Jean-marc Nicaud - Academia.edu (original) (raw)

Papers by Jean-marc Nicaud

Journal of Fungi, Jan 14, 2023

This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY

bioRxiv (Cold Spring Harbor Laboratory), Oct 30, 2020

Microbial production of lipids is one of the promising alternatives to fossil fuels with increasi... more Microbial production of lipids is one of the promising alternatives to fossil fuels with increasing environmental and energy concern. Odd-chain fatty acids (OCFA), a type of unusual lipids, are recently gaining a lot of interest as target compounds in microbial production due to their diverse applications in the medical, pharmaceutical, and chemical industries. In this study, we aimed to enhance the pool of precursors with three-carbon chain (propionyl-CoA) and fivecarbon chain (β-ketovaleryl-CoA) for the production of OCFAs in Yarrowia lipolytica. We evaluated different propionate-activating enzymes and the overexpression of propionyl-CoA transferase gene from Ralstonia eutropha increased the accumulation of OCFAs by 3.8 times over control strain, indicating propionate activation is the limiting step of OCFAs synthesis. It was shown that acetate supplement was necessary to restore growth and to produce a higher OCFA contents in total lipids, suggesting the balance of the precursors between acetyl-CoA and propionyl-CoA is crucial for OCFA accumulation. To improve β-ketovaleryl-CoA pools for further increase of OCFA production, we co-expressed the bktB encoding β-ketothiolase in the producing strain, and the OCFA production was increased by 33 % compared to control. Combining strain engineering and the optimization of the C/N ratio promoted the OCFA production up to 1.87 g/L representing 62% of total lipids, the highest recombinant OCFAs titer reported in yeast, up to date. This study provides a strong basis for the microbial production of OCFAs and its derivatives having high potentials in a wide range of applications.

The yeast Yarrowia lipolytica possesses multiple paralogues of genes dedicated to hydrophobic sub... more The yeast Yarrowia lipolytica possesses multiple paralogues of genes dedicated to hydrophobic substrates metabolization. Among them, 16 lipase encoding genes, involved in lipid or grease breakdown, were highlighted in the yeast genome. However, little information on all those paralogues has been yet obtained. Microarray data suggest that only a few of them could be expressed. Lipase synthesis seems to be dependent on the fatty acid or oil used as carbon source confirming the high adaptation of Y. lipolytica to different hydrophobic substrate. This review focuses on the biochemical characterization of those enzymes with special emphasis on the Lip2p lipase which is the isoenzyme mainly synthesized by Y. lipolytica. The 3D structure of this lipase established by homology modeling confirms that Lip2p is a lipase sensu stricto with a lid covering the active site of the enzyme in its closed conformation. Recent findings on enzyme conditioning in dehydrated or liquid formulation and in enzyme immobilization by entrapment in natural polymers from either organic or mineral origins are also discussed together with long-term storage strategies.

Selectable markers are a central component of genome edition technologies. In the yeast Yarrowia ... more Selectable markers are a central component of genome edition technologies. In the yeast Yarrowia lipolytica, these markers are traditionally based on antibiotic resistance (hygromycin B) or auxotrophy (e.g., leucine, uracil). However, the use of the former is impaired by a high level of spontaneous resistance, and the use of the latter by continuous complementation of the culture medium or restoration of prototrophy to the strains. As an alternative, genes related to the catabolism of carbon sources, or “catabolic selectable markers”, present the advantage of not being involved in essential metabolic pathways. The recently identified EYK1, encoding erythrulose kinase, can serve as an efficient catabolic selectable marker for genome editing in Y. lipolytica. Compared to auxotrophic markers such as URA3 and LEU2, EYK1 increases the growth rate of transformants on selective medium and the efficiency of genome edition. The utility of the marker EYK1 in a replicative vector was also demonstrated. Besides, the cloning-free strategy developed here simplifies the construction of disruption cassettes for genome editing in Y. lipolytica.Peer reviewe

In this study, Y. lipolytica was engineered to produce crotonic acid via the butanolforming route... more In this study, Y. lipolytica was engineered to produce crotonic acid via the butanolforming route. Firstly, the crotonase and 3-hydroxybutyryl-CoA dehydrogenase genes from Clostridium beijerinckii, and the thioesterase gene from Bacteroides thetaiotaomicron were heterologously expressed in Y. lipolytica, the engineered strain LZJ001 accumulated 62.3 ± 4.2 mg/L of crotonic acid. Secondly, the acetyl-CoA acetyltransferase from Saccharomyces cerevisiae was overexpressed, the derived recombinant strain LZJ002 produced 123.5 ± 6.8 mg/L of crotonic acid. Finally, the pyruvate dehydrogenase from Escherichia coli was additionally expressed, giving the fully engineered strain LZJ004 that produced 220.0 ± 8.2 mg/L of crotonic acid in shaking-flask culture, which represents a 3.5-fold increase over LZJ001 strain. The approach described here paves the way for environmentally friendly and large-scale industrial production of crotonic acid.

The non-conventional yeast Yarrowia lipolytica is widely investigated for its unusual metabolic p... more The non-conventional yeast Yarrowia lipolytica is widely investigated for its unusual metabolic properties. Among them is the ability of Y. lipolytica to adopt an ovoid or hyphal morphology according to environmental conditions. The mechanism of dimorphic transition involves numerous genes, which have been poorly documented to date. Here, we report on the isolation of a filamentous mutant from an insertion mutagenesis library, the subsequent identification of the mutated gene, and the use of this filamentous mutant in biofilm bioreactors.Peer reviewe

Yeast, Aug 8, 2019

Glutamate dehydrogenases (GDHs) are fundamental to cellular nitrogen and energy balance. Yet litt... more Glutamate dehydrogenases (GDHs) are fundamental to cellular nitrogen and energy balance. Yet little is known about these enzymes in the oleaginous yeast Yarrowia lipolytica. The YALI0F17820g and YALI0E09603g genes, encoding potential GDH enzymes in this organism, were examined. Heterologous expression in gdh-null Saccharomyces cerevisiae and examination of Y. lipolytica strains carrying gene deletions demonstrate that YALI0F17820g (ylGDH1) encodes a NADP-dependent GDH whereas YALI0E09603g (ylGDH2) encodes a NAD-dependent GDH enzyme. The activity encoded by these two genes accounts for all measurable GDH activity in Y. lipolytica. Levels of the two enzyme activities are comparable during logarithmic growth on rich medium, but the NADP-ylGDH1p enzyme activity is most highly expressed in stationary and nitrogen starved cells by threefold to 12-fold. Replacement of ammonia with glutamate causes a decrease in NADP-ylGdh1p activity, whereas NAD-ylGdh2p activity is increased. When glutamate is both carbon and nitrogen sources, the activity of NAD-ylGDH2p becomes dominant up to 18-fold compared with that of NADP-ylGDH1p. Gene deletion followed by growth on different carbon and nitrogen sources shows that NADP-ylGdh1p is required for efficient nitrogen assimilation whereas NAD-ylGdh2p plays a role in nitrogen and carbon utilization from glutamate. Overexpression experiments demonstrate that ylGDH1 and ylGDH2 are not interchangeable. These studies provide a vital basis for future consideration of how these enzymes function to facilitate energy and nitrogen homeostasis in Y. lipolytica.

Background: Yarrowia lipolytica, a non-conventional oleaginous yeast species, has attracted atten... more Background: Yarrowia lipolytica, a non-conventional oleaginous yeast species, has attracted attention due to its high lipid degradation and accumulation capacity. Y lipolytica is used as a chassis for the production of usual and unusual lipids and lipids derivatives. While genes involved in the intracellular transport and activation of fatty acids in the different cellular compartments have been characterized, no genes involved in fatty acid transport from the extracellular medium into the cell have been identified so far. In this study, we have identified secreted proteins involved in extracellular fatty acid binding.Results: The recent analysis of the Y. lipolytica secretome leads to the identification of a multi-gene family composed of four secreted proteins hereafter named UP1 to UP4. The protein products were efficiently over-expressed individually in native and multi-deletant strain (Q4: Δup1Δup2Δup3Δup4) backgrounds. Phenotype analysis demonstrated the involvement of those pr...

Yeast, 2019

Cyclopropane fatty acids, which can be simply converted to methylated fatty acids, are good unusu... more Cyclopropane fatty acids, which can be simply converted to methylated fatty acids, are good unusual fatty acid candidates for long‐term resistance to oxidization and low‐temperature fluidity useful for oleochemistry and biofuels. Cyclopropane fatty acids are present in low amounts in plants or bacteria. In order to develop a process for large‐scale biolipid production, we expressed 10 cyclopropane fatty acid synthases from various organisms in the oleaginous yeast Yarrowia lipolytica, a model yeast for lipid metabolism and naturally capable of producing large amounts of lipids.The Escherichia coli cyclopropane fatty acid synthase expression in Y. lipolytica allows the production of two classes of cyclopropane fatty acids, a C17:0 cyclopropanated form and a C19:0 cyclopropanated form, whereas others produce only the C17:0 form. Expression optimization and fed‐batch fermentation set‐up enable us to reach a specific productivity of 0.032 g·L−1·hr−1 with a genetically modified strain co...

Journal of Chromatography A, 2018

Please cite this article in press as: F. Merlier, et al., A gas chromatography full scan high res... more Please cite this article in press as: F. Merlier, et al., A gas chromatography full scan high resolution Orbitrap mass spectrometry method for separation and characterization of 3-hydroxymethyl pyridine ester of fatty acids at low levels,

Polish Journal of Food and Nutrition Sciences, 1997

The purpose of the investigation was to assess the dynamics of biomass and citric acid production... more The purpose of the investigation was to assess the dynamics of biomass and citric acid production in sucrose media by two invertase-positive (Suc + ) Yarrowia lipolytica transformants (W 29 ura3-302 and JM23/pINA169), originating from Laboratoire de Genetique Moleculaire et Cellulaire, INRA-CNRS. The industrial strain Y. lipolytica ATCC 20 460 (W29) with Suc - phenotype was used as a control. The growth of the two strains on sucrose was as good as on glucose under the conditions favourable for the expression of invertase activity in the transformants examined, i.e. in the presence of peptone and phosphate buffer at pH 6.8. The specific growth rates, μ max , were 0.5 - 0.536 h -1 and 0.492 - 0.565 h -1 , respectively. The biomass yield on sucrose was 6 to 19% higher than that on glucose. Satisfactory cell growth of the two transformants was also observed in production media (based on pure sucrose or sugar beet molasses) at N deficiency and low pH 5.5. However, only W29 ura 3-302, the strain derived from the industrial strain W29, was able to produce high levels of citric acid. Maximum accumulation of the citric acid in 20% molasses broth fermented by this strain reached 50.2 g/L, which can be considered promising. However, further studies are required to improve productivity of this bioprocess. Moreover, the results obtained in the present study show that the expression of foreign SUC2 gene in Y. lipolytica, under the control of the XPR2 promoter and using the alkaline extracellular protease signal sequence (encoded by the XPR2 gene), allowed the production of citric acid on carbohydrate materials.

HAL (Le Centre pour la Communication Scientifique Directe), 2016

Microbial Cell Factories, May 20, 2011

Background: The non conventional yeast Yarrowia lipolytica has aroused a strong industrial intere... more Background: The non conventional yeast Yarrowia lipolytica has aroused a strong industrial interest for heterologous protein production. However most of the studies describing recombinant protein production by this yeast rely on the use of complex media, such media are not convenient for large scale production particularly for products intended for pharmaceutical applications. In addition medium composition can also affect the production yield. Hence it is necessary to design an efficient medium for therapeutic protein expression by this host. Results: Five different media, including four minimal media and a complex medium, were assessed in shake flasks for the production of human interferon alpha 2b (hIFN α2b) by Y. lipolytica under the control of POX2 promoter inducible with oleic acid. The chemically defined medium SM4 formulated by Invitrogen for Pichia pastoris growth was the most suitable. Using statistical experimental design this medium was further optimized. The selected minimal medium consisting in SM4 supplemented with 10 mg/l FeCl 3 , 1 g/l glutamate, 5 ml/l PTM1 (Pichia Trace Metals) solution and a vitamin solution composed of myo-inositol, thiamin and biotin was called GNY medium. Compared to shake flask, bioreactor culture in GNY medium resulted in 416-fold increase of hIFN α2b production and 2-fold increase of the biological activity. Furthermore, SM4 enrichment with 5 ml/l PTM1 solution contributed to protect hIFN α2b against the degradation by the 28 kDa protease identified by zymography gel in culture supernatant. The screening of the inhibitory effect of the trace elements present in PTM1 solution on the activity of this protease was achieved using a Box-Behnken design. Statistical data analysis showed that FeCl 3 and MnSO 4 had the most inhibitory effect. Conclusion: We have designed an efficient medium for large scale production of heterologous proteins by Y. lipolytica. The optimized medium GNY is suitable for the production of hIFN α2b with the advantage that no complex nitrogen sources with non-defined composition were required.

HAL (Le Centre pour la Communication Scientifique Directe), Feb 10, 2017

Metabolic Engineering Communications, Jun 1, 2021

Microbial production of lipids is one of the promising alternatives to fossil resources with incr... more Microbial production of lipids is one of the promising alternatives to fossil resources with increasing environmental and energy concern. Odd-chain fatty acids (OCFA), a type of unusual lipids, are recently gaining a lot of interest as target compounds in microbial production due to their diverse applications in the medical, pharmaceutical, and chemical industries. In this study, we aimed to enhance the pool of precursors with three-carbon chain (propionyl-CoA) and five-carbon chain (β-ketovaleryl-CoA) for the production of OCFAs in Yarrowia lipolytica. We evaluated different propionate-activating enzymes and the overexpression of propionyl-CoA transferase gene from Ralstonia eutropha increased the accumulation of OCFAs by 3.8 times over control strain, indicating propionate activation is the limiting step of OCFAs synthesis. It was shown that acetate supplement was necessary to restore growth and to produce a higher OCFA contents in total lipids, suggesting the balance of the precursors between acetyl-CoA and propionyl-CoA is crucial for OCFA accumulation. To improve β-ketovaleryl-CoA pools for further increase of OCFA production, we co-expressed the bktB encoding β-ketothiolase in the producing strain, and the OCFA production was increased by 33% compared to control. Combining strain engineering and the optimization of the C/N ratio promoted the OCFA production up to 1.87 g/L representing 62% of total lipids, the highest recombinant OCFAs titer reported in yeast, up to date. This study provides a strong basis for the microbial production of OCFAs and its derivatives having high potentials in a wide range of applications.