Isolation and characterization of Saccharomyces cerevisiae mutants with ornithine accumulation for value-added craft beer brewing.

Ornithine is a nonproteinogenic amino acid with various health benefits, making it a promising target for functional food development. In this study, we developed the yeast Saccharomyces cerevisiae strains with elevated intracellular ornithine and identified the genetic mutation responsible for this trait. An ornithine-rich mutant was successfully isolated by chemical mutagenesis and selection for resistance to canavanine, a toxic arginine analog. Whole-genome sequencing revealed a heterozygous point mutation in the ARG6 gene encoding N-acetylglutamate kinase, a key enzyme in ornithine biosynthesis. This mutation caused a Gly351Asp substitution located in a conserved linker region between functional domains. Introduction of this substitution, along with introducing alternative amino acids at the same site, consistently increased ornithine levels in various strain backgrounds. Structural modeling suggested that this substitution could affect the enzyme conformation or inter-domain interactions. These results establish a practical nongenetically modified breeding strategy for enhancing ornithine production in yeast, which will facilitate the development of ornithine-enriched fermented beverages such as craft beer. One-sentence summary The ARG6 mutant increases intracellular ornithine and enables value-added craft beer brewing.