Sirirungruang Sasilada, Blay Vincent, Rodriguez Elys P, Scott Yasmine F, Vuu Khanh M, Barnum Collin R, Opgenorth Paul H, Kong Fanzhou, Li Yuanyue, Fiehn Oliver, Shih Patrick M
Department of Plant and Microbial Biology, University of California, Berkeley, CA, USA.
Joint BioEnergy Institute, Emeryville, CA, USA.
Nat Commun. 2025 Jul 10;16(1):6366. doi: 10.1038/s41467-025-61530-6.
Plants have expanded various biosynthetic enzyme families to produce a wide diversity of natural products; however, most enzymes encoded in plant genomes remain uncharacterized, highlighting the need for new functional genomic approaches. Here, we report a platform enabling the rapid functional characterization of plant family 1 glycosyltransferases, which serve important roles in plant development, defense, and communication. Using substrate-multiplexed reactions, mass spectrometry, and automated analysis, we screen 85 enzymes against a diverse library of 453 natural products, for a total of nearly 40,000 possible reactions. The resulting dataset reveals a widespread promiscuity and a strong preference for planar, hydroxylated aromatic substrates among family 1 glycosyltransferases. We also characterize glycosyltransferases with an unusually wide substrate scope and with a non-canonical Cys-Asp catalytic dyad. This work establishes a widely-applicable enzymatic screening pipeline, reflects the immense glycosylation capability of plants, and has implications in biocatalysis, metabolic engineering, and gene discovery.
植物已经扩展了各种生物合成酶家族，以产生多种多样的天然产物；然而，植物基因组中编码的大多数酶仍未得到表征，这凸显了新的功能基因组学方法的必要性。在此，我们报告了一个平台，该平台能够快速对植物1型糖基转移酶进行功能表征，这些酶在植物发育、防御和通讯中发挥着重要作用。利用底物多重反应、质谱分析和自动化分析，我们针对包含453种天然产物的多样化文库筛选了85种酶，总共进行了近40000种可能的反应。所得数据集揭示了1型糖基转移酶中普遍存在的底物混杂现象以及对平面羟基化芳香族底物的强烈偏好。我们还对具有异常广泛底物范围和非典型半胱氨酸-天冬氨酸催化二元体的糖基转移酶进行了表征。这项工作建立了一个广泛适用的酶筛选流程，反映了植物巨大的糖基化能力，并对生物催化、代谢工程和基因发现具有重要意义。
