Summary. The kinetics and enzymology of o-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.

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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. I N T R O D U C T I O NDespite its well-known function as a reductant in anabolic metabolism, little is known about the quantitative aspects of NADPH production and consumption in micro-organisms. In contrast, the ATP balance in micro-organisms has been analysed in detail (Stouthamer, 1973(Stouthamer, , 1977. As for ATP, the amount of NADPH required for biosynthesis of cell constituents from central metabolic intermediates and ammonia is a constant. This requirement only depends on the relative amounts of monomers, i.e. amino acids, fatty acids, nucleotides and hexose phosphates. However, due to differences in NADPH production and consumption in the conversion of various carbon sources to, for example, triose phosphates, and of various nitrogen sources to ammonia, the NADPH requirement for biosynthesis will be strongly dependent on the carbon and nitrogen source used for growth. It is therefore evident that the NADPH balance of the cell must be carefully controlled, and that the NADPH-producing systems must be tuned to the NADPH-consuming processes in relation to environmental conditions.In this paper an attempt has been made to quantify the NADPH requirement for biomass synthesis from different carbon and nitrogen sources. Since in yeasts NADPH must be generated via intermediary pathways of carbon metabolism (see below) attention has been focused on the metabolic consequences of the NADPH requirement in terms of carbon flow over N A DP H-producing pathways .Before a quantitative estimation is made of the NADPH consumption in anabolism and NADPH production in catabolism, some relevant information on the nature of these processes in yeasts will be discussed.

Candida utilis CBS 621 was grown in chemostat cultures at D = 0.1 h-' 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.

Transcriptional gene fusions with the Escherichia coli ␤-glucuronidase gene (gusA) were used to study the medium-and growth-dependent expression of the divergently transcribed genes involved in proteinase production (prtP and prtM) of Lactococcus lactis SK11. The results show that both the prtP and prtM genes are controlled at the transcriptional level by the peptide content of the medium and, to a lesser extent, by the growth rate. A more than 10-fold regulation in ␤-glucuronidase activity was observed for both prtP and prtM promoters in batch and continuous cultures. The level of expression of the prtP and prtM promoters was high in whey permeate medium with relatively low concentrations of peptides, whereas at increased concentrations the expression of the promoters was repressed. The lowest level of expression was observed in peptide-and amino acid-rich laboratory media, such as glucose-M17 and MRS. The addition of specific dipeptides, such as leucylproline and prolylleucine, to the growth medium negatively affected the expression of the prtP-gusA fusions. The repression by dipeptides was not observed in mutants defective in the uptake of di-tripeptides, indicating that the internal concentration of dipeptides or derivatives is important in the regulation of proteinase production.