Metabolomic Profiling Revealed Diversion of Cytidinediphosphate-Diacylglycerol and Glycerol Pathway towards Denovo Triacylglycerol Synthesis in Rhodosporidium toruloides

Oleaginous yeast Rhodosporidium toruloides has great biotechnological potential and scientific interest, yet the molecular rationale of its cellular behavior to carbon and nitrogen ratios with concurrent lipid agglomeration remains elusive. Here, metabolomics adaptations of the R. toruloides in response to varying glucose and nitrogen concentrations have been investigated. In preliminary screening we found that 5% glucose (w/v) was optimal for further analysis in Rhodosporidium toruloides 3641. Hereafter, the effect of complementation to increase lipid agglomeration was evaluated with different nitrogen sources and their concentration. The results obtained illustrated that the biomass (13 g/L) and lipid (9.1 g/L) production were maximum on 5% (w/v) glucose and 0.12% (NH4)2SO4. Furthermore, to shed lights on lipid accumulation induced by nitrogen-limitation, we performed metabolomic analysis of the oleaginous yeast R. toruloides 3641. Significant changes were observed in metabolite concentrations by qualitative metabolomics through gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS), which were mapped onto the governing metabolic pathways. Notable finding in this strain concerns glycerol and CDP-DAG metabolism wherein reduced production of glycerol and phospholipids induced a bypass leading to enhanced de-novo triacylglyceride synthesis. Collectively, our findings help in understanding the central carbon metabolism of R. toruloides which may assist in developing rationale metabolic models and engineering efforts in this organism.

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Use of mass isotopomer distributions in secreted lipids to sample lipogenic acetyl-CoA pool in vivo in humans

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1991.261.4.e479 ◽

1991 ◽

Vol 261 (4) ◽

pp. E479-E486 ◽

Cited By ~ 1

Author(s):

M. K. Hellerstein ◽

C. Kletke ◽

S. Kaempfer ◽

K. Wu ◽

C. H. Shackleton

Keyword(s):

Mass Spectrometry ◽

De Novo ◽

Density Lipoprotein ◽

Gas Chromatography Mass Spectrometry ◽

Invasive Technique ◽

Acetyl Coa ◽

Precursor Pool ◽

Chromatography Mass Spectrometry ◽

Mass Isotopomer

Measurement of hepatic fatty acid (FA) and cholesterol synthesis has been limited by lack of access to the precursor pool, cytosolic acetyl-CoA. We present a method for inferring the enrichment of the true hepatic lipogenic precursor pool in humans using the frequency distribution of mass isotopomers within enriched circulating polymers of acetyl-CoA [very low-density lipoprotein (VLDL)-palmitate, VLDL-stearate]. Human subjects were infused intravenously (n = 16) with [1-13C]- or [2-13C]acetate. Oral sulfamethoxazole (SMX) was administered concurrently, and the acetylated conjugate (SMX acetate) was used to estimate independently the hepatic cytosolic acetyl-CoA enrichment. Isotopomer frequencies in VLDL-FA were determined by gas chromatography-mass spectrometry, whereas high-performance liquid chromatography-mass spectrometry was used to measure enrichments in SMX acetate. Based on the excess M2/excess M1 ratio in VLDL-FA, calculated acetyl-CoA enrichments were 5.59 +/- 0.33 molar percent excess (MPE), whereas SMX acetate enrichments were 5.38 +/- 0.31 MPE (the 2 methods were not significantly different). Mass isotopomer-calculated and SMX acetate-measured estimates of acetyl-CoA enrichments correlated very closely in individual subjects (r2 = 0.93; P less than 0.0001). De novo hepatic lipogenesis can be measured using isotopomer-calculated precursor enrichments compared with measured incorporation in specific isotopomers of VLDL-FA. In summary, excess isotopomer frequencies in secreted lipids provide a non-invasive technique for estimating hepatic cytosolic acetyl-CoA enrichments in humans in vivo and correlate closely with enrichments observed using the xenobiotic probe technique. Isotopomeric distributions represent a new strategy for accurate measurement of macromolecule synthesis that may be applicable to other classes of molecules besides lipids.

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Investigation of central carbon metabolism and the 2-methylcitrate cycle in Corynebacterium glutamicum by metabolic profiling using gas chromatography–mass spectrometry

Journal of Biotechnology ◽

10.1016/j.jbiotec.2007.04.026 ◽

2007 ◽

Vol 130 (4) ◽

pp. 354-363 ◽

Cited By ~ 25

Author(s):

Jens Plassmeier ◽

Aiko Barsch ◽

Marcus Persicke ◽

Karsten Niehaus ◽

Jörn Kalinowski

Keyword(s):

Mass Spectrometry ◽

Gas Chromatography ◽

Corynebacterium Glutamicum ◽

Carbon Metabolism ◽

Metabolic Profiling ◽

Central Carbon Metabolism ◽

Gas Chromatography Mass Spectrometry ◽

Chromatography Mass Spectrometry ◽

Central Carbon

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[15N]aspartate metabolism in cultured astrocytes. Studies with gas chromatography-mass spectrometry

Biochemical Journal ◽

10.1042/bj2410193 ◽

1987 ◽

Vol 241 (1) ◽

pp. 193-201 ◽

Cited By ~ 20

Author(s):

M Yudkoff ◽

I Nissim ◽

D Pleasure

Keyword(s):

Mass Spectrometry ◽

Gas Chromatography ◽

De Novo ◽

Adenine Nucleotides ◽

Gas Chromatography Mass Spectrometry ◽

Chromatography Mass Spectrometry ◽

Cultured Astrocytes ◽

Purine Nucleotide Cycle ◽

Citrulline Synthesis ◽

15N Urea

The metabolism of 2.5 mM-[15N]aspartate in cultured astrocytes was studied with gas chromatography-mass spectrometry. Three primary metabolic pathways of aspartate nitrogen disposition were identified: transamination with 2-oxoglutarate to form [15N]glutamate, the nitrogen of which subsequently was transferred to glutamine, alanine, serine and ornithine; condensation with IMP in the first step of the purine nucleotide cycle, the aspartate nitrogen appearing as [6-amino-15N]adenine nucleotides; condensation with citrulline to form argininosuccinate, which is cleaved to yield [15N]arginine. Of these three pathways, the formation of arginine was quantitatively the most important, and net nitrogen flux to arginine was greater than flux to other amino acids, including glutamine. Notwithstanding the large amount of [15N]arginine produced, essentially no [15N]urea was measured. Addition of NaH13CO3 to the astrocyte culture medium was associated with the formation of [13C]citrulline, thus confirming that these cells are capable of citrulline synthesis de novo. When astrocytes were incubated with a lower (0.05 mM) concentration of [15N]aspartate, most 15N was recovered in alanine, glutamine and arginine. Formation of [6-amino-15N]adenine nucleotides was diminished markedly compared with results obtained in the presence of 2.5 mM-[15N]aspartate.

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Characterization of fatty acid desaturases reveals stress-induced synthesis of C18 unsaturated fatty acids enriched in triacylglycerol in the oleaginous alga Chromochloris zofingiensis

Biotechnology for Biofuels ◽

10.1186/s13068-021-02037-2 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Tao Wu ◽

Lihua Yu ◽

Yu Zhang ◽

Jin Liu

Keyword(s):

Mass Spectrometry ◽

Fatty Acids ◽

Unsaturated Fatty Acids ◽

De Novo ◽

Membrane Lipids ◽

Membrane Lipid ◽

Gas Chromatography Mass Spectrometry ◽

Transcription Start Sites ◽

Chromatography Mass Spectrometry ◽

Chromochloris Zofingiensis

Abstract Background The green microalga Chromochloris zofingiensis is capable of producing high levels of triacylglycerol rich in C18 unsaturated fatty acids (UFAs). FA desaturation degree is regulated by FA desaturases (FADs). Nevertheless, it remains largely unknown regarding what FADs are involved in FA desaturations and how these FADs collaborate to contribute to the high abundance of C18 UFAs in triacylglycerol in C. zofingiensis. Results To address these issues, we firstly determined the transcription start sites of 11 putative membrane-bound FAD-coding genes (CzFADs) and updated their gene models. Functional validation of these CzFADs in yeast and cyanobacterial cells revealed that seven are bona fide FAD enzymes with distinct substrates. Combining the validated functions and predicted subcellular compartments of CzFADs and the FA profiles of C. zofingiensis, the FA desaturation pathways in this alga were reconstructed. Furthermore, a multifaceted lipidomic analysis by systematically integrating thin-layer chromatography, gas chromatography–mass spectrometry and liquid chromatography–mass spectrometry techniques was conducted, unraveling profiles of polar membrane lipids in C. zofingiensis and major desaturation steps occurring in these lipids. By correlating transcriptional patterns of CzFAD genes and changes of lipids upon abiotic stress conditions, our results highlighted collaboration of CzFADs for C18 UFA synthesis and supported that both de novo FA synthesis and membrane lipid remodeling contributed C18 UFAs to triacylglycerol for storage. Conclusions Taken together, our study for the first time elucidated the pathways of C18 FA desaturations and comprehensive profiles of polar membrane lipids in C. zofingiensis and shed light on collaboration of CzFADs for the synthesis and enrichment of C18 UFAs in triacylglycerol.

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