O-methyltransferase mutants and uses thereof

Abstract

The present application relates to catechol O The present application relates to a vanillin production method, and belongs to the technical field of metabolic engineering. The present application uses catechol methyltransferase mutants to produce vanillin. Leptospira interrogans ​ The present application relates to a vanillin production method, and belongs to the technical field of metabolic engineering. The present application uses catechol methyltransferase mutants to produce vanillin. O The present application relates to a vanillin production method, and belongs to the technical field of metabolic engineering. The present application uses catechol methyltransferase mutants to produce vanillin. The present application relates to a vanillin production method, and belongs to the technical field of metabolic engineering. The present application uses catechol methyltransferase mutants to produce vanillin.

Description

Technical Field

[0001] This invention relates to catechins O The methyltransferase mutant and its application in vanillin production belong to the field of metabolic engineering technology. Background Technology

[0002] Vanillin, hailed as the "King of Flavors," boasts a vast market potential. In the food industry, vanillin, with its intense sweetness and rich, creamy vanilla flavor, is used as a multifunctional flavor additive. Beyond enhancing the flavor of food and beverages, vanillin is also used as a biological preservative due to its antibacterial, antioxidant, and antimutagenic activities. Synthetic vanillin is also used in the manufacture of numerous household products, such as deodorants, air fresheners, floor polishes, and herbicides. Furthermore, it is used in the pharmaceutical field as a precursor to dopamine and other compounds.

[0003] Vanillin production methods include plant extraction, chemical synthesis, and microbial synthesis. Natural vanillin is primarily extracted from the orchid *Vanilla veitchii*. Plant extraction is complex and costly, and its yield is extremely low due to the influence of plant growth cycles and climate change, accounting for less than 0.2% of the vanillin market. Chemical and microbial synthesis are currently the main methods in the market. While chemical synthesis reduces costs, its production process easily leads to environmental pollution and inevitably involves the use of harmful and toxic compounds, potentially endangering human health, thus its use is strictly limited. Microbial synthesis of vanillin can use inexpensive and readily available natural substances as substrates, synthesizing vanillin through cell factories or bio-enzyme catalysis. Vanillin obtained through microbial synthesis has high purity and the activity of natural vanillin, effectively solving the problems of large land occupation, slow plant growth cycles, and low yields associated with plant extraction, while avoiding the pollution and toxicity of chemical synthesis.

[0004] In the microbial production of vanillin, compounds such as ferulic acid, eugenol, and isoeugenol are usually used as precursors. Among them, ferulic acid is the most common raw material for vanillin production and has good vanillin conversion efficiency. However, ferulic acid is expensive, which leads to excessively high production costs of vanillin. In addition, it is highly toxic to the production strains, which is not conducive to large-scale industrial production.

[0005] The biosynthesis of vanillin from simple carbon sources such as glucose and glycerol is more attractive because these sources are cheaper and more readily available. *E. coli* has become one of the preferred production hosts due to its advantages such as rapid growth, simple culture conditions, ease of genetic manipulation, and good characterization. The natural shikimic acid pathway in *E. coli* enables the production of 3-dehydroshikimic acid from glucose. The production pathway from 3-dehydroshikimic acid to vanillin involves three steps, catalyzed by four different enzymes. First, 3-dehydroshikimic acid dehydratase (DSD) converts 3-dehydroshikimic acid to protocatechuic acid, and then, catechins... O 3-Dehydroshikimic acid (COMT) is converted from protocatechuic acid to vanillic acid, and finally, carboxylic acid reductase (CAR) and phosphopanylthioethylaminotransferase (SFP) convert vanillic acid to vanillin. In this metabolic pathway, COMT is the rate-limiting enzyme and plays a decisive role in the conversion efficiency of 3-dehydroshikimic acid to vanillin. However, existing technologies show that naturally derived COMT enzymes have relatively low activity, poor conversion rates, and insufficient expression levels, necessitating modification.

[0006] Currently, *E. coli* possesses well-established and easily operable gene editing and enzyme expression systems, while also offering advantages such as a short growth cycle and simple cell structure. Using *E. coli* for the microbial synthesis of aromatic compounds can accelerate the construction and optimization of vanillin metabolic pathways. Summary of the Invention

[0007] The purpose of this invention is to provide a catechin with enhanced enzyme activity. O A methyltransferase mutant was developed and applied to the vanillin biotransformation pathway to overcome the problem of low vanillin yield in de novo biosynthesis using glucose as a substrate.

[0008] This invention uses a hook-tip helix ( Leptospira interrogans Catechins from O The methyltransferase-encoding gene was used as a starter gene for molecular modification, and protocatechuic acid was used as a substrate to screen for catechins that enhance vanillin production. O A methyltransferase mutant was obtained, which led to the development of a recombinant strain that produces high levels of vanillin.

[0009] The technical approach adopted by the present invention to achieve the above objectives is as follows:

[0010] One of the technical solutions provided by this invention is a catechin. O -Methyltransferase mutant, said mutant is the wild-type catechol shown in SEQ ID NO.1 O It was obtained by mutations of N5W, Q39I and D190M on the basis of α-methyltransferase; Furthermore, the catechols OThe -methyltransferase mutant is the COMT-N5W / Q39I / D190M mutant, and its amino acid sequence is shown in SEQ ID NO.3.

[0011] The second technical solution provided by this invention is a biomaterial, which includes any one of the following: A1) One of the coding technology solutions for catechins O -Methyltransferase mutant nucleic acid molecules; A2) An expression cassette containing the nucleic acid molecules described in A1); A3) A recombinant vector containing the nucleic acid molecules described in A1); A4) Recombinant microorganisms containing the nucleic acid molecules described in A1); Furthermore, all of the biological materials can express the nucleic acid molecules described in A1).

[0012] In the above-mentioned biological materials, the nucleic acid molecule described in A1) includes a DNA molecule with a coding sequence as shown in SEQ ID NO:4; Furthermore, the nucleic acid molecule may also include a nucleic acid molecule obtained by codon preference modification based on the nucleotide sequence shown in SEQ ID NO: 4; The nucleic acid molecules mentioned in this article can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecules can also be RNA, such as mRNA or hnRNA.

[0013] In the aforementioned biological materials, the recombinant vector described in A3) can be constructed using an expression vector. The structure of expression vectors is well known to those skilled in the art. Expression vectors typically contain elements required for target gene expression, such as promoters, multiple cloning sites, terminators, and ribosome binding sites, and may also contain selection marker genes (such as kanamycin resistance gene kanr, neomycin resistance gene neo, hygromycin resistance gene hyg, chloramphenicol resistance gene cat, streptomycin resistance gene str, bleomycin resistance gene ble, etc.). Expression vectors can be constructed using any method known in the art (such as recombination technology, synthesis technology, etc.) or can be commercially purchased. For example, in one or more embodiments of the present invention, the expression vector used for the recombinant vector is the pBR322 plasmid.

[0014] The recombinant vector can be a recombinant expression vector obtained by cloning a nucleic acid molecule encoding the COMT-N5W / Q39I / D190M mutant into an expression vector (such as a prokaryotic expression vector or a eukaryotic expression vector). Although the expression vector used in the embodiments provided in this invention is the pBR322 plasmid, this invention is not limited to this specific vector. Those skilled in the art can use other suitable vectors (e.g., pETM6, pUC18, pGEX, etc.), as long as the vector can express the catechol.O A methyltransferase mutant is sufficient.

[0015] In the above-mentioned biological materials, the recombinant microorganisms described in A4) include: recombinant Escherichia coli, recombinant Bacillus subtilis, or recombinant yeast; the preferred hosts are Escherichia coli and Bacillus subtilis, such as Escherichia coli JM109, Bacillus subtilis 168, or the Escherichia coli BW25113△adhE△yjgB△eutG△yqhD△yahK (i.e., BW△5), BW25113△adhE△yjgB△eutG△yqhD△yahK△yeaE△aroE (i.e., BW△7) constructed in this invention, etc. Preferably, in the above-mentioned biological materials, the recombinant microorganism in A4) is a recombinant Escherichia coli; the recombinant Escherichia coli is a recombinant Escherichia coli obtained by mutating the gene encoding the protein with the amino acid sequence shown in SEQ ID NO:1 in the host; the protein expressed after mutation includes one of the following mutations relative to SEQ ID NO:1: N5W, Q39I and D190M; Preferably, in the above-mentioned biological materials, the recombinant microorganism described in A4) is constructed in a host cell containing catechols described in one of the technical solutions. O The vanillin biosynthesis pathway of the methyltransferase mutant is obtained; further, the biosynthesis pathway includes catechol. O -Methyltransferase mutants, carboxylic acid reductase, and phosphate pantothenic acid ethylaminotransferase can achieve the biosynthesis from protocatechuic acid to vanillin; Furthermore, the catechins O -Methyltransferase mutant, encoding gene comt-N5W / Q39I / D190M The nucleotide sequence is shown in SEQ ID NO.4; the carboxylic acid reductase is from Nocardia and encodes the gene. car The nucleotide sequence is shown in SEQ ID NO. 6; the phosphate pantothenicotinamide ethylamine transferase is derived from Bacillus subtilis (…). Bacillus subtilis ), encoding genes sfp The nucleotide sequence is shown in SEQ ID NO.7; a structure containing catechol is constructed. O -Methyltransferase gene comt-N5W / Q39I / D190M Carboxylic acid reductase gene car and the phosphate pantothenic acid thioethylamine transferase gene sfp The recombinant vector was expressed in the host cell to obtain recombinant microorganisms, thus realizing the biosynthesis from protocatechuic acid to vanillin. Furthermore, the biosynthetic pathway also includes 3-dehydroshikimate dehydratase; the 3-dehydroshikimate dehydratase is derived from Pseudomonas and encodes a gene... dsdThe nucleotide sequence is shown in SEQ ID NO.5; a gene encoding 3-dehydroshikimate dehydratase was constructed. dsd、 Catechol O -Methyltransferase gene comt-N5W / Q39I / D190M Carboxylic acid reductase gene car and the phosphate pantothenic acid thioethylamine transferase gene sfp The recombinant vector was expressed in the host cell to obtain recombinant microorganisms, thus achieving the biosynthesis of vanillin from 3-dehydroshikimic acid.

[0016] The third technical solution provided by this invention is the catechin described in the first technical solution. O -Methyltransferase mutant, or the application of the nucleic acid molecule, expression cassette, recombinant vector, or recombinant microorganism described in technical solution two in any of the following: B1) Application in the catalytic conversion of protocatechuic acid to vanillic acid; B2) Application in increasing the yield of microbial vanillin; B3) Application in the preparation of vanillin or products containing vanillin; B4) Application in the construction of genetically engineered bacteria that produce vanillin.

[0017] The products include, but are not limited to, food, cosmetics, pharmaceuticals, animal feed, and daily chemical products.

[0018] Specifically, the application of the recombinant microorganisms described in technical solution two in the production of vanillin; the recombinant bacteria can synthesize vanillin using glucose as a substrate.

[0019] Beneficial effects: This invention provides a catechol methyltransferase mutant, COMT-N5W / Q39I / D190M, which, compared to the wild-type catechol methyltransferase, exhibits a more than fourfold increase in vanillin production capacity (wild-type strain BW-CC yields 115 mg / L vanillin with a conversion rate of 14.5%; the mutant strain BW-0716CC yields 3.207 mM vanillin with a conversion rate of 61.8%). This invention utilizes catechol... OThe methyltransferase mutant COMT-N5W / Q39I / D190M constructs a metabolic pathway from protocatechuic acid to vanillin, or from glucose to vanillin. This pathway overcomes the critical problem of low activity of key enzymes in this metabolic pathway, resulting in a higher ability to convert protocatechuic acid / glucose to produce vanillin. When this metabolic pathway is introduced into the vanillin-producing strain BW25113△adhE△yjgB△eutG△yqhD△yahK△yeaE△aroE(BW△7), the yield of vanillin produced using 0.8 g / L protocatechuic acid as a substrate is 0.488 g / L; and the yield of vanillin produced using 30 g / L glucose as a substrate reaches 0.223 g / L in shake flask mode. Attached Figure Description

[0020] Figure 1 Degradation of vanillin by BW25113△adhE△yjgB△eutG△yqhD (BW△4) with knockout of different aldehyde dehydrogenases.

[0021] Figure 2 This is a comparison chart of vanillin production yields of COMT-N5W / Q39I / D190M and LiCOMT in Escherichia coli BW△5 using protocatechuic acid as a substrate.

[0022] Figure 3 ppsA , tktA, talB, mtnN, luxS as well as aroG / aroD / aroB The effects of overexpression of key genes through pathways such as vanillin production.

[0023] Figure 4 This is a comparison chart showing the yield of vanillin produced by COMT-N5W / Q39I / D190M using glucose as a substrate in Escherichia coli strain BW△7 by varying the concentration of yeast extract. Detailed Implementation

[0024] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0025] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0026] The following embodiments are detailed descriptions and explanations of the technical solutions of the present invention, and are not intended to limit the present invention.

[0027] 1. The following definitions are used in this invention: (1) Nomenclature of amino acids and DNA nucleic acid sequences The IUPAC nomenclature, a recognized system for naming amino acid residues, is used, employing single-letter or three-letter codes. DNA nucleic acid sequences are named using the IUPAC nomenclature.

[0028] (2) CatecholO Identifier of methyltransferase mutants Catechins are represented using the formula "original amino acid + position + substituted amino acid". O -Methyltransferase mutants contain mutated amino acids. For example, N5W indicates that the 5th amino acid is mutated from asparagine to tryptophan, and the position number corresponds to the amino acid sequence number of wild-type LiCOMT in SEQ ID NO. 1.

[0029] In this invention, lowercase italics comt wild-type catechins O The gene encoding the methyltransferase LiCOMT (lowercase italics) comt-N5W / Q39I / D190M The coding gene for the mutant COMT-N5W / Q39I / D190M is shown in the table below.

[0030]

[0031] Wild-type catechins described in this invention O The amino acid sequence of the methyltransferase LiCOMT is shown in SEQ ID NO.1: MSRKNISLTESLEEYIFRNSVREPDSFLKLRKETGTLAQANMQISPEEGQFLNILTKISGAKRIIEIGTFTGYSSLCFASALPEDGKILCCDVSEEWTNVARKYWKENGLENKIF LKLGSALETLQVLIDSKSAPSWASDFAFGPSSIDLFFLDADKENYPNYYPLILKLLKPGGLLIADNVLWDGSVADLSHQEPSTVGIRKFNELVYNDSLVDVSLVPIADGVSLVRKR

[0032] The amino acid sequence of the catechol methyltransferase mutant COMT-N5W / Q39I / D190M described in this invention is shown in SEQ ID NO.3: MSRKWISLTESLEEYIFRNSVREPDSFLKLRKETGTLAIANMQISPEEGQFLNILTKISGAKRIIEIGTFTGYSSLCFASALPEDGKILCCDVSEEWTNVARKYWKENGLENKIF LKLGSALETLQVLIDSKSAPSWASDFAFGPSSIDLFFLDADKENYPNYYPLILKLLKPGGLLIADNVLWDGSVAMLSHQEPSTVGIRKFNELVYNDSLVDVSLVPIADGVSLVRKR

[0033] 2. Primer sequence listing Table 1. Some primers and sequences involved in the embodiments of the present invention.

[0034] Escherichia coli BW25113 in the present invention and embodiments Escherichia coli The strain has been extensively disclosed in existing technologies, such as in the literature "Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol. 2006;2:2006.0008. doi: 10.1038 / msb4100050. Epub 2006 Feb 21. PMID: 16738554; PMCID:PMC1681482." It is also a commonly used model organism in the field of biotechnology, and can be obtained by the public from the Enzyme and Metabolic Engineering Laboratory of Beijing Institute of Technology.

[0035] The present invention will be further explained and illustrated below through specific embodiments.

[0036] Example 1: Construction of Escherichia coli BW△5 and BW△7 (1) In this embodiment, Escherichia coli BW25113 was used as the starting strain. In order to avoid the degradation of synthesized aldehydes by the endogenous enzyme system of the strain, four alcohol dehydrogenase genes were knocked out simultaneously by gene knockout technology, targeting the application scenario of chassis cells for aldehyde production. adhE (Gene ID: 945837) yjgB (Gene ID:948802) eutG (Gene ID:946233) and yqhD(Gene ID: 947493) The aldehyde degradation-deficient engineered strain BW△4 was constructed.

[0037] (2) In the study of de novo vanillin synthesis in Escherichia coli, Escherichia coli strain BW△4 was used as the starting strain, and five genes on the genome were analyzed respectively: yeaE (As shown in SEQ ID NO.8) , rutE (As shown in SEQ ID NO.9) , ybbO (As shown in SEQ ID NO.10) , yahK (As shown in SEQ ID NO.11) , aroE (Shown SEQ ID NO.12) Knockout. Its core lies in eliminating side reactions in the synthetic pathway, blocking competitive metabolic branches, reducing the degradation or enzymatic modification of intermediates / products, and optimizing the targeted supply of aromatic precursors, thereby improving the system's fidelity control and production adaptability for key intermediates such as aldehydes / vanillin, and ultimately improving the synthesis efficiency and yield of vanillin.

[0038] The five genes knocked out in the genome of Escherichia coli BW△4 strain and their functions are shown in Table 2: Table 2

[0039] Genes were constructed based on the above-mentioned genomic fragments using CRISPR / Cas9 technology on the Escherichia coli BW△4 strain. yeaE, rutE, ybbO, yahK, aroE Knockout strain BW△4△ yeaE, BW△4△ rutE, BW△4△ ybbO, BW△4△ yahK, BW△4△ aroE The specific steps are as follows: (3) Construction of CRISPR / Cas9 editing plasmids: The target gene sequence was input into the CHOPCHOP website (https: / / chopchop.cbu.uib.no / ) to predict the N20 efficiency, and five N20 sequences were identified: N20-1, N20-2, N20-3, N20-4 and N20-5.

[0040] The N20-1 sequence is: gccgaaatgtatgccgatgg; the N20-2 sequence is: acatgctcacaatatcagcg; the N20-3 sequence is: atccagatcgatcaacacgc; the N20-4 sequence is: ggcgatttatgcggtgtcgc; and the N20-5 sequence is: ggtgccaacacgcgcccata.

[0041] (4) Design primers ΔyeaE-N20-1-1, ΔyeaE-N20-1-2, ΔrutE-N20-2-1, ΔrutE-N20-2-2, ΔybbO-N20-3-1, ΔybbO-N20-3-2, ΔyahK-N20-4-1, ΔyahK-N20-4-2, ΔaroE-N20-5-1, and ΔaroE-N20-5-2; use pTargetF as a template and amplify with the above primers to obtain backbones containing different N20 fragments.

[0042] N20-1 is the overlapping region of ΔyeaE-N20-1-1 and ΔyeaE-N20-1-2. N20-2 is the overlapping region of ΔrutE-N20-2-1 and ΔrutE-N20-2-2. N20-3 is the overlapping region of ΔybbO-N20-3-1 and ΔybbO-N20-3-2. N20-4 is the overlapping region of ΔyahK-N20-4-1 and ΔyahK-N20-4-2. N20-5 is the overlapping region of ΔaroE-N20-5-1 and ΔaroE-N20-5-2. Homologous sequences (5HA and 3HA) were amplified using the BW25113 genome as a template. The primers used for amplification were ΔyeaE-5HA-1 and ΔyeaE-5HA-2, ΔrutE-5HA-3 and ΔrutE-5HA-4, ΔybbO-5HA-5 and ΔybbO-5HA-6, ΔyahK-5HA-7 and ΔyahK-5HA-8, ΔaroE-5HA-9 and ΔaroE-5HA-10, ΔyeaE-3HA-1 and ΔyeaE-3HA-2, ΔrutE-3HA-3 and ΔrutE-3HA-4, ΔybbO-3HA-5 and ΔybbO-3HA-6, ΔyahK-3HA-7 and ΔyahK-3HA-8, and ΔaroE-3HA-9 and ΔaroE-3HA-10.

[0043] The corresponding backbone containing the N20 fragment, 5HA, and 3HA fragments were connected using the Gibson assembly method, and chemical transformation was performed by incubation at 50°C for 50 minutes; gene knockout plasmid was successfully constructed.

[0044] (5) CRISPR / Cas9 editing: The successfully constructed plasmids were transferred into BWΔ4 competent cells containing Cas9 and placed on ice for 25 min. After the ice bath, the centrifuge tubes were placed at 42℃ for 1 min 30 s water bath heat shock. After the heat shock, the centrifuge tubes were placed back on ice for 2 min. After the ice bath, 900 μL of SOC medium was added to the centrifuge tubes, and then the tubes were placed in a shaker at 30℃ and 220 rpm for 45 min for recovery. The recovered cells were then plated on ampicillin sodium and kanamycin resistance plates and placed in a 30℃ incubator for overnight growth.

[0045] (6) Detection of CRISPR / Cas9 gene editing results: Single colonies were randomly selected from LB editing plates containing kanamycin and ampicillin sodium, and positive colonies were screened by colony PCR using two pairs of primers designed for the deleted gene fragment. Sanger sequencing was then performed to further verify the genome editing status.

[0046] The PCR products were subjected to agarose gel electrophoresis and compared with the DNA marker. If the size of the target band was consistent with the DNA marker, it indicated that the colonies were successfully edited.

[0047] (7) Elimination of edited plasmids: After successful gene editing, the edited plasmids within the bacteria need to be eliminated for subsequent gene manipulation. Correctly edited positive mutants are inoculated into antibiotic-free LB liquid medium and cultured overnight at 50°C and 220 rpm. A sterile inoculating loop is used to draw a suitable amount of bacterial culture and streaked onto antibiotic-free LB agar plates, then incubated at 42°C until single colonies appear. These single colonies are simultaneously inoculated into LB liquid medium containing kanamycin, LB medium containing ampicillin sodium, and antibiotic-free LB medium to verify plasmid elimination. If the same colony grows normally on antibiotic-free LB plates but not on kanamycin and ampicillin sodium LB plates, the colony has successfully lost the edited plasmid. After plasmid removal, the resulting strains are named *Escherichia coli* BW△4△yeaE, BW△4△rutE, ​​BW△4△ybbO, BW△4△yahK, and BW△4△aroE.

[0048] (8) The obtained knockout strains were inoculated into a shake-flask culture system, and 1.5 g / L vanillin was added to the culture medium. Samples were taken at 12 h at the set culture time point, and the changes in vanillin residue in the culture medium were detected by HPLC to assess the degradation of vanillin by each knockout strain. The results are as follows: Figure 1 As shown.

[0049] (9) According to Figure 1 Data shows that, compared with the control group, knocking out the chassis gene... yeaE andyahK It has a significant effect on inhibiting the degradation of vanillin. Among these effects, knockout... yahK The best results are achieved. Therefore, BW△4△yahK (i.e., BW△5) is selected as the optimal base for COMT screening.

[0050] in addition, aroE Encoding shikimate dehydrogenase, through knockout aroE This can block the loss of 3-dehydroshikimic acid to the shikimic acid main pathway at the metabolic level, thereby enhancing the flux of 3-dehydroshikimic acid to the vanillin branch, achieving the goals of "precursor-directed supply" and "reducing competitive branches," ultimately improving vanillin synthesis efficiency and yield; gene knockout yeaE It has a positive effect on inhibiting vanillin degradation. Therefore, gene knockout based on BW△5 is performed. yeaE and aroE, The vanillin de novo synthesis chassis strain BW△7 was obtained.

[0051] Example 2: Screening and Validation of COMT Mutants The conversion of protocatechuic acid to vanillin requires the expression of the following genes: catechins from Leptospira. O- Methyltransferase gene comt (SEQ ID NO.2), Phosphopantoylthioethylamine transferase from Bacillus subtilis sfp (SEQ ID NO. 7) and carboxyl reductase from Nocardia car (SEQ ID NO. 6). Due to the low conversion efficiency of COMT, a COMT mutant library was constructed to screen for COMT mutants with higher conversion efficiency, thereby improving the conversion efficiency of protocatechuic acid to vanillin. Details are as follows: (1) Gene synthesis comt, sfp, car Gene sequence fragments, comt, sfp and car The pBR322 plasmid was used to construct pBR-CC for the production of vanillin from protocatechuic acid via PCR ligation. The PCR reaction system is as follows:

[0052] Prepare the linear plasmid backbone fragment CC-G in the corresponding PCR tubes as shown in the table above. comt Gene fragments, sfp Gene fragments and car The PCR reaction system for gene fragments was vortexed and mixed before being placed in a PCR instrument for PCR reaction.

[0053] (2) After the PCR reaction was completed, CC-G was obtained. comt, sfp and carThe gene fragments were subjected to agarose gel electrophoresis and compared with DNA markers to obtain the target size bands shown in the table above.

[0054] (3) The remaining reaction products in the PCR tubes were recovered as DNA to obtain the purified linear fragments of the DNA amplification products, namely CC-G, comt, sfp and car Gene fragments.

[0055] (4) The four purified DNA fragments were ligated using Gibson ligation. The ligation products were then transferred into 100 μL of E. coli JM109 competent cells and placed on ice for 30 min. After the ice bath, the centrifuge tubes were placed in a water bath at 42℃ for 1 min 30 s for heat shock. After the heat shock, the centrifuge tubes were placed back on ice for 2 min. After the ice bath, 900 μL of LB medium was added to the centrifuge tubes, and the tubes were placed in a shaker at 37℃ for 45 min at 220 rpm. The resulting plates were then plated on ampicillin sodium resistant plates and incubated overnight at 37℃.

[0056] (5) The next day, a single colony was picked for PCR to verify whether the plasmid was successfully constructed.

[0057] After the PCR reaction was completed, agarose gel electrophoresis was performed, and the bands were compared with DNA markers. The correct target bands were obtained in the table above, indicating that the vanillin production plasmid pBR-CC was successfully constructed.

[0058] (6) To improve the conversion rate of vanillin, a COMT mutant library was constructed using PCR mutagenesis. The fragment size was 696 bp. The PCR reaction system was as follows:

[0059] (7) After the PCR reaction is complete, the following results are obtained: comt Gene mutation library. The gene mutations were subjected to agarose gel electrophoresis and compared with DNA markers to obtain the target size bands shown in the table above.

[0060] (8) The purified target fragment was obtained through DNA recovery, and the wild-type fragment was deleted. comtThe pBR-CC vector backbone was ligated using Gibson chromatography. The ligation product was then transferred into 100 μL of BW△5 competent cells and placed on ice for 30 min. After the ice bath, the centrifuge tubes were placed in a 42°C water bath for 1 min 30 s for heat shock. After the heat shock, the centrifuge tubes were placed back on ice for 2 min. After the ice bath, 900 μL of LB medium was added to the centrifuge tubes, and the tubes were placed in a 37°C shaker at 220 rpm for 45 min for recovery. The recovered cells were then plated onto ampicillin sodium resistance plates and incubated overnight at 37°C.

[0061] (12) The next day, single colonies grew on the plate. All the single colonies on the plate were inoculated into 96-well plates containing 1 ml of LB medium and 1‰ ampicillin sodium, and cultured overnight in a shaker at 37°C as seed culture. The following day, the seed culture was re-inoculated at a ratio of 1% into 96-well plates containing 1 ml of M9Y medium (M9Y medium + glucose formulation: 6 g / L Na2HPO4, 3 g / L KH2PO4, 0.5 g / L NaCl, 1 g / L NH4Cl, 2 g yeast extract. The reagents were dissolved in distilled water and brought to a final volume of 950 mL. The plates were sterilized at 121 °C for 20 minutes, cooled to room temperature, and then mixed with 50 mL of sterilized 40% glucose solution, 1 mL of 1 M MgSO4 stock solution, 1 mL of 0.1 M CaCl2 stock solution, and 1 mL of 10 mg / mL VB1 stock solution) and 1‰ ampicillin sodium. After 4 hours (OD... 600 When the concentration was approximately 0.6 g / L, protocatechuic acid was added to bring the final concentration to 0.8 g / L. The strain carrying LiCOMT was used as a control, specifically the BW△5 strain carrying the pBR-CC plasmid, which was the control strain BW-CC. The plates were cultured in a shaker at 30°C. After 12 h, 500 mL of the bacterial culture was added to a new deep-well plate, along with 500 mL of the culture containing the pBR-CC plasmid from the COMT mutant library, which had reached the logarithmic growth phase. After 4 h, the RFP / OD600 ratio was measured. Strains with high response values ​​were selected for shake-flask fermentation verification.

[0062] (13) The results are as follows Figure 2As shown, the wild-type strain BW-CC produced 115 mg / L of vanillin with a conversion rate of 14.5%; one mutant strain, BW-0716CC, produced 488 mg / L of vanillin with a conversion rate of 61.8%. Sequencing revealed that the COMT mutant carried by strain BW-0716CC had three amino acid mutations compared to LiCOMT shown in SEQ ID NO.1: the first mutation was from asparagine to tryptophan at position 5; the second mutation was from glutamine to isoleucine at position 39; and the third mutation was from aspartic acid to methionine at position 190. This mutant was named COMT-N5W / Q39I / D190M, and its amino acid sequence is shown in SEQ ID NO.3 (the coding gene is shown in SEQ ID NO.4). The mutant plasmid carried by strain BW-0716CC was named pBR-0716CC.

[0063] Example 3: Metabolic pathway modification for de novo synthesis of vanillin To obtain a complete expression plasmid for the 3-dehydroshikimic acid to vanillin pathway, the Pseudomonas gene encoding the 3-dehydroshikimic acid dehydratase was further expressed. dsd (SEQ ID NO.5) was integrated into plasmid pBR-0716CC.

[0064] To improve the de novo synthesis efficiency of vanillin using glucose as a carbon source, a carbon flow direction enhancement strategy was constructed by strengthening the supply capacity of aromatic precursors and key intermediates. ppsA , tktA, talB as well as aroG / aroD / aroB Key genes are overexpressed through pathways such as [list of pathways]. ppsA To enhance the ability of pyruvate to generate PEP and increase the supply of PEP required by the shikimic acid pathway; tktA and talB Enhanced carbon skeleton rearrangement in the pentose phosphate pathway increases the generation of key precursors such as E4P; simultaneously, overexpression of aroG / aroD / aroB further increases upstream flux in the shikimic acid pathway, promoting the formation and accumulation of aromatic skeletons. Through this synergistic regulation, more glucose carbon flux can be diverted from basal metabolism to the vanillin synthesis pathway, reducing precursor limitations and flux bottlenecks, thereby improving the efficiency and yield of vanillin synthesis.

[0065] Overexpression of the gene phosphoenolpyruvate synthase ppsA (Gene ID:946209) 、 Thiamine triphosphate-binding transketolase tktA (Gene ID:947420) 、 transaldolase talB(Gene ID:944748) 、 and key enzyme genes in the SAM cycle mtnN (Gene ID:938153) luxS (Gene ID:947168) and aroG (Gene ID:945605) aroB (Gene ID:947927) aroD (Gene ID:946210) ， The overexpressed gene was integrated into the production plasmid pBR-0716CC, then transferred to BW△7 competent cells, plated, and cultured overnight. The next day, the seed culture was inoculated, and after a period of culture, it was transferred to a shake flask for shake flask verification of vanillin de novo synthesis yield (method as in Example 2, without the addition of protocatechuic acid).

[0066] The results are as follows Figure 3 As shown, expression on plasmid pBR-0716CC dsd Based on this, further overexpression of pathway genes ppsA, tktA, talB, mtnN, luxS and aroGBD The yield of de novo vanillin synthesis reached 121 mg / L. It was also expressed on plasmid pBR-0716CC. dsd, ppsA, tktA, talB, mtnN, luxS and aroGBD The recombinant plasmid obtained was named pYSY91.

[0067] Example 4: Optimizing culture medium conditions to increase vanillin de novo synthesis yield This example illustrates that changing the concentration of yeast extract in Escherichia coli can increase the yield of vanillin synthesized de novo.

[0068] (1) M9Y medium + glucose formulation: 6 g / L Na2HPO4, 3 g / L KH2PO4, 0.5 g / L NaCl, 1 g / L NH4Cl, 2 g yeast extract. Dissolve the above reagents in distilled water, bring the volume to 950 mL, sterilize at 121 °C for 20 minutes, cool to room temperature, and then add 50 mL of sterilized 60% glucose solution, 1 mL of 1 M MgSO4 stock solution, 1 mL of 0.1 M CaCl2 stock solution and 1 mL of 10 mg / mL VB1 stock solution and mix well.

[0069] (2) Change the concentration of yeast extract: keep other conditions unchanged, reduce the concentration of yeast extract from 2 g / L to 1 g / L.

[0070] (3) Take out the BW△7 strain containing the pYSY91 plasmid stored in the -80℃ refrigerator, place it on ice, and use an inoculation loop to take a small amount of bacterial solution in a clean bench and streak it on solid culture medium. Then place it in an incubator at 37℃ and incubate overnight until single colonies grow. Randomly pick single colonies from the plates and inoculate them into 4 mL of fresh LB medium and incubate overnight at 37℃ and 220 rpm as seed culture. The next day, add 20 mL of culture medium (the culture medium described in steps (1) and (2)), 20 μL of ampicillin sodium and 200 μL of seed culture to the shake flasks, and incubate at 37℃ and 220 rpm. When the OD600 reaches 0.5, transfer all shake flasks to 30℃ and incubate at 220 rpm. Take samples after 12, 24, 28, 32, 36 and 48 h to detect the yield of vanillin in the fermentation broth.

[0071] (3) The results are as follows Figure 4 As shown, vanillin yield varied with different fermentation times, reaching its highest value at 32 h. Vanillin gradually degraded with increasing fermentation time. Specifically, with a yeast extract concentration of 1 g / L, the de novo vanillin yield after 32 h of fermentation was 223 mg / L.

[0072] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various changes, modifications, substitutions and variations in form and detail to these embodiments without departing from the spirit and principles of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. A type of catechin O -Methyltransferase mutant, characterized by The mutant was obtained by N5W, Q39I and D190M mutations in the wild-type catechol methyltransferase shown in SEQ ID NO.

1.

2. A catechin as described in claim 1 O -Methyltransferase mutant, characterized by The catechol methyltransferase mutant is the COMT-N5W / Q39I / D190M mutant, and its amino acid sequence is shown in SEQ ID NO.

3.

3. A biomaterial, characterized in that, The biomaterial includes any of the following: A1) Encoding the catechins of claim 1 O -Methyltransferase mutant nucleic acid molecules; A2) An expression cassette containing the nucleic acid molecules described in A1); A3) A recombinant vector containing the nucleic acid molecules described in A1); A4) Recombinant microorganisms containing the nucleic acid molecules described in A1).

4. A biomaterial as described in claim 3, characterized in that, The recombinant microorganisms used as expression hosts include: Escherichia coli, Bacillus subtilis, or yeast; The recombinant vectors used include the following expression vectors: pBR322 plasmid, pETM6 plasmid, pUC18 plasmid, and pGEX plasmid.

5. A biomaterial as described in claim 4, characterized in that, The recombinant microorganism is constructed in a host cell containing the catechols of claim 1. O The vanillin biosynthesis pathway of the methyltransferase mutant is obtained; the biosynthesis pathway includes catechol. O -Methyltransferase mutants, carboxylic acid reductase, and phosphate pantothenic acid ethylaminotransferase can achieve the biosynthesis of protocatechuic acid to vanillin.

6. A biomaterial as described in claim 5, characterized in that, The biosynthetic pathway also includes 3-dehydroshikimic acid dehydratase, and the obtained recombinant microorganisms can achieve the biosynthesis of 3-dehydroshikimic acid to vanillin.

7. A biomaterial as described in claim 6, characterized in that, Further overexpression of phosphoenolpyruvate synthase in the aforementioned biological pathway ppsA, Thiamine triphosphate-binding transketolase tktA、 transaldolase talB、 Key enzyme genes in the SAM cycle mtnN , luxS and aroGBD Then, it was transferred into the chassis bacteria BW△7 to obtain recombinant microorganisms; The aforementioned BW△7 strain is derived from Escherichia coli BW25113, with four alcohol dehydrogenase genes knocked out. adhE , yjgB , eutG and yqhD and NADPH-dependent aldehyde reductase yahK、 Shikimate dehydrogenase encoding gene aroE and the gene encoding methylglyoxal enzyme hydrolysis yeaE get.

8. A biomaterial as described in claim 7, characterized in that, The catechins O -Methyltransferase mutant, encoding gene comt-N5W / Q39I / D190M The nucleotide sequence is shown in SEQ ID NO.4; the 3-dehydroshikimate dehydratase encodes the gene. dsd The nucleotide sequence is shown in SEQ ID NO.5; the carboxylic acid reductase, encoding gene car The nucleotide sequence is shown in SEQ ID NO.6; The phosphate pantothenicotinyl ethylamine transferase, encoding gene sfp The nucleotide sequence is shown in SEQ ID NO.

7.

9. The catechins of claim 1 O - The use of the methyltransferase mutant or the biomaterial of claim 3 in any of the following: B1) Application in the catalytic conversion of protocatechuic acid to vanillic acid; B2) Application in increasing the yield of microbial vanillin; B3) Application in the preparation of vanillin or products containing vanillin; B4) Application in the construction of genetically engineered bacteria that produce vanillin.

10. The application as described in claim 9, characterized in that, This refers to the application of the recombinant microorganisms in the synthesis of vanillin using glucose as a substrate.

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