Comparative Genomic Analysis Of Plant Growth-promoting Rhizobacteria In Pseudomonas And Construction Of Dominant Genome-reduced P. Chlororaphis Strains

With the rapid development of synthetic biology,construction of biological chassis for the use of biotechnology is quite promising.And microbial genome reduction and modification are important strategies for constructing cellular chassis.Some strains of the genus Pseudomonas exhibit obvious metabolic and physiological diversities,so they are pre-endowed with remarkable capacity to endure both endogenous and exogenous stresses,strong ability to synthesize many kinds of bioactive compounds and adapt to different physicochemical and nutritional niches.Meanwhile,there is more and more genomic information of pseudomonads,which makes their genetic background more clear.Therefore,environmental Pseudomonas strains can serve as model organisms to implement genome reduction and genetic modification.Then the ideal biological chassis can be used for the production of important chemicals and medicines in industrial,argricultural and medical areas,which are demanded by current and future synthetic biology and biotechnological needs.P.chlororaphis GP72 isolated from green pepper rhizosphere is a biocontrol agent mainly due to the production of phenazines and other biocontrol related secondary metabolites.In this dissertation,we used the sequence of P.chlororaphis GP72 as a reference genome to carry out comparative genomic analysis.These results not only will be utilized to improve the biocontrol activity.but also provide information for engineering genome-reduced strains with high production of phenazine compounds.Based on the requirenments of constructing chassis cell,comparative genomics approach was usd to identify genes related to plant growth-promoting activity,essential genes,and important functional and regulatory genes of P.chlororaphis.Then to implement the sequential simplification of the genome,a markerless deletion method was applied to reduce the genome.Finally,the multiple-deletion series(MDS)strains were assessed from multiple aspects.So this work provided detailed theory and optimized technical support for the further synthetic biology research and industrial production.The details and key conclusions are described as follows:Firstly,the genome of strain GP72 was sequenced via the high throughput sequencing technology,which was the first genome sequence of the P.chlororaphis group.The genome consisted of a single circular chromosome of 6,663,241 base pairs with a G+C content of 63.1%.Genome annotation revealed the genetic basis for the biocontrol activity of this strain.Secondly,comparative genomic analysis of four representative plant growth-promoting rhizobacteria(PGPR)in Pseudomonas was conducted to systematically clarify the mechanisms of plant growth-promoting activity.Those included two non-pathogenic biocontrol agents P.fluorescens Pf-5 and P.chlororaphis GP72,a P.aeruginosa strain M18 isolated from sweet melon rhizosphere with biocontrol activity,and the nitrogen-fixing bacteria P.stutzeri A1501.Comparisons among the four Pseudomonas species revealed 603 conserved genes in GP72,illustrating common plant growth-promoting traits shared among these PGPR.While the strain-specific characters in strain GP72 included synthesis of different kinds of secondary metabolites,versatile heavy metal resistance(cus operon)and the additional pilus biosynthesis system(tad operon).Those may confer the strain with high adaptability in the environment and specific biological function.Thirdly,some preliminary findings were provided on the essential gene set of P.chlororaphis.For each homology search,BLAST was run against sets of essential genes from different gram-negative bacteria.We speculated that there were at least 215 essential genes in the genome of strain GP72,which layed the foundation for work of the genome reduction and modification.Fourthly,comparative genomic approach together with top-down strategy was used to construct dominant genome-simplified strains in Pseudomonas.We applied a homologous recombination method mediated by the suicide plasmid to sequentially delete 22large-scale genome regions,resulting in a 10%reduced genome size.Among the 22 MDS strains,strain MDS22 with the minimal genome had 4.4 times higher production of2-OH-PHZ(99.1 mg/L)and showed slower growth than that of the parental strain.While mutant MDS10 preserved good growth profiles and exhibited highest phenazine production(852.0 mg/L)among all the studied strains.Further transcriptome analysis between high phenazine-producing mutant MDS10 and the parental strain showed that the higher production of phenazines may relate to its secretion system,and the ferric ion made great contribution to the growth and phenazine production.In conclusion,from the perspective of synthetic biology,we used the principle and method of comparative genomics to construct genome-reduced P.chlororaphis strains with enhanced phenazine production,Therefore,this study may provide theoretical and technical basis for designing ideal chassis cells to realize the synthetic biology research and biotechnology application.