Rewiring the methanol assimilation pathway in the methylotrophic yeast Pichia pastoris for high-level production of erythritol.

Methanol, which is non-food competing, abundant, inexpensive, and potentially renewable, serves as an ideal alternative feedstock for biomanufacturing. Although engineered methylotrophic yeasts have successfully achieved gram-scale production of C2 (acetyl-CoA), C3 (pyruvate), and C6 (fructose-6-phosphate) building blocks from methanol, the production of C4-based (i.e. erythrose-4-phosphate) chemicals remains unexplored. This study demonstrates high-level methanol-to-erythritol production by rewiring the methanol assimilation pathway of Pichia pastoris, achieved through trimming and strengthening the carbon rearrangement network (CRN). Notably, we introduced a bacterial ribulose monophosphate (RuMP) pathway in addition to the native xylulose monophosphate (XuMP) pathway of P. pastoris, creating a hybrid network that significantly improved erythritol production and reduced pentitol byproduct formation. Combining these strategies generated a high-producing recombinant strain, achieving titers up to 31.5 g/L in fermentor culture. This study validates the feasibility of engineering P. pastoris for the efficient conversion of methanol to valuable erythrose-4-phosphate (E4P)-based chemicals. The CRN rewiring strategies employed here offer a valuable reference for engineering methylotrophic cell factories for the production of a wide range of chemicals from methanol.