The Mechanism Of The Prodigiosin Synthesis Regulated By Temperature In Serratia Marcecens

Prodigiosin (PG), one of the prodiginines family, is a tripyrrole chemical and a secondarymetabolite which is produced by some Serratia, Actinomyces, Pseudomonas, Ocean bacteriaand so on. PG which is considered to be an important natural red-pigment can be applied extensively in medicine, chemical, dye and some other industry. This thesis will focus on the studying of the synthesis of PG controlled by temperature in Serratia marcecens JNB5-1, a bacterium which is screened earlier in our lab for high-yield production of PG. The main contentsand results are as follows:1) Based on the analysis results of two-dimensional gel electrophoresis and matrix-assistedlaser desorption/ionization-time of flight-mass spectrometry,16different expressionproteins were identified. The results indicated that the expression of prodigiosinbiosynthetic enzymes (O-methyl transferase and Oxidoreductase) and proteins relevant toprodigiosin biosynthesis were significantly decreased at37oC compared with those at28oC. However, the expression of heat shock protein(Hsp60) and superoxidedismutase(SOD) were increased at37oC.2) The different expression proteins were further analyzed by RT-qPCR. The results ofRT-qPCR showed that the transcriptional levels of o-methyl transferase, oxidoreductaseand transketolase at37oC were lower than that at28oC, which was consistent with theresults obtained by the2-DE, and there was a significant positive correlation between thetranscriptional levels and the expression levels.3) Promoter probe vector was constructed and the promoter characteristics was estimatedaccording the Chloramphenicol acetyltransferase(CAT) activity in the host cell. Theresults showed that P-pigF had a strong promoter activity, and the activity of CAT at28oC was2.1times higher than that at37oC. P-pigN have no the promoter activity. Theresults above indicated that the P-pigF was regulated by temperature at transcriptionallevel.4) The gene pigF from S. marcecens JNB5-1was cloned and heterologous expressed inE.coli, and the properties of PigF were characterized for the first time. The resultsindicated that the enzyme showed high activity under pH ranging from6.0to9.0andtemperature ranging from-20oC to28oC, and its optimum pH and temperature for thecatalytic activity was about7.0and30oC, respectively. The Kmand Vmaxvalues of PigFwere14.6mmol·L-1and0.58mmol·L-1·min-1, respectively. PigF activity was stimulatedby Mg2+, while it was inhibited by Fe3+、Mn2+、Zn2+、K+、Ni2+、Ca2+、Na+、Hg2+、Cu2+and EDTA.These results above provided intimations about the effect of temperature on prodigiosinsynthesis in S. marcecens JNB5-1. They also lay a foundation for futher studies onprodigiosin biosynthesis as well as a theoretical guidance for constructing high-yieldingprodigiosin strains.