Yarrowia Lipolytica Construction For Heterologous Synthesis Of ?-santalene And Fermentation Exploration

Sandalwood oil is a valuable spice derived from plant Santalum album L.,which has antibacterial,cosmetic and sedative effects.?-santalene is the precursor of a-santalol,the main component of sandalwood oil.Yarrowia lipolytica is generally regarded as safe Generally regarded as safe(GRAS),Yarrowia lipolytica is an oleaginous yeast and has been metabolically engineered to produce many valuable compounds such as terpenoids and biofuel.This paper presents a method for the heterologous synthesis of a-santalene by Y.lipolytica.Using Y.lipolytica ATCC 201249 as the starting strain,the codon-optimized plant-origin a-santalene synthase STS was integrated into the genome,and a yield of 5.19 mg/L for a-santalene was obtained after fermentation.Overespressing of the upstream genes in the MVA pathway by plasmids construction(ERG8,ERG10,ERG12,ERG13,ERG19,HMG1 and tHMGl),we found that the key genes ERG8,HMG1,and tHMG1 can increase the supply of FPP and increase the yield of a-santalene.The overexpression of the rest of the genes perfer to achieve the synthesis of squalene.The ERG8 and HMG1 were overexpressed inmultiple-copy formats,and the plasmid pERG8HMG1 and ERG8-HMG1 expression module were optimized as single-copy and multiple-copy formats,respectively.The overexpression of single-copy plasmid pERG8HMG1 make ?-santalene yield reach at 13.31mg/L.The effect of n-dodecane addition amount,addition time,and fermentation duration on product accumulation are explored in two-phase fermentation optimization.The addition of 10%n-dodecane at 6 h of fermentation resulted in the best growth of the bacteria and product accumulation over a period of 5 days.The optimal feeding strategy was determined by four carbon source optimizations and five feeding methods.Using 50 g/L glucose as the initial carbon source,maintaining the carbon source concentration at 5-20 g/L during the feeding process is most conducive to the increase of production.These results were verified in 5-L fermenter by batch and fed-batch fermentation.In the end,the OD value of fed-batch fermentation broth reached 80,and the production of a-santalene reached 26.4 mg/L,which was 2.56 times of the initial yield without by-products farnesol and trans-?-bergamotene.