Study On Screening Genes Regulating The Expression Of Lipase LipA From Pseudomonas Protegens Pf-5 And Their Corresponding Regulation Mechanism

As one group of the important industrial enzymes,lipases are widely used in bio-energy,cosmetics,fine chemicals,leather,oil,feed,food process and many other industrial fields.Almost all microorganisms,animals and plants possess lipases.However,microorganisms provide most of the commercial lipases.Compared with fungal lipases,bacterial lipases have not only wider catalytic reactions in organic phase and aqueous phase,but also have higher stability and reactivity.Among bacterial lipases,Pseudomonas lipases perform the best.But the production of bacterial lipases is usually relatively low.Till now,little progress in enhancing their production has been achieved through conventional culture,breeding and other technologies.Therefore,it is necessary to fully learn the molecular regulation mechanism of bacterial lipase gene expression,then it is possible to obtain effective strategy for genetical manipulation.Pseudomonas protegens Pf-5 is a good antibacterial strain.Nowadays,scholars have been paying more and more attention to Pf-5.In spite of this,the study on the lipases of P.protegens Pf-5 is still very limited.Therefore,in this study,we have taken P.protegens Pf-5 as the research object,towards the regulation mechanism of whose lipase LipA expression we have carried out a series of studies to understand the regulatory network of Pseudomonas lipase gene expression so as to establish a solid theoretical basis for the construction of highly effictive lipase genetical engineering strain.The main work and innovations of this study are summarized below:1.The screening method of lipase lip A regulatory genes was successfully constructed in P.rotegens Pf-5.Taking gfmut2 as the reporter gene,we used the method of random insertion mutagenesis by transposon pRL27 to screen the regulation genes of lipA and established a capacity of about 15,000 strains of the transposon insertion mutant library.A total of 4 candidate mutants were screened and their locations were found.Then,a bioinformatics analysis was conducted for the four genes to explore their effects on the expression of lipA,and further predicted their functions and structures.By the screening system,we could easily observe the phenotypic differences of green fluorescence by,the naked eye after adding carbon powder in the LB plates,which greatly facilitated the subsequent screening of mutant strains and resulted in less false positive.The system provided a powerful tool for the fully studying the regulation of lipase expression.Moreover,it could also be used to investigate the regulating mechanism of the expression of other genes in Pseudomonas,The method established in this study provides an effective platform for the study of following-up gene expression regulation mechanism.2.Through the knockout of algR,hfq,rsmY,rsmZ,the over expression of algR,hfq,rsmY,rsmZ,the fusion expression of lipA '-'lacZ and lipA-lacZ,qRT-PCR analysis,EMSA and lipase activity assay,for the first time,it was proved that AlgR could activate lipA by directly binding with the promoter region of rsmZ,and it mainly functioned at transcription level.While Hfq was directly combined with rsmYto affect the translation of lipA by maintaining the stability of rsmY.It was also for the first time to learn that AlgR and Hfq could influence the expression of lipA in Pf-5.Furthermore,whether AlgR or Hfq,all play their roles via sRNAs and Rsm system.3.Through the knockout of rsmY,rsmZ,the fusion expression of lipA'-'lacZ and lipA-lacZ,qRT-PCR analysis,the fusion expression of rsmA'-'lacZ and rsmE'-'lacZ,the analysis of the effects of rsmY and rsmZ on the expression of lipase lipA,the effects of rsmYand rsmZ on the expression of rsmA and rsmE were explored.It was found that rsmY and rsmZ regulated the expression of lipA mainly through rsmE.4.The above results show that,in P.protegens Pf-5,the expression of lipA is regulated by multiple genes.AlgR is through the pathway of AlgR-rsmZ-rsmE-lipA and Hfq is through the pathway of Hfq-rsmY-rsmE-lipA to regulate of lipase lipA expression.The study also shows that both AlgR and Hfq play their regulatory roles via Rsm system.These results are of great significance to understand the mechanism of bacterial lipase expression and provide new basis and clues for the construction of highly effective lipase genentical engineering strains in the future.