Engineering Saccharomyces Cerevisiae For Production Of Limonene And Perillyl Alcohol

Limonene and its derivative perillyl alcohol are natural products of plant origin with fragrance.They have antibacterial,anthelmintic,anti-cancer,anti-cough and stone-dissolving activities and are widely used in food,pharmaceuticals and cosmetics industries.The development of synthetic biology and metabolic engineering and the construction of engineered yeast have opened up new methods for the efficient and sustainable supply of limonene and perillyl alcohol.In this study,we provided the combinatorial engineering of S.cerevisiae for improved the production and yield of limonene with combination of modified chassis and optimized limonene synthesis pathway.First,the orthogonal limonene biosynthetic pathway was integrated into the chromosome.Engineering the PDH bypass and found that limonene production was decreased when overexpressing of endogenous ADH2,ALD6 and ACS2,while the overexpressing of heterologous Se ACS^L641P had a positive effect and increased limonene production by 11.92%.Then,deleting genes outside terpenoids biosynthetic pathway such as ROX1,GDH1,GDH2,CIT2,and MLS1,and found that the production of limonene was increased by more than 10%respectively.Among them,deleting of CIT2 had the most significant effect in fed-feed fermentation,increasing by 17.81%.Finally,Se ACS^L641P was replaced the CIT2 and the limonene synthesis module was overexpressed to construct the engineering strain LMZ18.In shake-flask fed-batch fermentation,the production of limonene reached 2.23 g/L,and the productivity reached 13.27 mg/（L·h）.Designed and constructed perillyl alcohol engineering strain with limonene producing strain as the chassis.First,we tested the ability of catalyzing limonene to produce perillyl alcohol of Ctm ABE from Castellaniella defragrans,Cym AB from pseudomonas putida and CYP102A1^{A264V/A238V/L437F} and found that Cym AB was the most excellent one.Next,the medium composition and the extractant for the two-phase culture were optimized,and the optimal riboflavin concentration was 500μmol/L,and10%DNIP was the organic phase.Finally,the engineered strain was subjected to fed-batch fermentation under optimized conditions,and the yield of perillyl alcohol reached51.97 mg/L.This is the first report on the biosynthesis of perillyl alcohol by Saccharomyces cerevisiae.This study showed that metabolically engineered yeast possessed promising potential for industrial production of limonene,meanwhile,provided a meaningful strategy of combinatorial metabolic engineering of S.cerevisiae for production of monoterpenoids and derivative of limonene.