The physiological role of alcohol dehydrogenase 5 during chemostat cultivation of Saccharomyces cerevisiae James du Preez1, Laurinda Steyn1, Olga de Smidt2 and Jacobus Albertyn1 1Department of Microbial, Biochemical, and Food Biotechnology, University of the Free State, Bloemfontein, South Africa. 2School of Agriculture and Environmental Sciences, Central University of Technology, Bloemfontein, South Africa E-mail: dpreezjc@ufs.ac.za Introduction Materials & Methods Yeast strains used in this study Cultivation Chemically defined medium (per litre): 0.25 g citric acid, 5.0 g (NH4)2SO4, 2.5 g KH2PO4, 0.4 g MgSO4.7H2O, 0.02 g CaCl2.2H2O, 0.1 g NaCl and solutions of vitamins, trace elements and growth factors. Carbon source as specified. Carbon-limited chemostat: 30°C, pH 5.5, DOT > 30% of saturation, culture volume 650 ml with 8 g glucose l-1 or 4 g ethanol l-1. Analytical methods OD measured at 690 nm. Dry cell weight determined gravimetrically. Glucose, glycerol and acetic acid determined by HPLC; ethanol and acetaldehyde by GC. Oxygen and carbon dioxide content of the exhaust gas determined with a paramagnetic O2 analyser and an infrared CO2 analyser. Saccharomyces cerevisiae has five alcohol dehydrogenase isozymes, Adh1p to Adh5p, associated with glucose and ethanol metabolism. The alcohol dehydrogenase 5 (ADH5) gene was first identified through sequencing of chromosome II, and shares a 76%, 77%, and 70% sequence identity with the isozyme genes ADH1, ADH2 and ADH3, respectively [1, 2]. Kinetic studies revealed that, although Adh5p was capable of oxidizing C2 up to C10 alcohols, ethanol was the primary substrate [3]. The low Km value of Adh5p indicates a high affinity for ethanol, but its low Vmax value indicates that this enzyme is not very active in producing ethanol [3]. Batch cultivation of a quadruple adh deletion mutant of S. cerevisiae possessing only ADH5 demonstrated that Adh5p isozyme activity alone did not permit aerobic growth on ethanol, whereas growth on glucose was poor due to the accumulation of acetaldehyde to inhibitory concentrations. This suggested that the Adh5p activity for reducing acetaldehyde to ethanol was rate limiting [4]. The physiological role of the Adh5p isozyme in respect of ethanol metabolism in Saccharomyces cerevisiae strain W303-1A was further investigated, using a quadruple adh deletion mutant of this strain (designated strain Q5) with ADH5 as the only intact alcohol dehydrogenase gene. Data are presented on the growth characteristics of this mutant in aerobic carbon-limited continuous culture, used as a strategy to minimize inhibition of growth by the accumulation of acetaldehyde during cultivation. Yeast strain Genotype S. cerevisiae W303–1A(a) (parental strain) W303-1A, MATa, his3, leu2, trp1, ura3 Quadruple deletion mutant strain Q5 [4] W303-1A, MATa, ADH5, adh1Δ::LEU2, adh2Δ::URA3, adh3Δ::TRP1, adh4Δ::HIS3 Results Table 1 Growth parameters (maximum values) of S. cerevisiae W303-1A and quadruple deletion mutant Q5 in carbon-limited aerobic continuous culture with 8 g glucose l-1 as carbon substrate. In aerobic glucose-limited continuous culture the parental strain exhibited a much higher maximum specific growth rate (μmax) than mutant strain Q5, but the biomass yields were similar, and much higher than in batch cultures. (Table 1) Strain Q5 gave an ethanol yield coefficient of 0.22 at a dilution rate of 0.14 h-1, which was 3.6-fold higher than in batch culture. Strain W303-1A produced acetic acid, but no glycerol or acetaldehyde, whereas strain Q5 produced acetaldehyde and glycerol. (Table 1, Fig. 1). Under these carbon-limited conditions the low steady-state concentration of glucose resulted in a sufficiently low carbon flux that minimised acetaldehyde accumulation, permitting relatively good growth of strain Q5 on glucose. Strain Q5 was capable of slow growth on ethanol, in contrast to what was previously reported for batch cultivation of this strain, but only after a very long lag phase (Fig. 2A). In carbon-limited continuous culture with ethanol as substrate, a μmax of 0.17 h-1 was obtained, which was similar to the growth rate of strain Q5 on glucose (Table 1; Fig. 2B). The critical dilution rate for the onset of aerobic ethanol production (Crabtree effect) with these two yeast strains was about 24 to 28% of their respective μmax values (Fig. 1).. Parameter Batch cultivation Continuous cultivation W303-1A Q5 [4] Q5 µmax, h-1 0.44 0.17 0.50 0.18 Yx/s 0.15 0.05 0.59 0.42 Yp/s 0.38 0.06 0.37 0.22 Dcrit, h-1 0.12 Ethanol, g l-1 7.6 1.2 2.96 1.72 Glycerol, g l-1 0.48 3.1 2.69 Acetic acid, g l-1 Acetaldehyde, g l-1 0.61 Fig. 1 The critical dilution rate (Dc) and concentrations of biomass, ethanol and residual glucose in aerobic glucose-limited continuous culture of S. cerevisiae strains W303-1 (A) and Q5 (B) at steady state. Fig. 2 Determination of µmax of S. cerevisiae strain Q5 during batch cultivation (A) and by the wash-out procedure (B), using 4 g ethanol l-1 as carbon substrate. Conclusions The Adh5p isozyme as the only functional alcohol dehydrogenase in S. cerevisiae is sufficient for aerobic growth on glucose and ethanol, albeit at a lower growth rate than the parental strain and resulting in the accumulation of inhibitory concentrations of acetaldehyde. Adh5p did not substitute well for Adh2p and was insufficient for normal ethanolic fermentation of glucose. In strain Q5 grown on glucose, the oxidation of acetaldehyde to ethanol was apparently rate-limiting, as suggested by the accumulation of acetaldehyde. As a consequence, the rate of NADH re-oxidation via the acetaldehyde-ethanol pathway was probably also restricted, hence the accumulation of glycerol to mop up the excess of reducing equivalents. The use of a carbon-limited chemostat culture to facilitate growth of strain Q5 on ethanol proved successful, indicating that a previous observation of the inability of this strain to grow on ethanol was likely due to the accumulation of acetaldehyde in the culture. References Acknowledgements 1. Feldmann H, Aigle M, Aljinovic G, André B, Baclet MC, Barthe C, Baur A, Bécam AM, Biteau N, Boles E, Brandt T, Brendel M, Brückner M (1994) Complete DNA sequence of yeast chromosome II. EMBO J. 13, 5738-5745. 2. Ladriére JM, Georis I, Guérineau M, Vandenhaute J (2000) Kluyveromyces marxianus exhibits an ancestral Saccharomyces cerevisiae genome organization downstream of ADH2. Gene 255, 83-91. 3. Henn ME (2010) Over-expression, purification and characterization of Adh5p from Saccharomyces cerevisiae. M.Sc. dissertation, University of the Free State, Bloemfontein, South Africa. 4. de Smidt O (2007) Molecular and physiological aspects of alcohol dehydrogenases in the ethanol metabolism of Saccharomyces cerevisiae. Ph.D. thesis, University of the Free State, Bloemfontein, South Africa. The National Research Foundation, South Africa for financial support Mr. Sarel Marais for able technical assistance with the chromatographic analyses