Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, 100081 Beijing, People's Republic of China.
Natural Products Center, University of Arizona, Tucson, AZ 85706.
Proc Natl Acad Sci U S A. 2018 May 29;115(22):E4980-E4989. doi: 10.1073/pnas.1716046115. Epub 2018 May 14.
Glycosylation is a prominent strategy to optimize the pharmacokinetic and pharmacodynamic properties of drug-like small-molecule scaffolds by modulating their solubility, stability, bioavailability, and bioactivity. Glycosyltransferases applicable for "sugarcoating" various small-molecule acceptors have been isolated and characterized from plants and bacteria, but remained cryptic from filamentous fungi until recently, despite the frequent use of some fungi for whole-cell biocatalytic glycosylations. Here, we use bioinformatic and genomic tools combined with heterologous expression to identify a glycosyltransferase-methyltransferase (GT-MT) gene pair that encodes a methylglucosylation functional module in the ascomycetous fungus The GT is the founding member of a family nonorthologous to characterized fungal enzymes. Using combinatorial biosynthetic and biocatalytic platforms, we reveal that this GT is a promiscuous enzyme that efficiently modifies a broad range of drug-like substrates, including polyketides, anthraquinones, flavonoids, and naphthalenes. It yields both - and -glucosides with remarkable regio- and stereospecificity, a spectrum not demonstrated for other characterized fungal enzymes. These glucosides are faithfully processed by the dedicated MT to afford 4--methylglucosides. The resulting "unnatural products" show increased solubility, while representative polyketide methylglucosides also display increased stability against glycoside hydrolysis. Upon methylglucosidation, specific polyketides were found to attain cancer cell line-specific antiproliferative or matrix attachment inhibitory activities. These findings will guide genome mining for fungal GTs with novel substrate and product specificities, and empower the efficient combinatorial biosynthesis of a broad range of natural and unnatural glycosides in total biosynthetic or biocatalytic formats.
糖基化是一种通过调节药物样小分子支架的溶解性、稳定性、生物利用度和生物活性来优化其药代动力学和药效动力学性质的主要策略。可用于“糖衣”各种小分子受体的糖基转移酶已从植物和细菌中分离和鉴定出来，但直到最近，尽管一些真菌经常用于全细胞生物催化糖基化，但丝状真菌中的糖基转移酶仍然是隐藏的。在这里，我们使用生物信息学和基因组工具结合异源表达来鉴定一个糖基转移酶-甲基转移酶（GT-MT）基因对，该基因对在子囊菌真菌 中编码一个甲基葡萄糖基化功能模块。GT 是一个家族的创始成员，该家族与已鉴定的真菌酶没有同源性。使用组合生物合成和生物催化平台，我们揭示了该 GT 是一种混杂酶，能够有效地修饰广泛的药物样底物，包括聚酮、蒽醌、类黄酮和萘。它以显著的区域和立体特异性生成 - 和 - 葡萄糖苷，这是其他已鉴定的真菌酶所没有的。这些葡萄糖苷被专用的 MT 忠实地加工成 4-O-甲基葡萄糖苷。所得的“非天然产物”显示出增加的溶解度，而代表性的聚酮甲基葡萄糖苷也显示出增加的对糖苷水解的稳定性。在甲基糖基化后，发现特定的聚酮具有针对癌细胞系的特异性抗增殖或基质附着抑制活性。这些发现将指导具有新型底物和产物特异性的真菌 GT 的基因组挖掘，并为广泛的天然和非天然糖苷的高效组合生物合成提供总生物合成或生物催化格式。
