CN-122012438-A - Red bean alkali methyltransferase mutant and encoding gene, vector, recombinant bacterium and application thereof

Abstract

The invention discloses a ormosia alkali methyltransferase mutant and a coding gene, a vector, recombinant bacteria and application thereof, and belongs to the technical field of enzyme engineering. The invention mutates the variant EgtD M252V,E282A of the methyltransferase EgtD from M. SMEGMATIS ATCC700084 to mutate 34 Pro into Lys, 213 Thr into Ser and 284 Ser into Ala, and remarkably improves the methylation catalytic efficiency of taking L-red bean alkali as a substrate, and synthesizes L-erythrina alkali 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
• XIE ZIWEN
• SUN MINGXIN

Assignees

• 江南大学

Dates

Publication Date

20260512

Application Date

20260209

Claims (10)

1. 1. A mutant of the ormosia alkali methyltransferase is characterized in that the mutant of the ormosia alkali methyltransferase is characterized in that proline 34 of a parent sequence with an amino acid sequence shown as SEQ ID NO.1 is mutated to lysine, threonine 213 is mutated to serine, methionine 252 is mutated to valine, glutamic acid 282 is mutated to alanine, and serine 284 is mutated to alanine.
2. 2. A gene encoding the ormosia alkali 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 ormosia base 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 ormosia alkali methyltransferase mutant of claim 1 in biocatalysis for synthesizing L-erythrina.
8. 8. The use according to claim 7, wherein L-erythrina base is used as a substrate, the ormosia base methyltransferase mutant is used as a catalyst, or L-erythrina base is synthesized by catalytic synthesis, The application is that the red bean alkali methyltransferase mutant and methyltransferase catalyzing L-tryptophan L-erythrina alkali are used for combined catalysis, and L-tryptophan is used as a substrate to synthesize L-erythrina alkali.
9. 9. An enzyme preparation comprising the ormosia base 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

Red bean alkali methyltransferase mutant and encoding gene, vector, recombinant bacterium and application thereof Technical Field The invention relates to a ormosia alkali methyltransferase mutant and a coding gene, a vector, recombinant bacteria and application thereof, and belongs to the technical field of enzyme engineering. Background L-erythrina base (L-Hypaphorine, L-HYP) is a natural alkaloid existing in specific plants such as caragana microphylla and vaccinia, and is produced by taking L-tryptophan as a substrate and carrying out repeated three times of methylation under the action of methyltransferase. Can regulate plant growth process, such as regulating fungal and plant symbiosis, and increasing cytoplasmic calcium concentration to regulate cell signal passage, and can be used for treating animal and human diseases, including relieving dexamethasone-induced insulin resistance, and treating Alzheimer disease as acetylcholinesterase inhibitor. 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. Methyltransferases (MTases) are a class of enzymes that specifically catalyze the transfer of methyl groups from a methyl donor (mainly S-adenosylmethionine, SAM) to a specific atom (oxygen, nitrogen, carbon, etc.) of a substrate molecule. MSE is a SAM-dependent methyltransferase which transfers methyl groups to nitrogen atoms, an engineered variant obtained by site-directed mutagenesis of a M252V and E282A methyltransferase EgtD from Mycobacterium smegmatis (Mycobacterium smegmatis). The substitution of these two key residues significantly alters the substrate specificity of the enzyme, converting it from a native enzyme that would otherwise catalyze histidine methylation to an engineered enzyme that can efficiently catalyze L-tryptophan aminotrimethylation, thereby producing the product L-erythrina. The reaction mechanism is that after L-tryptophan is combined to an enzyme active center, a nucleophilic center of a substrate molecule attacks positively charged methyl sulfonium ions in SAM molecules, msE can sequentially transfer methyl on the SAM to an amino site of the L-tryptophan, three N-methylation reactions are continuously completed, and finally a trimethyl product L-erythrina base is generated. However, this catalytic mode of "single-binding-continuous methylation" tends to cause dissociation of the intermediate methylation product from the enzyme molecule, resulting in interruption of the methylation reaction, and thus, substantial accumulation of the monomethylated intermediate L-red bean base in the reaction system (L-red bean base, i.e., N, α -methyl-L-tryptophan, is a monomethyl product of L-Trp methylated by MSE). The 163-Thr mutation of the Wang et al focusing active pocket into Gly on the basis of EgtD M252V,E282A, and the obtained mutant EgtD T163G,M252V,E282A realizes the transformation of catalyzing and generating erythrina base in E.coli cells. However, the improvement of the catalytic efficiency of the mutant is accompanied by the simultaneous increase of the accumulation amount of L-red bean alkali, which indicates that the massive accumulation of intermediate products may be a key bottleneck for limiting the complete progress of the methylation reaction. In order to further improve the dimethyl efficiency of the enzyme, the research aims at modifying EgtD M252V,E282A, and the efficiency of catalyzing L-erythrina base to synthesize L-erythrina base by the mutant is improved by screening the optimal multiple mutant of the methyltransferase EgtD, and the mutant is used in combination with the methyltransferase for catalyzing L-tryptophan to generate L-erythrina base, so that the L-erythrina base is synthesized in vitro by using whole cells of the microorganism efficiently. Disclosure of Invention In order to solve the problem that in the prior art, an intermediate methylation product is dissociated from an enzyme molecule in the methylation process of L-tryptophan to interrupt the methylation reaction, so that a great amount of monomethylated intermediate L-ormosia alkali is accumulated in a reaction system, the invention firstly mutates 34 Pro in the amino acid sequence of a mutant EgtD M252V,E282A into Lys, 213 Thr into Ser and 284 Ser into Ala, and the mutant EgtD M252V,E282A is overexpressed in escherichia coli BL21 (