RecN: A tunable switch for DNA repair choice and stress tolerance in Zymomonas mobilis.

Abstract

Engineering polyploid industrial microorganisms is hindered by their intrinsic capacity to repair induced mutations, limiting the efficiency of genome editing and directed evolution. Using the ethanologenic bacterium Zymomonas mobilis- a polyploid alphaproteobacterium that exhibits exceptionally efficient microhomology-mediated end joining (MMEJ)- we demonstrate that RecN is essential for MMEJ and homologous recombination (HR) in vivo. Strikingly, a specialized mutant RecN-K35A, with strongly impaired ATP hydrolysis, specifically blocks MMEJ while leaving HR fully intact. The physiological importance of RecN-mediated MMEJ is highlighted by the cell elongation phenotype and increased stress sensitivity observed in the RecN-K35A mutant. Based on this connection, we developed a high-phosphorus cultivation strategy that increases cellular DNA content and significantly enhanced ethanol fermentation efficiency under industrial stress conditions. In summary, this work defines RecN as a key ATP-dependent effector of MMEJ and positions it as a potential engineering target for modulating DNA repair pathway choice and stress tolerance in Z. mobilis. Moreover, the essential role of RecN in both HR and MMEJ suggests that RecN-deficient polyploid strains could facilitate directed evolution by preventing repair of newly introduced mutations, offering a new strategy for strain improvement.