The enzymatic biosynthesis of acylated steroidal glycosides and their cytotoxic activity

Abstract

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.

Combinatorial mutation on the β-glycosidase specific to 7-β-xylosyltaxanes and increasing the mutated enzyme production by engineering the recombinant yeast

“…Glycoside hydrolases (GHs, EC 3.2.1), also known as glycosidases, are enzymes that can hydrolyze the glycosidic bonds in glycoconjugates1., 2., 3., 4.. GHs can be categorized into over 100 families based on the amino acid sequences of different species.…”

Transcriptome-Wide Identification of an Aurone Glycosyltransferase with Glycosidase Activity from Ornithogalum saundersiae

“…Ornithogalum saundersiae is a medicinal plant rich in glycosides, suggesting this species contains glycosyltransferase responsible for the biosynthesis of glycosides [ 22 , 23 ]. Hence, the transcriptome sequencing of O. saundersiae was performed previously in our laboratory [ 17 ]. The extensive transcriptome mining was carried out with the aim of retrieving unigenes encoding GTs in this investigation.…”

“…The transcriptome of O. saundersiae was sequenced previously and the resultant database was kept in our laboratory [ 17 ]. The unigenes in this database were first aligned by Blast X algorithm to protein databases, such as Non-Redundant Dataset (NR), Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Cluster of Orthologous Groups (COG) ( e -value < 0.00001) for functional annotation.…”

“…The formation of new glycosides was monitored by high performance liquid chromatography (HPLC). The HPLC conditions have been detailed as described by Yuan et al [ 16 ] and Liu and Kong [ 17 ]. The resulting glycosides were collected by reversed-phase HPLC and then injected into a TripleTOF 5600 MS instrument (AB Sciex, Concord, ON, Canada) operated in positive mode with a DuoSpray ion source.…”

Characterization of two flavonol synthases with iron-independent flavanone 3-hydroxylase activity from Ornithogalum caudatum Jacq

“…The resultant pET28a-OcFLS2 was transformed into E. coli Transetta (DE3) (TransGen Co. Ltd., Beijing, China) for the recombinant expression. The other plasmid pET28a-OcFLS1 was co-transformed into E. coli BL21(DE3) strain with a chaperone plasmid pGro7 (Takara Biotechnology Co., Ltd., Dalian, China) to improve soluble expression as previously reported [42,51]. These recombinant cells were cultivated until the optical density at 600 nm (OD 600 ) reached 0.6 and was induced at 20°C overnight by the addition of isopropyl-D-thiogalactopyranoside (IPTG) to reach a final concentration of 0.3 mM.…”