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bioRxiv · 10.1101/2022.02.25.482030

Natural Variation Meets Synthetic Biology: Promiscuous Trichome Expressed Acyltransferases from Nicotiana acuminata

Schenck, C.·Anthony, T. M.·Jacobs, M. L.·Jones, A. D.·Last, R. L.
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Abstract

Acylsugars are defensive, trichome-synthesized sugar esters produced in plants across the Solanaceae (nightshade) family. Although assembled from simple metabolites and synthesized by a relatively short core biosynthetic pathway, tremendous within- and across-species acylsugar structural variation is documented across the family. To advance our understanding of the diversity and the synthesis of acylsugars within the Nicotiana genus, trichome extracts were profiled across the genus coupled with transcriptomics-guided enzyme discovery and in vivo and in vitro analysis. Differences in the types of sugar cores, numbers of acylations, and acyl chain structures contributed to over 300 unique annotated acylsugars throughout Nicotiana. Placement of acyl chain length into a phylogenetic context revealed that an unsaturated acyl chain type was detected in a few closely-related species. A comparative transcriptomics approach identified trichome-enriched Nicotiana acuminata acylsugar biosynthetic candidate enzymes. > 25 acylsugar variants could be produced in a single enzyme assay with four acylsugar acyltransferases (NacASAT1-4) together with structurally diverse acyl-CoAs and sucrose. Liquid chromatography coupled with mass spectrometry screening of in vitro products revealed the ability of these enzymes to make acylsugars not present in Nicotiana plant extracts. In vitro acylsugar production also provided insights into acyltransferase acyl donor promiscuity and acyl acceptor specificity as well as regiospecificity of some ASATs. This study suggests that promiscuous Nicotiana acyltransferases can be used as synthetic biology tools to produce novel and potentially useful metabolites. ONE SENTENCE SUMMARYAnalysis of Nicotiana glandular trichome metabolites and BAHD acyltransferases revealed diverse sucrose and glucose based acylesters.

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Schenck, C., Anthony, T. M., Jacobs, M. L., Jones, A. D., Last, R. L.. 2022-02-26. Natural Variation Meets Synthetic Biology: Promiscuous Trichome Expressed Acyltransferases from Nicotiana acuminata. https://doi.org/10.1101/2022.02.25.482030

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