Fatty Acid Distribution in Triglycerides of Yeasts Grown on Glucose or n-Alkanes

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184

124

361The biochemical explanation for lipid accumulation was investigated principally in Candida 107 and, for comparison, in the non-oleaginous yeast Candida utilis. There were no significant differences between these two yeasts in their control of glucose uptake; in both yeasts, the rates of glucose uptake were independent of the growth rate and were higher in carbon-limited chemostat cultures than in nitrogen-limited cultures. There was no lipid turnover in either yeast, as judged from [14C]acetate uptake and subsequent loss of 14C from the lipid of steady-state chemostat cultures. Acetyl-CoA carboxylase from both yeasts was similar in most characteristics except that from Candida 107 was activated by citrate (40 0 4 activation at 1 mM). The enzyme from Candida 107 was relatively unstable and, when isolated from nitrogen-limited (lipid-accumulating) cultures, was accompanied by a low molecular weight inhibitor.The reason for lipid accumulation is attributed to the decrease in the intracellular concentration of AMP as cultures become depleted of nitrogen. As the NAD+-dependent isocitrate dehydrogenase of Candida 107, but not C. utilis, requires AMP for activity, the metabolism of citrate through the tricarboxylic acid cycle in the mitochondria becomes arrested. In Candida 107, but not in C. utilis, there is an active ATP:citrate lyase which converts the accumulating citrate, when it passes into the cytosol, into acetyl-CoA and oxaloacetate. The former product is then available for fatty acid biosynthesis which is stimulated by the high ATP concentration within the cells, by the activation of acetyl-CoA carboxylase by citrate and by the provision of NADPH generated as oxaloacetate is converted via malate to pyruvate. Similar characteristics were evident in oleaginous strains of Rhodotorula glutinis and Mucor circinelloides but not in non-oleaginous representatives of these species.

Section: Results a N D Discussionmentioning

confidence: 99%

Section: Lipid Turnovermentioning

confidence: 99%

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

Botham

Ratledge

1979

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184

124

“…Under appropriate conditions, Candida 107 can produce up to 40 7; of its weight as lipid when grown with either carbohydrates, such as glucose or sucrose, or with n-alkanes as source of carbon (Ratledge, 1968(Ratledge, ,1970Thorpe & Ratledge, 1972). The oxidation of n-alkanes is constitutive in this organism and, furthermore, when n-alkanes are added to the yeast actively growing on glucose, immediate inhibition of glucose transport and metabolism occurs (Gill & Ratledge,I 973a).…”

Section: Introductionmentioning

confidence: 99%

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

Whitworth¹,

Ratledge²

1975

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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.

“…In yeasts, n-alkanes are oxidized, via n-alkanols, to fatty acids which then, depending on chain-length, are either incorporated into triglycerides and phospholipids or are degraded to small carbon fragments (Thorpe & Ratledge, 1972; R. F. Thorpe & C. Ratledge, unpublished work). Fatty acids from alkanes with a chain-length of CI3 or less are not incorporated into lipid with Candida 107 and, significantly, it was alkanes of such chain-lengths which were the most potent inhibitors of glucose incorporation.…”

Section: Discussionmentioning

confidence: 99%

Inhibition of Glucose Assimilation and Transport by n-Decane and Other n-Alkanes in Candida 107

Gill

Ratledge

1973

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S U M M A R YCaridida 107 constitutively oxidizes n-nonane and longer alkanes. Decane and longer alkanes have little effect on endogenous respiration (measured as 14C02 expiration from yeast grown on [ U-14C]glucose). Alkane-grown yeast assimilated [ i-14C]decane more rapidly than glucose-grown yeast though without a lag in both cases. Glucose did not inhibit these incorporations. [U-14C]Glucose was assimilated without a lag into glucose-grown yeast and, at a quarter of this rate, into alkanegrown yeast; if the latter yeast was pre-incubated with glucose for 60 min, it took up [ U-14C]glucose at the higher rate. n-Decane (0.5 to I ' 0 mg/mg yeast dry wt) severely and rapidly inhibited glucose assimilation. Longer chain alkanes were progressively less effective. All alkanes were more inhibitory with alkane-grown yeast. The rate of glucose assimilation was inversely proportional to the concentration of decane and directly proportional to the yeast concentration. Transport of glucose, as distinct from its assimilation, was also inhibited by decane. Uptake of fiicose, a nonmetabolizable deoxyhexose, was similarly affected. Passive transport of glucose did not occur. Alkanes did not prevent glucose access to the yeast surface as pristane, a non-metabolizable paraffin, had no effect on glucose assimilation in Candida I 07 and dodecane was not inhibitory against carbohydrate transport in Saccharon?yces carlsberget~sis. Alkanes probably inhibit by causing accumulation of fatty acids or acyl CoA esters which may, either through feedback inhibitions or by further metabolism, cause build-up of ATP, acetyl CoA, and glucose 6-phosphate, thus leading to cessation of glucose transport and metabolism.