Optimization of a cytochrome P450 oxidation system for enhancing protopanaxadiol production in Saccharomyces cerevisiae

Abstract

Ginsenosides, the major bioactive components of Panax ginseng, are regarded as promising high-value pharmaceutical compounds. In ginseng, ginsenosides are produced from their precursor protopanaxadiol. Recently, an artificial biosynthetic pathway of protopanaxadiol was built in Saccharomyces cerevisiae by introducing a P. ginseng dammarenediol-II synthase, a P. ginseng cytochrome P450-type protopanaxadiol synthase (PPDS), and a Arabidopsis thaliana NADPH-cytochrome P450 reductase (ATR1). In this engineered yeast strain, however, the low metabolic flux through PPDS resulted in a low productivity of protopanaxadiol. Moreover, health of the yeast cells was significantly affected by reactive oxygen species released by the pool coupling between PPDS and ATR1. To overcome the obstacles in protopanaxadiol production, PPDS was modified through transmembrane domain truncation and self-sufficient PPDS-ATR1 fusion construction in this study. The fusion enzymes conferred approximately 4.5-fold increase in catalytic activity, and 71.1% increase in protopanaxadiol production compared with PPDS and ATR1 co-expression. Our in vivo experiment indicated that the engineered yeast carrying fusion protein effectively converted 96.8% of dammarenediol-II into protopanaxadiol. Protopanaxadiol production in a 5 L bioreactor in fed-batch fermentation reached 1436.6 mg/L. Our study not only improved protopanaxadiol production in yeast, but also provided a generic method to improve activities of plant cytochrome P450 monooxygenases. This method is promising to be applied to other P450 systems in yeast. Biotechnol. Bioeng. 2016;113: 1787-1795. © 2016 Wiley Periodicals, Inc.

A modular engineering strategy for high‐level production of protopanaxadiol from ethanol by Saccharomyces cerevisiae

“…Among them, HMGR and ERG1 are two key rate‐limiting enzymes, and both of them are subjected to feedback regulation at transcriptional and protein levels . In cases where tHMG1 has been overexpressed in yeast, squalene accumulates in yeast cells, and further overexpressing ERG1 could completely convert squalene . However, in this study, overexpression of all genes in MVA pathway significantly improved carbon flux to squalene.…”

“…Previously, high expressions of genes DS , PPDS‐ATR1 , ERG1 , ERG9 , ERG20 , and tHMG1 were necessary for efficient production of PPD . In this study, these genes were controlled under promoters we selected, and their expression cassettes were integrated into yeast W0 resulting in strain WLT.…”

“…As secondary metabolites, they exist in plant tissues at low level, so the extraction yield was usually very low. In recent years, S. cerevisiae has been used as a heterologous host for many triterpenes production, for example, β‐amyrin, oleanolic acid, ursolic acid, betulinic acid, PPD, and so forth. However, high‐level productions of triterpenes in microbes remain challenging.…”

“…The strains used in this study are listed in Table . For strains construction, DS , PPDS ‐ ATR1 , farnesyl diphosphate synthase gene ( ERG20 ), squalene synthase gene ( ERG9 ), ERG1 , and MVA pathway genes as well as yeast endogenous promoters and terminators were amplified from the genome of strain W3a . A synthetic, codon‐optimized version of acsSE L641P specific to S. cerevisiae was obtained from SynbioML@TJU (http://www.synbioml.org/index.jsp).…”

“…Dai et al firstly engineered S. cerevisiae for de novo biosynthesis of PPD, and they found that the intermediates DMD accumulates largely during fermentation process . Then our group developed several PPD synthase and ATR1 fusions (PPDS‐ATR1) which efficiently improve DMD conversion up to 96.8%, allowing production of 1.4 g/L of PPD in 5 L fermenter . In our recent study, engineering oxidative stress defense pathways was conducted, and a robust PPD producing strain was obtained with the PPD production reaching about 4 g/L .…”