Papers by Jean-marc Nicaud
Poly(ethylene terephthalate) (PET) degradation by Yarrowia lipolytica: Investigations on cell growth, enzyme production and monomers consumption
Process Biochemistry, Aug 1, 2020
Abstract The development of technologies for poly(ethylene terephthalate) (PET) depolymerization,... more Abstract The development of technologies for poly(ethylene terephthalate) (PET) depolymerization, such as biocatalysis, has been pointed as a very promising alternative to chemical hydrolysis processes. This work aims to understand the behavior of Yarrowia lipolytica, a robust yeast for diverse applications, in the presence of molecules from the PET production chain such as monoethylene glycol (MEG), terephthalic acid (TPA), bis (2-hydroxyethyl) terephthalate (BHET), PET oligomers, amorphous PET and post-consumer PET. The yeast was cultivated in rich media with and without glucose addition, in order to compare monomers release or consumption. TPA and MEG were consumed more intensely in the absence of glucose. The addition of the diester BHET yielded a 3-fold increased lipase production both at 160 rpm (118 U/L) and 250 rpm (385 U/L). In addition, 250 rpm agitation also provided a higher consumption of TPA (26% increase). The mono(2-hydroxyethyl) terephthalate (MHET) was the main intermediate released during polymer hydrolysis, followed by TPA and BHET. Thus, the use of Yarrowia lipolytica, which is capable of catalyzing the PET hydrolysis is of great potential to reduce the environmental impacts caused by unappropriated disposal of packages.
How do engineered Yarrowia lipolytica strains secrete free fatty acids: hints from comparative transcriptomics
Fems Yeast Research, May 8, 2023
Yarrowia lipolytica has been considered one of the most promising platforms for the microbial pro... more Yarrowia lipolytica has been considered one of the most promising platforms for the microbial production of fatty acids and derived products. The deletion of the faa1 gene coding for an acyl-CoA synthetase leads to the accumulation and secretion of free fatty acids (FFAs) into the extracellular space. The secretion of products is beneficial for the development of microbial cell factories to avoid intracellular inhibitory effects and reduce downstream processing costs. However, the mechanism behind the secretion of fatty acids is not well known. As a starting point, we compared the transcriptome of this mutant showing FFA secretion to a wildtype-like strain not showing this phenotype. The 12 most upregulated genes were evaluated for involvement in FFA secretion by the creation of deletion and overexpression mutants, among them MCH2, YMOH, three cell wall proteins CWP3, CWP4, and CWP11, M12B, and three proteins with unknown functions YUP1, YUP2, and YUP3. None of these proteins take a clear or isolated role in FFA export. As the transcriptomic data revealed an overrepresentation of cell wall-related proteins, some of them were further examined on a theoretical and experimental way. Surprisingly, overexpression of Ygpi led to the production of FFAs in the wildtype-like genetic background. Finally, some of the evaluated genes showed involvement in resistance to FFA toxicity.
Frontiers in Bioengineering and Biotechnology, Jan 22, 2020
Microbial oils are regarded as promising alternatives to fossil fuels as concerns over environmen... more Microbial oils are regarded as promising alternatives to fossil fuels as concerns over environmental issues and energy production systems continue to mount. Odd-chain fatty acids (FAs) are a type of valuable lipid with various applications: they can serve as biomarkers, intermediates in the production of flavor and fragrance compounds, fuels, and plasticizers. Microorganisms naturally produce FAs, but such FAs are primarily even-chain; only negligible amounts of odd-chain FAs are generated. As a result, studies using microorganisms to produce odd-chain FAs have had limited success. Here, our objective was to biosynthesize odd-chain FAs de novo in Yarrowia lipolytica using inexpensive carbon sources, namely glucose, without any propionate supplementation. To achieve this goal, we constructed a modular metabolic pathway containing seven genes. In the engineered strain expressing this pathway, the percentage of odd-chain FAs out of total FAs was higher than in the control strain (3.86 vs. 0.84%). When this pathway was transferred into an obese strain, which had been engineered to accumulate large amounts of lipids, odd-chain fatty acid production was 7.2 times greater than in the control (0.05 vs. 0.36 g/L). This study shows that metabolic engineering research is making progress toward obtaining efficient cell factories that produce odd-chain FAs.
HAL (Le Centre pour la Communication Scientifique Directe), Mar 1, 2000
We reported previously on the function of acyl coenzyme A (acyl-CoA) oxidase isozymes in the yeas... more We reported previously on the function of acyl coenzyme A (acyl-CoA) oxidase isozymes in the yeast Yarrowia lipolytica by investigating strains disrupted in one or several acyl-CoA oxidase-encoding genes (POX1 through POX5) (H. Wang et al., J. Bacteriol. 181:5140-5148, 1999). Here, these mutants were studied for lactone production. Monodisrupted strains produced similar levels of lactone as the wild-type strain (50 mg/liter) except for ⌬pox3, which produced 220 mg of ␥-decalactone per liter after 24 h. The ⌬pox2 ⌬pox3 doubledisrupted strain, although slightly affected in growth, produced about 150 mg of lactone per liter, indicating that Aox2p was not essential for the biotransformation. The ⌬pox2 ⌬pox3 ⌬pox5 triple-disrupted strain produced and consumed lactone very slowly. On the contrary, the ⌬pox2 ⌬pox3 ⌬pox4 ⌬pox5 multidisrupted strain did not grow or biotransform methyl ricinoleate into ␥-decalactone, demonstrating that Aox4p is essential for the biotransformation.
Triacylgylcerol synthases of the oleaginous yeast Yarrowia lipolytica
Contribution à l'étude de mécanisme de sécrétion de protéines par les bactéries à Gram négatif : étude de la sécrétion d'enzymes par Myxococcus xanthus et étude de la sécrétion d'hémolysine par Escherichia coli
La secretion de proteines par les microorganismes represente un outil important pour etudier la m... more La secretion de proteines par les microorganismes represente un outil important pour etudier la migration des proteines. De telles etudes peuvent aussi avoir des repercussions en biotechnologie pour produire des proteines etrangeres dans le milieu de culture. La production de nombreuses proteines par Myxococcus xanthus et d’hemolysine par Escherichia coli representent deux exemples pour aborder la secretion de proteines dans le milieu de culture par bacteries a Gram negatif. Chez Myxococcus xanthus, nous avons determine la production de l’activite phosphatase acide periplasmique et des activites bacteriolytique et proteasique extracellulaires au cours de la croissance, parallelement aux proteines totales extracellulaires. Ces resultats ont demontre que la presence d’enzymes dans le milieu de culture resulte bien d’un mecanisme de secretion. L’obtention et l’etude biochimique et genetique de mutants de transposition affectes dans la production d’enzymes extracellulaires a revele l’existence d’un mecanisme de secretion complexe, regulant la production coordonnee d’une cinquantaine de proteines produites pendant la phase de croissance exponentielle. Chez Escherichia coli nous avons montre que l’hemolysine est une proteine 107Kd secretee en phase exponentielle par des souches possedant un determinant HLY. Un determinant HLY se compose de quatre genes contigus appeles hlyC, hlyA, hlyB et hlyD. Le gene hlyA code pour une proteine non hemolytique de 107Kd activee par le produit du gene hlyC. Les formes active ou inactive sont transportees grâce a un mecanisme de transport specifiques post-traductionnel dont les fonctions sont codees par les genes hlyB et hlyD. Ces proteines reconnaissent une « sequence topo-genique » de la partie COOH-terminal de la proteine de 107Kd et permettent le passage de la proteine directement du cytoplasme dans le milieu de culture. Les mecanismes de secretion chez ces deux bacteries a Gram negatif peuvent representer des alternatives, pour la secretion de proteines d’interet industriel, aux systemes actuellement utilises.
Yarrowia lipolytica
Wiley-VCH Verlag GmbH & Co. KGaA eBooks, May 19, 2005
ABSTRACT
<i>Yarrowia lipolytica</i> chassis strains engineered to produce aromatic amino acids via the shikimate pathway
Microbial biotechnology, Dec 30, 2020
SummaryYarrowia lipolyticais widely used as a microbial producer of lipids and lipid derivatives.... more SummaryYarrowia lipolyticais widely used as a microbial producer of lipids and lipid derivatives. Here, we exploited this yeast’s potential to generate aromatic amino acids by developing chassis strains optimized for the production of phenylalanine, tyrosine and tryptophan. We engineered the shikimate pathway to overexpress a combination ofY. lipolyticaand heterologous feedback‐insensitive enzyme variants. Our best chassis strain displayed high levels ofde novoEhrlich metabolite production (up to 0.14 g l−1in minimal growth medium), which represented a 93‐fold increase compared to the wild‐type strain (0.0015 g l−1). Production was further boosted to 0.48 g l−1when glycerol, a low‐cost carbon source, was used, concomitantly to high secretion of phenylalanine precursor (1 g l−1). Among these metabolites, 2‐phenylethanol is of particular interest due to its rose‐like flavour. We also established a production pathway for generating protodeoxyviolaceinic acid, a dye derived from tryptophan, in a chassis strain optimized for chorismate, the precursor of tryptophan.We have thus demonstrated thatY. lipolyticacan serve as a platform for the sustainablede novobio‐production of high‐value aromatic compounds, and we have greatly improved our understanding of the potential feedback‐based regulation of the shikimate pathway in this yeast.
Gene, Jun 1, 1998
A shuttle mutagenesis system was developed for the dimorphic yeast Yarrowia lipolytica. This syst... more A shuttle mutagenesis system was developed for the dimorphic yeast Yarrowia lipolytica. This system combines transposon insertions generated in Escherichia coli with the transformation of yeast with the Tn-mutagenized DNA. The mini-transposon mTn-3xHA/GFP, used in Saccharomyces cerevisiae for producing stable insertions, was adapted for use in the yeast Y. lipolytica. The mTnYl1 transposon (for mini-Tn of Y. lipolytica) confers resistance to tetracycline in E. coli. It also contains the Y. lipolytica URA3 gene for selection of yeast transformants, and the coding sequence for the S65T mutant form of GFP. The rare cutter endonuclease, I-SceI, restriction site, which enables identification of the chromosomal localization of mutagenized genes, was also incorporated. mTnYl1 was first tested on the ACO1 gene, which encodes an Acyl CoA oxidase isozyme. The mutagenesis system was further validated on a Y. lipolytica genomic DNA library constructed in a pHSS6 derivative vector. Mutants with a particular morphology or defective for alkane, fatty acids and oil degradation were obtained.
Infection and Immunity, Apr 1, 1986
Escherichia coli hemolysin is secreted as a water-soluble polypeptide of Mr 107,000. After bindin... more Escherichia coli hemolysin is secreted as a water-soluble polypeptide of Mr 107,000. After binding to target erythrocytes, the membrane-bound toxin resembled an integral membrane protein in that it was refractory towards extraction with salt solutions of low ionic strength. Toxin-induced hemolysis could be totally inhibited by addition of 30 mM dextran 4 (mean Mr, 4,000; molecular diameter -3 nm) to the extracellular medium. Uncharged molecules of smaller size (e.g., sucrose, with a molecular diameter of 0.9 nm, or raffinose, with a molecular diameter of 1.2 to 1.3 nm) did not afford such protection. Treatment of erythrocytes suspended in dextran-containing buffer with the toxin induced rapid efflux of cellular K+ and influx of 45Ca2+, as well as influx of ["4C]mannitol and [3H]sucrose. [3H]inulin only slowly permeated into toxin-treated cells, and [3H]dextran uptake was virtually nil. Membranes lysed with high doses of E. coli hemolysin exhibited no recognizable ultrastructural lesions when examined by negative-staining electron microscopy. Sucrose density gradient centrifugation of deoxycholate-solubilized target membranes led to recovery of the toxin exclusively in monomer form. Incubation of toxin-treated cells with trypsin caused limited proteolysis with the generation of membrane-bound, toxin-derived polypeptides of Mr -80,000 without destroying the functional pore. We suggest that E. coli hemolysin may damage cell membranes by partial insertion into the lipid bilayer and generation of a discrete, hydrophilic transmembrane pore with an effective diameter of -3 nm. In contrast to the structured pores generated by cytolysins of gram-positive bacteria such as staphylococcal a-toxin and streptolysin 0, pore formation by E. coli hemolysin may be caused by the insertion of toxin monomers into the target lipid bilayers.
bioRxiv (Cold Spring Harbor Laboratory), Jul 6, 2022
Dysregulation of lipid metabolism is associated with obesity, metabolic diseases but there is als... more Dysregulation of lipid metabolism is associated with obesity, metabolic diseases but there is also increasing evidences of a relationship between lipid bodies (LBs) excess and some cancer. LBs synthesis requires diacylglycerol acyltransferases (DGATs) which catalyses the last step of triacylglycerol (TAG) synthesis, the main storage lipid form in lipid bodies. DGATs and in particular DGAT2, are therefore considered as potential therapeutic targets for the control of these pathologies. Here, the murine and the human DGAT2 were overexpressed in the oleaginous yeast Yarrowia lipolytica deleted for all DGATs activities, for evaluating the functionality of the enzymes in this heterologous host and to evaluate DGAT activity inhibitors. This work provide evidence that mammalian DGATs expressed in Y. lipolytica is a useful tool for screening chemical libraries to identify potential inhibitors or activators of these enzymes of therapeutic interest.
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Papers by Jean-marc Nicaud