Adaptive evolution and reverse engineering to explore the low pH tolerance mechanisms of <i>Streptomyces albulus</i>

<i>Streptomyces albulus</i> 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 <i>S. albulus</i>, leading to a decrease in the efficiency of ε-PL biosynthesis. To enhance the low pH tolerance of <i>S. albulus</i> and investigate its low pH tolerance mechanisms, we employed adaptive laboratory evolution (ALE) technology to evolve the <i>S. albulus</i> 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 <i>desA</i>, <i>gatD</i>, and <i>mamU</i> 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 <i>S. albulus</i> cell membrane and cell wall under low pH stress. This study enhances the understanding of low pH tolerance in the <i>Streptomyces</i> 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 <i>Streptomyces albulus</i> 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 <i>S. albulus</i> under low pH conditions.