Investigation of the contribution of Complex I to respiratory energy-coupling in glucose-grown cultures of the Crabtree-negative yeast Ogataea parapolymorpha

The methylotrophic, thermotolerant yeast Ogataea parapolymorpha (formerly Hansenula polymorpha) is an industrially relevant production host and exhibits a respiratory metabolism in the presence of oxygen. It possesses a branched respiratory chain with multiple entry points for NADH-derived electrons that differ in complexity and degree of energy conservation: proton-translocating respiratory Complex I and three putative alternative NADH dehydrogenases. To investigate the physiological importance of Complex I, wild type O. parapolymorpha and a Complex I-disrupted mutant were cultured in glucose-grown bioreactor experiments in batch, chemostat and retentostat cultivations which allowed quantitative characterization of the strains over a wide range of growth rates in the presence and absence of excess substrate. In batch cultures, both strains exhibited a respiratory metabolism with no change in growth rate or biomass yield, indicating a negligible role of Complex I in NADH oxidation under these conditions. In glucose-limited chemostat cultures both strains also exhibited a respiratory metabolism, but the absence of functional Complex I considerably reduced biomass yields on substrate and oxygen, which is consistent with a lower respiratory efficiency of the mutant strain. In retentostat cultures that reached specific growth rates of ~0.001 h-1, both O. parapolymorpha strains remained highly viable and exhibited a 'stringent response'-like behavior, decreasing their maintenance energy requirements ~3-fold compared to their estimated maintenance requirements at higher growth rates. Quantitative transcriptome and proteome analyses indicated condition-dependent expression of Complex I and the alternative NADH dehydrogenases on the transcriptional and translational level and were consistent with the physiological observations.