Othmar Käppeli

An Expanded Concept for the Glucose Effect in the Yeast Saccharomyces uvarum: Involvement of Short- and Long-term Regulation

SUMMARY: When Saccharomyces uvarum was cultivated in continuous culture, it exhibited the typical growth behaviour of a glucose-sensitive yeast. Metabolic changes related to glucose-repressed growth were assessed by an analysis of overall culture parameters (biomass formation, ethanol and acetate production and gas exchange rates) and by measuring the mitochondrial cytochrome content. These functions were mainly affected by the glucose effect; the steady state values of these variables were first established in the chemostat as a function of dilution rate. The short- and long-term regulation taking place when the cells were submitted to repression was assessed by administering glucose pulses and by shifts in the dilution rate. The primary response of the cells to the initiation of repressed growth was the formation of ethanol and acetate. Since there was no repression of oxygen uptake rate or cytochrome content prior to this response, it was concluded that ethanol and acetate formation was not the consequence of repression of respiratory activity, but resulted from the regulation of pyruvate dehydrogenase and pyruvate decarboxylase activities. Long-term adaptation of the cells occurred within 24 to 48 h of the initiation of repressed growth as manifested by a decrease of mitochondrial cytochrome content to the steady state value corresponding to that of repressed growth.

Biosurfactants from Bacillus licheniformis : structural analysis and characterization

SummaryThe surface-active compounds of the strain Bacillus licheniformis were isolated and their structure was elucidated. The high surface-active capacity of the crude extract was basically due to traces of long-chain saturated fatty acids, especially of palmitic and stearic acids, to a mixture of small amounts of hydrocarbons with chain lengths of 20 and 22 carbons, and mainly to appreciable amounts of four slightly different lipopeptides. The lipopeptides were found to be a mixture of four closely related compounds. The lipophilic part consisting of i-, n-C14 or i-, ai-C15 β-OH fatty acids was linked to the hydrophilic peptide moiety, which contained seven amino acids (Glu, Asp, Val, three Leu and Ile) by a lactone linkage. Fifteen milligrams per litre of the purified lipopeptide product decreased the surface tension of water from 72 mN m−1 to 27 mN m−1, characterizing the product as a powerful surface-active agent that compares favourably to other (bio)surfactants. Antibiotic activity was demonstrated against bacteria and yeasts.

Transient Responses of Saccharomyces uvarum to a Change of the Growth-limiting Nutrient in Continuous Culture

SUMMARY: The transition of Saccharomyces uvarum from oxidative to oxido-reductive glucose metabolism was characterized by the immediate formation of ethanol and the gradual adaptation of cells to a new physiological state, as expressed by a decrease in mitochondrial cytochrome content and a loss of malate dehydrogenase activity. Shifting the cells from carbon-limited to oxygen-, nitrogen- and iron-limited media in a continuous culture growing oxidatively led to the initiation of the same effects. The results lend support to the concept of a limited respiratory capacity as the basis for the occurrence of oxido-reductive glucose metabolism. Limitation of growth by a nutrient other than glucose results in an imbalance between glucose flux and biosynthetic potential of the cells and causes oxido-reductive glucose breakdown.

Structure of the cell surface of the yeast Candida tropicalis and its relation to hydrocarbon transport.

The surface structure of the hypdrocarbon-utilizing yeast Candida tropicalis was investigated by scanning and transmission electron microscopy (SEM and TEM respectively). The sample preparation technique was based on a rapid cryofixation without any addition of cryoprotectants. In subsequently freeze-dried samples the surface structure was analysed by scanning electron microscopy. Thin sections were prepared from freeze substituted samples. Both techniques revealed hair-like structures at the surface of hydrocarbon-grown cells. The hairy surface structure of the cells was less expressed in glucose-grown cells and it was absent completely after proteolytic digestion of the cells. When cells were incubated with hexadecane prior to cyryofixation a contrast-rich region occured in the hair fringe of thin sections as revealed by TEM. Since these structures were characteristic for hexadecane-grown cells and could not be detected in glucose-grown or proteasetreated cells it was concluded that they originate from hexadecane adhering to the cell surface and are functionally related to hexadecane transport. The structure of the surface and its relation to hydrocarbon transport are discussed in view of earlier results on the chemical composition of the surface layer of the cell wall.

Convenient procedure for the isolation of highly enriched, cytochrome P-450-containing microsomal fraction from Candida tropicalis

Abstract The suitability of Ca2+ ions for the precipitation of the microsomal fraction from the hydrocarbon-grown yeast Candida tropicalis was evaluated. In the final procedure the microsomes were precipitated by the addition of 16 m m CaCl2. Crude extracts obtained from cells via spheroplast lysis were centrifuged at 12,000g for 15 min and at 25,000g for 15 min prior to precipitation. The cytochrome P-450 content of the fraction was between 0.22 and 0.35 nmol mg−1 protein. The isolated microsomes exhibited both hexadecane hydroxylation activity and NADPH-cytochrome c reductase activity.

Chemostat studies on the hexadecane assimilation by the yeastCandida tropicalis

SummaryThe yeastCandida tropicalis was grown on glucose or hexadecane in batch cultures and on hexadecane in chemostat cultures. The cytochrome content and activities of several enzymes were determined in harvested cells.Cytochrome P-450 was induced and long chain alcohol and aldehyde hydrogenase were derepressed when hexadecane was the sole carbon source instead of glucose. Therefore, these enzymes are functionally related to hexadecane oxidation.A remarkable relationship was observed between the relative content of cytochrome P-450 and Qs in the chemostat and this relationship was more obvious when the cells grew in conditions of oxygen limitation. Alcohol and aldehyde dehydrogenase showed no regulation and therefore the primary hydroxylation of hexadecane by cytochrome P-450 is presumed to be the rate limiting step in hexadecane uptake and subsequent oxidation to palmitate.The specific activity of cytochromec oxidase was also remarkably higher when the cells were grown under conditions of oxygen limitation. Limitation of the electron transport chains by the oxidases, in conditions of oxygenlimitation, is discussed.

The Respirative Breakdown of Glucose by Saccharomyces cerevisiae: an Assessment of a Physiological State

Cells of Saccharomyces cerevisiae exhibiting respirative glucose metabolism in continuous culture were able to use ethanol as a co-substrate. The ethanol uptake rate was dependent on the residual respirative capacity of the cells. The activities of gluconeogenic enzymes and of malate dehydrogenase were higher in cells degrading glucose respiratively than in cells metabolizing glucose respiro-fermentatively, but were lower than in cells growing on ethanol only. The pattern of distribution of the mitochondrial cytochromes was similar but the differences were less distinct. In synchronously growing cells, the activities of gluconeogenic enzymes and of malate dehydrogenase oscillated, with activities increasing during the budding phase. The increase was preceded by the appearance of ethanol in the culture medium.

Genetic transformation of the filamentous yeast, Trichosporon cutaneum, using dominant selection markers

An efficient transformation system for the filamentous yeast, Trichosporon cutaneum, has been developed. Transformation was obtained with plasmids carrying either the Escherichia coli hygromycin B phosphotransferase-encoding gene (hph) or the Streptoalloteichus hindustanus phleomycin-resistance gene (ble), as dominant selection markers. Expression of both resistance-conferring genes was controlled by the gpd promoter and the trpC terminator, from Aspergillus nidulans. The transformation frequency was up to 500 colonies/micrograms of transforming DNA, using the ble gene, and up to 100 colonies/micrograms of transforming DNA, using the hph gene. Co-transformation frequencies using unselected DNA varied between 50 and 65%. The transforming DNA was found to consist of multiple tandem plasmid copies of high Mr. This polymeric structure, in nonselective media, was mitotically unstable, possibly indicating that it existed in an episomal state.

The distinction of different types of cytochromes P-450 from the yeasts Candida tropicalis and Saccharomyces uvarum☆

The distinction between two types of cytochromes P-450 originating from microsomes of Candida tropicalis grown on glucose and on alkane was achieved. Criteria of differentiation between these two cytochrome P-450 forms were based on the characteristics of reduced carbon monoxide difference spectra, on substrate specificity, and on binding and inhibition kinetics of the fungistatic compound propiconazole. One cytochrome P-450 form catalyzed the 14 alpha-demethylation of lanosterol and bound propiconazole with an equimolar ratio. This form was present in microsomes from glucose-grown cells and shared similar characteristics with the cytochrome P-450 originating from Saccharomyces uvarum grown on the same carbon source. The other cytochrome P-450 form catalyzed the terminal hydroxylation of aliphatic hydrocarbons and showed a less specific binding ratio with propiconazole (10(3) mol propiconazole for 1 mol cytochrome P-450). This type of cytochrome P-450 was only present in the microsomes of C. tropicalis grown on alkane.

A comparative study of carbon energy reserve metabolism ofC. tropicalis growing on glucose and on hydrocarbons

SummaryGlycogen was markedly accumulated inCandida tropicalis growing on glucose with increasing limitation of external substrate supply. The same effect was caused by a N-free medium. The lipid content did not show any significant change in either case. On the hydrocarbon substrate, lipid increased as substrate availability decreased, whereas glycogen accumulation was only slight. However, the increase of lipid content on hydrocarbons did not reach the same level of accumulation as glycogen on glucose. In N-free medium both glycogen and lipids were accumulated, indicating that glycogen is not substituted by lipids as the carbon energy reserve on a hydrocarbon substrate.In addition, a refined shift technique is described. The disturbing influence of excess substrate at the beginning of a shift from glucose to hydrocarbons is avoided by a portioned substrate feeding according to the cell activity.