Fitness Of Amorphadiene Production Functional Modules And Yeast Chassis

As the key precursor of the antimalaria agent artemisinin, the heterologousprocuction of armorphadiene in microbial cells has drawn lots of attention in the pastfew years. Heterologous production of armophadiene has been very successful. Thefitness of heterologous functional modules and chassis, which is the key to construct-ing functional yeast cells, may further improve the amorphadiene production.Firstly, two functional modules constructed with centromere vector and episomalvector were introduced into two engineered chassis overexpressing key genes (atruncated3-hydroxyl-3-methylglutaryl-CoA reductase gene tHMGR and farnesyldiphosphate synthase gene ERG20) in mevalonate pathway. The highestamorphadiene production was11.2mg/L in the functional yeast cell with betterfitness. Episomal vector and engineered W303a chassis were chosen for furtherresearch to improve the fitness of heterologous functional modules and chassis.Then fusion enzyme modules were constructed by fusion amorphadiene synthasegene ADS and ERG20under the control of different promoters (TDH1p, TDH3p,TEF1p, PGK1p). The fusion enzyme (ERG20-ADS) module under the control ofPGK1p fit the chassis better, resulting in a5.4-fold improvement in amorphadieneproduction (71.8mg/L).At last,20strians with gene deletions in BY4742were chosen as the chassis tostudy the impact of gene deletions on amorphadiene production. Results showed that16gene deletions had significant influence in amorphadiene production, indicatingthat these gene deletions may influence the amorphadiene production indirectly.In conclusion, the fitness between functional modules and chassis were studied toimprove amorphadiene production in functional yeast cells, by optimization of vectors,protein expression, and promoters in the functional modules introduced into differentchassis. The results of the study in the heterologous production of terpenoids couldoffer more information for future research. The study of amorphadiene production ingene deletion strains may help the improvement of heterologous amorphadieneproduction and the research of genes of unverified functions in yeast.