Physiology of the yeast Kluyveromyces marxianus during batch and chemostat cultures with glucose as the sole carbon source

Growth, substrate consumption, metabolite formation, biomass composition and respiratory parameters of Kluyveromyces marxianus ATCC 26548 were determined during aerobic batch and chemostat cultivations, using mineral medium with glucose as the sole carbon source, at 30 degrees C and pH 5.0. Carbon balances closed within 95-101% in all experiments. A maximum specific growth rate of 0.56 h(-1), a biomass yield on glucose of 0.51 g g(-1), and a maximum specific consumption of oxygen of 11.1 mmol g(-1) h(-1) were obtained during batch cultures. The concentration of excreted metabolites was very low at the culture conditions applied, representing 6% of the consumed carbon at most. Acetate and pyruvate were excreted to a larger extent than ethanol under the batch conditions, and the protein content accounted for 54.6% of the biomass dry weight. Steady states were obtained during chemostats at dilution rates of 0.1, 0.25 and 0.5 h(-1). At the two former dilution rates, cells grew at carbon limitation and the biomass yield on glucose was similar to that obtained under the batch conditions. Metabolite formation was rather low, accounting for a total of 0.005 C-mol C-mol(-1) substrate. At 0.5 h(-1), although the biomass yield on glucose was similar to the value obtained under the above-mentioned conditions, the cultivation was not under carbon limitation. Under this condition, 2-oxoglutarate, acetate, pyruvate and ethanol were the prevalent metabolites excreted. Total metabolite formation only accounted to 0.056 C-mol C-mol(-1) of substrate. A very high protein and a low carbohydrate content (71.9% and 9.6% of biomass dry weight, respectively) were measured in cells under this condition. It is concluded that K. marxianus aligns with the so-called aerobic-respiring or Crabtree-negative yeasts. Furthermore, it has one of the highest growth rates among yeasts, and a high capacity of converting sugar into biomass, even when carbon is not the limiting nutrient. These results provide useful data regarding the future application of K. marxianus in processes aimed at the production of biomass-linked compounds, with high yields and productivities.     

Network identification and flux quantification in the central metabolism of Saccharomyces cerevisiae under different conditions of glucose repression

The network structure and the metabolic fluxes in central carbon metabolism were characterized in aerobically grown cells of Saccharomyces cerevisiae. The cells were grown under both high and low glucose concentrations, i.e., either in a chemostat at steady state with a specific growth rate of 0.1 h(-1) or in a batch culture with a specific growth rate of 0.37 h(-1). Experiments were carried out using [1-(13)C]glucose as the limiting substrate, and the resulting summed fractional labelings of intracellular metabolites were measured by gas chromatography coupled to mass spectrometry. The data were used as inputs to a flux estimation routine that involved appropriate mathematical modelling of the central carbon metabolism of S. cerevisiae. The results showed that the analysis is very robust, and it was possible to quantify the fluxes in the central carbon metabolism under both growth conditions. In the batch culture, 16.2 of every 100 molecules of glucose consumed by the cells entered the pentose-phosphate pathway, whereas the same relative flux was 44.2 per 100 molecules in the chemostat. The tricarboxylic acid cycle does not operate as a cycle in batch-growing cells, in contrast to the chemostat condition. Quantitative evidence was also found for threonine aldolase and malic enzyme activities, in accordance with published data. Disruption of the MIG1 gene did not cause changes in the metabolic network structure or in the flux pattern.     

Protection against diabetes and improved NK/NKT cell performance in NOD.NK1.1 mice congenic at the NK complex

The NK1.1 cell surface receptor, which belongs to the NKR-P1 gene cluster, has been bred onto nonobese diabetic (NOD) mice for two purposes. The first was to tag NK and NKT cells for easier experimental identification of those subsets and better analysis of their implication in type 1 diabetes. The second was to produce a congenic strain carrying Idd6, a susceptibility locus that has been repeatedly mapped in the vicinity of the NKR-P1 gene cluster and the NK complex, to explore the impact of this locus upon autoimmune diabetes. NOD.NK1.1 mice express the NK1.1 marker selectively on the surface of their NK and NKT cell subsets. In addition, the mice manifest reduced disease incidence and improved NK and NKT cell performance, as compared with wild-type NOD mice. The association of those two features in the same congenic strain constitutes a strong argument in favor of Idd6 being associated to the NK complex. This could explain at the same time the multiple alterations of innate immunity reported in NOD mice and the fact that disease onset can be readily modified by boosting the innate immune system of the mouse.     

Ethanol formation and enzyme activities around glucose-6-phosphate in Kluyveromyces marxianus CBS 6556 exposed to glucose or lactose excess

The aim of this work was to investigate the physiology of Kluyveromyces marxianus CBS 6556 in terms of its low tendency to form ethanol under exposure to sugar excess, and the split of carbon flux which takes place at the level of glucose-6-phosphate. Measurements were performed in batch cultivations, and after a glucose or a lactose pulse applied to chemostat-grown respiring cells (with a dilution rate of 0.1 h(-1)). No ethanol formation was observed in batch cultivations or during pulse experiments, unless the oxygen supply was shut down, indicating that this organism is more strictly Crabtree-negative than its close relative K. lactis and other known Crabtree-negative yeasts. During the pulse experiments, activities of phosphoglucoisomerase, glucose-6-phosphate dehydrogenase and phosphoglucomutase in cell-free extracts remained rather constant, at higher levels than those of Saccharomyces cerevisiae grown at similar conditions. When cells were exposed to glucose concentrations as high as 26 gl(-1), the activity of phosphoglucomutase was higher than that in cells exposed to 14 gl(-1) glucose, whereas the activities of phosphoglucoisomerase and glucose-6-phosphate dehydrogenase did not change. Our results suggest that the low tendency for ethanol formation in K. marxianus might be a consequence of this yeast's capacity of keeping the glycolytic flux constant, due at least in part to the diversion of carbon flux towards the biosynthesis of carbohydrates and towards the pentose phosphate pathway.     

Application of the Cre-loxP system for multiple gene disruption in the yeast Kluyveromyces marxianus

The yeast Kluyveromyces marxianus presents several interesting features that make this species a promising industrial yeast for the production of several compounds. In order to take full advantage of this yeast and its particular properties, proper tools for gene disruption and metabolic engineering are needed. The Cre-loxP system is a very versatile tool that allows for gene marker rescue, resulting in mutant strains free of exogenous selective markers, which is a very important aspect for industrial application. As the Cre-loxP system works in some non-conventional yeasts, namely Kluyveromyces lactis, we wished to know whether it also works in K. marxianus. Here, we report the validation of this system in K. marxianus CBS 6556, by disrupting two copies of the LAC4 gene, which encodes a beta-galactosidase activity.