Background: SomePseudomonasstrains function as predominant plant growth-promoting rhizobacteria (PGPR). Within this group,Pseudomonas chlororaphisandPseudomonas fluorescensare non-pathogenic biocontrol agents, and somePseudomonas aeruginosaandPseudomonas stutzeristrains are PGPR.P.chlororaphisGP72 is a plant growth-promoting rhizobacterium with a fully sequenced genome. We conducted a genomic analysis comparing GP72 with three other pseudomonad PGPR:P.fluorescensPf-5,P.aeruginosaM18, and the nitrogen-fixing strainP.stutzeriA1501. Our aim was to identify the similarities and differences among these strains using a comparative genomic approach to clarify the mechanisms of plant growth-promoting activity.Results: The genome sizes of GP72, Pf-5, M18, and A1501 ranged from 4.6 to 7.1 M, and the number of protein-coding genes varied among the four species. Clusters of Orthologous Groups (COGs) analysis assigned functions to predicted proteins. The COGs distributions were similar among the four species. However, the percentage of genes encoding transposases and their inactivated derivatives (COG L) was 1.33% of the total genes with COGs classifications in A1501, 0.21% in GP72, 0.02% in Pf-5, and 0.11% in M18. A phylogenetic analysis indicated that GP72 and Pf-5 were the most closely related strains, consistent with the genome alignment results. Comparisons of predicted coding sequences (CDSs) between GP72 and Pf-5 revealed 3544 conserved genes. There were fewer conserved genes when GP72 CDSs were compared with those of A1501 and M18. Comparisons among the fourPseudomonasspecies revealed 603 conserved genes in GP72, illustrating common plant growth-promoting traits shared among these PGPR. Conserved genes were related to catabolism, transport of plant-derived compounds, stress resistance, and rhizosphere colonization. Some strain-specific CDSs were related to different kinds of biocontrol activities or plant growth promotion. The GP72 genome contained thecusoperon (related to heavy metal resistance) and a gene cluster involved in type IV pilus biosynthesis, which confers adhesion ability.Conclusions: Comparative genomic analysis of four representative PGPR revealed some conserved regions, indicating common characteristics (metabolism of plant-derived compounds, heavy metal resistance, and rhizosphere colonization) among these pseudomonad PGPR. Genomic regions specific to each strain provide clues to its lifestyle, ecological adaptation, and physiological role in the rhizosphere.

The biocontrol strain Pseudomonas chlororaphis GP72 isolated from the green pepper rhizosphere synthesizes three antifungal phenazine compounds, 2-Hydroxyphenazine (2-OH-PHZ), 2-hydroxy-phenazine-1-carboxylic acid (2-OH-PCA) and phenazine-1-carboxylic acid (PCA). PCA has been a commercialized antifungal pesticide registered as “Shenqinmycin” in China since 2011. It is found that 2-OH-PHZ shows stronger fungistatic and bacteriostatic activity to some pathogens than PCA. 2-OH-PHZ could be developed as a potential antifungal pesticide. But the yield of 2-OH-PHZ generally is quite low, such as P. chlororaphis GP72, the production of 2-OH-PHZ by the wide-type strain is only 4.5 mg/L, it is necessary to enhance the yield of 2-OH-PHZ for its application in agriculture. Different strategies were used to improve the yield of 2-OH-PHZ: knocking out the negative regulatory genes, enhancing the shikimate pathway, deleting the competing pathways of 2-OH-PHZ synthesis based on chorismate, and improving the activity of PhzO which catalyzes the conversion of PCA to 2-OH-PHZ, although the last two strategies did not give us satisfactory results. In this study, four negative regulatory genes (pykF, rpeA, rsmE and lon) were firstly knocked out of the strain GP72 genome stepwise. The yield of 2-OH-PHZ improved more than 60 folds and increased from 4.5 to about 300 mg/L. Then six key genes (ppsA, tktA, phzC, aroB, aroD and aroE) selected from the gluconeogenesis, pentose phosphate and shikimate pathways which used to enhance the shikimate pathway were overexpressed to improve the production of 2-OH-PHZ. At last a genetically engineered strain that increased the 2-OH-PHZ production by 99-fold to 450.4 mg/L was obtained. The 2-OH-PHZ production of P. chlororaphis GP72 was greatly improved through disruption of four negative regulatory genes and overexpression of six key genes, and it is shown that P. chlororaphis GP72 could be modified as a potential cell factory to produce 2-OH-PHZ and other phenazine biopesticides by genetic and metabolic engineering.

Pseudomonas chlororaphis HT66, a plant growth-promoting rhizobacterium that produces phenazine-1-carboxamide with high yield, was compared with three genomic sequenced P. chlororaphis strains, GP72, 30-84 and O6. The genome sizes of four strains vary from 6.66 to 7.30 Mb. Comparisons of predicted coding sequences indicated 4833 conserved genes in 5869-6455 protein-encoding genes. Phylogenetic analysis showed that the four strains are closely related to each other. Its competitive colonization indicates that P. chlororaphis can adapt well to its environment. No virulence or virulence-related factor was found in P. chlororaphis. All of the four strains could synthesize antimicrobial metabolites including different phenazines and insecticidal protein FitD. Some genes related to the regulation of phenazine biosynthesis were detected among the four strains. It was shown that P. chlororaphis is a safe PGPR in agricultural application and could also be used to produce some phenazine antibiotics with high-yield.

Phenazine-1-carboxamide (PCN), a phenazine derivative, is strongly antagonistic to fungal phytopathogens. The high PCN biocontrol activity fascinated researcher’s attention in isolating and identifying novel bacterial strains combined with engineering strategies to target PCN as a lead molecule. The chemical route for phenazines biosynthesis employs toxic chemicals and display low productivities, require harsh reaction conditions, and generate toxic by-products. Phenazine biosynthesis using some natural phenazine-producers represent remarkable advantages of non-toxicity and possibly high yield in environmentally-friendlier settings. A biocontrol bacterium with antagonistic activity towards fungal plant pathogens, designated as strain HT66, was isolated from the rice rhizosphere. The strain HT66 was identified as Pseudomonas chlororaphis based on the colony morphology, gas chromatography of cellular fatty acids and 16S rDNA sequence analysis. The secondary metabolite produced by HT66 strain was purified and identified as PCN through mass spectrometry, and 1H, 13C nuclear magnetic resonance spectrum. The yield of PCN by wild-type strain HT66 was 424.87 mg/L at 24 h. The inactivation of psrA and rpeA increased PCN production by 1.66- and 3.06-fold, respectively, which suggests that psrA and rpeA are PCN biosynthesis repressors. qRT-PCR analysis showed that the expression of phzI, phzR, and phzE was markedly increased in the psrA and rpeA double mutant than in psrA or rpeA mutant. However, the transcription level of rpeA and rpeB in strain HT66ΔpsrA increased by 3.52- and 11.58-folds, respectively. The reduced psrA expression in HT66ΔrpeA strain evidenced a complex regulation mechanism for PCN production in HT66. In conclusion, the results evidence that P. chlororaphis HT66 could be modified as a potential cell factory for industrial-scale biosynthesis of PCN and other phenazine derivatives by metabolic engineering strategies.

The current chassis organisms or various types of cell factories have considerable advantages and disadvantages. Therefore, it is necessary to develop various chassis for an efficient production of different bioproducts from renewable resources. In this context, synthetic biology offers unique potentialities to produce value-added products of interests. Microbial genome reduction and modification are important strategies for constructing cellular chassis and cell factories. Many genome-reduced strains from Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum and Streptomyces, have been widely used for the production of amino acids, organic acids, and some enzymes. Some Pseudomonas strains could serve as good candidates for ideal chassis cells since they grow fast and can produce many valuable metabolites with low nutritional requirements and strong environmental adaptability. Pseudomonas chlororaphis GP72 is a non-pathogenic plant growth-promoting rhizobacterium that possesses capacities of tolerating various environmental stresses and synthesizing many kinds of bioactive compounds with high yield. These include phenazine-1-carboxylic acid (PCA) and 2-hydroxyphenazine (2-OH-PHZ), which exhibit strong bacteriostatic and antifungal activity toward some microbial pathogens. We depleted 685 kb (10.3% of the genomic sequence) from the chromosome of P. chlororaphis GP72(rpeA-) by a markerless deletion method, which included five secondary metabolic gene clusters and 17 strain-specific regions (525 non-essential genes). Then we characterized the 22 multiple-deletion series (MDS) strains. Growth characteristics, production of phenazines and morphologies were changed greatly in mutants with large-fragment deletions. Some of the genome-reduced P. chlororaphis mutants exhibited more productivity than the parental strain GP72(rpeA-). For example, strain MDS22 had 4.4 times higher production of 2-OH-PHZ (99.1 mg/L) than strain GP72(rpeA-), and the specific 2-OH-PHZ production rate (mmol/g/h) increased 11.5-fold. Also and MDS10 had the highest phenazine production (852.0 mg/L) among all the studied strains with a relatively high specific total phenazine production rate (0.0056 g/g/h). In conclusion, P. chlororaphis strains with reduced genome performed better in production of secondary metabolites than the parent strain. The newly developed mutants can be used for the further genetic manipulation to construct chassis cells with the less complex metabolic network, better regulation and more efficient productivity for diverse biotechnological applications.

Arbutin is a plant-derived glycoside with potential antioxidant, antibacterial and anti-inflammatory activities. Currently, it is mainly produced by plant extraction or enzymatic processes, which suffers from expensive processing cost and low product yield. Metabolic engineering of microbes is an increasingly powerful method for the high-level production of valuable biologicals. Since Pseudomonas chlororaphis has been widely engineered as a phenazine-producing platform organism due to its well-characterized genetics and physiology, and faster growth rate using glycerol as a renewable carbon source, it can also be engineered as the cell factory using strong shikimate pathway on the basis of synthetic biology. In this work, a plasmid-free biosynthetic pathway was constructed in P. chlororaphis P3 for elevated biosynthesis of arbutin from sustainable carbon sources. The arbutin biosynthetic pathway was expressed under the native promoter Pphz using chromosomal integration. Instead of being plasmid and inducer dependent, the metabolic engineering approach used to fine-tune the biosynthetic pathway significantly enhanced the arbutin production with a 22.4-fold increase. On the basis of medium factor optimization and mixed fed-batch fermentation of glucose and 4-hydroxybenzoic acid, the engineered P. chlororaphis P3-Ar5 strain led to the highest arbutin production of 6.79 g/L with the productivity of 0.094 g/L/h, with a 54-fold improvement over the initial strain. The results suggested that the construction of plasmid-free synthetic pathway displays a high potential for improved biosynthesis of arbutin and other shikimate pathway derived biologicals in P. chlororaphis.