Engineering Rhodosporidium toruloides for limonene production

Area of Science:

• Microbial biotechnology
• Synthetic biology
• Metabolic engineering

Background:

• Limonene is a versatile monoterpene with broad applications in food, pharmaceuticals, and biofuels.
• Engineering microbial cell factories is crucial for sustainable production of valuable chemicals.

Purpose of the Study:

• To engineer the yeast Rhodosporidium toruloides as an efficient cell factory for limonene production.
• To explore novel metabolic engineering strategies for enhancing limonene biosynthesis in R. toruloides.

Main Methods:

• Overexpression of key enzymes in the mevalonate pathway, including limonene synthase (LS) and geranyl pyrophosphate synthase (NPPS).
• Introduction of heterologous genes from Enterococcus faecalis and Methanosarcina mazei to optimize the mevalonate route.
• Construction of a chimeric NPPS-LS fusion protein and expression in a carotenogenesis-deficient R. toruloides strain.

Main Results:

• Established a baseline for limonene production in R. toruloides by optimizing the native mevalonate pathway.
• Achieved a maximum limonene titer of 393.5 mg/L through metabolic engineering and introduction of novel enzymes.
• Demonstrated the efficacy of the engineered mevalonate pathway and the chimeric NPPS-LS fusion protein.

Conclusions:

• Successfully engineered R. toruloides for limonene production, marking the first report using this yeast for limonene biosynthesis via the NPP pathway.
• Validated R. toruloides as a viable microbial platform for the production of limonene and potentially other valuable monoterpenes.
• Provided valuable insights into the microbial production of monoterpenes through advanced metabolic engineering techniques.