Importance of the pentose phosphate pathway for <scp>d</scp>-glucose catabolism in the obligatory aerobic yeast <i>Rhodotorula gracilis</i>

Höfer, Milan; Brand, Karl; Deckner, K; Becker, Jörn‐Ullrich

doi:10.1042/bj1230855

Cited by 33 publications

(7 citation statements)

References 10 publications

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“…An alternative must exist to explain the high molar growth and lipid yields of this organism (Gill, Hall & Ratledge, 1977). The same conclusion was reached by Hofer and co-workers (Hofer, 1968;Hofer et a/., 1971) and by Brady & Chambliss ( I 967) working with another fat-accumulating yeast, Rhodotorula gracilis (now Rhodosporidium toruloides), in which phosphofructokinase also seemed to be absent. Hofer et a/.…”

Section: Introductionsupporting

confidence: 63%

“…This enzyme had been assumed by Hofer et al (1971) to be present in Rhodotorulagracilis to account for glucose metabolism, though apparently no attempt was made to find it. Xylulose-5-phosphate phosphoketolase when assayed in extracts of Candida 107 grown on glucose in batch or continuous culture had an activity of only 2 nmol acetyl-CoA formed min-l (mg protein)-l confirming the results of Whitworth & Ratledge (1977).…”

Section: Results a N D Discussionmentioning

confidence: 99%

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Pathways of Glucose Metabolism in Candida 107, a Lipid-accumulating Yeast

Ratledge

Botham

1977

Journal of General Microbiology

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Phosphofructokinase was not detected in extracts of Candida 107 prepared in a variety of ways but was highly active in cells treated with toluene. Disruption of these cells destroyed activity of phosphofructokinase indicating that the enzyme is extremely labile. As patterns of labelling from [~-~~C]glucose and [6-14C]glucose showed that 60 % of glucose was metabolized via the pentose cycle, augmentation of this cycle is necessary to account for the high molar growth yields of this yeast. Phosphoketolases, reacting with xylulose 5-phosphate and fructose 6-phosphate, were found but the extent to which they contribute to glucose metabolism was not assessed. I N T R O D U C T I O NWe have previously examined the intermediary metabolism of an oleaginous (fatproducing) yeast, Candida 107, and, because of the apparent absence of phosphofructokinase, concluded that glucose may b: metabolized via the pentose cycle (Whitworth & Ratledge, 1975a). Metabolism of glucose by this cycle, although providing NADPH for fatty acid biosynthesis, would be inefficient with only I mol acetyl-CoA arising from I mol glucose. An alternative must exist to explain the high molar growth and lipid yields of this organism (Gill, Hall & Ratledge, 1977). The same conclusion was reached by Hofer and co-workers (Hofer, 1968;Hofer et a/., 1971) and by Brady & Chambliss ( I 967) working with another fat-accumulating yeast, Rhodotorula gracilis (now Rhodosporidium toruloides), in which phosphofructokinase also seemed to be absent. Hofer et a/. (1969) suggested that phosphoketolase may be present to split xylulose 5-phosphate (formed by the transaldolase and transketolase reactions) into glyceraldehyde 3-phosphate and acetyl-CoA but did not look for this enzyme. However, Mazon, Gancedo & Gancedo (1974) found a phosphofructokinase in Rhodotorula glutinis which was active only under carefully controlled conditions, indicating that caution was needed in interpreting the previous results. When we repeated the latter workers' assay with extracts of Candida 107 we still failed to detect this enzyme and therefore began an examination of glucose metabolism in this yeast. Our results are presented here. M E T H O D SOrganisms and growth. Candida 107 Ruinen 8c Deinema 1964 and, as a control organism, Saccharomyces cerevisiae were grown at 30 "C in a medium consisting of (g 1-9: glucose, 12; KH2P04, 7; Na,HPO,, 2 ; NH,CI, 1-7; MgS04.7Ho0, 1.5; yeast extract, 1.5; CaC1,.6H20, 0 . 1 ; FeCI3.6H,O, 0.05; ZnS04.7H,0, 0-0001; with a final pH of 5-5. The yeasts were usually grown in 100 ml medium in 250 ml conical flasks but occasionally in a chemostat as previously described (Gill et al., 1977). Yeasts were harvested at the end of active growth by centrifuging at 10000 g for 5 to 10 min, washed once with 50 mwphosphate buffer, pH 7-5, containing 2 mM-MgC1, and then resuspended in the same buffer solution to about 300 mg dry wt ml-l.Preparation of cell-free extracts. Conventional procedures were followed for disrupting cells (Hughes,

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Section: Introductionsupporting

confidence: 63%

Section: Results a N D Discussionmentioning

confidence: 99%

Pathways of Glucose Metabolism in Candida 107, a Lipid-accumulating Yeast

Ratledge

Botham

1977

Journal of General Microbiology

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“…Earlier views that the central pathways in micro-organisms for the degradation of carbohydrates were relatively invariable in activity, usually with a major and a minor pathway functioning (Wang et al, 1958(Wang et al, , 1959Stern et al, 1960;Goldman & Blumenthal, 1963;Zagallo & Wang, 1967;Kersters & De Ley, 1968;Hofer et al, 1971), and that the EM pathway was characteristically absent from pseudomonads (Tiwari & Campbell, 1969 ;Lynch et al, 1975) have been proved by more recent studies to be oversimplifications. The discovery of fructose 1,6-bisphosphate formation by PEP-dependent phosphorylation of fructose to fructose 1-phosphate (by PEP-fructose phosphotransferase) and 1 -phosphofructokinase (1 -PFK) in Arthrobacter pyridinolis (Sobel & Krulwich, 1973) and Pseudomonas doudorofl (Baumann & Baumann, 1975) suggested that a modified EM pathway could function in these organisms.…”

Section: Discussionmentioning

confidence: 99%

Carbohydrate Degradation Pathways in Thiobacillus A2 Grown on Various Sugars

Wood¹,

Kelly²

1980

Microbiology

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A detailed radiorespirometric and enzymological analysis was made of the wild-type and GFI strains of Thiobacillus A2 grown on numerous sugars. The wild-type used the EmbdenMeyerhof, Entner-Doudoroff and pentose phosphate pathways for glucose oxidation only after culture on glucose. The fast-growing strain GFI used all three pathways after growth on glucose, maltose or fructose. The wild-type grown on a wide range of pentoses, hexoses, disaccharides, a trisaccharide and a mixture of glucose and maltose oxidized glucose by means of the Entner-Doudoroff pathway (68 to 90%) and pentose phosphate pathway (10 to 32 yo). The key enzyme determining the presence or absence of the Embden-Meyerhof pathway was 6-phosphofructokinase. Other mechanisms, such as a phosphoketolase pathway, were shown to be unimportant, even during growth on pentoses. Arabinose was metabolized via hexose phosphate synthesis. The mechanisms of regulation of sugar metabolism and the energetic significance of alternative pathways are discussed.

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“…PFK was originally thought to be absent in high lipid-producing species of Rhodotorula and Candida 107 (Brady & Chambliss, 1967;Hofer et al, 1969Hofer et al, , 1971Whitworth & Ratledge, 1975). It was eventually detected in the red yeast Rhodotorula glutinis and Candida 107 using the toluene permeabilization method of Serrano et al (1973) and shown to be extremely labile (Mazon et al, 1974;Ratledge & Botham, 1977).…”

Section: Discussionmentioning

confidence: 99%

Phosphofructokinase and the Regulation of the Flux of Carbon from Glucose to Lipid in the Oleaginous Yeast Rhodosporidium toruloides

Evans

Ratledge

1984

Microbiology

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Changes in cell composition of Rhodosporidium toruloides CBS 14 were monitored during growth of batch cultures with NH4Cl and glutamate as nitrogen sources. Carbohydrate was synthesized at the expense of lipid in NHZ-grown cells, whereas in glutamate-grown cells lipid accumulation was predominant. Total biomass and protein concentration were similar in both cultures. Uptake of [U-14C], [1-I4C] and [6-14C]glucose, and evolution of ' T O 2 from these sources, by washed suspensions of cells grown on glutamate revealed the flux of carbon during glucose dissimilation was principally via the Embden-Meyerhof pathway (72%), with 28 % being metabolized by the pentose phosphate pathway. Both urea and glutamate, when added to the cell suspensions, significantly stimulated glucose catabolism, with the flux of carbon via the former pathway increasing to about 89% of the total. Phosphofructokinase (PFK) was implicated as the likely controlling enzyme to explain these events.PFK was only detected in extracts prepared from the yeast grown in a carbon-limited (nitrogen-excess) medium; no activity was detected in extracts of cells grown in nitrogen-limited medium. The presence of a protease in these latter extracts was revealed. PFK was purified 92-fold to a final specific activity (in the presence of 10 mM-NH,+) of 4.2 units (mg protein)-' and exhibited one broad band on polyacrylamide gel electrophoresis. The apparent mol. wt (Mr) of the enzyme was approx. 700000. The major properties of the enzyme were examined to determine its regulatory role during lipid biosynthesis. Unlike the enzyme from Saccharomyces cerevisiae, no inhibition was found with 10 mM-ATP. ADP was not inhibitory either. NHS ions increased activity 1 1-fold by increasing the affinity of the enzyme for both fructose 6-phosphate and ATP. K+ ions also stimulated activity but to a lesser extent. Activity was severely inhibited by citrate, isocitrate and cis-aconitate but this inhibition was dramatically alleviated by NH:. Inhibition by citrate was competitive with fructose 6-phosphate in the absence of NH,+ ions. The K , values for citrate were 1.0 mM (with no NH;) and 7.2 mM (with 10 mM-NH,+). Long-chain fatty acyl-CoA esters had no significant inhibitory effect. It is concluded that the interplay between the prevailing intracellular concentrations of NHZ and citrate is the major determinant of the activity of PFK in riro and thus governs the extent to which glucose is converted either to lipid or carbohydrate.

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Importance of the pentose phosphate pathway for d-glucose catabolism in the obligatory aerobic yeast Rhodotorula gracilis

Cited by 33 publications

References 10 publications

Pathways of Glucose Metabolism in Candida 107, a Lipid-accumulating Yeast

Pathways of Glucose Metabolism in Candida 107, a Lipid-accumulating Yeast

Carbohydrate Degradation Pathways in Thiobacillus A2 Grown on Various Sugars

Phosphofructokinase and the Regulation of the Flux of Carbon from Glucose to Lipid in the Oleaginous Yeast Rhodosporidium toruloides

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