Recombinant Escherichia coli for de novo synthesis of anisic acid, and method and application of producing anisic acid

Abstract

The present invention provides a recombinant Escherichia coli for synthesizing anisic acid from scratch, wherein at least one of the following improvements is made to the starting strain of Escherichia coli: (1) overexpressing O-methyltransferase PSA-OMT1 from Pleurotus sapidus; overexpressing chorismate lyase UbiC from E. coli MG1655 or Providencia rustigianii; (2) overexpressing a feedback inhibition mutant AroG of 3-deoxy-D-arabino-heptanoic acid 7-phosphate (DAHP) synthase from E. coli MG1655 on the basis of (1); D146N (3) Based on (2), the enzyme PpsA derived from phosphoenolpyruvate (PEP) synthase was overexpressed, and the pykF gene encoding pyruvate kinase I was knocked out. Thus, the recombinant E. coli of the present invention can achieve accumulation of anisic acid in the fermentation broth during the fermentation process, and the maximum anisic acid yield of the recombinant E. coli in a shake flask can reach 284.9 mg / L, laying the foundation for the subsequent construction of high-yield anisic acid engineered bacteria.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering and bioengineering, and in particular to a recombinant Escherichia coli for synthesizing anisic acid from scratch, and a method and application for producing anisic acid. Background Art

[0002] Anisic acid's chemical name is 4-methoxybenzoic acid (4MeBA), also known as p-anisic acid and p-methoxybenzoic acid. Its CAS registration number is 100-09-4. Anisic acid is primarily used as an intermediate in the synthesis of many pharmaceuticals and fragrances, such as aniracetam, a drug for treating cerebrovascular sequelae and Alzheimer's disease, the antiarrhythmic drug amiodarone, and methyl anisate, used in fragrances. Furthermore, anisic acid is commonly used as a fragrance and preservative in cosmetic formulations and in food flavorings. Currently, anisic acid is primarily produced by chemical synthesis, with some also being produced through chemical and microbial conversion using trans-anethole and anise oil. However, there have been no reports of de novo biosynthesis of anisic acid using synthetic biology techniques. Chemical synthesis, however, suffers from high production costs, demanding process conditions, numerous byproducts, and potential pollution, and is gradually being replaced by de novo synthesis using microbial fermentation. Summary of the Invention

[0003] The main purpose of the present invention is to provide a recombinant Escherichia coli for synthesizing anisic acid from scratch and a method and application for producing anisic acid, so as to at least solve the problems of high cost and large number of by-products in the prior art of synthesizing anisic acid by chemical synthesis.

[0004] To achieve the above objectives, the present invention provides a recombinant Escherichia coli for de novo synthesis of anisic acid, wherein the starting strain of Escherichia coli is improved by at least one of the following:

[0005] (1) Overexpression of PSA-OMT1, an O-methyltransferase derived from Pleurotus sapidus; overexpression of UbiC, a chorismate lyase derived from E. coli MG1655 or Providencia rustigianii;

[0006] (2) Based on (1), overexpression of the feedback inhibition mutant AroG of 3-deoxy-D-arabino-heptanoic acid 7-phosphate (DAHP) synthase from E. coli MG1655 D146N ;

[0007] (3) Based on (2), PpsA derived from phosphoenolpyruvate (PEP) synthase was overexpressed, and the pykF gene encoding pyruvate kinase I was knocked out;

[0008] Wherein, the nucleotide sequence of the codon-optimized O-methyltransferase PSA-OMT1 is shown in SEQ ID No. 1;

[0009] The nucleotide sequence of the chorismate lyase UbiC from E. coli MG1655 is shown in SEQ ID No. 2;

[0010] The nucleotide sequence of the codon-optimized chorismate lyase UbiC from Providencia rustigianii is shown in SEQ ID No. 3;

[0011] The 3-deoxy-D-arabino-heptanoic acid 7-phosphate (DAHP) synthase feedback inhibition mutant AroG D146N The nucleotide sequence is shown in SEQ ID No. 4;

[0012] The nucleotide sequence of the phosphoenolpyruvate (PEP) synthase PpsA is shown in SEQ ID No.5.

[0013] Optionally, pTrc99A is used as an expression vector to express the O-methyltransferase PSA-OMT1 and chorismate lyase UbiC.

[0014] Alternatively, pTrc99A or pBBR1Tac is used as an expression vector to express the 3-deoxy-D-arabino-heptanoic acid 7-phosphate (DAHP) synthase feedback inhibition mutant AroG D146N .

[0015] Optionally, pCDFTac is used as an expression vector to express the phosphoenolpyruvate (PEP) synthase PpsA.

[0016] Optionally, the recombinant Escherichia coli uses Escherichia coli MG1655 as a starting strain.

[0017] The present invention also provides a method for producing anisic acid, which comprises using the recombinant Escherichia coli described in the present application as a fermentation strain and glucose as a substrate to ferment and produce anisic acid.

[0018] Optionally, the method includes: inoculating the recombinant Escherichia coli into a fermentation medium, fermenting and culturing at 28-32° C. and 100-500 rpm for 48-72 hours, adding isopropyl-β-D-thiogalactopyranoside (IPTG) with a final concentration of 0.1-1.0 mM for induction when OD600 = 0.5-0.8, and collecting the supernatant after the fermentation to separate and purify anisic acid.

[0019] Optionally, the fermentation medium comprises glucose, MgSO4·7H2O, KH2PO4, (NH4)2HPO4, citric acid, L-methionine, trace metal salt solution, pH 6.5-7.5, and deionized water.

[0020] The present invention also provides an application of the method for producing anisic acid in preparing anisic acid-containing products by microbial fermentation.

[0021] The present invention provides a recombinant Escherichia coli for synthesizing anisic acid from scratch, wherein at least one of the following improvements is made to the starting strain of Escherichia coli: (1) overexpressing the O-methyltransferase PSA-OMT1 from Pleurotus sapidus; overexpressing the chorismate lyase UbiC from E. coli MG1655 or Providencia rustigianii; (2) overexpressing the feedback inhibition mutant AroG of 3-deoxy-D-arabino-heptanoic acid 7-phosphate (DAHP) synthase from E. coli MG1655 on the basis of (1). D146N ; (3) Based on (2), the phosphoenolpyruvate (PEP) synthase PpsA is overexpressed, and the pykF gene encoding pyruvate kinase I is knocked out; wherein, the nucleotide sequence of the codon-optimized O-methyltransferase PSA-OMT1 is shown in SEQ ID No.1; the nucleotide sequence of the chorismate lyase UbiC from the E. coli MG1655 is shown in SEQ ID No.2; the nucleotide sequence of the codon-optimized chorismate lyase UbiC from Providencia rustigianii is shown in SEQ ID No.3; the feedback inhibition mutant of the 3-deoxy-D-arabino-heptanoic acid 7-phosphate (DAHP) synthase AroG D146N The nucleotide sequence of the enzyme is shown in SEQ ID No. 4; the nucleotide sequence of the phosphoenolpyruvate (PEP) synthase PpsA is shown in SEQ ID No. 5. Thus, the recombinant E. coli of the present invention can achieve accumulation of anisic acid in the fermentation broth during fermentation, and the maximum anisic acid yield in shake flasks of the recombinant E. coli can reach 284.9 mg / L, laying the foundation for the subsequent construction of high-yield anisic acid-producing engineered bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0023] Figure 1 This is a schematic diagram of an anisic acid biosynthesis pathway and genetic engineering modification in Escherichia coli that can be constructed according to an embodiment of the present invention;

[0024] Figure 2 is a map of plasmid pLPAA01 which can be selected according to an embodiment of the present invention;

[0025] Figure 3 is a map of plasmid pLPAA02 that can be selected according to an embodiment of the present invention;

[0026] Figure 4 is a map of plasmid pLPAA03 which can be selected according to an embodiment of the present invention;

[0027] Figure 5 is a map of plasmid pLPAA04, which can be selected according to an embodiment of the present invention;

[0028] Figure 6 is a map of plasmid pLPAA05, which can be selected according to an embodiment of the present invention;

[0029] Figure 7 is a map of plasmid pLPAA06, which can be selected according to an embodiment of the present invention;

[0030] Figure 8 1. The fermentation production of anisic acid by strains LPAA01 to LPAA05 and the corresponding plasmids according to the embodiments of the present invention are shown in FIG.

[0031] Figure 9 1 is a liquid phase peak diagram of optional p-hydroxybenzoic acid and anisic acid standards and LPAA03 fermentation sample according to an embodiment of the present invention;

[0032] Figure 10 The figures show the fermentation production of anisic acid by the strains LPAA05 and LPAA06 selected according to the embodiments of the present invention under different conditions. DETAILED DESCRIPTION

[0033] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments in this application can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0034] like Figure 1As shown, glucose in Escherichia coli first undergoes glycolysis and the pentose phosphate pathway to produce phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P), respectively. Most of PEP is converted to pyruvate (PYR) catalyzed by pyruvate kinase I (PykF) and pyruvate kinase II (PykA), and then enters the tricarboxylic acid cycle (TCA cycle). PYR is catalyzed to PEP by phosphoenolpyruvate synthase (PpsA). Therefore, knocking out the pykF gene and overexpressing the ppsA gene can enhance the synthesis of intracellular PEP. PEP and E4P are catalyzed by 3-deoxy-D-arabino-heptanoic acid 7-phosphate (DAHP) synthase AroG to produce DAHP, which is then catalyzed to chorismate (CHA) by six enzymes in the shikimate pathway. AroG mutants D146N This can relieve the feedback inhibition of L-phenylalanine and increase the carbon flux to chorismate. The chorismate lyase UbiC, derived from Escherichia coli or Providencia rustigianii, cleaves chorismate into PYR and p-hydroxybenzoic acid (4HBA). The g12203 gene from Pleurotus sapidus encodes an O-methyltransferase, PSA-OMT1, which uses S-adenosyl-L-methionine (SAM) as a methyl donor to convert p-hydroxybenzoic acid to anisic acid, simultaneously generating S-adenosyl-L-homocysteine ​​(SAH). Therefore, constructing this biosynthesis pathway in E. coli and enhancing the carbon flux to this pathway is a promising approach for developing microbial anisic acid producers and holds considerable promise for future applications.

[0035] Specifically, the present application makes at least one of the following improvements to E. coli MG1655:

[0036] (1) The O-methyltransferase PSA-OMT1 from Pleurotus sapidus was expressed using the pTrc99A plasmid as an expression vector, and the chorismate lyase UbiC from E. coli MG1655 or Providencia rustigianii was expressed using the pTrc99A plasmid as an expression vector, thereby preliminarily constructing a recombinant E. coli strain capable of synthesizing anisic acid from scratch using glucose as a substrate. The nucleotide sequence of the codon-optimized O-methyltransferase PSA-OMT1 (g12203 opt) is shown in SEQ ID No.1; the nucleotide sequence of the endogenous chorismate lyase UbiC of E. coli MG1655 is shown in SEQ ID No.2; the nucleotide sequence of the codon-optimized chorismate lyase UbiC from Providencia rustigianii is shown in SEQ ID No.3.

[0037] (2) Based on (1), the pBBR1Tac plasmid was used as an expression vector to express the feedback inhibition mutant AroG of 3-deoxy-D-arabino-heptanoic acid 7-phosphate (DAHP) synthase from E. coli MG1655. D146N , to enhance the carbon flow of the shikimate pathway and increase the concentration of the precursor chorismate. 3-deoxy-D-arabino-heptanoic acid 7-phosphate (DAHP) synthase feedback inhibition mutant AroG D146N The nucleotide sequence is shown in SEQ ID No.4.

[0038] (3) Based on (2), the pCDFTac plasmid was used as an expression vector to express PpsA, a phosphoenolpyruvate (PEP) synthase, and the pykF gene encoding pyruvate kinase I was knocked out to increase the concentration of PEP, a precursor of DAHP. The nucleotide sequence of PpsA is shown in SEQ ID No. 5.

[0039] The present application also proposes a method for producing anisic acid, comprising using the recombinant Escherichia coli constructed in the present application as a fermentation strain and fermenting anisic acid with glucose as a substrate.

[0040] After obtaining recombinant E. coli, each recombinant E. coli was used as a fermentation strain, and glucose was used as a substrate to ferment and produce anisic acid. The specific process is as follows: the anisic acid-producing bacteria were inoculated into a fermentation medium, and fermentation was carried out at 28-32°C and 100-500 rpm for 48-72 hours. After the fermentation, the supernatant was separated and purified to obtain anisic acid. The fermentation medium contained glucose, MgSO4·7H2O, KH2PO4, (NH4)2HPO4, citric acid, L-methionine, and a trace metal salt solution; the pH was 6.5-7.5, and the solvent was deionized water. The composition of the trace metal salt solution is: 10g / L FeSO4·7H2O, 2.65g / L CaCl2·2H2O, 2.2g / L ZnSO4·7H2O, 0.58g / L MnSO4·5H2O, 1g / L CuSO4·5H2O, 0.1g / L (NH4)6Mo7O 24 ·4H2O, 0.02g / LNa2B4O7·10H2O, 10mL / L35% HCl, the solvent is deionized water.

[0041] Before fermentation, the recombinant E. coli strain was inoculated into LB medium and cultured overnight on a shaker at 37°C and 200 rpm. The inoculum was then added to the fermentation medium at a concentration of 1-5% by volume. The total fermentation time was 60 hours. The recombinant E. coli was inoculated into the fermentation medium and cultured to an OD600 of 0.5-0.8, at which point IPTG was added to induce the fermentation reaction at a final concentration of 0.1-1.0 mM.

[0042] The method for producing anisic acid in the present application can be applied to the preparation of anisic acid-containing products by microbial fermentation.

[0043] The present invention is further illustrated by the following examples.

[0044] In the following examples, ampicillin (Ap) was used at a final concentration of 100 mg / L, chloramphenicol (Cm) was used at a final concentration of 34 mg / L, and streptomycin (Sm) was used at a final concentration of 50 mg / L. The E. coli competent cells used in the examples were E. coli DH5a, restriction endonucleases were purchased from TAKARA, and the seamless cloning kit used in the recombination reaction was purchased from Abotek, but the examples are not limited to these companies.

[0045] Example 1: Construction of pLPAA01 to pLPAA03 plasmids

[0046] The pTrc99A plasmid was digested with restriction endonuclease NcoI to obtain a linearized plasmid vector fragment. At the same time, PSA-OMT1_F and PSA-OMT1_R were used as primers to clone the plasmid containing g12203 containing the fully synthesized O-methyltransferase PSA-OMT1. opt The plasmid of the gene was used as a template to amplify g12203 opt Gene (sequence shown in SEQ ID No. 1). opt The gene fragment was subjected to agarose gel electrophoresis, and the correct band was excised and purified using a gel extraction kit. Recombination was then performed using a seamless cloning kit. The reaction product was transformed into competent E. coli cells and plated onto LB plates containing ampicillin sodium. Single clones were verified by colony PCR using primers Seq-pTrc99A_F and Seq-pTrc99A_R. Positive clones were sent to a sequencing company for sequencing to confirm that the ligated plasmid pLPAA01 was successfully constructed.

[0047] The pLPAA01 plasmid was digested with restriction endonuclease XbaI to obtain a linearized plasmid vector fragment; ubiC_F and ubiC_R were used as primers and the Escherichia coli genome was used as a template to amplify the ubiC gene (sequence shown in SEQ ID No. 2); PrubiC_F and PrubiC_R were used as primers to amplify the ubiC gene containing the fully synthesized PrubiC opt The plasmid of the gene was used as a template to amplify PrubiC opt Gene (sequence shown in SEQ ID No. 3). The linearized pLPAA01 fragment after enzyme digestion and the amplified ubiC gene fragment, PrubiC opt The gene fragment was subjected to agarose gel electrophoresis, the correct band was cut out, and the fragment was purified using a gel recovery kit. The ubiC gene fragment and the purified pLPAA01 fragment were recombined using a seamless cloning kit. The reaction product was transformed into E. coli competent cells and then spread on LB plates containing ampicillin sodium. Using Seq-pTrc99A_F and Seq-pTrc99A_R as primers, colony PCR was performed to verify the single clone. The positive clone was sent to a sequencing company for sequencing to confirm the successful connection of the plasmid pLPAA02. opt The gene fragment was recombined with the purified pLPAA01 fragment using a seamless cloning kit. The reaction product was transformed into competent E. coli cells and plated on LB plates containing ampicillin sodium. Single clones were verified by colony PCR using primers Seq-pTrc99A_F and Seq-pTrc99A_R. Positive clones were sent to a sequencing company for sequencing, confirming that the ligated plasmid pLPAA03 was successfully obtained.

[0048] Example 2: Construction of pLPAA04-pLPAA06 plasmids

[0049] The linearized plasmid fragment was amplified by PCR using pBBR1Tac plasmid as template and pBBR1Tac_F and pBBR1Tac_R as primers. Meanwhile, aroG1_F and aroGf1_R, aroGf2_F and aroG2_R were used as primers and the Escherichia coli genome was used as template to amplify the aroG D146N(Sequence shown in SEQ ID No. 4) fragments aroG-F1 and aroG-F2. Linearized pBBR1Tac, aroG-F1, and aroG-F2 fragments were subjected to agarose gel electrophoresis. The correct bands were excised and purified using a gel recovery kit. Recombination reactions were then performed using a seamless cloning kit. The reaction products were transformed into competent E. coli cells and plated onto LB plates containing chloramphenicol. Using primers Seq-pBBR1Tac_F and Seq-pTrc99A_R, colony PCR was performed to verify single clones. Positive clones were sent to a sequencing company for sequencing to confirm the successful ligation of plasmid pLPAA05.

[0050] The pLPAA02 plasmid was double-digested with restriction endonucleases KpnⅠ and HindⅢ to obtain a linearized plasmid vector fragment; using ubiCT_F and ubiCT_R as primers and the pLPAA02 plasmid as a template, the ubiC gene and rrnB terminator were amplified; using tac-aroG_F and tac-aroG_R as primers and the pLPAA05 plasmid as a template, the tac-aroG D146N The linearized pLPAA02 fragment after enzyme digestion and the amplified ubiC gene and rrnB terminator fragment, tac-aroG D146N The fragments were subjected to agarose gel electrophoresis, and the correct bands were excised and purified using a gel extraction kit. Recombination reactions were then performed using a seamless cloning kit. The reaction products were transformed into competent E. coli cells and plated onto LB plates containing ampicillin sodium. Single clones were verified by colony PCR using primers Seq-pTrc99A_F and Seq-pTrc99A_R. Positive clones were sent to a sequencing company for sequencing, confirming that the ligated plasmid pLPAA04 was successfully constructed.

[0051] The pCDFTac plasmid was double-digested with restriction endonucleases EcoRI and SacI to obtain a linearized plasmid vector fragment. Simultaneously, the ppsA gene (sequence shown in SEQ ID No. 5) was amplified using primers ppsA_F and ppsA_R and the E. coli genome as a template. The linearized pCDFTac fragment and the amplified ppsA gene fragment were subjected to agarose gel electrophoresis. The correct bands were excised and purified using a gel recovery kit. Recombination was then performed using a seamless cloning kit. The reaction products were transformed into competent E. coli cells and plated on LB plates containing streptomycin sulfate. Single clones were verified by colony PCR using primers Seq-pCDFTac_F and Seq-pTrc99A_R. Positive clones were sent to a sequencing company for sequencing, confirming the successful ligation of the plasmid pLPAA06.

[0052] Example 3: Knockout of the pykF gene in E. coli MG1655

[0053] (1) Preparation of linearized fragment KO-ΔpykF

[0054] Using the E. coli genome as a template, 500 bp upstream and downstream sequences of the target gene were amplified using primers ΔpykF-up_F and ΔpykF-up_R, and ΔpykF-down_F and ΔpykF-down_R, respectively, to generate the DNA fragments ΔpykF-up and ΔpykF-down. Using the pUTrc plasmid as a template and primers KO_F and KO_R, a lox71-CmR-lox66 gene fragment, designated fKO, was amplified. Overlap extension PCR was performed using ΔpykF-up and fKO as templates and ΔpykF-up_F and KO_R as primers to generate the fragment up-fKO. Furthermore, overlap extension PCR was performed using up-fKO and ΔpykF-down as templates and primers ΔpykF-up_F and ΔpykF-down_R to generate the fragment KO-ΔpykF.

[0055] (2) Introduction of auxiliary plasmid pKD46

[0056] Preparation of electrocompetent E. coli MG1655: Inoculate laboratory-stored E. coli MG1655 into a test tube containing 5 mL of LB and shake at 37°C, 200 rpm, for 12-16 hours. Inoculate 2% of the total volume into 100 mL of fresh LB medium and add L-arabinose to a final concentration of 10 mM. Shake at 37°C, 200 rpm, for 1.5 hours until the OD600 reaches 0.4-0.6. Centrifuge and discard the supernatant. Wash twice with 10% glycerol. Finally, add a sufficient amount of 10% glycerol to a final volume of 600-800 μL. Aliquot 100 μL into 1.5 mL centrifuge tubes and store in a -80°C freezer.

[0057] To introduce the helper plasmid pKD46: Remove a competent E. coli MG1655 cell from a -80°C freezer, thaw on ice, mix thoroughly with approximately 50 ng of plasmid pKD46, transfer to a pre-chilled 1 mm electroporation cuvette, and incubate on ice for 10 minutes. Wipe the outside of the cuvette dry with paper, place in an electroporator, and electroporate once at setting Ec1. Immediately add 600 μL of pre-chilled SOC medium, gently mix, tilt the cuvette, and transfer the entire mixture to a sterile 1.5 mL centrifuge tube. Shake at 200 rpm at 30°C for 80 minutes. Spread 80 μL of the mixture onto an LB plate containing ampicillin sodium and incubate overnight at 30°C.

[0058] (3) Insertion of the linearized fragment KO-ΔpykF

[0059] Single colonies from the plate in step (2) were inoculated into a test tube containing 5 mL of LB and sodium ampicillin and shaken at 30°C, 200 rpm for 16-20 h. 2% of the volume was inoculated into 15 mL of fresh LB medium and shaken at 30°C, 200 rpm for 2 h. When OD600 reached 0.4-0.6, the supernatant was discarded after centrifugation and washed twice with 10% glycerol. Finally, a certain amount of 10% glycerol was added to make the final volume 100 uL. The cells were transferred to a 1.5 mL centrifuge tube and thoroughly mixed with approximately 500 ng of the KO-ΔpykF fragment. The cells were transformed by electroporation in the same manner as in step (2). The recovered culture mixture was then spread onto an LB plate containing sodium ampicillin and chloramphenicol and cultured overnight at 30°C. Colony PCR was performed on the single colonies on the plate using Seq-ΔpykF_F and Seq-KO_R as primers to screen for positive clones.

[0060] (4) Elimination of auxiliary plasmid pKD46

[0061] The positive clones from step (3) were streaked onto LB plates containing chloramphenicol and cultured overnight at 42°C. Single clones from the plates were transferred to 5 mL LB tubes containing or without ampicillin sodium to verify whether plasmid pKD46 was eliminated.

[0062] (5) Elimination of screening marker CmR

[0063] The strain with plasmid pKD46 eliminated was inoculated into a test tube containing 5 mL of LB and chloramphenicol and shaken at 37°C, 200 rpm for 12-16 hours. 2% of the volume was inoculated into 15 mL of fresh LB medium and shaken at 37°C, 200 rpm for 1.5 hours. When OD600 reached 0.4-0.6, the supernatant was discarded after centrifugation and washed twice with 10% glycerol. Finally, a certain amount of 10% glycerol was added to make the final volume 100 uL. The strain was transferred to a 1.5 mL centrifuge tube and thoroughly mixed with approximately 50 ng of plasmid pjw168. The strain was transformed by electroporation in the same manner as in step (2) and plated onto an LB plate containing sodium ampicillin and 1 mM IPTG. The strain was cultured at 30°C overnight. Single clones on the plate were subjected to colony PCR using primers Seq-ΔpykF_F and Seq-ΔpykF_R to screen for positive clones.

[0064] (6) Elimination of auxiliary plasmid pjw168

[0065] Streak the positive clones from step (5) onto LB plates and culture overnight at 42°C. Transfer the single colonies from the plates to 5 mL LB tubes containing or without ampicillin sodium to verify the elimination of plasmid pjw168. The strain with successful elimination is designated strain MG1655ΔpykF.

[0066] Table 1 Genotypes of strains and plasmids

[0067]

[0068]

[0069] Table 2 Primer sequence list

[0070]

[0071]

[0072] Example 4: Construction of LPAA01 to LPAA06 strains

[0073] Inoculate E. coli MG1655 into a test tube containing 5 mL of LB and shake at 37°C, 200 rpm, for 12-16 hours. Inoculate 2% of the total volume into 100 mL of fresh LB medium and shake at 37°C, 200 rpm, for 1.5 hours. When OD600 reaches 0.4-0.6, centrifuge and discard the supernatant. Wash twice with 10% glycerol. Finally, add a sufficient amount of 10% glycerol to a final volume of 500 μL. Aliquot 100 μL into a 1.5 mL centrifuge tube. Four of the competent cells were fully mixed with approximately 50 ng of plasmids pTrc99A, pLPAA02, pLPAA03, and pLPAA04, respectively, and electroporated in the same manner as step (2) in Example 3, and plated on an LB plate containing sodium ampicillin, and cultured overnight at 37°C to obtain LPAA01, LPAA02, LPAA03, and LPAA04, respectively; one competent cell was fully mixed with 50 ng each of plasmids pLPAA02 and pLPAA05, and electroporated in the same manner as step (2) in Example 3, and plated on an LB plate containing sodium ampicillin and chloramphenicol, and cultured overnight at 37°C to obtain LPAA05.

[0074] MG1655ΔpykF was made competent as described above, and plasmids pLPAA02, pLPAA05, and pLPAA06 were introduced in sequence, with attention paid to adding corresponding resistance in each culture medium, to finally obtain strain LPAA06.

[0075] Example 5: Fermentation of LPAA01 to LPAA05 strains

[0076] Seed culture: Pick a single clone from the corresponding plate and inoculate it into a test tube containing 5 mL of seed culture medium (LB medium) and culture it at 37°C and 200 rpm overnight;

[0077] Shake flask fermentation: Inoculate 50 mL of fermentation medium in a 250 mL baffled shake flask at 2% seed solution at 2% inoculum. Incubate at 30°C, shaking at 200 rpm for 60 h. Add IPTG to a final concentration of 1 mM 8 h after inoculation (OD600 = 0.5-0.8). Perform three biological replicates per group.

[0078] The shake flask fermentation medium consisted of 10 g / L glucose, 0.8 g / L MgSO4·7H2O, 6.67 g / L KH2PO4, 4 g / L (NH4)2HPO4, 0.8 g / L citric acid, and 5 mL / L trace metal salt solution, pH 7.0. The glucose and MgSO4·7H2O stock solutions were prepared at concentrations of 500 g / L and 80 g / L, respectively, and sterilized separately by moist heat. The trace metal salt solution consisted of 10 g / L FeSO4·7H2O, 2.65 g / L CaCl2·2H2O, 2.2 g / L ZnSO4·7H2O, 0.58 g / L MnSO4·5H2O, 1 g / L CuSO4·5H2O, and 0.1 g / L (NH4)6Mo7O. 24 ·4H2O, 0.02g / LNa2B4O7·10H2O, 10mL / L 35% HCl, the solvent is deionized water.

[0079] After the fermentation was completed, 1 mL of fermentation broth was taken, 100 μL of which was used to dilute 10 times to detect the OD value of the bacterial solution. The remaining 900 μL was centrifuged at 16000 × g for 10 min, the supernatant was filtered through a 0.22 μm aqueous filter membrane, and diluted 5 times. The results were detected by high performance liquid chromatography. Figure 8 As shown, LPAA02 produced 167.9 mg / L of anisic acid, while LPAA05 produced 145.4 mg / L. Residual p-hydroxybenzoic acid (52.0 mg / L) was also observed, along with a low OD600 value. This may be due to excessively high IPTG concentrations. Furthermore, insufficient SAM cofactor supply may also be a factor limiting anisic acid production.

[0080] The HPLC system was Shimadzu LC-20 series, and the chromatographic column was Agilent Poroshell 120EC-C18 (4.6×150 mm). Phase A was ddH2O containing 0.1% trifluoroacetic acid, and phase B was methanol. The flow rate was 0.65 mL / min according to the following program: 0-1 min, 10% B; 1-21 min, linear gradient increase of B from 10% to 50%; 21-22 min, linear gradient increase of B from 50% to 70%; 22-24 min, 70% B; 24-26 min, linear gradient decrease of B from 70% to 10%; 26-35 min, 10% B. The injection volume was 5 uL, the column oven was at 30°C, and the detection wavelength of the UV-Vis spectrophotometer was 246 nm. Figure 9 As shown, peak 1 is p-hydroxybenzoic acid standard, with an elution time of 15.225 min; peak 2 is anisic acid standard, with an elution time of 26.738 min. Peaks 3 and 4 are samples of LPAA3, identified as p-hydroxybenzoic acid and anisic acid, respectively.

[0081] Example 6: Fermentation of LPAA05 and LPAA06 under different conditions

[0082] LPAA05 was fermented in a shake flask in the same manner as in Example 5, but IPTG was added at final concentrations of 0.10 mM, 0.25 mM, 0.50 mM, and 0.75 mM 8 h after inoculation (OD600 = 0.5-0.8). Figure 10 As shown, when the IPTG concentration was 0.1 mM, the anisic acid production reached 222.3 mg / L, and 22.2 mg / L of p-hydroxybenzoic acid remained.

[0083] LPAA05 and LPAA06 were fermented in shake flasks in the same manner as in Example 5, except that 8 h after inoculation (OD600 = 0.5-0.8), IPTG was added to a final concentration of 0.10 mM and L-methionine was added to a stock solution of 50 g / L. The L-methionine solution was sterilized by filtration. The results were shown in the following table. Figure 9 As shown, under these conditions, LPAA05 and LPAA06 produced 281.0 mg / L of anisic acid, respectively, and 284.9 mg / L, with virtually no residual para-hydroxybenzoic acid. In particular, the OD600 of LPAA05 increased significantly after addition of L-methionine, indicating that insufficient SAM supply also affected strain growth.

[0084] After genetic engineering, the genetically engineered strain LPAA06 achieved an anisic acid yield of 284.9 mg / L in shake flask fermentation. Therefore, the anisic acid-producing genetically engineered bacteria constructed in the present invention can effectively accumulate anisic acid in the fermentation broth during fermentation, laying the foundation for the construction of genetically engineered strains that produce high anisic acid yields.

[0085] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

[0086]

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Claims

1. A recombinant Escherichia coli for de novo synthesis of anisic acid, characterized in that: At least one of the following improvements has been made to the starting E. coli strain: (1) Overexpression of PSA-OMT1, an O-methyltransferase from Pleurotus sapidus; overexpression of UbiC, a chorismate lyase from E. coli MG1655 or Providencia rustigianii; (2) Based on (1), the feedback inhibition mutant AroG of 3-deoxy-D-arabino-heptanoic acid 7-phosphate (DAHP) synthase from E. coli MG1655 was overexpressed. D146N ; (3) Based on (2), PpsA derived from phosphoenolpyruvate (PEP) synthase was overexpressed, and the pykF gene encoding pyruvate kinase I was knocked out; Wherein, the nucleotide sequence of the O-methyltransferase PSA-OMT1 is shown in SEQ ID No. 1; The nucleotide sequence of the chorismate lyase UbiC from E. coli MG1655 is shown in SEQ ID No. 2; The nucleotide sequence of the chorismate lyase UbiC from Providencia rustigianii is shown in SEQ ID No. 3; The 3-deoxy-D-arabino-heptanoic acid 7-phosphate (DAHP) synthase feedback inhibition mutant AroG D146N The nucleotide sequence is shown in SEQ ID No. 4; The nucleotide sequence of the phosphoenolpyruvate (PEP) synthase PpsA is shown in SEQ ID No.

5.

2. The recombinant Escherichia coli according to claim 1, characterized in that pTrc99A was used as an expression vector to express the O-methyltransferase PSA-OMT1 and chorismate lyase UbiC.

3. The recombinant Escherichia coli according to claim 1, characterized in that The 3-deoxy-D-arabino-heptanoic acid 7-phosphate (DAHP) synthase feedback inhibition mutant AroG was expressed using pTrc99A or pBBR1Tac as an expression vector. D146N .

4. The recombinant Escherichia coli according to claim 1, characterized in that The phosphoenolpyruvate (PEP) synthase PpsA was expressed using pCDFTac as an expression vector.

5. The recombinant Escherichia coli according to claim 1, characterized in that The recombinant Escherichia coli uses Escherichia coli MG1655 as the starting strain.

6. A method for producing anisic acid, characterized in that: The method comprises using the recombinant Escherichia coli according to any one of claims 1 to 5 as a fermentation strain and glucose as a substrate to ferment and produce anisic acid.

7. The method for producing anisic acid according to claim 6, wherein The recombinant Escherichia coli is inoculated into a fermentation medium, and fermented at 28-32° C. and 100-500 rpm for 48-72 hours. When OD600 is 0.5-0.8, isopropyl-β-D-thiogalactopyranoside (IPTG) is added to induce the culture at a final concentration of 0.1-1.0 mM. After the fermentation is completed, the supernatant is collected and separated and purified to obtain anisic acid.

8. The method for producing anisic acid according to claim 7, wherein The fermentation medium comprises glucose, MgSO4·7H2O, KH2PO4, (NH4)2HPO4, citric acid, l-methionine, trace metal salt solution, pH value of 6.5-7.5, and deionized water.

9. Use of the method for producing anisic acid according to claim 6 in preparing anisic acid-containing products by microbial fermentation.

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