Identification of a Strong Quorum Sensing- and Thermo-Regulated Promoter for the Biosynthesis of a New Metabolite Pesticide Phenazine-1-carboxamide in Pseudomonas strain PA1201.

Phenazine-1-carboxamide (PCN) produced by multifarious Pseudomonas strains represents a promising candidate as a new metabolite pesticide due to its broad-spectrum antifungal activity and capacity to induce systemic resistance in plants. The rice rhizosphere Pseudomonas strain PA1201 contains two reiterated gene clusters, phz1 and phz2, for phenazine-1-carboxylic acid (PCA) biosynthesis; PCA is further converted into PCN by this strain using a functional phzH-encoding glutamine aminotransferase. However, PCN levels in PA1201 constitute approximately one-fifth of PCA levels and the optimal temperature for PCN synthesis is 28 °C. In this study, the phzH open reading frame (ORF) and promoter region were investigated and reannotated. phzH promoter PphzH was found to be a weak promoter, and PhzH levels were not sufficient to convert all of the native PCA into PCN. Following RNA Seq and promoter-lacZ fusion analyses, a strong quorum sensing (QS)- and thermo-regulated promoter PrhlI was identified and characterized. The activity of PphzH is approximately 1% of PrhlI in PA1201. After three rounds of promoter editing and swapping by PrhlI, a new PCN-overproducing strain UP46 was generated. The optimal fermentation temperature for PCN biosynthesis in UP46 was increased from 28 to 37 °C and the PCN fermentation titer increased 179.5-fold, reaching 14.1 g/L, the highest ever reported.