Strain Development of Enzymes in Actinomycetes by Single Cell Mutant Selection

Abstract

Actinomycetes are filamentous Gram-positive bacteria that play an important role in soil nutrient cycling due to their robust secretion of hydrolytic enzymes, which facilitate the breakdown of complex environmental polysaccharides. However, the inherent physiological complexity and slow growth rates of these organisms necessitate specialized strain development methodologies to optimize enzyme yields for commercial applications. In this study, five enzymes were selected for parallel strain development in Streptomyces lavendulae. Single cell mutant selection was applied through iterative cycles of mutagenesis, fluorescence‑activated cell sorting, and enzymatic assays. Fluorescence intensity increased throughout strain development cycles, indicating effective enrichment of high-promoter-activity mutants. Enzymatic assays confirmed enhanced enzyme production in several candidate strains, although improvements varied between the five enzymes. The increases in fluorescence levels and enzyme activity demonstrated a positive correlation between fluorescence intensity and protein production, although the correlation was not strictly linear and the improvement varied between enzymes. The varied results highlight the importance of secondary selection methods alongside fluorescence-based selection. Overall, the study shows that SCMS is a promising high-throughput strategy for strain development of actinomycetes, enabling efficient enrichment of high-producing mutants. Further optimization may be required to improve strain robustness and consistency across different enzyme targets.