Christopher A. Boulton

Correlation of Lipid Accumulation in Yeasts with Possession of ATP: Citrate Lyase

ATP: citrate lyase has been found in 13 strains of yeast (representing six genera) which are capable of accumulating lipid to above 20% of their biomass. The enzyme is absent in 10 other yeasts which do not accumulate lipid. The presence of the enzyme is therefore directly correlated to the phenomenon of oleaginicity. The enzyme is located in the cytosol fraction of the yeasts and is probably the sole means of producing acetyl-CoA in most oleaginous yeasts. The specific activity of the enzyme correlates with the specific rate of lipid synthesis as determined in nitrogen-limited chemostat cultures of Lipomyces starkeyi, though not with the lipid content of the cells. From this and by calculation, it may be inferred that the enzyme is possibly the rate-limiting reaction for lipid biosynthesis.

Partial Purification and Some Properties of ATP:Citrate Lyase from the Oleaginous Yeast Lipomyces starkeyi

Summary: ATP:citrate lyase has been purified some 20-fold from the oleaginous yeast, Lipomyces starkeyi CBS 1809. The enzyme, which is labile, was stabilized by the addition of ATP and citrate to extraction buffers. Michaelis-Menten kinetics were exhibited towards ATP and citrate. The molecular weight of the enzyme was determined to be approximately 510000. There was a specific requirement for ATP for activity. No activity was observed in the absence of Mg2+, although Co2+ and Mn2+ could partially substitute for Mg2+. ADP inhibited activity (50% inhibition at 1 mM), as did long-chain fatty acyl-CoA esters (50% inhibition with 4μm oleoyl-CoA). The possibility of control of ATP:citrate lyase activity by fluctuations in the prevailing energy charge or by changes in the intracellular concentrations of long-chain fatty acyl-CoA esters is discussed.

Regulatory Studies on Citrate Synthase in Candida 107, an Oleaginous Yeast

The kinetic properties of citrate synthase from Candida 107 were examined. Adenine nucleotides were inhibitory to enzyme activity, the order of efficacy being ATp>ADp>AMP. Inhibition by ATP was competitive with respect to acetyl-CoA and mixed with respect to oxaloacetate. Combinations of adenine nucleotides giving simulated energy charge values were also inhibitory, though the total adenine nucleotide concentration was of greater significance than the relative proportions of each in determining the degree of inhibition. When Mg2+was added at a concentration sufficient to saturate the adenine nucleotides, the inhibition was almost entirely relieved. The apparent absence of any rigorous control of citrate synthase by adenine nucleotides in oleaginous micro-organisms is consistent with previous observations that the flow of carbon through the glycolytic and pentose phosphate pathways to pyruvate and thence to citrate should be uninterrupted during the process of lipid accumulation.

Use of Transition Studies in Continuous Cultures of Lipomyces starkeyi, an oleaginous yeast, to Investigate the Physiology of Lipid Accumulation

Summary: The physiological changes that occurred in chemostat cultures of an oleaginous yeast, Lipomyces starkeyi CBS 1809 undergoing transitions from steady-state carbon-limiting to steady-state nitrogen-limiting conditions have been investigated. A sequence of events has been identified initiated by the presentation of excess glucose to the culture and culminating in the accumulation of substantial quantities of intracellular lipid. The intracellular concentrations of adenine nucleotides and citrate have been determined and their possible regulatory significance in the control of catabolic and anabolic pathways is discussed.

Cryptococcus terricolus, an oleaginous yeast re-appraised

SummaryThree strains of Cryptococcus terricolus have been examined for their ability to accumulate intra-cellular lipid. Of those only C. terricolus IFO 1322 was capable of accumulating substantial amounts (35–40% w/w) when cultivated in batch culture. The pattern of lipid accumulation was unusual in that the maximum rate of lipid synthesis was observed to occur during the exponential phase of growth when exogenous nitrogen was still available. Thus these results confirmed the earlier observations of Pedersen (1961; 1962a, b, c) that lipid levels in this organism were constitutively high. A preliminary biochemical analysis has failed to indicate why this unusual pattern of lipid accumulation occurs. The potential value of C. terricolus IFO 1322 for use in the production of single cell oil is discussed.

ATP: Citrate Lyase - The Regulatory Enzyme for Lipid Biosynthesis in Lipomyces starkeyi?

ATP: citrate lyase from Lipomyces starkeyi was inhibited by oleoyl-CoA (50% inhibition at approx. 3 μM) and other long-chain fatty acyl-CoA esters but not by other fatty acyl compounds. The inhibition was readily reversed by adding bovine serum albumin. The molecular size of the protein was not altered even in the presence of 200 μM-oleoyl-CoA. It is concluded that this is a genuine feedback inhibition effect which may be significant in regulating the amount of triacylglycerol being produced by an oleaginous yeast.

Synthesis of Certain Assimilatory and Dissimilatory Enzymes during Bacterial Adaptation to Growth on Trimethylamine

Summary: During the adaptation of Pseudomonas aminovorans from growth on succinate to growth on trimethylamine, the following enzymes were synthesized in the lag phase before exponential growth on trimethylamine began: trimethylamine and dimethylamine mono-oxygenases, trimethylamine-N-oxide aldolase (demethylase), glutathione- and NAD-dependent formaldehyde dehydrogenase, dye-linked formaldehyde dehydrogenase, hydroxypyruvate reductase and N-methylglutamate dehydrogenase. Differential plots suggested that the rate of enzyme synthesis in the lag phase exceeded the rate of synthesis during exponential growth. The evidence suggests that the enzymes discussed are essential for growth on trimethylamine, while the NADPH-dependent N-methylalanine dehydrogenase is not involved.

Properties of Pseudomonas AM1 primary-amine dehydrogenase immobilized on agarose

1. The primary-amine dehydrogenase of Pseudomonas AM1 (primary amine:(acceptor) oxidoreductase (deaminating), EC 1.4.99.-) was purified by an improved method and covalently attached to cyanogen bromide-activated Sepharose 4B. The immobilized enzyme showed very little change in its sensitivity to heat and to inhibition by semicarbazide as compared with the soluble enzyme, but had enhanced stability at 0 degrees C. The pH optimum of the immobilized enzyme remained unchanged at pH 7.4. 2. A new type of spectrophotometric assay is described in which sedimentation of the immobilized enzyme in the cuvette is prevented by increasing the viscosity by the presence of 10% (w/w) polyethylene glycol (M1 20 000). Detailed kinetic analysis using this assay showed only insignificant differences in the Km values for n-butylamine and phenazine methosulphate between the soluble and Agarose-bound enzymes. The results are compared with those for other oxidoreductase enzymes immobilized on Sepharose.

Oxidation of N-alkyl- and NN-dialkylhydroxylamines by partially purified preparations of trimethylamine mono-oxygenase from Pseudomonas aminovorans

and of dimethylamine to formaldehyde and another product which was not methylamine [1]. In the present paper we present evidence suggesting that the unknown oxidation product is N-methylhydroxylamine. We also show that NN-dimethylhydroxylamine is a substrate for the enzyme, and gives the same oxidation products as dimethylamine. The reaction product N-methylhydroxylamine is itself slowly oxidized by preparation in the presence of NADPH and oxygen to an unidentified product which may possibly be formamide. The corresponding N-ethyland NN-diethylhydroxylamines are also oxidized by the enzyme. We suggest the following reaction sequence to explain our observations: