Physiological and transcriptional responses to high concentrations of lactic acid in anaerobic chemostat cultures of Saccharomyces cerevisiae

Abstract

Based on the high acid tolerance and the simple nutritional requirements of Saccharomyces cerevisiae, engineered strains of this yeast are considered biocatalysts for industrial production of high-purity undissociated lactic acid. However, high concentrations of lactic acid are toxic to S. cerevisiae, thus limiting its growth and product formation. Physiological and transcriptional responses to high concentrations of lactic acid were studied in anaerobic, glucose-limited chemostat cultures grown at different pH values and lactic acid concentrations, resulting in a 50% decrease in the biomass yield. At pH 5, the yield decrease was caused mostly by osmotically induced glycerol production and not by the classic weak-acid action, as was observed at pH 3. Cultures grown at pH 5 with 900 mM lactic acid revealed an upregulation of many genes involved in iron homeostasis, indicating that iron chelation occurred at high concentrations of dissociated lactic acid. Chemostat cultivation at pH 3 with 500 mM lactate, resulting in lower anion concentrations, showed an alleviation of this iron homeostasis response. Six of the 10 known targets of the transcriptional regulator Haa1p were strongly upregulated in lactate-challenged cultures at pH 3 but showed only moderate induction by high lactate concentrations at pH 5. Moreover, the haa1Delta mutant exhibited a growth defect at high lactic acid concentrations at pH 3. These results indicate that iron homeostasis plays a major role in the response of S. cerevisiae to high lactate concentrations, whereas the Haa1p regulon is involved primarily in the response to high concentrations of undissociated lactic acid.

FIG. 1.

Effect of lactic acid on biomass formation in glucose-limited anaerobic chemostat cultures of S. cerevisiae CEN.PK 113-7D. Each data point represents an independent chemostat culture which was grown to steady state at a dilution rate of 0.10 h⁻¹. Based on the data obtained at pH 5.0 (•), the Henderson-Hasselbach equation was used to estimate an equivalent stress at pH 3.5 (dashed line), assuming that the undissociated acid was solely responsible for the observed decrease of the biomass concentration. Experimental data obtained at pH 3.5 (○) did not correlate with this prediction.

FIG. 2.

Relative free-iron concentration, as indicated by an optical density at 570 nm (OD_570nm) in complete synthetic medium supplemented with increasing concentrations of lactic acid at pH 5 (•) and pH 3 (○). For each condition, 150 μM FeSO₄ was added, and unbound Fe²⁺ was detected with ferrozine (250 μM), which results in absorbance at 570 nm upon interaction with Fe²⁺ (47). Decreased OD_570nm indicates decreased availability of Fe²⁺ in growth medium containing increasing concentrations of the lactate anion. However, the quantitative relationship between free Fe²⁺ and OD_570nm is unknown.

FIG. 3.

Growth of the S. cerevisiae mutant strain (aft1Δ) in glucose synthetic medium containing 900 mM lactic acid. Iron sulfate concentrations in the synthetic medium were 10 μM (standard concentration, •), 100 μM (○), and 250 μM (▪). In contrast to the aft1Δ strain, the reference strain (CEN.PK 113-7D) did not exhibit a growth deficiency in the presence of lactic acid with the standard concentration of iron (□). The pH of all shake flasks was set to 5, and urea was utilized as the nitrogen source to prevent acidification of the growth medium (21).