Improving The Production Of Active Secondary Metabolites In Pseudomonas Protegens Pf-5 By Using Ribosome Engineering

Pseudomonas protegens Pf-5 is an important plant commensal bacterium which has an excellent capacity to produce a wide variety of antimicrobial active metabolites including Pyoluteorin(Plt), 2,4-diacetylphloroglucinol(Phl), Pyrrolnitrin(Prn) and siderophore, these secondary metabolites which can suppress various pathogens. However,the wild-type P. protegens is imperfect on account of its weak biosynthesis ability of active secondary metabolites. Nowadays it has proved to be a effective way to increase the production of secondary metabolites of Ribosome engineering.In order to improve the yield of secondary metabolites produced by P. protegens Pf-5, we used Ribosome engineering to screen the Rifampicin resistant mutants. Under a high inhibitory Rifampicin concentration, 60 spontaneous rifampicin resistant mutants were isolated.By comparing the inhibitory effect among the Pf-5 and the mutant strains,seven mutants exhibited a higher anti-fungal capability than the original strain. DNA sequencing showed that all of the 7 mutants there existed a same point mutations in the conserved regions(rif-cluster I) of rpoB gene that encodes RNA polymerase beta subunit. The mutations revealed a C to A transversion mutations occurred at nucleotide pair 1591, which resulted in the mutations of Histidine(531) to Asparagine. So we selected the one of the mutant R55 was selected for the later study as it has show best inhibitory effect in 7 mutants. The yields of secondary metabolites were then analyzed and compared with parent strain. We found the synthesis of two major metabolites displayed a larger increase in R55,which were 2.5 and 1.4 fold higher than in the parent strain. MetabolitesB and D were identificated as Pyoluteorin(Plt), 2,4-diacetylphloroglucinol(Phl) respectivly by mass spectrometry. It also shows different degrees of inhibitory effects upon the tested strains. In addition, we attempted to overexpress the ropBR gene from R55 in the original strain Pf-5,and constructed the engineering strain Pf-rpoBR. The engineering bacteria can grow in high concentrations of rifampicin resistant plate, and CAS agar plate show the siderophore production has greatly improved.In this study, the Ribosome engineering was applied to introduce rifampicin resistance mutation to P. protegens Pf-5, then a mutant with two kinds of secondary metabolites production significantly increased was obtainedand, and its antibacterial activity greatly enhanced. Mutation sites were also successfully identified in mutants. And R55 exhibited genetic stability even after multiple passage. This study provides a foundation for ribosome engineering breeding of P. protegens and makes sure that ribosome engineering technology have a significant potential application value in gram negative bacteria.