Title:
PHOSPHINOTHRICIN DEHYDROGENASE MUTANT, GENETICALLY ENGINEERED BACTERIUM, AND ONE-POT MULTI-ENZYME SYNCHRONOUS DIRECTED EVOLUTION METHOD
Document Type and Number:
WIPO Patent Application WO/2022/001038
Kind Code:
A1
Abstract:
A phosphinothricin dehydrogenase mutant, a genetic engineering bacterium, and a one-pot multi-enzyme synchronous directed evolution method. The phosphinothricin dehydrogenase mutant is obtained by mutating the 164th amino acid from alanine to glycine, mutating the 205th arginine to lysine and mutating the 332nd threonine to alanine of phosphinothricin dehydrogenase derived from Pseudomonas fluorescens, and an amino acid sequence is as shown in SEQ ID No.1. The genetically engineered bacterium is obtained by introducing a gene of the phosphinothricin dehydrogenase mutant into a host cell. A coding gene of glucose dehydrogenase or a coding gene of formate dehydrogenase can also be introduced into the host cell to perform simultaneous directed evolution to overexpress the double genes. The one-pot multi-enzyme synchronous directed evolution method can obtain, by means of screening, a genetically engineered bacterium having greatly improved activity. Compared with catalytic processes such as transaminase, the L-PPT preparation method has a relatively simple process, a high conversion rate of raw materials, a conversion rate of up to 100%, and high stereo-selectivity.
Inventors:
XUE YAPING (CN)
CHENG FENG (CN)
LI JUMOU (CN)
LI QINGHUA (CN)
ZHENG YUGUO (CN)
ZOU SHUPING (CN)
XU JIANMIAO (CN)
CHENG FENG (CN)
LI JUMOU (CN)
LI QINGHUA (CN)
ZHENG YUGUO (CN)
ZOU SHUPING (CN)
XU JIANMIAO (CN)
Application Number:
PCT/CN2020/139770
Publication Date:
January 06, 2022
Filing Date:
December 26, 2020
Export Citation:
Assignee:
UNIV ZHEJIANG TECHNOLOGY (CN)
International Classes:
C12N1/21; C12N9/06; C12N15/53; C12N15/70; C12P13/04; C12R1/19
Domestic Patent References:
| WO2019192505A1 | 2019-10-10 |
Foreign References:
| CN111621482A | 2020-09-04 | |||
| CN110592036A | 2019-12-20 | |||
| CN108588045A | 2018-09-28 | |||
| CN110885803A | 2020-03-17 | |||
| CN109609475A | 2019-04-12 |
Other References:
YIN, XJ. ET AL.: "Efficient Reductive Amination Process for Enantioselective Synthesis of L-Phosphinothricin Applying Engineered Glutamate Dehydrogenase", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 102, no. 10, 16 March 2018 (2018-03-16), XP036493130, DOI: 10.1007/s00253-018-8910-z
CHENG, F. ET AL.: "Tuning amino acid dehydrogenases with featured sequences for L-phosphinothricin synthesis by reductive amination", JOURNAL OF BIOTECHNOLOGY, vol. 312, 20 March 2020 (2020-03-20), XP086092028, DOI: 10.1016/j.jbiotec.2020.03.001
XU JIAN: "Directed Evolution of Enzymes for the Regulation of Selectivity and Catalytic Promiscuity", CHINESE DOCTORAL DISSERTATIONS FULL-TEXT DATABASE, ENGINEERING SCIENCE & TECHNOLOGY I, no. 8, 15 August 2019 (2019-08-15), XP055883610
CHENG, F. ET AL.: "Tuning amino acid dehydrogenases with featured sequences for L-phosphinothricin synthesis by reductive amination", JOURNAL OF BIOTECHNOLOGY, vol. 312, 20 March 2020 (2020-03-20), XP086092028, DOI: 10.1016/j.jbiotec.2020.03.001
XU JIAN: "Directed Evolution of Enzymes for the Regulation of Selectivity and Catalytic Promiscuity", CHINESE DOCTORAL DISSERTATIONS FULL-TEXT DATABASE, ENGINEERING SCIENCE & TECHNOLOGY I, no. 8, 15 August 2019 (2019-08-15), XP055883610
Attorney, Agent or Firm:
HANGZHOU TIANQIN INTELLECTUAL PROPERTY AGENCY CO., LTD. (CN)
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