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Establishing Modular Cell-Free Expression System for the Biosynthesis of bicyclomycin from a Chemically Synthesized Cyclodipeptide

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Chinese Journal of Chemistry 2024年第4期42卷 384-390页

作者： Yi-Pei Liu Yu-Heng Zhao Wen-Qi Zhang Lian Wu Linjuan Huang Gong-Li Tang Jun-Bin He Shaanxi Natural Carbohydrate Resource Engineering Research Center College of Food Science and TechnologyNorthwest UniversityXi'anShaanxi710069 China State Key Laboratory of Chemical Biology Shanghai Institute of Organic ChemistryUniversity of Chinese Academy of Sciences(CAS)CASShanghai200032 China School of Chemistry and Materials Science Hangzhou Institute for Advanced StudyUniversity of CAS1 Sub-lane XiangshanHangzhouZhejiang310024 China

Cell-free expression systems have emerged as a versatile and powerful platform for metabolic engineering,biosynthesis and synthetic biology studies.Nevertheless,successful examples of the synthesis of complex natural products using this system are still limited.bicyclomycin,a structurally unique and complex diketopiperazine alkaloid,is a clinically promising antibiotic that selectively inhibits the transcription termination factor Rho.Here,we established a modular cell-free expression system with cascade catalysis for the biosynthesis of bicyclomycin from a chemically synthesized cyclodipeptide.The six cell-free expressed biosynthetic enzymes,including five iron-andα-ketoglutarate-dependent dioxygenases and one cytochrome P450 monooxygenase,were active in converting their substrates to the corresponding products.The co-expressed enzymes in the cell-free module were able to complete the related partial pathway.In vitro biosynthesis of bicyclomycin was also achieved by reconstituting the entire biosynthetic pathways(i.e.,six enzymes)using the modular cell-free expression system.This study demonstrates that the modular cell-free expression system can be used as a robust and promising platformforthe biosynthesis of complex antibiotics.

关键词： Natural products bicyclomycin Biosynthesis Iron-andα-ketoglutarate-dependent dioxygenases Cytochrome P450 monooxygenase Cell-freeprotein synthesis Enzyme catalysis

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