P. M. Bruinenberg

NADH-linked aldose reductase: the key to anaerobic alcoholic fermentation of xylose by yeasts

SummaryThe kinetics and enzymology of d-xylose utilization were studied in aerobic and anaerobic batch cultures of the facultatively fermentative yeasts Candida utilis, Pachysolen tannophilus, and Pichia stipitis. These yeasts did not produce ethanol under aerobic conditions. When shifted to anaerobiosis cultures of C. utilis did not show fermentation of xylose; in Pa. tannophilus a very low rate of ethanol formation was apparent, whereas with Pi. stipitis rapid fermentation of xylose occurred. The different behaviour of these yeasts ist most probably explained by differences in the nature of the initial steps of xylose metabolism: in C. utilis xylose is metabolized via an NADPH-dependent xylose reductase and an NAD+-linked xylitol dehydrogenase. As a consequence, conversion of xylose to ethanol by C. utilis leads to an overproduction of NADH which blocks metabolic activity in the absence of oxygen. In Pa. tannophilus and Pi. stipitis, however, apart from an NADPH-linked xylose reductase also an NADH-linked xylose reductase was present. Apparently xylose metabolism via the NADH-dependent reductase circumvents the imbalance of the NAD+/NADH redox system, thus allowing fermentation of xylose to ethanol under anaerobic conditions. The finding that the rate of xylose fermentation in Pa. tannophilus and Pi. stipitis corresponds with the activity of the NADH-linked xylose reductase activity is in line with this hypothesis. Furthermore, a comparative study with various xylose-assimilating yeasts showed that significant alcoholic fermentation of xylose only occurred in those organisms which possessed NADH-linked aldose reductase.

The role of redox balances in the anaerobic fermentation of xylose by yeasts

SummaryThe kinetics of glucose and xylose utilization by batch cultures of Candida utilis were studied under aerobic and anaerobic conditions during growth in complex media. Rapid ethanol formation occurred during growth on glucose when aerobic cultures were shifted to anaerobic conditions. However, with xylose as a substrate, transfer to anaerobiosis resulted in an immediate cessation of metabolic activity, as evidenced by the absence of both ethanol formation and xylose utilization. The inability of the yeast to ferment xylose anaerobically was not due to the absence of key enzymes of the fermentation pathway, since the addition of glucose to such cultures resulted in the immediate conversion of glucose to ethanol. Furthermore, when the enzyme xylose isomerase was added to an anaerobic xylose culture, immediate conversion of xylose to ethanol was observed. This indicates that the inability of the yeast to form ethanol from xylose under anaerobic conditions is caused by metabolic events associated with the conversion of xylose to xylulose. A hypothesis is put forward which explains that ethanol production from xylose by yeast under anaerobic conditions is negligible. It is suggested that the failure to ferment xylose anaerobically is due to a discrepancy between production and consumption of NADH in the overall conversion of xylose to ethanol. When a hydrogen acceptor (i.e. acetoin) was added to anaerobic cultures of C. utilis, xylose utilization resumed, and ethanol and acetate were produced with the concomitant stoicheiometric reduction of acetoin to 2,3-butanediol.

An enzymic analysis of NADPH production and consumption in Candida utilis

Candida utilis CBS 621 was grown in chemostat cultures at D = 0.1 h-1 on glucose, xylose, gluconate, acetate, or ethanol as the growth-limiting substrate with ammonia or nitrate as the nitrogen source and analysed for NADPH-producing and NADPH-consuming enzyme activities. Nitrate and nitrite reductases were strictly NADPH-dependent. For all carbon sources, growth with nitrate resulted in elevated levels of HMP pathway enzymes. NADP+-linked isocitrate dehydrogenase did not vary significantly with the NADPH requirement for biosynthesis. Growth on ethanol strongly enhanced activity of NADP+-linked aldehyde dehydrogenase. Neither NADP+-linked malic enzyme nor transhydrogenase activities were detectable under any of the growth conditions. The absence of transhydrogenase was confirmed by the enzyme profiles of cells grown on mixtures of glucose and formate. It is concluded that the HMP pathway and possibly NADP+-linked isocitrate dehydrogenase are the major sources of NADPH in Candida utilis.

A theoretical analysis of NADPH production and consumption in yeasts

Theoretical calculations of the NADPH requirement for yeast biomass formation reveal that this parameter is strongly dependent on the carbon and nitrogen source. The data obtained have been used to estimate the carbon flow over the NADPH-producing pathways in these organisms, namely the hexose monophosphate pathway and the NADP+-linked isocitrate dehydrogenase reaction. It was calculated that during growth of yeasts on glucose with ammonium as the nitrogen source at least 2% of the glucose metabolized has to be completely oxidized via the hexose monophosphate pathway for the purpose of NADPH synthesis. This figure increases to approximately 20% in the presence of nitrate as the nitrogen source. Not only during growth on glucose but also on other substrates such as xylose. methanol, or acetate the operation of the hexose monophosphate pathway as a source of NADPH is essential, since the N ADP+-isocitrate dehydrogenase reaction alone cannot meet the NADPH demand for anabolism. NADPH production via these pathways requires an expenditure of ATP. Therefore, the general assumption made in calculations of the ATP demand for biomass formation that generation of NADPH does not require energy is, at least in yeasts, not valid.

Phosphorus-31 nuclear magnetic resonance studies of intracellular pH, phosphate compartmentation and phosphate transport in yeasts

Abstract31P NMR spectra were obtained from suspensions of Candida utilis, Saccharomyces cerevisiae and Zygosaccharomyces bailii grown aerobically on glucose. Direct introduction of substrate into the cell suspension, without interruption of the measurements, revealed rapid changes in pH upon addition of the energy source. All 31P NMR spectra of the yeasts studied indicated the presence of two major intracellular inorganic phosphate pools at different pH environments. The pool at the higher pH was assigned to cytoplasmic phosphate from its response to glucose addition and iodoacetate inhibition of glycolysis. After addition of substrate the pH in the compartment containing the second phosphate pool decreased. A parallel response was observed for a significant fraction of the terminal and penultimate phosphates of the polyphosphate observed by 31P NMR. This suggested that the inorganic phosphate fraction at the lower pH and the polyphosphates originated from the same intracellular compartment, most probably the vacuole. In this vacuolar compartment, pH is sensitive to metabolic conditions. In the presence of energy source a pH gradient as large as 0.8 to 1.5 units could be generated across the vacuolar membrane. Under certain conditions net transport of inorganic phosphate across the vacuolar membrane was observed during glycolysis: to the cytoplasm when the cytoplasmic phosphate concentration had become very low due to sugar phosphorylation, and into the vacuole when the former concentration had become high again after glucose exhaustion.

Utilization of formate as an additional energy source by glucose-limited chemostat cultures ofCandida utilis CBS 621 andSaccharomyces cerevisiae CBS 8066

Candida utilis CBS 621 andSaccharomyces cerevisiae CBS 8066 were grown in glucose-limited chemostat cultures with formate as an additional energy source. In both yeasts formate was oxidized via a cytoplasmic NAD+-linked formate dehydrogenase. Other formate-oxidizing enzymes could not be detected.WithCandida utilis the steady-state cell yield on glucose increased with increasing amounts of formate in the medium until growth became carbon-limited. The maximum growth yield on glucose in the presence of excess formate was dependent on the nitrogen source used for growth. With ammonium and nitrate the maximum yields were 0.69 and 0.56 g cells/g glucose, respectively. Calculations showed that this difference correlates with the NADPH requirement for biomass formation with these two nitrogen sources. This implies that the NADH produced from formate oxidation cannot replace the NADPH needed for biomass formation. It therefore is concluded that inCandida utilis transhydrogenase activity is absent.AlsoSaccharomyces cerevisiae was capable of oxidizing formate in glucose-limited chemostat cultures. However, in contrast toCandida utilis utilization of formate by this yeast did not enhance the cell yield on glucose.

In vivo 31P NMR studies on the role of the vacuole in phosphate metabolism in yeasts

Abstract31P NMR was used to study the dynamics of phosphate pools during substrate utilization by aerobic and anaerobic suspensions of the yeast Candida utilis and by aerobic suspensions of the yeast Brettanomyces intermedius. In both yeast, the cytoplasmic pH was monitored; in C. utilis also the vacuolar pH could be measured. When glucose was used as a substrate for C. utilis, the vacuolar store of inorganic phosphorus (both orthophosphate and polyphosphate) was mobilized to replenish cytoplasmic phosphate which had become very low due to the build-up of high sugar phosphate levels. The hydrolysis of polyphosphate was glucose-dependent; it did not occur with ethanol as the substrate. After glucose depletion resynthesis of polyphosphate occurred only under aerobic conditions; anaerobic C. utilis cells continued to hydrolyze vacuolar polyphosphate. This difference between the aerobic and anaerobic suspension could be related to differences in cellular ATP levels. When ethanol was employed as a substrate, both Candida utilis and Brettanomyces intermedius exhibited a substantial increase in polyphosphate levels. These observations suggested a dual role for polyphosphate in yeasts both as a phosphate and an energy store. The cytoplasmic pH in C. utilis displayed characteristic responses to metabolic changes during glucose degradation. B. intermedius experienced a strong cytoplasmic acidification upon ethanol utilization due to acetic acid formation. The mechanism of transport of Pi across the vacuolar membrane in C. utilis appeared to be different from that reported for the plasma membrane.

Oxidation of NADH and NADPH by Mitochondria from the Yeast Candida utilis

Mitochondria were isolated from Candida utilis CBS 621 grown in carbon-limited continuous cultures on glucose, gluconate, xylose, ethanol or acetate as the carbon source and ammonia or nitrate as the nitrogen source. In all cases mitochondria were isolated which could oxidize exogenous NADH and NADPH via a cyanide- and antimycin A-sensitive but rotenone-insensitive respiratory chain. Oxidation of NADH and NADPH was coupled to energy conservation as evidenced by high respiratory control values. Different respiratory control values of mitochondria with NADH and NADPH as well as variations in the ratio of NADH and NADPH oxidase activities indicate that separate systems exist for the oxidation of exogenous redox equivalents by mitochondria of C. utilis. Variation of the NADPH requirement for biomass formation by applying differnt growth conditions did not result in significant changes in NADPH oxidase activities of mitochondria. It is concluded that in C. utilis NADPH can be used in dissimilatory processes for the generation of ATP.

The NADP(H) redox couple in yeast metabolism

Theoretical calculations of the NADPH requirement for biomass formation indicate that in yeasts this parameter is strongly dependent on the carbon and nitrogen sources used for growth. Enzyme surveys of NADPH-generating metabolic pathways and radiorespirometric studies demonstrate that in yeasts the HMP pathway is the major source of NADPH. Furthermore, radiorespirometric data suggest that in yeasts the HMP pathway activities are close to the theoretical minimum. It may be concluded that the mitochondrial NADPH oxidation, which in yeasts may yield ATP, is quantitatively not an important process.The inability of C. utilis to utilize the NADH produced in formate oxidation as an extra source of NADPH strongly suggests that transhydrogenase activity is absent. Furthermore, the absence of xylose utilization under anaerobic conditions in most facultatively fermentative yeasts indicates that also in these organisms transhydrogenase activity is absent. This conclusion is supported by the observation that anaerobic xylose utilization is observed only in those yeasts which possess a high activity of an NADH-linked xylose reductase. Hence in these organisms the redox-neutral conversion of xylose to ethanol is possible, since the second step in xylose metabolism is mediated by an NAD+-linked xylitol dehydrogenase.

Critical Parameters in the Isolation of Mitochondria from Candida utilis Grown in Continuous Culture

The successive steps in the isolation of mitochondria from chemostat-grown Candida utilis have systematically been investigated for their effects on organelle integrity. Growth rate had a profound effect on the susceptibility of carbon-limited cells towards Zymolyase, whereas the nature of the carbon source had no effect. Stabilization of spheroplasts with at least 2M-sorbitol was required to prevent premature lysis. This was concluded from the amounts of glucose-6-phosphate dehydrogenase liberated during Zymolyase treatments. The influence of the method for disruption of spheroplasts on the quality of the mitochondria was analysed with particular emphasis on respiratory control values and the distribution of marker enzymes among the cell fractions. Disruption by osmotic shock resulted in mitochondria without respiratory control and a high degree of solubilization of NADH and NADPH dehydrogenase activities. Only a gradual decrease of the osmotic value of the medium, preferably by dialysis against a hypotonic buffer, in combination with mechanical disruption with a Potter-Elvehjem homogenizer yielded mitochondria with high respiratory control values and a high retention of NADH dehydrogenase in the organelle. It is concluded that, for the quality of mitochondrial preparations from yeasts, the distribution of NADH dehydrogenase among the cell fractions is a more reliable measure than that of the usual marker enzymes.