Biomedical subjects

Jack T Pronk

Publications and source records attributed to Jack T Pronk.

39 records · Page 3Linked to original sources

PubMed2002

Novel pathway for alcoholic fermentation of delta-gluconolactone in the yeast Saccharomyces bulderi.

Under anaerobic conditions, the yeast Saccharomyces bulderi rapidly ferments delta-gluconolactone to ethanol and carbon dioxide. We propose that a novel pathway for delta-gluconolactone fermentation operates in this yeast. In this pathway, delta-gluconolactone is first reduced to glucose via an NADPH-dependent glucose dehydrogenase (EC 1.1.1.47). After phosphorylation, half of the glucose is metabolized via the pentose phosphate pathway, yielding the NADPH required for the glucose-dehydrogenase reaction. The remaining half of the glucose is dissimilated via glycolysis. Involvement of this novel pathway in delta-gluconolactone fermentation in S. bulderi is supported by several experimental observations. (i) Fermentation of delta-gluconolactone and gluconate occurred only at low pH values, at which a substantial fraction of the substrate is present as delta-gluconolactone. Unlike gluconate, the latter compound is a substrate for glucose dehydrogenase. (ii) High activities of an NADP(+)-dependent glucose dehydrogenase were detected in cell extracts of anaerobic, delta-gluconolactone-grown cultures, but activity of this enzyme was not detected in glucose-grown cells. Gluconate kinase activity in cell extracts was negligible. (iii) During anaerobic growth on delta-gluconolactone, CO(2) production exceeded ethanol production by 35%, indicating that pyruvate decarboxylation was not the sole source of CO(2). (iv) Levels of the pentose phosphate pathway enzymes were 10-fold higher in delta-gluconolactone-grown anaerobic cultures than in glucose-grown cultures, consistent with the proposed involvement of this pathway as a primary dissimilatory route in delta-gluconolactone metabolism.

Anaerobiosis↗

PubMed1998

Inactivation of the Kluyveromyces lactis KlPDA1 gene leads to loss of pyruvate dehydrogenase activity, impairs growth on glucose and triggers aerobic alcoholic fermentation.

The KlPDA1 gene, encoding the E1alpha subunit of the mitochondrial pyruvate-dehydrogenase (PDH) complex was isolated from a Kluyveromyces lactis genomic library by screening with a 1.1 kb internal fragment of the Saccharomyces cerevisiae PDA1 gene. The predicted amino acid sequence encoded by KlPDA1 showed 87% similarity and 79% identity to its S. cerevisiae counterpart. Disruption of KIPDA1 resulted in complete absence of PDH activity in cell extracts. The maximum specific growth rate on glucose of null mutants was 3.5-fold lower than that of the wild-type, whereas growth on ethanol was unaffected. Wild-type K. lactis CBS 2359 exhibits a Crabtree-negative phenotype, i.e. no ethanol was produced in aerobic batch cultures grown on glucose. In contrast, substantial amounts of ethanol and acetaldehyde were produced in aerobic cultures of an isogenic Klpda1 null mutant. A wild-type specific growth rate was restored after introduction of an intact KlPDA1 gene but not, as previously found for S. cerevisiae pda1 mutants, by cultivation in the presence of leucine. The occurrence of aerobic fermentation and slow growth of the Klpda1 null mutant indicate that, although present, the enzymes of the PDH bypass (pyruvate decarboxylase, acetaldehyde dehydrogenase and acetyl-CoA synthetase) could not efficiently replace the PDH complex during batch cultivation on glucose. Only at relatively low growth rates (D = 0.10 h(-1)) in aerobic, glucose-limited chemostat cultures, could the PDH bypass completely replace the PDH complex, thus allowing fully respiratory growth. This resulted in a lower biomass yield [g biomass (g glucose)-1] than in the wild-type due to a higher consumption of ATP in the PDH bypass compared to the formation of acetyl-CoA via the PDH complex.

Aerobiosis↗

PubMed1996

Metabolic fluxes in chemostat cultures of Schizosaccharomyces pombe grown on mixtures of glucose and ethanol.

Simultaneous utilization of glucose and ethanol by the yeast Schizosaccharomyces pombe CBS 356 was studied in aerobic chemostat cultures. In glucose-limited cultures, respirofermentative metabolism occurred at growth rates above 0.16 h-1. Although Sch. pombe lacks a functional glyoxylate cycle and therefore cannot utilize ethanol as a sole carbon source, ethanol was co-consumed by glucose-limited chemostat cultures. As a result, biomass yields increased, but not up to the theoretical value [0.92 g biomass (g glucose)-1] expected if all of the acetyl-CoA produced from glucose was instead synthesized from ethanol. When ethanol accounted for more than 30% of the substrate carbon in the mixed feed, it was incompletely utilized. In mixed-substrate cultures with a saturating ethanol fraction in the feed, the increase of the biomass yield as a result of ethanol consumption was highest at low dilution rates. This was not due to an increased specific rate of ethanol consumption at low growth rates; rather, the longer residence times at low dilution rates allowed Sch. pombe to utilize a larger fraction of the available ethanol, part of which was oxidized to acetate. Activities of gluconeogenic and glyoxylate-cycle enzymes were not detected in cell-free extracts of any of the cultures. Activities of acetaldehyde dehydrogenase and acetyl-CoA synthetase were low and of the same order of magnitude as the in vivo rates of acetate activation to acetyl-CoA. The results show that ethanol is a poor substrate for Sch. pombe, even as an auxiliary energy source.

Adenosine Triphosphate↗