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- Title
A synthetic cell-free 36-enzyme reaction system for vitamin B<sub>12</sub> production.
- Authors
Kang, Qian; Fang, Huan; Xiang, Mengjie; Xiao, Kaixing; Jiang, Pingtao; You, Chun; Lee, Sang Yup; Zhang, Dawei
- Abstract
Adenosylcobalamin (AdoCbl), a biologically active form of vitamin B12 (coenzyme B12), is one of the most complex metal-containing natural compounds and an essential vitamin for animals. However, AdoCbl can only be de novo synthesized by prokaryotes, and its industrial manufacturing to date was limited to bacterial fermentation. Here, we report a method for the synthesis of AdoCbl based on a cell-free reaction system performing a cascade of catalytic reactions from 5-aminolevulinic acid (5-ALA), an inexpensive compound. More than 30 biocatalytic reactions are integrated and optimized to achieve the complete cell-free synthesis of AdoCbl, after overcoming feedback inhibition, the complicated detection, instability of intermediate products, as well as imbalance and competition of cofactors. In the end, this cell-free system produces 417.41 μg/L and 5.78 mg/L of AdoCbl using 5-ALA and the purified intermediate product hydrogenobyrate as substrates, respectively. The strategies of coordinating synthetic modules of complex cell-free system describe here will be generally useful for developing cell-free platforms to produce complex natural compounds with long and complicated biosynthetic pathways. Adenosylcobalamin (AdoCbl) is an essential vitamin whose production is limited to bacterial fermentation. Here, the authors report an alternative method for the synthesis of AdoCbl based on a cell-free reaction system integrating more than 30 biocatalytic reactions to produce AdoCbl from 5-aminolevulinic acid.
- Subjects
COMPLEX compounds; FERMENTATION
- Publication
Nature Communications, 2023, Vol 14, Issue 1, p1
- ISSN
2041-1723
- Publication type
Article
- DOI
10.1038/s41467-023-40932-4