Abstract
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 and grows on organic feedstocks that have competing uses in the production of food and animal feed. In a step toward more sustainable industrial processes, we describe the conversion of P. pastoris into an autotroph that grows on CO(2). By addition of eight heterologous genes and deletion of three native genes, we engineer the peroxisomal methanol-assimilation pathway of P. pastoris into a CO(2)-fixation pathway resembling the Calvin-Benson-Bassham cycle, the predominant natural CO(2)-fixation pathway. The resulting strain can grow continuously with CO(2) 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. This engineered P. pastoris strain may promote sustainability by sequestering the greenhouse gas CO(2), and by avoiding consumption of an organic feedstock with alternative uses in food production.