Genomic And Biochemical Study Of The Catabolism Of Aromatic Compounds By Pseudomonas

Polycyclic aromatic hydrocarbons?PAHs?including nitrogen,oxygen,and sulfur heterocycles,pose serious harm to the environment and human health.As the engine of the Earth's carbon cycle,microbial decomposition of these pollutants is essential for processes of bioremediation of contaminated sites.As a kind of non-pathogenic environmentally friendly strain,Pseudomonas putida strains exhibit obvious metabolic properties of aromatic compounds,so they have been regarded as one of the best choices to eliminate the PAHs pollutants.Pseudomonas putida B6-2 is such a naturally occurring super bacterium capable of efficiently and extensively degrading a series PAHs,even dioxins.Strain B6-2 could also simultaneously degrade each of such aromatic polycycles and their derivatives when they were present as mixtures,and tolerate high concentrations of extremely toxic solvents.However,the mechanism of the metabolism of aromatic compounds in strain B6-2 is still scarce.In this dissertation,based on multi-omics data analysis and construction of transposon library,the biochemical,genetic and evolutionary aspects of P.putida are investigated.The main results and key conclusions are given as following:Comprehensive analysis of whole genome,proteomics and transcriptomics of strain B6-2.The complete genome sequence was obtained by 3rd generation sequencing technology,and the genome comprised a circular chromosome DNA of 6,377,291 bp with5,832 coding genes.Through the RAST annotation and BLAST alignment analysis,the location and organization of genes?clusters?related to metabolic network of aromatic compounds in strain B6-2 was analysed.Functional clustering of transcriptome data revealed obvious correlation between gene transcription profiles and different carbon sources,indicating the reliability of transcriptome data.Genes belonging to the subsystems‘biphenyl degradation pathway',‘benzoate degradation pathway',‘catechol branch of beta-ketoadipate pathway'and‘central meta-cleavage pathway of aromatic compound degradation'were significantly up-regulated when cultured in biphenyl medium.The proteomic data were compared and analyzed among different groups,and the numbers of up-regulated proteins belonging to categories O?posttranslational modification,protein turnover,chaperones?,J?translation,ribosomal structure,and biogenesis?,E?amino acid transport and metabolism?,and C?energy production and conversion?were higher than proteins in other categories.In addition,proterins belongs to K?transcription?and U?intracellular trafficking,secretion,and vesicular transport?were presumed to play an important role in B6-2 co-metabolism of non-available carbon sources.Screening of novel genes for the metabolism of aromatic compounds in Pseudomonas.A transposon library was constructed,and 9 mutants were screened and the insertion site of the transposon were identified.Based on the alignment analysis of correspongding interrupted genes,it was found that 7 mutant strains belonged to auxotrophic mutations,and the disrupted genes of the other two mutant strains C4 and N25 may be involved in biphenyl metabolism.Total 19 target genes related to chemotaxis,redox,outer membrane-protein and transport were knocked out in strain B6-2.The biphenyl metabolic capacity of mutants B6-2?2426&2429,B6-2?2445 and B6-2?4751were affected by the deletion of the corresponding gene.After 100 generations of cultivation,48%of the strains lost the biphenyl metabolism ability when subcultured in LB medium.By constract,the biphenyl metabolic gene cluster in KF707 was quite stable.The stability of the B6-2 biphenyl metabolic gene cluster was modetate between the two strains KF715 and KF707.No phenomenon of the self-mobilization of the bph was found by horizontal gene transfer.The transcriptional analysis of bph in Pseudomonas.The GntR family gene bphR1was located upstream of bph gene cluster,and the mutant strain with deletion of bphR1gene did not lose the biphenyl metabolism ability.The LysR family gene bphR2 was located upstream of sal gene cluster,and the mutants containing the deletion of bphR2gene failed to metabolize salicylate.RT-qPCR data speculated that BphR1 may be an inducible activator of bph gene cluster,but BphR1 was essential for biphenyl catabolism.The transcription level of gene salA in B6-2dbphR2 was very low when cultured in salicylate culture,indicating that BphR2 significantly activated the transcription of the downstream sal gene cluster.The transcription strart sites of bphR1,bphA1,bphR2 and salA were identifide by the 5'RACE method.Conversion of nitrogen heterocyclic contaminants to high value compouds by Pseudomonas.Using whole-cell biocatalyst of engineered Pseudomonas,the preparation of high-value intermediate 3-succinylpyridine was performed by using low-grade tobacco leaves as the raw material.The efficient and specific biocatalyst could be easily prepared using the reusable whole cells of Pseudomonas,and the catalytic activity of the whole-cell biocatalyst can still retain over 80%activeity after repeated use for three times.Under the optimal reaction conditions,SP was prepared via batch and fed-batch fermentation to the high oncentration of 9.8 g l^-1.Under the optimal biotransformation process,the isolation yield of crystal compound SP was 54.2%.