Cascaded de novo biosynthesis of lacto-proteins from CO2 by engineered Pichia pastoris

Food proteins are key nutrients, which play an important role in our diet. However, the acquisition of food protein mainly depends on the agroindustry, resulting in environmental pollution, high land usage and the consumption of water resources. Here, we describe a chemo-biocascade catalysis (CBCC) system that combines spatially separated CO2 thermo-catalysis with yeast fermentation to efficiently convert CO2 to lacto-proteins. First, by uncovering and breaking the rate limiting step, we constructed a Pichia pastoris strain that efficiently synthesized human lactoferrin (hLF), able to reach a production of 5.4 g L-1 in bioreactor, the highest titer reported so far. Then, CuZnAl catalyst was used to stably synthesize the methanol from CO2 in a continuous-flow reactor. The collected liquid-phase products consisted of high-purity methanol of 99% in water with a yield of 6.7% (mol/mol). The hLF synthesis titer by P. pastoris using thermally generated methanol as the sole carbon source reached 56.2 mg L-1, with a methanol conversion rate of 1.1 mg g(-1), and a carbon atoms conversion rate of 0.17%. Additionally, the developed CBCC was also used to produce other dairy proteins including osteopontin and lactalbumin. These results illustrate the potential of tandem chemocatalysis and biocatalysis for the conversion of renewable resources into food proteins. This methodology paves the way towards the development of sustainable alternatives for protein manufacturing that may allow us to meet the growing demand.