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 haa1⌬ 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.

Nuclear Localization of Haa1, Which Is Linked to Its Phosphorylation Status, Mediates Lactic Acid Tolerance in Saccharomyces cerevisiae

“…Despite its tolerance for acid, high levels of undissociated lactic acid (Ͼ6%) are severely toxic to yeast (13) (Fig. 1).…”

“…Lactate chelates metal cations, especially the iron that is present in culture media, which are required for growth (13,14). As a response, Aft1, a transcriptional activator involved in iron utilization, is translocated to the nucleus upon exposure to lactic acid (12) and induces its target genes (12,13).…”

“…Another transcriptional factor, Haa1, has been implicated in the mechanisms of S. cerevisiae resistance to acid stress (13,15,16). Haa1, a homolog of the copper-regulated transcriptional factor Ace1 (17), was initially reported to be required for rapid adaptation of yeast cells to acetic, propionic, and butyric acids (18).…”

“…The Haa1-mediated acetic acid resistance could be regulated by Yak1, as this protein kinase, which activates the stress-responsive tran-scriptional factors Hsf1 and Msn2/Msn4, was suggested to be involved (22). Analysis of transcriptional responses to high concentrations of lactic acid of S. cerevisiae at pH 3, when more undissociated acid is present, and at pH 5, when lactate is the prominent form, indicated that Haa1 regulates the response to undissociated lactic acid, while at pH 5, the main response was related to iron homeostasis (13).…”

Saccharomyces cerevisiae Genome Shuffling through Recursive Population Mating Leads to Improved Tolerance to Spent Sulfite Liquor

“…It has been shown recently that evolutionarily engineered strains of S. cerevisiae, adapted to increased acetic acid concentrations, demonstrated strongly inducible rather than constitutive acetic acid tolerance (41). It was hypothesized that this might arise because acetic acid occurs in the natural S. cerevisiae environment, and the native inducible acetic acid tolerance mechanisms, such as the acetate-induced HAA1 regulon, may be affected (1,9). R57 performed noticeably better than the WT, R311, and R511 on NaCl at 3 and 7% (wt/vol) ( Fig.…”