Interrogation of Benzomalvin Biosynthesis Using Fungal Artificial Chromosomes with Metabolomic Scoring (FAC-MS): Discovery of a Benzodiazepine Synthase Activity

Kenneth D. Clevenger, Rosa Ye, Jin Woo Bok, Paul M. Thomas, Md Nurul Islam, Galen P. Miley, Matthew T. Robey, Cynthia Chen, Kahoua Yang, Michael Swyers, Edward Wu, Peng Gao, Chengcang C. Wu*, Nancy P. Keller, Neil L. Kelleher

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

13 Scopus citations

Abstract

The benzodiazepine benzomalvin A/D is a fungally derived specialized metabolite and inhibitor of the substance P receptor NK1, biosynthesized by a three-gene nonribosomal peptide synthetase cluster. Here, we utilize fungal artificial chromosomes with metabolomic scoring (FAC-MS) to perform molecular genetic pathway dissection and targeted metabolomics analysis to assign the in vivo role of each domain in the benzomalvin biosynthetic pathway. The use of FAC-MS identified the terminal cyclizing condensation domain as BenY-CT and the internal C-domains as BenZ-C1 and BenZ-C2. Unexpectedly, we also uncovered evidence suggesting BenY-CT or a yet to be identified protein mediates benzodiazepine formation, representing the first reported benzodiazepine synthase enzymatic activity. This work informs understanding of what defines a fungal CT domain and shows how the FAC-MS platform can be used as a tool for in vivo analyses of specialized metabolite biosynthesis and for the discovery and dissection of new enzyme activities.

Original languageEnglish (US)
Pages (from-to)3237-3243
Number of pages7
JournalBiochemistry
Volume57
Issue number23
DOIs
StatePublished - Jun 12 2018

ASJC Scopus subject areas

  • Biochemistry

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