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

Abstract

The invention provides recombinant escherichia coli for de novo synthesis of anisic acid. At least one of the following improvements is carried out on an escherichia coli starting strain: (1) overexpression of O-methyltransferase PSA-OMT1 from Pleuromyces sapidus, (2) overexpression of O-methyltransferase PSA-OMT1 from Pleuromyces sapidus, and (3) overexpression of O-methyltransferase PSA-OMT1 from Pleuromyces sapidus; the method comprises the following steps: carrying out overexpression on chorismate lyase UbiC derived from E.coli MG1655 or Providencia rustiana, and carrying out the overexpression on the chorismate lyase UbiC derived from E.coli MG1655 or Providencia rustiana; (2) carrying out overexpression on a 3-deoxy-D-arabic-heptanone acid 7-phosphate (DAHP) synthase feedback inhibition mutant AroGD146N derived from E.coli MG1655 on the basis of the step (1); and (3) overexpressing synthase PpsA derived from phosphoenolpyruvate (PEP) on the basis of the step (2), and knocking out a pykF gene for coding pyruvate kinase I. Therefore, in the fermentation process of the recombinant escherichia coli, the accumulation of the anisic acid in the fermentation liquor can be realized, the maximum anisic acid shake flask yield of the recombinant escherichia coli can reach 284.9 mg / L, and a foundation is laid for the subsequent construction of high-yield anisic acid engineering 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] The chemical name of anisic acid is 4-methoxybenzoic acid (4MeBA), also known as p-anisic acid, p-methoxybenzoic acid, etc. Its CAS registration number is 100-09-4. Anisic acid is mainly used as an intermediate in the synthesis of many drugs and spices, such as aniracetam, a drug for treating cerebrovascular sequelae and Alzheimer's disease, antiarrhythmic drug ethiophenone, and methyl anisate for preparing flavors. In addition, anisic acid is often used as a fragrance and preservative in cosmetic formulas, and is also used in food flavors. At present, anisic acid is mainly produced by chemical synthesis, and it is also produced by chemical transformation and microbial transformation using trans-anethole and anise oil as raw materials. However, there has been no report on the de novo biosynthesis of anisic acid using synthetic biology technology. The chemical synthesis method has problems such as high production cost, harsh process conditions, more by-products, and easy pollution, and will gradually be replaced by de novo synthesis by 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 problem of high cost and large number of by-products in the prior art of synthesizing anisic acid by chemical synthesis.

[0004] In order to achieve the above object, 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:

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

[0006] (2) Based on (1), the 3-deoxy-D-arabino-heptanoic acid 7-phosphate (DAHP) synthase feedback inhibition mutant AroG from E. coli MG1655 was overexpressed. 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 derived from the 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 the 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 100-500rpm for 48-72h, adding isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.1-1.0mM for induction when OD600=0.5-0.8, and after the fermentation is completed, separating and purifying the supernatant to obtain anisic acid.

[0019] Optionally, the components of the fermentation medium include: glucose, MgSO4·7H2O, KH2PO4, (NH4)2HPO4, citric acid, L-methionine, trace metal salt solution; pH 6.5-7.5, 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 technical scheme of the present invention is 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 AroG, a feedback inhibition mutant 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), overexpression of phosphoenolpyruvate (PEP) synthase PpsA and knockout of pyruvate kinase I encoding pykF gene; wherein the nucleotide sequence of the codon-optimized O-methyltransferase PSA-OMT1 is as shown in SEQ ID No.1; the nucleotide sequence of the chorismate lyase UbiC from E.coliMG1655 is as shown in SEQ ID No.2; the nucleotide sequence of the codon-optimized chorismate lyase UbiC from Providencia rustigianii is as 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 of is shown in SEQ ID No. 4; the nucleotide sequence of the phosphoenolpyruvate (PEP) synthase PpsA is shown in SEQ ID No. 5. Therefore, the recombinant Escherichia coli of the present invention can achieve the accumulation of anisic acid in the fermentation broth during the fermentation process, and the maximum anisic acid shake flask yield of the recombinant Escherichia coli can reach 284.9 mg / L, which lays a foundation for the subsequent construction of high-yield anisic acid engineering bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0023] Figure 1 This is a schematic diagram of 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 which 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 It is a schematic diagram of fermentation production of anisic acid by strains LPAA01 to LPAA05 and corresponding plasmids selected according to an embodiment of the present invention;

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

[0032] Fig.10 The following are the results of fermentation 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 in the embodiments of the present 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 first passes through the glycolysis pathway and the pentose phosphate pathway in Escherichia coli to generate phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P), respectively. Most of PEP is converted into pyruvate (PYR) by pyruvate kinase I (PykF) and pyruvate kinase II (PykA), and then enters the tricarboxylic acid cycle (TCA cycle). PYR can be catalyzed into PEP by phosphoenolpyruvate synthase (PpsA). Therefore, by knocking out the pykF gene and overexpressing the ppsA gene, the synthesis of intracellular PEP can be enhanced. PEP and E4P are catalyzed by 3-deoxy-D-arabino-heptanoic acid 7-phosphate (DAHP) synthase AroG to generate DAHP, and then DAHP is catalyzed into chorismate (CHA) by 6 enzymes in sequence through the shikimate pathway. AroG mutant AroG D146N It can relieve the feedback inhibition of L-phenylalanine and increase the carbon flow to chorismic acid. The chorismic acid lyase UbiC from Escherichia coli or Providencia rustigianii can cleave chorismic acid into PYR and p-hydroxybenzoic acid (4HBA). The g12203 gene from Pleurotus sapidus encodes an O-methyltransferase PSA-OMT1, which can convert p-hydroxybenzoic acid into anisic acid using S-adenosyl-L-methionine (SAM) as a methyl donor and generate S-adenosyl-L-homocysteine ​​(SAH). Therefore, constructing this synthetic pathway in E. coli and enhancing the carbon flow to this pathway is a good way to construct microbial production of anisic acid, which has certain application prospects.

[0035] Specifically, the present application makes at least one of the following improvements to Escherichia coli 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 O-methyltransferase PSA-OMT1 after codon optimization (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. AroG, a feedback inhibition mutant of 3-deoxy-D-arabino-heptanoic acid 7-phosphate (DAHP) synthase D146N The nucleotide sequence is shown in SEQ ID No.4.

[0038] (3) Based on (2), pCDFTac plasmid was used as an expression vector to express PpsA derived from phosphoenolpyruvate (PEP) synthase, and the pykF gene encoding pyruvate kinase I was knocked out, thereby increasing the concentration of PEP, the 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, which comprises 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 the recombinant Escherichia coli, each recombinant Escherichia coli is used as a fermentation strain, and glucose is used as a substrate to ferment and produce anisic acid. The specific process is: the anisic acid-producing bacteria are inoculated into a fermentation medium, and the fermentation culture is carried out at 28-32°C and 100-500rpm for 48-72 hours. After the fermentation is completed, the supernatant is separated and purified to obtain anisic acid; the fermentation medium contains: glucose, MgSO4·7H2O, KH2PO4, (NH4)2HPO4, citric acid, L-methionine, and trace metal salt solution; pH 6.5-7.5, and the solvent is deionized water. The trace metal salt solution consists of: 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 first inoculated into LB medium, cultured overnight on a shaker at 37°C and 200 rpm, and then inoculated into the fermentation medium at a volume concentration of 1-5%. The total fermentation time was 60 hours. Among them, the fermentation was inoculated into the fermentation medium by inoculating the recombinant E. coli to OD600 = 0.5-0.8, and IPTG was added at a final concentration of 0.1-1.0 mM for induction.

[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, the final concentration of ampicillin (Ap) was 100 mg / L, the final concentration of chloramphenicol (Cm) was 34 mg / L, and the final concentration of streptomycin (Sm) was 50 mg / L. The E. coli competent cells used in the examples were E. coli DH5a, the restriction endonucleases were purchased from TAKARA, and the seamless cloning kit used in the recombination reaction was purchased from Abotek, but 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. PSA-OMT1_F and PSA-OMT1_R were used as primers to generate a 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 as shown in SEQ ID No. 1). opt The gene fragment was subjected to agarose gel electrophoresis, the correct band was cut out, and the gel recovery kit was used for purification. The seamless cloning kit was used for recombination reaction. After the reaction product was transformed into E. coli competent cells, it was spread on LB plates containing ampicillin sodium. Seq-pTrc99A_F and Seq-pTrc99A_R were used as primers, colony PCR was used to verify the single clone, and the positive clone was sent to the sequencing company for sequencing to confirm the successful connection of the plasmid pLPAA01.

[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 fully synthesized PrubiC opt The plasmid of the gene was used as a template to amplify PrubiC opt The linearized pLPAA01 fragment after enzyme digestion and the amplified ubiC gene fragment and PrubiC opt The gene fragment was subjected to agarose gel electrophoresis, the correct band was cut out, and the gel recovery kit was used for purification. The ubiC gene fragment and the purified pLPAA01 fragment were recombined using a seamless cloning kit. After the reaction product was transformed into E. coli competent cells, it was spread on an LB plate containing ampicillin sodium. Seq-pTrc99A_F and Seq-pTrc99A_R were used as primers, and colony PCR was used 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 and the purified pLPAA01 fragment were recombined using a seamless cloning kit. After the reaction product was transformed into E. coli competent cells, it was spread on an LB plate containing ampicillin sodium. Seq-pTrc99A_F and Seq-pTrc99A_R were used as primers, and colony PCR was used to verify the single clone. The positive clone was sent to a sequencing company for sequencing to confirm the successful connection of the plasmid pLPAA03.

[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 as shown in SEQ ID No.4) fragments aroG-F1 and aroG-F2. The linearized pBBR1Tac fragment, aroG-F1 fragment and aroG-F2 fragment were subjected to agarose gel electrophoresis, the correct band was cut out, and the gel recovery kit was used for purification, and then the seamless cloning kit was used for recombination reaction. After the reaction product was transformed into E. coli competent cells, it was coated on an LB plate containing chloramphenicol. Using Seq-pBBR1Tac_F and Seq-pTrc99A_R as primers, colony PCR was used to verify the single clone, and the positive clone was sent to the sequencing company for sequencing to determine the successfully connected plasmid pLPAA05.

[0050] The pLPAA02 plasmid was double-digested with restriction endonucleases KpnⅠ and HindⅢ to obtain a linearized plasmid vector fragment; the ubiC gene and rrnB terminator were amplified using primers ubiCT_F and ubiCT_R and the pLPAA02 plasmid as a template; the tac-aroG_F and tac-aroG_R were used as primers and the pLPAA05 plasmid as a template to amplify the tac-aroG D146N The linearized pLPAA02 fragment after restriction digestion and the amplified ubiC gene and rrnB terminator fragment, tac-aroG D146N The fragments were subjected to agarose gel electrophoresis, the correct bands were cut out, and the gel recovery kit was used for purification. The seamless cloning kit was used for recombination reaction. After the reaction products were transformed into E. coli competent cells, they were spread on LB plates containing ampicillin sodium. Seq-pTrc99A_F and Seq-pTrc99A_R were used as primers, and colony PCR was used to verify the single clone. The positive clones were sent to the sequencing company for sequencing to confirm the successful connection of the plasmid pLPAA04.

[0051] The pCDFTac plasmid was double-digested with restriction endonucleases EcoRⅠ and SacⅠ to obtain a linearized plasmid vector fragment; at the same time, ppsA_F and ppsA_R were used as primers and the Escherichia coli genome was used as a template to amplify the ppsA gene (sequence shown in SEQ ID No.5). The linearized pCDFTac fragment after enzyme digestion and the amplified ppsA gene fragment were subjected to agarose gel electrophoresis, the correct band was cut out, and the gel recovery kit was used for purification, and then the seamless cloning kit was used for recombination reaction. After the reaction product was transformed into Escherichia coli competent cells, it was coated on an LB plate containing streptomycin sulfate. Seq-pCDFTac_F and Seq-pTrc99A_R were used as primers, and colony PCR was used to verify the single clone. The positive clone was sent to a sequencing company for sequencing to confirm the successful connection 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 Escherichia coli genome as a template, ΔpykF-up_F and ΔpykF-up_R, ΔpykF-down_F and ΔpykF-down_R as primers, 500bp sequences upstream and downstream of the target gene were amplified as homology arms to obtain DNA fragments ΔpykF-up and ΔpykF-down. Using the pUTrc plasmid as a template, KO_F and KO_R as primers, the lox71-CmR-lox66 gene fragment was amplified, denoted as fKO. Using ΔpykF-up and fKO as templates, ΔpykF-up_F and KO_R as primers, overlap extension PCR was performed to amplify the fragment up-fKO; then using up-fKO and ΔpykF-down as templates, ΔpykF-up_F and ΔpykF-down_R as primers, overlap extension PCR was performed to amplify the fragment KO-ΔpykF.

[0055] (2) Introduction of auxiliary plasmid pKD46

[0056] Preparation of E.coli MG1655 electroporation competent state: Inoculate the laboratory-stored E.coli MG1655 into a test tube containing 5mL LB, shake at 37℃, 200rpm for 12-16h. Inoculate into 100mL fresh LB medium at 2% volume, and add L-arabinose with a final concentration of 10mM, shake at 37℃, 200rpm for 1.5h, OD600 = 0.4-0.6, centrifuge and discard the supernatant, wash twice with 10% glycerol, and finally add a certain amount of 10% glycerol to make the final volume 600-800uL, and dispense 100uL into a 1.5mL centrifuge tube, and freeze in a -80℃ refrigerator.

[0057] Introduction of auxiliary plasmid pKD46: Take an E. coli MG1655 electroporation competent cell from a -80℃ refrigerator, thaw on ice, mix thoroughly with about 50ng of plasmid pKD46, transfer to a pre-cooled 1mm electroporation cup, and place on ice for 10 minutes. Wipe the water mist on the outside of the electroporation cup with paper, place it in the electroporator, electroporate once using the Ec1 gear, immediately add 600uL of pre-cooled SOC culture medium, mix gently, tilt the electroporation cup, and transfer all the mixed solution to a sterile 1.5mL centrifuge tube. Shake at 30℃, 200rpm, for 80min, take 80uL and spread it on an LB plate containing ampicillin sodium, and culture overnight at 30℃.

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

[0059] Pick the single clone on the plate of step (2) and inoculate it into a test tube containing 5mL LB and sodium ampicillin, shake at 30℃, 200rpm for 16-20h. Inoculate it into 15mL fresh LB medium at 2% volume, shake at 30℃, 200rpm for 2h, OD600 = 0.4-0.6, centrifuge and discard the supernatant, wash twice with 10% glycerol, finally add a certain amount of 10% glycerol to make the final volume 100uL, transfer it to a 1.5mL centrifuge tube, mix it thoroughly with about 500ng fragment KO-ΔpykF, perform electroporation transformation in the same way as step (2), and spread the mixed solution after recovery culture on the LB plate containing sodium ampicillin and chloramphenicol, and culture it at 30℃ overnight. Using Seq-ΔpykF_F and Seq-KO_R as primers, perform colony PCR on the single clone on the plate to screen positive clones.

[0060] (4) Elimination of auxiliary plasmid pKD46

[0061] The positive clones in step (3) were streaked onto LB plates containing chloramphenicol and cultured overnight at 42° C. The single clones on the plates were transferred to 5 mL LB test tubes containing or not containing 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 LB and chloramphenicol, and shaken at 37°C, 200 rpm for 12 to 16 hours. 2% volume was inoculated into 15 mL fresh LB medium, and shaken at 37°C, 200 rpm for 1.5 hours. OD600 = 0.4 to 0.6, centrifuged and discarded the supernatant, washed twice with 10% glycerol, and finally added a certain amount of 10% glycerol to make the final volume 100uL, transferred to a 1.5 mL centrifuge tube, and fully mixed with about 50 ng of plasmid pjw168, and electroporated in the same way as step (2), and coated on an LB plate containing ampicillin sodium and 1 mM IPTG, and cultured at 30°C overnight. Using Seq-ΔpykF_F and Seq-ΔpykF_R as primers, colony PCR was performed on the single clones on the plate to screen positive clones.

[0064] (6) Elimination of auxiliary plasmid pjw168

[0065] The positive clones in step (5) were streaked onto LB plates and cultured overnight at 42°C. The single clones on the plates were transferred to 5 mL LB tubes containing or without ampicillin sodium to verify whether the plasmid pjw168 was eliminated. The strain that was successfully eliminated was 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 5mL LB, shake at 37℃, 200rpm for 12-16h. Inoculate 2% volume into 100mL fresh LB medium, shake at 37℃, 200rpm for 1.5h, OD600 = 0.4-0.6, centrifuge and discard the supernatant, wash twice with 10% glycerol, and finally add a certain amount of 10% glycerol to make the final volume 500uL, and dispense 100uL into a 1.5mL centrifuge tube. Four of the competent cells were fully mixed with about 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 of plasmids pLPAA02 and pLPAA05, respectively, 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, and attention was paid to adding corresponding resistance in each culture medium, and finally strain LPAA06 was obtained.

[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, 200 rpm overnight;

[0077] Shake flask fermentation: Inoculate the seed solution at 2% inoculum into a 250 mL baffled shake flask containing 50 mL fermentation medium, 30°C, 200 rpm shaking culture, fermentation cycle 60 h; 8 h after inoculation (OD600 = 0.5-0.8), add IPTG at a final concentration of 1 mM. Three biological replicates were performed for each group.

[0078] The composition of the shake flask fermentation medium is: glucose 10g / L, MgSO4·7H2O 0.8g / L, KH2PO46.67 g / L, (NH4)2HPO44g / L, citric acid 0.8g / L, trace metal salt solution 5mL / L, pH 7.0. The mother liquor concentrations of glucose and MgSO4·7H2O are 500g / L and 80g / L, respectively, and they are sterilized by wet heat independently. The trace metal salt solution is composed of: 10g / L FeSO4·7H2O, 2.65g / LCaCl2·2H2O, 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 / L 35% HCl, the solvent is deionized water.

[0079] After the fermentation, 1 mL of fermentation liquid was taken, 100 uL of which was used to dilute 10 times to detect the OD value of the bacterial liquid, and the remaining 900 uL 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 in the figure, the anisic acid production of LPAA02 reached 167.9 mg / L; the anisic acid production of LPAA05 reached 145.4 mg / L, with 52.0 mg / L of p-hydroxybenzoic acid remaining, and the OD600 value was low, which may be caused by the high concentration of inducer IPTG. At the same time, the insufficient supply of cofactor SAM may also be a factor limiting the production of anisic acid.

[0080] The HPLC system was Shimadzu LC-20 series, and the chromatographic column was Agilent Poroshell 120EC-C18 (4.6×150mm). Phase A was ddH2O containing 0.1% trifluoroacetic acid, and phase B was methanol, flowing at 0.65mL / min according to the following program: 0-1min, 10% B; 1-21min, B increased linearly from 10% to 50%; 21-22min, B increased linearly from 50% to 70%; 22-24min, 70% B; 24-26min, B decreased linearly from 70% to 10%; 26-35min, 10% B. The injection volume was 5uL, the column oven was 30°C, and the detection wavelength of the UV-Vis spectrophotometer was 246nm. Fig. 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, which are 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). Fig.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, but IPTG with a final concentration of 0.10 mM and 1.5 g / L L-methionine were added 8 h after inoculation (OD600 = 0.5-0.8), and the concentration of L-methionine mother liquor was 50 g / L, and sterilized by filtration. The same method as in Example 5 was used for detection, and the results are shown in FIG. Fig. 9 As shown in the figure, under these conditions, the anisic acid production of LPAA05 reached 281.0 mg / L, and the anisic acid production of LPAA06 reached 284.9 mg / L, and almost no p-hydroxybenzoic acid remained. In particular, after the addition of L-methionine, the OD600 of LPAA05 increased significantly, indicating that insufficient SAM supply also affected the growth of the strain.

[0084] After genetic engineering, the anisic acid yield of the genetically engineered bacteria LPAA06 reached 284.9 mg / L by shake flask fermentation. Therefore, the anisic acid genetically engineered bacteria constructed by the present invention can achieve effective accumulation of anisic acid in the fermentation broth during the fermentation process, laying a foundation for the construction of a genetically engineered strain with high anisic acid production.

[0085] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0086]

[0087]

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[0090]

Claims

1. A recombinant Escherichia coli for de novo synthesis of anisic acid, characterized in that: The starting strain of E. coli has been improved by at least one of the following: (1) Overexpression of O-methyltransferase PSA-OMT1 from Pleurotus sapidus; Overexpression of chorismate lyase UbiC 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 codon-optimized nucleotide sequence of the O-methyltransferase PSA-OMT1 is shown in SEQ ID No. 1; The nucleotide sequence of the chorismate lyase UbiC derived 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 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 the chorismate lyase UbiC.

3. The recombinant Escherichia coli according to claim 1, characterized in that 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 .

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, characterized in that: The recombinant Escherichia coli is inoculated into a fermentation medium, and fermented and cultured for 48 to 72 hours at 28 to 32° C. and 100 to 500 rpm. When OD600 is 0.5 to 0.8, isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.1 to 1.0 mM is added for induction. After the fermentation is completed, the supernatant is separated and purified to obtain anisic acid.

8. The method for producing anisic acid according to claim 7, characterized in that: 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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