Identification, synthesis and regulatory function of the N-acylated homoserine lactone signals produced by Pseudomonas chlororaphis HT66

Abstract Background Pseudomonas chlororaphis HT66 isolated from the rice rhizosphere is an important plant growth-promoting rhizobacteria that produce phenazine-1-carboxamide (PCN) in high yield. Phenazine production is regulated by a quorum sensing (QS) system that involves the N-acylated homoserine lactones (AHLs)—a prevalent type of QS molecule. Results Three QS signals were detected by thin layer chromatography (TLC) and high-performance liquid chromatography–mass spectrometry (HPLC–MS/MS), which identified to be N-(3-hydroxy hexanoyl)-l-homoserine lactone (3-OH-C6-HSL), N-(3-hydroxy octanoyl)-l-homoserine lactone (3-OH-C8-HSL) and N-(3-hydroxy decanoyl)-l-homoserine lactone (3-OH-C10-HSL). The signal types and methods of synthesis were different from that in other phenazine-producing Pseudomonas strains. By non-scar deletion and heterologous expression techniques, the biosynthesis of the AHL-signals was confirmed to be only catalyzed by PhzI, while other AHLs synthases i.e., CsaI and HdtS were not involved in strain HT66. In comparison to wild-type HT66, PCN production was 2.3-folds improved by over-expression of phzI, however, phzI or phzR mutant did not produce PCN. The cell growth of HT66∆phzI mutant was significantly decreased, and the biofilm formation in phzI or phzR inactivated strains of HT66 decreased to various extents. Conclusion In conclusion, the results demonstrate that PhzI–PhzR system plays a critical role in numerous biological processes including phenazine production

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

Abstract Background 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. Results 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. Conclusions 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

Comparative genomic analysis and phenazine production of Pseudomonas chlororaphis, a plant growth-promoting rhizobacterium

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

Enhanced biosynthesis of phenazine-1-carboxamide by engineered Pseudomonas chlororaphis HT66

Abstract Background 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. Results 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. Conclusion 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

Research on Optical and Mechanical Compatible Design Technology of Multilayer Films

The stress’s accumulation will critically affect optical device stability, leading to components’ deviation and film peeling. Stress control techniques based on strain cancelation cannot precisely match film stress, and it is difficult to reduce the total stress of multilayer film to ultra-low levels. Existing software for film system design is mainly based on 2D design concepts such as wavelength-spectrum and does not consider 3D design concepts such as wavelength-spectrum-stress, but the stress parameter plays an essential role in film performance, and it is necessary to use it as an optimization index for film design. In this paper, we introduce mechanical synchronization design into film system design. The multilayer film’s optimal structure is precisely matched by the genetic algorithm, and an ultra-low stress and wide-spectrum optical film element is developed under the dual requirements of optical performance and mechanical performance. This technique incorporates computer-aided design and automatically seeks an optimal combination of layered film nanostructures through self-compiled optical and mechanical compatible design software. While ensuring invariant optical properties, it can effectively reduce the total stress of multilayer film. This can provide a new idea for film stress control

Research on Optical and Mechanical Compatible Design Technology of Multilayer Films

The stress’s accumulation will critically affect optical device stability, leading to components’ deviation and film peeling. Stress control techniques based on strain cancelation cannot precisely match film stress, and it is difficult to reduce the total stress of multilayer film to ultra-low levels. Existing software for film system design is mainly based on 2D design concepts such as wavelength-spectrum and does not consider 3D design concepts such as wavelength-spectrum-stress, but the stress parameter plays an essential role in film performance, and it is necessary to use it as an optimization index for film design. In this paper, we introduce mechanical synchronization design into film system design. The multilayer film’s optimal structure is precisely matched by the genetic algorithm, and an ultra-low stress and wide-spectrum optical film element is developed under the dual requirements of optical performance and mechanical performance. This technique incorporates computer-aided design and automatically seeks an optimal combination of layered film nanostructures through self-compiled optical and mechanical compatible design software. While ensuring invariant optical properties, it can effectively reduce the total stress of multilayer film. This can provide a new idea for film stress control