Performance Optimization Of Saccharomyces Cerevisiae For Dammarendiol-Ⅱ Production

Terpenoids in natural plant sources play a key role in people’s life, among them ginsenosides, a triterpene compound from ginseng, have high medicinal value for its anti-tumor and anti-inflammatory activity. Dammarendiol-Ⅱ is an important precursor of ginsenosides. In this research, the protein sturcture of dammarendiol-Ⅱ synthase(DS) has been studied, and we introduced heterologous DS module to construct a dammarendiol-Ⅱ producing engineered yeast and successfully enhanced dammarendiol-Ⅱ level by optimizing pathway of Dammarendiol-Ⅱ production in Saccharomyces cerevisiae.In our study, two truncated DS mutant strains W303tDS0 and W303tDS4 were constructed for obtaining free DS protein based on prediction of its transmembrane region. We made alignment of nine dammarendiol synthases and found 29 different sites. In order to investigate how these sites will influence DS enzyme activity, four sites were selected and mutated to obtain W303DSΔa, W303DSΔ29, W303DSΔ252 and W303DSΔ624. All of these mutant strains led to varying degrees decline of dammarendiol-Ⅱ production, which means these mutations have great influence on DS enzyme activity.A 3D model of DS protein was built by homologous modeling based on I-TASSER and MODELLER, and optimized to remove clashes by Chrion. The model showed that DS consisted of two α barrel domains, which connected by loops and three smaller b-structures. The active site cavity was in the centre of the protein structure between two α domains. Catalytic active sites Asp488, Cys489 and Cys568 were predicted based on our DS model. And Trp421, Phe477, Trp538, Trp616, Tyr263 and Tyr732 may be responsible for stabilizing 2,3-oxidosqualene, Cys264, Tyr268, Ile559 may involve in comformation of substrate passage.An optimized DS gene expression module together with an overexpressed ERG1 module was integrated into W303 TE to produce dammarendiol-Ⅱ in Saccharomyces cerevisiae platform. The obtained engineered yeast WDS produced 77.05 mg/L dammarendiol-Ⅱ for flask fermentation and reduced intracellular precursor squalene level to 50%. Based on WDS, four genes involved in MVA pathway including truncated 8-hydroxyl-3-methylglutaryl-CoA reductase tHMGR, mutant allele of global transcription factor regulating the biosynthesis of sterols upc2.1, 2,3-oxidosqualene synthase gene ERG1, squalene synthase ERG9 were overexpressed to increase the precursor supply. The engineered strain WDS-TU19 had a higher grow rate and produced 211.52 mg/L dammarendiol-Ⅱ for flask fermentation, which was 2.7-fold higher than WDS. Moreover, the intracellular squalene level in WDS-TU19 was only 0.365 mg/L.