Revealing the endogenous homoserine cycle for the effective methanol conversion in Pichia pastoris

Methanol, being electron rich and derivable from methane or CO2, is a very promising feedstock for biomanufacturing. Nevertheless, challenges still remain in methanol metabolism for value-added chemicals overproduction. Here, we performed gene mining and revealed the endogenous homoserine cycle for methanol conversion in Pichia pastoris for the first time. In addition, by manipulation of the concentration of methanol, adding the amino acids, and overexpressing the pathway genes, we identified the key substrates and rate-limiting enzymes (SDA and TS) affecting the homoserine cycle. Hence, the metabolic fluxes were rewired toward homoserine cycle and the methanol conversion was stepwise promoted through the introduction of cytoplasmic methanol oxidation pathway, overexpression of the key genes in the glycine and serine synthesis routes, and multicopy integration of the SDA and TS expression cassettes. Eventually, in combination with pathway revelation and metabolic engineering, the rebuilding methylotroph with strengthened homoserine cycle was able to produce 33.9 mg/L/OD β-carotene with a titer of 173.9 mg/L from methanol. The unit cell titer of β-carotene was 145.7% higher than that of the initial strain (Kp16). This study will lay a good foundation for optimizing methylotrophic platform to promote industrial utilization.