Metabolic Engineering Of Saccharomyces Cerevisiae For Zerumbone And 13R-manoyl Oxide Production

Saccharomyces cerevisiae is the typical eukaryotic model microorganism used to produce terpenoids.The heterology terpenoids biosynthetic pathway was found to influence the physiological and genetic characteristics of S.cerevisiae,such as cell growth inhibition and genetic instability,and these influences could further decrease its production efficiency.How to balance the engineered cell growth and product synthesis is the key problem to improve terpenoids production efficiency.Based on this problem,the sesquiterpene zerumbone and diterpene 13R-manoyl oxide（13R-MO）was selected to synthesize in S.cerevisiae and the high-efficiency yeast cell factories were constructed according to the metabolic engineering and synthetic biology strategies including the construction of metabolic modules and the adaptation between modules and between modules and S.cerevisiae.Zerumbone is a sesquiterpene nature product and it exhibits diverse pharmacological properties,such as anti-carcinogenic and obesity prevention and treatment effects.In order to produce zerumbone in S.cerevisiae,an autoregulatory product synthesis strategy was constructed based on the substrate concentration changing in the fermentation process according to the glucose metabolism features of S.cerevisiae and the regulation of MVA pathway key genes to balance the engineered yeast cells growth andα-humulene production;the titer ofα-humulene increased by12.8 fold to 62.86 mg/L.To further improve the production ofα-humulene,eight MVA pathway genes responsible for converting acetyl-Co A to FPP andα-humulene synthase ZSS1 were imported to the peroxisome.Theα-humulene production was further increased to 160 mg/L.Five cytochrome P450 reductases（CPRs）from different sources were selected for CYP71BA1 adaptability tests,and At CPR1 from A.thaliana was found to be the optimal,producing 113.16μg/L of8-hydroxy-α-humulene.Multicopy integration of CYP71BA1,At CPR1,ZSS1 and ICE2（type III membrane protein）genes increased the production of8-hydroxy-α-humulene by 134-fold to 15.2 mg/L.Multicopy integration of ZSD1^S114Aincreased the production of zerumbone to 20.6 mg/L.13R-manoyl oxide（13R-MO）is the precursor of forskolin which is a cyclicAMP booster.Based on the autoregulatory product synthesis strategy,an efficient13R-MO yeast cell factory was constructed via stepwise metabolic engineering strategies,including Bts1p and Erg20^F96Cp fusion and N-terminal plastid transit peptide sequences excision of Cf TPS2 and Cf TPS3（13R-MO synthesis pathway genes）and the 13R-MO titer was increased by 142-fold to 328.15 mg/L in the engineered strain.Theα-humulene titer in engineered yeast cell factory increased by 352.4 fold to1726.78 mg/L and the 13R-MO titer in engineered yeast cell factory increased by1408 fold to 3 g/L via 5-L bioreactor fermentation compared to their origional strains.At the same time,sesquiterpenoid zerumbone was de novo synthesized by engineered Saccharomyces cerevisiae for the first time.The engineered S.cerevisiae and the constructed autoregulatory product synthesis strategy in this study could also be used to produce other sesquiterpenes,diterpenes and even tetraterpenes.