Metabolic bottleneck at TCA cycle entry point and competition with trehalose production hinder growth of methylotrophic yeasts during methanol assimilation.

Methylotrophic yeasts are promising hosts for bioproduction from methanol, but their growth is significantly reduced in the presence of methanol. In this study, we investigated metabolic bottlenecks in Komagataella phaffii, Ogataea polymorpha, and Candida boidinii during methanol assimilation through comparative metabolome analysis. We found a common decrease in α-ketoglutarate-derived amino acids and tricarboxylic acid (TCA) cycle intermediates in all three species during methanol assimilation, suggesting a metabolic bottleneck around the TCA cycle entry. Additionally, K. phaffii and O. polymorpha accumulate trehalose in the presence of methanol. ΔΔG analysis suggested reduced activity of pyruvate kinase and TCA cycle entry enzymes as potential rate-limiting steps. Biosynthesis of TCA cycle-derived amino acids, including Glu, Arg, and Pro, is a limiting factor because supplementation with these amino acids significantly enhanced growth rates and final cell density for all three species. Furthermore, disruption of the trehalose biosynthesis gene (TPS2) in K. phaffii further improved growth, indicating that trehalose accumulation negatively affects methanol-based growth. Amino acid supplementation with TPS2 disruption in K. phaffii resulted in a 1.2-fold increase in the specific growth rate and a 2.73-fold increase in final cell density. These findings provide insights into the metabolic limitations of methylotrophic yeast growth in the presence of methanol and offer strategies for improving bioproduction efficiency.