Analysis of ATP-citrate lyase and malic enzyme mutants of Yarrowia lipolytica points out the importance of mannitol metabolism in fatty acid synthesis

AbstractThe role of the two key enzymes of fatty acid (FA) synthesis, ATP-citrate lyase (Acl) and malic enzyme (Mae), was analyzed in the oleaginous yeast Yarrowia lipolytica. In most oleaginous yeasts, Acl and Mae are proposed to provide, respectively, acetyl-CoA and NADPH for FA synthesis. Acl was mainly studied at the biochemical level but no strain depleted for this enzyme was analyzed in oleaginous microorganisms. On the other hand the role of Mae in FA synthesis in Y. lipolytica remains unclear since it was proposed to be a mitochondrial NAD(H)-dependent enzyme and not a cytosolic NADP(H)-dependent enzyme. In this study, we analyzed for the first time strains inactivated for corresponding genes. Inactivation of ACL1 decreases FA synthesis by 60 to 80%, confirming its essential role in FA synthesis in Y. lipolytica. Conversely, inactivation of MAE1 has no effects on FA synthesis, except in a FA overaccumulating strain where it improves FA synthesis by 35%. This result definitively excludes Mae as a major key enzyme for FA synthesis in Y. lipolytica. During the analysis of both mutants, we observed a negative correlation between FA and mannitol level. As mannitol and FA pathways may compete for carbon storage, we inactivated YlSDR, encoding a mannitol dehydrogenase converting fructose and NADPH into mannitol and NADP+. The FA content of the resulting mutant was improved by 60% during growth on fructose, demonstrating that mannitol metabolism may modulate FA synthesis in Y. lipolytica

Identification and characterization of DGA2, an acyltransferase of the DGAT1 acyl-CoA:diacylglycerol acyltransferase family in the oleaginous yeast Yarrowia lipolytica. New insights into the storage lipid metabolism of oleaginous yeasts

Triacylglycerols (TAG) and steryl esters (SE) are the principal storage lipids in all eukaryotic cells. In yeasts, these storage lipids accumulate within special organelles known as lipid bodies (LB). In the lipid accumulation-oriented metabolism of the oleaginous yeast Yarrowia lipolytica, storage lipids are mostly found in the form of TAG, and only small amounts of SE accumulate. We report here the identification of a new DAG acyltransferase gene, DGA2, homologous to the ARE genes of Saccharomyces cerevisiae. This gene encodes a member of the type 1 acyl-CoA:diacylglycerol acyltransferase family (DGAT1), which has not previously been identified in yeasts, but is commonly found in mammals and plants. Unlike the Are proteins in S. cerevisiae, Dga2p makes a major contribution to TAG synthesis via an acyl-CoA-dependent mechanism and is not involved in SE synthesis. This enzyme appears to affect the size and morphology of LB, suggesting a direct role of storage lipid proteins in LB formation. We report that the Are1p of Y. lipolytica was essential for sterol esterification, as deletion of the encoding gene (ARE1) completely abolished SE synthesis. Unlike its homologs in yeasts, YlARE1 has no DAG acyltransferase activity. We also reconsider the role and function of all four acyltransferase enzymes involved in the final step of neutral lipid synthesis in this oleaginous yeast

Transfert du carbone au cours de l'infection du tournesol par le champignon nécrotophe B. cinerea (des hexoses de la plante au mannitol fongique)

Our work was completed on Botrytis cinerea, a necrotrophic ascomycete fungus. The first stage of our work consisted in drawing up an inventory of the soluble carbon metabolites present in partners of infection: Botrytis cinerea and sunflower cotyledons and to follow their evolution during the infection. During the colonization of the cotyledons of sunflower, hexoses from plant disappear, whereas mannitol of fungal origin is accumulated. In order to highlight the elements implied in the disappearance of hexoses in the plant, we sought genes potentially implied in the transport of hexoses in the genome of B. cinerea. The bioinformatic analyses made it possible to characterize eighteen sequences like transmembrane proteins implied in the transport of hexoses. Transcriptional profiles of these genes indicate a great flexibility of expression during infection as during in vitro development. Once transported, plant s hexoses are metabolized by fungi. In order to know to become to it hexoses, we followed their assimilation thanks to the use of marked sugars. This approach was coupled with an analysis of the way of synthesis of the mannitol, metabolite carbonaceous prevalent accumulated by B. cinerea in answer to the assimilation of sugars. It exists two mannitol biosynthesis pathways in Ascomycetes fungi and the results obtained with B. cinerea, propose new prospects of operation and make it possible to better determine the importance of each way in the metabolism of the mannitolNos travaux ont été réalisés sur Botrytis cinerea, champignon nécrotrophe ascomycète. La première étape de notre travail a consisté à dresser un état des lieux des métabolites carbonés solubles présents chez les partenaires de l infection : Botrytis cinerea et le cotylédons de tournesol et de suivre leur évolution au cours de l infection. Au cours de la colonisation des cotylédons de tournesol, les hexoses d origine végétale disparaissent, alors que du mannitol d origine fongique est accumulé. Afin de mettre en évidence les éléments impliqués dans la disparition des hexoses dans la plante, nous avons recherché les gènes potentiellement impliqués dans le transport d hexoses dans le génome de B. cinerea. Les analyses bioinformatiques ont permis de caractériser dix-huit séquences comme des protéines transmembranaires impliquées dans le transport des hexoses. Les profils transcriptionnels de ces gènes indiquent une grande flexibilité d expression aussi bien durant l infection que pendant le développement in vitro du pathogène. Une fois transportés, les hexoses d origine végétale sont métabolisés par le champignon. Afin de connaitre le devenir des hexoses, nous avons suivi leur assimilation grâce à l utilisation de sucres marqués. Cette approche a été couplée à une analyse de la voie de synthèse du mannitol, métabolite carboné prédominant accumulé par B. cinerea en réponse à l assimilation des sucres. Il existe deux voies de biosynthèse du mannitol chez les champignons Ascomycètes et les résultats obtenus chez B. cinerea, proposent de nouvelles perspectives de fonctionnement et permettent de mieux cerner l importance de chacune des voies dans le métabolisme du mannitolLYON1-BU.Sciences (692662101) / SudocSudocFranceF

Role of Pex11p in Lipid Homeostasis in Yarrowia lipolytica

Peroxisomes are essential organelles in the cells of most eukaryotes, from yeasts to mammals. Their role in beta-oxidation is particularly essential in yeasts; for example, in Saccharomyces cerevisiae, fatty acid oxidation takes place solely in peroxisomes. In this species, peroxisome biogenesis occurs when lipids are present in the culture medium, and it involves the Pex11p protein family: ScPex11p, ScPex25p, ScPex27p, and ScPex34p. Yarrowia lipolytica has three Pex11p homologues, which are YALI0C04092p (YlPex11p), YALI0C04565p (YlPex11C), and YALI0D25498p (Pex11/25p). We found that these genes are regulated by oleic acid, and as has been observed in other organisms, YlPEX11 deletion generated giant peroxisomes when mutant yeast were grown in oleic acid medium. Moreover, Delta Ylpex11 was unable to grow on fatty acid medium and showed extreme dose-dependent sensitivity to oleic acid. Indeed, when the strain was grown in minimum medium with 0.5% glucose and 3% oleic acid, lipid body lysis and cell death were observed. Cell death and lipid body lysis may be partially explained by an imbalance in the expression of the genes involved in lipid storage, namely, DGA1, DGA2, and LRO1, as well as that of TGL4, which is involved in lipid remobilization. TGL4 deletion and DGA2 overexpression resulted in decreased oleic acid sensitivity and delayed cell death of Delta Ylpex11, which probably stemmed from the release of free fatty acids into the cytoplasm. All these results show that YlPex11p plays an important role in lipid homeostasis in Y. lipolytica

Lipid production by the oleaginous yeast Yarrowia lipolytica using industrial by-products under different culture conditions

Background: Microbial lipid production using renewable feedstock shows great promise for the biodiesel industry. Results: In this study, the ability of a lipid-engineered Yarrowia lipolytica strain JMY4086 to produce lipids using molasses and crude glycerol under different oxygenation conditions and at different inoculum densities was evaluated in fed-batch cultures. The greatest lipid content, 31% of CDW, was obtained using a low-density inoculum, a constant agitation rate of 800 rpm, and an oxygenation rate of 1.5 L/min. When the strain was cultured for 450 h in a chemostat containing a nitrogen-limited medium (dilution rate of 0.01 h(-1); 250 g/L crude glycerol), volumetric lipid productivity was 0.43 g/L/h and biomass yield was 60 g CDW/L. The coefficient of lipid yield to glycerol consumption (Y-L/gly) and the coefficient of lipid yield to biomass yield (Y-L/X) were equal to 0.1 and 0.4, respectively. Conclusions: These results indicate that lipids may be produced using renewable feedstock, thus providing a means of decreasing the cost of biodiesel production. Furthermore, using molasses for biomass production and recycling glycerol from the biodiesel industry should allow biolipids to be sustainably produced

Optimization of odd chain fatty acid production by Yarrowia lipolytica

Background Odd chain fatty acids (odd FAs) have a wide range of applications in therapeutic and nutritional industries, as well as in chemical industries including biofuel. Yarrowia lipolytica is an oleaginous yeast considered a preferred microorganism for the production of lipid-derived biofuels and chemicals. However, it naturally produces negligible amounts of odd chain fatty acids. Results The possibility of producing odd FAs using Y. lipolytica was investigated. Y. lipolytica wild-type strain was shown able to grow on weak acids; acetate, lactate, and propionate. Maximal growth rate on propionate reached 0.24 ± 0.01 h−1 at 2 g/L, and growth inhibition occurred at concentration above 10 g/L. Wild-type strain accumulated lipids ranging from 7.39 to 8.14% (w/w DCW) depending on the carbon source composition, and odd FAs represented only 0.01–0.12 g/L. We here proved that the deletion of the PHD1 gene improved odd FAs production, which reached a ratio of 46.82% to total lipids. When this modification was transferred to an obese strain, engineered for improving lipid accumulation, further increase odd FAs production reaching a total of 0.57 g/L was shown. Finally, a fed-batch co-feeding strategy was optimized for further increase odd FAs production, which generated 0.75 g/L, the best production described so far in Y. lipolytica. Conclusions A Y. lipolytica strain able to accumulate high level of odd chain fatty acids, mainly heptadecenoic acid, has been successfully developed. In addition, a fed-batch co-feeding strategy was optimized to further improve lipid accumulation and odd chain fatty acid content. These lipids enriched in odd chain fatty acid can (1) improve the properties of the biodiesel generated from Y. lipolytica lipids and (2) be used as renewable source of odd chain fatty acid for industrial applications. This work paves the way for further improvements in odd chain fatty acids and fatty acid-derived compound production

Amino acid changes during sunflower infection by the necrotrophic fungus B. cinerea

Addendum to: Dulermo T, Rascle C, Chinnici G, Gout E, Bligny R, Cotton P. Dynamic carbon transfer during pathogenesis of sunflower by the necrotrophic Fungus Botrytis cinerea: from plant hexoses to mannitol. New Phytol (2009) 183(4): 1149-1162International audienceMetabolic changes that occur in host tissues during a necrotrophic plant/fungal interaction have been poorly investigated. Whereas carbon metabolism reprogramming and photosynthesis disturbances have been studied,1 data on plant amino acids stores during infection are scarce. Here we report an analysis of sunflower cotyledon amino acid content during infection with the necrotrophic fungus Botrytis cinerea, by using 13C-NMR spectroscopy. A rapid disappearance of plant amino acids was observed, most probably due to fungal assimilation. In order to explore amino acid changes due to host reaction, we investigated the amino acid content in healthy and invaded region of infected leaves. During the course of infection, glutamate and total amino acid stores were affected at distance in the non invaded region. Glutamate depletion was correlated to an enhanced sunflower glutamate dehydrogenase (GDH) transcription level in the area invaded by pathogen. Our data suggest that glutamate could be transferred to the invaded region to supply nitrogen. Such a strategy could delay cell death, and consequently disturb fungal progression in plant tissues

A genome-scale metabolic model of the lipid-accumulating yeast <it>Yarrowia lipolytica</it>

<p>Abstract</p> <p>Background</p> <p><it>Yarrowia lipolytica</it> is an oleaginous yeast which has emerged as an important microorganism for several biotechnological processes, such as the production of organic acids, lipases and proteases. It is also considered a good candidate for single-cell oil production. Although some of its metabolic pathways are well studied, its metabolic engineering is hindered by the lack of a genome-scale model that integrates the current knowledge about its metabolism.</p> <p>Results</p> <p>Combining <it>in silico</it> tools and expert manual curation, we have produced an accurate genome-scale metabolic model for <it>Y. lipolytica</it>. Using a scaffold derived from a functional metabolic model of the well-studied but phylogenetically distant yeast <it>S. cerevisiae</it>, we mapped conserved reactions, rewrote gene associations, added species-specific reactions and inserted specialized copies of scaffold reactions to account for species-specific expansion of protein families. We used physiological measures obtained under lab conditions to validate our predictions.</p> <p>Conclusions</p> <p><it>Y. lipolytica</it> iNL895 represents the first well-annotated metabolic model of an oleaginous yeast, providing a base for future metabolic improvement, and a starting point for the metabolic reconstruction of other species in the <it>Yarrowia</it> clade and other oleaginous yeasts.</p

MOESM2 of Engineering Yarrowia lipolytica to produce biodiesel from raw starch

Additional file 2: Figure S1

Using a vector pool containing variable-strength promoters to optimize protein production in Yarrowia lipolytica

Background The yeast Yarrowia lipolytica is an increasingly common biofactory. To enhance protein expression, several promoters have been developed, including the constitutive TEF promoter, the inducible POX2 promotor, and the hybrid hp4d promoter. Recently, new hp4d-inspired promoters have been created that couple various numbers of UAS1 tandem elements with the minimal LEU2 promoter or the TEF promoter. Three different protein-secretion signaling sequences can be used: preLip2, preXpr2, and preSuc2. Results To our knowledge, our study is the first to use a set of vectors with promoters of variable strength to produce proteins of industrial interest. We used the more conventional TEF and hp4d promoters along with five new hybrid promoters: 2UAS1-pTEF, 3UAS1-pTEF, 4UAS1-pTEF, 8UAS1-pTEF, and hp8d. We compared the production of RedStar2, glucoamylase, and xylanase C when strains were grown on three media. As expected, levels of RedStar2 and glucoamylase were greatest in the strain with the 8UAS1-pTEF promoter, which was stronger. However, surprisingly, the 2UAS1-pTEF promoter was associated with the greatest xylanase C production and activity. This finding underscored that stronger promoters are not always better when it comes to protein production. We therefore developed a method for easily identifying the best promoter for a given protein of interest. In this gateway method, genes for YFP and α-amylase were transferred into a pool of vectors containing different promoters and gene expression was then analyzed. We observed that, in most cases, protein production and activity were correlated with promoter strength, although this pattern was protein dependent. Conclusions Protein expression depends on more than just promoter strength. Indeed, promoter suitability appears to be protein dependent; in some cases, optimal expression and activity was obtained using a weaker promoter. We showed that using a vector pool containing promoters of variable strength can be a powerful tool for rapidly identifying the best producer for a given protein of interest