The Functional Study On The P450 Monooxygenase AveE Of Streptomyces Avermitilis

Avermectin is a 16-membered macrolide compound with insecricidal and acaricidal activity produced by Streptomyces avermitilis,whose great value has been acknowledged by the Nobel Priza in Physiology or Medicine in 2015.The research on avermectin biosynthesis started from more than 20 years ago.Due to the important role avermectin plays in practical applications,its biosynthesis mechanism has also been a hot issue in the field of microbial genetics and biochemistry.In the biosynthetic pathway of avermectins,the cytochrome P450 monooxygenase AveE is believed to be involved in the first step of catalyzing the post-modification of avermectin precursor,but the function of AveE is only predicted by gene knockout in vivo,and its in vitro enzyme activity remains to be elucidated.Cytochrome P450 belongs to heme-iron containing protein superfamily with wide distribution and functional versatility,which is known as "diversal catalyst" in nature.As the most abundant redox partner system,Class ? redox partners system,mainly distributed in bacteria and eukaryotes mitochondria,including Ferredoxin Reductase(FdR)containing FAD and Ferredoxin(Fdx)with iron-sulf,which sequencially transfer two electrons from cofactor NADPH through "NAD(P)H ? FdR ? Fdx ? P450" to the catalytic center of P450 to activate molecular oxygen to complete the catalytic reaction.The P450 enzyme AveE,which is involved in the formation of the avermectin furan ring,is named CYP171A1 with total length of 456 amino acids?In this study,aveE was knocked out to obtain the substrate of AveE,then AveE and avermectin redox partner proteins were expressed in E.coli.The enzyme activity of AveE was reconstructed in vitro and the function of AveE was verified.All these studies will not only deepen the understanding of the formation mechanism of oxygenated heterocycles in the secondary metabolites of microorganisms,but also provide theoretical and experimental basis of vital important for the engineering transformation of AveE and its redox partner proteins to further increase the production of high-yield avermectin industrial production strains.To elucidate the catalytic mechanism of the formation of furan rings between C-6 and C-8a,the research is aimed to provide important insights into the functionality of AveE through isolating the intermediates absent of furan ring from the ?aveE knockout strain and reconstituting the in vitro activity of AveE.In this research,the in vitro enzyme activity reconstruction was completed by isolating the intermediate products of the aveE knock-out strain and the expression of AveE in E.coli.Meanwhile,the redox partner proteins of AveE that are better at supporting activitiy of AveE were determined between endogenous redox partners from Streptomyces avermitilis and a selected group of heterologous redox partners.This research will not only further our understanding on the catalytic mechanism of AveE,but also provide theoretical and experimental basis for future industrial strain improvement via engineering AveE and/or its redox system.