Using the formose reaction for methanol assimilation in Komagataella phaffii

Abstract

Methylotrophic yeast Komagataella phaffii (P. pastoris) can assimilate methanol, which makes it a promising microbial host for sustainable biomass and protein production. The methanol assimilation pathway (XuMP) requires at least 10 enzymes to enable the conversion from methanol to dihydroxyacetone. In addition, three moles of ATP are required for the conversion of three moles of formaldehyde to one mol of DHAP. The aim of this study was to implement a recombinant metabolic pathway in K. phaffii that allows conversion of formaldehyde to biomass via the formose reaction. This alternative pathway only requires one mol of ATP to form DHAP, which is an intermediate of the central carbon metabolism. The synthetic enzyme formolase (FLS) was computationally designed to establish in vivo pathways for bacteria like Escherichia coli to convert CO2 (Siegel et al., 2015), however, a full carbon conversion into biomass via the formolase reaction was not reported so far. Here, an improved version of the enzyme, FLS-M3 (Cai et al., 2021) was targeted to the peroxisome of K. phaffii by fusing a peroxisomal targeting sequence to the C-terminus of the protein. Different inducible and constitutive promoters were used to express the recombinant FLS protein, while a strain with a blocked methanol assimilation pathway was used as a parental strain. On minimal media plates containing only methanol as a carbon source, the strains retained their ability to grow. The FLS-expressing strains showed an increase in optical density during cultivation in synthetic minimal media with methanol. In the cell free extract, formolase enzymatic activity was detected. This innovative system is predicted to produce less heat and increase product yield but also be able to convert C1 molecules more effectively into biomass.