In vitro and in vivo characterization of a new-to-nature pathway for formaldehyde assimilation in methylotrophic yeast Komagataella phaffii

Description

Abstract (English)

Formolase (FLS) is the first synthetic enzyme to catalyse the formose reaction, wherein formaldehyde is converted to dihydroxyacetone (DHA). It is thus uniquely suited for the construction of synthetic methanol assimilation cascades, proceeding via methanol oxidation to formaldehyde condensation to DHA, and finally ATP-dependent conversion to dihydroxyacetonephosphate (DHAP). Compared to the native xylulose monophosphate (XuMP) cycle of methylotrophic yeasts, this pathway produces DHAP in fewer catalytic steps, without the need for acceptor recycling and at the cost of less ATP. Here, we implement FLS-based formaldehyde assimilation in Komagataella phaffii, a methylotrophic yeast used on an industrial scale to produce bioproducts, particularly proteins. To this end, an optimized FLS gene with a peroxisomal targeting signal (PTS1) under the control of a methanol-inducible promoter was integrated into the genome of a XuMP-deficient K. phaffii strain. Transformants with high copy numbers of the FLS gene produced up to 53.65 ± 2.15 µM·min-1 DHA in cell-free extract (CFE).  In small-scale bioreactor cultivation in minimal media with methanol as the carbon source, the FLS strains outgrew their parental strain, confirming the in vivo functionality of this linear methanol assimilation pathway. FLS strains also exhibited sustained growth on methanol following DHA growth phases in continuous turbidostat cultivation. This was in stark contrast to the parental strain (XuMP–), which exhibited a reduction in OD600. In the fed-batch phase of cultivations on methanol feed, the FLS-producing strain showed a biomass yield on methanol of 0.27 ± 0·09 g·g-1 and a biomass formation rate of 0.0113 g·h-1. This work lays the foundation for the implementation of a more energy-efficient methanol assimilation pathway as the basis for sustainable bioproduction in yeasts.