Expression Of Prodiginine Gene Cluster In Heterologous Actinomycetes

Secondary metabolites play an important role in many aspects of our lives,particularly in the medical field,where nearly two-thirds of the current therapeutic drugs are derived from directly isolated secondary metabolites or related derivatives.With the development of gene sequencing technology,the accumulation of actinomycete genome data,the production potential of large secondary metabolites of actinomycetes were detected.However,most of the secondary metabolic genes contained in actinomycetes are in a silent state,or the expression yield is too low to be genetically manipulated to improve the yield and so on,so that the production potential of actinomycetes cannot be effectively developed.Therefore,the heterologous expression technology generated by introducing a heterologous genome into a host cell for further development of the secondary metabolite production potential of actinomycetes is an important means for activating silent gene clusters.The genome of strain NA03103 was analyzed by antiSMASH to obtain 23 secondary metabolic gene cluster information.The gene cluster ucp was found to be highly similar to the biosynthetic gene cluster mar of the compound marineosins by gene alignment.In order to exclude the influence of the complex metabolic background of the original strain on the detection of the gene cluster product,a method for constructing the expression plasmid in a heterologous host was selected to confirm its function.The cosmid library of strain NA03103 was successfully constructed,and the cosmid plasmid 23H-3 containing the gene necessary for biosynthesis of the gene cluster was screened by PCR,and finally the product was obtained by expressing the product undecylprodigiosin and butyl-meta-cycloheptylprodiginine in the heterologous host S.ablus.When the heterologous expression was successfully obtained,the host S.ablus sensitive to prodiginine was found to be resistant.Therefore,plasmids containing different fragment of gene cluster ucp were screened again by PCR to express the presence of a resistance gene ucpE which may encode an acyltransferase.In addition,in order to enrich the secondary metabolite resources that can be used for heterologous expression research in the future,the strain library constructed by our research group was screened by different culture conditions,and then the results were analyzed,the products were isolated.We finally got7 compounds of different types from 3 different strains.