Gene mining method combining functional sequence and structure simulation, NADH-preferring phosphinothricin dehydrogenase mutant and application thereof

US11939599B2 · kind B2 · utility

Assignee

• ZHEJIANG UNIVERSITY OF TECHNOLOGY · CN

Inventors

• Yaping Xue · Dongguan, CN
• Feng Cheng · Zhejiang, CN
• Jiamin Zhang · Vancouver, US
• Shuping Zou · Hangzhou City, CN
• Jianmiao Xu · Hangzhou City, CN
• Yuguo Zheng · Zhejiang, CN

Key dates

• Filing date: Dec 30, 2021
• Grant date: Mar 26, 2024
• Expiry date: Dec 30, 2041

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12Y104/01004
• WIPO field: Biotechnology
• WIPO sector: Chemistry

Abstract

Disclosed are a gene mining method combining functional sequence and structure simulation, an NADH-preferring phosphinothricin dehydrogenase mutant and an application thereof. The gene mining method comprises the following steps: (1) analyzing a characteristic sequence which an NADH-type glutamate dehydrogenase should have; (2) searching a gene library based on the characteristic sequence; (3) performing clustering analysis and protein structure simulation on genes obtained by the searching; (4) selecting genes that feature high gene aggregation and a protein structure similar to that of the known phosphinothricin dehydrogenase as candidate genes. A wild-type phosphinothricin dehydrogenase with an amino acid sequence as set forth in SEQ ID No.2 derived from Lysinibacillus composti is obtained through the gene mining, and then mutated, and an NADH-preferring phosphinothricin dehydrogenase mutant is screened out, which has a mutation site selected from one of the following: (1) A144G-V375F-M91A; (2) A144G-V345A-M91A; (3) A144G. This mutant enzyme can be used for catalytic reaction with an inexpensive coenzyme NAD.