Sonia Abreu

Les acyles gras (FAs) représentent le principal élément constitutif des lipides complexes (glycérolipides / phospholipides / sphingolipides). Leur structure varie en fonction de leur fonctionnalisation, nombre de carbones, nombre et position d'insaturations. Cette variété est à l'origine du très grand nombre d'espèces lipidiques et de la complexité de leur analyse.Les travaux menés au cours de cette thèse ont démontré, au travers de trois publications, que l'évaluation de la distribution FAs des glycérolipides et phospholipides était accessible grâce à l'intensité des ions fragments [monoacylglycérol+H- H2O]+ (ions B) produits en source APPI/APPI-HRMS, lors du couplage à la FIA ou la NPLC.Les bases de ce nouveau concept ont été posées via l'étude des triglycérides d'huiles végétales.Il a été observé que la distribution des intensités des ions B était directement reliée à la distribution des FAs, sauf pour le FA polyinsaturé C18:3.Des coefficients de pondération des intensités des ions B ont été calculés pour compenser les différences d'ionisation. Globalement, un coefficient deux fois plus important a été appliqué aux intensités des ions B(18 :3) pour compenser sa plus faible intensité.Le concept a ensuite été étendu aux phospholipides (PLs). Pour les extraits contenant uniquement des diacyl-PLs, la transposition est également directe. La nature de la tête polaire n'a pas eu d'influence sur la formation des ions B.Pour les extraits contenant à la fois des diacyl-PLs et des plasmalogènes (fonction éther vinylique) une séparation préalable doit être réalisée. En effet, la formation des ions B n'a pas lieu au même degré pour ces deux sous-classes.Une hydrolyse par des vapeurs d'acide chlorhydrique a permis de rompre spécifiquement les liaisons éthers vinyliques. Les plasmalogènes sont alors transformés en Lyso-PLs et aldéhydes.Ces extraits ont également la particularité de contenir des teneurs importantes de FAs polyinsaturées. Par analogie avec l'étude des TGs, un coefficient pondérateur de deux a été appliqué aux intensités des ions B de ces FAs.Pour accéder à la distribution des alkényls gras des plasmalogènes, un concept similaire à celui des ions B a été mis en œuvre avec succès.De plus, un schéma de fragmentation a été proposé pour les plasmalogènes PC lors de l'utilisation de la source APCI.Enfin, notre nouveau concept a été mis en œuvre sur des extraits lipidiques de souches Yarrowia Lipolytica (levure oléagineuse) modifiées pour favoriser la production de FAs à chaînes impaires. La distribution des FAs au sein de chaque classe de lipides a été évaluée et le concept a été étendu à deux autres classes de lipide (Diglycérides-DG et glucuronosyl diacylglycerol-GlcADG).

Since nitrogen limitation is known to be an important trigger of triacylglycerol (TAG) accumulation in most microorganisms, we first assessed the global lipid content of 21 strains derived from Streptomyces coelicolor M145 deleted for genes involved in nitrogen metabolism. Seven of these strains deleted for genes encoding proteins involved in polyamine (GlnA2/SCO2241, GlnA3/SCO6962, GlnA4/SCO1613), or protein (Pup/SCO1646) degradation, in the regulation of nitrogen metabolism (GlnE/SCO2234 and GlnK/SCO5584), or the global regulator DasR/SCO5231 that controls negatively the degradation of N-acetylglucosamine, a constituent of peptidoglycan, had a higher TAG content than the original strain, whereas five of these strains (except the glnA2 and pup mutants) had a lower cardiolipin (CL) content. The production of the blue polyketide actinorhodin (ACT) was totally abolished in the dasR mutant in both Pi conditions, whereas the deletion of pup, glnA2, glnA3, and glnA4 was correlated with a significant increase in total ACT production, but mainly in Pi limitation. Unexpectedly, ACT production was strongly reduced in the glnA3 mutant in Pi proficiency. Altogether, our data suggest that high TAG and ACT biosynthesis and low CL biosynthesis might all contribute to the lowering of oxidative stress resulting from nitrogen limitation or from other causes.

In order to assess the impact of nitrogen availability on lipid metabolism of Streptomyces coelicolor, we first assessed the global lipid content of 21 strains derived from S. coelicolor M145 deleted for at least one gene involved in nitrogen metabolism. This study revealed that only 7 strains deleted either for genes encoding, proteins involved in polyamine (GlnA2/SCO2241, GlnA3/SCO6962, GlnA4/SCO1613) or protein (Pup/SCO1646) degradation, for the global regulator DasR/SCO5231 that controls negatively the degradation of N acetylglucosamine, a constituant of peptidoglycan, or for two proteins involved in the regulation of nitrogen metabolism, GlnE/SCO2234 and GlnK/SCO5584, had a higher triacylglycerol content than the original strain whereas 5 of these strains (with the exception of glnA2 and pup mutant strains) had a lower cardiolipin content than the original strain. Furthermore, the determination of the level of production of the blue polyketide, actinorhodin (ACT), of these 7 strains grown either in phosphate (Pi) limitation or proficiency, revealed that ACT production was totally abolished in the dasR mutant in both Pi conditions whereas the deletion of pup, glnA2, glnA3 and glnA4 was correlated with a significant increase of total ACT production, in Pi limitation. However, in Pi proficiency, ACT production was only slightly enhanced in the pup, glnA2 and glnA4 mutants and was strongly reduced in the glnA3 mutant. Altogether our data suggest that high TAG and ACT biosynthesis and low cardiolipin biosynthesis might all contribute to the lowering of oxidative stress resulting from nitrogen limitation or from other causes.

Streptomyces coelicolor M145 is a model strain extensively studied to elucidate the regulation of antibiotic biosynthesis in Streptomyces species. This strain abundantly produces the blue polyketide antibiotic, actinorhodin (ACT), and has a low lipid content. In a process designed to delete the gene encoding the isocitrate lyase (sco0982) of the glyoxylate cycle, an unexpected variant of S. coelicolor was obtained besides bona fide sco0982 deletion mutants. This variant produces 7- to 15-fold less ACT and has a 3-fold higher triacylglycerol and phosphatidylethanolamine content than the original strain. The genome of this variant was sequenced and revealed that 704 genes were deleted (9% of total number of genes) through deletions of various sizes accompanied by the massive loss of mobile genetic elements. Some deletions include genes whose absence could be related to the high total lipid content of this variant such as those encoding enzymes of the TCA and glyoxylate cycles, enzymes involved in nitrogen assimilation as well as enzymes belonging to some polyketide and possibly trehalose biosynthetic pathways. The characteristics of this deleted variant of S. coelicolor are consistent with the existence of the previously reported negative correlation existing between lipid content and antibiotic production in Streptomyces species.

Yarrowia lipolytica is a promising oleaginous yeast for producing unusual lipids, such as odd-chain fatty acids (OCFA). Their diverse applications and low natural production make OCFA particularly interesting. In recent studies, inhibiting the catabolic pathway of precursor, boosting precursor pools, and optimizing substrate combination greatly improved the production of OCFA in Y. lipolytica. We explored the lipid readjustment of OCFA in engineered Y. lipolytica strains. NPLC-Corona-CAD^® evidenced a time-dependent overproduction of free fatty acids, diglycerides, and phosphatidylcholine (PC) in obese LP compared to obese L. Phosphatidylethanolamine (PE) and phosphatidylinositol, largely overproduced in obese LP at 72 h compared to obese L, vanished at 216 h. The fatty acyls (FAs) composition of glycero- and glycerophospholipids was determined by NPLC-APPI⁺-HRMS from in-source generated monoacylglycerol-like fragment ions. C18:1 and C17:1 were predominant acylglycerols in obese L and obese LP, respectively. Phosphatidic acid, PE, and PC exhibited similar FAs composition but differed in their molecular species distributions. Cardiolipin (CL) is known to contain mostly C18:2 FAs corresponding to the composition in obese L, 50% of C18:2, and 35% of C18:1. In obese LP, both FAs dropped to drop to 20%, and C17:1 were predominant, reaching 55%. We hypothesize that CL-modified composition in obese LPs may alter mitochondrial function and limit lipid production.

In this issue we demonstrated that the phospholipid content of Streptomyces lividans varies greatly with Pi availability being was much lower in Pi limitation than in Pi proficiency whereas that of Streptomyces coelicolor varied little with Pi availability. In contrast the content in phosphate free ornithine lipids was enhanced in both strains in condition of phosphate limitation. Ornithine lipids biosynthesis starts with the N-acylation of ornithine to form lyso-ornithine that is then O-acylated to yield ornithine lipid. The operon sco1222-23 was proposed to be involved in the conversion of specific amino acids into ornithine in condition of phosphate limitation whereas the sco0921-20 operon encoding N- and O-acyltransferase, respectively, was shown to be involved in the biosynthesis of these lipids. The expression of these two operons was shown to be under the positive control of the two components system PhoR/PhoP and thus induced in phosphate limitation. The expression of phoR/phoP being weak in S. coelicolor, the poor expression of these operons resulted into a fivefold lower ornithine lipids content in this strain compared to S. lividans. In the deletion mutant of the sco0921-20 operon of S. lividans, lyso-ornithine and ornithine lipids were barely detectable and TAG content was enhanced. The complementation of this mutant by the sco0921-20 operon or by sco0920 alone restored ornithine lipids and TAG content to wild type level and was correlated with a twofold increase in the cardiolipin content. This suggested that SCO0920 bears, besides its broad O-acyltransferase activity, an N-acyltransferase activity and this was confirmed by the detection of lyso-ornithine in this strain. In contrast, the complementation of the mutant by sco0921 alone had no impact on ornithine lipids, TAG nor cardiolipin content but was correlated with a high lyso-ornithine content. This confirmed that SCO0921 is a strict N-acyltransferase. However, interestingly, the over-expression of the sco0921-20 operon or of sco0921 alone in S. coelicolor, led to an almost total disappearance of phosphatidylinositol that was correlated with an enhanced DAG and TAG content. This suggested that SCO0921 also acts as a phospholipase C, degrading phosphatidylinositol to indirectly supply of phosphate in condition of phosphate limitation.

Cyclosporin A (CsA) is a molecule with well-known immunosuppressive properties. As it also acts on the opening of mitochondrial permeability transition pore (mPTP), CsA has been evaluated for ischemic heart diseases (IHD). However, its distribution throughout the body and its physicochemical characteristics strongly limit the use of CsA for intravenous administration. In this context, nanoparticles (NPs) have emerged as an opportunity to circumvent the above-mentioned limitations. We have developed in our laboratory an innovative nanoformulation based on the covalent bond between squalene (Sq) and cyclosporin A to avoid burst release phenomena and increase drug loading. After a thorough characterization of the bioconjugate, we proceeded with a nanoprecipitation in aqueous medium in order to obtain SqCsA NPs of well-defined size. The SqCsA NPs were further characterized using dynamic light scattering (DLS), cryogenic transmission electron microscopy (cryoTEM), and high-performance liquid chromatography (HPLC), and their cytotoxicity was evaluated. As the goal is to employ them for IHD, we evaluated the cardioprotective capacity on two cardiac cell lines. A strong cardioprotective effect was observed on cardiomyoblasts subjected to experimental hypoxia/reoxygenation. Further research is needed in order to understand the mechanisms of action of SqCsA NPs in cells. This new formulation of CsA could pave the way for possible medical application.

Streptomycetes are well known antibiotic producers and are among the rare prokaryotes able to store carbon as lipids. Previous comparative studies of the weak antibiotic producer Streptomyces lividans with its ppk mutant and with Streptomyces coelicolor, which both produce antibiotics, suggested the existence of a negative correlation between total lipid content and the ability to produce antibiotics. To determine whether such a negative correlation can be generalized to other Streptomyces species, fifty-four strains were picked randomly and grown on modified R2YE medium, limited in phosphate, with glucose or glycerol as the main carbon source. The total lipid content and antibiotic activity against Micrococcus luteus were assessed for each strain. This study revealed that the ability to accumulate lipids was not evenly distributed among strains and that glycerol was more lipogenic than glucose and had a negative impact on antibiotic biosynthesis. Furthermore, a statistically significant negative Pearson correlation between lipid content and antibiotic activity could be established for most strains, but a few strains escape this general law. These exceptions are likely due to limits and biases linked to the type of test used to determine antibiotic activity, which relies exclusively on Micrococcus luteus sensitivity. They are characterized either by high lipid content and high antibiotic activity or by low lipid content and undetectable antibiotic activity against Micrococcus luteus. Lastly, the comparative genomic analysis of two strains with contrasting lipid content, and both named Streptomyces antibioticus (DSM 41,481 and DSM 40,868, which we found to be phylogenetically related to Streptomyces lavenduligriseus), indicated that some genetic differences in various pathways related to the generation/consumption of acetylCoA could be responsible for such a difference.