Jacobus Thomas Pronk

Glucose uptake kinetics and transcription of HXT genes in chemostat cultures of Saccharomyces cerevisae

The kinetics of glucose transport and the transcription of all 20 members of the HXT hexose transporter gene family were studied in relation to the steady statein situ carbon metabolism of Saccharomyces cerevisiae CEN.PK113-7D grown in chemostat cultures. Cells were cultivated at a dilution rate of 0.10 h−1 under various nutrient-limited conditions (anaerobically glucose- or nitrogen-limited or aerobically glucose-, galactose-, fructose-, ethanol-, or nitrogen-limited), or at dilution rates ranging between 0.05 and 0.38 h−1 in aerobic glucose-limited cultures. Transcription ofHXT1–HXT7 was correlated with the extracellular glucose concentration in the cultures. Transcription of GAL2, encoding the galactose transporter, was only detected in galactose-limited cultures. SNF3 and RGT2, two members of the HXT family that encode glucose sensors, were transcribed at low levels. HXT8–HXT17 transcripts were detected at very low levels. A consistent relationship was observed between the expression of individual HXT genes and the glucose transport kinetics determined from zero-transinflux of 14C-glucose during 5 s. This relationship was in broad agreement with the transport kinetics of Hxt1–Hxt7 and Gal2 deduced in previous studies on single-HXT strains. At lower dilution rates the glucose transport capacity estimated from zero-trans influx experiments and the residual glucose concentration exceeded the measured in situ glucose consumption rate. At high dilution rates, however, the estimated glucose transport capacity was too low to account for the in situ glucose consumption rate.

Reductions of 3-oxo esters by baker's yeast : Current status

The objective of the current review is to present a mechanism and process engineering approach of stereospecific reductions of 3-oxo esters by bakers yeast. The stereospecific outcome of a reduction by bakers yeast depends on the kind of 3-oxo ester reductases involved and their specific activity. Various competing 3-oxo ester reductases are present in a yeast cell. An important aspect for efficient biotransformations with whole cells is the regeneration of NADH and NADPH cofactors. Use of different electron donors leads to the involvement of different metabolic routes influencing the reduction process. Optimization of the process conditions such as aeration, immobilization of cells, use of additives, or use of two phases, will enhance re-use of bakers yeast, yield, stereospecific outcome and scale up. Since the genome of bakers yeast is known, genetic engineering will soon increase the possibilities of stereoselective reductions.

Oxidation of reduced sulphur compounds by intact cells of Thiobacillus acidophilus

Oxidation of reduced sulphur compounds by Thiobacillus acidophilus was studied with cell suspensions from heterotrophic and mixotrophic chemostat cultures. Maximum substrate-dependent oxygen uptake rates and affinities observed with cell suspensions from mixotrophic cultures were higher than with heterotrophically grown cells. ph Optima for oxidation of sulphur compounds fell within the pH range for growth (pH 2–5), except for sulphite oxidation (optimum at pH 5.5). During oxidation of sulphide by cell suspensions, intermediary sulphur was formed. Tetrathionate was formed as an intermediate during aerobic incubation with thiosulphate and trithionate. Whether or not sulphite is an inter-mediate during sulphur compound oxidation by T. acidophilus remains unclear. Experiments with anaerobic cell suspensions of T. acidophilus revealed that trithionate metabolism was initiated by a hydrolytic cleavage yielding thiosulphate and sulphate. A hydrolytic cleavage was also implicated in the metabolism of tetrathionate. After anaerobic incubation of T. acidophilus with tetrathionate, the substrate was completely converted to equimolar amounts of thiosulphate, sulphur and sulphate. Sulphide- and sulphite oxidation were partly inhibited by the protonophore uncouplers 2,4-dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) and by the sulfhydryl-binding agent N-ethylmaleimide (NEM). Oxidation of elemental sulphur was completely inhibited by these compounds. Oxidation of thiosulphate, tetrathionate and trithionate was only slightly affected. The possible localization of the different enzyme systems involved in sulphur compound oxidation by T. acidophilus is discussed.

Role of NADP-dependent and quinoprotein glucose dehydrogenases in gluconic acid production by Gluconobacter oxydans

Abstract Gluconobacter oxydans, an organism used for the industrial production of gluconic acid, contains two glucose dehydrogenases (GDHs) catalysing the direct oxidation of glucose to gluconic acid. In addition to a quinoprotein (PQQ-dependent), membrane-bound GDH (EC 1.1.99.17), an NADP-dependent, cytoplasmic GDH is present. From three types of experiments, evidence is presented that the quinoprotein GDH is the enzyme responsible for gluconic acid production by G. oxydans. In cell-free extracts, the activity of quinoprotein GDH was 30-fold higher than the activity of NADP-dependent GDH. A kinetic analysis of glucose-dependent oxygen uptake showed that a system with an affinity constant similar to the Km of quinoprotein GDH is involved in the process of gluconic acid production. The conclusion that gluconic acid production is essentially an extracytoplasmic process catalysed by quinoprotein GDH was confirmed in uptake experiments, which demonstrated that inhibition of glucose transport does not result in inhibition of gluconic acid production.

Heterotrophic Growth of Thiobacillus-Acidophilus in Batch and Chemostat Cultures

Heterotrophic growth of the facultatively chemolithoautotrophic acidophile Thiobacillus acidophilus was studied in batch cultures and in carbon-limited chemostat cultures. The spectrum of carbon sources supporting heterotrophic growth in batch cultures was limited to a number of sugars and some other simple organic compounds. In addition to ammonium salts and urea, a number of amino acids could be used as nitrogen sources. Pyruvate served as a sole source of carbon and energy in chemostat cultures, but not in batch cultures. Apparently the low residual concentrations in the steady-state chemostat cultures prevented substrate inhibition that already was observed at 150 μM pyruvate. Molar growth yields of T. acidophilus in heterotrophic chemostat cultures were low. The Ymax and maintenance coefficient of T. acidophilus grown under glucose limitation were 69 g biomass · mol−1 and 0.10 mmol · g−1 · h−1, respectively. Neither the Ymax nor the maintenance coefficient of glucose-limited chemostat cultures changed when the culture pH was increased from 3.0 to 4.3. This indicates that in T. acidophilus the maintenance of a large pH gradient is not a major energy-requiring process. Significant activities of ribulose-1,5-bisphosphate carboxylase were retained during heterotrophic growth on a variety of carbon sources, even under conditions of substrate excess. Also thiosulphate- and tetrathionate-oxidising activities were expressed under heterotrophic growth conditions.

Effects of cultivation conditions on the production of heterologous α-galactosidase by Kluyveromyces lactis

Growth conditions relevant for the large-scale production of heterologous proteins with yeasts were studied on a laboratory scale. A strain of Kluyveromyces lactis, containing 15 copies of an expression cassette encoding guar α-galactosidase integrated into its ribosomal DNA, was used as a model. By using urea as a nitrogen source, it was possible to produce active extracellular α-galactosidase in shake-flask cultures grown on a defined mineral medium. Inclusion of urea instead of ammonium sulphate prevented unwanted acidification of cultures. With urea-containing mineral medium, enzyme production in shake flasks was comparable to that in complex media containing peptone. In contrast, the presence of peptone was required to achieve high productivity in chemostat cultures. The low productivity in chemostat cultures growing on mineral media was not due to loss oft the expression cassette, since addition of peptone to such cultures resulted in an immediate high rate of α-galactosidase production. The discrepancy between the behaviour of shake-flask and chemostat cultures during growth on mineral medium illustrates the necessity of physiological studies for the scalling-up of heterologous protein production from laboratory to production scale.

Preparation of D-xylulose from D-xylose

A simple method is described for the preparation of D-xylulose. It consists of the isomerization of D-xylose with xylose isomerase (EC 5.3.1.5), yielding an equilibrium mixture of o-xylulose and D-xylose. This is followed by the quantitative oxidation of residual o-xylose to o-xylonic acid with immobilized A. calcoaceticus cells. A combination of methanol precipitation and ion exchange is used for the removal of xylonic acid. This procedure offers many advantages over existing methods for the preparation of o-xylulose. The purity of the final product compares favorably to that of a commercial o-xylulose preparation.

ENERGETICS OF MIXOTROPHIC AND AUTOTROPHIC C1-METABOLISM BY THIOBACILLUS ACIDOPHILUS

Although the facultatively autotrophic acidophile Thiobacillus acidophilus is unable to grow on formate and formaldehyde in batch cultures, cells from glucose-limited chemostat cultures exhibited substrate-dependent oxygen uptake with these C1-compounds. Oxidation of formate and formaldehyde was uncoupler-sensitive, suggesting that active transport was involved in the metabolism of these compounds. Formate- and formaldehyde-dependent oxygen uptake was strongly inhibited at substrate concentrations above 150 and 400 μM, respectively. However, autotrophic formate-limited chemostat cultures were obtained by carefully increasing the formate to glucose ratio in the reservoir medium of mixotrophic chemostat cultures. The molar growth yield on formate (Y=2.5 g ·mol-1 at a dilution rate of 0.05 h-1) and RuBPCase activities in cell-free extracts suggested that T. acidophilus employs the Calvin cycle for carbon assimilation during growth on formate. T. acidophilus was unable to utilize the C1-compounds methanol and methylamine. Formate-dependent oxygen uptake was expressed constitutively under a variety of growth conditions. Cell-free extracts contained both dye-linked and NAD-dependent formate dehydrogenase activities. NAD-dependent oxidation of formaldehyde required reduced glutathione. In addition, cell-free extracts contained a dye-linked formaldehyde dehydrogenase activity. Mixotrophic growth yields were higher than the sum of the heterotrophic and autotrophic yields. A quantitative analysis of the mixotrophic growth studies revealed that formaldehyde was a more effective energy source than formate.

Regulation of alcohol-oxidizing capacity in chemostat cultures of Acetobacter pasteurianus

Acetobacter pasteurianus LMG 1635 was studied for its potential application in the enantioselective oxidation of alcohols. Batch cultivation led to accumulation of acetic acid and loss of viability. These problems did not occur in carbon-limited chemostat cultures (dilution rate = 0.05 h−1) grown on mineral medium supplemented with ethanol, L-lactate or acetate. Nevertheless, biomass yields were extremely low in comparison to values reported for other bacteria. Cells exhibited high oxidation rates with ethanol and racemic glycidol (2,3-epoxy-1-propanol). Ethanol- and glycidol-dependent oxygen-uptake capacities of ethanol-limited cultures were higher than those of cultures grown on lactate or acetate. On all three carbon sources, A. pasteurianus expressed NAD-dependent and dye-linked ethanol dehydrogenase activity. Glycidol oxidation was strictly dye-linked. In contrast to the NAD-dependent ethanol dehydrogenase, the activity of dye-linked alcohol dehydrogenase depended on the carbon source and was highest in ethanol-grown cells. Cell suspensions from chemostat cultures could be stored at 4°C for over 30 days without significant loss of ethanol- and glycidol-oxidizing activity. It is concluded that ethanol-limited cultivation provides an attractive system for production of A. pasteurianus biomass with a high and stable alcohol-oxidizing activity.

Energization of Solute Transport by Pqq-Dependent Glucose-Dehydrogenase in Membrane-Vesicles of Acinetobacter Species

Most Acinetobacter strains can not grow on glucose or gluconic acid as a sole carbon source. However, when glucose is added to the growth medium, it is quantitatively oxidized to gluconic acid. The enzyme responsible for this reaction is a membrane-bound glucose dehydrogenase (GDH, EC 1.1.99.17) which contains PQQ as a prosthetic group. GDH synthesis is constitutive in A. calcoaceticus. Strains of A. lwoffi, on the other hand, are unable to oxidize glucose but nevertheless contain apo-GDH under all growth conditions. Addition of the prosthetic group PQQ is required to restore GDH activity in A. lwoffi (Van Schie et al., 1984). In order to study the functional coupling of GDH to the respiratory chain we developed a procedure for the isolation of membrane vesicles from bacteria grown under well-defined conditions. Cells were collected at 4 ~ from an acetate-limited continuous culture. The method for the isolation of membrane vesicles was a modification of the procedure described for Pseudomonas aeruginosa by Stinnett et al. (1973). Membrane vesicles prepared from carbon-limited chemostat cultures of A. calcoaceticus LMD 79.41 and A. lwoffi LMD 83.25 exhibited active transport of alanine energized by GDH. In case of A. lwoffi both oxidation of glucose and active transport energized by glucose oxidation were dependent on the presence of PQQ. The rate of alanine uptake when energized with GDH was comparable with the rate of uptake energized by the artificial electron donor system ascorbate phenazine methosulphate (Table l).