Abstract

The take-all disease of wheat poses a significant threat to global food security, underscoring the need for effective biocontrol agents. 2-Hydroxyphenazine (2-OH-PHZ) shows superior antifungal activity against the take-all disease of wheat pathogen over the commercial biopesticide phenazine-1-carboxylic acid (PCA). However, the biosynthetic production of 2-OH-PHZ is constrained by three critical limitations: the low hydroxylation efficiency of the flavin-dependent monooxygenase PhzO, inadequate intracellular supply of the precursor PCA, and the long fermentation process. To systematically address these interconnected challenges, we developed and implemented a Cofactor-Pathway-Process (CPP) engineering strategy in Pseudomonas chlororaphis LX24. First, cofactor engineering was employed to enhance PhzO activity by improving the supply of FADH 2 and NADPH, which increased the hydroxylation efficiency from 22% to over 85%. Subsequently, pathway optimization was applied to overcome the precursor limitation by enhancing phenazine biosynthesis, which resulted in a 2.18-fold increase in 2-OH-PHZ accumulation to 988.25 mg/L. Combined with medium optimization and phzO overexpression, the titer of 2-OH-PHZ reached 2291.56 mg/L in shake flasks and 2663.12 mg/L in a 5-L bioreactor within 144 h, which is the highest production reported to date. Finally, a two-stage temperature-shift fermentation process was introduced to accelerate the decarboxylation of the intermediate 2-hydroxyphenazine-1-carboxylic acid, reducing the total fermentation time by 36 h and significantly improving process efficiency and sustainability. In summary, the integrated CPP strategy successfully overcomes multiple bottlenecks in 2-OH-PHZ biosynthesis, culminating in record-high productivity and underscoring its value as a versatile blueprint for the sustainable bioproduction of phenazine derivatives and