Adaptive evolution and reverse engineering to explore the low pH tolerance mechanisms of Streptomyces albulus

Streptomyces albulus is well-known as a cell factory for producing ε-poly-L-lysine (ε-PL), but its ability to produce effectively requires an environment with a pH of about 4.0. Unfortunately, prolonged exposure to low pH environment compromises the cellular integrity of S. albulus, leading to a decrease in the efficiency of ε-PL biosynthesis. To enhance the low pH tolerance of S. albulus and investigate its low pH tolerance mechanisms, we employed adaptive laboratory evolution (ALE) technology to evolve the S. albulus GS114 strain by progressively lowering the environmental pH. This process ultimately yielded the mutant strain ALE3.6, which exhibited significantly improved low pH tolerance at pH 3.6 and achieved a 37.9% increase in ε-PL production compared to the parental GS114 strain under the optimal fermentation condition. The physiological evaluation of the mutant strain ALE3.6 indicated a pronounced enhancement in the integrity of its cell membrane and cell wall under low pH conditions. To identify the key genes involved in low pH tolerance, we employed whole-genome resequencing and quantitative real-time PCR, which pinpointed desA, gatD, and mamU as critical contributors. We further validated the roles of these genes through reverse engineering, which improved both low pH tolerance and ε-PL production efficiency. Finally, we elucidated the response mechanisms of the S. albulus cell membrane and cell wall under low pH stress. This study enhances the understanding of low pH tolerance in the Streptomyces species, particularly regarding the production of valuable biochemical products under challenging environmental conditions.IMPORTANCEIn this study, we improved the viability and ε-poly-L-lysine production efficiency of Streptomyces albulus at low pH by staged adaptive laboratory evolution while simplifying the previously studied fed-batch fermentation strategy. We identified key genes associated with the mutant strains' cell membrane and cell wall phenotypes by utilizing whole-genome resequencing and reverse engineering. Subsequently, we validated the cell membrane and cell wall response mechanisms in S. albulus under low pH conditions.