Protein Engineering of Nicotinamide Riboside Kinase Based on a Combinatorial Semirational Design Strategy for Efficient Biocatalytic Synthesis of Nicotinamide Mononucleotides.

Autor: Mao XA; Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, PR China.; National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, School of Biotechnology, Jiangnan University, Wuxi 214122, PR China., Zhang P; Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, PR China.; National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, School of Biotechnology, Jiangnan University, Wuxi 214122, PR China., Gong JS; Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, PR China.; Institute of Future Food Technology, JITRI, Yixing 214200, PR China., Marshall GL; Seragon Biosciences, Inc., 400 Spectrum Center Drive, 16th Floor, Irvine, California 92618, United States., Su C; Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, PR China.; Institute of Future Food Technology, JITRI, Yixing 214200, PR China., Qin ZQ; Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, PR China.; National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, School of Biotechnology, Jiangnan University, Wuxi 214122, PR China., Li H; Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, PR China., Xu GQ; National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, School of Biotechnology, Jiangnan University, Wuxi 214122, PR China.; Institute of Future Food Technology, JITRI, Yixing 214200, PR China., Xu ZH; National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, School of Biotechnology, Jiangnan University, Wuxi 214122, PR China.; Institute of Future Food Technology, JITRI, Yixing 214200, PR China.; College of Biomass Science and Engineering, Sichuan University, Chengdu 610065, PR China., Shi JS; Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, PR China.; Institute of Future Food Technology, JITRI, Yixing 214200, PR China.
Jazyk: angličtina
Zdroj: Journal of agricultural and food chemistry [J Agric Food Chem] 2024 Nov 13; Vol. 72 (45), pp. 25210-25218. Date of Electronic Publication: 2024 Oct 31.
DOI: 10.1021/acs.jafc.4c05520
Abstrakt: Industrial biosynthesis of β-nicotinamide mononucleotide (β-NMN) lacks a highly active nicotinamide riboside kinase for the phosphorylation process. Cumbersome preprocessing steps and excessive ATP addition contribute to its increased cost. To tackle these challenges, a docking combination simulation (DCS) semirational mutagenesis strategy was designed in this study, combining various modification strategies to obtain a mutant NRK-TRA with 2.9-fold higher enzyme activity. Molecular dynamics simulations and structural analysis demonstrate the enhancement of its structural stability. High-density fermentation was achieved through a 5 L fermentation tank, with a titer reaching 208.3 U/mL, the highest in the current report. An ATP-cycling whole-cell catalytic system was employed and optimized by introducing a polyphosphate kinase 2 (PPK2) recombinant strain, and 15.16 g/L β-NMN was obtained through a series of batch transformation experiments. This study provides a new strategy for the efficient screening of highly active enzyme variants and offers a green and promising biotransformation system for NMN production.
Databáze: MEDLINE
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