Bradykinetic alcohol dehydrogenases make yeast fitter for growth in the presence of allyl alcohol

Abstract

Previous studies showed that fitter yeast (Saccharomyces cerevisiae) that can grow by fermenting glucose in the presence of allyl alcohol, which is oxidized by alcohol dehydrogenase I (ADH1) to toxic acrolein, had mutations in the ADH1 gene that led to decreased ADH activity. These yeast may grow more slowly due to slower reduction of acetaldehyde and a higher NADH/NAD+ ratio, which should decrease the oxidation of allyl alcohol. We determined steady-state kinetic constants for three yeast ADHs with new site-directed substitutions and examined the correlation between catalytic efficiency and growth on selective media of yeast expressing six different ADHs. The H15R substitution (a test for electrostatic effects) is on the surface of ADH and has small effects on the kinetics. The H44R substitution (affecting interactions with the coenzyme pyrophosphate) was previously shown to decrease affinity for coenzymes 2-4-fold and turnover numbers (V/Et) by 4-6-fold. The W82R substitution is distant from the active site, but decreases turnover numbers by 5-6-fold, perhaps by effects on protein dynamics. The E67Q substitution near the catalytic zinc was shown previously to increase the Michaelis constant for acetaldehyde and to decrease turnover for ethanol oxidation. The W54R substitution, in the substrate binding site, increases kinetic constants (K’s, by > 10-fold) while decreasing turnover numbers by 2-7-fold. Growth of yeast expressing the different ADHs on YPD plates (yeast extract, peptone and dextrose) plus antimycin to require fermentation, was positively correlated with the log of catalytic efficiency for the sequential bi reaction (V1/KiaKb = KeqV2/KpKiq, varying over 4 orders of magnitude, adjusted for different levels of ADH expression) in the order: WT H15R > H44R > W82R > E67Q > W54R. Growth on YPD plus 10 mM allyl alcohol was inversely correlated with catalytic efficiency. The fitter yeast are “bradytrophs” (slow growing) because the ADHs have decreased catalytic efficiency.

Alcohol Dehydrogenase of a Novel Algae Fermentation Strain Meyerozyma guilliermondii

“…The result showed that alginate produced the highest enzyme activity. This conclusion was different from other literature 22 , which commonly used glucose as the carbon source of alcohol dehydrogenase fermentation. In the subsequent experiments, alginate was also used as fermentation medium carbon source.…”

“…In the process of enzyme fermentation, temperature control is very important, because the microbial growth and enzyme fermentation production need certain temperature conditions 23 . Different cells have their optimum growth temperature.…”

Macromolecular crowding effects on the kinetics of opposing reactions catalyzed by alcohol dehydrogenase

“…Thus, in the presence of allyl alcohol, yeast reduce YADH activity, even though it means slower growth [ 118 ]. Lower rates of acetaldehyde reduction result in an increased intracellular ratio of NADH/NAD + , thereby protecting the cell against acrolein toxicity [ 119 ]. Through genetic sequencing, specific YADH mutants were identified in yeast with improved fitness in allyl alcohol due to decreased acetaldehyde reduction [ 118 ].…”

Ethanol Dehydrogenase I Contributes to Growth and Sporulation Under Low Oxygen Condition via Detoxification of Acetaldehyde in Metarhizium acridum

“…In Thermoanaerobacter ethanolicus , three ADHs play different roles during ethanol formation ( Zhou et al, 2017 ). In yeast, ADH1 is responsible for ethanol production from acetaldehyde and NADH, contributing to fungal growth in the presence of alcohol ( Plapp et al, 2013 ). Yeast ADH2 converts the ethanol accumulating to acetaldehyde under anaerobic conditions ( de Smidt et al, 2008 ).…”