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

Abstract

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.

Fermentation Characteristics of Mortierella alpina in Response to Different Nitrogen Sources

“…The DCW and total lipids obtained from organic nitrogen source were higher than that obtained from inorganic nitrogen source, suggesting that M. alpina hardly assimilate inorganic nitrogen source and require amino acid or protein for cell growth and total lipid accumulation. It has been known that organic nitrogen source has a higher stimulating effect on the lipid synthesis in some oleaginous yeast as inorganic nitrogen source [24]. Organic nitrogen source substrates of a complex chemical structure supported an intensive lipid accumulation, and the nature of the nitrogen source influences the amount and the composition of M. alpina CCF 185 lipid produced [25].…”

Kinetics of continuous cultivation of the oleaginous yeast Rhodosporidium toruloides

“…The oleaginous yeast Rhodosporidium toruloides has been widely employed for microbial lipid study. It has been demonstrated that the products of catabolism of the medium nitrogen can influence the rate of lipid production (Evans and Ratledge, 1984a) and that intracellular ammonia can affect the extent of conversion of glucose to lipids (Evans and Ratledge, 1984b). Very high lipid content was obtained when it was cultivated in media with an initial carbon-to-nitrogen (C/N) ratio of 77 (Turcotte and Kosaric, 1989).…”

Partial purification and properties of pyruvate kinase and its regulatory role during lipid accumulation by the oleaginous yeast Rhodosporidium toruloides CBS 14

“…Evans et al 1983a;Boulton and Ratledge 1983a) would be sufficient to inhibit pyruvate kinase activity and so decrease the flux of carbon to citrate (and hence acetyl-CoA) for lipid production. Intracellulr concentrations of citric acid varying from 2 to 8 m M have been recorded with R. toruloides (Evans and Ratledge 1984a), which could be completely inhibitory to activity but this would depend largely on the availability of cytosolic ADP, N H~, and F1,6-P2. The intracellular concentration of ADP during lipogenesis, as measured in other oleaginous yeasts (Boulton and Ratledge 1983a;Botham and Ratledge 1979), would serve to saturate pyruvate kinase.…”

“…The studies with pyruvate kinase, coupled with those of phosphofructokinase (Evans and Ratledge 1984c), indicate that glycolysis in this yeast is under stringent control such that an alteration of the glycolytic flux could have a significant bearing on the final lipid content of the ce11 (see Scheme 1). Thus, carbon can be channelled into lipid efficiently only when the various "glycolytic effectors" are at sufficient concentration within the appropriate cellular compartment.…”

“…Rhodosporidium toruloides CBS 14 was cultivated in a 1-L chemostat run at a dilution rate 0.05 h-l. For details of media and chemostat operation, see Evans and Ratledge (1983).…”