Highly efficient assimilation of lactose by a metabolically engineered strain of Saccharomyces cerevisiae.

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 beta-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.

On-line estimation of biomass through pH control analysis in aerobic yeast fermentation systems.

The amount of acid or base consumed in yeast cultures has been recently assigned to the pathway of nitrogen assimilation under respiratory conditions with no contribution by carbon metabolism (Castrillo et al., 1995). In this investigation, experiments under respirofermentative conditions have shown that production or consumption of ethanol does not contribute significantly to the specific rate of proton production (qH+), thus extending the previously obtained relationships for all aerobic conditions in which other major acid/base contributions are not involved. Tests in batch and chemostat culture confirm the validity of qH+ as a formal control parameter in aerobic fermentations.

Proton production and consumption pathways in yeast metabolism. A chemostat culture analysis.

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 (meq 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. Sine 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.

Purification of functionally sealed cytoplasmic side-out plasma membrane vesicles from Saccharomyces cerevisiae.

Highly purified plasma membrane vesicles were prepared from yeast protoplasts by a combination of osmotic lysis, differential centrifugation, and separation in an aqueous dextran/polyethylene glycol two-phase system. The vesicles were predominantly (85-90%) of cytoplasmic side-out orientation and displayed large ATP-dependent proton pumping activity which was inhibited by vanadate (100 microM) but not by bafilomycin or nitrate. The preparation presented a distinct polypeptide profile with respect to the total membrane fraction and was enriched in the 110-kDa polypeptide corresponding to the plasma membrane H(+)-ATPase. This preparation of native plasma membranes vesicles is especially suitable for functional studies in vitro.

A general model of yeast energy metabolism in aerobic chemostat culture.

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.

Preparation and characterization of whey protein hydrolysates: applications in industrial whey bioconversion processes.

A whey protein hydrolysate was prepared by incubation of reconstituted whey or a whey protein concentrate with Alcalase 0.6L. The proteolytic degradation of alpha-lactalbumin and beta-lactoglobulin initially resulted in 6-kDa and, later, 2.5-kDa degradation products, quickly followed by the appearance of multiple peptides of 1 kDa or smaller. The hydrolysate showed a steady increase in solubility and a biphasic change in foaming characteristics with decreasing peptide size. At the highest degree of hydrolysis achieved (22%), the majority of the peptides were smaller than 1 kDa and could be efficiently assimilated by the yeast Kluyveromyces marxianus growing in a defined medium.

Calcium-induced conidiation in Penicillium cyclopium: calcium triggers cytosolic alkalinization at the hyphal tip.

Addition of Ca2+ (1 to 10 mM) to submerged cultures of Penicillium cyclopium induces conidiation. Ca2+ induced an increase in cytosolic pH from approximately 7.00 to > 7.60 in less than 10 min, as determined with the fluorescent pH probe fluorescein. Measurement of the H(+)-ATPase activity in total membrane fractions did not show any stable activation in vivo as a result of Ca2+ treatment. By fluorescence ratio imaging microscopy, it was observed that vegetative hyphae exhibit a tip-to-base pH gradient, with the tip being more acidic. Ca2+ caused this gradient to dissipate within 10 min. The effect of several agents that are supposed to cause internal acidification, by different means, on conidiation was tested. Concentrations of these agents that did not significantly affect growth but inhibited Ca(2+)-induced conidiation also prevented the intracellular alkalinization observed after exposure to the cation. Calcium channel blockers (lanthanum, cobalt, verapamil, and nifedipine) were not able to inhibit Ca(2+)-induced conidiation, although their effect on calcium uptake was not evaluated. However, the combined results point towards externally bound Ca2+ as the primary agent of conidiation induction, causing changes in plasma membrane function which disrupt the pH gradient observed during apical growth.

Preparation of right-side-out plasma membrane vesicles from Penicillium cyclopium: a critical assessment of markers.

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.

Calcium binding and induction of conidiation in protoplasts of Penicillium cyclopium.

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. Exposure of the protoplasts to Ca2+ for periods of 2 min, followed by incubation in calcium-free medium for 24 hours, was sufficient to induce conidiation with morphogenetic events parallel to those found in cultures containing calcium throughout the incubation period, and similar to those reported in cultures inoculated from conidia. The conidiation event caused by short exposure to calcium could be reversed, within 2 hours of Ca2+ addition, by a brief treatment with the specific calcium chelating agent BAPTA (100 microM), which removed 65 to 75% of the total cell calcium. The results implicate the membrane-bound calcium fraction in the process of conidiation induction.

Properties of mitochondria from Penicillium cyclopium and their response to calcium and other divalent cations.

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.