Study of Synthesis Pathways of the Essential Polyunsaturated Fatty Acid 20:5n-3 in the Diatom Chaetoceros Muelleri Using 13C-Isotope Labeling

Abstract

The present study sought to characterize the synthesis pathways producing the essential polyunsaturated fatty acid (PUFA) 20:5n-3 (EPA). For this, the incorporation of 13C was experimentally monitored into 10 fatty acids (FA) during the growth of the diatom Chaetoceros muelleri for 24 h. Chaetoceros muelleri preferentially and quickly incorporated 13C into C18 PUFAs such as 18:2n-6 and 18:3n-6 as well as 16:0 and 16:1n-7, which were thus highly 13C-enriched. During the experiment, 20:5n-3 and 16:3n-4 were among the least-enriched fatty acids. The calculation of the enrichment percentage ratio of a fatty acid B over its suspected precursor A allowed us to suggest that the diatom produced 20:5n-3 (EPA) by a combination between the n-3 (via 18:4n-3) and n-6 (via 18:3n-6 and 20:4n-6) synthesis pathways as well as the alternative ω-3 desaturase pathway (via 20:4n-6). In addition, as FA from polar lipids were generally more enriched in 13C than FA from neutral lipids, particularly for 18:1n-9, 18:2n-6 and 18:3n-6, the existence of acyl-editing mechanisms and connectivity between polar and neutral lipid fatty acid pools were also hypothesized. Because 16:3n-4 and 20:5n-3 presented the same concentration and enrichment dynamics, a structural and metabolic link was proposed between these two PUFAs in C. muelleri.

Identification of Polyunsaturated Fatty Acids Synthesis Pathways in the Toxic Dinophyte Alexandrium minutum Using 13C-Labelling

“…This experiment was conducted following the experimental design described by Remize et al [ 33 ]. Briefly, the toxic Dinophyte A. minutum (strain AM89BM, isolated from a bloom in the Bay of Morlay, France) were cultured in batch (total volume of 2 L) under continuous light (24 h light cycle, 100 µmoles photons m −2 ·s −1 at 20 °C).…”

“…Samples (40–70 mL) for Particulate Organic Carbon (POC) concentration and stable isotopic composition were processed as described in Remize et al [ 33 ]. The POC concentrations of all samples were measured using a CE Elantech NC2100 (ThermoScientific, Lakewood, NJ, USA) according to the protocol of the United States Environmental Protection Agency [ 67 ] with acetanilide (99.9% purity, C 8 H 19 NO CASRN 103-84-4) as a standard.…”

“…Samples for the Dissolved Inorganic Carbon (DIC) concentration and stable isotopic composition were collected from the filtrate of POC samples and processed as described in Remize et al [ 33 ] and following the protocol of Assayag et al [ 68 ]. The DIC was measured using a gas bench coupled with a Delta Plus mass spectrometer from Thermo Scientific, Bremen, Germany (GB-IRMS).…”

“…The different pathways described above are not always active or even present in all phytoplankton or micro-zooplankton species, resulting in the different fatty acid compositions found in marine primary producers. Diatoms, which are suspected to preferentially use the “conventional” routes [ 33 ], are naturally richer in 20:5n-3, while Dinophytes, suspected to use both the “conventional” and PKS pathways, are richer in 22:6n-3. Some species such as Chlorophytes taxa or Cyanobacteria do not synthesize them or do so only in very small proportions [ 34 ].…”

“…The principle is to monitor the incorporation of an isotopic label, introduced as a substrate, into targeted organic macromolecules such as fatty acids [ 49 , 50 , 51 , 52 ] to quantify lipid production and identify synthesis pathways. This had already been applied in E. coli [ 53 ], yeast [ 52 ], and microalgae [ 33 , 54 , 55 , 56 ]. In compound-specific isotopic analysis (CSIA), these techniques couple gas chromatography with mass spectrometry (GC-c-IRMS) and allow the direct resolution of the isotopic composition of organic macromolecules, including fatty acids [ 57 , 58 , 59 , 60 ].…”