Chlororaphin

Pseudomonas chlororaphis has been demonstrated as a valuable source of antimicrobial metabolites for plant disease biocontrol and biopesticide development. Although phenazine-1-carboxylic acid (PCA) secreted by P. chlororaphis has been commercialized as an antifungal biopesticide, it shows poor antibacterial activity. Questiomycin A, with versatile antibacterial activities, is mainly discovered in some well-known phenazine-producing strains but not in Pseudomonas. Its low titer hinders practical applications. In this work, a metabolite was first identified as Questiomycin A in P. chlororaphis-derived strain HT66ΔphzBΔNat. Subsequently, Questiomycin A has been elucidated to share the same biosynthesis process with PCA by gene deletion and in vitro assays. Through rational metabolic engineering, heterologous phenoxazinone synthase introduction, and medium optimization, the titer reached 589.78 mg/L in P. chlororaphis, the highest production reported to date. This work contributes to a better understanding of Questiomycin A biosynthesis and demonstrates a promising approach to developing a new antibacterial biopesticide in Pseudomonas.

Quorum sensing is a population density-dependent gene regulation mechanism. N-Acyl-l-homoserine lactone (AHL) has been identified as a signal compound in quorum sensing in gram-negative bacteria. Phenazine derivatives are bacterial secondary metabolites known for their broad-spectrum antifungal activity. Pseudomonas chlororaphis has been demonstrated to be a biocontrol strain, and most of its species can produce phenazine derivatives under AHL-mediated quorum sensing. Although P. chlororaphis is divided into four subspecies, the relationship between phenazine production and quorum sensing has not been investigated in two of the subspecies, P. chlororaphis subsp. chlororaphis and piscium. Two luxI/luxR homolog gene sets, phzI and phzR and csaI and csaR, were found in the complete genome sequences of the type strains of P. chlororaphis subsp. chlororaphis JCM 2778T and P. chlororaphis subsp. piscium DSM 21509T. Two major AHLs, N-(3-hydroxyhexanoyl)-l-homoserine lactone and N-(3-hydroxyoctanoyl)-l-homoserine lactone, were detected in JCM 2778 and DSM 21509 samples. PhzI synthesized all AHLs; however, CsaI could not perform AHL biosynthesis in JCM 2778 and DSM 21509. In both strains, disruption of the phzI caused complete disappearance of phenazine-1-carboxylic acid (PCA) and phenazine-1-carboxamide (PCN) production; however, disruption of csaI did not induce significant changes in PCA and PCN production. Phenazine derivatives produced by JCM 2778 and DSM 21509 under quorum sensing are crucial for the control of the plant pathogenic fungi, Rhizoctonia solani, Fusarium graminearum, and Fusarium nirenbergiae. These results demonstrated that PhzI/PhzR quorum-sensing system play an important role in production of phenazine derivatives and biocontrol activity.

The biocontrol rhizobacterium Pseudomonas chlororaphis is one of the bacterial species of the P. fluorescens group where insecticide fit genes have been found. Fit toxin, supported with other antimicrobial compounds, gives the bacterial the ability to repel and to fight against eukaryotic organisms, such as nematodes and insect larvae, thus protecting the plant host and itself. Pseudomonas chlororaphis PCL1606 is an antagonistic rhizobacterium isolated from avocado roots and show efficient biocontrol against fungal soil-borne disease. The main antimicrobial compound produced by P. chlororaphis PCL606 is 2-hexyl-5-propyl resorcinol (HPR), which plays a crucial role in effective biocontrol against fungal pathogens. Further analysis of the P. chlororaphis PCL1606 genome showed the presence of hydrogen cyanide (HCN), pyrrolnitrin (PRN), and homologous fit genes. To test the insecticidal activity and to determine the bases for such activity, single and double mutants on the biosynthetic genes of these four compounds were tested in a Galleria mellonella larval model using inoculation by injection. The results revealed that Fit toxin and HPR in combination are involved in the insecticide phenotype of P. chlororaphis PCL1606, and additional compounds such as HCN and PRN could be considered supporting compounds.