Design And Construction Of Lycopene-overproducing Saccharomyces Cerevisiae And Fermentation Process Optimization

The compatibility between heterologous metabolic pathway and host cell is critical to heterologous microbial production.Here,with the aim of lycopene overproduction in Saccharomyces cerevisiae,both host cell and heterologous pathway were delicately designed and combinatorially optimized,and consequently lycopene output and purity were drastically improved.In the meanwhile,the effect of YPL062 W,a key genetic target engineered in this study,on terpenoids production was systematically elucidated,and the biological function of YPL062 W was re-annotated as well.Firstly,S.cerevisiae CEN.PK2 was used as host cell and inducible heterologous lycopene pathway was integrated.By comparing two strategies of increasing the induction efficiency of GAL expression system(Δgal1 Δgal7 Δgal10 and Δgal80),Δgal1 Δgal7 Δgal10 was found to be more efficient in lycopene production.On this basis,deletion of ypl062 w was found to improve lycopene yield by 1.5-fold,reduce acetate accumulation tremendously and increase the content of acetyl-Co A,the vital precursor of mevalonate(MVA)pathway,by approximately 1.0-fold,thus the ypl062 w deletion strain was selected as the host cell for subsequent engineering.In addition,deletion of ypl062 w was found to generally boost the output of diverse terpenoids in S.cerevisiae,including monoterpenoid,sesquiterpenoid,diterpenoid,triterpenoid and tetraterpenoid.Through transcriptome analysis,deletion of ypl062 w was found to benefit heterologous terpenoids production from upregulation of acetyl-CoA anabolism,MVA pathway,heterologous gene expression,energy anabolism and the biosynthesis of key compositions of cytomembrane.Moreover,it was found that YPL062 W is not transcribed but belongs to the core-promoter of ALD6,and the expression level of ALD6 was negatively correlated to terpenoids production capacity.Therefore,YPL062 W functions as a crucial regulatory element in terpenoids biosynthesis primarily through regulating ALD6 transcription.On the basis of the host cell with enhanced precursor supply,lycopene biosynthesis pathway was then optimized,through combinatorial screening of five CrtE,two CrtB and three CrtI from diverse species,heterologous pathway flux was amplified and an optimal carotenogenic gene combination was obtained;through fine-tuning the rate-limiting enzyme Crt I from Blakeslea trispora,lycopene proportion in overall carotenoids was increased.Finally,host cell was further engineered,different cell mating types and distant genetic loci were evaluated to enhance lycopene yield;INO2,a stress-responsive transcription factor,was upregulated to confer cellular tolerance toward lycopene;VHb,a heterologous hemoglobin encoding gene,was introducted to increase biomass and product yields.Through above successive modifications,lycopene yield was stepwise increased by approximately 30-fold(from 2.43 mg/g DCW to 66.14 mg/g DCW),and lycopene proportion in total carotenoids reached to 91.41%.Eventually through fermentation process(media,feeding mode,dissolved oxygen control)optimization,lycopene titer reached to 2.75 g/L after fed-batch fermentation in 50 L bioreactors,which represents the highest titer in S.cerevisiae to date.