Biochemical and molecular analysis of LC-PUFA biosynthesis in the microalga Nannochloropsis

Abstract

The present research project comprised both basic research and biotechnology and focused on investigating the role of peroxisomes in lipid metabolism in the lipid-rich microalgae, Phaeodactylum and Nannochloropsis, and on trying to optimize their productivity in synthesizing omega-3 fatty acids for Norwegian aquaculture by genetic engineering and technology-assisted breeding. The starting point of the present thesis was to develop the fundamental microalgal methodology in the Reumann group and to obtain important preliminary research data on peroxisome biology and fatty acid metabolism in order to establish the research group in this field and potentially qualify for research funding in the new BIOTEK 2021 program of the Norwegian Research Council. The objectives of this M. Sc. study were the following: a) establishment of basic culturing techniques and analytical methodology; b) prediction and analysis of the PTS1/2 proteome of soluble matrix proteins for Nannochloropsis gaditana; and c) cloning of full-length cDNAs or C-terminal exons of predicted PTS1 proteins in N. gaditana for subsequent subcellular localization and functional studies. After the fulfillment of extensive empirical and theoretical work, basic culturing techniques for Nannochloropsis were established, including those for analysis and optimization of growth conditions, subculturing and harvesting. The methodology for visualizing the accumulation of neutral lipids in lipid bodies by fluorescence microscopy upon nitrogen starvation was developed, and a protocol for DNA isolation and single-exon cloning was set-up. Among six Nannochloropsis species investigated, N. gaditana showed the most promising growth and productivity characteristics, followed by N. oceanica. The comprehensive analysis of the PTS1/2 proteome of soluble matrix proteins was carried out for Nannochloropsis gaditana and N. oceanica, combining various bioinformatic methods. The identified PTS1 proteins included not only conserved orthologs of known Arabidopsis PTS1 proteins, but also several novel genus- and species-specific proteins. To validate the predictions, two full-length cDNAs and one C-terminal exon of predicted PTS1 proteins in N. gaditana were cloned for subsequent subcellular localization. In addition nuclear transformation of N. oceanica was successfully attempted, as indicated by antibiotic resistance conferred by a given plasmid. In summary, the present Master project made major and highly significant contributions to the establishment of basic and applied microalgal research in the plant peroxisome group by Prof. Reumann at UiS and to National funding of the 10-Mio NOK BIOTEK2021 project “Microalgae 2021” in June 2013.