Metabolic Engineering Of Streptomyces Bingchenggensis For Efficient Production Of Milbemycin

Milbemycins(MILs)are a group of 16-membered macrolides.Owing to their high efficiency,low toxicity,easy degradation,and environmental friendliness,milbemycins have been approved for pest control of crops and flowers in 43 countries and regions including the United States and Italy,and milbemycin oxime has been evaluated as the safest antiparasitic drug for the prevention and treatment of parasitic diseases in cats and dogs.However,the significant lower yield of MILs than avermectins restrict their wide application.In this study,using the complete genome and transcriptome sequence of Streptomyces bingchenggensis,the MIL industrial strain,we sketched the temporal expression profiles of all potential BGCs in S.bingchenggensis,and a type ? PKS gene cluster kel was identified to be responsible for the biosynthesis of a kelly-green compound.Then we found that disruption of the core-biosynthetic genes kelCD and the regulatory gene kelR have the obverse effect on MIL production.The mil and kel clusters were co-activated by the SARP family regulator KelR from kel cluster and they are competing for the metabolic precursors at late fermentation stage.Then two other co-expressed BGCs,clusterl-typel PKS and cluster41-otherKS,were confirmed to possess the co-regulation and metabolic competition relationship with mil cluster.Based on those,an engineering strategy was proposed for MIL overproduction by overexpressing regulatory gene and disrupting the core-biosynthetic genes.Eventually,the production of MIL A3/A4 was increased to 4058.2±71.0 mg/L mg/L.In conclusion,this study identified three secondary metabolite biosynthetic gene clusters co-expressed with mil cluster,and comprehensively deciphered the metabolic competition and transcriptional co-regulation among the BGCs in S.bingchenggensis,offered an engineering strategy of overexpressing regulatory gene and disrupting the core-biosynthetic genes for MIL overproduction.Besides,this study firstly found and characterized a new bottom-layer activator of MIL biosynthesis,providing the theoretical basis for the research of the regulatory mechanism of MIL biosynthesis.