Construction Of Artificial Saccharomyces Cerevisiae For Producing Amorphadiene And Fermentation Optimization

Armorphadiene is the key precursor forthe anti-malaria agent artemisinin.Based on synthetic biology strategy,the research fileds about producing armorphadiene in an appropriate microbial chassis cell has developed well in the past decades.In this study,The armorphadiene biosynthesis pathway was successfully constructed in Saccharomyces cerevisiae by overexpressing two key homologous genes(a truncated 3-hydroxyl-3-methylglutaryl-CoA reductase gene(tHMGR)and farnesyl diphosphate synthase gene(ERG20)and introduceing a heterologous armorphadiene synthetase ADS into yeast genome at the multiple copies Delta sequence.It was found that the production of amorphadiene and cell growth under the same culture condition varied in strains from different S.cerevisiae strains as the chassis.According to the results,S.cerevisiae strain CEN.PK2-1C was selected as the chassis cell due to the excellent performance on strain growth and armorphadiene production.An amorphadiene titer of 225.3mg·L-1 was obtained in SyBE_Sc00011101 at 50 mL flask level.For process optimization,we firstly investigated the cell growth and amorphadiene production under different culture medium(YPD and SC-drop out culture medium)with different concentration of glucose,nitrogen source and essential amino acids.It was found that SC-drop out culture medium with 4g·L-1 glucose was selected as the culture medium due toits better performance on armorohadiene production.We also found that the cell growth and production of amorphadiene was influenced by C/N ratio and essential amino acids concentration.Dissolved oxygen in fermentator was identified as an important parameter for amorphadiene production.Finally,the armorphadiene production reached 1.05g·L-1 in 5L fermentatorafter optimizing.Consequently,the expression of squalene systhase gene ERG9,the critical gene involved in FPP competing pathway,was downregulated.It was observed that the production of amorphadiene increased by 1.35 times and the accumulation of squalene decreased to 0.16 times compared to the control one.Our study gets amorphadiene producing strain by genome integration at Delta sites.This strategy can be used for other isoprenoids overproduction in microbial organism.