Heterologous Biosynthesis Of Patchoulol In Saccharomyces Cerevisiae

With the rapid development of metabolic engineering and synthetic biology,heterologous biosynthesis of natural products in microorganism caused great attention.Patchoulol,extracted from patchouli oil,is a natural sesquiterpene compound,has important economic value and is widely used in perfumes and cosmetics.It also has neuroprotective,anti-inflammatory,anti-cancer activities.Saccharomyces cerevisiae is an important platform organism for the synthesis of natural products.This study constructed the synthesis of patchoulol in S.cerevisiae,and constructed recombinant strain which produced high-yield patchoulol.Firstly,the synthetic pathway of patchouli alcohol was constructed.Patchoulol synthase is a kind of famesyl pyrophosphate(FPP)cyclases which catalyzes the formation of patchoulol by FPP,was codon-optimized and introduced into Saccharomyces cerevisiae by high-copy plamid pESC-URA,resulting in the synthesis of patchoulol,approximately 1.02mg/L.In order to increase the yield of patchouli alcohol,metabolic engineering strategy was adopted to optimize the synthetic pathway of patchoulol.1.In order to increase the utilization of precursor FPP,a gene fusion strategy was adopted.The fusion of farnesyl diphosphate synthase and patchoulol synthase increased the production of patchoulol up to to 2.01mg/L.2.The gene tHMGR,IDI1,and upc2-1 were integrated into the yeast genome to enhance the rate-limiting step of the mevalonate pathway in Saccharomyces cerevisiae,and ERG9 was weaked by glucose concentration-related promoter PHXT1 to reduce metabolic flux from FPP to ergosterol,which increased the yield of patchoulol to 59.16 mg/L.3.The genes DPP1,LPP1,related to the synthesis of farnesol,was knocked out,resulting in the decrease of farnseol,but the yield of patchoulol has no increase.The well-characterized strain REL003-FPTS was amplified in a 5-liter bioreactor,two-phase fermentation and medium-feeding strategy were used to achieve a high production of patchoulol to 466.81 mg/L,which was much higher than the reported up to date.Morever,in order to increase the water-solubility and reduce the volatility of patchoulol,the glycosylation reaction of patchoulol was also explored.