A CRISPR–Cas9 System-Mediated Genetic Disruption and Multi-fragment Assembly in Starmerella bombicola

Gene editing technology plays an extremely significant role in synthetic biology and metabolic engineering. Traditional genetic manipulation methods, such as homologous recombination, however, are inefficient, time-consuming, and barely feasible when disrupting multiple genes simultaneously. Starmerella bombicola, a nonconventional yeast that overproduces sophorolipids, lacks convenient genetic tools for engineering strains. Here, we developed an efficient CRISPR–Cas9 genome editing technology by combining molecular element mining and expression system optimization for S. bombicola. This CRISPR–Cas9 system improved the efficiency of gene-integration/target gene-introducing disruption by homology-directed repair and realized the multi-gene simultaneous disruptions. Based on this CRISPR–Cas9 system, we also further constructed an engineered strain via the in vivo assembly of multiple DNA fragments (10 kb) that can produce acid-type sophorolipids. These results showed that the CRISPR–Cas9 system may be an efficient and convenient strategy to perform genetic manipulation in S. bombicola.