Enhancement of Free Fatty Acids Production in <i>Rhodotorula toruloides</i> Using the CRISPR/Cas9-Based Base Editor

<i>Rhodotorula toruloides</i> is a promising cell factory to produce various value-added chemicals, including fatty acid derivatives. However, their metabolic engineering development has been hindered by the limited availability of genetic tools. In this study, an accurate and specific gene-editing tool, CRISPR/Cas-based cytidine base editor (CBE) system, was developed for the first time in <i>R. toruloides</i> to broaden its genetic toolbox. The target gene was disrupted by introducing a premature stop codon via C to T mutation. This system achieved single-gene disruption efficiencies of up to 90% and successfully disrupted four genes in parallel with 5% efficiency, marking a breakthrough in multiplexed editing for this yeast. To enable iterative engineering, an inducible Cre-loxP system was integrated, achieving an over 70% selection marker recycling efficiency. Application of this system enabled the construction of uracil-auxotrophic strains. Furthermore, the CBE system was employed to disrupt four genes involved in lipid metabolism, resulting in an engineered strain capable of producing 512.3 mg/L of free fatty acids, thereby demonstrating the utility of the CBE system as an efficient genome editing tool in <i>R. toruloides</i>. The study provides valuable tools to expand the genetic toolbox of <i>R. toruloides</i> and paves the way for fully exploiting its metabolic engineering potential.