MOESM2 of Genetic engineering of Pseudomonas chlororaphis GP72 for the enhanced production of 2-Hydroxyphenazine

Additional file 2: Figure S2. Simplified scheme of the steps required in the construction of the BglBric plasmid pBbB5K-aroE-aroD-aroB-phzC-tktA-ppsA

MOESM3 of Genetic engineering of Pseudomonas chlororaphis GP72 for the enhanced production of 2-Hydroxyphenazine

Additional file 3: Table S1. Primers used in this study

Additional file 1 of Nonlinear relationship between viral load and TCT in single/multiple HPV52 infection

Additional file 1: Table S1. HPV 52 LOAD tertile

MOESM3 of Engineering and systems-level analysis of Pseudomonas chlororaphis for production of phenazine-1-carboxamide using glycerol as the cost-effective carbon source

Additional file 3. Primers used in this study

Reaction Kinetics for the Biocatalytic Conversion of Phenazine-1-Carboxylic Acid to 2-Hydroxyphenazine

<div><p>The phenazine derivative 2-hydroxyphenazine (2-OH-PHZ) plays an important role in the biocontrol of plant diseases, and exhibits stronger bacteriostatic and fungistatic activity than phenazine-1-carboxylic acid (PCA) toward some pathogens. PhzO has been shown to be responsible for the conversion of PCA to 2-OH-PHZ, however the kinetics of the reaction have not been systematically studied. Further, the yield of 2-OH-PHZ in fermentation culture is quite low and enhancement in our understanding of the reaction kinetics may contribute to improvements in large-scale, high-yield production of 2-OH-PHZ for biological control and other applications. In this study we confirmed previous reports that free PCA is converted to 2-hydroxy-phenazine-1-carboxylic acid (2-OH-PCA) by the action of a single enzyme PhzO, and particularly demonstrate that this reaction is dependent on NADP(H) and Fe³⁺. Fe³⁺ enhanced the conversion from PCA to 2-OH-PHZ and 28°C was a optimum temperature for the conversion. However, PCA added in excess to the culture inhibited the production of 2-OH-PHZ. 2-OH-PCA was extracted and purified from the broth, and it was confirmed that the decarboxylation of 2-OH-PCA could occur without the involvement of any enzyme. A kinetic analysis of the conversion of 2-OH-PCA to 2-OH-PHZ in the absence of enzyme and under different temperatures and pHs <i>in vitro</i>, revealed that the conversion followed first-order reaction kinetics. In the fermentation, the concentration of 2-OH-PCA increased to about 90 mg/L within a red precipitate fraction, as compared to 37 mg/L within the supernatant. The results of this study elucidate the reaction kinetics involved in the biosynthesis of 2-OH-PHZ and provide insights into <i>in vitro</i> methods to enhance yields of 2-OH-PHZ.</p></div

MOESM1 of Identification, synthesis and regulatory function of the N-acylated homoserine lactone signals produced by Pseudomonas chlororaphis HT66

Additional file 1: Figure S1. a Colonial morphology changes in wild-type HT66, HT66∆phzI and HT66∆phzR strains during 7 days and b Influence of antifungal activity of HT66 and its mutants on the growth of Pythium ultimum. P. ultimum was spotted at the left of the PDA plate, whereas HT66 and its mutants were inoculated on the right side

Additional file 4: of Developing genome-reduced Pseudomonas chlororaphis strains for the production of secondary metabolites

Primers used in this study. (XLSX 14 kb

Complemented mechanism of the conversion of PCA to 2-OH-PHZ: PhzO hydroxylates PCA to 2-OH-PCA in the presence of NADP(H), Fe³⁺ enhanced the activity of PhzO.

<p>Subsequently, the intermediate product 2-OH-PCA spontaneously decarboxylates to form 2-OH-PHZ followed first-order reaction kinetics. The chemical structure of phenazine-1-carboxylic acid (PCA) (a), 2-hydroxy phenazine-1-carboxylic acid (2-OH-PCA) (b) and 2-hydroxyphenazine (2-OH-PHZ) (c). Arrows indicate positive direction of reaction, lines with flat ends indicate negative regulation.</p

The influence of enzymes in cells extracts on the conversion of 2-OH-PCA to 2-OH-PHZ.

1<p>The control group was 30 mg/L 2-OH-PCA in PBS without any enzyme.</p><p>The experiment was performed three times and means±SE of triplicate experiments from same culture are plotted.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p