Abstract
Synthetic biology approach has been frequently applied to produce plant rare bioactive compounds in microbial cell factories by fermentation. However, to reach an ideal manufactural efficiency, it is necessary to optimize the microbial cell factories systemically by boosting sufficient carbon flux to the precursor synthesis and tuning the expression level and efficiency of key bioparts related to the synthetic pathway. We previously developed a yeast cell factory to produce ginsenoside Rh2 from glucose. However, the ginsenoside Rh2 yield was too low for commercialization due to the low supply of the ginsenoside aglycone protopanaxadiol (PPD) and poor performance of the key UDP-glycosyltransferase (UGT) (biopart UGTPg45) in the final step of the biosynthetic pathway. In the present study, we constructed a PPD-producing chassis via modular engineering of the mevalonic acid pathway and optimization of P450 expression levels. The new yeast chassis could produce 529.0 mg/L of PPD in shake flasks and 11.02 g/L in 10 L fed-batch fermentation. Based on this high PPD-producing chassis, we established a series of cell factories to produce ginsenoside Rh2, which we optimized by improving the C3–OH glycosylation efficiency. We increased the copy number of UGTPg45, and engineered its promoter to increase expression levels. In addition, we screened for more efficient and compatible UGT bioparts from other plant species and mutants originating from the direct evolution of UGTPg45. Combining all engineered strategies, we built a yeast cell factory with the greatest ginsenoside Rh2 production reported to date, 179.3 mg/L in shake flasks and 2.25 g/L in 10 L fed-batch fermentation. The results set up a successful example for improving yeast cell factories to produce plant rare natural products, especially the glycosylated ones.
Highlights
High-efficiency production of artemisinic acid via yeast fermentation has become a milestone for the application of synthetic biology to the production of natural plant products[1,2]
Dammarenediol II (DM) production decreased to 72.6 mg/L, representing only 12.1% of the total triterpenoid (PPD + DM) production (Fig. 2). These results suggest that the increasing expression of key P450 genes could enhance the efficient conversion of DM into PPD
UGT73C10 can catalyze the C3–OH glycosylation of DM to produce 3-O-glucosyl-dammarenediol II (3-DMG) in vitro, as shown in Supplementary Fig. S3, and the yield of 3-DMG in strain ZWDRH2-5 was 9.3 mg/L. These results demonstrated that the introduction of UGTPn50 led to a higher ginsenoside Rh2 yield than UGTPg45, resulting in a 27.7% increase in production
Summary
High-efficiency production of artemisinic acid via yeast fermentation has become a milestone for the application of synthetic biology to the production of natural plant products[1,2]. Ginsenoside Rh2, derived from Panax species, is a promising candidate for cancer prevention and therapy[5,6,7]. The ginsenoside Rh2 accounts for less than 0.01% of dried Panax ginseng roots[8]. Available ginsenoside Rh2 is currently produced mainly via the deglycosylation of major protopanaxadiol (PPD)-type ginsenosides isolated from Panax spp. using chemical or biotransformation approaches[9,10,11]. Such approaches rely heavily on the cultivation of Panax plants and the
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