Synthetic Carbon Dioxide Assimilation in Yeast for Autotrophic Growth

Global warming, caused by rapidly increasing atmospheric carbon dioxide levels, calls for novel measures to sequester CO₂. Assimilation of CO₂ into biomass and bulk chemicals is an attractive option. Besides the use of photosynthesizing organisms, synthetic biology offers the change to develop strains for a novel CO₂ based biotechnology. The methylotrophic yeast Pichia pastoris is widely used in the manufacture of industrial enzymes and pharmaceuticals. Like most biotechnological production hosts, P. pastoris is heterotrophic, growing on organic feedstocks. By addition of eight heterologous genes and deletion of three native genes, we engineered the peroxisomal methanol-assimilation pathway of P. pastoris into a CO₂ fixation pathway resembling the Calvin-Benson-Bassham (CBB) cycle, the predominant natural CO₂ fixation pathway. Due to the modular metabolic design which separates carbon assimilation from energy production, any NADH yielding energy source can be used. At present methanol oxidation is employed. The resulting strain can grow continuously with CO₂ as a sole carbon source at a µ_max of 0.008 h^-1. The specific growth rate was further improved to 0.018 h^-1 by adaptive laboratory evolution. Mutations in evolved strains point at the balancing of CBB cycle reactions and NADH supply as critical steps for efficient growth. This engineered P. pastoris strain may promote sustainability by sequestering the greenhouse gas CO₂ and by avoiding consumption of an organic feedstock with alternative uses in food production.