Biosintesi di PUFA in Fragilariopsis cylindrus: una chiave per comprendere il metabolismo adattato al freddo dei lipidi biologicamente attivi

The PhD project investigated the metabolic pathway for n-3 polyunsaturated fatty acid (PUFA) biosynthesis in the diatom Fragilariopsis cylindrus - a model cold-adapted eukaryote - with a focus on how temperature variations can influence PUFA production in view of Global Warming scenarios. First, putative gene sequences encoding the enzymes involved in this pathway were identified in silico. Sequence and structural analyses revealed features characteristic of cold-adapted proteins. F. cylindrus was then cultured in photobioreactors at 1°C – mimicking a normal Antarctic summer – and 7°C. The cells underwent genomic, transcriptomic, and lipidomic analyses. Genes associated with the PUFA biosynthetic pathway were amplified via PCR. Their cloning and sequencing uncovered multiple genetic variants not previously reported. Targeted fatty acid profiling provided insights into the composition and abundance of seven key metabolites in the PUFA biosynthetic pathway, suggesting that F. cylindrus maintains a stable PUFA accumulation mechanism, regardless of environmental temperature fluctuations. qRT-PCR analysis revealed downregulation of four out of the seven pathway genes in cells grown at 7°C, potentially contributing to metabolite homeostasis. CpG islands, which may serve as potential sites for epigenetic modifications and transcriptional regulation, were identified in four of the genes. To validate enzyme functions within the pathway, the enzymes were recombinantly expressed in Escherichia coli and Pichia pastoris, and enzyme assays utilizing whole-cell systems and analysed via mass spectrometry were performed using their putative substrates. For three enzymes, their roles in the PUFA biosynthetic pathway were experimentally confirmed. In an industrial placement at DNAPhone s.r.l. (PR, Italy), three analytical methods were developed to spectroscopically measure peroxide value, free fatty acids, and conjugated trienes in oils. The resulting kits were produced as test lots and distributed to beta-testers for evaluation. A kit for determining total polyphenol content is under development. This PhD program was co-financed by the National Operational Programme Research and Innovation, supported by ESF REACT-EU resources. The project was conducted in collaboration with the National Research Council (CNR) in Naples, the Scottish Association for Marine Science (SAMS, Oban, UK), the British Antarctic Survey (BAS, UK) and the company DNAPhone s.r.l. (Parma, Italy).