Anaerobic and aerobic batch cultivations of Saccharomyces cerevisiae mutants impaired in glycerol synthesis

Abstract

Glycerol is formed as a by-product in production of ethanol and baker's yeast during fermentation of Saccharomyces cerevisiae under anaerobic and aerobic growth conditions, respectively. One physiological role of glycerol formation by yeast is to reoxidize NADH, formed in synthesis of biomass and secondary fermentation products, to NAD + . The objective of this study was to evaluate whether introduction of a new pathway for reoxidation of NADH, in a yeast strain where glycerol synthesis had been impaired, would result in elimination of glycerol production and lead to increased yields of ethanol and biomass under anaerobic and aerobic growth conditions, respectively. This was done by deletion of GPD1 and GPD2, encoding two isoenzymes of glycerol 3-phosphate dehydrogenase, and expression of a cytoplasmic transhydrogenase from Azotobacter vinelandii, encoded by cth. In anaerobic batch fermentations of strain TN5 (gpd2-D1), formation of glycerol was signi®cantly impaired, which resulted in reduction of the maximum speci®c growth rate from 0.41/h in the wild-type to 0.08/h. Deletion of GPD2 also resulted in a reduced biomass yield, but did not affect formation of the remaining products. The modest effect of the GPD1 deletion under anaerobic conditions on the maximum speci®c growth rate and product yields clearly showed that Gdh2p is the important factor in glycerol formation during anaerobic growth. Strain TN6 (gpd1-D1 gpd2-D1) was unable to grow under anaerobic conditions due to the inability of the strain to reoxidize NADH to NAD + by synthesis of glycerol. Also, strain TN23 (gpd1-D1 gpd2-D1 YEp24-PGKp-cth-PGKt) was unable to grow anaerobically, leading to the conclusion that the NAD + pool became limiting in biomass synthesis before the nucleotide levels favoured a transhydrogenase reaction that could convert NADH and NADP + to NADPH and NAD + . Deletion of either GPD1 or GPD2 in the wild-type resulted in a dramatic reduction of the glycerol yields in the aerobic batch cultivations of strains TN4 (gpd1-D1) and TN5 (gpd2-D1) without serious effects on the maximum speci®c growth rates or the biomass yields. Deletion of both GPD1 and GPD2 in strain TN6 (gpd1-D1 gpd2-D1) resulted in a dramatic reduction in the maximum speci®c growth rate and in biomass formation. Expression of the cytoplasmic transhydrogenase in the double mutant, resulting in TN23, gave a further decrease in m max from 0.17/h in strain TN6 to 0.09/h in strain TN23, since the transhydrogenase reaction was in the direction from NADPH and NADP + to NADH and NADP + . Thus, it was not possible to introduce an alternative pathway for reoxidation of NADH in the cytoplasm by expression of the transhydrogenase from A. vinelandii in a S. cerevisiae strain with a double deletion in GPD1 and GPD2.

Distinct Intracellular Localization of Gpd1p and Gpd2p, the Two Yeast Isoforms of NAD+-dependent Glycerol-3-phosphate Dehydrogenase, Explains Their Different Contributions to Redox-driven Glycerol Production

“…3), and growth rate (Fig. 2), as is previously reported (17,18,35), whereas removal of GPD2 in the cox18⌬ background is completely growth inhibiting under aerobic as well as anaerobic conditions.…”

“…These findings suggest a minor role for GPD1 in the aerobic redoxadjusting glycerol production of cox18⌬ mutants. The observation that cox18⌬ strains lacking GPD2 experience a complete growth arrest indicates that GPD2 is given a more important role for relieving the redox constraints in respiratory deficient mutants than reported for cells that are deprived of respiration due to anaerobic conditions (17,18,35).…”

NADPH-dependent glutamate dehydrogenase in Penicillium chrysogenum is involved in regulation of β-lactam production

“…These findings are in accordance with similar studies in S. cerevisiae, where decreases of 22 % or 46 % were found during aerobic and anaerobic conditions, respectively (Moreira dos Santos et al, 2004;Nissen et al, 2000). As expected, the m max of the gdhAdisrupted strains could be restored by supplementing glutamate to the medium and by overexpression of gdhB.…”

“…1), and it is therefore possible to shift the co-factor requirements through genetic engineering. This has been demonstrated previously, and the co-factor switch successfully led to increased bioethanol production by Saccharomyces cerevisiae (Nissen et al, 2000).…”

Alcoholic fermentation of carbon sources in biomass hydrolysates by Saccharomyces cerevisiae: current status

“…Indeed, the authors suggest that the redox factor balance might be restored by a transhydrogenase activity that would interconvert NADH/NADP + and NAD + /NADPH. However, Nissen et al (2000) showed that the in vivo cofactor concentrations in S. cerevisiae probably would not allow the reaction to proceed in the desired direction. Another way that has been proposed to ameliorate problems with cofactor imbalances is expression of the aldose reductase gene from Pi.…”