Construction Of Integrated Saccharomyces Cerevisiae Strain For Santalol Production And Subsequent Fermentation Optimization

Santalol is a sesquiterpene alcohol,generated from its precursor substance santalene through a monooxygenation.Because of its unique fragrance and characteristics,santalol is widely used in the cosmetics industry and healthcare industry with good biosecurity.Due to excessive development,the price of sandalwood oil becomes expensive,therefore,extensive research is being carried out to produce santalol through microorganisms.The purpose of the study is to construct a strain of cerevisiae for genome-integrated santalol production.The santalene synthase gene,CYP736A167 and its reductase CPR2 were integrated into multiple copy sites of S.cerevisiae chromosome by CRISPR/Cas9 gene editing technology.Finally,we increased the production of sandalwood in a 10 L bench-top fermentor.Firstly,inducibly and constitutively expressing santalene synthase gene SaSSy and tHMG1 genes were integrated into the delta site of S.cerevisiae.It was verified that the yield of inducible sandalwood production strains was higher than that of constitutive strains.In YPD(Gal)medium,the production of sandalwood reached 128.0 mg/L,which was 4.17 times of that in plasmid form.Then,CYP736A167 and its reductase gene CPR2-inducible expression module was integrated into the rDNA site of Saccharomyces cerevisiae.And in YPD(Gal),the production of z-?-sandalol reached 119.2 mg/L,and the fermentation yield after adding glycerol reached 160.5 mg/L with new induction mode.Finally,the constructed strain was domesticated serially,which was used to produce in the 10 liter fermenter.By adopting the control of the carbon source ratio of the initial culture medium and the feedback feeding method,the final cell dry weight reached 15 g/L,and our target product reached 269.3 mg/L.The above research paid a base for the improvement of this natural product in the future.