Construction and application of escherichia coli for producing shikimic acid by using formic acid

Abstract

The invention discloses construction and application of escherichia coli for producing shikimic acid by using formic acid, and belongs to the technical field of biological engineering. According to the invention, four modules, i.e., construction of a C1-formic acid assimilation path, enhancement of an energy system, construction of an auxotrophic strain and design of a product synthesis path, are constructed. According to the synthesis route, after 13C isotope labeling discovers, it is detected that shikimic acid is labeled by 13C isotope from a reaction system. The invention realizes green biosynthesis of shikimic acid by using formic acid as a substrate, and provides a new method for synthesis of drug intermediates.

Description

Technical Field

[0001] The present invention relates to the construction and application of Escherichia coli for producing shikimic acid using formic acid, and belongs to the technical field of bioengineering. Background Art

[0002] In recent years, with the development and application of microbial cell factories, the biorefining of one-carbon resources such as carbon dioxide (CO2), carbon monoxide (CO), methane (CH4), methanol, and formic acid has attracted much attention. Compared with fossil fuels and traditional carbon sources, one-carbon resources are rich in natural reserves and relatively low in production cost. Therefore, one-carbon resources are expected to become the next-generation raw materials for preparing high-value-added chemicals such as bioenergy, bio-based materials, and biomedicines, thereby promoting the sustainable recycling of carbon resources.

[0003] Formic acid is an important chemical raw material, widely existing in nature, and is widely used in the pesticide, leather, textile, printing and dyeing, pharmaceutical, and rubber industries, etc. It can also be used to prepare various solvents, plasticizers, rubber coagulants, animal feed additives, and new process synthetic insulin, etc. As a one-carbon raw material, formic acid can be simply and effectively synthesized from CO2 through various chemical processes such as using metal catalysts or electricity, and formic acid has the advantages of being easily soluble in water, convenient for storage and transportation, and having a faster biological assimilation rate than CO2. Therefore, it is expected to be developed into a green and sustainable biomass resource for chemical production.

[0004] Shikimic acid (SA) is an essential intermediate in the synthetic metabolism pathway of hydroaromatic compounds. Since it has an important pharmaceutical function in the chemical synthesis of the anti-influenza drug oseltamivir phosphate (OSP, an effective antiviral inhibitor for treating seasonal influenza viruses A and B, human influenza virus H1N1, and avian influenza virus H5N1), and can be assembled into different bioactive compounds, shikimic acid has become an important precursor for pharmaceuticals and industrial products.

[0005] It is reported in the literature that microorganisms have limited metabolic ability for formic acid, and formic acid has certain toxicity, which limits the growth and metabolic efficiency of microorganisms. At present, although there have been studies on metabolic engineering to transform microorganisms to improve their tolerance and utilization efficiency of formic acid, the formic acid metabolic ability of existing strains is still insufficient to meet the requirements of industrial applications. Currently, the biofuels and chemicals synthesized by microorganisms using formic acid include polyhydroxyalkanoates (PHA), butanol, acetic acid, and succinic acid, etc., and the high-value-added biological products synthesized using formic acid include lactic acid, indigo, and 5-aminolevulinic acid, etc. However, there is currently no research report on synthesizing the aromatic product shikimic acid using formic acid as a raw material. Therefore, the present invention aims to develop a recombinant bacterium that can produce shikimic acid using formic acid. Compared with traditional fermentation for producing shikimic acid, it can not only realize the resource utilization of CO2, but also synthesize important pharmaceutical intermediates and provide medicinal value. SUMMARY OF THE INVENTION

[0006] To solve the above problems, the present invention provides a method for synthesizing shikimic acid using formic acid as a substrate, coupling the reductive glycine pathway with metabolic engineering to transform Escherichia coli, enabling the strain to use formic acid and glucose as co-substrates to synthesize the high-value product shikimic acid.

[0007] The present invention provides a recombinant Escherichia coli, which expresses methylenetetrahydrofolate cyclohydrolase (Fhs), methylenetetrahydrofolate dehydrogenase (FchA), methylenetetrahydrofolate reductase (FolD), the gcvTHP gene encoding the glycine cleavage (Gcv) system, formate dehydrogenase (Fdh), and lipoamide dehydrogenase (Lpd), and knocks out phosphoglycerate dehydrogenase (SerA), pyruvate formate-lyase (PlfB), and the repressor gene (gcvR).

[0008] In one embodiment, the recombinant Escherichia coli selects the laboratory high-yield strain E. coli SA09 as the starting strain.

[0009] In one embodiment, the genes fhs encoding methylenetetrahydrofolate cyclohydrolase, fchA encoding methylenetetrahydrofolate dehydrogenase, and folD encoding methylenetetrahydrofolate reductase are overexpressed at the plasmid level.

[0010] In one embodiment, the nucleotide sequence encoding the gcvTHP enzyme is as shown in SEQ ID NO.1; the nucleotide sequence of the serA gene encoding phosphoglycerate dehydrogenase is as shown in SEQ ID NO.2; the nucleotide sequence of the pflB gene encoding pyruvate formate-lyase is as shown in SEQ ID NO.3; the nucleotide sequence of the gcvR gene is as shown in SEQ ID NO.4; the nucleotide sequence of the methylenetetrahydrofolate cyclohydrolase-encoding gene is as shown in SEQ ID NO.5; the nucleotide sequence of the methylenetetrahydrofolate dehydrogenase-encoding gene is as shown in SEQ ID NO.6; the nucleotide sequence of the methylenetetrahydrofolate reductase-encoding gene is as shown in SEQ ID NO.7; the nucleotide sequence of the lipoamide dehydrogenase-encoding gene is as shown in SEQ ID NO.8; the nucleotide sequence of the formate dehydrogenase-encoding gene is as shown in SEQ ID NO.9.

[0011] In one embodiment, the recombinant Escherichia coli uses PET series vectors, pETDuet-1 vectors, pGEX series vectors, and pTrcHis series vectors as expression vectors;

[0012] In one embodiment, the chassis cell is E. coli SA09;

[0013] In one embodiment, pBR322 was used as the expression vector, and the genes gcvTHP of the glycine cleavage (Gcv) system were overexpressed at the plasmid level.

[0014] In one embodiment, pBR322 was used as the expression vector, and the lpd gene was overexpressed at the plasmid level.

[0015] In one embodiment, pCDR was used as the expression vector, and the fdh gene was overexpressed at the plasmid level.

[0016] In one embodiment, the nucleotide sequence of the gene gcvTHP is shown in SEQ ID NO.1; the nucleotide sequence of the gene Fhs is shown in SEQ ID NO.5; the nucleotide sequence of the gene FchA is shown in SEQ ID NO.6; the nucleotide sequence of the gene folD encoding methylenetetrahydrofolate reductase (FolD) is shown in SEQ ID NO.7; the nucleotide sequence of the gene lpd is shown in SEQ ID NO.8; the nucleotide sequence of the gene fdh is shown in SEQ ID NO.9.

[0017] The present invention also provides a method for improving the ability of Escherichia coli to utilize formic acid. The method is to knock out the serA gene encoding phosphoglycerate dehydrogenase, the pflB gene encoding pyruvate formate-lyase, and the gcvR gene on the genome of E. coli S09, and overexpress methylenetetrahydrofolate cyclohydrolase, methylenetetrahydrofolate reductase, methylenetetrahydrofolate dehydrogenase, gcvTHP, lipoamide dehydrogenase, and formate dehydrogenase.

[0018] Preferably, the nucleotide sequence of the gene encoding the gcvTHP enzyme is shown in SEQ ID NO.1; the nucleotide sequence of the gene encoding methylenetetrahydrofolate cyclohydrolase is shown in SEQ ID NO.5; the nucleotide sequence of the gene encoding methylenetetrahydrofolate dehydrogenase is shown in SEQ ID NO.6; the nucleotide sequence of the gene encoding methylenetetrahydrofolate reductase is shown in SEQ ID NO.7; the nucleotide sequence of the gene encoding lipoamide dehydrogenase is shown in SEQ ID NO.8; the nucleotide sequence of the gene encoding formate dehydrogenase is shown in SEQ ID NO.9;

[0019] The nucleotide sequence of the serA gene encoding phosphoglycerate dehydrogenase is shown in SEQ ID NO.2; the nucleotide sequence of the pflB gene encoding pyruvate formate-lyase is shown in SEQ ID NO.3; the nucleotide sequence of the gcvR gene is shown in SEQ ID NO.4.

[0020] The present invention also provides the application of the recombinant Escherichia coli in the production of shikimic acid using formic acid.

[0021] In one embodiment, the application is to culture the recombinant Escherichia coli in TB medium for at least 16 h.

[0022] In one embodiment, the application is to culture the recombinant Escherichia coli in TB medium at 37 °C until the OD 600 is 0.6 - 0.8, induce it overnight with IPTG, collect the cells by centrifugation, and then add formic acid for whole-cell catalysis for at least 24 h.

[0023] In one embodiment, the addition amount of IPTG is 0.5 mM.

[0024] The present invention also provides a method for preparing shikimic acid using formic acid and glucose as co-substrates. The method is to use the above-mentioned recombinant Escherichia coli and ferment to prepare shikimic acid with formic acid and glucose as co-substrates.

[0025] In one embodiment, the addition amount of formic acid is: 1 g / L - 6 g / L; the addition amount of glucose is: 10 g / L - 20 g / L; the fermentation conditions are: 33 °C - 40 °C; the addition amount of recombinant Escherichia coli is: 0.2 - 0.4.

[0026] In one embodiment, the recombinant Escherichia coli is added to the reaction system in the form of a bacterial agent for fermentation culture; the preparation method of the bacterial agent is: (1) inoculate the SA-5 strain into LB medium and grow it at 35 - 37 °C and a rotation speed of 200 - 220 rpm for 10 - 12 hours to obtain a seed liquid; (2) transfer the prepared seed liquid to an M9 medium containing 4 - 5 g / L glucose, 4 - 5 g / L sodium formate and antibiotics (100 μg / ml ampicillin and 33 μg / ml streptomycin) according to an inoculation amount with an initial OD 600 value of 0.1, transfer the bacterial liquid to a new M9 basal medium every 12 h, and keep the initial OD 600 value at 0.1 each time. Reduce 0.5 g / L glucose for each transfer; after the 10th transfer, the initial OD 600The value was maintained at 0.1. The bacterial liquid was transferred to a new M9 minimal medium (supplemented with 4 - 5 g / L sodium formate, 100 μg / ml ampicillin, and 33 μg / ml streptomycin) every 12 h, glucose was no longer added, and the culture continued; the culture temperature was 37°C, and the total culture time was 260 h; a culture solution was obtained; after centrifuging the culture solution obtained in step (2), all the precipitate (bacteria) was inoculated into a TB liquid medium containing 10 - 12 g / L glucose, 150 - 170 mM formic acid, 0.1 mM IPTG, and appropriate antibiotics, and grown at 37°C and 200 rpm for 6 h to obtain a culture solution; then, with an initial inoculum OD600 of 0.3 - 0.4, the culture solution was transferred to an M9 medium supplemented with 10 - 12 g / L glucose for high-density fermentation, and fermented at 35 - 37°C for 56 h, and concentrated to an OD 600 value of approximately 28 - 30;

[0027] The present invention also provides a method for improving the tolerance of Escherichia coli to formic acid. The method is to knockout the serA gene encoding phosphoglycerate dehydrogenase, the pflB gene encoding pyruvate formate-lyase, and the gcvR gene on the genome of E. coli S09, and overexpress methylenetetrahydrofolate cyclohydrolase, methylenetetrahydrofolate reductase, methylenetetrahydrofolate dehydrogenase, gcvTHP, lipoamide dehydrogenase, and formate dehydrogenase;

[0028] Preferably, the nucleotide sequence encoding the gcvTHP enzyme is as shown in SEQ ID NO.1; the nucleotide sequence of the methylenetetrahydrofolate cyclohydrolase-encoding gene is as shown in SEQ ID NO.5; the nucleotide sequence of the methylenetetrahydrofolate dehydrogenase-encoding gene is as shown in SEQ ID NO.6; the nucleotide sequence of the methylenetetrahydrofolate reductase-encoding gene is as shown in SEQ ID NO.7; the nucleotide sequence of the lipoamide dehydrogenase-encoding gene is as shown in SEQ ID NO.8; the nucleotide sequence of the formate dehydrogenase-encoding gene is as shown in SEQ ID NO.9;

[0029] The nucleotide sequence of the serA gene encoding phosphoglycerate dehydrogenase is as shown in SEQ ID NO.2; the nucleotide sequence of the pflB gene encoding pyruvate formate-lyase is as shown in SEQ ID NO.3; the nucleotide sequence of the gcvR gene is as shown in SEQ ID NO.4.

[0030] The present invention also provides the use of the above-mentioned recombinant Escherichia coli or the above method in the preparation of shikimic acid or products containing shikimic acid.

[0031] Beneficial effects

[0032] The present invention designs a metabolic pathway for synthesizing shikimic acid using formic acid as a substrate; the expression and purification of pathway enzymes can be used to construct a formic acid assimilation pathway based on the reductive glycine pathway in vitro, and the detection shows that the yield of shikimic acid generated reaches 3 g / L; alternatively, the pathway can be introduced into Escherichia coli, and the successful utilization of formic acid by microorganisms to synthesize shikimic acid is achieved. The strain constructed by the present invention provides a green biological manufacturing method for the biosynthesis of shikimic acid. Description of the Drawings

[0033] Figure 1 It is a design diagram of the formic acid assimilation pathway.

[0034] Figure 2 It is a design diagram of the shikimic acid pathway. Detailed Description of the Invention

[0035] Reagents and Materials:

[0036] The ClonExpress one-step directional cloning kit and the gel extraction kit were purchased from Vazyme Biotech (Nanjing). The plasmid extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. Various analytical pure reagents were purchased from the Sinopharm Group. In the present invention, except for NADH oxidase purchased from Sigma, other enzymes were obtained by prokaryotic expression according to genetic engineering methods.

[0037] The genotype of the starting strain E. coli SA09 involved in the following examples is shown in Table 2-2, and the construction method is described in detail in the paper "Systems engineering of Escherichia coli for high-level shikimate production".

[0038] Construction of the expression vector pCDR: The pCDR plasmid is a plasmid obtained by modifying the promoter region of pCDM4 (purchased from addgene, #49796). The gene sequence of the pCDR plasmid is shown as SEQ ID NO.6 on page 6 of the sequence listing of Patent CN110951660B.

[0039] Culture Medium and Reaction System:

[0040] LB medium (g / L): Yeast extract 5, NaCl 10, Peptone 10; made up to the corresponding volume with deionized water and sterilized at 121 °C for 20 min for use; about 2% agar powder needs to be added before sterilizing the solid medium.

[0041] TB medium (g / L): glycerol 4, yeast powder (Angel 802) 24, tryptone 12, KH2PO4 2.31, K2HPO4·3H2O 16.42; made up to the corresponding volume with deionized water and sterilized at 121 °C for 15 - 20 min for later use.

[0042] Composition of M9 medium (pH 7.0): 6.8 g / L Na2HPO4·7H2O, 3 g / L KH2PO4, 0.5 g / L NaCl, 2 g / L NH4Cl, 4.2 g / L NaHCO3, 0.8 g / L MgSO4·7H2O, 0.24 g / L IPTG, 0.05 g / L ethylenediaminetetraacetic acid (EDTA), 0.002 g / L thiamine·HCl and 5 ml of trace metal solution. The trace metal solution contains (per liter of 0.5 M HCl) 10 g FeSO4·7H2O, 2 g CaCl2, 2.2 g ZnSO4·7H2O, 0.5 g MnSO4·4H2O, 1 g CuSO4·5H2O, 0.1 g (NH4)6Mo7O 24 ·4H2O and 0.02 g Na2B4O7·10H2O, and the pH value of M9 medium was adjusted with 1 M NaOH solution.

[0043] The whole-cell catalytic reaction system (30 mL) for the formic acid assimilation pathway based on the reductive glycine pathway contains: The reaction system (30 mL) contains: 50 mM KPi (potassium phosphate buffer, pH = 8.0), 2 mM MgCl2, 0.2 mM TPP (thiamine pyrophosphate), 2 mM ATP, 3 mM NADH, 0.2 mM DTT (dithiothreitol), 50 mM NH4 + , 50 mM HCO3 - , 0.02 mM THF (tetrahydrofuran), 20 mM formic acid and 20 g / L cells. After reacting at 37 °C for 24 h, the reaction was terminated, and the supernatant was taken by centrifugation for HPLC analysis to detect the synthesis of glycine. The sample needed to be pre-column derivatized with 2,4-dinitrofluorobenzene (DNFB).

[0044] The whole-cell catalytic reaction system (30 mL) of the carboxylic acid reductase pathway (NiCAR-FLS-DHAK) and the formaldehyde enzyme pathway (ACS-ACDH-FLS-DHAK) constructed based on the existing HWLS pathway in the laboratory contains: 50 mM KPi (pH = 8.0), 2 mM MgCl2, 3 mM NADPH, 0.2 mM TPP, 2 mM ATP, 0.2 mM DTT, 20 mM formic acid, and 20 g / L cells. After reacting at 37 °C for 12 h, the reaction was terminated, and the supernatant was taken by centrifugation for kit enzyme-linked immunosorbent assay (ELISA) and HPLC analysis to detect the synthesis of dihydroxyacetone phosphate (DHAP).

[0045] The whole-cell catalytic reaction system (30 mL) of the formic acid assimilation pathway based on the reduced glycine pathway contains: 50 mM KPi (pH = 8.0), 2 mM MgCl2, 0.2 mM TPP, 2 mM ATP, 3 mM NADH, 0.2 mM DTT, 50 mM NH4 + ,50 mM HCO3 - ,0.02 mM THF, 20 mM formic acid, and 20 g / L cells.

[0046] The HPLC detection conditions are as follows:

[0047] Chrom Core C18 chromatographic column (specification: 4.6 × 150 mm, 5 μm) was used, mobile phase A: 50% acetonitrile, mobile phase B: 50 mmol / L sodium acetate (pH adjusted to 6.4 with glacial acetic acid), flow rate was 0.5 mL / min, the detector was an ultraviolet detector, detection wavelength: 360 nm, column temperature: 33 °C, injection volume: 10 μL, and gradient elution was used.

[0048] Table 1-1 HPLC detection method

[0049]

[0050]

[0051] Plasmids, strains, and primers involved in the examples:

[0052] Table 1-2: Plasmids used in this application

[0053]

[0054] Table 2-1: Strains involved in this application

[0055]

[0056] Table 2-2: Genotype of E. coli SA09

[0057]

[0058] Table 3: Primers and Sequences

[0059]

[0060]

[0061] The above pTarget plasmid, pCas9 plasmid, and E. coli TOP10 strain are all commercial plasmids and strains.

[0062] Example 1: Design of the Formate Assimilation Pathway

[0063] To better assimilate formate into central carbon metabolism, three pathways were designed based on mining databases such as Brenda and Uniport and literature research, as Figure 1 shown below:

[0064] Pathway 1: Based on the HWLS carbon fixation pathway constructed in the early stage of the laboratory, carboxylic acid reductase (CAR) was introduced to catalyze the conversion of formate into formaldehyde. Carboxylic acid reductase (CAR) is a class of multifunctional enzymes that can efficiently catalyze the reaction from carboxylic acid to aldehyde. The most optimally soluble-expressed CAR was assembled with FDH, FLS, and DHAK to construct a new CO2 fixation cascade pathway. The cascade reaction generates dihydroxyacetone phosphate (DHAP). This cascade pathway only requires four catalytic reactions to fix CO2 into glycolytic metabolism.

[0065] Pathway 2: Based on the combination of the HWLS carbon fixation pathway and the formaldehyde enzyme pathway constructed in the early stage of the laboratory, acetyl-CoA synthetase (ACS: P27550·ACSA_ECOLI), acetaldehyde dehydrogenase (ACDH: Q47146 FADE_ECOLI), formaldehyde enzyme (FLS: nucleotide sequence as shown in SEQ ID NO.10), and glycerol kinase (DHAK: P43550·DAK2_YEAST) were assembled, and the cascade reaction generates dihydroxyacetone phosphate (DHAP).

[0066] Pathway 3: The design of the reductive glycine pathway is to introduce heterologous methylenetetrahydrofolate cyclohydrolase (Fhs), methylenetetrahydrofolate dehydrogenase (FchA), and methylenetetrahydrofolate reductase (FolD) to assemble and generate glycine together.

[0067] Example 2: In Vitro Enzyme Cascade Verification of the Feasibility of the Three Pathways

[0068] The three pathways designed in Example 1 were verified by in vitro enzyme cascade.

[0069] 1. Verification of Pathway 1:

[0070] (1) Screening of Carboxylic Acid Reductase (CAR)

[0071] The carboxylic acid reductases NiCAR (Q6RKB1 CAR_NOCIO) from Nocardia iowensis, MsCAR from Mycolicibacterium smegmatis (A0A653FAQ0 A0A653FAQ0_MYCSM), MmCAR from Mycobacterium marinum (B2HN69 CAR_MYCMM), SrCAR from Segniliparus rotundus (D6ZDT1 D6ZDT1_SEGRD), and NcCAR from Neurosporacrassa (Q7RW48·CAR_NEUCR) were respectively ligated to the pET28a expression vector and then transformed into Escherichia coli BL21(DE3) to prepare recombinant bacteria respectively. The prepared recombinant bacteria were respectively inoculated into TB medium and cultured at 37 °C until the cell density reached OD 600 When it reached 0.6 - 0.8, isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.5 mM was added, and after inducing for 16 hours at 16 °C, the cells were collected by centrifugation. After cell disruption, the proteins were obtained and the expression levels of the proteins were respectively detected. The results showed that the expression level of the carboxylic acid reductase from Nocardia iowensis was the best. The carboxylic acid reductase from Nocardia iowensis with the best expression level was used for subsequent experiments after purifying the protein through a nickel column.

[0072] (2) Preparation of Formate Dehydrogenase (FDH), Formaldehyde Lyase (FLS), and Glycerol Dehydrogenase Kinase (DHAK): The formate dehydrogenase (FDH), formaldehyde lyase (FLS, SEQ ID NO.10) from Candida boidinii, and glycerol dehydrogenase kinase (DHAK) from Saccharomyces cerevisiae were chemically synthesized, ligated to the pET28a expression vector, and then transformed into Escherichia coli BL21(DE3) respectively to prepare recombinant bacteria; according to the method of step (1), pure enzymes of formate dehydrogenase (FDH), formaldehyde lyase (FLS), and glycerol dehydrogenase kinase (DHAK) were respectively prepared;

[0073] (3) In vitro enzyme cascade verification: The 1 mL reaction system contains (at final concentration): 50 mM KPi (pH = 8.0), 2 mM MgCl2, 3 mM NADPH, 0.2 mM TPP, 2 mM ATP, 0.2 mM DTT, 20 mM formic acid, and 1 mg / ml pure enzyme solution (1 mg / ml FDH, 1 mg / ml FLS, 1 mg / ml DHAK, 1 mg / ml CAR). React at 37 °C for 8 hours, and sample every 2 hours to detect dihydroxyacetone phosphate (DHAP) in the reaction system. Quantitative detection by high-performance liquid chromatography found that no dihydroxyacetone phosphate (DHAP) was synthesized.

[0074] 2. Verification of Path 2

[0075] Chemically synthesize acetyl-CoA synthetase (ACS) from Escherichia coli, acetaldehyde dehydrogenase (ACDH) from Escherichia coli, formolase (FLS, SEQ ID NO.10), and dihydroxyacetone kinase (DHAK) from Saccharomyces cerevisiae, ligate them to the pET28a expression vector, and transform them into Escherichia coli BL21(DE3) respectively to obtain recombinant bacteria; according to the method of step (1) in step 1, prepare pure enzymes of acetyl-CoA synthetase (ACS), acetaldehyde dehydrogenase (ACDH), formolase (FLS), and dihydroxyacetone kinase (DHAK) respectively; the 1 mL reaction system contains (at final concentration): 50 mM KPi (pH = 8.0), 2 mM MgCl2, 3 mM NADPH, 0.2 mM TPP, 2 mM ATP, 0.2 mM DTT, 20 mM formic acid, and 1 mg / ml pure enzyme solution (1 mg / ml ACS, 1 mg / ml ACDH, 1 mg / ml FLS, 1 mg / ml DHAK). React at 37 °C for 8 hours, and sample every 2 hours to detect dihydroxyacetone phosphate (DHAP) in the reaction system. Quantitative detection by high-performance liquid chromatography found that no dihydroxyacetone phosphate (DHAP) was synthesized.

[0076] 3. Verification of Path 3

[0077] Chemically synthesized methyltetrahydrofolate cyclohydrolase (Fhs) (nucleotide sequence shown in SEQ ID NO: 5), methylene tetrahydrofolate dehydrogenase (FchA) (nucleotide sequence shown in SEQ ID NO: 6), and methylene tetrahydrofolate reductase (FolD) (nucleotide sequence shown in SEQ ID NO: 7) were respectively prepared to obtain pure Fhs enzyme, pure FchA enzyme, and pure FolD enzyme according to the method of step (1) in step 1; the 1 mL reaction system contained (at final concentration): 50 mM KPi (pH = 8.0), 2 mM MgCl2, 0.2 mM TPP, 2 mM ATP, 3 mM NADH, 0.2 mM DTT, 50 mM NH4 + , 50 mM HCO3 - , 0.02 mM THF, 20 mM formic acid, and 1 mg / ml pure enzyme solution (1 mg / ml Fhs, 1 mg / ml FchA, 1 mg / ml FolD). After the sample from the above in vitro multi-enzyme catalytic reaction was boiled to remove proteins, the supernatant was taken by centrifugation, and mass spectrometry analysis was performed using a UPLC-TripleTOF6600 liquid chromatography-mass spectrometry instrument in the positive ion mode. After library search and comparison analysis, it was confirmed to be glycine.

[0078] It can be seen that the reductive glycine pathway (path three) designed in Example 1 can utilize formic acid to produce glycine, a key precursor of shikimic acid.

[0079] Example 3: Construction of auxotrophic strains (construction of strains SA-1 and SA-2)

[0080] Based on literature reports, the construction of the assimilation of formic acid into the central carbon metabolism pathway is designed around the reductive glycine pathway. Therefore, in order to enable the strain to grow using formic acid, the reductive glycine pathway was used as the basis and optimized to achieve efficient assimilation of formic acid. The design of the pathway was mainly divided into four modules: construction of the C1-formic acid assimilation pathway, strengthening of the energy system, construction of auxotrophic strains, and design of the product synthesis pathway, as Figure 2 shown.

[0081] To confirm that the strain can assimilate formic acid into central carbon metabolism, the serA gene encoding phosphoglycerate dehydrogenase (nucleotide sequence shown in SEQ ID NO:2, protein number P0A9T0) was knocked out to obtain the serine auxotrophic Escherichia coli strain SA-1. The serine synthesis of this strain is limited under the condition of only glucose, thus affecting growth. To strengthen the central carbon metabolic flux, on the one hand, the pflB gene encoding pyruvate formate-lyase (nucleotide sequence shown in SEQ ID NO:3, protein number P09373) was knocked out to prevent pyruvate from being cleaved into formic acid; on the other hand, the gcvR gene (nucleotide sequence shown in SEQ ID NO:4, protein number P0A9I3) that inhibits the reductive glycine pathway was knocked out to promote the conversion of 5,10-methylenetetrahydrofolate to glycine, and strain SA-2 was obtained.

[0082] Using the laboratory high-yield strain E.coli SA09 as the starting strain, the engineered strain SA-1 with serA knocked out was constructed, and then pflB and gcvR were further knocked out on the basis of SA-1 to obtain the engineered strain SA-2. All gene editing operations were completed using the CRISPR-Cas9 gene editing technology. The specific construction method is as follows:

[0083] (1) Using the genome of the laboratory high-yield strain E.coli S09 (SA-0) as a template, the upstream and downstream homologous arms of the serA gene were amplified using the serA knockout primers P1 and P2, P3 and P4 in Table 3, and then the upstream and downstream arms were fused into the serA knockout cassette using primers P1 and P4;

[0084] (2) Using the pTarget plasmid as a template and P5 and P6 as primers, a linearized fragment was amplified, transformed into the cloning host E.coli TOP10 by homologous recombination, and the pTarget-serA plasmid was extracted;

[0085] (3) The verified pTarget-serA plasmid and the serA knockout cassette were electrotransformed into the strain SA-0 containing the pCas9 plasmid. After electrotransformation, it was spread on an LB plate containing 50 ng / μL kanamycin and 30 ng / μL spectinomycin and cultured under the culture condition of 30 °C;

[0086] (4) Colony PCR was performed using the verification primers P7 and P8, and the correct transformants were screened by sequencing;

[0087] (5) The correct transformants were transferred to an LB medium containing 0.5 mM IPTG and 50 ng / μL kanamycin and cultured under the culture condition of 30 °C to eliminate the pTarget plasmid, and the correct transformants were screened by streaking on the plate;

[0088] (6) Transfer the correct transformant to LB medium and culture it at 42 °C to eliminate the pCas9 plasmid. Streak on a plate to screen for the correct transformant, obtaining the recombinant strain SA-1 (E. coli SA09ΔserA).

[0089] (7) Following the above steps, iteratively knockout the pflB and gcvR genes (primer sequences are shown in Table 3) based on the recombinant strain SA-1 to obtain the recombinant strain SA-2 (E. coli SA09ΔserAΔpflBΔgcvR).

[0090] Example 4: In vivo verification of pathway feasibility

[0091] Based on the in vitro construction of the reductive glycine pathway, further verify its effectiveness in vivo. The specific steps are as follows:

[0092] 1. Construction of SA-3

[0093] (1) Construction of the recombinant vector

[0094] Construct the heterologous enzymes methylenetetrahydrofolate cyclohydrolase (Fhs, SEQ ID NO:5), methylenetetrahydrofolate dehydrogenase (FchA, SEQ ID NO:6), and methylenetetrahydrofolate reductase (FolD, SEQ ID NO:7) from Example 2 onto the pBR322 plasmid. Use primers P1 and P2 to amplify the pBR322 vector, and after purification, use the DpnⅠ digestion enzyme from TaKaRa to remove the template plasmid. At the same time, use primers P5 and P6 to amplify the gene fragment fchA, primers P7 and P8 to amplify the gene fragment fhs, and primers P9 and P10 to amplify the gene fragment folD. Homologously recombine the purified gene fragments fchA, fhs, folD with the digested pBR322 vector, then transform into E. coli Top10 competent cells, and extract to obtain the single plasmid pBR322-fhs-folD-fchA.

[0095] (2) Construction of the recombinant strain

[0096] Introduce the plasmid pBR322-fhs-folD-fchA into the SA-2 (E. coli SA09ΔserAΔpflBΔgcvR) strain prepared in Example 2 to obtain the strain SA-3 (E. coli SA09ΔserAΔpflBΔgcvR / pBR322-fhs-folD-fchA).

[0097] (3) Growth of the recombinant strain

[0098] Inoculate SA-3 into LB medium for activation and culture at 37 °C until the cells grow to OD 600is 0.6 to 0.8. Then, with an initial inoculum OD 600 of 0.1, it was transferred to M9 medium. Then, a sodium formate solution was added to the medium to a final concentration of 20 mM, and it was cultured at 37 °C with a rotation speed of 200 rpm.

[0099] Samples were taken every 4 h to measure the absorbance at OD 600 to test the growth of cells within 48 h. The results showed that strain SA-3 cannot grew under the conditions of formic acid and CO2 (without glucose addition).

[0100] 2. Construction of SA-4

[0101] (1) Construction of the recombinant vector

[0102] The pathway enzyme GcvTHP (nucleotide sequence as shown in SEQ ID NO: 1) was constructed onto the pBR322-fhs-folD-fchA plasmid. The pBR322 vector was amplified using primers P1 and P2. After purification, the template plasmid was removed using the DpnⅠ digestion enzyme from TaKaRa. At the same time, the gene fragment fchA was amplified using primers P5 and P6, the gene fragment fhs was amplified using primers P7 and P8, the gene fragment folD was amplified using primers P9 and P10, and the gene fragment gcvTHP was amplified using primers P11 and P12. The purified gene fragments fhs, fchA, folD, and gcvTHP were subjected to homologous recombination with the digested pBR322 vector, and then transformed into Escherichia coli Top10 competent cells. The plasmid pBR322-fhs-folD-fchA-gcvTHP was obtained by extraction.

[0103] (2) Construction of the recombinant bacterium

[0104] To strengthen the reductive glycine pathway, the gene gcvTHP was overexpressed in strain SA-3 (E. coli SA09ΔserAΔpflBΔgcvR / pBR322-fhs-folD-fchA). That is, the recombinant vector prepared in step (1) was then transformed into strain SA-2 (E. coli SA09ΔserAΔpflBΔgcvR) to obtain strain SA-4 (E. coli SA09ΔserAΔpflBΔgcvR / pBR322-fhs-folD-fchA-gcvTHP).

[0105] (3) Growth of the recombinant bacterium

[0106] SA-4 was first inoculated into LB medium for activation and cultured at 37 °C until the cells grew to OD 600 of 0.6 to 0.8. Then, with an initial inoculum OD600 Transfer 0.1 to M9 medium, then add sodium formate solution to the medium to make the final concentration 20 mM, and culture at 37 °C with a rotation speed of 200 rpm;

[0107] Take samples every 8 h to measure the absorbance at OD 600 to test the growth of cells within 72 h. It was found that the growth of strain SA-4 was slightly improved under the conditions of formic acid and CO2 (without glucose addition), and the initial inoculum OD 600 was transferred to M9 medium at 0.1. The maximum OD of strain SA-4 was measured after 72 h of cultivation 600 and the value was 0.24 .

[0108] Example 5: Enhancement of the energy system (construction of strain SA-5)

[0109] As an energy source, formic acid can not only provide NADPH but also NADH. Specifically, the fdh gene encoding formate dehydrogenase and the lpd gene encoding lipoamide dehydrogenase can promote the conversion of NAD + to generate NADH. Insufficient NADH will affect the normal operation of the reductive glycine cleavage system. Therefore, the fdh gene and the lpd gene were overexpressed to solve the problem of insufficient NADH supply.

[0110] 1. Screening of enzymes

[0111] Screen different sources of formate dehydrogenase (FDH). After obtaining the protein sequence on the NCBI website (https: / / www.ncbi.nlm.nih.gov), send it to GenScript Biotech Corporation for codon optimization and gene synthesis; clone the codon-optimized formate dehydrogenase Fdh gene sequence into the pET28a expression vector; prepare the recombinant vector: pET28a-Fdh; then transform the recombinant vector into Escherichia coli BL21(DE3) (Invitrogen, Carlsbad, CA) to prepare the recombinant bacterium: E. coli BL21(DE3) / pET28a-Fdh; inoculate the prepared recombinant bacterium into TB medium and culture at 37 °C until the cells grow to OD 600 When it is 0.6 - 0.8, add isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.5 mM, induce at 16 °C for 16 hours, centrifuge to collect the cells, break them to obtain the protein, and detect the expression level of the protein respectively. The formate dehydrogenase from Candida boidinii with the best expression level was purified by nickel column and used for subsequent experiments. Finally, it was found that the formate dehydrogenase (FDH) from Candida boidinii could efficiently catalyze NAD +Generate NADH, and the codon-optimized nucleotide sequence is as shown in SEQ ID NO:9.

[0112] 2. Construction of SA-5

[0113] The gene fragment fdh (SEQ ID NO:9) encoding formate dehydrogenase (FDH) from Candida boidinii and the gene fragment lpd (SEQ ID NO:8) encoding lipoamide dehydrogenase were constructed onto the pCDR plasmid. The specific steps are as follows:

[0114] The pCDR vector was amplified using primers P15 and P16. After purification, the template plasmid was removed using DpnⅠ digestion enzyme from TaKaRa. At the same time, the gene fragment fdh was amplified using primers P11 and P12, and the gene fragment lpd was amplified using primers P13 and P14. The purified gene fragments fdh and lpd were subjected to homologous recombination with the digested pCDR vector, and then transformed into Escherichia coli Top10 competent cells. The plasmid pCDR-fdh-lpd was obtained by extraction. Subsequently, this plasmid pCDR-fdh-lpd was transformed into SA-4 strain to obtain strain SA-5 (E.coli SA09ΔserAΔpflBΔgcvR / pBR322-fhs-folD-fchA-gcvTHP / pCDR-fdh-lpd).

[0115] 3. Cultivation of the strain

[0116] The culture medium components and culture conditions of SA-5 strain were the same as those of SA-3 in the previous step for culturing and verification.

[0117] SA-5 was first inoculated into LB medium for activation and cultured at 37°C until the cell growth reached OD 600 of 0.6 - 0.8. Then, with an initial inoculum OD 600 of 0.1, it was transferred to M9 medium. Subsequently, a sodium formate solution was added to the medium to a final concentration of 20 mM, and the culture was carried out at 37°C and a rotation speed of 200 rpm.

[0118] Samples were taken every 8 h to measure the absorbance value at OD 600 to test the cell growth within 72 h. The results showed that the growth of strain SA-5 was slightly improved under the conditions of formic acid and CO2. With an initial inoculum OD 600 of 0.1, it was transferred to M9 medium. The maximum OD of strain SA-4 was measured after 72 h of cultivation 600 and the value was 0.8

[0119] Example 6: Isotope analysis of SA-3, SA-4, and SA-5 strains

[0120] 1. Use the following procedure for the isotope analysis of SA-3 and SA-4 strains.

[0121] (1) Inoculate SA-3 and SA-4 strains separately into 10 ml of LB medium and culture at 200 rpm and 37 °C for 12 hours; prepare the culture solution; after centrifuging the culture solution, take the precipitate;

[0122] Transfer the cells (precipitate) to M9 minimal medium containing 5 g / L glucose, 4 g / L formate, and 100 μg / ml ampicillin, and culture at 200 rpm and 37 °C for 24 hours.

[0123] (2) After centrifuging the culture solution prepared in step (1), take the cell precipitate; inoculate the cell precipitate into a baffled conical flask containing M9 minimal medium supplemented with 0.1 g / L [U- 13 C] glucose, 4 g / L formate, and 100 μg / ml ampicillin. Use a 100 ml baffled conical flask to culture the cells (working volume is 30 ml), culture at 200 rpm and 37 °C, and introduce gas with a composition of 10 mol / mol% CO2, 18 mol / mol% O2, and 72 mol / mol% N2. After culturing for 72 hours, add 0.1 g / L [U- 13 C] glucose again.

[0124] (3) After culturing the conical flask for 120 hours, collect the cell samples for 13 C isotope analysis, inoculate the cells into 10 ml of LB medium containing appropriate antibiotics (100 μg / ml ampicillin and 33 μg / ml streptomycin) and culture for 12 hours. Next, transfer the cells to M9 minimal medium supplemented with 5 g / L [U- 13 C] glucose, 4 g / L 13 C-labeled formate, 100 μg / ml ampicillin, and 33 μg / ml streptomycin and culture for 24 hours.

[0125] After that, inoculate the pre-cultured cells (OD 600 is 2 - 3) into M9 minimal medium containing 4 g / L formate to make the initial OD 600 be 0.1. Before inoculation, wash the cells once with fresh M9 minimal medium. After culturing the culture flask for 100 hours, collect the cell samples for 13 C isotope analysis (1 ml OD 600 is ~3). All cultures are carried out using 300 ml baffled flasks with a working volume of 50 ml.

[0126] (4) Wash the cells collected in step (3) with Tris-HCl (pH 7.6) and hydrolyze them with 1 ml of 6 M HCl in a dry heating block at 105 °C for 24 h. Dry the hydrolyzate in an oven at 85 °C. For the 12 C ratio in the protamine amino acids, it is calculated using the following formula. To test whether the cells can maintain growth using only formic acid and carbon dioxide, when formic acid is exhausted during the cultivation, formic acid is added to the M9 medium.

[0127] The results show that: Through 13 C isotope labeling, it was found that 1.5% pyruvate was detected in SA-3 strain to be 13 C-labeled, The SA-4 strain was detected with 4.5% of pyruvate being 13 C-labeled .

[0128] 2. Next, perform isotope analysis on the SA-5 strain. The culture components and culture conditions are the same as those of the SA-3 and SA-4 strains. The plasmid pBR322-fhs-folD-fchA-gcvTHP and pCDR-fdh-lpd are simultaneously introduced into the defective strain to obtain the SA-5 strain. It was found that 32.6% of the carbon flux of pyruvate detected in strain SA-5 was 13 C-labeled .

[0129] Example 7: Production of shikimic acid by SA-5 using formic acid and glucose as co-substrates

[0130] 1. Preparation of the bacterial agent

[0131] (1) Preparation of the seed solution

[0132] Inoculate the SA-5 strain into 10 ml of LB medium containing appropriate antibiotics (100 μg / ml ampicillin and 33 μg / ml streptomycin), and grow it at 37 °C and a rotation speed of 200 rpm for 12 h to prepare the seed solution.

[0133] (2) Cultivation of the strain

[0134] Transfer the prepared seed solution to the M9 minimal medium containing 5 g / L glucose, 4 g / L sodium formate, and appropriate antibiotics (100 μg / ml ampicillin and 33 μg / ml streptomycin) according to an inoculum size with an initial OD 600 value of 0.1. Transfer the bacterial solution to a new M9 minimal medium (supplemented with 4.5 g / L glucose, 4 g / L sodium formate, 100 μg / ml ampicillin, 33 μg / ml streptomycin) every 12 h. Each time of transfer, the initial OD 600 value is maintained at 0.1, and 0.5 g / L glucose is reduced each time of transfer (i.e., according to 4.5 g / L, 4 g / L, 3.5 g / L, 3 g / L, 2.5 g / L, 2 g / L, 1.5 g / L, 1 g / L, 0.5 g / L, 0);

[0135] After the 10th transfer (after 120 h of cultivation), the initial OD 600 value remained at 0.1. The bacterial liquid was transferred to a new M9 minimal medium (supplemented with 4 g / L sodium formate, 100 μg / ml ampicillin, and 33 μg / ml streptomycin) every 12 h, glucose was no longer added, and the cultivation continued;

[0136] The cultivation temperature was 37 °C, and the total cultivation time was 260 h.

[0137] Finally, after 260 h of cultivation at 37 °C without glucose addition, the OD 600 value in the culture broth was measured to be 0.6.

[0138] (3) Preparation of bacterial agent by high-density fermentation

[0139] After centrifuging the culture broth obtained in step (2), all the precipitate (bacterial cells) was inoculated into a TB liquid medium containing 10 g / L glucose, 150 mM formic acid, 0.1 mM IPTG, and appropriate antibiotics, and grown at 37 °C and 200 rpm for 6 h to obtain a culture broth;

[0140] Then, with an initial inoculum OD600 of 0.34, the culture broth was transferred to an M9 medium supplemented with 10 g / L glucose for high-density fermentation, and fermented at 37 °C for 56 h, concentrated to an OD 600 value of approximately 28. As needed, glucose (10 g / L) was fed every 12 h to ensure that glucose was never depleted.

[0141] 2. Production of shikimic acid using formic acid and glucose as co-substrates

[0142] (1) Preparation of the culture medium

[0143] Fermentation medium: The composition includes a standard fermentation medium (NBS medium, 1 L), containing K2HPO4 (8.5 g), ferric ammonium (III) citrate (0.4 g), citric acid monohydrate (2.3 g), L-phenylalanine (0.7 g), L-tyrosine (0.7 g), L-tryptophan (0.35 g), concentrated H2SO4 (1.2 mL). Before autoclaving the medium, concentrated ammonia water was added to adjust the fermentation medium to pH 7.0.

[0144] (2) Before the start of fermentation, add to the fermentation medium in step (1):

[0145] 20 g / L glucose, MgSO4 (0.24 g / L), formic acid (1.8 g / L), p-hydroxybenzoic acid (0.010 g / L), potassium p-aminobenzoate (0.010 g / L), 2,3-dihydroxybenzoic acid (0.010 g / L), and trace minerals (NH4)6(Mo7O24)·4H2O (0.0037 g / L), ZnSO4·7H2O (0.0029 g / L), H3BO3 (0.0247 g / L), CuSO4·5H2O (0.0025 g / L), MnCl2·4H2O (0.0158 g / L), methyl-α-d-glucopyranoside with a final concentration of 1 mM; separately autoclave the glucose, MgSO4, and methyl-α-d-glucopyranoside solutions, and sterilize the aromatic vitamins and trace mineral solutions with a 0.22 μM membrane. Before adding to the fermentation medium, adjust the pH to 7.0 with KOH and sterilize through a 0.22 μm membrane; add an antifoaming agent (Sigma 204) as needed;

[0146] (3) Detect the fermentation performance of SA-5 strain in a 500 mL shake flask; the specific steps are as follows:

[0147] Inoculate the bacterial agent prepared in step 1 into the fermentation medium obtained in step (2), and the inoculation amount is: 2.5 mL (5%); the fermentation conditions are: 38 °C, 480 rpm, pH 7.0, (after detection, the initial OD of the strain fermentation 600 is 0.3), the aeration rate is 1 vvm, and the total fermentation time is 80 h.

[0148] The results show that: at the end of fermentation, the shikimic acid yield is 3 g / L.

[0149] In summary, we designed an Escherichia coli strain that can grow alone on CO2 and formic acid, and the SA-5 strain increases the assimilation of formic acid and the generation of energy through metabolic engineering. Detection found that, according to the same method, the shikimic acid yield of SA-4 strain is 0 g / L, while the shikimic acid yield of SA-5 strain reaches 3 g / L through high-density fermentation method.

[0150] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications 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. A recombinant Escherichia coli capable of producing shikimic acid using formic acid, characterized in that, The recombinant Escherichia coli uses Escherichia coli with high shikimic acid production using glucose as a substrate as the chassis cell, knocking out the serA gene encoding phosphoglycerate dehydrogenase, the pflB gene encoding pyruvate formate-lyase, and the gcvR gene on the genome, and overexpressing methylenetetrahydrofolate cyclohydrolase, methylenetetrahydrofolate reductase, methylenetetrahydrofolate dehydrogenase, gcvTHP, lipoamide dehydrogenase, and formate dehydrogenase.

2. The recombinant Escherichia coli according to claim 1, characterized in that, The nucleotide sequence encoding the gcvTHP enzyme is as shown in SEQ ID NO.1; the nucleotide sequence of the serA gene encoding phosphoglycerate dehydrogenase is as shown in SEQ ID NO.2; the nucleotide sequence of the pflB gene encoding pyruvate formate-lyase is as shown in SEQ ID NO.3; the nucleotide sequence of the gcvR gene is as shown in SEQ ID NO.4; The nucleotide sequence of the methylenetetrahydrofolate cyclohydrolase-encoding gene is as shown in SEQ ID NO.5; the nucleotide sequence of the methylenetetrahydrofolate dehydrogenase-encoding gene is as shown in SEQ ID NO.6; the nucleotide sequence of the methylenetetrahydrofolate reductase-encoding gene is as shown in SEQ ID NO.7; the nucleotide sequence of the lipoamide dehydrogenase-encoding gene is as shown in SEQ ID NO.8; the nucleotide sequence of the formate dehydrogenase-encoding gene is as shown in SEQ ID NO.

9.

3. The recombinant Escherichia coli according to claim 1 or 2, characterized in that, The recombinant Escherichia coli uses PET series vectors, pETDuet-1 vector, pGEX series vectors, and pTrcHis series vectors as expression vectors; Preferably, the chassis cell is E. coli SA09.

4. A method for improving the ability of Escherichia coli to utilize formic acid, characterized in that, The method is to knock out the serA gene encoding phosphoglycerate dehydrogenase, the pflB gene encoding pyruvate formate-lyase, and the gcvR gene on the genome of E. coli S09, and overexpress methylenetetrahydrofolate cyclohydrolase, methylenetetrahydrofolate reductase, methylenetetrahydrofolate dehydrogenase, gcvTHP, lipoamide dehydrogenase, and formate dehydrogenase.

5. The method according to claim 4, wherein The nucleotide sequence encoding the gcvTHP enzyme is as shown in SEQ ID NO.1; the nucleotide sequence of the serA gene encoding phosphoglycerate dehydrogenase is as shown in SEQ ID NO.2; the nucleotide sequence of the pflB gene encoding pyruvate formate-lyase is as shown in SEQ ID NO.3; the nucleotide sequence of the gcvR gene is as shown in SEQ ID NO.4; The nucleotide sequence of the methylenetetrahydrofolate cyclohydrolase-encoding gene is as shown in SEQ ID NO.5; the nucleotide sequence of the methylenetetrahydrofolate dehydrogenase-encoding gene is as shown in SEQ ID NO.6; the nucleotide sequence of the methylenetetrahydrofolate reductase-encoding gene is as shown in SEQ ID NO.7; the nucleotide sequence of the lipoamide dehydrogenase-encoding gene is as shown in SEQ ID NO.8; the nucleotide sequence of the formate dehydrogenase-encoding gene is as shown in SEQ ID NO.

9.

6. A method for preparing shikimic acid using formic acid and glucose as co-substrates, characterized in that, The method is to use the recombinant Escherichia coli described in any one of claims 1 to 3, with formic acid and glucose as co-substrates, to ferment and prepare shikimic acid.

7. The method according to claim 6, wherein The addition amount of the formic acid is: 1 g / L to 6 g / L; the addition amount of the glucose is: 10 g / L to 20 g / L.

8. The method according to claim 6 or 7, characterized in that, The conditions for the fermentation are: 33 °C to 40 °C; Preferably, the addition amount of recombinant Escherichia coli (initial OD 600 ) is: 0.2 to 0.

4.

9. A method for improving the formic acid tolerance of Escherichia coli, characterized in that, The method is to knockout the serA gene encoding phosphoglycerate dehydrogenase, the pflB gene encoding pyruvate formate-lyase, and the gcvR gene on the genome of E. coli S09, and overexpress methylenetetrahydrofolate cyclohydrolase, methylenetetrahydrofolate reductase, methylenetetrahydrofolate dehydrogenase, gcvTHP, lipoamide dehydrogenase, and formate dehydrogenase; Preferably, the nucleotide sequence encoding the gcvTHP enzyme is as shown in SEQ ID NO.1; the nucleotide sequence of the methylenetetrahydrofolate cyclohydrolase-encoding gene is as shown in SEQ ID NO.5; the nucleotide sequence of the methylenetetrahydrofolate dehydrogenase-encoding gene is as shown in SEQ ID NO.6; the nucleotide sequence of the methylenetetrahydrofolate reductase-encoding gene is as shown in SEQ ID NO.7; the nucleotide sequence of the lipoamide dehydrogenase-encoding gene is as shown in SEQ ID NO.8; the nucleotide sequence of the formate dehydrogenase-encoding gene is as shown in SEQ ID NO.9; The nucleotide sequence of the serA gene encoding phosphoglycerate dehydrogenase is as shown in SEQ ID NO.2; the nucleotide sequence of the pflB gene encoding pyruvate formate-lyase is as shown in SEQ ID NO.3; the nucleotide sequence of the gcvR gene is as shown in SEQ ID NO.

4.

10. Use of the recombinant Escherichia coli described in claims 1 to 3, or the method described in any one of claims 4 to 9 in the preparation of shikimic acid or a product containing shikimic acid.

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