An Analysis of Intermediary Metabolism and its Control in a Fat-synthesizing Yeast (Candida 107) Growing on Glucose or Alkanes

Abstract

Enzymes of glycolysis, pentose phosphate pathway, gluconeogenesis, tricarboxylate acid cycle, glyoxylate by-pass and fatty-acid biosynthesis were assayed in extracts from Candida 107 grown continuously on glucose under carbon limitation, nitrogen limitation and on n-alkanes. The yeast was therefore either in a lipogenic or lipolytic state. Phosphofructokinase was absent under all conditions whereas enzymes of gluconeogenesis, including fructose I ,6-bisphosphatase and the pentose phosphate cycle, were all present. Glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were specific for NADP+ and were inhibited in a non-competitive manner by NADPH and NADH. Phosphoenolpyruvate, citrate, ATP and acetyl CoA had no inhibitory effects. Thus glucose metabolism appears to be by the pentose phosphate pathway which will rapidly produce NADPH. This can readily be consumed during fatty-acid biosynthesis and, as there appears to be no inhibition of the flow of carbon from glucose to acetyl CoA, fatty-acid synthesis can continue for as long as there is a supply of glucose. These results help to explain the probable causes of fat build-up to high concentrations (about 40 7; of the cell dry weight) in this and other organisms. In alkane-grown cells, lipogenesis is repressed and carbon is able to flow from the alkanes via acetyl CoA, oxaloacetate and pyruvate into pentoses and hexoses in a unidirectional manner, because of the strong repression of pyruvate kinase and the increased activities of phosphoenolpyruvate kinase and fructose I ,6-bisphosphatase under these conditions. Although there was little change in the total activity of the TCA cycle enzymes under the various growth conditions, isocitrate lyase was induced under lipolytic conditions.

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

“…In extracts of both nitrogen-and carbon-limited cells of Candida 107, ATP: citrate lyase activity was low, which agreed with the findings of Whitworth & Ratledge (1975) for Candida 107. AMP was partially inhibitory when added at 0.4 mM or greater (Fig.…”

“…The oxaloacetate arising from citrate cleavage will probably be converted to malate and then decarboxylated to pyruvate. The former reaction is catalysed by malate dehydrogenase which is highly active in Candida 107 (Whitworth & Ratledge, 1975). The latter reaction is catalysed by malic enzyme which is also present in this yeast (Whitworth & Ratledge, 1975) and which, moreover, will produce COz and NADPH.…”

“…The former reaction is catalysed by malate dehydrogenase which is highly active in Candida 107 (Whitworth & Ratledge, 1975). The latter reaction is catalysed by malic enzyme which is also present in this yeast (Whitworth & Ratledge, 1975) and which, moreover, will produce COz and NADPH. The COz will be used, amongst other reactions, by the acetyl-CoA carboxylase and the NADPH will be used by the fatty acid synthetase.…”

“…Before we commenced these studies, we examined the pathways of glucose metabolism in our model oleaginous yeast, Candida 107, as there had been a previous suggestion that in this yeast, as well as in the oleaginous yeast Rhodotorula glutinis, there was no phosphofructokinase and thus no Embden-Meyerhof pathway (Whitworth & Ratledge, 1975 ;Hofer et al, 1969). 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).…”

Activities of Regulatory Enzymes in Alkane-utilizing and Lipid-accumulating Yeasts and Moulds

“…Observations with Candida 107, a lipid-accumulating, alkane-utilizing yeast, similar to Rhodotorula in possessing enzymes of the pentose pathway and lacking phosphofructokinase (Whitworth & Ratledge, 1975), led us to investigate whether the activities of regulatory enzymes of the pentose phosphate and glycolytic pathways are similar throughout lipid-accumulating or alkane-utilizing micro-organisms.…”