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

作者：Yi Yu, Quan Yuan, Zihe Liu, Baisong Tong, Shuobo Shi · 发表于：ACS Synthetic Biology · 年份：2025 · DOI：10.1021/acssynbio.5c00359 · 被引用次数：7 · 研究领域：CRISPR and Genetic Engineering、Microbial Metabolic Engineering and Bioproduction、Viral Infectious Diseases and Gene Expression in Insects

Rhodotorula toruloides 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 R. toruloides 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 R. toruloides . The study provides valuable tools to expand the genetic toolbox of R. toruloides and paves the way for fully exploiting its metabolic engineering potential.