Complete integration of carbene-transfer chemistry into biosynthesis

Jing Huang, Andrew Quest, Pablo Cruz-Morales, Kai Deng, Jose Henrique Pereira, Devon Van Cura, Ramu Kakumanu, Edward E.K. Baidoo, Qingyun Dan, Yan Chen, Christopher J. Petzold, Trent R. Northen, Paul D. Adams, Douglas S. Clark*, Emily P. Balskus, John F. Hartwig*, Aindrila Mukhopadhyay*, Jay D. Keasling*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review


Biosynthesis is an environmentally benign and renewable approach that can be used to produce a broad range of natural and, in some cases, new-to-nature products. However, biology lacks many of the reactions that are available to synthetic chemists, resulting in a narrower scope of accessible products when using biosynthesis rather than synthetic chemistry. A prime example of such chemistry is carbene-transfer reactions1. Although it was recently shown that carbene-transfer reactions can be performed in a cell and used for biosynthesis2,3, carbene donors and unnatural cofactors needed to be added exogenously and transported into cells to effect the desired reactions, precluding cost-effective scale-up of the biosynthesis process with these reactions. Here we report the access to a diazo ester carbene precursor by cellular metabolism and a microbial platform for introducing unnatural carbene-transfer reactions into biosynthesis. The α-diazoester azaserine was produced by expressing a biosynthetic gene cluster in Streptomyces albus. The intracellularly produced azaserine was used as a carbene donor to cyclopropanate another intracellularly produced molecule—styrene. The reaction was catalysed by engineered P450 mutants containing a native cofactor with excellent diastereoselectivity and a moderate yield. Our study establishes a scalable, microbial platform for conducting intracellular abiological carbene-transfer reactions to functionalize a range of natural and new-to-nature products and expands the scope of organic products that can be produced by cellular metabolism.

Original languageEnglish
Issue number7960
Pages (from-to)403-408
Number of pages6
Publication statusPublished - 2023


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