Efficient C25-Hydroxylation of Vitamin D3 Utilizing an Artificial Self-Sufficient Whole-Cell Cytochrome P450 Biocatalyst DOI
Ziqi Liang, Qiao Zhou, Yicheng Li

et al.

Journal of Agricultural and Food Chemistry, Journal Year: 2025, Volume and Issue: unknown

Published: March 20, 2025

Cytochrome P450 enzymes (P450s) are promising candidates for the biosynthesis of 25-hydroxyvitamin D3 (25(OH)VD3). However, their industrial application is limited by challenges, such as low stability, inefficient catalysis, and uncoupling reactions. The construction self-sufficient P450s offers a strategic solution to these limitations, but requires linker optimization regulate interdomain conformational dynamics. In this study, we integrated whole-cell biocatalyst screening with systematic reaction conditions, including cosolvents, cell concentrations, plasmid selection, enhance catalytic performance. Under optimized heme domain Vdh-K1 achieved 91.6% conversion efficiency was subsequently selected chimeric enzyme assembly. By employing local energetic frustration analysis evaluate protein flexibility allosteric dynamics, identified variants highly frustrated linkers. optimal variant, VK1-CYP116B46-L21, exhibited improved thermostability, activity, coupling efficiency, achieving yield 4.89 mM (1.96 g/L) 25(OH)VD3 in Escherichia coli catalysis─the highest reported date. This work underscores utility computational refining dynamics multidomain establishes scalable, cost-effective framework advance systems high-value compounds.

Language: Английский

Efficient C25-Hydroxylation of Vitamin D3 Utilizing an Artificial Self-Sufficient Whole-Cell Cytochrome P450 Biocatalyst DOI
Ziqi Liang, Qiao Zhou, Yicheng Li

et al.

Journal of Agricultural and Food Chemistry, Journal Year: 2025, Volume and Issue: unknown

Published: March 20, 2025

Cytochrome P450 enzymes (P450s) are promising candidates for the biosynthesis of 25-hydroxyvitamin D3 (25(OH)VD3). However, their industrial application is limited by challenges, such as low stability, inefficient catalysis, and uncoupling reactions. The construction self-sufficient P450s offers a strategic solution to these limitations, but requires linker optimization regulate interdomain conformational dynamics. In this study, we integrated whole-cell biocatalyst screening with systematic reaction conditions, including cosolvents, cell concentrations, plasmid selection, enhance catalytic performance. Under optimized heme domain Vdh-K1 achieved 91.6% conversion efficiency was subsequently selected chimeric enzyme assembly. By employing local energetic frustration analysis evaluate protein flexibility allosteric dynamics, identified variants highly frustrated linkers. optimal variant, VK1-CYP116B46-L21, exhibited improved thermostability, activity, coupling efficiency, achieving yield 4.89 mM (1.96 g/L) 25(OH)VD3 in Escherichia coli catalysis─the highest reported date. This work underscores utility computational refining dynamics multidomain establishes scalable, cost-effective framework advance systems high-value compounds.

Language: Английский

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