Directed Evolution for Catalytic Activity of Formate Dehydrogenase and Its Overexpression

doi:10.19894/j.issn.1000-0518.200271

Chinese Journal of Applied Chemistry ›› 2021, Vol. 38 ›› Issue (6): 704-712.DOI: 10.19894/j.issn.1000-0518.200271

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Directed Evolution for Catalytic Activity of Formate Dehydrogenase and Its Overexpression

ZHANG Zhen-Hua¹, XIE Yu-Li¹, WANG Tie-Jun¹, ZHAO Hong², TANG Cun-Duo^1*, KAN Yun-Chao¹, YAO Lun-Guang¹

¹Henan Provincial Engineering Laboratory of Insect Bio-reactor and Henan Key Laboratory of Ecological Security for Water Source Region of Mid-line of South-to-North, Nanyang Normal University, Nanyang 473061, China
²Henan Life Pharmaceutical Co. LTD, Nanyang 473061, China

Received:2020-09-04 Revised:2020-12-31 Published:2021-06-01 Online:2021-08-01
Supported by:
Scientific and Technological Project of China (Nos.31900916, 31870917), the Science & Technology Innovation Talents in Universities of Henan Province (No.21HASTIT041) and the Innovative Research Team of Science and Technology in University of Henan Province (No.20IRTSTHN024)

Abstract

Abstract: Formate dehydrogenase (FDH) is a D-2-hydroxy acid dehydrogenase, and catalyzes the oxidation of formate to carbon dioxide, coupled with reduction of NAD⁺(oxidized nicotinamide adenine dinucleotide (NAD)) to NADH (reduced NAD) that plays a key role in the process of NADH regeneration. In order to obtain highly active formate dehydrogenase mutants, the Candida boidinii formate dehydrogenase CbFDH^C23S was used as the parent to conduct two rounds of directional evolution, and a mutant M2 was obtained. The specific activity of M2 is about 4 times more than the parent and M2 and is more suitable for coenzyme regeneration under physiological conditions. Then, the molecular mechanism of the temperature characteristic and the catalytic efficiency change was preliminarily elucidated by the computer aided method. Finally, with the help of the co-expression strategy, the expression level of mutant M2 in Escherichia coli is further improved, and the formate dehydrogenase activity in the ultrasonic lysate reaches 45.85 U/mL, which is far higher than the expression level of the parent single copy. This study laid a theoretical foundation for the green biosynthesis of food additives such as chiral alcohols and amino acid derivatives catalyzed by FDH coupling to enhance the regeneration capacity of NADH, reduce the regeneration cost of NADH, and achieve high efficiency and low cost.

Key words: Formate dehydrogenase, Catalytic efficiency, Directed evolution, Molecular docking, Overexpression

CLC Number:

O629.8

ZHANG Zhen-Hua, XIE Yu-Li, WANG Tie-Jun, ZHAO Hong, TANG Cun-Duo, KAN Yun-Chao, YAO Lun-Guang. Directed Evolution for Catalytic Activity of Formate Dehydrogenase and Its Overexpression[J]. Chinese Journal of Applied Chemistry, 2021, 38(6): 704-712.

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Directed Evolution for Catalytic Activity of Formate Dehydrogenase and Its Overexpression

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