Thesis

Characterization of neutral lipid production of Nostoc punctiforme

Nostoc punctiforme is a member of the cyanobacteria that forms filaments of cells and is capable of forming lipid droplets in the cytoplasm. References to lipid droplets in cyanobacteria are scattered throughout the cyanobacterial literature, and are mostly limited to merely describing their presence in electron micrographs. The goal of this work is characterize neutral lipid production in the cyanobacterium Nostoc punctiforme, and to alter metabolic pathways that might favor its production. Neutral lipids of Nostoc punctiforme were characterized by extraction, separation using appropriate solvent systems unique for separation of diacylglycerol (DAG) and triacylglycerol (TAG) by thin layer chromatography, and visualized using iodine. Results indicate that Nostoc punctiforme produces both DAG and TAG. Neutral lipids of Nostoc punctiforme were also turned in to fatty acid methyl esters (FAME) and analyzed using gas chromatography and mass spectrometry (GC-MS). GC-MS results suggest that neutral lipids of Nostoc punctiforme are mainly composed of C16, C16:1, C18, C18:2 and C18:3 fatty acid chains. Wild type cells grown in different growth conditions suggest that the composition of the neutral fatty acids could change with the growth media. Whole-cell staining with BODIPY and visualization by laser confocal microscopy indicated neutral lipids were contained in lipid globules within cells. The quantity of neutral lipid globules increases with the age and the growth condition of the culture. Log phase has considerably fewer and smaller neutral lipid globules compared to the stationary phase. Cells grown in Mops/Nitrate/Fructose had the highest concentration of neutral lipids per cell area in log phase and cells grown in Mops/Nitrate had the highest concentration of neutral lipids per cell area in stationary phase cultures. A strain that overproduces neutral lipids was developed using buoyancy as selection. Sonicated filaments were layered on a Percoll gradient and centrifuged. Upper fractions that contain the most buoyant filaments were selected, re-grown to early stationary phase, and subjected to additional rounds of selection. After eight rounds of Percoll selection, quantification of lipid area per cell from digital images indicated that the selected strain produced twice as many neutral lipids as the wild type strain under similar growth conditions. Individual clones arising from this culture are being screened to identify those with maximal lipid globule production. To see if neutral lipid production could be further increased by genetic modification, gene deletion plasmids were been constructed to block non-essential carbon storage pathways. A glucose-1-phosphate adenylyltransferase deletion plasmid was created by PCR mediated ligation and conjugated into Nostoc punctiforme to create a mutant unable to synthesize glycogen. A second mutation in cyanophycin synthatase, expected to block cyanophycin production (a non-ribosomally produced amino acid polymer used for carbon and nitrogen storage in cyanobacteria) is also in progress.

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