Study On Interaction Between Trans Acyltransferase And Polyketide Synthase In The Synthesis Of Oxazolomycin And Preliminary Study Of Its Function

Type Ⅰ polyketide synthase(PKS)can synthesze many useful polyketides,such as antibiotic erythromycin,immunosuppressant tacrolimus(FK506),etc.Since the modularity of type I PKS has been identified,researchers are hoping that genetic mechanisms can be engineered to synthesize more useful polyketides by studying the mechanism of its synthesis.The acyltransferase(AT)less type I PKS as a special branch of type I PKS can lead to more complex polyketide compounds,trans-AT outside PKS functions to transfer acyl-CoA in place of cis-AT.In general,trans AT deliver malonylCoA to ACP and have no specific specificity for the ACP domain,for example,LnmG can deliver malonyl-CoA to all ACPs of PKS in the synthesis of leinamycin.The studies proves that trans-ATs are well-tolerated for the non-natural substrates of ACP.Therefore,the researchers wanted to generate diversified compounds by performing mutation of the cis-AT domain in the type I PKS and replacing trans-AT with its function to incorporate different extension units into the polyketide backbone.Therefore,it is helpful to understand whether trans-AT interacts with PKS module when delivering acyl-CoA to the ACP domain,which helps to design new target products through structural matching rationality.Oxazolomycin can be produced by various strains of Streptomyces and has antibacterial,antitumor and anti-HIV activity.Zhao Chunhua et al.found that none of the PKS modules contained AT domain in the oxazolomycin gene cluster of Streptomyces albus JA3453,but had two trans AT-like genes ozmM and ozmC outside the PKS gene.The deletions and complements of ozmM genes,as well as the deletions and replenishment of the ozmC gene,have demonstrated that these two genes are necessary for the synthesis of oxazolomycin.The function of both trans ATs is to deliver malonyl-CoA(OzmM)and methoxymalonyl ACP(OzmC)to ACP.Purified OzmM and OzmQ-ACP were used for in vitro experiments to verify that OzmM can transfer malonyl-CoA to ACP.Using the affinity chromatography copurification and biolayer interferometry(BLI)technology,we further found that OzmM had interaction with OzmK and OzmQ of PKS protein.When the substrate exists,the interaction between proteins becomes weaker.We speculate that when OzmM transfers the substrate to ACP,the protein structure of the two proteins changes,and after the disruption of interaction,OzmM protein no longer participates in the condensation reaction between the extension unit and the polyketone backbone,facilitating multiple uses.The weak interaction between trans AT(OzmM or OzmC)and PKS is necessary for this function of trans AT to rapidly transfer the extension unit between ACP and PKS.In this study,a preliminary exploration of the broadness of the OzmM protein substrate was performed.The OzmM protein also delivered methylmalonyl-CoA to OzmQ-ACP.When studying protein interactions,we found that another trans AT OzmC protein interacts with the OzmK and OzmQ proteins of the PKS module.According to Zhao Chunhua et al.,the OzmC protein is responsible for transferring the methoxymalonylACP to the OzmJ-ACP.There should be no interaction with OzmK and OzmQ proteins.So we speculate that OzmC protein can also transfer malonyl-CoA to ACP.Through in vitro experiments we proved that OzmC protein can also deliver malonyl-CoA to OzmQ-ACP.In this study we verified the interaction between trans AT OzmM and PKS protein.And when malonyl-CoA is present,the interaction between the two proteins is weak.In addition,we have explored in vitro the substrate tolerance of trans AT OzmM and OzmC.OzmM protein can transfer methylmalonyl-CoA to ACP,and OzmC protein can transfer malonyl-CoA to ACP.