Establishing Modular Cell‐Free Expression System for the Biosynthesis of Bicyclomycin from a Chemically Synthesized Cyclodipeptide

• Published in: Chinese journal of chemistry Vol. 42; no. 4; pp. 384 - 390
• Main Authors: Liu, Yi‐Pei, Zhao, Yu‐Heng, Zhang, Wen‐Qi, Wu, Lian, Huang, Linjuan, Tang, Gong‐Li, He, Jun‐Bin
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY‐VCH Verlag GmbH & Co. KGaA 15.02.2024; Wiley Subscription Services, Inc
• Subjects: Antibiotics; Bicozamycin; Bicyclomycin; Biosynthesis; Catalysis; Cell‐free protein synthesis; Cytochrome P450; Cytochrome P450 monooxygenase; Cytochromes P450; Enzyme catalysis; Enzymes; Iron‐ and α‐ketoglutarate‐dependent dioxygenases; Metabolic engineering; Modular systems; Natural products; Substrates; Transcription termination; Transcription termination factor RHO
• ISSN: 1001-604X; 1614-7065
• DOI: 10.1002/cjoc.202300496

Comprehensive Summary 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‐ and α‐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 platform for the biosynthesis of complex antibiotics. A modular cell‐free expression system was established for the biosynthesis of bicyclomycin from a chemically synthesized cyclodipeptide.