Catalase Overexpression Reduces Lactic Acid-Induced Oxidative Stress in Saccharomyces cerevisiae

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 cyb2Δ 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.Lactic acid is an organic acid with a wide range of applications. In the food industry, lactic acid has traditionally been used as an antimicrobial as well as a flavor enhancer. Besides having applications in textile, cosmetic, and pharmaceutical industries (5), lactic acid has been applied for the manufacture of lactic acid polymers (11, 40). These polymers have properties that are similar to those of petroleum-derived plastics. Skyrocketing oil prices caused by dwindling fossil fuel reserves coupled with pressures to tackle environmental issues are creating increased demand for bioderived, and often biodegradable, polymers, such as poly-lactic acid.Current industrial lactic acid fermentations are based on different species of lactic acid bacteria. These bacteria have complex nutrient requirements due to their limited ability to synthesize B vitamins and amino acids (8) and are intolerant to acidic conditions with a pH between 5.5 and 6.5 required for growth (40). Acidification of the growth medium during lactic acid fermentation is typically counteracted by the addition of neutralizing agents (e.g., CaCO₃), resulting in the formation of large quantities of insoluble salts, such as gypsum, during downstream processing.Saccharomyces cerevisiae has received attention as a possible alternative biocatalyst. This organism is relatively tolerant to low pH and has simple nutrient requirements. The production of lactic acid with metabolically engineered S. cerevisiae was achieved by introducing a NAD⁺-dependent lactate dehydrogenase, leading to the simultaneous formation of both ethanol and lactate (1a, 12, 31, 32, 36). Further improvements were made by constructing a pyruvate decarboxylase-negative (Pdc⁻) S. cerevisiae strain (1a, 31, 44) that converted glucose to lactic acid as the sole fermentation product.Although the redox balance and ATP generation in lactic acid fermentation are analogous to those in alcoholic fermentation, engineered homolactic S. cerevisiae strains could not sustain anaerobic growth (44). In addition, the lactate formation rate under anaerobic conditions in the presence of excess glucose was significantly lower than the specific ethanol production rate of the wild-type strain. Moreover, exposure of the anaerobic cell suspension to oxygen immediately led to a 2.5-fold increase in the lactate formation rate. The stimulatory effect of oxygen on lactic acid fermentation may reflect an energetic constraint in lactate fermentation, probably as a consequence of energy-dependent product export (42, 44). In agreement with this hypothesis, intracellular ATP concentrations and the related energy charge decrease rapidly during anaerobic homolactic fermentation by S. cerevisiae (1). Consequently, industrial production of lactic acid with S. cerevisiae may require (micro)aerobic conditions to allow for the generation of sufficient ATP to enable cell growth and, even under nongrowing conditions, maintenance.The formation of reactive oxygen species (ROS) during cellular respiration is an unavoidable side effect of aerobic life relying on oxygen as the final electron acceptor. At least 2% of oxygen consumed in mitochondrial respiration undergoes only one electron reduction, mainly by the semiquinone form of coenzyme Q, generating superoxide radicals (O₂⁻) (26). In addition, the prooxidant effects of organic acids have been demonstrated using sod mutants (30). An in vitro study by Ali et al. (3) also linked ROS formation to weak organic acids and showed enhanced hydroxy radical (OH) generation in the presence of lactic acid.Among different ROS, the hydroxy radical that originates from H₂O₂ in the metal-mediated Fenton/Haber-Weiss reactions is especially reactive. It indiscriminately oxidizes intracellular proteins, nucleic acids, and lipids in the cell membranes (4, 38). Lactate interacts with the ferric ion (Fe³⁺) to form a stable complex of Fe³⁺-lactate at a molar ratio of 1:2. This complex then reacts with H₂O₂ to enhance the OH generation via the Fenton reaction (2, 3). Although a similar in vivo mechanism has not yet been proven, previous research indicates that lactic acid and other weak organic acids enhance oxidative stress of aerobic yeast cultures.Like other eukaryotic organisms, S. cerevisiae possesses enzymatic defense mechanisms, including several crucial antioxidant enzymes, such as catalase and superoxide dismutase (SOD). SOD removes O₂⁻ by converting it to H₂O₂, which, in turn, can be disproportionated to water by catalase or glutathione peroxidase. Cytosolic catalase, Ctt1p, is thought to play a general role, as CTT1 expression is regulated by various stresses, including oxidative stress, osmotic stress, and starvation (15, 23, 33). More recently, catalase has also been implicated in response to acetic acid tolerance and acetic acid-induced programmed cell death (17, 47).The goals of the present study were to assess the in vivo relevance of lactate-mediated oxidative stress in S. cerevisiae and to investigate whether its effects could be ameliorated by enhanced expression of catalase.