Application Of Red Recombinant System In Serratia Marcescens And Preliminary Screening Of Regulating Genes Of Synthesis Of Prodigiosin

Prodigiosin(PG)is a kind of natural pigments with polypyrrole ring,which has many biological activities,such as antibacterial,algal removal,anti-tumor,anti-malaria and immunosuppressive.Because PG has a huge application prospect,its industrial mass production has become an inevitable trend.However,the chemical synthesis method of PG is very complicated and the yield is very low,so at present,PG is mainly produced by the fermentation of microorganisms such as serratia marcescens.The research of increasing PG yield mainly focuses on two aspects:the optimization of fermentation medium and the modification of PG anabolic genes.In this study,we will start from the direction of modification of PG anabolic genes to improve PG yield.The main methods and research results are as follows:(1)Red homologous recombination was introduced into serratia marcescens,in order to genetically modify it.The pig A and nuc A genes in serratia marcescens were successfully replaced by Gm ~Rresistance genes through double-stranded recombination,Later,10kb(partial pig gene cluster)and 21kb(whole pig gene cluster)were replaced by Gm ~Rsuccessfully.The Gm ~R-kil selection reverse selection system initiated by the p BAD was inserted into the coding region of the pig A gene.Under the induction of 0.4%arabinose kil the gene expression lethality to serratia marcescens.At the same time,the colony was white due to pig A gene mutation caused by Gm ~R-kil insertion.Finally,Gm ~R-kil insertion-induced mutations were repaired by DNA single-stranded recombination recombination to achieve seamless modification.By double-stranded recombination and single-stranded recombination,we have successfully introduced Red homologous recombination into the serratia marcescens,It is expected that this platform can provide favorable help for the study of biological synthesis and metabolic engineering of serratia marcescens.(2)Red homologous recombination was used to replace the promoter of the pig gene cluster in Serratia marcescens with p BAD(arabinose promoter),which enabled the transcription of the pig gene cluster to synthesize PG under the regulation of the strong promoter.The constructed FC-1 not only increased the yield of PG,but also laid a foundation for further study of the temperature regulation mechanism of PG.(3)At present,researchers do not have a thorough understanding of the synthetic regulatory network of PG in serratia marcescens.This study obtained the mutant strain of serratia marcescens by Tn5 transposon insertion technique,screened out the mutant strain with high or low production of PG,and then Tn5 inserted the gene locus of the mutant strain through reverse PCR analysis.The genes of positive regulation of Env Z/Omp R、Rsc A、Mar R and negative regulation of Yee Z、Nuo B、que C have been screened.The mechanism of the above genes to regulate PG will be further studied,so as to improve the network of synthetic and metabolic regulation of PG,and it will also be beneficial to the gene modification of serratia marcescens in the future.