To select a Saccharomyces cerevisiae reference strain amenable to experimental techniques used in (molecular) genetic, physiological and biochemical engineering research, a variety of properties were studied in four diploid, prototrophic laboratory strains. The following parameters were investigated: 1) maximum specific growth rate in shake-flask cultures; 2) biomass yields on glucose during growth on defined media in batch cultures and steady-state chemostat cultures under controlled conditions with respect to pH and dissolved oxygen concentration; 3) the critical specific growth rate above which aerobic fermentation becomes apparent in glucose-limited accelerostat cultures; 4) sporulation and mating efficiency; and 5) transformation efficiency via the lithium-acetate, bicine, and electroporation methods. On the basis of physiological as well as genetic properties, strains from the CEN.PK family were selected as a platform for cell-factory research on the stoichiometry and kinetics of growth and product formation.
Tre~lo~6-phosphate (P) ~mpetitively inhibited the hexokinases from ~~cc~omyee~ cerevisiae. The strongest inhibition was observed upon hexokinase II, with a K, of 40 FM, while in the case of hexoklnase I the K, was 200 NM. Glucokinase was not inhibited by trehalose-6-P up to 5 mM. This inhibition appears to have physiological significance, since the intracellular levels of trehalose-6-P were about 0.2 mM. Hexokinases from other organisms were also inhibited, while glucokinases were unaffected. The hexokinase from the yeast, Yarrowia iipolytica, was particularly sensitive to the inhibition by trehalose-6-P: when assayed with 2 mM fructose an apparent K, of 5 PM was calculated. Two 5. cerevisiae mutants with abnormal levels of trehalose-6-P exhibited defects in glucose metabolism. It is concluded that trehalose-6-P plays an important role in the regulation of the first steps of yeast glycolysis, mainly through the inhibition of hexokinase II.Trehalose-6-phosphate; Hexokinase; Glycolysis; Yeast PRODUCTIONControl of the glycolytic flux in Succhurowryces cerevisicze has been considered to occur mainly at the level of phosphofructokinase and pyruvate kinase. Phosphofructokinase is regulated through activation by fructose-2,6-bisphosphate, phosphate (P) and AMP, and inhibition by ATP, while pyruvate kinase activity is modulated by its activation by fructose-l,(i-bisphosphate (for a review see [l]). However, since phosphof~~tokinase does not catalyze the first irreversible step in the utilization of glucose, its regulation is not sufficient to control the rate of glucose utilization, and thus some mechanism should exist to regulate the rate of glucose transport, phosphorylation, or both. Regulation of glucose transport by glucose-6-P was suggested by Sols [2], but experimental results obtained using mutants affected in phosphoglucose isomerase activity did not support this idea [3,4]. Besides, none of the yeast kinases that phosphorylate glucose are sensitive to glucose-6-P, in contrast with the mammalian hexokinase PI-The need for a regulation of the first steps of yeast glycolysis is illustrated by the pattern of ambulation of metabolites in certain yeast mutants upon addition of glucose [6-81. Yeast strains carrying the mutations fdpl [6] or cifl [7] do not grow on glucose, although the glycolytic enzymes are operative. These mutations turned out to be allelic [9], and strains bearing them become depleted of ATP upon addition of glucose and accumulate fructose-l ,6-bisphosphate up to 20 mM [6,7,10], suggesting that the rate of the first glycolytic steps exceeds the capacity of the glycolytic pathway. The sequence of the CIFZ gene [lo] encodes the small subunit of the trehalose-6-P synthase/trehalose-6-P phosphatase complex [ 11,123. A plausible explanation for the growth behaviour and the metabolic defects of cifl strains could be that either trehalose or trehalose-6-P play a role in the regulation of the yeast glycolytic flux. We show in this article that trehalose-6-P inhibits sugar phospho~lation.The stron~st inh...
SummaryAn Arabidopsis thaliana cDNA clone, AtTPS1, that encodes a trehalose-6-phosphate synthase was isolated. The identity of this protein is supported by both structural and functional evidence. On one hand, the predicted sequence of the protein encoded by AtTPS1 showed a high degree of similarity with trehalose-6-phosphate synthases of different organisms. On the other hand, expression of the AtTPS1 cDNA in the yeast tps1 mutant restored its ability to synthesize trehalose and suppressed its growth defect related to the lack of trehalose-6-phosphate. Genomic organization and expression analyses suggest that AtTPS1 is a single-copy gene and is expressed constitutively at very low levels.
The view of the role of trehalose in yeast has changed in the last few years. For a long time considered a reserve carbohydrate, it gained new importance when its function in the acquisition of thermotolerance was demonstrated. More recently the cellular processes in which the trehalose biosynthetic pathway has been implicated range from the control of glycolysis to sporulation and infectivity by certain fungal pathogens. There is now enough experimental evidence to conclude that trehalose 6-phosphate, an intermediate of trehalose biosynthesis, is an important metabolic regulator in such different organisms as yeasts or plants. Its inhibition of hexokinase plays a key role in the control of the glycolytic flux in Saccharomyces cerevisiae but other, likely important, sites of action are still unknown. We present examples of the phenotypes produced by mutations in the two steps of the trehalose biosynthetic pathway in different yeasts and fungi, and whenever possible examine the molecular explanations advanced to interpret them.
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