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Engineering and systems-level analysis of Pseudomonas chlororaphis for production of phenazine-1-carboxamide using glycerol as the cost-effective carbon source.

ABSTRACT: Glycerol, an inevitable byproduct of biodiesel, has become an attractive feedstock for the production of value-added chemicals due to its availability and low price. Pseudomonas chlororaphis HT66 can use glycerol to synthesize phenazine-1-carboxamide (PCN), a phenazine derivative, which is strongly antagonistic to fungal phytopathogens. A systematic understanding of underlying mechanisms for the PCN overproduction will be important for the further improvement and industrialization.We constructed a PCN-overproducing strain (HT66LSP) through knocking out three negative regulatory genes, lon, parS, and prsA in HT66. The strain HT66LSP produced 4.10 g/L of PCN with a yield of 0.23 (g/g) from glycerol, which was of the highest titer and the yield obtained among PCN-producing strains. We studied gene expression, metabolomics, and dynamic 13C tracer in HT66 and HT66LSP. In response to the phenotype changes, the transcript levels of phz biosynthetic genes, which are responsible for PCN biosynthesis, were all upregulated in HT66LSP. Central carbon was rerouted to the shikimate pathway, which was shown by the modulation of specific genes involved in the lower glycolysis, the TCA cycle, and the shikimate pathway, as well as changes in abundances of intracellular metabolites and flux distribution to increase the precursor availability for PCN biosynthesis. Moreover, dynamic 13C-labeling experiments revealed that the presence of metabolite channeling of 3-phosphoglyceric acid to phosphoenolpyruvate and shikimate to trans-2,3-dihydro-3-hydroxyanthranilic acid in HT66LSP could enable high-yielding synthesis of PCN.The integrated analysis of gene expression, metabolomics, and dynamic 13C tracer enabled us to gain a more in-depth insight into complex mechanisms for the PCN overproduction. This study provides important basis for further engineering P. chlororaphis for high PCN production and efficient glycerol conversion.

SUBMITTER: Yao R

PROVIDER: S-EPMC5934903 | biostudies-other | 2018

REPOSITORIES: biostudies-other

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Engineering and systems-level analysis of <i>Pseudomonas chlororaphis</i> for production of phenazine-1-carboxamide using glycerol as the cost-effective carbon source.

Yao Ruilian R Pan Keli K Peng Huasong H Feng Lei L Hu Hongbo H Zhang Xuehong X

Biotechnology for biofuels 20180504

<h4>Background</h4>Glycerol, an inevitable byproduct of biodiesel, has become an attractive feedstock for the production of value-added chemicals due to its availability and low price. <i>Pseudomonas chlororaphis</i> HT66 can use glycerol to synthesize phenazine-1-carboxamide (PCN), a phenazine derivative, which is strongly antagonistic to fungal phytopathogens. A systematic understanding of underlying mechanisms for the PCN overproduction will be important for the further improvement and indust ...[more]

PMID: 29755589

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Project description:BackgroundPhenazine-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.ResultsA 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.ConclusionIn 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.

Project description:The recently characterized strain Pseudomonas orientalis F9, an isolate from apple flowers in a Swiss orchard, exhibits antagonistic traits against phytopathogens. At high colonization densities, it exhibits phytotoxicity against apple flowers. P. orientalis F9 harbors biosynthesis genes for the siderophore pyoverdine as well as for the antibiotics safracin and phenazine. To elucidate the role of the three compounds in biocontrol, we screened a large random knockout library of P. orientalis F9 strains for lack of pyoverdine production or in vitro antagonism. Transposon mutants that lacked the ability for fluorescence carried transposons in pyoverdine production genes. Mutants unable to antagonize Erwinia amylovora in an in vitro double-layer assay carried transposon insertions in the safracin gene cluster. As no phenazine transposon mutant could be identified using the chosen selection criteria, we constructed a site-directed deletion mutant. Pyoverdine-, safracin-, and phenazine mutants were tested for their abilities to counteract the fire blight pathogen Erwinia amylovoraex vivo on apple flowers or the soilborne pathogen Pythium ultimumin vivo in a soil microcosm. In contrast to some in vitro assays, ex vivo and in vivo assays did not reveal significant differences between parental and mutant strains in their antagonistic activities. This suggests that, ex vivo and in vivo, other factors, such as competition for resources or space, are more important than the tested antibiotics or pyoverdine for successful antagonism of P. orientalis F9 against phytopathogens in the performed assays.IMPORTANCEPseudomonas orientalis F9 is an antagonist of the economically important phytopathogen Erwinia amylovora, the causal agent of fire blight in pomme fruit. On King's B medium, P. orientalis F9 produces a pyoverdine siderophore and the antibiotic safracin. P. orientalis F9 transposon mutants lacking these factors fail to antagonize E. amylovora, depending on the in vitro assay. On isolated flowers and in soil microcosms, however, pyoverdine, safracin, and phenazine mutants control phytopathogens as clearly as their parental strains.

Dataset Information

Engineering and systems-level analysis of Pseudomonas chlororaphis for production of phenazine-1-carboxamide using glycerol as the cost-effective carbon source.

Publications

Engineering and systems-level analysis of <i>Pseudomonas chlororaphis</i> for production of phenazine-1-carboxamide using glycerol as the cost-effective carbon source.

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