Derepression of galactose metabolism in melibiase producing bakers' and distillers' yeast

Beet molasses is widely used as a growth substrate for bakers' and distillers' yeast in the production of biomass and ethanol. Most commercial yeasts do not fully utilise the carbohydrates in molasses since they are incapable of hydrolysing the disaccharide melibiose to glucose and galactose. Also, expression of genes encoding enzymes for the utilisation of carbon sources that are alternatives to glucose is tightly regulated, sometimes rates of yeast growth and/or ethanol production. The GAL genes are regulated by specific induction by galactose and repression during growth on glucose. In an industrial distillers' yeast, two genes interacting synergistically in glucose repression of galactose utilization, MIG1 and GAL80, have been disrupted with MEL1, encoding melibiase. The physiology of the wild-type strain and the recombinant strains was investigated on mixtures of glucose and galactose and on molasses. The recombinant strain started to ferment galactose when 9.7 g 1(-1) glucose was still present during a batch fermentation, whereas the wild-type strain did not consume any galactose in the presence of glucose. The ethanol yield in the recombinant strain was 0.50 g ethanol g sugar (-1) in an ethanol fermentation on molasses, compared with 0.48 g ethanol g sugar (-1) for the wild-type strain. The increased ethanol yield was due to utilization of melibiose in the molasses.     

Catabolite repression by galactose in overexpressed GAL4 strains of Saccharomyces cerevisiae

Catabolite repression by galactose was investigated in several strains of Saccharomyces cerevisiae grown on different carbon sources. Galactose repressed as much as glucose; raffinose was less effective. Full derepression was achieved with lactate. The functions tested were L-lactate ferricytochrome c oxidoreductase, NAD-glutamate dehydrogenase, and respiration. Galactose repression was observed only in the GAL4 but not in the gal4 strain. The presence of multiple copies of the GAL4 gene enhanced the repression by galactose. Different alleles of the GAL4 gene and the copy number did not affect glucose repression.     

Comparative study on the production of guar alpha-galactosidase by Saccharomyces cerevisiae SU50B and Hansenula polymorpha 8/2 in continuous cultures.

Saccharomyces cerevisiae SU50B and Hansenula polymorpha 8/2, both carrying a multicopy integrated guar alpha-galactosidase, have been cultivated in continuous cultures, using various mixtures of carbon sources and cultivation conditions. Both S. cerevisiae SU50B and H. polymorpha 8/2 are stable and produce high levels of extracellular alpha-galactosidase in continuous cultures for more than 500 h. For these expression systems the strong inducible promoter systems GAL7 and methanol oxidase, respectively, were used. The induction of alpha-galactosidase synthesis by galactose in SU50B is limited by the low galactose uptake. Apart from that, at high dilution rates, the glucose repression is substantial, and a maximal expression level of 28.6 mg of extracellular alpha-galactosidase.g (dry weight) of biomass-1 could be obtained. In H. polymorpha, the induction of alpha-galactosidase synthesis, in addition to methanol oxidase synthesis using formaldehyde, is very effective up to 42 mg of extracellular alpha-galactosidase.g (dry weight) of biomass-1. Productivities in terms of specific production rate enable a good comparison with those of other heterologous expression systems in the literature. The productivities found with S. cerevisiae SU50B and H. polymorpha, 3.25 and 5.5 mg of alpha-galactosidase.g of biomass-1.liter-1.h-1, respectively, rank among the highest reported in the literature. Enzyme production and secretion in H. polymorpha are more complex. A two-peaked optimum is found in enzyme production. No clear explanation of this phenomenon can be given.     

The roles of galactitol, galactose-1-phosphate, and phosphoglucomutase in galactose-induced toxicity in Saccharomyces cerevisiae

The uptake and catabolism of galactose by the yeast Saccharomyces cerevisiae is much lower than for glucose and fructose, and in applications of this yeast for utilization of complex substrates that contain galactose, for example, lignocellulose and raffinose, this causes prolonged fermentations. Galactose is metabolized via the Leloir pathway, and besides the industrial interest in improving the flux through this pathway it is also of medical relevance to study the Leloir pathway. Thus, genetic disorders in the genes encoding galactose-1-phosphate uridylyltransferase or galactokinase result in galactose toxicity both in patients with galactosemia and in yeast. In order to elucidate galactose related toxicity, which may explain the low uptake and catabolic rates of S. cerevisiae, we have studied the physiological characteristics and intracellular metabolite profiles of recombinant S. cerevisiae strains with improved or impaired growth on galactose. Aerobic batch cultivations on galactose of strains with different combinations of overexpression of the genes GAL1, GAL2, GAL7, and GAL10, which encode proteins that together convert extracellular galactose into glucose-1-phosphate, revealed a decrease in the maximum specific growth rate when compared to the reference strain. The hypothesized toxic intermediate galactose-1-phosphate cannot be the sole cause of galactose related toxicity, but indications were found that galactose-1-phosphate might cause a negative effect through inhibition of phosphoglucomutase. Furthermore, we show that galactitol is formed in S. cerevisiae, and that the combination of elevated intracellular galactitol concentration, and the ratio between galactose-1-phosphate concentration and phosphoglucomutase activity seems to be important for galactose related toxicity causing decreased growth rates.     

Impairment by hexoses of the utilization of maltose by Saccharomyces cerevisiae1

The effect of hexoses with different transport and phosphorylation systems on the utilization of maltose by a galactose constitutive mutant of Saccharomyces cerevisiae has been studied. Galactose, mannose and fructose inhibit both the entrance of maltose in the cells and the phosphorylation of the glucose generated by intracellular hydrolysis of maltose. Transport of maltose is less affected than glucose phosphorylation and, once inside the cell, maltose is hydrolysed and the sparing glucose subsequently excreted. In addition to the well known inactivating effect of glucose, we have found that galactose inactivates the maltose transporter and that this inactivation is enhanced by maltose, which fails to inactivate the system by itself. As reported for glucose, inactivation by galactose involves proteolysis. Other strains of yeast with inducible pathways for both galactose and maltose behave similarly to the galactose constitutive mutant, with some minor changes. The use of maltose as a source of intracellular glucose has allowed to find the existence of mutual interferences in the utilization of hexoses by yeast at the phosphorylation step, that otherwise would have remained unnoticed.     

Increasing galactose consumption by Saccharomyces cerevisiae through metabolic engineering of the GAL gene regulatory network

Increasing the flux through central carbon metabolism is difficult because of rigidity in regulatory structures, at both the genetic and the enzymatic levels. Here we describe metabolic engineering of a regulatory network to obtain a balanced increase in the activity of all the enzymes in the pathway, and ultimately, increasing metabolic flux through the pathway of interest. By manipulating the GAL gene regulatory network of Saccharomyces cerevisiae, which is a tightly regulated system, we produced prototroph mutant strains, which increased the flux through the galactose utilization pathway by eliminating three known negative regulators of the GAL system: Gal6, Gal80, and Mig1. This led to a 41% increase in flux through the galactose utilization pathway compared with the wild-type strain. This is of significant interest within the field of biotechnology since galactose is present in many industrial media. The improved galactose consumption of the gal mutants did not favor biomass formation, but rather caused excessive respiro-fermentative metabolism, with the ethanol production rate increasing linearly with glycolytic flux.     

Fluorescence based assay of GAL system in yeast Saccharomyces cerevisiae

The GAL1 promoter is one of the strongest inducible promoters in the yeast Saccharomyces cerevisiae. In order to improve recombinant protein production we have developed a fluorescence based method for screening and evaluating the contribution of various gene deletions to protein expression from the GAL1 promoter. The level of protein synthesis was determined in 28 selected mutant strains simultaneously, by direct measurement of fluorescence in living cells using a microplate reader. The highest, 2.4-fold increase in GFP production was observed in a gal1 mutant strain. Deletion of GAL80 caused a 1.3-fold increase in fluorescence relative to the isogenic strain. GAL3, GAL4 and MTH1 gene deletion completely abrogated GFP synthesis. Growth of gal7, gal10 and gal3 also exhibited reduced fitness in galactose medium. Other genetic perturbations affected the GFP expression level only moderately. The fluorescence based method proved to be useful for screening genes involved in GAL1 promoter regulation and provides insight into more efficient manipulation of the GAL system.     

Computational analysis of GAL pathway pinpoints mechanisms underlying natural variation

Quantitative traits are measurable phenotypes that show continuous variation over a wide phenotypic range. Enormous effort has recently been put into determining the genetic influences on a variety of quantitative traits with mixed success. We identified a quantitative trait in a tractable model system, the GAL pathway in yeast, which controls the uptake and metabolism of the sugar galactose. GAL pathway activation depends both on galactose concentration and on the concentrations of competing, preferred sugars such as glucose. Natural yeast isolates show substantial variation in the behavior of the pathway. All studied yeast strains exhibit bimodal responses relative to external galactose concentration, i.e. a set of galactose concentrations existed at which both GAL-induced and GAL-repressed subpopulations were observed. However, these concentrations differed in different strains. We built a mechanistic model of the GAL pathway and identified parameters that are plausible candidates for capturing the phenotypic features of a set of strains including standard lab strains, natural variants, and mutants. In silico perturbation of these parameters identified variation in the intracellular galactose sensor, Gal3p, the negative feedback node within the GAL regulatory network, Gal80p, and the hexose transporters, HXT, as the main sources of the bimodal range variation. We were able to switch the phenotype of individual yeast strains in silico by tuning parameters related to these three elements. Determining the basis for these behavioral differences may give insight into how the GAL pathway processes information, and into the evolution of nutrient metabolism preferences in different strains. More generally, our method of identifying the key parameters that explain phenotypic variation in this system should be generally applicable to other quantitative traits.