The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Jean-marc Nicaud - One of the best experts on this subject based on the ideXlab platform.
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A modular Golden Gate toolkit for Yarrowia Lipolytica synthetic biology
Microbial Biotechnology, 2019Co-Authors: Macarena Larroudé, Jean-marc Nicaud, Young Kyoung Park, Paul Soudier, Monika Kubiak, Tristan RossignolAbstract:The oleaginous yeast Yarrowia Lipolytica is an established host for the bio-based production of valuable compounds and an organism for which many genetic tools have been developed. However, to properly engineer Y. Lipolytica and take full advantage of its potential, we need efficient, versatile, standardized and modular cloning tools. Here, we present a new modular Golden Gate toolkit for the one-step assembly of three transcription units that includes a selective marker and sequences for genome integration. Perfectly suited to a combinatorial approach, it contains nine different validated promoters, including inducible promoters, which allows expression to be fine-tuned. Moreover, this toolbox incorporates six different markers (three auxotrophic markers, two antibiotic-resistance markers and one metabolic marker), which allows the fast sequential construction and transformation of multiple elements. In total, the toolbox contains 64 bricks, and it has been validated and characterized using three different fluorescent reporter proteins. Additionally, it was successfully used to assemble and integrate a three-gene pathway allowing xylose utilization by Y. Lipolytica. This toolbox provides a powerful new tool for rapidly engineering Y. Lipolytica strains and is available to the community through Addgene.
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engineering Yarrowia Lipolytica to enhance lipid production from lignocellulosic materials
Biotechnology for Biofuels, 2018Co-Authors: Xochitl Niehus, Jean-marc Nicaud, Rodrigo Ledesmaamaro, Annemarie Crutzle Coq, Georgina SandovalAbstract:Yarrowia Lipolytica is a common biotechnological chassis for the production of lipids, which are the preferred feedstock for the production of fuels and chemicals. To reduce the cost of microbial lipid production, inexpensive carbon sources must be used, such as lignocellulosic hydrolysates. Unfortunately, lignocellulosic materials often contain toxic compounds and a large amount of xylose, which cannot be used by Y. Lipolytica. In this work, we engineered this yeast to efficiently use xylose as a carbon source for the production of lipids by overexpressing native genes. We further increased the lipid content by overexpressing heterologous genes to facilitate the conversion of xylose-derived metabolites into lipid precursors. Finally, we showed that these engineered strains were able to grow and produce lipids in a very high yield (lipid content = 67%, titer = 16.5 g/L, yield = 3.44 g/g sugars, productivity 1.85 g/L/h) on a xylose-rich agave bagasse hydrolysate in spite of toxic compounds. This work demonstrates the potential of metabolic engineering to reduce the costs of lipid production from inexpensive substrates as source of fuels and chemicals.
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Synthetic biology tools for engineering Yarrowia Lipolytica
Biotechnology Advances, 2018Co-Authors: Macarena Larroudé, Jean-marc Nicaud, Tristan Rossignol, Rodrigo Ledesma AmaroAbstract:The non-conventional oleaginous yeast Yarrowia Lipolytica shows great industrial promise. It naturally produces certain compounds of interest but can also artificially generate non-native metabolites, thanks to an engineering process made possible by the significant expansion of a dedicated genetic toolbox. In this review, we present recently developed synthetic biology tools that facilitate the manipulation of Y. Lipolytica, including 1) DNA assembly techniques, 2) DNA parts for constructing expression cassettes, 3) genome-editing techniques, and 4) computational tools.
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Enhancing erythritol productivity in Yarrowia Lipolytica using metabolic engineering
Metabolic Engineering, 2017Co-Authors: F. Carly, Jean-marc Nicaud, M. Vandermies, Samuel Telek, Sébastien Steels, Stephane Thomas, Patrick FickersAbstract:Erythritol (1,2,3,4-butanetetrol) is a four-carbon sugar alcohol with sweetening properties that is used by the agrofood industry as a food additive. In this study, we demonstrated that metabolic engineering can be used to improve the production of erythritol from glycerol in the yeast Yarrowia Lipolytica. The best results were obtained using a mutant that overexpressed GUT1 and TKL1, which encode a glycerol kinase and a transketolase, respectively, and in which EYK1, which encodes erythrulose kinase, was disrupted; the latter enzyme is involved in an early step of erythritol catabolism. In this strain, erythritol productivity was 75% higher than in the wild type; furthermore, the culturing time needed to achieve maximum concentration was reduced by 40%. An additional advantage is that the strain was unable to consume the erythritol it had created, further increasing the process's efficiency. The erythritol productivity values we obtained here are among the highest reported thus far.
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Metabolic engineering of Yarrowia Lipolytica to produce chemicals and fuels from xylose
Metabolic Engineering, 2016Co-Authors: Rodrigo Ledesma Amaro, Zhongpeng Guo, Zbigniew Lazar, Magdalena Rakicka, Florian Fouchard, Anne-marie Le Coq, Jean-marc NicaudAbstract:Yarrowia Lipolytica is a biotechnological chassis for the production of a range of products, such as microbial oils and organic acids. However, it is unable to consume xylose, the major pentose in lignocellulosic hydrolysates, which are considered a preferred carbon source for bioprocesses due to their low cost, wide abundance and high sugar content. Here, we engineered Y. Lipolytica to metabolize xylose to produce lipids or citric acid. The overexpression of xylose reductase and xylitol dehydrogenase from Scheffersomyces stipitis were necessary but not sufficient to permit growth. The additional overexpression of the endogenous xylulokinase enabled identical growth as the wild type strain in glucose. This mutant was able to produce up to 80g/L of citric acid from xylose. Transferring these modifications to a lipid-overproducing strain boosted the production of lipids from xylose. This is the first step towards a consolidated bioprocess to produce chemicals and fuels from lignocellulosic materials.
Hal S. Alper - One of the best experts on this subject based on the ideXlab platform.
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metabolic engineering in the host Yarrowia Lipolytica
Metabolic Engineering, 2018Co-Authors: A M Abdelmawgoud, Nian Liu, Gregory Stephanopoulos, Kelly A Markham, Claire M Palmer, Hal S. AlperAbstract:The nonconventional, oleaginous yeast, Yarrowia Lipolytica is rapidly emerging as a valuable host for the production of a variety of both lipid and nonlipid chemical products. While the unique genetics of this organism pose some challenges, many new metabolic engineering tools have emerged to facilitate improved genetic manipulation in this host. This review establishes a case for Y. Lipolytica as a premier metabolic engineering host based on innate metabolic capacity, emerging synthetic tools, and engineering examples. The metabolism underlying the lipid accumulation phenotype of this yeast as well as high flux through acyl-CoA precursors and the TCA cycle provide a favorable metabolic environment for expression of relevant heterologous pathways. These properties allow Y. Lipolytica to be successfully engineered for the production of both native and nonnative lipid, organic acid, sugar and acetyl-CoA derived products. Finally, this host has unique metabolic pathways enabling growth on a wide range of carbon sources, including waste products. The expansion of carbon sources, together with the improvement of tools as highlighted here, have allowed this nonconventional organism to act as a cellular factory for valuable chemicals and fuels.
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synthetic biology expands the industrial potential of Yarrowia Lipolytica
Trends in Biotechnology, 2018Co-Authors: Kelly A Markham, Hal S. AlperAbstract:The oleaginous yeast Yarrowia Lipolytica is quickly emerging as the most popular non-conventional (i.e., non-model organism) yeast in the bioproduction field. With a high propensity for flux through tricarboxylic acid (TCA) cycle intermediates and biological precursors such as acetyl-CoA and malonyl-CoA, this host is especially well suited to meet our industrial chemical production needs. Recent progress in synthetic biology tool development has greatly enhanced our ability to rewire this organism, with advances in genetic component design, CRISPR technologies, and modular cloning strategies. In this review we investigate recent developments in metabolic engineering and describe how the new tools being developed help to realize the full industrial potential of this host. Finally, we conclude with our vision of the developments that will be necessary to enhance future engineering efforts.
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enabling xylose utilization in Yarrowia Lipolytica for lipid production
Biotechnology Journal, 2016Co-Authors: Hal S. AlperAbstract:The conversion of lignocellulosic sugars, in particular xylose, is important for sustainable fuels and chemicals production. While the oleaginous yeast Yarrowia Lipolytica is a strong candidate for lipid production, it is currently unable to effectively utilize xylose. By introducing a heterologous oxidoreductase pathway and enabling starvation adaptation, we obtained a Y. Lipolytica strain, E26 XUS, that can use xylose as a sole carbon source and produce over 15 g/L of lipid in bioreactor fermentations (29.3% of theoretical yield) with a maximal lipid productivity of 0.19 g/L/h. Genomic sequencing and genetic analysis pointed toward increases in genomic copy number of the pathway and resulting elevated expression levels as the causative mutations underlying this improved phenotype. More broadly, many regions of the genome were duplicated during starvation of Yarrowia. This strain can form the basis for further engineering to enhance xylose catabolic rates and conversion. Finally, this study also reveals the flexibility and dynamic nature of the Y. Lipolytica genome, and the means at which starvation can be used to induce genomic duplications.
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Metabolic engineering of Yarrowia Lipolytica for itaconic acid production.
Metabolic engineering, 2015Co-Authors: John Blazeck, Jarrett Miller, Anny Pan, Mariam Jamoussi, Andrew Hill, Hal S. AlperAbstract:Itaconic acid is a naturally produced organic acid with diverse applications as a replacement for petroleum derived products. However, its industrial viability as a bio-replacement has been restricted due to limitations with native producers. In this light, Yarrowia Lipolytica is an excellent potential candidate for itaconic acid production due to its innate capacity to accumulate citric acid cycle intermediates and tolerance to lower pH. Here, we demonstrate the capacity to produce itaconic acid in Y. Lipolytica through heterologous expression of the itaconic acid synthesis enzyme, resulting in an initial titer of 33 mg/L. Further optimizations of this strain via metabolic pathway engineering, enzyme localization, and media optimization strategies enabled 4.6g/L of itaconic acid to be produced in bioreactors, representing a 140-fold improvement over initial titer. Moreover, these fermentation conditions did not require additional nutrient supplementation and utilized a low pH condition that enabled the acid form of itaconic acid to be produced. Overall yields (0.058 g/g yield from glucose) and maximum productivity of 0.045 g/L/h still provide areas for future strain improvement. Nevertheless, this work demonstrates that Y. Lipolytica has the potential to serve as an industrially relevant platform for itaconic acid production.
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Draft Genome Sequence of the Oleaginous Yeast Yarrowia Lipolytica PO1f, a Commonly Used Metabolic Engineering Host.
Genome announcements, 2014Co-Authors: Leqian Liu, Hal S. AlperAbstract:ABSTRACT The draft genome sequence of the oleaginous yeast Yarrowia Lipolytica stain PO1f, a commonly used metabolic engineering host, is presented here. The approximately 20.3-Mb genome sequence of PO1f will greatly facilitate research efforts in metabolic engineering of Yarrowia Lipolytica for value-added chemical production.
Rodrigo Ledesma Amaro - One of the best experts on this subject based on the ideXlab platform.
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Synthetic biology tools for engineering Yarrowia Lipolytica
Biotechnology Advances, 2018Co-Authors: Macarena Larroudé, Jean-marc Nicaud, Tristan Rossignol, Rodrigo Ledesma AmaroAbstract:The non-conventional oleaginous yeast Yarrowia Lipolytica shows great industrial promise. It naturally produces certain compounds of interest but can also artificially generate non-native metabolites, thanks to an engineering process made possible by the significant expansion of a dedicated genetic toolbox. In this review, we present recently developed synthetic biology tools that facilitate the manipulation of Y. Lipolytica, including 1) DNA assembly techniques, 2) DNA parts for constructing expression cassettes, 3) genome-editing techniques, and 4) computational tools.
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Metabolic engineering of Yarrowia Lipolytica to produce chemicals and fuels from xylose
Metabolic Engineering, 2016Co-Authors: Rodrigo Ledesma Amaro, Zhongpeng Guo, Zbigniew Lazar, Magdalena Rakicka, Florian Fouchard, Anne-marie Le Coq, Jean-marc NicaudAbstract:Yarrowia Lipolytica is a biotechnological chassis for the production of a range of products, such as microbial oils and organic acids. However, it is unable to consume xylose, the major pentose in lignocellulosic hydrolysates, which are considered a preferred carbon source for bioprocesses due to their low cost, wide abundance and high sugar content. Here, we engineered Y. Lipolytica to metabolize xylose to produce lipids or citric acid. The overexpression of xylose reductase and xylitol dehydrogenase from Scheffersomyces stipitis were necessary but not sufficient to permit growth. The additional overexpression of the endogenous xylulokinase enabled identical growth as the wild type strain in glucose. This mutant was able to produce up to 80g/L of citric acid from xylose. Transferring these modifications to a lipid-overproducing strain boosted the production of lipids from xylose. This is the first step towards a consolidated bioprocess to produce chemicals and fuels from lignocellulosic materials.
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Metabolic Engineering for Expanding the Substrate Range of Yarrowia Lipolytica.
Trends in Biotechnology, 2016Co-Authors: Rodrigo Ledesma Amaro, Jean-marc NicaudAbstract:Economically viable biotechnology processes must be characterized by a favorable ratio between the production costs and the product market price. In the bioproduction of bulk chemicals, costs must be minimized so that the process is competitive relative to petroleum-based production. The substrate costs must thus be reduced by employing inexpensive carbon sources, such as industrial wastes. Unfortunately, the most convenient microorganisms for a bioconversion are typically unable to degrade such substrates. Fortunately, the discovery of new enzymes together with advances in synthetic biology has moved metabolic engineering forward, expanding substrate ranges. Here we review the latest advances made using the industrial yeast Yarrowia Lipolytica, which can exploit various carbon sources to produce biofuels and chemicals.
Gregory Stephanopoulos - One of the best experts on this subject based on the ideXlab platform.
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engineering Yarrowia Lipolytica for the utilization of acid whey
Metabolic Engineering, 2020Co-Authors: Junichi Mano, Nian Liu, John H Hammond, Devin H Currie, Gregory StephanopoulosAbstract:Abstract Acid whey, a byproduct in cheese and yogurt production, demands high costs in disposal at large quantities. Nonetheless, it contains abundant sugars and nutrients that can potentially be utilized by microorganisms. Here we report a novel platform technology that converts acid whey into value-added products using Yarrowia Lipolytica. Since wild type strains do not assimilate lactose, a major carbon source in whey, a secreted β-galactosidase was introduced. Additionally, to accelerate galactose metabolism, we overexpressed the relevant native four genes of the Leloir pathway. The engineered strain could achieve rapid total conversion of all carbon sources in acid whey, producing 6.61 g/L of fatty acids (FAs) with a yield of 0.146 g-FAs/g-substrates. Further engineering to introduce an omega-3 desaturase enabled the synthesis of α-linolenic acid from acid whey, producing 10.5 mg/gDCW within a short fermentation time. Finally, PEX10 knockout in our platform strain was shown to minimize hyphal formation in concentrated acid whey cultures, greatly improving fatty acid content. These results demonstrate the feasibility of using acid whey as a previously untapped resource for biotechnology.
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holistic approaches in lipid production by Yarrowia Lipolytica
Trends in Biotechnology, 2018Co-Authors: Zbigniew Lazar, Nian Liu, Gregory StephanopoulosAbstract:Concerns about climate change have driven research on the production of lipid-derived biofuels as an alternative and renewable liquid fuel source. Using oleaginous yeasts for lipid synthesis creates the potential for cost-effective industrial-scale operations due to their ability to reach high lipid titer, yield, and productivity resulting from their unique metabolism. Yarrowia Lipolytica is the model oleaginous yeast, with the best-studied lipid metabolism, the greatest number of genetic tools, and a fully sequenced genome. In this review we highlight multiomics studies that elucidate the mechanisms allowing this yeast to achieve lipid overaccumulation and then present several major metabolic engineering efforts that enhanced the production metrics in Y. Lipolytica. Recent achievements that applied novel engineering strategies are emphasized.
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metabolic engineering in the host Yarrowia Lipolytica
Metabolic Engineering, 2018Co-Authors: A M Abdelmawgoud, Nian Liu, Gregory Stephanopoulos, Kelly A Markham, Claire M Palmer, Hal S. AlperAbstract:The nonconventional, oleaginous yeast, Yarrowia Lipolytica is rapidly emerging as a valuable host for the production of a variety of both lipid and nonlipid chemical products. While the unique genetics of this organism pose some challenges, many new metabolic engineering tools have emerged to facilitate improved genetic manipulation in this host. This review establishes a case for Y. Lipolytica as a premier metabolic engineering host based on innate metabolic capacity, emerging synthetic tools, and engineering examples. The metabolism underlying the lipid accumulation phenotype of this yeast as well as high flux through acyl-CoA precursors and the TCA cycle provide a favorable metabolic environment for expression of relevant heterologous pathways. These properties allow Y. Lipolytica to be successfully engineered for the production of both native and nonnative lipid, organic acid, sugar and acetyl-CoA derived products. Finally, this host has unique metabolic pathways enabling growth on a wide range of carbon sources, including waste products. The expansion of carbon sources, together with the improvement of tools as highlighted here, have allowed this nonconventional organism to act as a cellular factory for valuable chemicals and fuels.
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engineering Yarrowia Lipolytica for poly 3 hydroxybutyrate production
Journal of Industrial Microbiology & Biotechnology, 2017Co-Authors: Kangjian Qiao, Nian Liu, Gregory StephanopoulosAbstract:Strains of Yarrowia Lipolytica were engineered to express the poly-3-hydroxybutyrate (PHB) biosynthetic pathway. The genes for β-ketothiolase, NADPH-dependent acetoacetyl-CoA reductase, and PHB synthase were cloned and inserted into the chromosome of Y. Lipolytica. In shake flasks, the engineered strain accumulated PHB to 1.50 and 3.84% of cell dry weight in complex medium supplemented with glucose and acetate as carbon source, respectively. In fed-batch fermentation using acetate as sole carbon source, 7.35 g/l PHB (10.2% of cell dry weight) was produced. Selection of Y. Lipolytica as host for PHB synthesis was motivated by the fact that this organism is a good lipids producer, which suggests robust acetyl-CoA supply also the precursor of the PHB pathway. Acetic acid could be supplied by gas fermentation, anaerobic digestion, and other low-cost supply route.
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lipid production in Yarrowia Lipolytica is maximized by engineering cytosolic redox metabolism
Nature Biotechnology, 2017Co-Authors: Kangjian Qiao, Thomas M Wasylenko, Kang Zhou, Gregory StephanopoulosAbstract:Microbial factories have been engineered to produce lipids from carbohydrate feedstocks for production of biofuels and oleochemicals. However, even the best yields obtained to date are insufficient for commercial lipid production. To maximize the capture of electrons generated from substrate catabolism and thus increase substrate-to-product yields, we engineered 13 strains of Yarrowia Lipolytica with synthetic pathways converting glycolytic NADH into the lipid biosynthetic precursors NADPH or acetyl-CoA. A quantitative model was established and identified the yield of the lipid pathway as a crucial determinant of overall process yield. The best engineered strain achieved a productivity of 1.2 g/L/h and a process yield of 0.27 g-fatty acid methyl esters/g-glucose, which constitutes a 25% improvement over previously engineered yeast strains. Oxygen requirements of our highest producer were reduced owing to decreased NADH oxidization by aerobic respiration. We show that redox engineering could enable commercialization of microbial carbohydrate-based lipid production.
Tristan Rossignol - One of the best experts on this subject based on the ideXlab platform.
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A modular Golden Gate toolkit for Yarrowia Lipolytica synthetic biology
Microbial Biotechnology, 2019Co-Authors: Macarena Larroudé, Jean-marc Nicaud, Young Kyoung Park, Paul Soudier, Monika Kubiak, Tristan RossignolAbstract:The oleaginous yeast Yarrowia Lipolytica is an established host for the bio-based production of valuable compounds and an organism for which many genetic tools have been developed. However, to properly engineer Y. Lipolytica and take full advantage of its potential, we need efficient, versatile, standardized and modular cloning tools. Here, we present a new modular Golden Gate toolkit for the one-step assembly of three transcription units that includes a selective marker and sequences for genome integration. Perfectly suited to a combinatorial approach, it contains nine different validated promoters, including inducible promoters, which allows expression to be fine-tuned. Moreover, this toolbox incorporates six different markers (three auxotrophic markers, two antibiotic-resistance markers and one metabolic marker), which allows the fast sequential construction and transformation of multiple elements. In total, the toolbox contains 64 bricks, and it has been validated and characterized using three different fluorescent reporter proteins. Additionally, it was successfully used to assemble and integrate a three-gene pathway allowing xylose utilization by Y. Lipolytica. This toolbox provides a powerful new tool for rapidly engineering Y. Lipolytica strains and is available to the community through Addgene.
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Synthetic biology tools for engineering Yarrowia Lipolytica
Biotechnology Advances, 2018Co-Authors: Macarena Larroudé, Jean-marc Nicaud, Tristan Rossignol, Rodrigo Ledesma AmaroAbstract:The non-conventional oleaginous yeast Yarrowia Lipolytica shows great industrial promise. It naturally produces certain compounds of interest but can also artificially generate non-native metabolites, thanks to an engineering process made possible by the significant expansion of a dedicated genetic toolbox. In this review, we present recently developed synthetic biology tools that facilitate the manipulation of Y. Lipolytica, including 1) DNA assembly techniques, 2) DNA parts for constructing expression cassettes, 3) genome-editing techniques, and 4) computational tools.
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Alternative Splicing Regulates Targeting of Malate Dehydrogenase in Yarrowia Lipolytica
DNA Research, 2012Co-Authors: Affoue Philomen Kabran Gnankon, Jean-marc Nicaud, Claude Gaillardin, Tristan Rossignol, Cécile NeuvégliseAbstract:Alternative pre-mRNA splicing is a major mechanism contributing to the proteome complexity of most eukaryotes, especially mammals. In less complex organisms, such as yeasts, the numbers of genes that contain introns are low and cases of alternative splicing (AS) with functional implications are rare. We report the first case of AS with functional consequences in the yeast Yarrowia Lipolytica. The splicing pattern was found to govern the cellular localization of malate dehydrogenase, an enzyme of the central carbon metabolism. This ubiquitous enzyme is involved in the tricarboxylic acid cycle in mitochondria and in the glyoxylate cycle, which takes place in peroxisomes and the cytosol. In Saccharomyces cerevisiae, three genes encode three compartment-specific enzymes. In contrast, only two genes exist in Y. Lipolytica. One gene (YIMDH1, YALI0D16753g) encodes a predicted mitochondrial protein, whereas the second gene (YIMDH2, YALI0E14190g) generates the cytosolic and peroxisomal forms through the alternative use of two 3'-splice sites in the second intron. Both splicing variants were detected in cDNA libraries obtained from cells grown under different conditions. Mutants expressing the individual YlMdh2p isoforms tagged with fluorescent proteins confirmed that they localized to either the cytosolic or the peroxisomal compartment.
