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

Area of Science:

• Microbiology
• Synthetic Biology
• Biotechnology

Background:

• * Rhodotorula toruloides is a valuable yeast for producing chemicals like fatty acids.
• * Limited genetic tools have restricted its metabolic engineering potential.
• * Efficient gene editing is crucial for unlocking R. toruloides' capabilities.

Purpose of the Study:

• * To develop and validate a CRISPR/Cas-based cytidine base editor (CBE) system for R. toruloides.
• * To establish multiplexed gene editing and marker recycling for iterative engineering.
• * To engineer R. toruloides for enhanced free fatty acid production.

Main Methods:

• * Development of a CRISPR/Cas-based cytidine base editor (CBE) for C-to-T base editing in R. toruloides.
• * Integration of an inducible Cre-loxP system for selection marker recycling.
• * Application of CBE for single and multiplex gene disruptions, including genes involved in lipid metabolism.

Main Results:

• * Achieved single-gene disruption efficiencies up to 90% using the CBE system.
• * Demonstrated successful parallel disruption of four genes with 5% efficiency.
• * Enabled selection marker recycling with over 70% efficiency and constructed uracil-auxotrophic strains.
• * Engineered a strain producing 512.3 mg/L of free fatty acids by disrupting lipid metabolism genes.

Conclusions:

• * The developed CBE system is an accurate and specific genome editing tool for R. toruloides.
• * This toolset significantly expands the genetic engineering capabilities for R. toruloides.
• * The study paves the way for enhanced metabolic engineering and chemical production in R. toruloides.