Discovery And Application Of Glycosyltransferase-Methyltransferase Biosynthetic Module From Beauveria Bassiana

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.Plant and bacterial glycosyltransferases capable of decorating various privileged scaffolds have been extensively studied,but those from kingdom Fungi still remain underexploited.Here,bioinformatic and genomic tools combined with heterologous expression were used to identify a glycosyltransferase–methyltransferase(GT–MT)gene pair that encodes a methylglucosylation functional module in the ascomycetous fungus Beauveria bassiana ARSEF 2860,and combinatorial biosynthetic and biocatalytic platforms were used to analyze the function and application of methylglucosylation biosynthetic module.The contents are as follows:1.Four novel glycosyltransferase-methyltransferase(GT-MT)functional modules from B.bassiana ARSEF 2860 were identified with combination of genome mining and heterologous expression techniques,and methylglucosylation of desmethyl-Lasiodiplodin(DLD)was achieved by heterologous expression in Saccharomyces cerevisiae BJ5464-Npg A.2.Feeding phenolic compounds or using combinatorial biosynthesis with ?-type fungal polyketide synthase genes,Bb GT86 display decent substrate promiscuity and regiospecificity,that efficiently modifies a broad range of drug-like substrates,including polyketides,anthraquinones,flavonoids,and naphthalenes.It yields both O-and N-glucosides with remarkable regio-and stereospecificity.These glucosides are faithfully processed by the dedicated Bb MT85 to afford 4-O-methylglucosides.3.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 linespecific antiproliferative or matrix attachment inhibitory activities.And GT–MT pair is functionally independent from other biosynthetic pathways.4.In addition,four novel GT-MT functional modules were identified from Hypocreales fungi,including Metarhizium robertsii,Isaria fumosorosea,Cordyceps militaris and Claviceps purpurea.Native GT-MT modules can be split up and regrouped into hybrid modules with similar or even improved efficacy in methylglucosylation of kaempferol specifically as compared with native pairs.And also find that methylglucosylation of kaempferol considerably improves its insecticidal activity against the larvae of oriental armyworm Mythimna separata(Walker).In this study,through genome mining with heterologous expression to identify the methylglucosylation functional module in B.bassiana.These findings will assist further 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.