Engineering Saccharomyces Cerevisiae For Efficient Production Of Limonene

Limonene,a plant-derived natural monoterpene product with various biological and physicochemical properties,is widely used in pharmaceutical,food and cosmetics industries.The production of limonene from plants is always inefficient for seasonal cycles and environmental concerns for the solid waste discharge from the chemical extraction process.Metabolic engineering and synthetic biology of industrial microbes facilitates an alternative route for the effective production of limonene.Saccharomyces cerevisiae is considered as a promising cell factory for inexpensive production of terpenoids.In this study,we firstly constructed the engineered S.cerevisiae strain with the coexpression of geranyl diphosphate synthase(GPPS)and truncated limonene synthases(t LSs)to produce limonene.The unbalanced metabolism between cellular growth and biosynthesis of limonene greatly leads to low titer of limonene.Therefore,we reconstituted a biosynthetic orthogonal pathway for limonene production by screening nine different plant-derived t LSs combined with neryl diphosphate synthase(NPPS),instead of GPPS.And through this orthogonal pathway,the titer of limonene was achieved 28.9 mg/L in shake-flask fermentation.Then we dynamically regulated the metabolic flow between the cell growth and production pathway by engineering glucose sensitive promoter of ERG20.This glucose-sensing promoter P_HXT1 greatly enhanced biosynthesis of limonene and slightly effect on the cell growth of engineered yeast strain.Finally,the HXT1 promoter strain Lim Y28 achieved 917.7 mg/L of limonene in fed-batch culture,representing the highest production titer reported to date.The result of this study demonstrated that orthogonal pathway coupled with dynamic controller was effective strategies for improving the production of monoterpenes in S.cerevisiae and the engineered strain may serve a platform microbial cell factory for efficient production of limonene and its derivatives.