New fatty acids, oxylipins and volatiles in microalgae

Abstract

The main objective of the present thesis was the identification of microalgae producing compounds of high value, in particular polyunsaturated fatty acids (PUFAs) and oxylipins which might be useful for commercial applications. Therefore all available microalgal strains of the Culture Collection of Algae at the University of Göttingen (SAG) were analysed for their long chain FAs (C14-C24) via appropriate techniques (gas-chromatography and mass spectrometry). The large number of data obtained was added into a database, which finally contained 2347 microalgal strains and their corresponding FAs. In total 87 different substances were detected, whereby 77 represent methyl ethers of FAs. As an additional aspect of this investigation the lipid screening was examined for its suitability as a chemotaxonomic marker. An initial comparison of FA profiles of different microalgal strains showed that FA profiles were similar among the same species but different among genera and classes. For example, the polyunsaturated fatty acid (PUFA) 18:5n-3 was found to be specific for the marine algal classes, Dinophyceae, Haptophyceae and Raphidophyceae, whereas the PUFA pinolenic acid was found to be specific for the green algae Chlamydomonas and Chlamydocapsa.The data obtained of FA profiles were then used to identify microalgal strains, which harbour new or interesting polyunsaturated fatty acids (PUFAs), like 18:5n-3 in high amounts. In order to identify novel desaturases and elongases involved in the synthesis of certain FAs, the biosynthetic pathway of 18:5n-3 was investigated by analysing the fate of exogenously fed labelled FAs in Prymnesium parvum. The results obtained indicate that 18:5n-3 may be synthesised via a Δ3-desaturase which introduces a double bond into 18:4n-3.Furthermore the metabolism of PUFAs, in particular the oxylipin formation, was analysed in selected algae. A search of the genomic sequence of the cyanobacteria Nostoc sp. PCC 7120 (identical strain SAG 25.80) and Nostoc punctiforme PCC 73102, suggested one open reading frame encoding putative lipoxygenases (LOXs), which catalyse the initial step of the oxylipin pathway. For N. sp. PCC 7120 a LOX with an amino-terminal extension named NspLOX was identified whereas for N. punctiforme two open reading frames encoding two LOXs named NpLOX1 and NpLOX2 were identified. Individual analysis of recombinant proteins NpLOX1 and NpLOX2 revealed enzymatic activity as a linoleate (13S)-LOX. Analysis of the NspLOX-protein revealed, that the C-terminal LOX domain showed enzymatic activity as a (9R)-LOX. The amino-terminal extension however, showed linoleate diol synthase activity, generating (10E,12E)-9,14-dihydroxy-10,12-octadecadienoic acid as the main product from linoleic acid and (! 10E,12E,14E)-9,16-dihydroxy-10,12,14-octadecatrienoic acid as the main product from α-linolenic acid substrates, respectively. Additionally the function of the LOXs and the oxylipin pathway within these microalgae were analysed in vivo. Soluble extracts of N. sp. and N. punctiforme contained more LOX-derived hydroperoxides in sonified than in non-sonified cells, suggesting an increase of LOX activity upon stress. Furthermore in both strains only hydroperoxy fatty acids were detectable as endogenous oxylipins, indicating that these organisms were only capable to form oxylipins that derive directly from (13S) or (9R)-LOX.