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 cyb2⌬ 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., CaCO 3 ), 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 ϩ -dependent lactate dehydrogenase, leading to the simultaneous formation of both ethanol and lactate (1a, 12, 31, 32, 36). Further improvements were made by constr...

bTo improve the economic viability of microalgal biodiesel, it will be essential to optimize the productivity of fuel molecules such as triacylglyceride (TAG) within the microalgal cell. To understand some of the triggers required for the metabolic switch to TAG production, we studied the effect of the carbon supply (acetate or CO 2 ) in Chlamydomonas reinhardtii (wild type and the starchless sta6 mutant) grown under low N availability. As expected, initial rates of TAG production were much higher when acetate was present than under strictly photosynthetic conditions, particularly for the sta6 mutant, which cannot allocate resources to starch. However, in both strains, TAG production plateaued after a few days in mixotrophic cultures, whereas under autotrophic conditions, TAG levels continued to rise. Moreover, the reduced growth of the sta6 mutant meant that the greatest productivity (measured as mg TAG liter ؊1 day ؊1 ) was found in the wild type growing autotrophically. Wild-type cells responded to low N by autophagy, as shown by degradation of polar (membrane) lipids and loss of photosynthetic pigments, and this was less in cells supplied with acetate. In contrast, little or no autophagy was observed in sta6 mutant cells, regardless of the carbon supply. Instead, very high levels of free fatty acids were observed in the sta6 mutant, suggesting considerable alteration in metabolism. These measurements show the importance of carbon supply and strain selection for lipid productivity. Our findings will be of use for industrial cultivation, where it will be preferable to use fast-growing wild-type strains supplied with gaseous CO 2 under autotrophic conditions rather than require an exogenous supply of organic carbon.

BackgroundTemperature strongly affects microbial growth, and many microorganisms have to deal with temperature fluctuations in their natural environment. To understand regulation strategies that underlie microbial temperature responses and adaptation, we studied glycolytic pathway kinetics in Saccharomyces cerevisiae during temperature changes.ResultsSaccharomyces cerevisiae was grown under different temperature regimes and glucose availability conditions. These included glucose-excess batch cultures at different temperatures and glucose-limited chemostat cultures, subjected to fast linear temperature shifts and circadian sinoidal temperature cycles. An observed temperature-independent relation between intracellular levels of glycolytic metabolites and residual glucose concentration for all experimental conditions revealed that it is the substrate availability rather than temperature that determines intracellular metabolite profiles. This observation corresponded with predictions generated in silico with a kinetic model of yeast glycolysis, when the catalytic capacities of all glycolytic enzymes were set to share the same normalized temperature dependency.ConclusionsFrom an evolutionary perspective, such similar temperature dependencies allow cells to adapt more rapidly to temperature changes, because they result in minimal perturbations of intracellular metabolite levels, thus circumventing the need for extensive modification of enzyme levels.