A diploid strain of Saccharomyces cerevisiae able to metabolize lactose with high efficiency has been obtained. Haploid strains of Saccharomyces able to grow on lactose were constructed by cotransformation with two genes of Kluyveromyces lactis required for the utilization of the sugar, LAC4 and LAC12, encoding β-galactosidase and lactose permease respectively. Both genes were placed under the control of a galactose-inducible promoter and targeted to the rDNA encoding region (RDN1 locus) of the Saccharomyces genome. Lac+ transformants were selected on medium with lactose as the only carbon source. These transformants were mitotically stable, they maintained the Lac+ phenotype after growing in non-selective medium for more than 60 generations, but their growth was slow. We found that this lack of vigour was caused by their genetic background and not by a deficient expression of the heterologous genes. Therefore, their performance could be improved by crossing with a wild-type strain. Among the offspring of the crosses, two strains of opposite mating type were selected and mated to obtain a fast-growing Lac+ diploid. This diploid strain showed the typical fermentative behaviour of S. cerevisiae when it was grown in aerated liquid medium with glucose. In lactose medium, it exhibited a respiro-fermentative metabolism similar to that of K. lactis, with low ethanol production and high biomass yield. © 1998 John Wiley & Sons, Ltd.

In this investigation, a method for the accurate quantitative determination of net proton production or consumption in biological cultures has been devised. Cells are cultured under constant pH conditions. The specific rate of proton production or consumption by the culture (qH(+), mmol h(-1) per g biomass) is proportional to the mmol of base or acid required to maintain constant pH per unit time, and this equivalence is independent of the buffering capacity of the culture medium.The above method has been applied to chemostat cultures of Candida utilis growing on glucose or glycerol as carbon source, and different nitrogen sources. The results indicate that the nitrogen assimilation pathway alone determines the value of qH(+), and a fixed stoichiometric relationship between nitrogen uptake rate qN (meg h(-1) per g biomass) and qH(+) has been found for each nitrogen source employed. Thus, qH(+)/qN values of +1, 0 and -1 were found for ammonium ions, urea and nitrate respectively. Under oxidative metabolism, the contribution of carbon catabolism to the value of qH(+) was undetectable.Since qN may be related to growth and production of type 1 compounds in fermentation processes, the parameter qH(+) was incorporated into a model of growth and energy metabolism in chemostat culture (Castrillo and Ugalde, Yeast 10, 185-197, 1994), resulting in adequate simulations of experimentally observed culture performance. Thus, it is suggested that qH(+) may be employed as a simple and effective control parameter for biotechnological processes involving biomass-related products.

The pattern of energy metabolism of different types of yeasts (obligate aerobes and facultative anaerobes) in aerobic chemostat cultures has been evaluated and interpreted on the basis of a coupling of metabolic fluxes between glycolytic and oxidative components. A model has been formulated which defines glycolytic and oxidative subunits through which the substrate C-flux (gram-atom g-1 h-1) is calculated, stating that a relative imbalance between glycolytic flux and subsequent oxidative steps alone is sufficient to account for the onset of oxidoreductive metabolism in any type of yeast, irrespective of the maximum respiratory capacity. The model is able to reproduce the patterns of behaviour reported for the different types of yeasts, and the individual features of each strain are explained on the basis of metabolic differences which are defined by a set of normalized parameters. The model can be applied to different substrates and conditions, providing a methodological basis for more detailed studies of the steps controlling yeast energy metabolism.

A plasma membrane fraction was obtained by the combined use of differential centrifugation and aqueous polymer two-phase partitioning techniques. Vanadate-inhibited ATPase and glucan synthase activities were highly enriched in this fraction, although the presence of ATPase activity which was not inhibited by vanadate, nitrate, molybdate, anyimycin A or azide was also detected. Other intracellular membrane marker activities were present at very low or undetectable levels. A further separation step using Percoll density gradient centrifugation resulted in the separation of a fraction which exclusively contained vanadate-inhibited ATPase activity, and was enriched with silicotungstic-acid-staining membrane material. Latency tests performed on the plasma membrane markers showed that the membrane vesicles were in the right-side-out orientation.

The behavior of Kluyveromyces marxianus in aerobic chemostat cultures of deproteinated whey was studied in order to determine the characteristics of energy metabolism. The combined results show that K. marxianus presents a different pattern of behavior compared to Saccharomyces cereuisiae. A mathematical model has been constructed where energy metabolism is separated into two subunits: (a) a glycolytic subunit and (b) a respiratory subunit, through which the substrate C-flux (gram-atom g−1 h−1 is calculated. The coupling of fluxes assumes the existence of a respiratory pattern. Oxidoreductive metabolism appears as a consequence of an overflow effect resulting from relative increases in the substrate flux through the glycolytic subunit with respect to the respiratory unit, independent of the maximum respiratory capacity of the yeast. The model is able to reproduce the behavior of K. marxianus in the experimental conditions of culture implemented.

Summary: A new simple procedure for the production of protoplasts of Penicillium cyclopium with high regeneration rates and efficient transport activity is described, involving the use of a preparation of Novozym 234 with very low protease activity. The combination of a heat pretreatment at 55 °C for 15 min with the use of the protease inhibitor aprotinin resulted in a 97% reduction of Novozym 234 protease activity with respect to untreated controls. Polysaccharide-hydrolysing activity was inhibited much less, to 60% of the untreated Novozym 234 level. Protoplasts could be successfully produced with the new low-protease Novozym 234 preparation, showing a threefold increase in regeneration capacity compared to control protoplasts obtained with the original preparation. The rates of 3-O-methylglucose uptake and the capacity to accumulate this sugar analogue were also higher in protoplasts obtained by the new method.

A procedure for the production of plasma membrane vesicles, consisting of enzyme-assisted physical homogenization, differential centrifugation and partitioning in an aqueous polymer two phase system resulted in a fraction containing highly purified plasma membranes, as determined by vanadate-inhibited H+ ATPase and near absence of intracellular membrane marker activities. H+ ATPase latency tests indicated that at least 90% of the vesicles were oriented with the apoplastic surface facing outward (right-side-out orientation) in the upper phase, while inside out vesicles and other microsomal membrane material collected in the lower phase.

Cell wall-free protoplasts of P. cyclopium could regenerate a cell wall and form mycelia in liquid culture with high rates of viability. When calcium was added to the medium, protoplasts displayed biphasic accumulation with an immediate metabolism-independent adsorption phase, followed by slow metabolism-dependent uptake.

Penicillium notatum IMI 15378 sporulated profusely in submerged culture containing 10 mM-Ca 2+ , whilst the response of strain NRRL 832 was minimal up to 40 m m concentration of the cation. Calcium accumulation by mycelia of both strains was biphasic and involved rapid initial energy-independent binding, with the strains showing similar affinities for the cation, but with the responsive strain having a binding capacity four times greater than the nonresponsive strain. The second phase of Ca 2+ uptake was energy-dependent, involved ATPase activity, and showed saturation kinetics with K m values of 0.28 and 0.42 m m -Ca 2+ and V max values of 0.6 and 0.15 nmol (mg dry wt) −1 min −1 , for strains IMI 15378 and NRRL 832 respectively. Metabolically active preparations of protoplasts were free from wall contamination, and showed similar Ca 2+ binding affinities but these were lower than in the equivalent mycelia. The binding capacity of strain IMI 15378 was an order of magnitude greater than that of strain NRRL 832. Energy-dependent calcium transport was only demonstrated in strain IMI 15378 and followed saturation kinetics with a relatively low maximum influx rate. Isolated cell walls of both strains showed Ca 2+ binding characteristics similar to those of the respective mycelia.

The respiratory properties of isolated mitochondria from P. cyclopium were studied with particular attention to their response to calcium ions. The results obtained indicate concentration dependent stimulation of NADH oxidation by calcium ions. Similar effects could also be obtained with other divalent cations.

Uptake of calcium ions by Penicillium cyclopium in submerged culture was biphasic. The ion underwent rapid initial binding to the mycelium with a dissociation constant (KD) value of 0.114 mM-Ca2+ and binding capacity (Yt) of 3.5 nmol-Ca2+ (mg dry wt)−1. Metabolismindependent chemical adsorption of calcium was followed by metabolism-dependent active transport with a Km value of 1.88 m m and Vmax of 8.9 nmol-Ca2+ (mg dry wt)−1 min−1. Time-course experiments showed that exposures of mycelium to Ca2+ for periods as brief as 30 s were accompanied by substantial Ca2+ binding, but negligible active transport of the ion, and resulted in conidiation identical to that in cultures continuously exposed to Ca2+. The relationships between calcium binding and the mechanisms of conidiation in P. cyclopium are discussed.

Abstract. Recently much experimental evidence has accumulated concerning intracellular calcium and its fundamental role as a regulator in eukaryotic cells. The literature relating to Ca2+ in fungi is large and diverse and this paper draws together the available information and discusses the particular functions of the ion in this group of organisms.Uptake mechanisms in fungi are considered with special reference to the effect of Ca2+ on permeability and the systems responsible for transport of ions, sugars and amino acids. Discussion of the subcellular locations and distribution of Ca2+ is accompanied by a critique of methodology used in determination of subcellular sites of Ca2+ in fungi. The role of Ca2+ in morphogenesis in fungi is considered with particular reference to selected groups.

Intracellular sites of binding or accumulation of calcium ions, which are essential for induction of submerged conidiation in P. cyclopium , were determined by assay of 45 Ca in organelle fractions derived from mycelium which had previously been incubated with 45 Ca. The radionuclide was found principally in fractions rich in plasma membrane components and in intact mitochondria. Electron microscope autoradiography using 45 Ca was done using a modified fixation procedure which, by avoiding osmication, prevented extensive leaching of the isotope from the wall. Autoradiographs provided further evidence of calcium binding to the tonoplast in addition to its associations with the plasma membrane and the mitochondria. The physiological implications of the accumulation of calcium at subcellular sites are discussed with special reference to membrane binding and its relationship to conidiation.

Siliconization prevented sub-aerial accretions on the glass surface of shaken flasks and altered the pattern of pellet formation leading to improved growth and conidiation.