Bioinformatics-Based Screening Approach for the Identification and Characterization of Lipolytic Enzymes from the Marine Diatom Phaeodactylum tricornutum.

Oleaginous diatoms accumulate lipids of biotechnological interest when exposed to nutrient stress conditions such as nitrogen starvation. While accumulation mechanisms are well-known and have been engineered to improve lipid production, degradation mechanisms remain poorly investigated in diatoms. Identifying lipid-degrading enzymes is the initial step to understanding the catabolic processes. In this study, an in silico screening of the genome of Phaeodactylum tricornutum led to the identification of 57 putative triacylglycerol lipases (EC 3.1.1.3) grouped in 4 families. Further analysis revealed the presence of conserved domains and catalytic residues of lipases. Physico-chemical characteristics and subcellular localization predictions highlighted that a majority of these putative proteins are hydrophilic and cytosolic, suggesting they could be recruited to lipid droplets directly from the cytosol. Among the 57 identified putative proteins, three lipases were identified as possibly involved in lipophagy due to a potential vacuolar localization. The expression of the mRNA corresponding to the 57 proteins was then searched in 3 transcriptomic datasets obtained under nitrogen starvation. Nine genes were highly regulated and were considered as encoding enzymes with a probable important function in lipid catabolism. A tertiary structure prediction of these nine candidates yielded eight functional 3D models. Among those, two downregulated enzymes, Phatr3_J54974 and Phatr3_EG00720, were highlighted as good targets for future functional genomics and purification studies to investigate their role in lipid degradation.

Comparison of two Phaeodactylum tricornutum ecotypes under nitrogen starvation and resupply reveals distinct lipid accumulation strategies but a common degradation process.

Introduction: Phaeodactylum tricornutum is a model species frequently used to study lipid metabolism in diatoms. When exposed to a nutrient limitation or starvation, diatoms are known to accumulate neutral lipids in cytoplasmic lipid droplets (LDs). Those lipids are produced partly de novo and partly from the recycle of plastid membrane lipids. Under a nitrogen resupply, the accumulated lipids are catabolized, a phenomenon about which only a few data are available. Various strains of P. tricornutum have been isolated around the world that may differ in lipid accumulation patterns. Methods: To get further information on this topic, two genetically distant ecotypes of P. tricornutum (Pt1 and Pt4) have been cultivated under nitrogen deprivation during 11 days followed by a resupply period of 3 days. The importance of cytoplasmic LDs relative to the plastid was assessed by a combination of confocal laser scanning microscopy and cell volume estimation using bright field microscopy pictures. Results and discussion: We observed that in addition to a basal population of small LDs (0.005 µm3 to 0.7 µm3) present in both strains all along the experiment, Pt4 cells immediately produced two large LDs (up to 12 µm3 after 11 days) while Pt1 cells progressively produced a higher number of smaller LDs (up to 7 µm3 after 11 days). In this work we showed that, in addition to intracellular available space, lipid accumulation may be limited by the pre-starvation size of the plastid as a source of membrane lipids to be recycled. After resupplying nitrogen and for both ecotypes, a fragmentation of the largest LDs was observed as well as a possible migration of LDs to the vacuoles that would suggest an autophagic degradation. Altogether, our results deepen the understanding of LDs dynamics and open research avenues for a better knowledge of lipid degradation in diatoms.