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Construction and optimization of microbial cell factories for sustainable production of bioactive dammarenediol-II glucosides

Green Chem. 2019;

Zong-Feng Hu‡ , An-Di Gu‡ , Lan Liang , Yan Li , Ting Gong , Jing-Jing Chen , Tian-Jiao Chen , Jin-Ling Yang *and Ping Zhu

Products/Services Used	Details	Operation
Gene Synthesis	The genes encoding PgUGT74AE2 (GenBank: JX898529.1) and UGTPg1 (GenBank: KF377585.1) were amplified from the cDNA of P. ginseng prepared as described previously20 and subsequently cloned into pET-32a (Novagen, USA), resulting in pET-PgUGT74AE2 and pET-UGTPg1 for heterologous expression in E. coli BL21 (DE3). Additionally, the genes encoding PgUGT74AE2, UGTPg1 and DS (GenBank: AB265170.1) were also synthesized by GenScript (Nanjing, China) in Tianjin University (China) according to the codon bias ofS. cerevisiae for improved expression while DS was fused with GFP through overlap extension PCR (OE-PCR). Yeast promoters (TEF1,TDH3 and PGK1), terminators (CYC1, ADH1, TPI1 and ADH2) and genes encoding truncated HMG-CoA reductase (tHMG1), isopentenyl diphosphate isomerase (IDI1), farnesyl diphosphate synthase (ERG20), squalene synthase (ERG9), squalene epoxidase (ERG1), immunoglobulin-binding protein (BiP), transcriptional activator HAC1 and protein disulfide isomerase (PDI1) were amplified from the genomic DNA isolated from S.	Get A Quote

摘要

Ginsenosides, the predominant bioactive components of Panax species, are biosynthesized by glycosylation at C3–OH and/or C20–OH of protopanaxadiol (PPD), and C6–OH and/or C20–OH of protopanaxatriol (PPT). Dammarenediol-II (DM), the direct precursor of PPD, has two hydroxyls at C3 and C20 positions, but DM glucosides have scarcely been identified from Panaxspecies. Herein, we used two crude recombinant UDP-glycosyltransferases (UGTs), PgUGT74AE2 and UGTPg1 from Panax ginseng, to catalyze the glycosylation of DM with UDP-glucose (UDPG) as the sugar donor to produce DM glucosides 3-O-β-D-glucopyranosyl-dammar-24-ene-3β,20S-diol (3β-O-Glc-DM) and 20-O-β-D-glucopyranosyl-dammar-24-ene-3β,20S-diol (20S-O-Glc-DM), respectively. The in vitro and in vivo assays demonstrated that both 3β-O-Glc-DM and 20S-O-Glc-DM exhibited higher anti-colon cancer activities than natural ginsenosides. In order to produce DM glucosides in an economical, efficient and convenient way, we refactored the complete biosynthetic pathways of 3β-O-Glc-DM and 20S-O-Glc-DM by introducing the codon-optimized genes encoding DM synthase (DS) together with PgUGT74AE2 or UGTPg1 into Saccharomyces cerevisiae, respectively. Furthermore, multistep metabolic engineering strategies were applied, including optimization of chassis cell, multi-copy integration of heterologous genes via the CRISPR/Cas9 system, increase of precursor supply by overexpressing rate-limiting enzymes, down-regulation of the competitive pathway to redirect the metabolic flux towards the target products, and overexpression of the transcriptional activator. Finally, the titers of 2.4 g L−1 3β-O-Glc-DM and 5.6 g L−1 20S-O-Glc-DM were achieved through fed-batch fermentation in a 3 L bioreactor. This is the first study to demonstrate the anti-colon cancer activities of DM glucosides and to achieve the de novo biosynthesis of DM glucosides with high titers in microbial cell factories. This study has established a green and sustainable approach for the industrial production of DM glucosides, which provides promising candidates for new drug research and development.