Excavation And Functional Analysis Of Novel Regulatory Factors For Prodigiosin Biosynthesis In Serratia Marcescens

Prodigiosin?PG?,a red linear tripyrrole pigment and most prominent member of the prodiginine family,is a natural secondary metabolite produced by different microorganisms.Studies on prodigiosin have found that it has important antimicrobial,anticancer,and immunosuppressive properties,hence prodigiosin has great application value in the fields of pharmaceutical development,environmental management,and dye preparation.Therefore,fermentation of S.marcescens to produce prodigiosin has been extensively studied for improving prodigiosin production in recent years.However,our understanding of the regulatory mechanism behind prodigiosin biosynthesis in S.marcescens is still limited,which restricts the development speed of the microbial fermentation production industry of prodigiosin.Based on the preliminary work in our laboratory,the prodigiosin-producing strain S.marcescens JNB5-1 was used as the research object in our study.By constructing a Tn5G transposon insertion mutation library,the novel regulatory factors BVG90?02415?Dac A?,BVG90?22495?Met R?,BVG90?13250?Rcs B?,BVG90?13250?Psr A?and BVG90?04085?Psr B?important for prodigiosin biosynthesis were identified and the molecular mechanism how these regulatory factors influence prodigiosin biosynthesis in strain JNB5-1 was investigated.Also,the important roles of these regulatory factors in affecting other cellular processes in strain JNB5-1 were also clarified.On this basis,a prodigiosin high-producing strain,here we called SK68?Met R-P_{SMWW4vlc02160}-Psr A-Psr B was constructed via the metabolic engineering strategy.The main research contents and results of this study are as follows:?1?Novel regulatory factors important for prodigiosin biosynthesis were revealed based on the Tn5G transposon insertion mutation technology.By using E.coli/p RK2013 Tn5G as the donor strain and S.marcescens JNB5-1 as the recipient strain,a Tn5G transposon insertion mutation library was constructed and numbers of novel regulator factors related to prodigiosin biosynthesis were identified in S.marcescens,such as BVG90?02415,BVG90?22495,BVG90?13250,BVG90?12635 and BVG90?04085.For regulator factor BVG90?02415?Dac A?,accompanied by confirming that D-Ala-D-Ala carboxypeptidase Dac A negatively regulation of prodigiosin biosynthesis in S.marcescens JNB5-1,we also found that Dac A affected the expression level of the prodigiosin biosynthesis-related pig A gene,cell morphology,and cell membrane permeability in strain JNB5-1.These results suggested that the molecular mechanism how Dac A affecting the biosynthesis of prodigiosin in strain JNB5-1 was probably that the deletion of dac A gene enhanced prodigiosin leakage,which in turn alleviated inhibition effect of prodigiosin on the pig gene cluster,thus the expression of pig gene cluster was increased,and ultimately enhanced the ability of strain JNB5-1 to synthesize prodigiosin.?2?Molecular mechanism and functional analysis of the transcriptional regulator Met R involved in the regulation of prodigiosin biosynthesis in S.marcescens.Sequence analysis and functional verification of the prodigiosin biosynthesis-related regulator BVG90?22495 isolated from the Tn5G transposon insertion mutation library revealed that the Lys R-type transcriptional regulator BVG90?22495 is Met R,and Met R is a novel regulator negatively controls prodigiosin biosynthesis in S.marcescens.Disruption of met R gene conferred a remarkably increased production of prodigiosin,which was 91.99%higher than that of the parent strain JNB5-1.By using real-time quantitative PCR?RT-q PCR?analysis,?-galactosidase assays,transcriptomics analysis,and electrophoretic mobility shift assays?EMSAs?,the molecular mechanism of Met R in regulation of prodigiosin biosynthesis in strain JNB5-1 was investigated.And results showed that the molecular mechanism how Met R negatively controls prodigiosin production in strain JNB5-1 was correlated with Met R directly binding to the promoter region of the prodigiosin-synthesis positive regulator Pig P and hence negatively regulated the expression of the prodigiosin-associated pig operon.Moreover,research on the function analysis of Met R on the cellular processes in strain JNB5-1 found that Met R,as a multifunctional transcriptional regulator,not only negatively regulated prodigiosin biosynthesis,but also contributes to some other important cellular processes in strain JNB5-1,such as L-methionine biosynthesis,H₂O₂tolerance,heat tolerance,exopolysaccharide synthesis,cell motility,and biofilm formation.?3?Molecular mechanism and functional analysis of the transcriptional regulator Rcs B involved in the regulation of prodigiosin biosynthesis in S.marcescens.Sequence analysis and functional verification of the prodigiosin biosynthesis-related regulator BVG90?13250 isolated from the Tn5G transposon insertion mutation library revealed that the transcriptional regulator BVG90?13250 is Rcs B,and is a negative regulator of prodigiosin biosynthesis in S.marcescens.We illustrated experimentally that compared with the wild-type strain JNB5-1,the rcs B mutant SK68 had an 128.35%increase in prodigiosin production in LB medium.Further,by using real-time quantitative PCR?RT-q PCR?analysis,?-galactosidase assays,comparative transcriptomics analysis,and electrophoretic mobility shift assays?EMSAs?,the molecular mechanism of Rcs B in regulation of prodigiosin biosynthesis in strain JNB5-1 was investigated.And results showed that the molecular mechanism of Rcs B in regulation of prodigiosin biosynthesis in strain JNB5-1 was that Rcs B negatively regulated the expression levels of the flh DC genes by directly binding to the conserved site?5'-TAAGATTATTCCTA-3'?in the flh DC genes promoter region,then negatively regulated the expression level of pig gene clusters,and ultimately influencing the ability of S.marcescens to synthesize prodigiosin.Moreover,more analyses revealed that Rcs B regulated some other important cellular processes,including cell motility,acid resistance,exopolysaccharide synthesis,and biofilm formation in S.marcescens.?4?Molecular mechanism of the transcriptional regulator Psr A and Psr B involved in the regulation of prodigiosin biosynthesis in S.marcescens and functional analysis of Psr A.Functional verification of the prodigiosin biosynthesis-related regulators Lys R family transcriptional regulator BVG90?12635 and Deo R family transcriptional regulator BVG90?04085 isolated from the Tn5G transposon insertion mutation library revealed that the transcriptional regulators BVG90?12635?here we called Psr A?and BVG90?04085?here we called Psr B?were positive regulators of prodigiosin biosynthesis in S.marcescens.With the absence of Psr A and Psr B,the ability of the strains to synthesize prodigiosin was only 0.05-times and 0.22-times that of the wild-type strain JNB5-1,respectively.Analysis of the molecular mechanism of Psr A and Psr B in regulating the biosynthesis of prodigiosin found that both Psr A and Psr B positively regulated the expression level of the pig gene cluster by directly binding on the promoter region of the prodigiosin biosynthesis-related pig gene cluster,then regulating the biosynthesis of prodigiosin in S.marcescens.Importantly,by further using transcriptome sequencing?RNA-Seq?analysis,we illustrated experimentally that the Psr A protein is not only a regulator involved in the regulation of prodigiosin biosynthesis,but also a novel regulator involved in extracellular polysaccharides production,biofilm formation,swarming motility,serrawettin W1 biosynthesis and T6SS-mediated antibacterial activity in S.marcescens.?5?Metabolic engineering modification of S.marcescens to produce prodigiosin.Based on the newly identified prodigiosin biosynthesis-related genes in our study,the prodigiosin high-producing strain SK68?Met R-Psr A-Psr B was constructed by integrating multiple gene mutations.That is,the S.marcescens rcs B mutant strain SK68 was chosen as the starting strain.With knocking out the met R gene,the psr A and psr B genes were overexpressed simultaneously.Then,based on the strain SK68?Met R-Psr A-Psr B,the promoter P_{SMWW4vlc02160} of the metalloproteinase encoding gene SMWW4?vlc02160 was used to replace the promoters of Ppsr A and Ppsr B of the psr A and psr B genes to obtain the strain SK68?Met R-P_{SMWW4vlc02160}-Psr A-Psr B.Shake flask fermentation analysis showed that the prodigiosin production of strains JNB5-1,SK68?Met R-Psr A-Psr B and SK68?Met R-P_{SMWW4vlc02160}-Psr A-Psr B was 5.33 g/L,7.45 g/L and 8.92 g/L,respectively and the productivity of these three strains was 0.074 g·L^-1·h^-1,0.103 g·L^-1·h^-1 and 0.124 g·L^-1·h^-1,respectively.