Enzyme Activities in Oleaginous Yeasts Accumulating and Utilizing Exogenous or Endogenous Lipids

Holdsworth, Jane E.; Veenhuis, Marten; Ratledge, Colin

doi:10.1099/00221287-134-11-2907

Cited by 56 publications

(66 citation statements)

References 13 publications

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“…alpina was cultivated on Tween 80, the metabolism of the fungus underwent changes identical to those previously described when both oleaginous yeasts and filamentous fungi were grown on exogenous triacylglycerol oils (Holdsworth et al, 1988 ;Wynn, 1994).…”

Section: Changes In Key Enzyme Activities When Mort Alpina Is Grown mentioning

confidence: 92%

Evidence that the rate-limiting step for the biosynthesis of arachidonic acid in Mortierella alpina is at the level of the 18:3 to 20:3 elongase

Wynn

Ratledge

2000

Microbiology

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Mortierella alpina, a fungus used commercially as a source of arachidonic acid, 20 :4(n-6), has been examined to see if growth on lipid-based carbon sources leads to repression of either fatty acid biosynthesis and/or fatty acid desaturation and elongation. Changes in the activities of ATP :citrate lyase, isocitrate lyase, carnitine acetyltransferase, malic enzyme, glucose-6-phosphate dehydrogenase and pyruvate kinase when the fungus was grown on fatty-acid-based (Tween) carbon sources were consistent with (i) the cells using the fatty acyl portion of the substrate as the sole carbon source, (ii) pyruvate kinase being the source of pyruvate for biosynthesis under these conditions and (iii) malic enzyme's major function being as a provider of NADPH for lipid biosynthesis. The abolition of fatty acid synthase activity when cells were grown on Tweens indicated the cessation of de novo fatty acid biosynthesis under these conditions. The fatty acyl composition of the lipid accumulated by the fungus grown on Tweens 20, 40 and 80 showed that desaturation and elongation of the substrate lipid still occurred. The absolute amount of arachidonic acid synthesized by Tween-grown cells was the same as for cells grown on glucose. The transformation of incorporated fatty acids into 20 :4(n-6) was, it appeared, limited at the elongation of 18 :3(n-6) to 20 :3(n-6) as, in every case, 18 :1, 18 :2 and 18 :3(n-6) increased in amount in the Tweengrown cells. These data show for the first time that fatty acid synthesis is regulated separately from fatty acid desaturation/elongation and that the latter reactions are not repressed by growth of the fungus on simple fatty acids. Furthermore, the data strongly implicate the elongation of 18 :3(n-6) to 20 :3(n-6) as the limiting step in arachidonic acid biosynthesis by Mort. alpina.

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Section: Changes In Key Enzyme Activities When Mort Alpina Is Grown mentioning

confidence: 92%

Evidence that the rate-limiting step for the biosynthesis of arachidonic acid in Mortierella alpina is at the level of the 18:3 to 20:3 elongase

Wynn

Ratledge

2000

Microbiology

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“…1 and 3A). It should be mentioned that in the case of oleaginous yeasts and molds, biodegradation of the previously produced storage lipid is frequently observed (14,21). Repression of storage lipid degradation can be achieved by genetic engineering (in the case of the ⌬gut2 ⌬pox1-6 mutant), as well as by cultivation conditions, such as using double (or multiply)-limited media for the cultivation of oleaginous microorganisms.…”

Section: Discussionmentioning

confidence: 99%

Control of Lipid Accumulation in the Yeast Yarrowia lipolytica

Béopoulos

Mrózová

Thevenieau

et al. 2008

Appl Environ Microbiol

362

266

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A genomic comparison of Yarrowia lipolytica and Saccharomyces cerevisiae indicates that the metabolism of Y. lipolytica is oriented toward the glycerol pathway. To redirect carbon flux toward lipid synthesis, the GUT2 gene, which codes for the glycerol-3-phosphate dehydrogenase isomer, was deleted in Y. lipolytica in this study. This ⌬gut2 mutant strain demonstrated a threefold increase in lipid accumulation compared to the wild-type strain. However, mobilization of lipid reserves occurred after the exit from the exponential phase due to ␤-oxidation. Y. lipolytica contains six acyl-coenzyme A oxidases (Aox), encoded by the POX1 to POX6 genes, that catalyze the limiting step of peroxisomal ␤-oxidation. Additional deletion of the POX1 to POX6 genes in the ⌬gut2 strain led to a fourfold increase in lipid content. The lipid composition of all of the strains tested demonstrated high proportions of FFA. The size and number of the lipid bodies in these strains were shown to be dependent on the lipid composition and accumulation ratio.

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“…To determine the macromolecular composition of C. protothecoides, the Lowry method (Holdsworth et al, 1988) was used to measure protein content, and amino acid composition was obtained with an L-8800 Amino Acid Analyzer (Hitachi); the content and composition of oil were measured by gas chromatography (GC)-MS as previously described (Xiong et al, 2010b). The 3,5-dinitrosalicylic acid method (Miller, 1959) was applied to determine intracellular carbohydrate and starch.…”

Section: Cultivation Conditions and Analytical Proceduresmentioning

confidence: 99%

Genome-Based Metabolic Mapping and 13C Flux Analysis Reveal Systematic Properties of an Oleaginous Microalga Chlorella protothecoides

Xiong

Dai

et al. 2014

Plant Physiology

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Integrated and genome-based flux balance analysis, metabolomics, and 13 C-label profiling of phototrophic and heterotrophic metabolism in Chlorella protothecoides, an oleaginous green alga for biofuel. The green alga Chlorella protothecoides, capable of autotrophic and heterotrophic growth with rapid lipid synthesis, is a promising candidate for biofuel production. Based on the newly available genome knowledge of the alga, we reconstructed the compartmentalized metabolic network consisting of 272 metabolic reactions, 270 enzymes, and 461 encoding genes and simulated the growth in different cultivation conditions with flux balance analysis. Phenotype-phase plane analysis shows conditions achieving theoretical maximum of the biomass and corresponding fatty acid-producing rate for phototrophic cells (the ratio of photon uptake rate to CO 2 uptake rate equals 8.4) and heterotrophic ones (the glucose uptake rate to O 2 consumption rate reaches 2.4), respectively. Isotope-assisted liquid chromatography-mass spectrometry/mass spectrometry reveals higher metabolite concentrations in the glycolytic pathway and the tricarboxylic acid cycle in heterotrophic cells compared with autotrophic cells. We also observed enhanced levels of ATP, nicotinamide adenine dinucleotide (phosphate), reduced, acetyl-Coenzyme A, and malonyl-Coenzyme A in heterotrophic cells consistently, consistent with a strong activity of lipid synthesis. To profile the flux map in experimental conditions, we applied nonstationary 13 C metabolic flux analysis as a complementing strategy to flux balance analysis. The result reveals negligible photorespiratory fluxes and a metabolically low active tricarboxylic acid cycle in phototrophic C. protothecoides. In comparison, high throughput of amphibolic reactions and the tricarboxylic acid cycle with no glyoxylate shunt activities were measured for heterotrophic cells. Taken together, the metabolic network modeling assisted by experimental metabolomics and 13 C labeling better our understanding on global metabolism of oleaginous alga, paving the way to the systematic engineering of the microalga for biofuel production.

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Enzyme Activities in Oleaginous Yeasts Accumulating and Utilizing Exogenous or Endogenous Lipids

Cited by 56 publications

References 13 publications

Evidence that the rate-limiting step for the biosynthesis of arachidonic acid in Mortierella alpina is at the level of the 18:3 to 20:3 elongase

Evidence that the rate-limiting step for the biosynthesis of arachidonic acid in Mortierella alpina is at the level of the 18:3 to 20:3 elongase

Control of Lipid Accumulation in the Yeast Yarrowia lipolytica

Genome-Based Metabolic Mapping and 13C Flux Analysis Reveal Systematic Properties of an Oleaginous Microalga Chlorella protothecoides

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