Genetically engineered bacterium for de novo synthesis of vanillin using glucose as substrate, and use thereof

Abstract

Disclosed are a genetically engineered bacterium for de novo synthesis of vanillin using glucose as a substrate, and a use thereof, belonging to the technical field of gene recombination and metabolic engineering. The genetically engineered bacterium for de novo synthesis of vanillin using glucose as a substrate disclosed in the present invention is a recombinant Corynebacterium glutamicum that has been modified by chassis microorganisms, and comprises a vanillin synthesis module and a methyl cycle regeneration module. The genetically engineered bacterium constructed in the present invention is safe and non-toxic, can produce natural vanillin de novo using glucose, and has low production costs, high yield and broad application prospects.

Description

A genetically engineered bacterium for synthesizing vanillin from scratch using glucose as a substrate and its application Technical Field

[0001] The present invention relates to the technical field of gene recombination and metabolic engineering, and more particularly to a genetically engineered bacterium for synthesizing vanillin from scratch using glucose as a substrate and its application. Background Art

[0002] Vanillin is an aromatic compound with hydroxyl, aldehyde, and oxymethyl substituents. Its CAS number is 121-33-5, molecular formula is C8H8O3, and molecular weight is 152.15. Vanillin typically appears as white or light yellow crystals with a melting point of 81-83°C and a boiling point of 284-285°C. It is insoluble in water but soluble in organic solvents such as methanol. As an important aromatic compound, vanillin has a wide range of applications. It is not only used as a food additive in various foods, but also in cosmetics such as perfumes and daily necessities such as soap and toothpaste, acting as a fixative and flavor enhancer. Vanillin is also used industrially in the production of plastics, rubber biocides, electroplating agents, and conductive agents. In recent years, vanillin has been widely used in the pharmaceutical field to produce various pharmaceutical intermediates. Vanillin may become one of the most valuable aromatic compounds in the pharmaceutical field.

[0003] As a major producer and user of vanillin, my country's annual demand for vanillin reaches 2,350 tons, and this demand continues to rise year by year. In addition to domestic consumption, my country's vanillin is also exported to Europe, the United States, Southeast Asia, and other countries, enjoying a good reputation globally. Given this situation, bio-fermentation is a promising method for producing vanillin, allowing for more efficient production of this compound from inexpensive sugars. Several studies have reported on the synthesis of vanillin using microbial fermentation, but these have all suffered from low yields. Among them, the earliest report on the de novo biosynthesis of vanillin was the de novo synthesis of vanillin in fission yeast (Schizosaccharomyces pombe) and baker's yeast (Saccharomyces cerevisiae) using protocatechuic acid as an intermediate in Schizosaccharomyces pombe and Saccharomyces cerevisiae, but the vanillin yield was only 65 and 45 mg / L (Hansen EH, et al. De novo biosynthesis of vanillin in fission yeast (Schizosaccharomyces pombe) and baker's yeast (Saccharomyces cerevisiae). [J]. Applied & Environmental Microbiology, 2009, 75(9): 2765-74.). Later, Ni et al. reported the de novo synthesis of 19.3 mg / L of vanillin in Escherichia coli using ferulic acid as an intermediate and glucose as a substrate. However, the yield of de novo vanillin synthesis was still very low (Ni J, et al. Mimicking a natural pathway for de novo biosynthesis: natural vanillin production from accessible carbon sources [J]. Rep, 2015, 5(1): 13670.). Subsequently, Chen et al. reported the de novo synthesis of 240.69 mg / L of vanillyl alcohol in Escherichia coli using glucose as a substrate (Chen Z, et al. Establishing an Artificial Pathway for De Novo Biosynthesis of Vanillyl Alcohol in Escherichia coli [J]. Acs Synthetic Biology, 2017: acssynbio.7b00129.).Then Yang et al. synthesized 559.4 mg / L of vanillyl alcohol from scratch in a shake flask using glucose as a substrate by using a mixed culture of Escherichia coli (Yang M, Meng H, Li X, et al. Coculture engineering for efficient production of vanillyl alcohol in Escherichia coli[J]. aBIOTECH, 2022, 3(4): 9.). However, vanillyl alcohol is an aldehyde reduction product of vanillin, and its product properties are very different from those of vanillin. In recent years, Kim et al. reported the de novo synthesis of 310 mg / L vanillin from glucose via the intermediate p-hydroxybenzoic acid in Corynebacterium glutamicum. The yield of de novo vanillin synthesis has been greatly improved (Kim HS, et al. Engineered Corynebacterium glutamicum as the Platform for the Production of Aromatic Aldehydes[J]. Frontiers in Bioengineering and Biotechnology, 2022, 10: 880277.), but there is still a certain gap from industrial scale-up production.

[0004] Therefore, providing a genetically engineered bacterium that can synthesize vanillin from scratch using glucose as a substrate and its application is an urgent problem that needs to be solved by those skilled in the art.

[0005] Summary of the Invention

[0006] In view of this, the present invention provides a genetically engineered bacterium for de novo synthesis of vanillin using glucose as a substrate and its application. In response to the problems existing in the current biological method for de novo synthesis of vanillin, a biological method for efficient de novo synthesis of vanillin has been developed, and a non-toxic food safety engineered strain capable of using cheap substrates such as glucose to produce natural vanillin has been constructed.

[0007] One of the purposes of the present invention is to provide a genetically engineered bacterium for synthesizing vanillin. The strain is safe and non-toxic, can produce vanillin by microbial fermentation, and has low production cost.

[0008] Specifically, a genetically engineered bacterium that synthesizes vanillin from scratch using glucose as a substrate, wherein the genetically engineered bacterium is a recombinant Corynebacterium glutamicum that has been modified from a chassis microorganism and includes a vanillin synthesis module and a methyl cycle regeneration module;

[0009] The chassis microorganism is transformed into a starting strain of Corynebacterium glutamicum, and the pcaHG (protocatechuate 3,4-dioxygenase subunit, protocatechuate 3,4-dioxygenase subunit beta (pcaH), protocatechuate 3,4-dioxygenase subunit alpha (pcaG)), van (vanillate demethylase), vdh (vanillin dehydrogenase) and fud (alcohol dehydrogenase (NADP+), alcohol dehydrogenase (NADP+), Zn-dependent alcohol dehydrogenases) genes are knocked out.

[0010] The vanillin synthesis module expresses a transketolase (Transketolase1) gene, a 3-deoxy-7-phosphoheptulonate synthase (3-deoxy-7-phosphoheptulonate synthase) gene, an O-methyltransferase (O-methyltransferase) gene, a carboxylic acid reductase (Carboxylic acid reductase) gene and a phosphopantetheinyl transferase (4'-phosphopantetheinyl transferase) gene.

[0011] Transketolase effectively enhances the synthesis of E4P in the HMP pathway, increasing DAHP production. 3-Deoxy-7-phosphoheptanoate synthase facilitates the synthesis of DAHP from PEP and E4P. O-methyltransferase catalyzes the conversion of protocatechuic acid to vanillic acid. Carboxylic acid reductase catalyzes the reduction of vanillic acid to vanillin. Because the carboxylic acid reductase expressed by Corynebacterium glutamicum is an apoenzyme, it becomes the holoenzyme after translational phosphorylation by phosphopantetheinyltransferase.

[0012] The methyl recycling module expresses a 5-methyltetrahydropteroyltriglutamate-homocysteine ​​methyltransferase gene, an S-adenosylmethionine synthase gene, a homoserine O-acetyltransferase gene, and an adenosylhomocysteinase gene.

[0013] Homoserine O-acyltransferase transfers an acyl group from acetyl-coenzyme A (CoA) or succinyl-CoA to the hydroxyl oxygen of homoserine, acylating homoserine to form O-acetyl-L-homoserine, which prepares for further sulfurization. The strain itself converts O-acetyl-L-homoserine to L-homocysteine. 5-Methyltetrahydrotriglutamate-homocysteine ​​methyltransferase helps catalyze the transfer of a methyl group from 5-methyltetrahydrofolate to L-homocysteine, resulting in the formation of methionine. It catalyzes the reaction of 5-methyltetrahydropteroyltri-L-glutamate with L-homocysteine ​​to produce L-methionine and tetrahydropteroyltri-L-glutamate. S-adenosylmethionine synthetase catalyzes the reaction of ATP and water with L-methionine to produce diphosphate, phosphate, and S-adenosyl-L-methionine. Adenosylhomocysteinease catalyzes the reaction of water and S-adenosyl-L-homocysteine ​​to produce adenosine and L-homocysteine.

[0014] Furthermore, the transketolase gene includes an endogenous Corynebacterium glutamicum transketolase gene tktA, and optionally an exogenous transketolase gene.

[0015] The 3-deoxy-7-phosphoheptone acid synthetase gene includes an endogenous Corynebacterium glutamicum 3-deoxy-7-phosphoheptone acid synthetase gene aroG and an optional exogenous 3-deoxy-7-phosphoheptone acid synthetase gene.

[0016] The O-methyltransferase gene includes an O-methyltransferase gene comt from Arabidopsis thaliana (Mouseear cress), an O-methyltransferase gene comt from coffee (Coffea canephora, Robusta coffee), an O-methyltransferase gene comt from Homo sapiens (Human), an O-methyltransferase gene comt from rat (Rattus norvegicus, Rat), an O-methyltransferase gene comt from mouse (Mus musculus, Mouse), and an optional O-methyltransferase gene comt.

[0017] The carboxylic acid reductase gene includes a carboxylic acid reductase gene car derived from Nocardia iowensis and a carboxylic acid reductase gene car derived from Mycobacterium marinum.

[0018] The phosphopantetheinyl transferase gene includes the phosphopantetheinyl transferase gene sfp derived from Nocardia iowensis, the phosphopantetheinyl transferase gene sfp derived from Mycobacterium marinum, and the phosphopantetheinyl transferase gene sfp derived from Bacillus subtilis.

[0019] The 5-methyltetrahydrotriglutamate-homocysteine ​​methyltransferase gene includes an endogenous Corynebacterium glutamicum 5-methyltetrahydrotriglutamate-homocysteine ​​methyltransferase gene metE and an optional exogenous 5-methyltetrahydrotriglutamate-homocysteine ​​methyltransferase gene.

[0020] The S-adenosylmethionine synthetase gene includes an endogenous Corynebacterium glutamicum S-adenosylmethionine synthetase gene metK, and an optional exogenous S-adenosylmethionine synthetase gene.

[0021] The homoserine-O-acetyltransferase gene includes the endogenous Corynebacterium glutamicum homoserine-O-acetyltransferase gene metX, and optionally an exogenous homoserine-O-acetyltransferase gene.

[0022] The adenosylhomocysteine ​​enzyme gene includes the endogenous Corynebacterium glutamicum adenosylhomocysteine ​​enzyme gene ahcY, and optionally an exogenous adenosylhomocysteine ​​enzyme gene.

[0023] The nucleotide sequence of the endogenous Corynebacterium glutamicum transketolase gene tktA is shown in SEQ ID NO.83.

[0024] The nucleotide sequence of the endogenous Corynebacterium glutamicum 3-deoxy-7-phosphoheptanoate synthase gene aroG is shown in SEQ ID NO.84.

[0025] The nucleotide sequence of the Arabidopsis thaliana-derived O-methyltransferase gene comt is shown as SEQ ID NO.85; the nucleotide sequence of the coffee-derived O-methyltransferase gene comt is shown as SEQ ID NO.122; the nucleotide sequence of the human-derived O-methyltransferase gene comt is shown as SEQ ID NO.105; the nucleotide sequence of the rat-derived O-methyltransferase gene comt is shown as SEQ ID NO.46; and the nucleotide sequence of the mouse-derived O-methyltransferase gene comt is shown as SEQ ID NO.137.

[0026] The nucleotide sequence of the carboxylic acid reductase gene car from Nocardia is shown as SEQ ID NO.47; the nucleotide sequence of the carboxylic acid reductase gene car from Mycobacterium marinum is shown as SEQ ID NO.86.

[0027] The nucleotide sequence of the phosphopantetheinyl transferase gene sfp from Nocardia is shown as SEQ ID NO.87; the nucleotide sequence of the phosphopantetheinyl transferase gene sfp from Mycobacterium marinum is shown as SEQ ID NO.48; and the nucleotide sequence of the phosphopantetheinyl transferase gene sfp from Bacillus subtilis is shown as SEQ ID NO.106.

[0028] The nucleotide sequence of the endogenous Corynebacterium glutamicum 5-methyltetrahydrotriglutamate-homocysteine ​​methyltransferase gene metE is shown in SEQ ID NO.49.

[0029] The nucleotide sequence of the endogenous Corynebacterium glutamicum S-adenosylmethionine synthetase gene metK is shown in SEQ ID NO.50.

[0030] The nucleotide sequence of the endogenous Corynebacterium glutamicum homoserine-O-acetyltransferase gene metX is shown in SEQ ID NO.51.

[0031] The nucleotide sequence of the endogenous Corynebacterium glutamicum adenosylhomocysteinease gene ahcY is shown in SEQ ID NO.52.

[0032] Furthermore, the exogenous transketolase gene is derived from Escherichia coli K12 (tktA, Gene ID: 947420), Saccharomyces cerevisiae S288C (tkl1, Gene ID: 856188) or Bacillus subtilis 168 (tktA, Gene ID: 937377).

[0033] The exogenous 3-deoxy-7-phosphoheptone synthase gene is derived from Escherichia coli K12 (nucleotide sequence shown in SEQ ID NO.45), Saccharomyces cerevisiae S288C (aro4, Gene ID: 852551) or Bacillus subtilis 168 (aroX, Gene ID: 937853).

[0034] The exogenous 5-methyltetrahydrotriglutamate-homocysteine ​​methyltransferase gene is derived from Escherichia coli K12 (metE, Gene ID: 948323), Saccharomyces cerevisiae S288C (met6, Gene ID: 856825) or Bacillus subtilis 168 (metE, Gene ID: 936480).

[0035] The exogenous S-adenosylmethionine synthetase gene is derived from Escherichia coli K12 (metK, Gene ID: 948323), Saccharomyces cerevisiae S288C (SAM2, Gene ID: 852113) or Bacillus subtilis 168 (metK, Gene ID: 937090);

[0036] The exogenous homoserine-O-acetyltransferase gene is derived from Saccharomyces cerevisiae S288C (met2, Gene ID: 855444) or Bacillus subtilis 168 (metAA, Gene ID: 939083).

[0037] Preferably, the exogenous transketolase gene is derived from Escherichia coli K12.

[0038] The exogenous 3-deoxy-7-phosphoheptanoate synthase gene is derived from Escherichia coli K12.

[0039] The O-methyltransferase gene is derived from rat.

[0040] The carboxylic acid reductase gene is derived from Nocardia.

[0041] The phosphopantethein aminotransferase gene is derived from marine mycobacterium.

[0042] The 5-methyltetrahydrotriglutamate-homocysteine ​​methyltransferase gene is derived from endogenous Corynebacterium glutamicum.

[0043] The S-adenosylmethionine synthetase gene is derived from endogenous Corynebacterium glutamicum.

[0044] The homoserine-O-acetyltransferase gene is derived from endogenous Corynebacterium glutamicum.

[0045] The adenosylhomocysteine ​​enzyme gene is derived from endogenous Corynebacterium glutamicum.

[0046] Furthermore, a method for constructing a genetically engineered bacterium for de novo synthesis of vanillin using glucose as a substrate comprises the following steps:

[0047] (1) Using Corynebacterium glutamicum as the starting strain, the pcaHG, van, vdh, and fud genes were knocked out to obtain a modified Corynebacterium glutamicum;

[0048] (2) Expressing the vanillin synthesis module and methyl cycle regeneration module in the modified Corynebacterium glutamicum.

[0049] The second object of the present invention is to provide the use of the above-mentioned genetically engineered bacteria for synthesizing vanillin in synthesizing vanillin.

[0050] Specifically, the use of the genetically engineered bacteria or the method in the production of vanillin.

[0051] Specifically, the application of the genetically engineered bacteria or the method in increasing the yield of vanillin.

[0052] Specifically, a method for producing vanillin comprises fermentation using the genetically engineered bacteria or the genetically engineered bacteria constructed by the method.

[0053] To this end, the present invention provides a biosynthetic pathway for vanillin ( FIG1 ), which comprises:

[0054] (1) Glucose is converted into phosphoenolpyruvate (PEP) through the microorganism's own glycolysis pathway, and into erythrose 4-phosphate (E4P) through the microorganism's own pentose phosphate pathway;

[0055] (2) Transketolase tktA catalyzes the reversible transfer of a dicarbon ketone group from the sugar heptose-7-phosphate to glyceraldehyde-3-phosphate, generating xylose-5-phosphate and ribose-5-phosphate;

[0056] (3) Phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) are catalyzed by 3-deoxy-D-arabinoheptulose-7-phosphate synthase aroG to produce 3-deoxy-D-arabinoheptulose-7-phosphate (DAHP);

[0057] (4) Protocatechuic acid (PCA) is produced by the shikimate pathway of Corynebacterium glutamicum itself;

[0058] (5) Protocatechuic acid (PCA) generates vanillic acid under the catalysis of O-methyltransferase comt;

[0059] (6) Vanillic acid generates vanillin under the catalysis of carboxylic acid reductase car;

[0060] (7) Carboxylate reductase car is converted into a holoenzyme by post-translational modification of the phosphopantetheinyltransferase sfp upon catalytic phosphorylation;

[0061] The above is the vanillin synthesis module.

[0062] (8) The strain produces homoserine endogenously;

[0063] (9) Homoserine-O-acetyltransferase metX transfers an acetyl group from acetyl-CoA to l-homoserine to form O-acetyl-l-homoserine;

[0064] (10) The strain itself converts O-acetyl-l-homoserine into L-homocysteine;

[0065] (11) 5-Methyltetrahydrofolate-homocysteine ​​methyltransferase metE catalyzes the transfer of a methyl group from 5-methyltetrahydrofolate to homocysteine, producing methionine;

[0066] (12) L-methionine is converted into S-adenosyl-L-methionine (SAM) under the catalysis of S-adenosyl-methionine synthetase metK;

[0067] (13) S-adenosyl-L-methionine donates a methyl group to protocatechuic acid under the catalysis of O-methyltransferase comt, generating S-adenosyl-L-homocysteine ​​(SAH) and vanillic acid;

[0068] (14) S-adenosyl-L-homocysteine ​​is converted into adenosine and L-homocysteine ​​under the catalysis of adenosylhomocysteine ​​enzyme ahcY;

[0069] The above is the methyl recycling module.

[0070] Chassis microbial modification: Using Corynebacterium glutamicum as the starting strain, the pcaHG, van, vdh, and fud genes were knocked out using the following methods:

[0071] pcaHG is a subunit of protocatechuate 3,4-dioxygenase; Corynebacterium glutamicum has a β-ketoadipate pathway that utilizes several aromatic compounds such as PCA as the sole carbon and energy source; protocatechuate degradation is initiated by protocatechuate 3,4-dioxygenase (PcaGH), which consists of two subunits encoded by pcaG and pcaH located in the pcaHGBC operon; knocking out pcaHG can prevent the consumption of protocatechuate by the β-ketoadipate pathway.

[0072] Van is a vanillin demethylase, which contains the vanillin demethylase subunit ABK; it can catalyze the conversion of vanillic acid to protocatechuic acid; knocking out van can prevent the demethylation of vanillic acid to protocatechuic acid and promote the utilization of protocatechuic acid.

[0073] VDH catalyzes the oxidation of vanillin to vanillic acid. It also significantly oxidizes 3,4-dihydroxybenzaldehyde (protocatechuic aldehyde) and 4-hydroxybenzaldehyde. It possesses both NAD+ and NADP+ activity. Due to the cytotoxicity of vanillin, VDH catalyzes the conversion of vanillin to vanillic acid. Knocking out VDH reduces vanillin oxidation and increases its accumulation.

[0074] Fud is an alcohol dehydrogenase that uses NADPH (instead of NADH) as a cofactor to convert vanillin into vanillyl alcohol. Knocking out fud can significantly reduce the process of vanillin reduction to vanillyl alcohol.

[0075] It can be seen from the above technical solution that, compared with the prior art, the present invention discloses a genetically engineered bacterium for synthesizing vanillin from scratch using glucose as a substrate and its application, wherein Corynebacterium glutamicum is modified from three aspects, including the construction and enhancement of the vanillin synthesis pathway (involving tktA, aroG, comt, car), the enhancement of the methyl regeneration system (involving metE, metK, metX, ahcY), and the knockout of the vanillin decomposition pathway and the by-product metabolism pathway (involving pcaHG, van, vdh and fud); finally, a recombinant Corynebacterium glutamicum that efficiently synthesizes vanillin is constructed.

[0076] The host strain, Corynebacterium glutamicum, selected in the present invention is a food-grade microorganism that is non-pathogenic to humans and animals. It exhibits superior growth advantages under equivalent culture conditions, allowing it to accumulate higher concentrations of vanillin than other strains. Repeated experiments have shown that shake-flask fermentation of the recombinant Corynebacterium glutamicum constructed in the present invention can produce a maximum vanillin concentration of 765.85 mg / L, the highest yield reported for de novo biosynthesis of vanillin in related research. This holds great promise for the industrialization of high-yield vanillin production using biosynthetic methods.

[0077] The genetically engineered bacteria constructed by the present invention are safe and non-toxic, can produce vanillin using glucose as a substrate by a microbial fermentation method, and have simple culture medium components, stable batches, and low production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0079] FIG1 is a diagram showing the biosynthesis pathway of vanillin according to the present invention.

[0080] Figure 2 shows the yield and biomass OD of vanillin synthesized by shake flask fermentation of the genetically engineered bacteria constructed in Example 1 of the present invention using glucose as a substrate at different times. 600 .

[0081] Figure 3 shows the yield and biomass OD of vanillin produced by shake flask fermentation of the genetically engineered bacteria constructed in Example 2 of the present invention using glucose as a substrate at different times. 600 .

[0082] Figure 4 shows the yield and biomass OD of vanillin synthesized by shake flask fermentation of the genetically engineered bacteria constructed in Example 3 of the present invention using glucose as a substrate at different times. 600 .

[0083] Figure 5 shows the yield and biomass OD of vanillin synthesized by shake flask fermentation of the genetically engineered bacteria constructed in Example 4 of the present invention using glucose as a substrate at different times. 600 .

[0084] Figure 6 shows the yield and biomass OD of vanillin synthesized by shake flask fermentation of the genetically engineered bacteria constructed in Example 5 of the present invention using glucose as a substrate at different times. 600 . DETAILED DESCRIPTION

[0085] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0086] LBHIS liquid medium: 5.0 g / L peptone, 2.5 g / L yeast extract, 5.0 g / L NaCl, 18.5 g / L brain heart extract (BHI), 91.0 g / L sorbitol. The corresponding LBHIS solid medium should be supplemented with 1.8%-2% agar. LBHIS medium is primarily used for culturing Corynebacterium glutamicum in test tubes and on solid plates.

[0087] EPO medium: peptone 10.0 g / L, yeast extract 5.0 g / L, NaCl 10.0 g / L, glycine 30.0 g / L, Tween 80 10.0 g / L. EPO medium is mainly used for the preparation of Corynebacterium glutamicum competent cells.

[0088] 30% sucrose medium: peptone 10.0 g / L, yeast powder 5.0 g / L, NaCl 10.0 g / L, sucrose 300.0 g / L.

[0089] 20% sucrose solid medium: 10.0 g / L peptone, 5.0 g / L yeast extract, 10.0 g / L NaCl, 200.0 g / L sucrose, 15.0 g / L agar. LB-sucrose medium (30% sucrose medium, 20% sucrose solid medium) is primarily used for homologous recombination screening in gene knockout experiments with Corynebacterium glutamicum.

[0090] LBG medium: 10.0 g / L peptone, 5.0 g / L yeast extract, 10.0 g / L NaCl, 20.0 g / L glucose. LBG medium is primarily used as a seed culture medium for Corynebacterium glutamicum fermentation.

[0091] Fermentation medium: glucose 65.0 g / L, urea 5.0 g / L, corn extract 8.0 g / L, biotin 4 × 10 -4 g / L, VB1 biotin 4×10 -4 g / L, K2HPO4 1.0g / L, KH2PO4 1.0g / L, CaCl2·2H2O 29.4mg / L, MgSO4·7H2O 1.2325g / L, trace element solution 0.2%. (Trace element solution preparation method: Weigh 1g FeSO4·7H2O, 1g MnSO4·H2O, 0.1g ZnSO4·7H2O, 0.2g CuSO4, 0.002g NiCl2·6H2O, add water to 100mL, then add 100μl concentrated hydrochloric acid to adjust the pH. Then filter through a microbial filter and add 0.2% to the fermentation medium.) The fermentation medium is primarily used for shake flask fermentation of Corynebacterium glutamicum.

[0092] Example 1

[0093] A method for constructing a genetically engineered bacterium for de novo synthesis of vanillin using glucose as a substrate comprises the following steps:

[0094] (1) Using genomic DNA from Corynebacterium glutamicum ATCC13032 as a template, primers pcaHG-L-up / pcaHG-L-down were used to amplify pcaHG-L (as shown in SEQ ID NO. 1), and primers pcaHG-R-up / pcaHG-R-down were used to amplify pcaHG-R (as shown in SEQ ID NO. 2), thereby obtaining a pcaHG upstream and downstream homology arms completely deleted. The primer sequences were:

[0095] pcaHG-L-up: 5'-ATGATTACGCCATCGCATTGCCGAAAAGC-3'; SEQ ID NO.3;

[0096] pcaHG-L-down: 5'-GGTCAATGCGAGACCTTTCTGCGTC-3'; SEQ ID NO.4;

[0097] pcaHG-R-up: 5'-AAAGGTCTCGCATTGACCCGATCTTTATACTCCGAC-3'; SEQ ID NO.5;

[0098] pcaHG-R-down: 5'-CTCAACGTTGACGGTGATGCCA-3'; SEQ ID NO. 6.

[0099] Gene amplification system: Primestar (Takara) 25 μL, upstream and downstream primers 2 μL each, template 1 μL, ddH2O 20 μL.

[0100] Gene amplification program: pre-denaturation at 98°C for 3 minutes; denaturation at 98°C for 10 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 10 seconds / kb, 35 cycles; extension at 72°C for 10 minutes.

[0101] The PCR products were subjected to agarose gel electrophoresis and product recovery to obtain gene fragments pcaHG-L and pcaHG-R.

[0102] The plasmid vector pk18mobsacb was linearized with primers pk-pcaHG-F and pk-pcaHG-R to obtain the pk-pcaHG vector. The primer sequences are as follows:

[0103] pk-pcaHG-F: 5'-ATCACCGTCAACGTTGAGCTCGGTAGATCCTCTAGAGT-3'; SEQ ID NO.7;

[0104] pk-pcaHG-R: 5'-AATGCGATGGCGTAATCATGTCATAGCTGTTTCCTG-3'; SEQ ID NO. 8.

[0105] Add 3 μL pcaHG-L, 3 μL pcaHG-R, 4 μL pk-pcaHG vector, and 10.0 μL Gibson ligase to a PCR tube. The ligation temperature is 50°C and the ligation time is 15 min. The total system is 20 μL.

[0106] All 20 μl of the Gibson ligation system was transformed into commercially competent E. coli Trans10 (Beijing Quanshijin Biotechnology) for culture and maintenance. Transformation procedures were performed strictly according to the manufacturer's instructions. After incubation at 37°C for 1 hour, the cells were plated onto LB plates (containing 50 μg / mL kanamycin) and incubated at 37°C for an additional 12 hours. Approximately 10-20 colonies were selected for colony PCR amplification and DNA sequencing verification. The primers for colony PCR amplification and DNA sequencing were:

[0107] Pklj-F: 5'-GCGGATAACAATTTCACACAGGA-3'; SEQ ID NO.9;

[0108] Pklj-R: 5'-cgggcctcttcgctattac-3'; SEQ ID NO. 10.

[0109] A correct single colony was selected and named Escherichia coli WJ001, which was expanded and plasmid extracted to obtain the WJ001 plasmid. The Escherichia coli WJ001 plasmid was electroporated into the competent cell of Corynebacterium glutamicum ATCC13032.

[0110] The competent cells of Corynebacterium glutamicum ATCC13032 were prepared as follows: glycerol-preserved strains of Corynebacterium glutamicum ATCC13032 were streaked onto LBHIS plates and cultured in a 30°C incubator for about 24-30 hours; colonies were picked from the plates and inoculated into test tubes containing LBHIS liquid medium and cultured for 12 hours, and a certain inoculum volume was inoculated into 20 ml of EPO medium to make the initial OD 600 When the OD is about 0.3, culture in a flask at 30℃ for about 3-5h. 600 When the pH reaches about 0.9, take the bacterial solution into a centrifuge tube and place it in an ice bath for 15 minutes to cool the bacteria to 0°C; collect the bacteria by refrigerated centrifugation at 4500 rpm for 10 minutes at 4°C (divide into 1.5 mL centrifuge tubes), discard the supernatant, and resuspend the bacteria with 100 μL of pre-cooled 10% glycerol (mix three tubes of bacteria into one tube); repeat the previous step three times; after washing, resuspend the bacteria with 100 μL of 10% sterile glycerol, divide into aliquots and use directly for electroporation.

[0111] Electroporation protocol: Add 2-4 μL of plasmid to each tube of competent cells, mix thoroughly, and incubate on ice for 5-10 minutes. Transfer the mixture to a pre-chilled 0.2 cm cuvette and electroporate at 1.8 kV for 5 ms at 50 μF and 100 Ω. Immediately after electroporation, add 800 μL of LBHIS liquid medium, mix gently, aspirate, and transfer to a 1.5 mL centrifuge tube. Incubate at 46°C in a water bath or metal bath for 6 minutes, then incubate at 30°C for 2-3 hours. Centrifuge at 8000 rpm for 1.5 minutes, discard the supernatant, and retain approximately 100-200 μL of the bacterial suspension. Mix thoroughly, spread on a plate containing LBHIS solid medium supplemented with 50 μg / mL kanamycin, and incubate at 30°C for 24-36 hours.

[0112] Single colonies grown on the plates were picked for verification of the sacB gene. Only colonies that could grow on kanamycin-resistant plates and amplify the sacB gene (band size of 999 bp) were considered to be strains that had undergone the first homologous recombination. The verification primers were as follows:

[0113] sac-F: 5'-CCCATATTACACGCCATGATATGCT-3'; SEQ ID NO.11;

[0114] sac-R: 5'-GCATGTAAATATCGTTAGACGTAATGCCG-3'; SEQ ID NO. 12.

[0115] The correct bacterial strain was selected and inoculated into 30% sucrose medium for 24 h. After the bacterial solution became turbid, it was streaked onto 20% sucrose solid medium. Single colonies on the plate were picked and subjected to colony PCR for the sacB gene. For single colonies that did not amplify the sacB gene, secondary colony PCR was performed using verification primers. The primers for pcaHG verification (band size 3376 bp) are as follows:

[0116] pcaHG-verification-F: 5'-CGCGACTTGCCATCACATC-3'; SEQ ID NO. 13;

[0117] pcaHG-verification-R: 5′-GTCAAGCAGGCTAAACCGGAG-3′; SEQ ID NO. 14.

[0118] According to the knockout principle of Corynebacterium glutamicum, after the second homologous recombination occurs, the entire knockout plasmid will fall off from the genome, thereby achieving the purpose of knockout. Therefore, the colony that has undergone secondary recombination will no longer contain the sacB gene; the bacteria that have been successfully knocked out by verification primers are inoculated into LBHIS liquid culture medium for culture, and streaked onto LBHIS solid culture medium plates for purification and re-verification. After confirmation, the bacteria can be preserved for use, and cgATCC13032ΔpcaHG is named Corynebacterium glutamicum CG001.

[0119] (2) Using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template, primers van-L-up / van-L-down were used to amplify van-L (as shown in SEQ ID NO. 15), and primers van-R-up / van-R-down were used to amplify van-R (as shown in SEQ ID NO. 16), thereby obtaining the upstream and downstream homology arms of a partially deleted van. The primer sequences are:

[0120] van-L-up: 5'-AACAGCTATGACATGATTACGATAAGCTGCTCATGCTTGGC-3'; SEQ ID NO. 17;

[0121] van-L-down: 5'-CTGATATCTCGACATTGCTGAGTTAATGTCAGCTGGGGTCTC-3'; SEQ ID NO. 18;

[0122] van-R-up: 5'-CAGCAATGTCGAGATATCAGTGG-3'; SEQ ID NO. 19;

[0123] van-R-down: 5'-AGAGGATCTACCGAGCTCTCATCAGTGTGTTTCGGGAGC-3'; SEQ ID NO. 20.

[0124] The plasmid vector PK-JL was linearized with primers pk-van-F and pk-van-R to obtain the pk-van vector. The primer sequences are as follows

[0125] pk-van-F: 5'-GAGCTCGGTAGATCCTCTAGAGT-3'; SEQ ID NO. 21;

[0126] pk-van-R: 5'-CGTAATCATGTCATAGCTGTTTCCTG-3'; SEQ ID NO. 22.

[0127] The gene amplification system and gene amplification procedure were the same as above to obtain van-L, van-R, and pk-van vectors. The ligation and transformation verification method was the same as above to obtain Escherichia coli WJ002. After expansion and plasmid extraction, the WJ002 plasmid was obtained.

[0128] The E. coli WJ002 plasmid was electroporated into competent cells of Corynebacterium glutamicum CG001. Competent cell preparation, electroporation, sacB verification, and sucrose culture were performed as above. The primers for partial van deletion verification (band size 4459 bp) were as follows:

[0129] van-verification-F: 5'-TTGATGGTGTCCGCAAAATCG-3'; SEQ ID NO. 23;

[0130] van-verification-R: 5′-ACCGACACGACCCATACCAA-3′; SEQ ID NO. 24.

[0131] The bacteria that have been successfully knocked out by verifying the primers are inoculated into LBHIS liquid medium for culture, and streaked onto LBHIS solid medium plates for purification and re-verification. After confirmation, the bacteria can be preserved for use, and cgATCC13032ΔpcaHGΔvan is named Corynebacterium glutamicum CG002.

[0132] (3) Using genomic DNA from Corynebacterium glutamicum ATCC13032 as a template, primers vdh-L-up / vdh-L-down were used to amplify vdh-L (as shown in SEQ ID NO. 25), and primers vdh-R-up / vdh-R-down were used to amplify vdh-R (as shown in SEQ ID NO. 26) to obtain a complete deletion of the upstream and downstream homology arms of vdh. The primer sequences were:

[0133] vdh-L-up: 5'-AACAGCTATGACATGATTACGCGACGGTATTCCTGCAGATGA-3'; SEQ ID NO. 27;

[0134] vdh-L-down: 5'-CTTTAGGAGACCTTCGCTAATCCGCTGAACAGG-3'; SEQ ID NO. 28;

[0135] vdh-R-up: 5'-GATTAGCGAAGGTCTCCTAAAGTTGATTGTGGATAC-3'; SEQ ID NO. 29;

[0136] vdh-R-down: 5'-AGCTCAGTATGGGAAGCATTGCTTGC-3'; SEQ ID NO. 30.

[0137] Plasmid vector PK-JL was linearized with primers pk-vdh-F and pk-vdh-R to obtain pk-vdh vector. The primer sequences are as follows:

[0138] pk-vdh-F: 5'-AATGCTTCCCATACTGAGCTCGGTAGATCCTCTAGAGT-3'; SEQ ID NO.31;

[0139] pk-vdh-R: 5'-CGTAATCATGTCATAGCTGTTTCCTG-3'; SEQ ID NO. 32.

[0140] The gene amplification system and gene amplification procedure were the same as above to obtain vdh-L, vdh-R, and pk-vdh vectors. The ligation transformation verification method was the same as above to obtain Escherichia coli WJ003. After expansion and plasmid extraction, the WJ003 plasmid was obtained.

[0141] The E. coli WJ003 plasmid was electroporated into competent cells of Corynebacterium glutamicum CG002. Competent cell preparation, electroporation, sacB verification, and sucrose culture were performed as above. The primers for vdh verification (band size 3653 bp) are as follows:

[0142] vdh-verification-F: 5'-GTGAATCAATCATTGCTGATTACCTTGTC-3'; SEQ ID NO. 33;

[0143] vdh-verification-R: 5′-CTTATGCCTTTGCGTAATGTTGATAGAAC-3′; SEQ ID NO. 34.

[0144] The bacteria that have been successfully knocked out by verifying the primers are inoculated into LBHIS liquid culture medium for culture, and streaked onto LBHIS solid culture medium plates for purification and re-verification. After confirmation, the bacteria can be preserved for use, and cgATCC13032ΔpcaHGΔvanΔvdh is named Corynebacterium glutamicum CG003.

[0145] (4) Using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template, primers fud-L-up / fud-L-down were used to amplify fud-L (as shown in SEQ ID NO. 35), and primers fud-R-up / fud-R-down were used to amplify fud-R (as shown in SEQ ID NO. 36) to obtain the upstream and downstream homology arms of fud. The primer sequences are:

[0146] fud-L-up: 5'-TTACGAATTCGAGCTCGGTAAAGCCACCGGATTTAGCGC-3'; SEQ ID NO.37;

[0147] fud-L-down: 5'-CCCAGTTAGCCTACCGAAAAATCAA-3'; SEQ ID NO.38;

[0148] fud-R-up: 5'-TTCGGTAGGCTAACTGGGCCCTGGCTTTGGAGAGTTACT-3'; SEQ ID NO.39;

[0149] fud-R-down: 5'-TCGACTCTAGAACACGATTCTCAAGCTTACTGCG-3'; SEQ ID NO. 40.

[0150] The plasmid vector PK-JL was linearized with primers pk-fud-F and pk-fud-R to obtain the pk-fud vector. The primer sequences are as follows:

[0151] pk-fud-F: 5'-AGAATCGTGTTCTAGAGTCGACCTGCAGGC-3'; SEQ ID NO.41;

[0152] pk-fud-R: 5'-TTTACCGAGCTCGAATTCGTAATCATGT-3'; SEQ ID NO. 42.

[0153] The gene amplification system and gene amplification procedure were the same as above to obtain fud-L, fud-R, and pk-fud vectors. The ligation transformation verification method was the same as above to obtain Escherichia coli WJ004. After expansion and plasmid extraction, the WJ004 plasmid was obtained.

[0154] The E. coli WJ004 plasmid was electroporated into competent cells of Corynebacterium glutamicum CG003. Competent cell preparation, electroporation, sacB verification, and sucrose culture were performed as described above. The primers for fud verification (band size 3431 bp) are as follows:

[0155] fud-verification-F: 5'-CTTTCTTGGGAAAGGCCCG-3'; SEQ ID NO. 43;

[0156] fud-verification-R: 5′-TGGCACCTACAACCTCACCAA-3′; SEQ ID NO. 44.

[0157] The bacteria that have been successfully knocked out by verifying the primers are inoculated into LBHIS liquid medium for culture, and streaked onto LBHIS solid medium plates for purification and re-verification. After confirmation, the bacteria can be preserved for use, and cgATCC13032ΔpcaHGΔvanΔvdhΔfud is named Corynebacterium glutamicum CG004.

[0158] (5) Using the genomic DNA of Escherichia coli K12 as a template, the primers tktA-up / tktA-down were used to amplify the fragment tktA (Gene ID: 947420) to obtain the E. coli-derived tktA fragment with seamless cloning homology arms connected to aroG and RBS (ribosome binding site).

[0159] The A base at position 436 of the aroG gene sequence derived from Escherichia coli K12 was mutated to a G base to obtain an anti-feedback inhibition gene sequence (as shown in SEQ ID NO.45); using the anti-feedback inhibition gene sequence as a template, the aroG fragment was amplified using primers aroG-up / aroG-down to obtain an Escherichia coli-derived aroG fragment with seamless cloning homology arms and RBS connected to the plasmid.

[0160] Using the codon-optimized comt fragment of rat (Rattus norvegicus, Rat) synthesized by BGI (as shown in SEQ ID NO.46) as a template, the comt fragment was amplified using primers comt-up / comt-down to obtain the rat-derived comt fragment with seamless cloned homology arms and RBS connected to tktA.

[0161] Using the codon-optimized car gene fragment of Nocardia iowensis synthesized by BGI (as shown in SEQ ID NO.47) as a template, the fragment car was amplified using primers car-up / car-down to obtain the Nocardia iowensis-derived car fragment with seamless cloning homology arms and RBS connected to metX.

[0162] Using the codon-optimized sfp gene fragment of Mycobacterium marinum synthesized by BGI (as shown in SEQ ID NO.48) as a template, the primers sfp-up / sfp-down were used to amplify the fragment sfp to obtain the sfp fragment derived from Mycobacterium marinum with seamless cloning homology arms and RBS connected to the car and plasmid.

[0163] Using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template, the metE fragment (as shown in SEQ ID NO.49) was amplified using primers metE-up / metE-down to obtain a metE fragment derived from Corynebacterium glutamicum with seamless cloning homology arms and RBS connected to ahcY and plasmid.

[0164] Using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template, the metK fragment (as shown in SEQ ID NO. 50) was amplified using primers metK-up / metK-down to obtain a metK fragment derived from Corynebacterium glutamicum with seamless cloning homology arms and RBS connected to the plasmid.

[0165] Using genomic DNA of Corynebacterium glutamicum ATCC13032 as a template, primers metX-up / metX-down were used to amplify the metX fragment (as shown in SEQ ID NO. 51), obtaining a metX fragment derived from Corynebacterium glutamicum with seamless cloned homology arms and RBS connected to metK.

[0166] Using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template, the primers ahcY-up / ahcY-down were used to amplify the fragment ahcY (as shown in SEQ ID NO. 52), obtaining the Corynebacterium glutamicum-derived ahcY fragment with seamlessly cloned homology arms and RBS connected to comt.

[0167] The primer sequences are:

[0168] tktA-up:

[0169] 5’-TAAAAGCGCGTCGCGGGTAAAAGGAGGATATACATATGTCCTCACGTAAAGAGCTTGC-3’;SEQ ID NO.53;

[0170] tktA-down:5’-TTACAGCAGTTCTTTTGCTTTCGC-3’;SEQ ID NO.54;

[0171] aroG-up:

[0172] 5’-GGAAACAGACCATGGAATTCAAGGAGGATATACATATGAATTATCAGAACGACGATTTACGC-3’;SEQ ID NO.55;

[0173] aroG-down:5’-TTACCCGCGACGCGCTTTTA-3’;SEQ ID NO.56;

[0174] comt-up:

[0175] 5’-AAAGCAAAAGAACTGCTGTAAAAGGAGGATATACATATGGGTGATACCAAAGAACAGCG-3’;SEQ ID NO.57;

[0176] comt-down:5’-TTAAGATTTATCAGGGCTACTAGGTCCC-3’;SEQ ID NO.58;

[0177] car-up:

[0178] 5’-CATCGAGTTCTACATCTAATCTAGAAAGGAGGATATACATATGGCAGTGGATTCCCCAG-3’;SEQ ID NO.59;

[0179] car-down:5’-TTACAGCAGCTGCAGCAGTTC-3’;SEQ ID NO.60;

[0180] sfp-up:

[0181] 5’-AACTGCTGCAGCTGCTGTAAGGATCCCCAAGGAGGATATACATATGACCGTGGGCACTCTG-3’;SEQ ID NO.61;

[0182] sfp-down:

[0183] 5’-CGCCAAAACAGCCAAGCTGAATTCGAGCTCGGTACCCTTACAGCACGATTGCAGTCAG-3’;SEQ ID NO.62;

[0184] metE-up:

[0185] 5’-CGGAGCACTACCGCTACTAAAAGGAGGATATACATATGACTTCCAACTTTTCTTCCACTG-3’;SEQ ID NO.63;

[0186] metE-down:

[0187] 5’-TCTAGAGGATCCCCGGGTACCGAGCTCTTAGATAGTTGCTCCGATTTTCTCACG-3’;SEQ ID NO.64;

[0188] metK-up:

[0189] 5’-TTAAGCTTGCATGCCTGCAGGTCGACAAGGAGGATATACATGTGGCTCAGCCAACCG-3’;SEQ ID NO.65;

[0190] metK-down:5’-TTAGGCCAACTTGAGGGCTG-3’;SEQ ID NO.66;

[0191] metX-up:

[0192] 5’-CAGCCCTCAAGTTGGCCTAAAAGGAGGATATACATATGCCCACCCTCGCG-3’;SEQ ID NO.67;

[0193] metX-down:5’-TCTAGATTAGATGTAGAACTCGATGTAGGTCG-3’;SEQ ID NO.68;

[0194] ahcY-up:

[0195] 5’-CCTAGTAGCCCTGATAAATCTTAAAAGGAGGATATACATATGGACTTCAAGGTTGCCGA-3’;SEQ ID NO.69;

[0196] ahcY-down: 5'-TTAGTAGCGGTAGTGCTCCG-3'; SEQ ID NO. 70.

[0197] The gene amplification system and gene amplification procedure are the same as above.

[0198] The PCR products were subjected to agarose gel electrophoresis and product recovery to obtain the corresponding gene products tktA, aroG, comt, car, sfp, metE, metK, metX and ahcY from different sources.

[0199] The plasmid vector pEC-XK99E was linearized with primers pec-F and pec-R to obtain the pec vector. The primer sequences are as follows:

[0200] pec-F: 5'-TACCCGGGGATCCTCTAGAGTC-3'; SEQ ID NO.71;

[0201] pec-R: 5'-GAATTCCATGGTCTGTTTCCTGTG-3'; SEQ ID NO. 72.

[0202] The amplification system and procedure are the same as those for gene amplification.

[0203] The plasmid vector pXMJ19 was linearized with primers px-F and px-R to obtain the pxmj vector. The primer sequences are as follows:

[0204] px-F: 5'-CGAATTCAGCTTGGCTGTTTGGCG-3'; SEQ ID NO.73;

[0205] px-R: 5'-TGCAGGCATGCAAGCTTAATTAATT-3'; SEQ ID NO. 74.

[0206] Add 1.5 μl tktA, 2.5 μl aroG, 1.0 μl comt, 1.5 μl metE, 1.5 μl ahcY, 2.0 μl pec vector from different sources, and 10.0 μl Gibson enzyme into a PCR tube.

[0207] The connection temperature was 50°C, the connection time was 15 min, and the total system volume was 20 μL.

[0208] All 20 μl of the Gibson ligation system was transformed into E. coli Trans10 commercial competent cells for culture and maintenance. Transformation procedures were performed strictly according to the manufacturer's instructions. After incubation at 37°C for 1 hour, the cells were plated onto LB plates (containing 50 μg / mL kanamycin) and incubated at 37°C for an additional 12 hours. Approximately 10-20 colonies were selected for colony PCR amplification and DNA sequencing verification. The primers for colony PCR amplification and DNA sequencing were:

[0209] Pec-F1: 5'-GGCTGTGCAGGGTCGTAAATCAC-3'; SEQ ID NO.75;

[0210] Pec-R1: 5'-AGTTCCCTACTCTCGCATGGG-3'; SEQ ID NO. 76.

[0211] A correct single colony was selected and named Escherichia coli WJ101. After multiplication and plasmid extraction, the Escherichia coli WJ101 plasmid was obtained.

[0212] Add 3.0 μl car, 1.0 μl sfp, 2.0 μl metK, 2.0 μl metX, 2.0 μl pxmj vector, and 10.0 μl Gibson enzyme to a PCR tube.

[0213] The connection temperature was 50°C, the connection time was 15 min, and the total system volume was 20 μL.

[0214] All 20 μl of the Gibson ligation system was transformed into commercially competent E. coli Trans10 (Beijing Quanshijin Biotechnology) for culture and maintenance. Transformation procedures were performed strictly according to the manufacturer's instructions. After incubation at 37°C for 1 hour, the cells were plated onto LB plates (containing 50 μg / mL kanamycin) and incubated at 37°C for an additional 12 hours. Approximately 10-20 colonies were selected for colony PCR amplification and DNA sequencing verification. The primers for colony PCR amplification and DNA sequencing were:

[0215] Pxmj-F: 5'-CTGTGGTATGGCTGTGCAGGTC-3'; SEQ ID NO.77;

[0216] Pxmj-R: 5'-ATGCCTGGCAGTTCCCTACT-3'; SEQ ID NO. 78.

[0217] A correct single colony was selected and named Escherichia coli WJ201. After multiplication and plasmid extraction, the Escherichia coli WJ201 plasmid was obtained.

[0218] Simultaneously electroporate 2.0 μl of E. coli WJ101 plasmid and 2.0 μl of E. coli WJ201 plasmid into competent cells of Corynebacterium glutamicum CG004. Prepare competent cells and perform electroporate as described above. Mix well, spread on LBHIS solid medium plates containing 50 μg / mL kanamycin and 5 μg / mL chloramphenicol, and incubate at 30°C for 24–36 hours.

[0219] Single colonies grown on the plates were picked for verification of the kana and cm genes. Only colonies that could grow on both kana- and chloramphenicol-resistant plates and amplify the kana and cm genes were the target strains, named Corynebacterium glutamicum CG101. The verification primers were as follows:

[0220] kana-F: 5'-ATGATTGAACAAGATGGATTGCACG-3'; SEQ ID NO.79;

[0221] kana-R: 5'-TCAGAAGAACTCGTCAAGAAGGC-3'; SEQ ID NO.80;

[0222] Cm-R: 5'-CCTGCCACTCATCGCAGTAC-3'; SEQ ID NO.81;

[0223] Cm-F: 5'-ATGGAGAAAAAAATCACTGGATATACCACC-3'; SEQ ID NO. 82.

[0224] The genetically engineered strain of Corynebacterium glutamicum "Corynebacterium glutamicum CG101" was fermented in a 50 mL system, using LBG medium for seed culture and a fermentation medium implementation plan for fermentation.

[0225] The fermentation of Corynebacterium glutamicum CG101 is divided into three steps:

[0226] Transfer 100 μL of Corynebacterium glutamicum CG101 stored at -80°C to a test tube containing 4 mL of LBHIS liquid medium (containing 50 μg / mL kanamycin and 5 μg / mL chloramphenicol) and culture at 30°C for 12-14 hours.

[0227] Inoculum: Remove the test tube after 12-14 hours of incubation, transfer 1 mL of the bacterial solution from the test tube to 20 mL of LBG liquid medium (container is a 100 mL unbaffled conical flask containing 50 μg / mL kanamycin and 5 μg / mL chloramphenicol), and incubate in a constant temperature shaker at 30°C, 200 rpm for 12-14 hours.

[0228] Fermentation medium: 50 mL of fermentation broth (container: 250 mL baffled conical flask, fermentation medium containing 50 μg / mL kanamycin and 5 μg / mL chloramphenicol) was inoculated with 5% of the total fermentation system. Concentrated ammonia was used to adjust the pH of the fermentation broth to neutral. The culture was placed in a constant temperature shaker at 30°C and 200 rpm for 96 h (0.8 mM IPTG was added for induction after 3 h of culture). Samples were taken every 24 h during the culture period to detect OD 600 And the concentration of target product vanillin. (1) OD 600 Detection: The fermentation liquid was injected into the cuvette, diluted 50 times with deionized water, and then the absorbance (600 nm) was measured using a UV spectrophotometer to measure the OD 600 (2) Determination of vanillin concentration in fermentation broth: The vanillin in the fermentation broth was detected using an UltiMate 3000 high performance liquid chromatograph (HPLC, Thermo Fisher Scientific). The detection method was as follows: the mobile phase included phase A (0.1% formic acid aqueous solution) and phase B (pure methanol). The chromatographic column used was a ZORBAX SB-C18 reversed phase column. The column temperature was set to 28°C, the flow rate of the mobile phase was set to 1.0 mL / min, and the detection was performed using an ultraviolet detector with an ultraviolet absorption wavelength set to 260 nm. Finally, the content of the product and other metabolites was calculated based on the data peak graphs of different fermentation samples according to the standard curve drawn from the standard sample. The results are shown in Table 1 and Figure 2.

[0229] Table 1

[0230] Example 2

[0231] A method for constructing a genetically engineered bacterium for de novo synthesis of vanillin using glucose as a substrate comprises the following steps:

[0232] Using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template, the primers tktA-up2 / tktA-down2 were used to amplify the fragment tktA (as shown in SEQ ID NO.83), obtaining a tktA fragment derived from Corynebacterium glutamicum with seamless cloning homology arms connected to aroG and RBS (ribosome binding site).

[0233] Using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template, the primers aroG-up2 / aroG-down2 were used to amplify the fragment aroG (as shown in SEQ ID NO.84), obtaining the Corynebacterium glutamicum-derived aroG fragment with seamless cloning homology arms and RBS connected to the plasmid.

[0234] Using the codon-optimized comt fragment of Arabidopsis thaliana (Mouseear cress) synthesized by BGI (as shown in SEQ ID NO.85) as a template, the comt fragment was amplified using primers comt-up2 / comt-down2 to obtain the Arabidopsis thaliana-derived comt fragment with seamless cloned homology arms and RBS connected to tktA.

[0235] Using the codon-optimized car gene fragment of Mycobacterium marinum synthesized by BGI (as shown in SEQ ID NO.86) as a template, the fragment car was amplified using primers car-up2 / car-down2 to obtain the marine Mycobacterium marinum-derived car fragment with seamless cloning homology arms and RBS connected to met2.

[0236] Using the codon-optimized sfp gene fragment of Nocardia iowensis synthesized by BGI (as shown in SEQ ID NO.87) as a template, the primers sfp-up2 / sfp-down2 were used to amplify the fragment sfp to obtain the Nocardia-derived sfp fragment with seamless cloning homology arms and RBS connected to the car and plasmid.

[0237] Using Escherichia coli K12 genomic DNA as a template, primers metE-up2 / metE-down2 were used to amplify the metE fragment (Gene ID: 948323) to obtain an E. coli-derived metE fragment with seamless cloning homology arms and RBS connected to ahcY and plasmid.

[0238] Using E. coli K12 genomic DNA as a template, primers metK-up2 / metK-down2 were used to amplify the metK fragment (Gene ID: 945389) to obtain an E. coli-derived metK fragment with seamless cloning homology arms and RBS connected to the plasmid.

[0239] The met2 fragment (Gene ID: 855444) was amplified using the genomic DNA of Saccharomyces cerevisiae S288C using primers met2-up2 / met2-down2 to obtain a Saccharomyces cerevisiae S288C-derived met2 fragment with seamlessly cloned homology arms and RBS connected to metK.

[0240] Using genomic DNA of Corynebacterium glutamicum ATCC13032 as a template, the primers ahcY-up2 / ahcY-down2 were used to amplify the fragment ahcY (as shown in SEQ ID NO. 52), obtaining an ahcY fragment derived from Corynebacterium glutamicum with seamlessly cloned homology arms and RBS connected to comt.

[0241] The primer sequences are:

[0242] tktA-up2:

[0243] 5'-ACGCCGAGCAGCAGCCAAGTAAAAGGAGGATATACATGTGGACACCAAGGCTGTAGA-3'; SEQ ID NO.88;

[0244] tktA-down2: 5'-TTAACCGTTAATGGAGTCCTGG-3'; SEQ ID NO.89;

[0245] aroG-up2:

[0246] 5'-GGAAACAGACCATGGAATTCAAGGAGGATATACATATGAGTTCTCCAGTCTCACTCG-3'; SEQ ID NO.90;

[0247] aroG-down2: 5'-TTACTTGGCTGCTGCTCG-3'; SEQ ID NO.91;

[0248] comt-up2:

[0249] 5'-CCAAGGACTCCATTAACGGTTAAAAGGAGGATATACATATGGGTAGCACCGCGG-3'; SEQ ID NO.92;

[0250] comt-down2: 5'-TTACAGTTTTTTCAGCAGTTCAATCAGG-3'; SEQ ID NO.93;

[0251] car-up2:

[0252] 5'-AAGAAGTTACCAACTGGTAGtctagaAAGGAGGATATACATATGTCCCCAATCACCCGC-3'; SEQ ID NO.94;

[0253] car-down2:5’-TTGGATCCTTACAGCAGGCCCAGCAGG-3’;SEQ ID NO.95;

[0254] sfp-up2:

[0255] 5’-CTGCTGGGCCTGCTGTAAggatccAAGGAGGATATACATATGATCGAAACCATCCTG-3’;SEQ ID NO.96;

[0256] sfp-down2:

[0257] 5’-CGCCAAAACAGCCAAGCTGAATTCGAGCTCGGTACCCTTATGCGTATGCGATTGCGGTC-3’;SEQ ID NO.97;

[0258] metE-up2:

[0259] 5’-CGGAGCACTACCGCTACTAAAAGGAGGATATACATATGACAATATTGAATCACACCCTCG-3’;SEQ ID NO.98;

[0260] metE-down2:

[0261] 5’-GACTCTAGAGGATCCCCGGGTACCGAGCTCTTACCCCCGACGCAAGTTC-3’;SEQ ID NO.99;

[0262] metK-up2:

[0263] 5’-TTAATTAAGCTTGCATGCCTGCAGGTCGACAAGGAGGATATACATATGGCAAAACACCTTTTTACGTC-3’;SEQ ID NO.100;

[0264] metK-down2:5’-TTACTTCAGACCGGCAGCA-3’;SEQ ID NO.101;

[0265] met2-up2:

[0266] 5’-GCTGCCGGTCTGAAGTAAAAGGAGGATATACATATGTCGCATACTTTAAAATCGAAAACG-3’;SEQ ID NO.102;

[0267] met2-down2: 5'-CTACCAGTTGGTAACTTCTTCGG-3'; SEQ ID NO.103;

[0268] ahcY-up2:

[0269] 5'-ATTGAACTGCTGAAAAAAACTGTAAAAGGAGGATATACATATGGACTTCAAGGTTGCCGA-3'; SEQ ID NO. 104;

[0270] ahcY-down2: Same as ahcY-down.

[0271] The gene amplification system and gene amplification procedure are the same as above.

[0272] The PCR products were subjected to agarose gel electrophoresis and product recovery to obtain the corresponding gene products tkt, aroG, comt, car, sfp, metE, metK, metX and ahcY from different sources.

[0273] The plasmid vector pEC-XK99E was linearized with primers pec-F and pec-R to obtain the pec vector, as in Example 1.

[0274] The ligation system, procedure, transformation and verification were the same as those in Example 1. A correct single colony was selected and named E. coli WJ102. After multiplication and plasmid extraction, the E. coli WJ102 plasmid was obtained.

[0275] The plasmid vector pXMJ19 was linearized with primers px-F and px-R to obtain the pxmj vector, as in Example 1.

[0276] The ligation system, procedure, transformation and verification were the same as those in Example 1. A correct single colony was selected and named E. coli WJ202. After multiplication and plasmid extraction, the E. coli WJ202 plasmid was obtained.

[0277] The E. coli WJ102 plasmid and the E. coli WJ202 plasmid were electroporated into competent cells of Corynebacterium glutamicum CG004 (prepared in Example 1), designated Corynebacterium glutamicum CG102. Competent cell preparation, electroporation, verification, and fermentation were performed as in Example 1. The results are shown in Table 2 and Figure 3.

[0278] Table 2

[0279] Example 3

[0280] A method for constructing a genetically engineered bacterium for de novo synthesis of vanillin using glucose as a substrate comprises the following steps:

[0281] The tkl1 fragment (Gene ID: 856188) was amplified using the genomic DNA of Saccharomyces cerevisiae S288C using primers tkl1up3 / tkl1-down3 to obtain a Saccharomyces cerevisiae S288C-derived tkl1 fragment with seamlessly cloned homology arms connected to aro4 and RBS (ribosome binding site).

[0282] The genomic DNA of Saccharomyces cerevisiae S288C was used as a template and primers aro4-up3 / aro4-down3 were used to amplify the fragment aro4 (Gene ID: 852551). The aro4 fragment derived from Saccharomyces cerevisiae S288C with seamlessly cloned homology arms and RBS connected to the plasmid was obtained.

[0283] Using the codon-optimized COMT fragment of Homo sapiens (Human) synthesized by BGI as a template (as shown in SEQ ID NO. 105), the COMT fragment was amplified using primers COMT-UP3 / COMT-DOWN3 to obtain a human-derived COMT fragment with seamless cloned homology arms and RBS connected to TKL1.

[0284] Using the codon-optimized car gene fragment of Nocardia iowensis synthesized by BGI (as shown in SEQ ID NO.47) as a template, the fragment car was amplified using primers car-up3 / car-down3 to obtain the Nocardia iowensis-derived car fragment with seamless cloning homology arms and RBS connected to met2.

[0285] Using the codon-optimized sfp gene fragment of Bacillus subtilis synthesized by BGI (as shown in SEQ ID NO.106) as a template, the primers sfp-up3 / sfp-down3 were used to amplify the fragment sfp to obtain the Bacillus subtilis-derived sfp fragment with seamless cloning homology arms and RBS connected to the car and plasmid.

[0286] The met6 fragment (Gene ID: 856825) was amplified using the genomic DNA of Saccharomyces cerevisiae S288C using primers met6-up3 / met6-down3 to obtain a Saccharomyces cerevisiae S288C-derived met6 fragment with seamlessly cloned homology arms and RBS connected to ahcY and plasmid.

[0287] The SAM2 fragment (Gene ID: 852113) was amplified using the genomic DNA of Saccharomyces cerevisiae S288C using primers SAM2-up3 / SAM2-down3 to obtain a SAM2 fragment derived from Saccharomyces cerevisiae S288C with seamlessly cloned homology arms and RBS connected to the plasmid.

[0288] The met2 fragment (Gene ID: 855444) was amplified using the genomic DNA of Saccharomyces cerevisiae S288C using primers met2-up3 / met2-down3 to obtain a Saccharomyces cerevisiae S288C-derived met2 fragment with seamlessly cloned homology arms and RBS connected to SAM2.

[0289] Using genomic DNA of Corynebacterium glutamicum ATCC13032 as a template, the primers ahcY-up3 / ahcY-down3 were used to amplify the fragment ahcY (as shown in SEQ ID NO. 52), obtaining an ahcY fragment derived from Corynebacterium glutamicum with seamlessly cloned homology arms and RBS connected to comt.

[0290] The primer sequences are:

[0291] tkl1up3:

[0292] 5'-AGAGAAGTTAACAAGAAATAGAAGGAGGATATACATATGACTCAATTCACTGACATTGATAAGC-3'; SEQ ID NO. 107;

[0293] tkl1-down3: 5'-TTAGAAAGCTTTTTTTCAAAGGAGAAATTAGC-3'; SEQ ID NO.108;

[0294] aro4-up3:

[0295] 5'-GGAAACAGACCATGGAATTCAAGGAGGATATACATATGAGTGAATCTCCAATGTTCGC-3'; SEQ ID NO. 109;

[0296] aro4-down3: 5'-CTATTTCTTGTTAACTTCTCTCTTTGTCTG-3'; SEQ ID NO.110;

[0297] comt-up3:

[0298] 5’-CTCCTTTGAAAAAAGCTTTCTAAAAGGAGGATATACATATGCCGGAAGCCCCG-3’; SEQ ID NO.111;

[0299] comt-down3: 5’-TTACGGACCCGCTTCACTACC-3’; SEQ ID NO.112;

[0300] car-up3:

[0301] 5’-AAGAAGTTACCAACTGGTAGTCTAGAAAGGAGGATATACATATGGCAGTGGATTCCCCAG-3’; SEQ ID NO.113;

[0302] car-down3: same as car-down;

[0303] sfp-up3:

[0304] 5’-CTGCTGCAGCTGCTGTAAGGATCCCCAAGGAGGATATACATATGAAGATCTACGGCATC-3’; SEQ ID NO.114;

[0305] sfp-down3:

[0306] 5’-CTGAATTCGAGCTCGGTACCCTTACAGCAGTTCTTCGTAGGACAC-3’; SEQ ID NO.115;

[0307] met6-up3:

[0308] 5’-CGGAGCACTACCGCTACTAAAAGGAGGATATACATATGGTTCAATCTGCTGTCTTAGG-3’; SEQ ID NO.116;

[0309] met6-down3:

[0310] 5’-TCTAGAGGATCCCCGGGTACCGAGCTCTTAATTCTTGTATTGTTCACGGAAGTACTTG-3’; SEQ ID NO.117;

[0311] SAM2-up3:

[0312] 5'-TTAAGCTTGCATGCCTGCAGGTCGACAAGGAGGATATACATATGTCCAAGAGCAAAACTTTCTTAT-3'; SEQ ID NO. 118;

[0313] SAM2-down3: 5'-TTAAAATTCCAATTTCTTTGGTTTTTCCC-3'; SEQ ID NO.119;

[0314] met2-up3:

[0315] 5'-CCAAAGAAATTGGAATTTTAAAAGGAGGATATACATATGTCGCATACTTTAAAATCGAAAACG-3'; SEQ ID NO. 120;

[0316] met2-down3: same as met2-down2;

[0317] ahcY-up3:

[0318] 5'-GGTAGTGAAGCGGGTCCGTAAAAGGAGGATATACATATGGACTTCAAGGTTGCCGA-3'; SEQ ID NO. 121;

[0319] ahcY-down3: Same as ahcY-down.

[0320] The gene amplification system and gene amplification procedure are the same as above.

[0321] The PCR products were subjected to agarose gel electrophoresis and product recovery to obtain the corresponding gene products tkl1, aro4, comt, car, sfp, met6, SAM2, met2 and ahcY from different sources.

[0322] The plasmid vector pEC-XK99E was linearized with primers pec-F and pec-R to obtain the pec vector, as in Example 1.

[0323] The ligation system, procedure, transformation and verification were the same as those in Example 1. A correct single colony was selected and named E. coli WJ103. After multiplication and plasmid extraction, the E. coli WJ103 plasmid was obtained.

[0324] The plasmid vector pXMJ19 was linearized with primers px-F and px-R to obtain the pxmj vector, as in Example 1.

[0325] The ligation system, procedure, transformation and verification were the same as those in Example 1. A correct single colony was selected and named E. coli WJ203. After multiplication and plasmid extraction, the E. coli WJ203 plasmid was obtained.

[0326] The E. coli WJ103 plasmid and the E. coli WJ203 plasmid were electroporated into competent cells of Corynebacterium glutamicum CG004 (prepared in Example 1), designated Corynebacterium glutamicum CG103. Competent cell preparation, electroporation, verification, and fermentation were performed as in Example 1. The results are shown in Table 3 and Figure 4.

[0327] Table 3

[0328] Example 4

[0329] A method for constructing a genetically engineered bacterium for de novo synthesis of vanillin using glucose as a substrate comprises the following steps:

[0330] Using Bacillus subtilis 168 genomic DNA as a template, the primers tktA-up4 / tktA-down4 were used to amplify the fragment tktA (Gene ID: 937377), obtaining a tktA fragment derived from Bacillus subtilis 168 with seamless cloning homology arms connected to aroX and RBS (ribosome binding site).

[0331] The genomic DNA of Bacillus subtilis 168 was used as a template and primers aroX-up4 / aroX-down4 were used to amplify the fragment aroX (Gene ID: 937853) to obtain the aroX fragment derived from Bacillus subtilis 168 with seamless cloning homology arms and RBS connected to the plasmid.

[0332] Using the codon-optimized comt fragment of coffee (Coffea canephora, Robusta coffee) synthesized by BGI (as shown in SEQ ID NO.122) as a template, the comt fragment was amplified using primers comt-up4 / comt-down4 to obtain the coffee-derived comt fragment with seamless cloned homology arms and RBS connected to tktA.

[0333] Using the codon-optimized car gene fragment of Nocardia iowensis synthesized by BGI (as shown in SEQ ID NO.47) as a template, the fragment car was amplified using primers car-up4 / car-down4 to obtain the Nocardia-derived car fragment with seamless cloning homology arms and RBS connected to metAA.

[0334] Using the codon-optimized sfp gene fragment of Mycobacterium marinum synthesized by BGI (as shown in SEQ ID NO.48) as a template, the primers sfp-up4 / sfp-down4 were used to amplify the fragment sfp to obtain the sfp fragment derived from Mycobacterium marinum with seamless cloning homology arms and RBS connected to CAR and plasmid.

[0335] The metE fragment (Gene ID: 936480) was amplified using the genomic DNA of Bacillus subtilis 168 as a template using primers metE-up4 / metE-down4 to obtain a metE fragment derived from Bacillus subtilis 168 with seamless cloning homology arms and RBS connected to ahcY and plasmid.

[0336] The metK fragment (Gene ID: 937090) was amplified using the genomic DNA of Bacillus subtilis 168 as a template using primers metK-up4 / metK-down4 to obtain a metK fragment derived from Bacillus subtilis 168 with seamless cloning homology arms and RBS connected to the plasmid.

[0337] The metAA fragment (Gene ID: 939083) was amplified using the genomic DNA of Bacillus subtilis 168 as a template using primers metAA-up4 / metAA-down4 to obtain the metAA fragment derived from Bacillus subtilis 168 with seamless cloned homology arms and RBS connected to metK.

[0338] Using genomic DNA of Corynebacterium glutamicum ATCC13032 as a template, the primers ahcY-up4 / ahcY-down4 were used to amplify the fragment ahcY (as shown in SEQ ID NO. 52), obtaining an ahcY fragment derived from Corynebacterium glutamicum with seamlessly cloned homology arms and RBS connected to comt.

[0339] The primer sequences are:

[0340] tktA-up4:

[0341] 5'-CAATGGTGAAAGTCAACGCTTAAAAGGAGGATATACATATGGATACAATTGAAAAGAAATCAGTTGC-3'; SEQ ID NO. 123;

[0342] tktA-down4: 5'-TTACTTATTGATTAATGCCTTAACTCGATTC-3'; SEQ ID NO. 124;

[0343] aroX-up4:

[0344] 5'-GGAAACAGACCATGGAATTCAAGGAGGATATACATATGAGCAACACAGAGTTAGAGC-3'; SEQ ID NO.125;

[0345] aroX-down4: 5'-TTAAGCGTTGACTTTCACCATTG-3'; SEQ ID NO.126;

[0346] comt-up4:

[0347] 5'-AGGCATTAATCAATAAGTAAAAGGAGGATATACATATGGCCGAAGAAGAAGCATG-3'; SEQ ID NO.127;

[0348] comt-down4: 5'-TTATTTACACAGTTCCATAATCCAGGTATTC-3'; SEQ ID NO.128;

[0349] car-up4:

[0350] 5'-TCCTTATGAATGGGACTAAtctagaAAGGAGGATATACATATGGCAGTGGATTCCCCAG-3'; SEQ ID NO. .129;

[0351] car-down4: same as car-down1;

[0352] sfp-up4: same as sfp-up 1;

[0353] sfp-down4: same as sfp-down 1;

[0354] metE-up4:

[0355] 5'-CGGAGCACTACCGCTACTAAAAGGAGGATATACATATGACAACCATCAAAACATCGAAT-3'; SEQ ID NO.130;

[0356] metE-down4:

[0357] 5'-TCTAGAGGATCCCCGGGTACCGAGCTCTTATACTAGCTGTGTCTGCTGTGC-3'; SEQ ID NO.131;

[0358] metK-up4:

[0359] 5'-TTAAGCTTGCATGCCTGCAGGTCGACAAGGAGGATATACATATGAGTAAAAATCGTCGTTTATTTACATC-3'; SEQ ID NO. 132;

[0360] metK-down4: 5'-TTATTCTCCTAACGCTTCTTTACGC-3'; SEQ ID NO.133;

[0361] metAA-up4:

[0362] 5'-AAGAAGCGTTAGGAGAATAAAAGGAGGATATACATTTGCCTATTAATATACCAACACACCTG-3'; SEQ ID NO. 134;

[0363] metAA-down4: 5'-TTAGTCCCATTCATAAGGAGTTTCTTG-3'; SEQ ID NO. 135;

[0364] ahcY-up4:

[0365] 5'-TTATGGAACTGTGTAAATAAAAGGAGGATATACATATGGACTTCAAGGTTGCCGA-3'; SEQ ID NO. 136;

[0366] ahcY-down4: Same as ahcY-down.

[0367] The gene amplification system and gene amplification procedure are the same as above.

[0368] The PCR products were subjected to agarose gel electrophoresis and product recovery to obtain the corresponding gene products tktA, aroX, comt, car, sfp, metE, metK, metAA and ahcY from different sources.

[0369] The plasmid vector pEC-XK99E was linearized with primers pec-F and pec-R to obtain the pec vector, as in Example 1.

[0370] The ligation system, procedure, transformation and verification were the same as those in Example 1. A correct single colony was selected and named E. coli WJ104. After multiplication and plasmid extraction, the E. coli WJ104 plasmid was obtained.

[0371] The plasmid vector pXMJ19 was linearized with primers px-F and px-R to obtain the pxmj vector, as in Example 1.

[0372] The ligation system, procedure, transformation and verification were the same as those in Example 1. A correct single colony was selected and named E. coli WJ204. After multiplication and plasmid extraction, the E. coli WJ204 plasmid was obtained.

[0373] The E. coli WJ104 plasmid and the E. coli WJ204 plasmid were electroporated into competent cells of Corynebacterium glutamicum CG004 (prepared in Example 1), designated Corynebacterium glutamicum CG104. Competent cell preparation, electroporation, verification, and fermentation were performed as in Example 1. The results are shown in Table 4 and Figure 5.

[0374] Table 4

[0375] Example 5

[0376] A method for constructing a genetically engineered bacterium for de novo synthesis of vanillin using glucose as a substrate comprises the following steps:

[0377] Using the codon-optimized comt fragment of mouse (Mus musculus, Mouse) synthesized by BGI (as shown in SEQ ID NO.137) as a template, the fragment comt was amplified using primers comt-up5 / comt-down4 to obtain the mouse-derived comt fragment with seamless cloned homology arms and RBS connected to tktA.

[0378] The primer sequences are as follows:

[0379] comt-up5:

[0380] 5'-AAAGCAAAAGAACTGCTGTAAAAGGAGGATATACATATGCTGCTGGCGGCC-3'; SEQ ID NO. 138;

[0381] comt-down4: 5'-TTAGCTTTTAACCGGGCTGCT-3'; SEQ ID NO. 139;

[0382] ahcY-up5:

[0383] 5'-AGCAGCCCGGTTAAAAGCTAAAAGGAGGATATACATATGGACTTCAAGGTTGCCGA-3'; SEQ ID NO. 140.

[0384] The remaining genes and primers are the same as in Example 1.

[0385] The gene amplification system and gene amplification procedure are the same as above.

[0386] The PCR products were subjected to agarose gel electrophoresis and product recovery to obtain the corresponding gene products tktA, aroG, comt, car, sfp, metE, metK, metX and ahcY from different sources.

[0387] The plasmid vector pEC-XK99E was linearized with primers pec-F and pec-R to obtain the pec vector, as in Example 1.

[0388] The ligation system, procedure, transformation and verification were the same as those in Example 1. A correct single colony was selected and named E. coli WJ105. After multiplication and plasmid extraction, the E. coli WJ105 plasmid was obtained.

[0389] The E. coli WJ105 plasmid and the E. coli WJ201 plasmid were electroporated into competent cells of Corynebacterium glutamicum CG004 (prepared in Example 1), designated Corynebacterium glutamicum CG105. Competent cell preparation, electroporation, verification, and fermentation were performed as in Example 1. The results are shown in Table 5 and Figure 6.

[0390] Table 5

[0391] The HPLC test results of the vanillin standard product showed a peak time of 17.231 min, and the peak time of the fermentation culture product of the genetically engineered strain prepared in Example 1-5 was 17.233 min. The peak time of the fermentation sample of the engineered strain was consistent with the peak time of the vanillin standard product, proving that the fermentation product of the engineered strain was the target product - vanillin.

[0392] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A genetically engineered bacterium for synthesizing vanillin from scratch using glucose as a substrate, characterized in that: The genetically engineered bacteria are recombinant Corynebacterium glutamicum that have been transformed from chassis microorganisms and include a vanillin synthesis module and a methyl cycle regeneration module; The chassis microorganism is transformed by using Corynebacterium glutamicum ATCC13032 as the starting strain, and knocking out the pcaHG, van, vdh and fud genes; The vanillin synthesis module expresses a transketolase gene, a 3-deoxy-7-phosphoheptanoate synthetase gene, an O-methyltransferase gene, a carboxylic acid reductase gene, and a phosphopantetheinyl transferase gene; The methyl cycle regeneration module expresses 5-methyltetrahydrotriglutamate-homocysteine ​​methyltransferase gene, S-adenosylmethionine synthetase gene, homoserine-O-acetyltransferase gene and adenosylhomocysteine ​​enzyme gene.

2. The genetically engineered bacterium for synthesizing vanillin from scratch using glucose as a substrate according to claim 1, characterized in that: The transketolase gene includes an endogenous Corynebacterium glutamicum transketolase gene or an exogenous transketolase gene; The 3-deoxy-7-phosphoheptalone synthetase gene includes an endogenous Corynebacterium glutamicum 3-deoxy-7-phosphoheptalone synthetase gene or an exogenous 3-deoxy-7-phosphoheptalone synthetase gene; The O-methyltransferase gene includes an O-methyltransferase gene from Arabidopsis thaliana (Arabidopsis thaliana, Mouseear cress), an O-methyltransferase gene from coffee (Coffea canephora, Robusta coffee), an O-methyltransferase gene from Homo sapiens (Human), an O-methyltransferase gene from rat (Rattus norvegicus, Rat), and an O-methyltransferase gene from mouse (Mus musculus, Mouse); The carboxylic acid reductase gene includes a carboxylic acid reductase gene derived from Nocardia iowensis and a carboxylic acid reductase gene derived from Mycobacterium marinum; The phosphopantetheinyl transferase gene includes a phosphopantetheinyl transferase gene derived from Nocardia iowensis, a phosphopantetheinyl transferase gene derived from Mycobacterium marinum, and a phosphopantetheinyl transferase gene derived from Bacillus subtilis; The 5-methyltetrahydrotriglutamate-homocysteine ​​methyltransferase gene includes an endogenous Corynebacterium glutamicum 5-methyltetrahydrotriglutamate-homocysteine ​​methyltransferase gene or an exogenous 5-methyltetrahydrotriglutamate-homocysteine ​​methyltransferase gene; The S-adenosylmethionine synthetase gene includes an endogenous Corynebacterium glutamicum S-adenosylmethionine synthetase gene and an optional exogenous S-adenosylmethionine synthetase gene; The homoserine-O-acetyltransferase gene includes an endogenous Corynebacterium glutamicum homoserine-O-acetyltransferase gene, and optionally an exogenous homoserine-O-acetyltransferase gene; The adenosylhomocysteine ​​enzyme gene includes an endogenous adenosylhomocysteine ​​enzyme gene of Corynebacterium glutamicum and an optional exogenous adenosylhomocysteine ​​enzyme gene; The nucleotide sequence of the endogenous Corynebacterium glutamicum transketolase gene is shown in SEQ ID NO.83; The nucleotide sequence of the endogenous Corynebacterium glutamicum 3-deoxy-7-phosphoheptoneate synthase gene is as shown in SEQ ID Shown in NO.84; The nucleotide sequence of the Arabidopsis thaliana-derived O-methyltransferase gene is shown in SEQ ID NO.85; the nucleotide sequence of the coffee-derived O-methyltransferase gene is shown in SEQ ID NO.122; the nucleotide sequence of the human-derived O-methyltransferase gene is shown in SEQ ID NO.105; the nucleotide sequence of the rat-derived O-methyltransferase gene is shown in SEQ ID NO.46; the nucleotide sequence of the mouse-derived O-methyltransferase gene is shown in SEQ ID NO.137; The nucleotide sequence of the carboxylic acid reductase gene derived from Nocardia is shown in SEQ ID NO.47; the nucleotide sequence of the carboxylic acid reductase gene derived from Mycobacterium marineum is shown in SEQ ID NO.86; The nucleotide sequence of the phosphopantetheinyl transferase gene from Nocardia is shown in SEQ ID NO.87; the nucleotide sequence of the phosphopantetheinyl transferase gene from Mycobacterium marineum is shown in SEQ ID NO.48; the nucleotide sequence of the phosphopantetheinyl transferase gene from Bacillus subtilis is shown in SEQ ID NO.106; The nucleotide sequence of the endogenous Corynebacterium glutamicum 5-methyltetrahydrotriglutamate-homocysteine ​​methyltransferase gene is shown in SEQ ID NO.49; The nucleotide sequence of the endogenous Corynebacterium glutamicum S-adenosylmethionine synthetase gene is shown in SEQ ID NO.50; The nucleotide sequence of the endogenous Corynebacterium glutamicum homoserine-O-acetyltransferase gene is shown in SEQ ID NO.51; The nucleotide sequence of the endogenous Corynebacterium glutamicum adenosylhomocysteine ​​enzyme gene is shown in SEQ ID NO.

52.

3. The genetically engineered bacterium for synthesizing vanillin from scratch using glucose as a substrate according to claim 2, characterized in that: The exogenous transketolase gene is derived from Escherichia coli K12, Saccharomyces cerevisiae S288C or Bacillus subtilis 168; the transketolase gene derived from Escherichia coli K12: Gene ID: 947420; the transketolase gene derived from Saccharomyces cerevisiae S288C: Gene ID: 856188; the transketolase gene derived from Bacillus subtilis 168: Gene ID: 937377; The exogenous 3-deoxy-7-phosphoheptone synthase gene is derived from Escherichia coli K12, Saccharomyces cerevisiae S288C or Bacillus subtilis 168; the nucleotide sequence of the 3-deoxy-7-phosphoheptone synthase gene derived from Escherichia coli K12 is shown in SEQ ID NO.45; the 3-deoxy-7-phosphoheptone synthase gene derived from Saccharomyces cerevisiae S288C: Gene ID: 852551; the 3-deoxy-7-phosphoheptone synthase gene derived from Bacillus subtilis 168: Gene ID: 937853; The exogenous 5-methyltetrahydrotriglutamate-homocysteine ​​methyltransferase gene is derived from Escherichia coli K12, Saccharomyces cerevisiae S288C or Bacillus subtilis 168; the 5-methyltetrahydrotriglutamate-homocysteine ​​methyltransferase gene derived from Escherichia coli K12: Gene ID: 948323; the 5-methyltetrahydrotriglutamate-homocysteine ​​methyltransferase gene derived from Saccharomyces cerevisiae S288C: Gene ID: 856825; the 5-methyltetrahydrotriglutamate-homocysteine ​​methyltransferase gene derived from Bacillus subtilis 168: Tetrahydrotriglutamate-homocysteine ​​methyltransferase gene: Gene ID: 936480; The exogenous S-adenosylmethionine synthetase gene is derived from Escherichia coli K12, Saccharomyces cerevisiae S288C or Bacillus subtilis 168; the S-adenosylmethionine synthetase gene derived from Escherichia coli K12: Gene ID: 948323; the S-adenosylmethionine synthetase gene derived from Saccharomyces cerevisiae S288C: Gene ID: 852113; the S-adenosylmethionine synthetase gene derived from Bacillus subtilis 168: Gene ID: 937090; The exogenous homoserine-O-acetyltransferase gene is derived from Saccharomyces cerevisiae S288C or Bacillus subtilis 168; the homoserine-O-acetyltransferase gene derived from Saccharomyces cerevisiae S288C: Gene ID: 855444; the homoserine-O-acetyltransferase gene derived from Bacillus subtilis 168: Gene ID: 939083.

4. The genetically engineered bacterium for synthesizing vanillin from scratch using glucose as a substrate according to claim 3, characterized in that: The exogenous transketolase gene is derived from Escherichia coli K12; The exogenous 3-deoxy-7-phosphoheptanoate synthase gene is derived from Escherichia coli K12; The O-methyltransferase gene is derived from rat; The carboxylic acid reductase gene is derived from Nocardia; The phosphopantethein aminotransferase gene is derived from marine mycobacterium; The 5-methyltetrahydrotriglutamate-homocysteine ​​methyltransferase gene is derived from endogenous Corynebacterium glutamicum; The S-adenosylmethionine synthetase gene is derived from endogenous Corynebacterium glutamicum; The homoserine-O-acetyltransferase gene is derived from endogenous Corynebacterium glutamicum; The adenosylhomocysteine ​​enzyme gene is derived from endogenous Corynebacterium glutamicum.

5. A method for constructing a genetically engineered bacterium for synthesizing vanillin from scratch using glucose as a substrate, characterized in that: The following steps are involved: (1) Using Corynebacterium glutamicum ATCC13032 as the starting strain, knocking out the pcaHG, van, vdh and fud genes to obtain a modified Corynebacterium glutamicum; (2) Expressing the vanillin synthesis module and the methyl cycle regeneration module described in claim 1 in the modified Corynebacterium glutamicum.

6. Use of the genetically engineered bacteria according to any one of claims 1 to 4 or the method according to claim 5 in producing vanillin.

7. Use of the genetically engineered bacteria according to any one of claims 1 to 4 or the method according to claim 5 in increasing the yield of vanillin.

8. A method for producing vanillin, characterized in that: Fermentation is carried out using the genetically engineered bacteria described in any one of claims 1 to 4 or the genetically engineered bacteria constructed by the method described in claim 5.

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