Herein we describe the discovery and functional characterization of a steroidal glycosyltransferase (SGT) from Ornithogalum saundersiae and a steroidal glycoside acyltransferase (SGA) from Escherichia coli and their application in the biosynthesis of acylated steroidal glycosides (ASGs). Initially, an SGT gene, designated as OsSGT1, was isolated from O. saundersiae. OsSGT1-containing cell free extract was then used as the biocatalyst to react with 49 structurally diverse drug-like compounds. The recombinant OsSGT1 was shown to be active against both 3β- and 17β-hydroxyl steroids. Unexpectedly, in an effort to identify OsSGT1, we found the bacteria lacA gene in lac operon actually encoded an SGA, specifically catalyzing the acetylations of sugar moieties of steroid 17β-glucosides. Finally, a novel enzymatic two-step synthesis of two ASGs, acetylated testosterone-17-O-β-glucosides (AT-17β-Gs) and acetylated estradiol-17-O-β-glucosides (AE-17β-Gs), from the abundantly available free steroids using OsSGT1 and EcSGA1 as the biocatalysts was developed. The two-step process is characterized by EcSGA1-catalyzed regioselective acylations of all hydroxyl groups on the sugar unit of unprotected steroidal glycosides (SGs) in the late stage, thereby significantly streamlining the synthetic route towards ASGs and thus forming four monoacylates. The improved cytotoxic activities of 3'-acetylated testosterone17-O-β-glucoside towards seven human tumor cell lines were thus observable.
Keywords: 6′-AE-17β-G, 6′-acetylated estradiol 17-O-β-glucoside; 6′-AT-17β-G, 6′-acetylated testosterone 17-O-β-glucoside; AE-17β-G, acetylated estradiol-17-O-β-glucoside; ASGs, acylated steroidal glycosides; AT-17β-G, acetylated testosterone-17-O-β-glucoside; Acylated steroidal glyco sides; E-17β-G, estradiol-17-O-β-glucoside; EcSGA1, E. coli steroidal glucoside acetyltransferase; HPLC—SPE—NMR, high-performance liquid chromatography–solid phase extraction–NMR spectroscopy; IPTG, isopropyl-β-D-thiogalactoside; LacA; ORF, open reading frame; Ornithogalum saunder siae; PSBD, putative steroid-binding domain; PSPG, plant secondary product glycosyltranferase box; RIN, RNA integrity number; RP-HPLC, reversed phase high-performance liquid chromatography; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SGAs, steroidal glycoside acyltransferases; SGEs, steroidal glycoside esters; SGTs, steroidal glycosyltransferases; SGs, steroidal glycosides; Steroidal glycoside acyl transferase; Steroidal glycosyltrans ferase; T-17β-G, testosterone-17-O-β-glucoside; UDP-Ara, UDP-l-arabinose; UDP-Gal, UDP-D-galactose; UDP-GalA, UDP-D-Galacturonic acid; UDP-Glc, UDP-D-glucose; UDP-GlcA, UDP-D-glucuronic acid; UDP-GlcNAc, UDP-N-acetylglucosamine; UDP-Xyl, UDP-D-xylose; UTR, untranslated region.