Transcriptomic Analyses during the Transition from Biomass Production to Lipid Accumulation in the Oleaginous Yeast Yarrowia lipolytica

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

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

Ingénierie génétique de la levure oléagineuse Yarrowia Lipolytica pour la production de lipides

Yarrowia lipolytica is an oleaginous yeast with a remarkable potential for growth and lipid accumulation in hydrophobic environments. In this study we have expanded our knowledge of lipid metabolism in this yeast with an emphasis on lipid accumulation and degradation capacity, and the regulation and interaction of the different metabolic pathways. The study of metabolic enzymes in oleaginous microorganisms and their functional specifications in comparison with the non-oleaginous microorganisms has led us to focus on the enzymes potentially conferring the oleaginous character. The objective of this study is to combine the knowledge of yeast's physiology with the existing genetic tools to use Y. lipolytica as a cell factory for the production of large amounts of lipids with an industrial interest. In order to redirect carbon flux towards lipid synthesis, the GUT2 gene coding for a glycerol-3-phosphate dehydrogenase isomer and the β-oxidation pathway were invalidated in Y. lipolytica leading to a strong increase of lipid accumulation. As to identify the limiting step of TAG synthesis and to construct strains with altered lipid profiles, we have modified the expression level of acyltransferases and desaturases. The relative contribution of acyltransferases depends on the growth phase and their affinity towards the acylation substrate is complementary. The modification of desaturases allowed the accumulation of specific fatty acids, thus demonstrating the possibility of modulating lipid profile in Y. lipolytica. This study has enabled us to construct hyper accumulative strains with added-value lipid profiles that could find biotechnological applications in the energy (biodiesel) field or in the oleochemical industry as alternative units for specific substrate production.Yarrowia lipolytica est une levure oléagineuse qui possède un remarquable potentiel de croissance et d'accumulation de lipides dans des milieux hydrophobes. Au cours de cette étude nous avons approfondi nos connaissances du métabolisme lipidique chez cette levure en mettant l'accent sur la capacité d'accumulation et de dégradation des lipides, la régulation et les interactions des différentes voies métaboliques. L'étude des enzymes du métabolisme des microorganismes oléagineux et de leurs particularités fonctionnelles, en comparaison avec celles des microorganismes non oléagineux, a permis de se focaliser sur des enzymes candidates pour être impliquées dans le caractère oléagineux. L'objectif de cette étude est de combiner les connaissances physiologiques sur cette levure avec les outils génétiques existants afin d'utiliser Y. lipolytica comme une usine cellulaire pour la production de grandes quantités de lipides ayant un intérêt industriel. Afin de rediriger les flux de carbone vers la synthèse de lipides, le gène GUT2, qui code l'un de deux isomères de la glycérol-3-phosphate déshydrogénase, ainsi que la voie de la β-oxydation ont été invalidés chez Y. lipolytica, conduisant à une forte augmentation de l'accumulation des lipides. Afin d'identifier l'étape limitante de la synthèse des TAG et de construire des souches aux profils lipidiques modifiés nous avons joué sur le niveau d'expression des acyltransférases et des désaturases. La contribution relative des acyltransférases dépend de la phase de croissance et leurs affinités vis-à-vis des substrats donneurs d'acyle sont complémentaires. La modification des désaturases a permis d'accumuler des acides gras spécifiques, démontrant ainsi qu'il est possible de moduler le profil des lipides accumulés chez Y. lipolytica. Cette étude nous a permis de construire des souches hyperaccumulatrices aux profils lipidiques tels qu'ils permettront de leur trouver des applications biotechnologiques dans la filière des biocarburants ou dans l'industrie oléochimique en tant qu'unités alternatives pour la production de substrats spécifiques

Yeast: A new oil producer?

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

Ingénierie génétique de la levure oléagineuse Yarrowia Lipolytica pour la production de lipides

en français : Yarrowia lipolytica est une levure oléagineuse qui possède un remarquable potentiel de croissance et d'accumulation de lipides dans des milieux hydrophobes. Au cours de cette étude nous avons approfondi nos connaissances du métabolisme lipidique chez cette levure en mettant l'accent sur la capacité d'accumulation et de dégradation des lipides, la régulation et les interactions des différentes voies métaboliques. L'étude des enzymes du métabolisme des microorganismes oléagineux et de leurs particularités fonctionnelles, en comparaison avec celles des microorganismes non oléagineux, a permis de se focaliser sur des enzymes candidates pour être impliquées dans le caractère oléagineux. L'objectif de cette étude est de combiner les connaissances physiologiques sur cette levure avec les outils génétiques existants afin d'utiliser Y. lipolytica comme une usine cellulaire pour la production de grandes quantités de lipides ayant un intérêt industriel. Afin de rediriger les flux de carbone vers la synthèse de lipides, le gène GUT2, qui code l'un de deux isomères de la glycérol-3-phosphate déshydrogénase, ainsi que la voie de la b-oxydation ont été invalidés chez Y. lipolytica, conduisant à une forte augmentation de l'accumulation des lipides. Afin d'identifier l'étape limitante de la synthèse des TAG et de construire des souches aux profils lipidiques modifiés nous avons joué sur le niveau d'expression des acyltransférases et des désaturases. La contribution relative des acyltransférases dépend de la phase de croissance et leurs affinités vis-à-vis des substrats donneurs d'acyle sont complémentaires. La modification des désaturases a permis d'accumuler des acides gras spécifiques, démontrant ainsi qu'il est possible de moduler le profil des lipides accumulés chez Y. lipolytica. Cette étude nous a permis de construire des souches hyperaccumulatrices aux profils lipidiques tels qu'ils permettront de leur trouver des applications biotechnologiques dans la filière des biocarburants ou dans l'industrie oléochimique en tant qu'unités alternatives pour la production de substrats spécifiques.en anglais : Yarrowia lipolytica is an oleaginous yeast with a remarkable potential for growth and lipid accumulation in hydrophobic environments. In this study we have expanded our knowledge of lipid metabolism in this yeast with an emphasis on lipid accumulation and degradation capacity, and the regulation and interaction of the different metabolic pathways. The study of metabolic enzymes in oleaginous microorganisms and their functional specifications in comparison with the non-oleaginous microorganisms has led us to focus on the enzymes potentially conferring the oleaginous character. The objective of this study is to combine the knowledge of yeast's physiology with the existing genetic tools to use Y. lipolytica as a cell factory for the production of large amounts of lipids with an industrial interest. In order to redirect carbon flux towards lipid synthesis, the GUT2 gene coding for a glycerol-3-phosphate dehydrogenase isomer and the b-oxidation pathway were invalidated in Y. lipolytica leading to a strong increase of lipid accumulation. As to identify the limiting step of TAG synthesis and to construct strains with altered lipid profiles, we have modified the expression level of acyltransferases and desaturases. The relative contribution of acyltransferases depends on the growth phase and their affinity towards the acylation substrate is complementary. The modification of desaturases allowed the accumulation of specific fatty acids, thus demonstrating the possibility of modulating lipid profile in Y. lipolytica. This study has enabled us to construct hyper accumulative strains with added-value lipid profiles that could find biotechnological applications in the energy (biodiesel) field or in the oleochemical industry as alternative units for specific substrate production.PARIS-AgroParisTech Centre Paris (751052302) / SudocSudocFranceF

Autonomic Nervous System Neuroanatomical Alterations Could Provoke and Maintain Gastrointestinal Dysbiosis in Autism Spectrum Disorder (ASD): A Novel Microbiome–Host Interaction Mechanistic Hypothesis

Dysbiosis secondary to environmental factors, including dietary patterns, antibiotics use, pollution exposure, and other lifestyle factors, has been associated to many non-infective chronic inflammatory diseases. Autism spectrum disorder (ASD) is related to maternal inflammation, although there is no conclusive evidence that affected individuals suffer from systemic low-grade inflammation as in many psychological and psychiatric diseases. However, neuro-inflammation and neuro–immune abnormalities are observed within ASD-affected individuals. Rebalancing human gut microbiota to treat disease has been widely investigated with inconclusive and contradictory findings. These observations strongly suggest that the forms of dysbiosis encountered in ASD-affected individuals could also originate from autonomic nervous system (ANS) functioning abnormalities, a common neuro–anatomical alteration underlying ASD. According to this hypothesis, overactivation of the sympathetic branch of the ANS, due to the fact of an ASD-specific parasympathetic activity deficit, induces deregulation of the gut–brain axis, attenuating intestinal immune and osmotic homeostasis. This sets-up a dysbiotic state, that gives rise to immune and osmotic dysregulation, maintaining dysbiosis in a vicious cycle. Here, we explore the mechanisms whereby ANS imbalances could lead to alterations in intestinal microbiome–host interactions that may contribute to the severity of ASD by maintaining the brain–gut axis pathways in a dysregulated state

Yarrowia lipolytica as a model for bio-oil production

International audienceThe 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

Control of Lipid Accumulation in the Yeast Yarrowia lipolytica▿

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 Δgut2 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 β-oxidation. Y. lipolytica contains six acyl-coenzyme A oxidases (Aox), encoded by the POX1 to POX6 genes, that catalyze the limiting step of peroxisomal β-oxidation. Additional deletion of the POX1 to POX6 genes in the Δgut2 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