Enhancing The Production Of Tacrolimus By Engineering Target Genes Identified In Important Primary And Secondary Metabolic Pathways

Tacrolimus has been widely used as a powerful novel immunosuppressant.The objective of this study was to improve the production of tacrolimus by en gineering the target genes of important primary and secondary metabolic pathw ays and feeding exogenous substances.Based on the metabonomics analysis,the shikimic acid pathway is an imp ortant primary metabolic pathway for the producing tacrolimus.Combined over expression of shikimate kinase and dehydroquinic acid synthetase genes led to a 33.1% enhancement of tacrolimus production compared to parent strain.In order to predict the most efficient targets in secondary metabolic pathw ays for improving the production of tacrolimus,a genome-scale dynamic metab olic network model was used.Combined with the overexpression of tryptophan e synthase and aspartate 1-decarboxylase genes led to a 29.8% enhancement of tacrolimus production compared to the parent strain.Finally,we investigated t he impact of the genetic manipulations on transcription levels,cell growth,cell morphology and production of tacrolimus by qRT-PCR and scanning electron microscopy to reveal the relationship between the growth of strains,the effects of engineering and fermentation.Optimize the fermentation medium of genetic modified high-yield strain rat ionally.The combined addition of shikimic acid,alanine and the oxygen carrier n-dodecane increased tacrolimus production by 49.5%.Reveal the action mech anism of exogenous substances by researching the relevant fermentation charact eristics.The insights obtained in this study will help further elucidate the mech anisms by which the identified target genes promote the activity of important p rimary and secondary metabolic pathways for tacrolimus biosynthesis and provi de a new feeding strategy to improve tacrolimus production.