[P002] Poster

1:00 PM - 1:50 PM JST(4:00 AM - 4:50 AM UTC)

[P01-036] Low-Cost Yeast Extract Production with Improved Umami Intensity via CRISPR-Based Chassis Strain and Glutaminase Complex

○Soojung Cho¹, Gahyun Nam¹, Yeonseo Nam¹ (1. Sungshin women's university (Korea))

Keywords:

Yeast engineering,CRISPR-Cas9,Low-cost medium,Enzyme complex design,Silico Protein engineering

Umami is a defining characteristic of Asian cuisine, and the efficient production of umami ingredients is a key objective in the food industry. Yeast extracts, rich in proteins, nucleotides, and vitamins, are widely utilized as flavor-enhancing agents in seasoning manufacturing. The primary umami component of yeast extract, glutamate, can be increased through glutaminase-mediated enzymatic conversion of glutamine. To this end, this study adopted a strategy of constructing a scaffold-based glutaminase complex to improve the glutamate content and flavor profile of yeast extract. The high cost of the rich medium is a major factor limiting the economic feasibility of large-scale yeast extract production. In this study, we developed and optimized a low-cost minimal medium composed of 10% molasses and 5% corn steep liquor (CSL), utilizing them as carbon and nitrogen sources, respectively. The resulting medium demonstrated yeast growth rates comparable to those of rich medium, at approximately 5% of the rich medium cost. The suitability of minimal medium for culturing both wild-type and chassis yeast strains is currently being validated. To enhance the production of umami-related compounds in Saccharomyces cerevisiae, a CRISPR-Cas9-based metabolic engineering strategy was applied to construct an engineered chassis strain. A pCas plasmid was introduced to generate a Cas9-expressing host strain, and target-specific gRNAs were designed using a gRNA-trp HyB vector. In particular, genes involved in suppressing glutamine production were selected as editing targets. Cas9-induced double-strand breaks at specific genomic loci were repaired by insertion of donor DNA fragments to inactivate target genes, and transformants were selected via antibiotic resistance screening. The engineered yeast was subjected to sequential enzymatic hydrolysis: endo-type peptidase cleaved proteins into polypeptides, followed by exo-type peptidase to generate free amino acids. Glutamine was subsequently converted to glutamic acid by glutaminase, and conversion efficiency was quantified using a glutamate assay and HPLC. Overall, the developed minimal medium holds strong potential to reduce production costs in yeast extract-based seasoning manufacturing. Future work will include quantitative comparison of growth rates and yeast extract yields between wild-type and engineered strains, as yeast extract yield serves as a practical indicator of umami compound biosynthetic capacity. In addition, enzyme stability and conversion efficiency will be further improved through the use of a scaffold-based enzyme complex, complemented by in silico protein engineering for structural stabilization and optimization.