Metabolic Engineering Of Saccharomyces Cerevisiae For Lycopene Production

Lycopene is a terpene with high commercial value.It has received extensive attention from researchers due to its good antioxidant properties and significant pharmacological activity.It has preventive effects on cardiovascular disease,ovarian cancer,and prostate cancer.In recent years,research into the production of lycopene using microbial factory fermentation has become more common.As a widely used microbial factory,Saccharomyces cerevisiae has high food safety and clear genetic background.In this study,we took S.cerevisiae YthmgⅠas the original strain.Through metabolic engineering methods,a recombinant strain of S.cerevisiae that efficiently synthesizes lycopene was constructed.The main research contents and results are as follows:（1）Overexpressing lipid carrier synthesis-related genes to increase the production of lycopene.Taking YthmgⅠas the original strain,GAL promoters was used to overexpress the lipid droplet binding protein gene LDP1 from Rhodosporidium toruloides,the bifunctional-farnesyl diphosphate synthase/dimethylallyltransferase gene ERG20^K197G,acetyl-CoA carboxylase gene ACC1,acyl-CoA:diacylglycerol acyltransferase gene DGA1,phosphatidic acid phosphatase gene PAH1 from S.cerevisiae.The results showed that after 96h of shake flask fermentation,the unit lycopene yield of recombinant strain LFD12 which overexpressed LDP1,DGA1 and PAH1 is highest,reached 45.76 mg×g^-1 DCW,which was 1.5times that of the original strain;the biomass reached 3.9 g×L^-1,which was 1.14 times that of the original strain.（2）Increasing the supply of precursor acetyl-CoA and metabolic regulation.Taking LFD12 as the original strain,we overexpressed the acetyl-CoA synthase gene ACS1^L641P from Salmonella enteritidis and the polyadenylation transferase gene PAP1 from S.cerevisiae,and knocked out YPL062W,GAL7 and GAL10 to obtain the recombinant strain LFD18.After 96 h of shake flask fermentation,the unit yield of lycopene reached 109.26 mg×g^-1 DCW,which was 3.36 times that of the original strain.Using ACT1 as the internal reference gene,the genes ACS1^L641P,LDP1,DGA1,PAH1,PAP1,crtE,crtB,crtI were detected by Real-time Quantitative PCR.The results showed that overexpressing PAP1 regulated the flux of MVA pathway and TAG pathway,which makes the lycopene production of the recombinant strain LFD18 significantly improved.（3）The recombinant strain LFD18 was cultured in a 5 L fermenter with fed-batch fermentation,and glucose and ethanol were fed at a constant rate as carbon sources.The results showed that the biomass of the recombinant strain increased when glucose was fed,and the unit yield of lycopene increased when ethanol was fed.The production of lycopene reached the maximum of 422.07 mg×L^-1 at 59 h of constant-flow ethanol fed-batch fermentation.The yield of lycopene per unit cell reached 124.14 mg×g^-1,while the biomass reached 3.6 g×L^-1.