Interrogation of Benzomalvin Biosynthesis Using Fungal Artificial Chromosomes with Metabolomic Scoring (FAC-MS): Discovery of a Benzodiazepine Synthase Activity
2018
Clevenger, Kenneth D. | Ye, Rosa | Bok, Jin Woo | Thomas, Paul M. | Nurul Islam, Md. | Miley, Galen P. | Robey, Matthew T. | Chen, Cynthia | Yang, KaHoua | Swyers, Michael | Wu, Edward | Gao, Peng | Wu, Chengcang C. | Keller, Nancy P. | Kelleher, Neil L.
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.
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