Whole-cell synthesis of nicotinamide mononucleotide by recombinant Saccharomyces cerevisiae from glucose and nicotinamide

IF 3.7 3区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Biochemical Engineering Journal Pub Date : 2024-10-10 DOI:10.1016/j.bej.2024.109528
Chaoguang Wang , Xiaohan Hui , George Marshall , Wenhan Xiao , Xiaomei Zhang , Jianying Qian , Jinsong Gong , Guoqiang Xu , Jinsong Shi , Zhenghong Xu
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Abstract

β-Nicotinamide mononucleotide is recognized as a significant bioactive nucleotide, which is can be used in the fields of health industries. Many studies on the synthesis of NMN have involved Escherichia coli and the current methods are limited by safety problems as well as the expense of the substrate. Herein, GRAS-grade Saccharomyces cerevisiae was chosen as chassis cells to synthesize NMN using the substrates glucose and nicotinamide. First, the gene for the key enzyme nicotinamide phosphoribosyltransferase (Nampt) was screened from different sources, and site-directed mutation was performed to improve the synthesis of NMN. The concentration of intracellular NMN in yeast expressing the D83N-Nampt mutant derived from Chitinophaga pinensis reached 413.4 mg/L, which was 3.7 times higher than that of yeast expressing wild enzymes. The synthesis of NMN was further enhanced by overexpressing Nampt combined with weakening of the further metabolism of NMN. Subsequently, the supply of precursor phosphate ribose pyrophosphate (PRPP) was increased by overexpressing the PRPP synthase mutant, which led to the concentration of intracellular NMN increased to 775.9 mg/L from 537.8 mg/L. Finally, the concentration of intracellular NMN reached 1.2 g/L at 6 h after whole-cell catalytic optimization, which is the highest titer achieved by S. cerevisiae from inexpensive substrate glucose and nicotinamide. This study provides the synthesis of NMN by S. cerevisiae with a new and promising method.
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重组酿酒酵母从葡萄糖和烟酰胺全细胞合成烟酰胺单核苷酸
β-烟酰胺单核苷酸被认为是一种重要的生物活性核苷酸,可用于健康产业领域。许多关于合成 NMN 的研究都涉及到大肠杆菌,目前的方法受到安全问题和底物昂贵的限制。本文选择 GRAS 级的酿酒酵母作为底盘细胞,以葡萄糖和烟酰胺为底物合成 NMN。首先,从不同来源筛选出关键酶烟酰胺磷酸核糖转移酶(Nampt)的基因,并进行定点突变以提高 NMN 的合成。在表达来自 Chitinophaga pinensis 的 D83N-Nampt 突变体的酵母中,细胞内 NMN 的浓度达到 413.4 mg/L,是表达野生酶的酵母的 3.7 倍。过表达 Nampt 进一步提高了 NMN 的合成,同时削弱了 NMN 的进一步代谢。随后,通过过表达 PRPP 合成酶突变体,增加了前体磷酸核糖焦磷酸(PRPP)的供应,从而使细胞内 NMN 的浓度从 537.8 mg/L 增加到 775.9 mg/L。最后,经过全细胞催化优化后,细胞内 NMN 的浓度在 6 h 时达到 1.2 g/L,这是 S. cerevisiae 从廉价底物葡萄糖和烟酰胺中获得的最高滴度。这项研究为利用酿酒酵母合成 NMN 提供了一种新的可行方法。
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来源期刊
Biochemical Engineering Journal
Biochemical Engineering Journal 工程技术-工程:化工
CiteScore
7.10
自引率
5.10%
发文量
380
审稿时长
34 days
期刊介绍: The Biochemical Engineering Journal aims to promote progress in the crucial chemical engineering aspects of the development of biological processes associated with everything from raw materials preparation to product recovery relevant to industries as diverse as medical/healthcare, industrial biotechnology, and environmental biotechnology. The Journal welcomes full length original research papers, short communications, and review papers* in the following research fields: Biocatalysis (enzyme or microbial) and biotransformations, including immobilized biocatalyst preparation and kinetics Biosensors and Biodevices including biofabrication and novel fuel cell development Bioseparations including scale-up and protein refolding/renaturation Environmental Bioengineering including bioconversion, bioremediation, and microbial fuel cells Bioreactor Systems including characterization, optimization and scale-up Bioresources and Biorefinery Engineering including biomass conversion, biofuels, bioenergy, and optimization Industrial Biotechnology including specialty chemicals, platform chemicals and neutraceuticals Biomaterials and Tissue Engineering including bioartificial organs, cell encapsulation, and controlled release Cell Culture Engineering (plant, animal or insect cells) including viral vectors, monoclonal antibodies, recombinant proteins, vaccines, and secondary metabolites Cell Therapies and Stem Cells including pluripotent, mesenchymal and hematopoietic stem cells; immunotherapies; tissue-specific differentiation; and cryopreservation Metabolic Engineering, Systems and Synthetic Biology including OMICS, bioinformatics, in silico biology, and metabolic flux analysis Protein Engineering including enzyme engineering and directed evolution.
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