Pathway-dependent reactive oxygen species accumulation accelerate genetic instability and drive loss of production

Abstract

Microbial bioproduction usually becomes unstable during prolonged scale-up fermentation because non-producing mutants can outcompete high-producing cells. As ubiquitous byproducts of aerobic metabolism, reactive oxygen species (ROS) can accumulate during production, yet their effects on production stability remain poorly understood. Here, we investigated the relationship among ROS accumulation, mutation rates, and production loss in engineered Escherichia coli . Using a fluorescent ROS-staining dye, we observed elevated ROS levels in E. coli strains engineered to produce betaxanthin or mussel foot protein (MFP), but not limonene, indicating that ROS accumulation depends on the biosynthetic pathway and the chemical properties of the product and its intermediates. We subsequently engineered ROS biosensors by screening six ROS-responsive promoters. Among them, the PgrxA –EGFP biosensor exhibited the most favorable dose–response characteristics and reproduced the ROS patterns detected by fluorescent staining. Fluctuation assays further demonstrated that cells with elevated ROS levels exhibited higher mutation rates, accelerating the acquisition of chromosomal antibiotic resistance. During prolonged fermentation, this resistance reduced the selective pressure for plasmid maintenance, thereby promoting plasmid loss and eventually causing a progressive decline in product titer. Together, these findings establish a mechanistic link among bioproduction-induced ROS accumulation, elevated mutation rates, and production instability, and suggest potential intervention strategies for developing more stable microbial cell factories.