Study On The Biosynthesis Of ?-pyrone In Marine-derived Nocardiopsis Sp.

?-pyrone is an unsaturated six-membered ring lactone,which is widely present in natural products,and has a variety of biological activities.In previous studies,various ?-pyrones were isolated and identified from marine source Nocardiopsis sp.SCSIO 04583 and SCSIO 10419.Bioinformatics analysis found that a type ? PKS gene cluster is responsible for the biosynthesis of ?-pyrone in both strains.The two synthetic gene clusters are highly homologous and heterologously expressed successfully.A type ? PKS gene responsible for the biosynthesis of phomapyrone C was identified in SCSIO 10419.Based on these previous results,this study will elucidate the biosynthetic pathways of ?-pyrones,and characterize the key enzyme reaction mechanisms,then reveal the diversity of the biosynthetic pathways of ?-pyrones.The main research contents and results are shown as follows:(1)Through heterologous expression of the two type ? PKS synthetic gene clusters responsible for ?-pyrone biosynthesis,we found that the heterologous expression of type ? PKS in SCSIO 10419 also produced new?-pyrone analogues.Two new ?-pyrone compounds and one known ?-pyrone compound were obtained through purification and identification.To determine the necessary genes of the two synthetic gene clusters,we determined the boundaries of the two synthetic gene clusters through bioinformatics analysis and in-frame deletion.Both the two synthetic gene clusters contain an oxidase gene.Combined with the chemical structure of?-pyrone isolated from the wild-type strain,we speculate that it may be related to the formation of side-chain hydroxyl group.Through reconstitution of in vitro enzymatic reaction,gene knockout,and biotransformation tests,the oxidase was shown that it can not catalyze the formation of side-chain hydroxyl group.Because the oxidase is critical for biosynthesis,and the module 2 of the two PKSs lack a KR domain responsible for the formation of hydroxyl,which is related to the cyclization of six-membraned of ?-pyrone,we therefore speculate that the oxidase genes in the two synthetic gene clusters participate in the formation of the six-membered ring of ?-pyrones through online modification.The function of oxidase will be unveiled in the future study.(2)A type ? PKS gene responsible for the biosynthesis of phomapyrone C(?-pyrone)in Nocardiopsis sp.SCSIO 10419 was identified.According to the type ? PKS enzyme reaction mechanism,combined with the phomapyrone C chemical structure,we used the corresponding starting unit and extension unit as substrates to analyze the type ? PKS enzyme reaction in vitro.The enzyme could not convert them into phomapyrone C.However,the type ? PKS can catalyze the reaction of other acyl-Co A to produce 2 new products,which are produced by 2 or3 steps of polyketone chain extension reaction,respectively.This study revealed the biosynthetic pathway of ?-pyrone from marine-derived Nocardiopsis sp.SCSIO 04583 and SCSIO 10419.This study fully demonstrates the diversity of the biosynthetic pathway of ?-pyrone.