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

Abstract

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,

A Biochemical Explanation for Lipid Accumulation in Candida 107 and Other Oleaginous Micro-organisms

“…However, we did find an active, though extremely labile, phosphofructokinase in both these yeasts and, from isotope-labelling studies with Candida 107, calculated that about 65% of the glucose as growth substrate was being metabolized via the pentose phosphate cycle, with the remainder going through the Embden-Meyerhof pathway (Ratledge & Botham, 1977). In both yeasts, these two pathways of glucose dissimilation were augmented to an unknown extent by hexose-and pentose-phosphoketolase(s) (Whitworth & which would serve to produce acetyl-CoA directly from glucose in the cytoplasm rather than in the mitochondria as usually happens.…”

“…The incorporation of radioactivity from [ l-14C]acetate was a measure of acetyl-CoA carboxylase and was not limited by the activity of acetyl-CoA synthetase which, being about 1.0 pmol h-l (mg protein)-l in both yeasts, was an order of magnitude higher than the activity of acetylCoA carboxylase. [Although there was a low activity of acetate kinase in both yeasts, there was no activity of phosphate acetyltransferase (see Ratledge & Botham, 1977) indicating that synthesis of acetyl-CoA was solely via acetyl-CoA synthetase.] The K, values for acetyl-CoA, calculated in the usual manner, were 4 mM in Candida 107 and 3 mM in C. utilis.…”

“…However, such extracts had low activities when assayed for acetyl-CoA carboxylase. The activity of this enzyme was increased considerably by first treating organisms bith toluene (Serrano et al, 1973 ;Ratledge & Botham, 1977) and then subjecting the lysed preparation to ultrasound (3 x 20 s at 5A). All acetyl-CoA carboxylase activities were assayed using extracts prepared by this modified method.…”

“…[Some minor supplementation of acetyl-CoA may occur in Candida 107 by the action of phosphoketolases on hexose and pentose phosphates (Whitworth & Ratledge, 1977;Ratledge & Botham, 1977).] The acetyl-CoA is then available for fatty acid biosynthesis, with acetyl-CoA carboxylase, the regulatory enzyme of this pathway, being stimulated by citrate.…”

“…On occasions, 4 pmol [l-14C]acetyl-CoA (0.6 pCi) was substituted for [l-14C]acetate, to ensure the activity detected by the assay was not limited by the activity of acetyl-CoA synthetase. [l-14CC]Acetyl-CoA was prepared from [l-14C]acetic anhydride by the method of Simon & Shemin (1953) (Ratledge & Botham, 1977). For both enzymes, activities are expressed as rates of product formation.…”