-2-pyrazinecarboxylic acid

[0050] Using the whole cells cultured with an IPTG final concentration of 0.25 mM in Example 3, in a 1 mL reaction system, 796.34 μL of PBS buffer (pH = 8, 100 mmol / L), HEPES buffer (pH = 8, 100 mmol / L), BIS-TRIS buffer (pH = 8, 100 mmol / L), add 200 μL of whole cells (final concentration 100 g / L), 3.66 μL of 5-methyl-2-formylpyrazine (final concentration 30 mM), and react at 220 rpm in a shaker at 20 °C for 5 h. Take 200 μL of the reaction solution, centrifuge at 12000 rpm for 1 min, and take the supernatant for HPLC detection of the product concentration. The detection method is the same as that in Example 1. The results are as Figure 4 shown. When the buffer is PBS, the highest amount of product is generated.

[0051] Example 6: Optimal temperature for the whole-cell catalyzed synthesis of 5-methyl-2-pyrazinecarboxylic acid

[0052] Using the whole cells cultured with an IPTG final concentration of 0.25 mM in Example 3, in a 1 mL reaction system, 796.34 μL of PBS buffer (pH = 8, 100 mmol / L), add 200 μL of whole cells (final concentration 100 g / L), 3.66 μL of 5-methyl-2-formylpyrazine (final concentration 30 mM), and react at 800 rpm in a constant temperature metal bath at 10, 20, 30, 40, 50, and 60 °C for 5 h. Take 200 μL of the reaction solution, centrifuge at 12000 rpm for 1 min, and take the supernatant for HPLC detection of the product concentration. The detection method is the same as that in Example 1. The results are as Figure 5 shown. When the reaction temperature is 30 °C, the highest amount of product is generated.

[0053] Example 7: Whole-cell catalyzed synthesis of 5-methyl-2-pyrazinecarboxylic acid

[0054] Using the whole cells cultured with an IPTG final concentration of 0.25 mM in Example 3, in a 1 mL PBS buffer (pH = 8, 100 mmol / L) reaction system, the final concentration of the whole-cell catalyst is 500 g / L, and the final concentration of 5-methyl-2-formylpyrazine is 125 mM. React at 220 rpm in a shaker at 30 °C for 24 h. End the reaction, take 50 μL of the reaction solution, dilute it 4 times with 150 μL of distilled water, centrifuge at 12000 rpm for 1 min, and take the supernatant for HPLC detection of the product concentration. The detection method is the same as that in Example 1. The yield of 5-methyl-2-pyrazinecarboxylic acid is 4.64 g / L.

[0055] Example 8: Preparation of the compound 6-methyl-2-carboxypyrazine as shown in the following formula

[0056]

[0057] Using the whole cells cultured with the final IPTG concentration of 0.25 mM in Example 3, in a 1 mL reaction system, 796.34 μL of PBS buffer (pH = 8, 100 mmol / L), add 200 μL of whole cells (final concentration 100 g / L), 3.66 μL of 6-methyl-2-formylpyrazine (final concentration 30 mM). After the above reaction system reacts on a shaker at 30 °C for 15 hours, the reaction is terminated, and analyzed by liquid chromatography, with a yield of 71%.

[0058] Example 9: Preparation of the compound 5-isopropylpyrazine-2-carboxylic acid of the following formula

[0059]

[0060] Using the whole cells cultured with the final IPTG concentration of 0.25 mM in Example 3, in a 1 mL reaction system, 795.02 μL of PBS buffer (pH = 8, 100 mmol / L), add 200 μL of whole cells (final concentration 100 g / L), 4.98 μL of 5-isopropylpyrazine-2-carboxaldehyde (final concentration 30 mM). After the above reaction system reacts on a shaker at 30 °C for 15 hours, the reaction is terminated, and analyzed by liquid chromatography, with a yield of 68%.

[0061] Example 10: Preparation of the compound 5-ethylpyrazine-2-carboxylic acid of the following formula

[0062]

[0063] Using the whole cells cultured with the final IPTG concentration of 0.25 mM in Example 3, in a 1 mL reaction system, 795.92 μL of PBS buffer (pH = 8, 100 mmol / L), add 200 μL of whole cells (final concentration 100 g / L), 4.08 μL of 5-ethylpyrazine-2-carboxaldehyde (final concentration 30 mM). After the above reaction system reacts on a shaker at 30 °C for 15 hours, the reaction is terminated, and analyzed by liquid chromatography, with a yield of 62%.

[0064] Example 11: Preparation of the compound 5-chloropyrazine-2-carboxylic acid of the following formula

[0065]

[0066] Using the whole cells cultured with an IPTG final concentration of 0.25 mM in Example 3, in a 1 mL reaction system, 795.72 μL of PBS buffer (pH = 8, 100 mmol / L), add 200 μL of whole cells (final concentration 100 g / L), 4.28 μL of 5-chloropyrazine-2-carbaldehyde (final concentration 30 mM). After the above reaction system reacts on a shaker at 30 °C for 15 hours, the reaction is terminated, and liquid chromatography is used for detection and analysis, with a yield of 69%.

[0067] Example 12: Preparation of the compound pyrrole-2-carboxylic acid of the following formula

[0068]

[0069] Using the whole cells cultured with an IPTG final concentration of 0.25 mM in Example 3, in a 1 mL reaction system, 796.67 μL of PBS buffer (pH = 8, 100 mmol / L), add 200 μL of whole cells (final concentration 100 g / L), 3.33 μL of pyrrole-2-carbaldehyde (final concentration 30 mM). After the above reaction system reacts on a shaker at 30 °C for 15 hours, the reaction is terminated, and liquid chromatography is used for detection and analysis, with a yield of 89%.

[0070] Example 13: Preparation of the compound pyrrole-3-carboxylic acid of the following formula

[0071]

[0072] Using the whole cells cultured with an IPTG final concentration of 0.25 mM in Example 3, in a 1 mL reaction system, 796.67 μL of PBS buffer (pH = 8, 100 mmol / L), add 200 μL of whole cells (final concentration 100 g / L), 3.33 μL of pyrrole-3-carbaldehyde (final concentration 30 mM). After the above reaction system reacts on a shaker at 30 °C for 15 hours, the reaction is terminated, and liquid chromatography is used for detection and analysis, with a yield of 82%.

[0073] Example 14: Preparation of the compound 3-furoic acid of the following formula

[0074]

[0075] Using the whole cells cultured with an IPTG final concentration of 0.25 mM in Example 3, in a 1 mL reaction system, 796.64 μL of PBS buffer (pH = 8, 100 mmol / L), add 200 μL of whole cells (final concentration 100 g / L), 3.36 μL of furan-3-carbaldehyde (final concentration 30 mM). After the above reaction system reacts on a shaker at 30 °C for 15 hours, the reaction is terminated, and liquid chromatography is used for detection and analysis, with a yield of 51%.

[0076] Example 15: Preparation of the compound 2-furoic acid of the following formula

[0077]

[0078] Using the whole cells cultured with an IPTG final concentration of 0.25 mM in Example 3, in a 1 mL reaction system, 796.64 μL of PBS buffer (pH = 8, 100 mmol / L), add 200 μL of whole cells (final concentration 100 g / L), 3.36 μL of furan-2-carbaldehyde (final concentration 30 mM). After the above reaction system was reacted on a shaker at 30 °C for 15 hours, the reaction was terminated, and analyzed by liquid chromatography, with a yield of 62%.

[0079] Example 16: Preparation of the compound 5H-pyrrolo[2,3-b]pyrazine-7-carboxylic acid of the following formula

[0080]

[0081] Using the whole cells cultured with an IPTG final concentration of 0.25 mM in Example 3, in a 1 mL reaction system, 795.59 μL of PBS buffer (pH = 8, 100 mmol / L), add 200 μL of whole cells (final concentration 100 g / L), 4.41 μL of 5H-pyrrolo[2,3-b]pyrazine-7-carbaldehyde (final concentration 30 mM). After the above reaction system was reacted on a shaker at 30 °C for 15 hours, the reaction was terminated, and analyzed by liquid chromatography, with a yield of 51%.

Claims

1. A method for producing heterocyclic acids by whole-cell catalysis, comprising the following steps: ； Using the heterocyclic aldehyde shown in Formula I as a reaction substrate, in a buffer reaction system, in the presence of Escherichia coli cells expressing aldehyde dehydrogenase, obtaining the heterocyclic acid product shown in Formula II.

2. The method according to claim 1, wherein The aldehyde dehydrogenase can be selected from the following different species sources: (1) Derived from Klebsiella pneumoniae Klebsiella pneumoniae succinic semialdehyde dehydrogenase Kp SSADH; (2) derived from Streptomyces laurentii Streptomyces laurentii aldehyde dehydrogenase Sla ADH; (3) derived from Acinetobacter lwoffii Acinetobacter vivianii benzaldehyde dehydrogenase Avi BADH; (4) Benzaldehyde dehydrogenase derived from Halococcus Egicoccus halophilus BADH; Eha BADH (5) derived from Rhodococcus opacus Rhodococcus opacus benzaldehyde dehydrogenase Rop BADH 3. The method according to claim 2, wherein The aldehyde dehydrogenase can be selected from any one of the following: (1) Succinic semialdehyde dehydrogenase derived from Klebsiella pneumoniae Klebsiella pneumoniae SSADH, whose amino acid sequence is shown in SEQ ID NO.1; Kp ​ (2) Derived from Streptomyces laurentii Streptomyces laurentii aldehyde dehydrogenase Sla ADH, with Genbank accession number BAU84253.1; (3) derived from Acinetobacter calcoaceticus Acinetobacter vivianii benzaldehyde dehydrogenase Avi BADH, whose Genbank number is GGI58546.1; (4) derived from Halococcus Egicoccus halophilus benzaldehyde dehydrogenase Eha BADH, whose Genbank number is GGI08474.1; (5) derived from Rhodococcus opacus Rhodococcus opacus benzaldehyde dehydrogenase Rop BADH, whose Genbank number is CAG7585578.1).

4. The method according to claim 3, characterized in that The nucleotide sequence of the succinic semialdehyde dehydrogenase is as shown in SEQ ID NO.

2.

5. The method according to any one of claims 1 to 4, characterized in that The substrate concentration is 30 - 125 mM.

6. The method according to claim 5, characterized in that The temperature of the reaction is 10 - 60 °C.

7. The method according to claim 5, characterized in that, The pH of the reaction system is 4.0 - 10.0; the reaction time is 6 - 24 h.

8. The method according to claim 5, wherein The buffer is any one of phosphate buffered saline, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, bis(2-hydroxyethyl)amino(trimethyl)methane, Tris-HCl buffer, citric acid-sodium citrate buffer, glycine-sodium hydroxide buffer.

9. The method according to claim 5, characterized in that, Using Escherichia coli cells expressing aldehyde dehydrogenase as a whole-cell catalyst, performing whole-cell catalysis on the heterocyclic aldehyde substrate in a buffer to obtain the heterocyclic acid product.

10. The method according to claim 5, wherein, Using 5-methyl-2-pyrazinecarboxaldehyde, 6-methyl-2-pyrazinecarboxaldehyde, 5-isopropylpyrazine-2-carbaldehyde, 5-ethylpyrazine-2-carbaldehyde, 5-chloropyrazine-2-carbaldehyde, pyrrole-2-carbaldehyde, pyrrole-3-carbaldehyde, 2-furaldehyde, 3-furaldehyde and 5H-pyrrolo[2,3-b]pyrazine-7-carbaldehyde as substrates to synthesize 5-methyl-2-pyrazinecarboxylic acid, 6-methyl-2-pyrazinecarboxylic acid, 5-isopropylpyrazine-2-carboxylic acid, 5-ethylpyrazine-2-carboxylic acid, 5-chloropyrazine-2-carboxylic acid, pyrrole-2-carboxylic acid, pyrrole-3-carboxylic acid, 2-furoic acid, 3-furoic acid and 5H-pyrrolo[2,3-b]pyrazine-7-carboxylic acid.

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