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

Phenazine-1-carboxamide (PCN) produced by multifarious 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 strain PA1201 contains two reiterated gene clusters, and , for phenazine-1-carboxylic acid (PCA) biosynthesis; PCA is further converted into PCN by this strain using a functional -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 open reading frame (ORF) and promoter region were investigated and reannotated. promoter P 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- fusion analyses, a strong quorum sensing (QS)- and thermo-regulated promoter P was identified and characterized. The activity of P is approximately 1% of P in PA1201. After three rounds of promoter editing and swapping by P, 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.