Identification Of Regulatory Genes Related To The Biosynthesis Of Prodigiosin From Serratia Plymuthica ACCC 02146

Prodigiosin is a secondary metabolite produced by Serritia sp.,Pseudomonas,and some actinomyces,which has anticancer,antimalarial,and antibacterial functions.It is a kind of natural red pigment and has great utilization value.At present,a lot of researchs are mainly focused on the production mechanism and the actions of prodigiosin,but there are few studies on the genes regulating the production of prodigiosin.In order to explore the characteristics of the Serratia sp.producing prodigiosin and the influence of external factors and regulatory genes on producing prodigiosin,We did the following research:In this study,15 strains were obtained from strains purchased from the Strain Preservation Center and strains stored in the laboratory.From the ability to produce enzymes,the degradation characteristics and ability were explored,and the antibiotic resistance of different strains was studied,as well as the influence of temperature,pH,and sodium concentration on Serratia sp.producing prodigiosin.The 16 S rRNA gene sequencing method was used to identify the strains,and the adjacent tree was used to classify the strains and compare the promoter sequence differences of different classes of prodigiosin biosynthetic gene clusters,to observe the chromogenic ability of each strain.The 16 S rDNA sequence and the sequence of the gene cluster promoter for the synthesis of prodigiosin in Serratia plymuthica ACCC 02146 were different from other strains.The transposon mutant library of ACCC 02146 was constructed,the clones with significantly changed chromogenic ability were screened and the transposon insertion mutant genes were identified,which laid a foundation for further study on the mechanism of synthesis of prodigiosin by Serratia sp.The contents and results to be studied are as follows:1.The ability to degrade protein was detected by skimmed milk method.The results showed that all strains could degrade protein,but the ability to degrade protein was different.The ability to degrade fat was detected by the Tween-20 degradation method,and 15 Serratia sp.strains were found to have the ability to degrade fat.The cellulase activity was identified by CMC-Na Congo red staining,and the results showed that none of them could degrade cellulose.The effects of various antibiotics,temperature,pH,and sodium concentration on 15 strains of Serratia sp.were studied.The results showed that kanamycin had the greatest effect on all strains,and actinomycin had the weakest effect.For most of the strains of Serratia sp.,the highest yield of prodigiosin was obtained at27℃,while for all of the strains,the lowest yield was obtained at 37℃.The effect of pH on 15 strains of Serratia sp.was weak,but the optimal pH of most strains of Serratia sp.was pH6,and the ability of all strains of Serratia sp.was the weakest under pH9 condition.The ability of Serratia sp.to produce prodigiosin was measured at six Na salt gradients of 0,1%,2%,3%,4%,and 5%.The optimal Na salt concentration of all strains was found to be concentrated at 0～2%.2.The strains were identified by morphological observation and 16 S rRNA gene sequencing,and the phylogenetic tree was established by the adjacency method.The strains were divided into four groups according to their branches,and the third group was Serratia marcescens.Serratia plymuthica was the first one.The promoter sequences of the prodigiosin biosynthetic gene cluster of different strains were compared to observe the chromogenic ability of each strain.It was found that the 16 S rDNA sequence of Serratia plymuthica ACCC 02146 and the sequence of the promoter of the gene cluster for the synthesis of prodigiosin were different from those of other strains.Therefore,the regulatory gene for the synthesis of prodigiosin in this strain was studied.3.The transposon mutant library of Serratia plymuthica ACCC02146 was constructed,the clones with significantly changed chromogenic ability were screened and corresponding transposon insertion mutants were identified.109 mutants in the mutant library showed changes in their ability to produce prodigiosin.Among them,38 transposon insertions occurred at pigA,pigB,pigC,pigD,and pigH,and71 transposon insertions were outside the gene cluster.The establishment of the mutant library and the identification of related genes will provide the direction for further research on the mechanism of synthesis of prodigiosin by Serratia sp..In conclusion,this study found that in addition to external factors affecting the ability of Serratia sp.to synthesize prodigiosin,and in addition to the genes that regulate the synthesis of prodigiosin,it is speculated that genes encoding related enzymes,transcriptional regulatory factors and some structural proteins outside the prodigiosin biosynthetic gene cluster regulate the synthesis of prodigiosin to varying degrees through direct or indirect pathways.This study provides theoretical support and potential target sites for further improving the synthesis of prodigiosin.