The high demand for new biomaterials makes synthesis of polyhydroxyalkanoates (PHA) in plants an interesting and desirable achievement. Production of polymers in plants is an example of application of biotechnology for improving the properties of plants, e.g. industrial properties, but it can also provide knowledge about plant physiology and metabolism. The subject of the present study was an industrially important plant: flax, Linum usitatissimum L., of a fibre cultivar (cv Nike). In the study the gene encoding PHA synthase from Pseudomonas aeruginosa, fused to a peroxisomal targeting signal, was expressed in flax plants with the aim of modifying the mechanical properties of plants. Medium-chain-length (mcl) hydroxy acids in flax plants from tissue cultures were detected by GC-FID and FTIR method. The introduced changes did not affect fatty acid content and composition in generated flax plants. Since mcl-PHA are known as elastomers, the mechanical properties of created plants were examined. Modified plants showed increases in the values of all measured parameters (except strain at break evaluated for one modified line). The largest increase was noted for tensile stiffness, which was 2- to 3-fold higher than in wild-type plants. The values estimated for another parameter, Young’s modulus, was almost at the same level in generated flax plants, and they were about 2.7-fold higher when compared to unmodified plants. The created plants also exhibited up to about 2.4-fold higher tensile strength. The observed changes were accompanied by alterations in the expression of selected genes, related to cell wall metabolism in line with the highest expression of phaC1 gene. Biochemical data were confirmed by spectroscopic methods, which also revealed that crystallinity index values of cellulose in modified flax plants were increased in comparison to wild-type flax plants and correlated with biomechanical properties of plants.

Conjugated linoleic acids (CLAs) have been found to have beneficial effects on human health when used as dietary supplements. However, their availability is limited because pure, chemistry-based production is expensive, and biology-based fermentation methods can only create small quantities. In an effort to enhance microbial production of CLAs, four genetically modified strains of the oleaginous yeast Yarrowia lipolytica were generated. These mutants presented various genetic modifications, including the elimination of β-oxidation (pox1-6∆), the inability to store lipids as triglycerides (dga1∆ dga2∆ are1∆ lro1∆), and the overexpression of the Y. lipolytica ∆12-desaturase gene (YlFAD2) under the control of the constitutive pTEF promoter. All strains received two copies of the pTEF-oPAI or pPOX-oPAI expression cassettes; PAI encodes linoleic acid isomerase in Propionibacterium acnes. The strains were cultured in neosynthesis or bioconversion medium in flasks or a bioreactor. The strain combining the three modifications mentioned above showed the best results: when it was grown in neosynthesis medium in a flask, CLAs represented 6.5% of total fatty acids and in bioconversion medium in a bioreactor, and CLA content reached 302 mg/L. In a previous study, a CLA degradation rate of 117 mg/L/h was observed in bioconversion medium. Here, by eliminating β-oxidation, we achieved a much lower rate of 1.8 mg/L/h.

Ricinoleic acid (RA) is an hydroxylated fatty acid of industrial interest currently extracted from castor oil. A microbiological alternative strategy to product RA was developed in order to overcome castorbean farming linked issues and to meet market demands. An engineered strain of the oleaginous yeast Yarrowia lipolytica was chosen for the RA production. This strain – OleoX combines 10 deletions which maximize the pool of available oleic acid which is the substrate used for bioconversion to RA. As a result, the fatty acid composition in the chassis OleoX strain contains mainly oleic acid (> 80%). Two hydroxylases originated from castorbean (RcFAH12) or fungi (CpFAH12) were expressed in OleoX. The strain expressing CpFAH12 was able to product RA (52%), with a coproduction of linoleic acid. Using promoter 4UAS-pTEF, stronger than pTEF, led to a strain producing RA up to 65% of its total lipid content. With the objective to optimise this chassis strain for library construction, a docking platform was introduced in OleoX strain to guide the integration of expression cassettes at a specific locus. The objectives are to improve both transformation efficiency and expression level reproducibility. This strain will be used to construct a library of mutants of FAH12.

Although there are numerous oleochemical applications for ricinoleic acid (RA) and its derivatives, their production is limited and subject to various safety legislations. In an effort to produce RA from alternative sources, we constructed a genetically modified strain of the oleaginous yeast Yarrowia lipolytica . This strain is unable to perform β-oxidation and is invalidated for the native triacylglycerol (TAG) acyltransferases (Dga1p, Dga2p, and Lro1p) and the ∆12 desaturase (Fad2p). We also expressed the Ricinus communis ∆12 hydroxylase (Rc FAH12 ) under the control of the TEF constitutive promoter in this strain. However, RA constituted only 7 % of the total lipids produced by this modified strain. By contrast, expression of the Claviceps purpurea hydroxylase Cp FAH12 in this background resulted in a strain able to accumulate RA to 29 % of total lipids, and expression of an additional copy of Cp FAH12 drove RA accumulation up to 35 % of total lipids. The co-expression of the C. purpurea or R. communis type II diacylglycerol acyltransferase (Rc DGAT2 or Cp DGAT2 ) had negative effects on RA accumulation in this yeast, with RA levels dropping to below 14 % of total lipids. Overexpression of the native Y. lipolytica PDAT acyltransferase (Lro1p) restored both TAG accumulation and RA levels. Thus, we describe the consequences of rerouting lipid metabolism in this yeast so as to develop a cell factory for RA production. The engineered strain is capable of accumulating RA to 43 % of its total lipids and over 60 mg/g of cell dry weight; this is the most efficient production of RA described to date.

tA novel approach to trigger lipid accumulation and/or citrate production in vivo through the inactivation of the 2-methyl-citrate dehydratase in Yarrowia lipolytica was developed. In nitrogen-limited cultures with biodiesel-derived glycerol utilized as substrate, the phd1 mutant (JMY1203) produced 57.7 g/L of total citrate, 1.6-fold more than the wild-type strain, with a concomitant glycerol to citrate yield of 0.91 g/g.Storage lipid in cells increased at the early growth stages, suggesting that inactivation of the 2-methyl-citrate dehydratase would mimic nitrogen limitation. Thus, a trial of JMY1203 strain was performed with glycerol under nitrogen-excess conditions. Compared with the equivalent nitrogen-limited culture,significant quantities of lipid (up to ≈ 31% w/w in dry weight, 1.6-fold higher than the nitrogen-limited experiment) were produced. Also, non-negligible quantities of citric acid (up to ≈ 26 g/L, though 0.57-fold lower than the nitrogen-limited experiment) were produced, despite remarkable nitrogen presence into the medium, indicating the construction of phenotype that constitutively accumulated lipid and secreted citrate in Y. lipolytica during growth on waste glycerol utilized as substrate.

In Yarrowia lipolytica, targeted gene replacement occurs only with long length (1 kb) homologous flanking fragments, as this yeast preferentially uses the non-homologous end-joining mechanism (NHEJ) for DNA repair over homologous recombination (HR). To improve the frequency of HR, we identified and disrupted the KU70 and KU80 genes responsible for double strand break repair in the NHEJ pathway in Y. lipolytica. In ku70∆ HR of URA3 marker at the ADE2 locus occurred with 43 % frequency with as little as 50 bp long flanking regions. The number of Ura+ transformants was reduced to 1 % of the Po1d (ura3-302) wild type-like strain level, regardless of the flanking fragment length. On the contrary, even though HR was not improved in ku80∆, Ura+ transformants was 60 % lower compared to the wild type.

Eukaryotes store lipids in a specialised organelle, the lipid body (LB), mainly as triglycerides (TAGs). Both the rates of synthesis and degradation contribute to the control of the accumulation of TAGs. The synthesis of TAGs in yeasts has been well documented, especially in the model yeast Saccharomyces cerevisiae and in the oleaginous yeast Yarrowia lipolytica. However, descriptions of the processes involved in TAG degradation are more scarce and mostly for S. cerevisiae. Here, we report the characterisation of two Y. lipolytica genes, YlTGL3 and YlTGL4, encoding intracellular lipases involved in TAG degradation. The two proteins are localised in lipid bodies, and YlTgl4 was mainly found at the interface between LBs. Surprisingly, the spatial organisation of YlTgl3 and YlTgl4 depends on the culture medium and on the physiological phase of the cell. Inactivation of one or both genes doubles the lipid accumulation capacity of Y. lipolytica, increasing the cell's capacity to accumulate TAGs. The amino acid sequence of YlTgl4 contains the consensus sequence motif (G/A)XSXG, typical of serine hydrolases, whereas YlTgl3 does not. Single and double mutants are unable to degrade TAGs, and higher expression of YlTgl4 correlates with TAG degradation. Therefore, we propose that YlTgl4 is the main lipase responsible for TAG degradation and that YlTgl3 may act as a positive regulator of YlTgl4 rather than a functional lipase. Thus, contrary to S. cerevisiae, Y. lipolytica possesses two intracellular lipases with distinct roles and with distinct localisations in the LB.

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.

The increasing demand of plant oils or animal fat for biodiesel and specific lipid derivatives for the oleochemical field (such as lubricants, adhesives or plastics) have created price imbalance in both the alimentary and energy field. Moreover, the lack of non-edible oil feedstock has given rise to concerns on land-use practices and on oil production strategies. Recently, much attention has been paid to the exploitation of microbial oils. Most of them present lipid profiles similar in type and composition to plants and could therefore have many advantages as are no competitive with food, have short process cycles and their cultivation is independent of climate factors. Among microorganisms, yeasts seem to be very promising as they can be easily genetically enhanced, are suitable for large-scale fermentation and are devoid of endotoxins. This review will focus on the recent understanding of yeasts lipid metabolism, the succeeding genetic engineering of the lipid pathways and the recent developments on fermentation techniques that pointed out yeasts as promising alternative producers for oil or plastic.

High energy prices, depletion of crude oil supplies, and price imbalance created by the increasing demand of plant oils or animal fat for biodiesel and specific lipid derivatives such as lubricants, adhesives, and plastics have given rise to heated debates on land-use practices and to environmental concerns about oil production strategies. However, commercialization of microbial oils with similar composition and energy value to plant and animal oils could have many advantages, such as being noncompetitive with food, having shorter process cycle and being independent of season and climate factors. This review focuses on the ongoing research on different oleaginous yeasts producing high added value lipids and on the prospects of such microbial oils to be used in different biotechnological processes and applications. It covers the basic biochemical mechanisms of lipid synthesis and accumulation in these organisms, along with the latest insights on the metabolic processes involved. The key elements of lipid accumulation, the mechanisms suspected to confer the oleaginous character of the cell, and the potential metabolic routes enhancing lipid production are also extensively discussed.

We previously developed a fermentation protocol for lipid accumulation in the oleaginous yeast Y. lipolytica. This process was used to perform transcriptomic time-course analyses to explore gene expression in Y. lipolytica during the transition from biomass production to lipid accumulation. In this experiment, a biomass concentration of 54.6 gCDW/l, with 0.18 g/gCDW lipid was obtained in ca. 32 h, with low citric acid production. A transcriptomic profiling was performed on 11 samples throughout the fermentation. Through statistical analyses, 569 genes were highlighted as differentially expressed at one point during the time course of the experiment. These genes were classified into 9 clusters, according to their expression profiles. The combination of macroscopic and transcriptomic profiles highlighted 4 major steps in the culture: (i) a growth phase, (ii) a transition phase, (iii) an early lipid accumulation phase, characterized by an increase in nitrogen metabolism, together with strong repression of protein production and activity; (iv) a late lipid accumulation phase, characterized by the rerouting of carbon fluxes within cells. This study explores the potential of Y. lipolytica as an alternative oil producer, by identifying, at the transcriptomic level, the genes potentially involved in the metabolism of oleaginous species.

The yeast Y. lipolytica has developed a very efficient mechanism in the degradation and utilization of hydrophobic substrates. In addition, its capacity to accumulate great amounts of lipids places it among the oleaginous yeasts. The completion of the sequence of the Y. lipolytica genome and the existence of genetic tools for gene manipulation allow the use of its metabolic functions for biotechnological applications. Examples are presented demonstrating that wild type and genetically engineered strains of Y. lipolytica can be used for fatty acid bioconversion, substrate valorization, polyunsaturated fatty acids production, oil polluted water bioremediation and single cell oil production. This review also presents the potential uses of this yeast as an alternative production (cell) factory in the oleochemical field.

Yarrowia lipolytica est une levure oleagineuse qui possede un remarquable potentiel de croissance et d'accumulation de lipides dans des milieux hydrophobes. Au cours de cette etude nous avons approfondi nos connaissances du metabolisme lipidique chez cette levure en mettant l'accent sur la capacite d'accumulation et de degradation des lipides, la regulation et les interactions des differentes voies metaboliques. L'etude des enzymes du metabolisme des microorganismes oleagineux et de leurs particularites fonctionnelles, en comparaison avec celles des microorganismes non oleagineux, a permis de se focaliser sur des enzymes candidates pour etre impliquees dans le caractere oleagineux. L'objectif de cette etude est de combiner les connaissances physiologiques sur cette levure avec les outils genetiques existants afin d'utiliser Y. lipolytica comme une usine cellulaire pour la production de grandes quantites de lipides ayant un interet industriel. Afin de rediriger les flux de carbone vers la synthese de lipides, le gene GUT2, qui code l'un de deux isomeres de la glycerol-3-phosphate deshydrogenase, ainsi que la voie de la β-oxydation ont ete invalides chez Y. lipolytica, conduisant a une forte augmentation de l'accumulation des lipides. Afin d'identifier l'etape limitante de la synthese des TAG et de construire des souches aux profils lipidiques modifies nous avons joue sur le niveau d'expression des acyltransferases et des desaturases. La contribution relative des acyltransferases depend de la phase de croissance et leurs affinites vis-a-vis des substrats donneurs d'acyle sont complementaires. La modification des desaturases a permis d'accumuler des acides gras specifiques, demontrant ainsi qu'il est possible de moduler le profil des lipides accumules chez Y. lipolytica. Cette etude nous a permis de construire des souches hyperaccumulatrices aux profils lipidiques tels qu'ils permettront de leur trouver des applications biotechnologiques dans la filiere des biocarburants ou dans l'industrie oleochimique en tant qu'unites alternatives pour la production de substrats specifiques.

The yeast Yarrowia lipolytica has developed very efficient mechanisms for breaking down and using hydrophobic substrates. It is considered an oleaginous yeast, based on its ability to accumulate large amounts of lipids. Completion of the sequencing of the Y. lipolytica genome and the existence of suitable tools for genetic manipulation have made it possible to use the metabolic function of this species for biotechnological applications. In this review, we describe the coordinated pathways of lipid metabolism, storage and mobilization in this yeast, focusing in particular on the roles and regulation of the various enzymes and organelles involved in these processes. The physiological responses of Y. lipolytica to hydrophobic substrates include surface-mediated and direct interfacial transport processes, the production of biosurfactants, hydrophobization of the cytoplasmic membrane and the formation of protrusions. We also discuss culture conditions, including the mode of culture control and the culture medium, as these conditions can be modified to enhance the accumulation of lipids with a specific composition and to identify links between various biological processes occurring in the cells of this yeast. Examples are presented demonstrating the potential use of Y. lipolytica in fatty-acid bioconversion, substrate valorization and single-cell oil production. Finally, this review also discusses recent progress in our understanding of the metabolic fate of hydrophobic compounds within the cell: their terminal oxidation, further degradation or accumulation in the form of intracellular lipid bodies.

The oleaginous yeast Yarrowia lipolytica is known to inhabit various lipid-containing environments. One of the most striking features in this yeast is the presence of several multigene families involved in the metabolic pathways of hydrophobic substrate utilization. The complexity and the multiplicity of these genes give Y. lipolytica a wide capability range towards hydrophobic substrate (HS) utilization and storage. The combination of the increasing knowledge of this yeast's metabolism and the development of more efficient genetic tools is offering new perspectives in using Y. lipolytica as a model organism to study the mechanisms involved in lipid metabolism associated to fat uptake, storage, deposition, mobilization and regulation. Nutrient status and culture conditions seem to play a major role in obesity.

A genomic comparison of Yarrowia lipolytica and Saccharomyces cerevisiae indicates that the metabolism of Y. lipolytica is oriented toward the glycerol pathway. To redirect carbon flux toward lipid synthesis, the GUT2 gene, which codes for the glycerol-3-phosphate dehydrogenase isomer, was deleted in Y. lipolytica in this study. This Delta gut2 mutant strain demonstrated a threefold increase in lipid accumulation compared to the wild-type strain. However, mobilization of lipid reserves occurred after the exit from the exponential phase due to beta-oxidation. Y. lipolytica contains six acyl-coenzyme A oxidases (Aox), encoded by the POX1 to POX6 genes, that catalyze the limiting step of peroxisomal beta-oxidation. Additional deletion of the POX1 to POX6 genes in the Delta gut2 strain led to a fourfold increase in lipid content. The lipid composition of all of the strains tested demonstrated high proportions of FFA. The size and number of the lipid bodies in these strains were shown to be dependent on the lipid composition and accumulation ratio.