Increasing biosensor-based cell selection pressure improves microbial biosynthesis of 4-hydroxybenzoate

Abstract

Biosensors are powerful tools for improving microbial biosynthesis. For example, biosensors can be used to establish feedback control mechanisms to inhibit the growth of low performing cells in a microbial population. In this study, a previously developed biosensor-assisted cell selection system was further engineered to increase the cell selection pressure for improving the bioproduction of 4-hydroxybenzoate (4HB) in E. coli. First, additional sensor protein PobR binding sites were introduced to reduce the binding of PobR controlling the expression of the selection marker gene (antibiotic tetracycline resistance tetA), which stimulated the engineered cells to produce more 4HB to compensate for the tetA expression reduction due to the loss of the PobR binding. Second, the degradation of the PobR sensor protein was promoted using an E. coli ssrA-SspB system to press the cells to make more 4HB. Third, the dosage of antibiotic tetracycline controlling the selection strengths was dynamically increased during the cultivation to align the selection pressure elevation with the cell growth. Our findings show these strategies all effectively improved the 4HB biosynthesis in E. coli and thus demonstrate their potential of wider applications in microbial biosynthesis.