Mitochondrial localization of key NAD+-dependent dehydrogenases explains the exceptionally high biomass yield on ethanol of the yeast Cyberlindnera jadinii.

UNLABELLED: Ethanol is an interesting renewable feedstock for microbial biotechnology. Compared to other yeasts, Cyberlindnera jadinii has an exceptionally high biomass yield on ethanol. Simulations with a stoichiometric metabolic model identified an intramitochondrial rather than cytosolic localization of NAD+-dependent alcohol and acetaldehyde dehydrogenases as a possible explanation for this high yield. This hypothesis was tested in a comparative study with Ogataea parapolymorpha, which, like C. jadinii, contains a proton-pumping Complex I NADH dehydrogenase, but shows a lower biomass yield on ethanol. Experiments with an aox null mutant showed that the lower biomass yield of O. parapolymorpha was not caused by in vivo alcohol oxidase activity. Proteome analyses showed that the most abundant ADH isozyme in C. jadinii and O. parapolymorpha contained and lacked a predicted mitochondrial targeting sequence, respectively. The ADH protein with the highest prevalence in ethanol-limited cultures of C. jadinii was predicted to be mitochondrial and accounted for almost 4% of the detected proteome. In subcellular fractionation studies with cells grown under ethanol limitation, ADH showed 10-fold lower specific activity in the mitochondria-enriched fraction of cell homogenates of O. parapolymorpha than in the cytosolic fraction. Conversely, C. jadinii showed a 50% higher ADH activity in the mitochondria-enriched fraction. A low overall recovery of ADH in mitochondria-enriched fractions of C. jadinii was attributed to disruption of the reticulate mitochondrial structures observed in ethanol-grown cells during preparation of cell homogenates. This study demonstrates how subcellular localization of an NAD+-dependent dehydrogenase can influence respiratory energy coupling in yeasts that harbor a Complex I NADH dehydrogenase.

IMPORTANCE: This study provides theoretical and experimental evidence that intracellular localization of NAD+-dependent dehydrogenases can be a key factor for respiratory energy coupling in yeasts. Based on this, alteration of the cellular localization of NAD+-dehydrogenases is identified as an interesting metabolic engineering strategy for improving respiratory energy coupling in eukaryotic cell factories.