Beyond alcohol oxidase: the methylotrophic yeast Komagataella phaffii utilizes methanol also with its native alcohol dehydrogenase Adh2

Abstract

Methylotrophic yeasts are considered to use alcohol oxidases to assimilate methanol, different to bacteria which employ alcohol dehydrogenases with better energy conservation. The yeast Komagataella phaffii carries two genes coding for alcohol oxidase, AOX1 and AOX2. The deletion of the AOX1 leads to the MutS phenotype and the deletion of AOX1 and AOX2 to the Mut– phenotype. The Mut– phenotype is commonly regarded as unable to utilize methanol. In contrast to literature, we found that the Mut– strain can consume methanol. This ability was based on the promiscuous activity of alcohol dehydrogenase Adh2, an enzyme ubiquitously found in yeast and normally responsible for ethanol consumption and production. Using 13C labeled methanol as substrate we could show that to the largest part methanol is dissimilated to CO2 and a small part is incorporated into metabolites, the biomass, and the secreted recombinant protein. Overexpression of the ADH2 gene in K. phaffii Mut– increased both the specific methanol uptake rate and recombinant protein production, even though the strain was still unable to grow. These findings imply that thermodynamic and kinetic constraints of the dehydrogenase reaction facilitated the evolution towards alcohol oxidase-based methanol metabolism in yeast.

The oxygen tolerant reductive glycine pathway in eukaryotes – a native methanol, formate and CO2 assimilation pathway in the yeast Komagataella phaffii

“…Flasks were incubated at 25 °C in atmosphere, at 30 °C and 5% CO 2, and at 25 °C at 10% CO 2 while shaking with 180 rpm. OD 600 and methanol or formate concentrations (HPLC measurements 17 ) were monitored every 2-3 days. The experiments were conducted in duplicates.…”

“…For experiments with metabolite sampling points at 2 h, 24 h and 72 h, the starting volume was set to 16 mL. 13 C-methanol was adjusted to 1% (v/v) after 24 h, 48 h and 58 h. 13 Cformate concentration was adjusted to 30 mmol L -1 after 7 h, 17 h, 22 h, 30 h, 41 h and 52 h (for methanol and formate feeding profiles see Supplementary information Figure S8, at each sample point HPLC measurements 17 were conducted to assess the carbon source concentration). Each metabolic sample had 3 mL, additional samples for methanol measurements 1 mL and for formate measurements 300 µL.…”

“…Hitherto unknown metabolic routes were recently found in K. phaffii . These routes are active even though their flux is too low to support growth 17 . These findings motivated us to seek for other hidden pathways, focusing on methanol, formate and CO 2 assimilation routes which are natively active.…”

“…We have recently described that Aox is not the only enzyme catalysing the first reaction step in the native methanol metabolism. The cytosolic alcohol dehydrogenase (Adh2), known for both synthesis and consumption of ethanol, is also oxidizing methanol to formaldehyde 17 . This reaction is energy conserving by the reduction of NAD + to NADH, but seems to have limitations, as growth was not observable with Adh only.…”

Development of a strategy induction AOX1 promoter methylotrophic yeast Komagataella phaffii

“…Дрожжевые системы экспрессии, в том числе Komagataellaphaffii способны к эффективной секреции рекомбинантных белков непосредственно в культуральную жидкость, что значительно облегчает последующие стадии очистки целевых белков от сопутствующих примесей и вторичных метаболитов [1,7,15].…”