Catalase overexpression reduces lactic acid-induced oxidative stress in Saccharomyces cerevisiae

Abstract

Industrial production of lactic acid with the current pyruvate decarboxylase-negative Saccharomyces cerevisiae strains requires aeration to allow for respiratory generation of ATP to facilitate growth and, even under nongrowing conditions, cellular maintenance. In the current study, we observed an inhibition of aerobic growth in the presence of lactic acid. Unexpectedly, the cyb2Delta reference strain, used to avoid aerobic consumption of lactic acid, had a specific growth rate of 0.25 h(-1) in anaerobic batch cultures containing lactic acid but only 0.16 h(-1) in identical aerobic cultures. Measurements of aerobic cultures of S. cerevisiae showed that the addition of lactic acid to the growth medium resulted in elevated levels of reactive oxygen species (ROS). To reduce the accumulation of lactic acid-induced ROS, cytosolic catalase (CTT1) was overexpressed by replacing the native promoter with the strong constitutive TPI1 promoter. Increased activity of catalase was confirmed and later correlated with decreased levels of ROS and increased specific growth rates in the presence of high lactic acid concentrations. The increased fitness of this genetically modified strain demonstrates the successful attenuation of additional stress that is derived from aerobic metabolism and may provide the basis for enhanced (micro)aerobic production of organic acids in S. cerevisiae.

FIG. 1.

Typical CO₂ off-gas profile for fermentations of the reference strain in the sequential batch reactor system. The batch fermentor was emptied and refilled automatically when the CO₂ concentration in the off-gas was ≥1.9%. The specific growth rates (h⁻¹) for each batch were calculated from the change in CO₂ production and are indicated above each peak.

FIG. 2.

Quantification of intracellular ROS in shake flask cultures of S. cerevisiae grown in synthetic medium at pH 3. The fluorescence of CM-H₂DCFDA was measured for unstressed control cultures (white bars) and in response to 0.5 mM H₂O₂ (gray bars) or 500 mM lactic acid (black bars). All values represent the fluorescence per milligram protein and are reported as percentages of values for the reference strain determined under unstressed conditions.

FIG. 3.

Average specific growth rates of aerobic cultivations performed in synthetic medium with glucose containing 500 mM lactic acid and controlled at pH 3. Each bar in this figure indicates the average growth rate of one independent sequencing batch reactor experiment consisting of five sequential batches. Error bars indicate standard deviations. Per strain, a total of 15 batch experiments were performed.