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CN-122012437-A - Methyltransferase mutant and encoding gene, vector, recombinant bacterium and application thereof

CN122012437ACN 122012437 ACN122012437 ACN 122012437ACN-122012437-A

Abstract

The invention discloses a methyltransferase mutant and a coding gene, a vector, recombinant bacteria and application thereof, belonging to the technical field of enzyme engineering. The invention mutates the variant EgtD T163G,M252V,E282A of the methyltransferase EgtD from M. SMEGMATIS ATCC700084, mutates Pro at position 35 into Cys, lys at position 36 into Arg, phe at position 38 into Leu and Thr at position 168 into Phe, remarkably improves the methylation catalytic efficiency of taking L-tryptophan as a substrate, and synthesizes L-erythrina with high efficiency. On the basis of the realized synthesis of the L-erythrina base by using E.coli whole cells, a further theoretical and technical basis is provided for establishing an industrial L-erythrina base production process with low production cost, high production intensity and small environmental pollution.

Inventors

  • XU JIANZHONG
  • TIAN SHUPING
  • SUN MINGXIN

Assignees

  • 江南大学

Dates

Publication Date
20260512
Application Date
20260209

Claims (10)

  1. 1. A methyltransferase mutant, wherein the methyltransferase mutant is characterized in that proline at position 35, lysine at position 36, phenylalanine at position 38, threonine at position 163, threonine at position 168, methionine at position 252 and glutamic acid at position 282 of a parent sequence shown in SEQ ID No.1 are mutated to cysteine, arginine, phenylalanine at position 38, glycine, phenylalanine at position 168, methionine at position 252 and alanine.
  2. 2. A gene encoding the methyltransferase mutant of claim 1.
  3. 3. An expression vector carrying the coding gene of claim 2.
  4. 4. The expression vector of claim 3, wherein the expression vector is a bacterial plasmid, a phage, a yeast plasmid, a plant cell virus, or a mammalian cell virus.
  5. 5. A recombinant bacterium expressing the methyltransferase mutant of claim 1.
  6. 6. The recombinant bacterium according to claim 5, wherein the recombinant bacterium is a bacterial, fungal, plant, insect or animal cell.
  7. 7. Use of the methyltransferase mutant of claim 1 or the recombinant bacterium of claim 5 or 6 for catalyzing synthesis of L-erythrina base from L-tryptophan or a derivative thereof.
  8. 8. The use according to claim 7, wherein the use is in the catalytic synthesis of L-erythrina using S-adenosyl-L-methionine as methyl donor, L-tryptophan or derivatives thereof as methyl acceptor, and the methyltransferase mutant or the recombinant bacterium as catalyst.
  9. 9. An enzyme preparation comprising the methyltransferase mutant of claim 1.
  10. 10. The enzyme preparation according to claim 9, characterized in that the enzyme preparation is a solid or a liquid enzyme preparation.

Description

Methyltransferase mutant and encoding gene, vector, recombinant bacterium and application thereof Technical Field The invention relates to a methyltransferase mutant, a coding gene thereof, a vector, recombinant bacteria and application thereof, belonging to the technical field of enzyme engineering. Background L-erythrina base (L-Hypaphorine, L-HYP) with molecular formula of C 14H18N2O2 is a natural small molecule indole alkaloid found in erythrina plant, also called trimethyl betaine and cloud Gan Ding, and is produced by using L-tryptophan as substrate and carrying out repeated methylation under the action of methyltransferase. L-erythrina base is slightly dissolved in organic solvents such as methanol, ethanol, DMSO and the like, and has a solubility of 5 mg/mL in PBS solution with pH of 7.2. Because of unique indole ring and trimethyl substituent groups in the molecular structure, the L-erythrina base shows various physiological activities, can regulate plant growth processes, such as regulating the symbiosis of fungi and plants, improving the concentration of cytosolic calcium to regulate cell signal paths, can be used for treating diseases of animals and humans, can relieve dexamethasone-induced insulin resistance, and can be used for treating Alzheimer's disease as an acetylcholinesterase inhibitor. Of particular importance, recent studies have established that L-erythrina is a novel antiviral compound against dengue virus. These abundant physiological functions and therapeutic potential highlight the important value of L-erythrina in various fields. Its widespread acquisition is by extraction from exogenous organisms or chemical synthesis. But the plant extraction yield is low and the chemical synthesis pollution is large. And the microbial contamination is small, the cost is low, the propagation is rapid, and the substrate preference of the key enzyme can be changed by carrying out site-directed mutagenesis on the key enzyme. Therefore, the development of a catalytic synthesis system and a whole-cell transformation platform based on the enzyme has important application prospect for realizing efficient green preparation of erythrina base. The process of directed production of L-erythrina from L-tryptophan requires the use of S-adenosyl-L-methionine (SAM) dependent methyltransferases to participate in an iterative catalytic three-fold transmethylation reaction, where SAM is the methyl donor and L-tryptophan and its derivatives are the methyl acceptor. Therefore, to realize the synthesis of L-erythrina base by using L-tryptophan as a substrate, it is important to screen or construct methyl transport enzyme capable of specifically catalyzing the methylation of L-tryptophan. Studies have reported that the methyl transferase EgtD from Mycolicibacterium smegmatisATCC700084 iteratively methylates ergothioneine with L-histidine as a substrate. It is worth pointing out that site-directed mutagenesis of amino acids 252 and 282 in the EgtD amino acid sequence from M. SMEGMATISATCC700084 to Val and Ala allows for iterative methylation of L-tryptophan as a substrate to L-erythrina. However, egtD (i.e., egtD M252V,E282A) carrying the 252 and 282 amino acid mutations can synthesize L-erythrina base using L-tryptophan as a substrate, but its catalytic efficiency is too low to satisfy industrial application. The 163 th Thr of the Wang et al focusing active pocket is mutated into Gly on the basis of EgtD M252V,E282A, and the obtained mutant EgtD T163G,M252V,E282A can realize basic conversion of catalyzing and generating erythrina in E.coli cells, but the conversion rate is still at a lower level. Disclosure of Invention In order to solve the defects in the prior art, the invention improves the efficiency of catalyzing L-tryptophan to synthesize L-erythrina base by the mutant through continuously modifying EgtD T163G,M252V,E282A, thereby realizing the in-vitro synthesis of L-erythrina base by using the whole cell of the microorganism. The invention mutates proline (Pro) at position 35 into cysteine (Cys), lysine (Lys) at position 36 into arginine (Arg), phenylalanine (Phe) at position 38 into leucine (Leu) and threonine (Thr) at position 168 into phenylalanine (Phe) in the amino acid sequence of mutant EgtD T163G,M252V,E282A, and the mutant EgtD T163G,M252V,E282A is over-expressed in escherichia coli BL21 (DE 3). As a result, the EgtD mutant is found to remarkably improve the capability of catalyzing and synthesizing L-erythrina base by using L-tryptophan as a substrate. The invention is realized by the following technical scheme: A first object of the present invention is to provide a methyltransferase mutant in which proline (Pro) at position 35, lysine (Lys) at position 36, phenylalanine (Phe) at position 38, threonine (Thr) at position 168, threonine (Phe) at position 163, methionine (Met) at position 252, and glutamic acid (Glu) at position 282 are mutated to alanine (Ala) of a parent sequence shown in SEQ ID NO