CRISPR-based synthetic biology toolkit development in Candida viswanthii and functional analysis of the stress responsive Ena1-like protein.

Industrial strains from the Candida genus have been applied in production of enzymes, biochemicals, and single-cell protein. However, the synthetic biology manipulation tools for Candida species remain underdeveloped. In this study, a high-efficiency genome editing strategy for C. viswanathii was established by combining the CRISPR/Cas9 and Cre/loxP systems. This approach achieved 100 % editing efficiency and supported rapid iterative editing cycles within 6 days. The system enables iterative genomic modifications and was successfully applied for multiplex editing and multicopy gene integration up to 7 copies. Leveraging this platform, g144, an Ena1-like protein that exhibited differential expression during dodecanedioic acid (DDA) fermentation, was functionally characterized. The results showed that g144 lacks Na+ transport activity, but both the disruption and overexpression strains showed increased sensitivity to alkaline pH and Na+ stress, as well as a decrease in DDA production. The genome editing toolkit reported here benefits further applications of Candida strains for sustainable bioproduction.