ScholarWorks@중앙대학교

초록

Enhancing methionine and protein content in Saccharomyces cerevisiae is essential for its use as single-cell protein. Here, we applied ethionine resistance–mediated adaptive laboratory evolution (ALE) to generate strains with improved resistance to this toxic methionine analog. Stepwise adaptation enabled growth at ethionine concentrations of up to 0.50 mM and yielded strains with progressively higher intracellular methionine levels and improved protein production efficiency. Whole-genome sequencing identified nonsynonymous SNPs in 32 genes, of which nine candidates were functionally validated. CRISPR/Cas9-based editing demonstrated that mutations in MDE1 and JJJ1 directly elevated free methionine levels, whereas most other mutations increased overall protein accumulation. Functional annotations linked these genes to RNA processing, protein degradation, methionine salvage, and amino acid uptake, highlighting RNA processing as a major target for global protein enhancement. These findings reveal that ethionine resistance–mediated ALE induces multifactorial adaptations. They also provide new insights into protein biosynthesis regulation and lay a foundation for future engineering of high-performance yeast strains.

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