Enhanced deoxyviolacein bioproduction via enzyme condensation with a yeast glycolytic enzyme-derived peptide tag

In microbial production, regulating endogenous and exogenous synthetic pathways is essential. Techniques to induce intracellular enzyme condensation have attracted attention as a means to increase apparent enzyme activity with minimal transcriptional and translational burden on the cell. Artificial enzyme condensation can be induced by tagging enzymes with specific proteins or peptides. In our previous study, we identified novel peptide tags derived from condensate-forming Saccharomyces cerevisiae glycolytic enzymes and their potential use in controlling intracellular metabolism by inducing artificial condensates in S. cerevisiae cells. Herein, we evaluated the condensate formation of two enzymes using peptide tags and the branched violacein biosynthetic pathway from Pseudoalteromonas luteoviolacea in S. cerevisiae, and tested the effects of modulating biosynthesis to increase the production of deoxyviolacein, a byproduct with antibacterial and anticancer activities. We used several protein and peptide tags with a simplified expression system, and all the tags successfully induced artificial condensate formation in the cell. Additionally, introducing a short peptide tag successfully increased deoxyviolacein production by approximately twofold, displaying a higher efficacy compared to FUS^N, a previously reported N-terminal 213 amino acid region with an intrinsically disordered property. These results demonstrate the potential use of peptide tags to enhance bioproduction through the regulation of endogenous and exogenous synthetic pathways. The methods contribute to the development of novel strategies for microorganisms to be used for bioproduction through the controlled condensation of metabolic enzymes in cells.