Engineering yeasts to Co-utilize methanol or formate coupled with CO₂ fixation

Area of Science:

• Synthetic biology
• Metabolic engineering
• Microbial biotechnology

Background:

• Microorganisms capable of utilizing one-carbon (C1) compounds like CO2, methanol, and formate are of significant interest for sustainable chemical production.
• Developing synthetic pathways for C1 assimilation in model yeasts like Pichia pastoris and Saccharomyces cerevisiae remains a key challenge.

Purpose of the Study:

• To engineer Pichia pastoris and Saccharomyces cerevisiae for synthetic methylotrophy and formatotrophy, enabling co-utilization of methanol or formate with CO2 fixation.
• To establish and validate a synthetic C1-compound assimilation pathway (MFORG pathway) in these yeast hosts.

Main Methods:

• Assembly of the MFORG pathway, comprising a methanol-formate oxidation module and the reductive glycine pathway.
• Genetic engineering of P. pastoris, including blocking native methanol assimilation and modularly engineering MFORG pathway genes.
• Introduction of the MFORG pathway into S. cerevisiae and confirmation of C1 compound utilization via 13C-tracer analysis.

Main Results:

• Successfully engineered P. pastoris to utilize both methanol and formate.
• Transferred the MFORG pathway to S. cerevisiae, establishing synthetic methylotrophy and formatotrophy with significant consumption rates.
• Demonstrated CO2 co-assimilation with methanol or formate in both engineered yeast strains.
• Produced 5-aminolevulinic acid and lactic acid using co-assimilation of methanol and CO2 in engineered yeasts.

Conclusions:

• The MFORG pathway enables synthetic methylotrophy and formatotrophy in P. pastoris and S. cerevisiae.
• Engineered yeasts can efficiently co-utilize C1 compounds (methanol/formate) with CO2 fixation.
• This work highlights the potential of the MFORG pathway for developing diverse microbial hosts for C1-based chemical production.