Construction And Optimization Of Recombinant Saccharomyces Cerevisiae For Production Of 20(S)-Protopanoxadiol

20（S）-protopanoxadiol（PPD）is a precursor of dammarane-type ginsenoside in ginseng and American ginseng.As an active triterpene compound,PPD presents antitumor,antidepressant and antioxidation effects.This study is focused on constructing a high-efficient PPD producing strain in Saccharomyces cerevisiae.Specifically,we optimized a cytochrome P450 monooxygenase catalytic process,employed the pull and push strategy to enhance PPD production,and engineered oxidative stress defense pathways to build a robust PPD production platform.In PPD biosynthesis pathway,conversion ratio of DMD was low,which means that the enzymatic activity of cytochrome P450-type PPD synthase（PPDS）is a limitation in this pathway.Based on the inspiration of a natural self-sufficient fatty acid hydroxylase,CYP102A1 from Bacillus megaterium,we built a PPDS-ATR1fusion protein.We found these fusion proteins all presented 4-5 folds increase in enzyme activity.Besides,DMD conversion rate reached to 96.8%.In addition,the copy number was regulated to balance DMD conversion and ROS release.In 5 L bioreactor,PPD production was 1.44 g/L.Here reported a synergistic effect of PPDS-ATR1 uncoupling and ethanol stress on ROS releasing,which reduced cells viability.To build a robust strain,a cell wall integrity associated gene SSD1 was high-expressed to improve ethanol tolerance,and ROS level decreased by 24.7%.Then,regulating the expression of an oxidative stress regulation gene YBP1 decreased 75.2%of ROS releasing,and improved cells viability from 71.3±1.3%to 88.3±1.4%at 84 h.Increased cells viability enables yeast to produce more PPD through feeding additional ethanol.In 5 L fermenter,PPD production of W3a-ssPy reached 4.25±0.18 g/L.Next,the MVA pathway genes were all overexpressed to boost precursor supply.After that,DMD,squalene and FPP accumulated largely.Simultaneously high expression of PPDS-ATR1,ERG1 mutation ERG1_K311R（to improve protein stability）and its reductase partner CPR could improve PPD production from 212.1 mg/L to432.6 mg/L.To decrease carbon flux to sterol pathway,we controlled the expression of ERG7 by engineering ERG7 promoter at different levels using TetR-TetO based gene regulation system.After that,PPD production reached 512.3 mg/L.In addition,intracellular Acetyl-CoA was improved by expressed ACS_SE^L641P（Salmonella typhi）and the PPD production reached 614.3 mg/L.Finally,5 L fed-batch bioreactors with ethanol feed resulted in 8.09 g/L of PPD production.This is the highest yield reported so far.An integrated fermentation and separation process was designed to recycle organic solvent and provide cost saving for protopanoxadiol（PPD）production.This bioprocess integrated four unit operations including fermentation,foam separation,extraction and resin chromatography.During this process,PPD removal was achieved via foam separation,and ethanol was used as PPD extractant and chromatography eluent for PPD purification.PPD product（purity 91%）was obtained and the recycled ethanol could compensate 81.3%of the total ethanol required for the fermentation.Yield of single cycle was about 75%,and the actual muiti-cycle yield was 85.78%.