摘要
Dodecanedioic acid (DDA) is a valuable monomer with broad industrial applications. The yeast Candida viswanathii can convert dodecane into 12-carbon DDA, making it an attractive host for metabolic engineering toward sustainable production. A recently developed miniature dCasMINI protein, when fused with the VPR activator, enables efficient CRISPR activation (CRISPRa) in mammalian cells, thereby simplifying gene cloning and advancing gene therapy applications. However, its potential for metabolic engineering in yeast has yet to be explored. Methods: We reconstituted the dCasMINI system in C. viswanathii and optimized it by modifying nuclear localization signals, sgRNA design, promoters and transcriptional activators. The redesigned system was applied to activate various endogenous genes. The impact of activating different transporter genes on DDA production was evaluated. Significant findings: The native dCasMINI-VPR system impaired C. viswanathii growth and failed to induce gene expression. By tuning design elements and replacing VPR with another yeast activator (Med2), we established a new dCasMINI-Med2 system that enabled robust activation of both constitutive and inducible genes while minimizing growth defects. Using this system, activation of transporter genes STL1_4 and HST6 enhanced DDA titers by 13-16 %. This study repurposes dCasMINI-based CRISPRa in C. viswanathii for metabolic engineering. The optimized dCasMINI-Med2 platform expands the CRISPRa toolbox for yeasts and demonstrates that transporter regulation can alleviate metabolic bottlenecks and improve DDA biosynthesis.