Method for catalytically synthesizing malonic acid by using whole cells

Abstract

The invention discloses a method for catalytic synthesis of malonic acid by using whole cells, and belongs to the field of bioengineering. According to the invention, escherichia coli BL21 (DE3) is taken as a host, an aspartic acid ammonia lyase gene aspA and an aspartic acid-alpha-decarboxylase gene panD, a succinate semialdehyde dehydrogenase gene yneI and a beta-alanine pyruvate transaminase gene, a beta-alanine pyruvate transaminase gene bauA and a malonyl-coenzyme A reductase gene mcr-C are expressed in modules, and recombinant escherichia coli is constructed. The malonic acid is produced by adopting a whole-cell catalysis method, so that the production period is shortened, the production efficiency of the malonic acid is improved, and the yield of the malonic acid can reach 51.0 g / L after the malonic acid is catalyzed for 24 hours.

Description

Technical Field

[0001] The present invention relates to a method for synthesizing malonic acid by using whole-cell catalysis, belonging to the field of bioengineering. Background Art

[0002] Malonic acid, also known as carrot acid, malic acid or beet acid, is one of the 30 bio-based platform compounds identified by the US Department of Energy. Its molecular structure has both active methylene and dicarboxyl functional groups, and can participate in complex organic synthesis through various types of reactions such as condensation and substitution. It is an important intermediate in the fields of medicine, pesticides and functional materials. Global market research shows that the market size of malonic acid reached 330 million yuan in 2022 and is expected to exceed 390 million yuan in 2029. Its demand growth is closely related to the development of emerging fields such as new energy materials (such as lithium battery additives) and biodegradable plastics (poly(malonic acid esters)).

[0003] Since the synthesis efficiency of the biological synthesis method of malonic acid is not sufficient to compete with the traditional chemical synthesis method, at present, the large-scale production of malonic acid is achieved by chemical methods. However, the chemical synthesis method faces problems such as raw materials, energy consumption, and environmental pollution, which are not conducive to sustainable development. Therefore, green and efficient production through industrial biotechnology has greater competitive advantages. Use renewable raw materials for biocatalysis / whole-cell methods to prepare malonic acid for the production and preparation of downstream chemicals and products. The natural biosynthetic pathway of malonic acid is still unknown. The known methods are through the malonyl-CoA pathway and the oxaloacetic acid pathway. However, the microbial fermentation method has problems such as long production cycle, low efficiency, complex culture medium components and by-product interference. Therefore, it is necessary to develop new methods to improve the production efficiency of malonic acid. Summary of the Invention

[0004] In view of the above problems, the present invention provides a recombinant Escherichia coli that can synthesize malonic acid through the fumarate-malonate semialdehyde pathway under the condition of adding exogenous fumaric acid; the recombinant Escherichia coli overexpresses aspartase (aspA), succinic semialdehyde dehydrogenase (yneI) and aspartate-α-decarboxylase (panD) from Escherichia coli in modules, two β-alanine pyruvate transaminases (pa0132, bauA) from Pseudomonas aeruginosa, and malonyl-CoA reductase (mcr-C) from Aspergillus oryzae.

[0005] In one embodiment, the modular expression is to express the genes aspA and panD on a common plasmid, express the genes yneI and pa0132 on a common plasmid, and express the genes bauA and mcr-C on a common plasmid.

[0006] In one embodiment, the modular expression is to express genes aspA and panD using pTrc99a as the expression vector, to express genes yne1 and pa0132 using pRSFDuet-1 as the expression vector, and to express genes bauA and mcr-C using pACYCDuet-1 as the expression vector.

[0007] In one embodiment, the nucleotide sequence of gene aspA is as shown in SEQ ID NO.1; the nucleotide sequence of gene panD is as shown in SEQ ID NO.2; the nucleotide sequence of gene yneI is as shown in SEQ ID NO.3; the nucleotide sequence of gene pa0132 is as shown in SEQ ID NO.4; the nucleotide sequence of gene bauA is as shown in SEQ ID NO.5; the nucleotide sequence of gene mcr-C is as shown in SEQ ID NO.6.

[0008] The present invention also provides a method for constructing the recombinant Escherichia coli, comprising the following steps:

[0009] (1) Using plasmid pTrc99a as the backbone vector, ligating gene fragments aspA and panD to obtain recombinant plasmid pTrc99a-aspA-panD;

[0010] (2) Using plasmid pRSFDuet-1 as the backbone vector, ligating gene fragments yneI and pa0132 to obtain recombinant plasmid pRSF-pa0132-yneI;

[0011] (3) Using plasmid pACYCDuet-1 as the backbone vector, ligating gene fragments bauA and mac-C to obtain recombinant plasmid pACYC-bauA-mcr-C;

[0012] (4) Co-transforming plasmids pTrc99a-aspA-panD, pRSF-yneI-pa0132 and pACYC-bauA-mcr-C into Escherichia coli BL21(DE3) to obtain recombinant Escherichia coli.

[0013] In one embodiment of the present invention, in step (1), primers are used to amplify the aspA and panD gene fragments, and primers are used to linearize the pTrc99A vector. First, the aspA fragment is ligated to the linearized pTrc99A vector using a homologous recombinase to obtain recombinant plasmid pTrc99a-aspA. Then, primers are used to linearize the pTrc99a-aspA plasmid, and the panD fragment is ligated to the linearized pTrc99a-aspA plasmid using a homologous recombinase to obtain recombinant plasmid pTrc99a-aspA-panD.

[0014] In an embodiment of the present invention, in step (2), primers are used to amplify the pa0132 and yneI gene fragments, and primers are used to linearize the pRSFDuet-1 vector. First, the homologous recombinase is used to ligate the pa0132 fragment with the linearized pRSFDuet-1 vector to obtain the recombinant plasmid pRSF-pa0132. Then, primers are used to linearize the pRSF-pa0132 plasmid, and the homologous recombinase is used to ligate the yneI fragment with the linearized pRSF-pa0132 plasmid to obtain the recombinant plasmid pRSF-pa0132-yneI.

[0015] In an embodiment of the present invention, in step (3), primers are used to amplify the bauA and mcr-C gene fragments, and primers are used to linearize the pACYCDuet-1 vector. First, the homologous recombinase is used to ligate the bauA fragment with the linearized pACYCDuet-1 vector to obtain the recombinant plasmid pACYC-bauA. Then, primers are used to linearize the pACYC-bauA plasmid, and the homologous recombinase is used to ligate the mcr-C fragment with the linearized pACYC-bauA plasmid to obtain the recombinant plasmid pACYC-bauA-mcr-C.

[0016] The present invention also provides a cell catalyst containing the recombinant Escherichia coli.

[0017] The present invention also provides a method for preparing the cell catalyst, which is to culture the strain in LB medium for a period of time, and induce it with IPTG with a final concentration of 1-1.6 mM to prepare the cell catalyst.

[0018] In one embodiment, the method is to culture the strain until OD 600 ≥2 and induce it with IPTG.

[0019] In one embodiment, the method is to induce the strain for 12-24 h and collect the bacterial cells.

[0020] In one embodiment, the method is to culture the strain in LB medium at 37 °C for 3-5 hours, add 1 mM IPTG and cool down to 30 °C for induction culture for 18 hours, and centrifuge at 5000 rpm to obtain the cell catalyst.

[0021] The present invention provides a method for producing malonic acid by whole-cell catalysis, using fumaric acid as a substrate, resuspending the recombinant Escherichia coli or the cell catalyst with a buffer solution, and catalytically preparing malonic acid.

[0022] In one embodiment, the pH of the reaction system is 6.8-7.5.

[0023] In one embodiment, fumaric acid with a final concentration of ≥25 g / L is added to the reaction system.

[0024] In one embodiment, the buffer is buffer-2 buffer, containing KH2PO4, K2HPO4, and NaHCO3.

[0025] In one embodiment, the buffer contains 4.17 g / L KH2PO4, 3.38 g / L K2HPO4, and 3 g / L NaHCO3.

[0026] In one embodiment, the reaction temperature is 30 - 37 °C.

[0027] In one embodiment, the method is to resuspend the biocatalyst with buffer-2 buffer to make the OD of the cells in the catalytic system 600 be 70, add the substrate fumaric acid with a final concentration of 70 g / L, and carry out the catalysis at 37 °C under the condition of pH 7.2.

[0028] The present invention also claims the application of the method in the preparation of malonic acid or its derivative products.

[0029] In one embodiment, the derivative products include but are not limited to diethyl malonate or dimethyl malonate.

[0030] In one embodiment, the derivative products include but are not limited to barbiturates, vitamin B1, or vitamin B6.

[0031] Beneficial effects: The present invention uses the whole-cell catalysis method to produce malonic acid. Compared with the chemical method and the shake-flask fermentation method, the production cycle is shortened, the production efficiency of malonic acid is improved, and the malonic acid yield can reach 51.0 g / L after 24 hours of catalysis.

[0032] The present invention uses fumaric acid as the substrate, which can effectively solve the environmental pollution problem caused by the chemical method. Compared with the microbial fermentation method, it has the following advantages: the enzyme activity is stable in the natural intracellular environment, the cofactors can be regenerated in situ through glycolysis and the tricarboxylic acid cycle, the high-cell-density catalytic system shortens the reaction time, simplifies the product separation process, can be operated under non-sterile conditions, and the process cost is significantly reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is the experimental results of catalysis using different substrates in Example 3.

[0034] Figure 2 It is the experimental results of constructing a whole-cell catalytic system using different buffers in Example 4.

[0035] Figure 3 It is the experimental results of catalysis using different concentrations of inducers in Example 5.

[0036] Figure 4 Optimization results of cell concentration during induction in Example 6.

[0037] Figure 5 Optimization results of cell collection time for preparing biocatalyst in Example 7.

[0038] Figure 6 Optimization experimental results of catalytic environment pH in Example 8.

[0039] Figure 7 Optimization experimental results of catalytic environment temperature in Example 9.

[0040] Figure 8 Catalytic experimental results of adding different concentrations of pyruvate in the catalytic system in Example 10.

[0041] Figure 9 Optimization results of cell concentration in the catalytic system in Example 11.

[0042] Figure 10 Optimization results of substrate concentration when the cell concentration in the catalytic system is 45 in Example 12.

[0043] Figure 11 Optimization results of substrate concentration when the cell concentration in the catalytic system is 70 in Example 13. Detailed implementation manners

[0044] Liquid-phase detection and result analysis of malonic acid:

[0045] Pretreatment: Centrifuge the fermentation sample at 12,000 rpm for 2 min to separate the fermentation broth from the cells. Treat the fermentation broth with a 0.22-μm filter membrane for HPLC (High Performance Liquid Chromatography, Aminex HPX-87H organic acid column from Bio-Rad, USA) detection. In HPLC detection, the mobile phase is 5 mM H2SO4, the column temperature is 30 °C, and a differential refractive index detector is used.

[0046] Table 1 Primers and sequences involved in the examples

[0047]

[0048] Example 1: Construction of recombinant plasmid and recombinant Escherichia coli

[0049] The aspA gene fragment (nucleotide sequence shown in SEQ ID NO.1) was amplified using PT-ASPA-F and PT-ASPA-R primers, the panD gene fragment (nucleotide sequence shown in SEQ ID NO.2) was amplified using PAND-F and PAND-R primers, and the pTrc99A vector was linearized using PTRC-F and PTRC-R primers. First, the aspA fragment was homologously recombined with the linearized pTrc99A vector using a homologous recombinase, and the recombinant product was transferred into competent E. coli JM109 cells to obtain the recombinant plasmid pTrc99a-aspA. Then, the pTrc99a-aspA plasmid was linearized using PT-AS-PAND-GR and PAC-BA-MCRC-GF primers, and the panD fragment was homologously recombined with the linearized pTrc99a-aspA plasmid using a homologous recombinase, and the recombinant product was transferred into competent E. coli JM109 cells to obtain the recombinant plasmid pTrc99a-aspA-panD.

[0050] The yneI gene fragment (nucleotide sequence shown in SEQ ID NO.3) was amplified using YNE1-F and YNE1-R primers, the pa0132 gene fragment (nucleotide sequence shown in SEQ ID NO.4) was amplified using PA0132-F and PA0132-R primers, and the pRSFDuet-1 vector was linearized using PR-PA0132-GF and PR-PA0132-GR primers. First, the pa0132 fragment was homologously recombined with the linearized pRSFDuet-1 vector using a homologous recombinase, and the recombinant product was transferred into competent E. coli JM109 cells to obtain the recombinant plasmid pRSF-pa0132. Then, the pRSF-pa0132 plasmid was linearized using PYNE1-R and PYNE1-F primers, and the yneI fragment was homologously recombined with the linearized pRSF-pa0132 plasmid using a homologous recombinase, and the recombinant product was transferred into competent E. coli JM109 cells to obtain the recombinant plasmid pRSF-pa0132-yneI.

[0051] The bauA gene fragment (nucleotide sequence shown in SEQ ID NO.5) was amplified using PAC-BAUA-MC-F and PAC-BAUA-MC-R primers, the mcr-C gene fragment (nucleotide sequence shown in SEQ ID NO.6) was amplified using PAC-BA-MCRC-R and PAC-BA-MCRC-F primers, and the pACYCDuet-1 vector was linearized using PAC-BAUA-MC-GF and PAC-BAUA-MC-GR primers. First, the bauA fragment was homologously recombined with the linearized pACYCDuet-1 vector using a homologous recombinase, and the recombinant product was transferred into competent E. coli JM109 cells to obtain the recombinant plasmid pACYC-bauA. Then, the pACYC-bauA plasmid was linearized using PAC-BA-MCRC-GF and PAC-BA-MCRC-GR primers, and the mcr-C fragment was homologously recombined with the linearized pACYC-bauA plasmid using a homologous recombinase, and the recombinant product was transferred into competent E. coli JM109 cells to obtain the recombinant plasmid pACYC-bauA-mcr-C.

[0052] The plasmids pTrc99a-aspA-panD, pRSF-pa0132-yneI, and pACYC-bauA-mcr-C were co-transformed into Escherichia coli BL21(DE3) to obtain recombinant Escherichia coli.

[0053] Example 2: Preparation of recombinant Escherichia coli cells

[0054] Seed solution preparation: The glycerol-preserved strain was streaked on a plate, and a single colony was picked and inoculated into a 250 ml conical flask containing 50 ml of LB liquid medium, and shaken overnight at 37 °C and 250 rpm.

[0055] Biocatalyst preparation conditions: The seed solution was inoculated into LB medium at an inoculation amount of 2% (2 mL), 4 g / L glucose was added to make its initial OD 600 be 0.1. The recombinant bacteria were induced with 1.0 mM IPTG after culturing at 37 °C and 250 rpm for 3 - 5 hours, the temperature was reduced to 30 °C, and the culture was continued at 250 rpm. After culturing for 12 h, the cells were collected at 5000 rpm.

[0056] Example 3: Selection of catalytic substrates

[0057] LB medium (g / L): Peptone 20, Yeast extract 5, MgCl2·6H2O 2.03, NaCl 0.5, KCl 0.186;

[0058] Substrates (25 g / L): Fumaric acid, Aspartic acid, and β-Alanine.

[0059] Biocatalytic system: The biocatalyst prepared in Example 2 was resuspended with buffer-1 buffer supplemented with the substrates fumaric acid, aspartic acid, or β-alanine, respectively, to make the OD of the cells in the catalytic system 600 reach 45, with the substrate concentration of 25 g / L, and the catalysis was carried out at pH 7.0 and 30 °C. The experiment was carried out for 48 h and detected every 12 h.

[0060] Result analysis: As Figure 1 shown, the highest malonic acid accumulation was observed in the reaction system with exogenous fumaric acid as the substrate, and the malonic acid accumulation reached 6.3 g / L at 24 h. The malonic acid accumulation in the reaction system with aspartic acid as the substrate was 0.67 g / L, and no malonic acid was detected in the reaction system with β-alanine as the substrate.

[0061] Example 4: Optimization of the buffer of the whole-cell catalytic system

[0062] LB medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl.

[0063] buffer-1 buffer: 4.17 g / L KH2PO4, 3.38 g / L K2HPO4, 3.3 g / L NaHCO3, 0.0124 g / L pyridoxal phosphate (PLP), 9 g / L (NH4)2SO4.

[0064] buffer-2 buffer: 4.17 g / L KH2PO4, 3.38 g / L K2HPO4, 3 g / L NaHCO3.

[0065] Preparation of biocatalyst: The biocatalyst was prepared according to Example 2.

[0066] Biocatalytic system: The biocatalyst was resuspended with LB medium, buffer-1 buffer, and buffer-2 buffer, respectively, to make the OD of the cells in the catalytic system 600 reach 45, and the substrate fumaric acid with a final concentration of 25 g / L was added, and the catalysis was carried out at pH 7.0 and 30 °C. Samples were taken for detection after the experiment was carried out for 24 h.

[0067] The results are as Figure 2 shown, the highest malonic acid accumulation was observed in the catalytic system prepared with buffer-2 buffer, and the highest malonic acid was 10.5 g / L at 24 h.

[0068] Example 5: Optimization of inducer concentration

[0069] The biocatalyst was prepared according to the method of Example 2, except that the IPTG concentration was adjusted to 0.5, 0.75, 1, 1.2, 1.6, 2 mM, respectively.

[0070] Biocatalytic system: Resuspend the biocatalyst with buffer-2 buffer to make the cell OD in the catalytic system 600 reach 45, and add the substrate fumaric acid with a final concentration of 25 g / L, and carry out the catalysis at pH 7 and 30 °C. Samples are taken for detection after the experiment is carried out for 24 h.

[0071] The results are as Figure 3 , when the IPTG concentration is 1.2 mM, the malonic acid accumulation amount can reach 13.1 g / L.

[0072] Example 6: Optimization of cell concentration when adding IPTG

[0073] Biocatalyst preparation conditions: Prepare the seed liquid according to the method of Example 2, inoculate the seed liquid at an inoculation amount of 2% (2 mL) into the LB medium containing 4 g / L glucose to make its initial OD 600 reach 0.1. Culture at 37 °C and 250 rpm until the OD 600 reaches 0.7, 1.5, 2, 2.5, 3, 3.5 respectively, add IPTG with a final concentration of 1.2 mM for induction, lower the temperature to 30 °C, and culture at 250 rpm. After culturing for 12 h, collect the cells at 5000 rpm to make the cell OD in the catalytic system 600 reach 45.

[0074] The biocatalytic system and reaction conditions are the same as those in Example 5.

[0075] The results are as Figure 4 , when the strain grows to OD 600 reach 2.5 for induction, the malonic acid accumulation amount can reach 13.8 g / L.

[0076] Example 7: Optimization of cell collection time

[0077] Cell collection time: 12, 18, 24, 30, 36 h after induction.

[0078] Biocatalyst preparation conditions: Prepare the seed liquid according to the method of Example 2, inoculate the seed liquid at an inoculation amount of 2% (2 mL) into the LB medium containing 4 g / L glucose to make its initial OD 600 reach 0.1. Culture at 37 °C and 250 rpm until the OD 600 reaches 2.5, add IPTG with a final concentration of 1.2 mM for induction, cool down to 30 °C and culture at 250 rpm. After culturing for 12, 18, 24, 30, 36 h respectively, collect the cells at 5000 rpm to make the cell OD in the catalytic system 600 reach 45.

[0079] The biocatalytic system and reaction conditions were the same as those in Example 5.

[0080] The results were as follows Figure 5 , when the biocatalyst was prepared after the strain grew for 18 h after induction, the malonic acid accumulation reached 15.1 g / L.

[0081] Example 8: Optimization of the pH condition of the catalytic environment

[0082] The biocatalyst preparation conditions were the same as those in Example 7.

[0083] Biocatalytic system: The biocatalyst was resuspended with buffer-2 buffer to make the OD of the cells in the catalytic system 600 be 45, and the substrate fumaric acid with a final concentration of 25 g / L was added, and the catalysis was carried out at 30 °C in environments with pH values of 6.0, 6.5, 6.8, 7.0, 7.2, 7.5, and 8.0 respectively. Samples were taken for detection after the experiment was carried out for 24 h.

[0084] The results were as follows Figure 6 , when the biocatalyst was prepared at an environmental pH of 7.2, the highest malonic acid accumulation was 17.3 g / L.

[0085] Example 9: Optimization of the temperature condition of the catalytic environment

[0086] The biocatalyst preparation conditions were the same as those in Example 7.

[0087] Biocatalytic system: The biocatalyst was resuspended with buffer-2 buffer to make the OD of the cells in the catalytic system 600 be 45, and the substrate fumaric acid with a final concentration of 25 g / L was added, and the catalysis was carried out at pH 7.2 in environments at 20, 25, 30, 37, and 42 °C respectively. Samples were taken for detection after the experiment was carried out for 24 h.

[0088] The results were as follows Figure 7 , when the environmental temperature was 37 °C, the malonic acid accumulation could reach 21.9 g / L.

[0089] Example 10: Optimization of the added pyruvic acid concentration

[0090] The biocatalyst preparation conditions were the same as those in Example 7.

[0091] Biocatalytic system: The biocatalyst was resuspended with buffer-2 buffer to make the OD of the cells in the catalytic system 600 be 45, and the substrate fumaric acid with a final concentration of 25 g / L and pyruvic acid with final concentrations of 0, 1, 3, 5, 8, 10, and 12 g / L respectively were added, and the catalysis was carried out at 37 °C in an environment with a pH of 7.2. Samples were taken for detection after the experiment was carried out for 24 h.

[0092] The results were as followsFigure 8 After adding pyruvic acid, the accumulation amount of malonic acid is lower than that without addition.

[0093] Example 11: Optimization of cell concentration in the catalytic system

[0094] The preparation conditions of the biocatalyst are the same as those in Example 7.

[0095] The biocatalytic system is the same as that in Example 10, except that the biocatalyst is resuspended with buffer-2 buffer to make the OD of the cells in the catalytic system 600 be 45, 70, 90, and 115 respectively. Samples are taken for detection after 24 h of the experiment.

[0096] The results are as Figure 9 shown. When the OD 600 is 70, the highest accumulation amount of malonic acid is 22.2 g / L.

[0097] Example 12: Optimization of substrate concentration when the cell concentration in the catalytic system is 45

[0098] The preparation conditions of the biocatalyst are the same as those in Example 7.

[0099] The biocatalytic system is the same as that in Example 10, except that substrates fumaric acid with final concentrations of 20, 25, 30, 50, 60, 70, and 80 g / L are added respectively. Samples are taken for detection after 24 h of the experiment.

[0100] The results are as Figure 10 shown. When the cell concentration in the catalytic system is 45, the highest accumulation amount of malonic acid is 36.7 g / L when the concentration of fumaric acid substrate is 50 g / L.

[0101] Example 13: Optimization of substrate concentration when the cell concentration in the catalytic system is 70

[0102] The preparation conditions of the biocatalyst are the same as those in Example 7.

[0103] Biocatalytic system: The biocatalyst is resuspended with buffer-2 buffer to make the OD of the cells in the catalytic system 600 be 70, and substrates fumaric acid with final concentrations of 20, 25, 30, 50, 60, 70, and 80 g / L are added respectively. The catalysis is carried out at 37 °C in an environment with a pH of 7.2. Samples are taken for detection after 24 h of the experiment.

[0104] The results are as Figure 11 shown. When the cell concentration in the catalytic system is 70, the highest accumulation amount of malonic acid is 51.0 g / L when the concentration of fumaric acid substrate is 70 g / L.

[0105] Although the present invention has been disclosed above in its preferred embodiments, it is not intended to limit the present invention. Any person skilled in this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. Recombinant Escherichia coli, characterized in that, Using pTrc99a as the expression vector to express the aspartate-α-decarboxylase gene panD and the aspartase gene aspA, using pRSFDuet-1 as the expression vector to express the β-alanine pyruvate transaminase gene pa0132 and the succinic semialdehyde dehydrogenase gene yneI, and using pACYCDuet-1 as the expression vector to express the β-alanine pyruvate transaminase gene bauA and the malonyl-CoA reductase gene mcr-C.

2. The recombinant Escherichia coli according to claim 1, characterized in that, Using Escherichia coli BL21(DE3) as the host.

3. A cell catalyst containing the recombinant Escherichia coli described in claim 1.

4. A method for preparing the cell catalyst according to claim 3, characterized in that, Culturing the recombinant Escherichia coli described in claim 1 or 2 in LB medium for a period of time, and inducing with IPTG at a final concentration of 1-1.6 mM to prepare a cell catalyst.

5. The method according to claim 4, characterized in that, Cultivate the recombinant Escherichia coli until OD 600 ≥2, induce with IPTG for 12 - 24 h, and collect the bacterial cells.

6. A method for producing malonic acid by whole-cell catalysis, characterized in that, Using fumaric acid as the substrate, and using the recombinant Escherichia coli described in claim 1 or 2 or the cell catalyst described in claim 3 to catalytically prepare malonic acid.

7. The method according to claim 6, wherein The pH of the reaction system is 6.8-7.

5.

8. The method according to claim 6 or 7, characterized in that, Adding fumaric acid with a final concentration ≥25 g / L to the reaction system.

9. The method according to any one of claims 6 to 8, characterized in that The reaction temperature is 30-37 °C.

10. Use of the recombinant Escherichia coli described in claim 1 or 2, or the cell catalyst described in claim 3, or any of the methods described in claims 4-9 in the preparation of malonic acid or its derivative products.

Images