Candida Intermedia

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Gunnar Liden - One of the best experts on this subject based on the ideXlab platform.

  • the glucose xylose facilitator gxf1 from Candida Intermedia expressed in a xylose fermenting industrial strain of saccharomyces cerevisiae increases xylose uptake in sscf of wheat straw
    Enzyme and Microbial Technology, 2011
    Co-Authors: Cesar Fonseca, Barbel Hahnhagerdal, Kim Olofsson, Carla Ferreira, David Runquist, Luis L Fonseca, Gunnar Liden
    Abstract:

    Ethanolic fermentation of lignocellulose raw materials requires industrial xylose-fermenting strains capable of complete and efficient D-xylose consumption. A central question in xylose fermentation by Saccharomyces cerevisiae engineered for xylose fermentation is to improve the xylose uptake. In the current study, the glucose/xylose facilitator Gxf1 from Candida Intermedia, was expressed in three different xylose-fermenting S. cerevisiae strains of industrial origin. The in vivo effect on aerobic xylose growth and the initial xylose uptake rate were assessed. The expression of Gxf1 resulted in enhanced aerobic xylose growth only for the TMB3400 based strain. It displayed more than a 2-fold higher affinity for D-xylose than the parental strain and approximately 2-fold higher initial specific growth rate at 4 g/L D-xylose. Enhanced xylose consumption was furthermore observed when the GXF1-strain was assessed in simultaneous saccharification and co-fermentation (SSCF) of pretreated wheat straw. However, the ethanol yield remained unchanged due to increased by-product formation. Metabolic flux analysis suggested that the expression of the Gxf1 transporter had shifted the control of xylose catabolism from transport to the NAD(+) dependent oxidation of xylitol to xylulose. (C) 2011 Elsevier Inc. All rights reserved. (Less)

  • The glucose/xylose facilitator gxf1 from Candida Intermedia expressed in a xylose-fermenting industrial strain of Saccharomyces cerevisiae increases xylose uptake in SSCF of wheat straw.
    Enzyme and Microbial Technology, 2011
    Co-Authors: Cesar Fonseca, Kim Olofsson, Carla Ferreira, David Runquist, Luis L Fonseca, Bärbel Hahn-hägerdal, Gunnar Liden
    Abstract:

    Ethanolic fermentation of lignocellulose raw materials requires industrial xylose-fermenting strains capable of complete and efficient D-xylose consumption. A central question in xylose fermentation by Saccharomyces cerevisiae engineered for xylose fermentation is to improve the xylose uptake. In the current study, the glucose/xylose facilitator Gxf1 from Candida Intermedia, was expressed in three different xylose-fermenting S. cerevisiae strains of industrial origin. The in vivo effect on aerobic xylose growth and the initial xylose uptake rate were assessed. The expression of Gxf1 resulted in enhanced aerobic xylose growth only for the TMB3400 based strain. It displayed more than a 2-fold higher affinity for D-xylose than the parental strain and approximately 2-fold higher initial specific growth rate at 4 g/L D-xylose. Enhanced xylose consumption was furthermore observed when the GXF1-strain was assessed in simultaneous saccharification and co-fermentation (SSCF) of pretreated wheat straw. However, the ethanol yield remained unchanged due to increased by-product formation. Metabolic flux analysis suggested that the expression of the Gxf1 transporter had shifted the control of xylose catabolism from transport to the NAD(+) dependent oxidation of xylitol to xylulose. (C) 2011 Elsevier Inc. All rights reserved. (Less)

  • A mutated xylose reductase increases bioethanol production more than a glucose/xylose facilitator in simultaneous fermentation and co-fermentation of wheat straw
    AMB Express, 2011
    Co-Authors: Kim Olofsson, David Runquist, Bärbel Hahn-hägerdal, Gunnar Liden
    Abstract:

    Genetically engineered Saccharomyces cerevisiae strains are able to ferment xylose present in lignocellulosic biomass. However, better xylose fermenting strains are required to reach complete xylose uptake in simultaneous saccharification and co-fermentation (SSCF) of lignocellulosic hydrolyzates. In the current study, haploid Saccharomyces cerevisiae strains expressing a heterologous xylose pathway including either the native xylose reductase (XR) from P. stipiti s, a mutated variant of XR (mXR) with altered co-factor preference, a glucose/xylose facilitator (Gxf1) from Candida Intermedia or both mXR and Gxf1 were assessed in SSCF of acid-pretreated non-detoxified wheat straw. The xylose conversion in SSCF was doubled with the S. cerevisiae strain expressing mXR compared to the isogenic strain expressing the native XR, converting 76% and 38%, respectively. The xylitol yield was less than half using mXR in comparison with the native variant. As a result of this, the ethanol yield increased from 0.33 to 0.39 g g^-1 when the native XR was replaced by mXR. In contrast, the expression of Gxf1 only slightly increased the xylose uptake, and did not increase the ethanol production. The results suggest that ethanolic xylose fermentation under SSCF conditions is controlled primarily by the XR activity and to a much lesser extent by xylose transport.

  • The glucose/xylose facilitator Gxf1 from Candida Intermedia expressed in a xylose-fermenting industrial strain of Saccharomyces cerevisiae increases xylose uptake in SSCF of wheat straw.
    Enzyme and microbial technology, 2011
    Co-Authors: Cesar Fonseca, Kim Olofsson, Carla Ferreira, David Runquist, Luis L Fonseca, Bärbel Hahn-hägerdal, Gunnar Liden
    Abstract:

    Ethanolic fermentation of lignocellulose raw materials requires industrial xylose-fermenting strains capable of complete and efficient D-xylose consumption. A central question in xylose fermentation by Saccharomyces cerevisiae engineered for xylose fermentation is to improve the xylose uptake. In the current study, the glucose/xylose facilitator Gxf1 from Candida Intermedia, was expressed in three different xylose-fermenting S. cerevisiae strains of industrial origin. The in vivo effect on aerobic xylose growth and the initial xylose uptake rate were assessed. The expression of Gxf1 resulted in enhanced aerobic xylose growth only for the TMB3400 based strain. It displayed more than a 2-fold higher affinity for D-xylose than the parental strain and approximately 2-fold higher initial specific growth rate at 4 g/L D-xylose. Enhanced xylose consumption was furthermore observed when the GXF1-strain was assessed in simultaneous saccharification and co-fermentation (SSCF) of pretreated wheat straw. However, the ethanol yield remained unchanged due to increased by-product formation. Metabolic flux analysis suggested that the expression of the Gxf1 transporter had shifted the control of xylose catabolism from transport to the NAD(+) dependent oxidation of xylitol to xylulose.

Akihiko Kondo - One of the best experts on this subject based on the ideXlab platform.

Lisbeth Olsson - One of the best experts on this subject based on the ideXlab platform.

  • Genomic and transcriptomic analysis of Candida Intermedia reveals the genetic determinants for its xylose-converting capacity.
    Biotechnology for Biofuels, 2020
    Co-Authors: Cecilia Geijer, Antonio D Moreno, Simon Stenberg, S Mazurkewich, Fábio Faria-oliveira, Lisbeth Olsson
    Abstract:

    Background: An economically viable production of biofuels and biochemicals from lignocellulose requires microorganisms that can readily convert both the cellulosic and hemicellulosic fractions into product. The yeast Candida Intermedia displays a high capacity for uptake and conversion of several lignocellulosic sugars including the abundant pentose d-xylose, an underutilized carbon source since most industrially relevant microorganisms cannot naturally ferment it. Thus, C. Intermedia constitutes an important source of knowledge and genetic information that could be transferred to industrial microorganisms such as Saccharomyces cerevisiae to improve their capacity to ferment lignocellulose-derived xylose. Results: To understand the genetic determinants that underlie the metabolic properties of C. Intermedia, we sequenced the genomes of both the in-house-isolated strain CBS 141442 and the reference strain PYCC 4715. De novo genome assembly and subsequent analysis revealed C. Intermedia to be a haploid species belonging to the CTG clade of ascomycetous yeasts. The two strains have highly similar genome sizes and number of protein-encoding genes, but they differ on the chromosomal level due to numerous translocations of large and small genomic segments. The transcriptional profiles for CBS 141442 grown in medium with either high or low concentrations of glucose and xylose were determined through RNA-sequencing analysis, revealing distinct clusters of co-regulated genes in response to different specific growth rates, carbon sources and osmotic stress. Analysis of the genomic and transcriptomic data also identified multiple xylose reductases, one of which displayed dual NADH/NADPH co-factor specificity that likely plays an important role for co-factor recycling during xylose fermentation. Conclusions: In the present study, we performed the first genomic and transcriptomic analysis of C. Intermedia and identified several novel genes for conversion of xylose. Together the results provide insights into the mechanisms underlying saccharide utilization in C. Intermedia and reveal potential target genes to aid in xylose fermentation in S. cerevisiae.

  • genomic and transcriptomic analysis of Candida Intermedia reveals the genetic determinants for its xylose converting capacity
    Biotechnology for Biofuels, 2020
    Co-Authors: Cecilia Geijer, Antonio D Moreno, Fabio Fariaoliveira, Simon Stenberg, S Mazurkewich, Lisbeth Olsson
    Abstract:

    An economically viable production of biofuels and biochemicals from lignocellulose requires microorganisms that can readily convert both the cellulosic and hemicellulosic fractions into product. The yeast Candida Intermedia displays a high capacity for uptake and conversion of several lignocellulosic sugars including the abundant pentose d-xylose, an underutilized carbon source since most industrially relevant microorganisms cannot naturally ferment it. Thus, C. Intermedia constitutes an important source of knowledge and genetic information that could be transferred to industrial microorganisms such as Saccharomyces cerevisiae to improve their capacity to ferment lignocellulose-derived xylose. To understand the genetic determinants that underlie the metabolic properties of C. Intermedia, we sequenced the genomes of both the in-house-isolated strain CBS 141442 and the reference strain PYCC 4715. De novo genome assembly and subsequent analysis revealed C. Intermedia to be a haploid species belonging to the CTG clade of ascomycetous yeasts. The two strains have highly similar genome sizes and number of protein-encoding genes, but they differ on the chromosomal level due to numerous translocations of large and small genomic segments. The transcriptional profiles for CBS 141442 grown in medium with either high or low concentrations of glucose and xylose were determined through RNA-sequencing analysis, revealing distinct clusters of co-regulated genes in response to different specific growth rates, carbon sources and osmotic stress. Analysis of the genomic and transcriptomic data also identified multiple xylose reductases, one of which displayed dual NADH/NADPH co-factor specificity that likely plays an important role for co-factor recycling during xylose fermentation. In the present study, we performed the first genomic and transcriptomic analysis of C. Intermedia and identified several novel genes for conversion of xylose. Together the results provide insights into the mechanisms underlying saccharide utilization in C. Intermedia and reveal potential target genes to aid in xylose fermentation in S. cerevisiae.

  • evolutionary engineered Candida Intermedia exhibits improved xylose utilization and robustness to lignocellulose derived inhibitors and ethanol
    Applied Microbiology and Biotechnology, 2019
    Co-Authors: Antonio D Moreno, Lisbeth Olsson, Antonella Carbone, Rosita Pavone, Cecilia Geijer
    Abstract:

    The development of robust microorganisms that can efficiently ferment both glucose and xylose represents one of the major challenges in achieving a cost-effective lignocellulosic bioethanol production. Candida Intermedia is a non-conventional, xylose-utilizing yeast species with a high-capacity xylose transport system. The natural ability of C. Intermedia to produce ethanol from xylose makes it attractive as a non-GMO alternative for lignocellulosic biomass conversion in biorefineries. We have evaluated the fermentation capacity and the tolerance to lignocellulose-derived inhibitors and the end product, ethanol, of the C. Intermedia strain CBS 141442 isolated from steam-exploded wheat straw hydrolysate. In a mixed sugar fermentation medium, C. Intermedia CBS 141442 co-fermented glucose and xylose, although with a preference for glucose over xylose. The strain was clearly more sensitive to inhibitors and ethanol when consuming xylose than glucose. C. Intermedia CBS 141442 was also subjected to evolutionary engineering with the aim of increasing its tolerance to inhibitors and ethanol, and thus improving its fermentation capacity under harsh conditions. The resulting evolved population was able to ferment a 50% (v/v) steam-exploded wheat straw hydrolysate (which was completely inhibitory to the parental strain), improving the sugar consumption and the final ethanol concentration. The evolved population also exhibited a better tolerance to ethanol when growing in a xylose medium supplemented with 35.5 g/L ethanol. These results highlight the potential of C. Intermedia CBS 141442 to become a robust yeast for the conversion of lignocellulose to ethanol.

  • genomic and transcriptomic analysis of Candida Intermedia reveals genes for utilization of biotechnologically important carbon sources
    35th International Specialised Symposium on Yeasts Antalya, 2019
    Co-Authors: Fabio Luis Da Silva Faria Oliveira, Lisbeth Olsson, Cecilia Geijer
    Abstract:

    A future biobased society relies on efficient industrial microorganisms that can convert all sugars from agricultural, forestry and industrial waste streams into fuels, chemicals and materials. To be able to tailor-make such potent cell factories, we need a far better understanding of the proteins responsible for the assimilation of biotechnologically important carbon sources including pentoses, disaccharides and oligomers. The yeast Candida Intermedia, known for its superior growth on xylose owing to its efficient uptake and conversion systems, can also utilize a range of other important carbon sources such as cellobiose, galactose and lactose. The aim of this project was to identify the genomic determinants for the utilization of these mono- and disaccharides in our in-house isolated C. Intermedia strain CBS 141442. Genome sequencing and transcriptional (RNA seq) data analysis during growth in defined medium supplemented with glucose, xylose, galactose, lactose or cellobiose, revealed numerous distinct clusters of coregulated genes. By scanning the CBS 141442 genome for genes encoding Major Facilitator Superfamily (MFS) sugar transporters, and the RNA-seq dataset for the corresponding transcriptional profiles, we identified several novel genes encoding putative xylose transporters and multiple Lac12-like transporters likely involved in the uptake of disaccharides in C. Intermedia. We also found that the yeast possesses no less than three genes encoding aldose reductases with different transcriptional profiles, and heterologous expression of the genes in Saccharomyces cerevisiae showed that the aldose reductases have different substrate and co-factor specificities, suggesting diverse physiological roles. Taken together, the results of this study provide insights into the mechanisms underlying carbohydrate metabolism in C. Intermedia, and reveals several genes with potential future applications in cell factory development.

  • split marker recombination for efficient targeted gene deletions in Candida Intermedia
    Non-conventional Yeasts: from Basic Research to Application Rzeszow, 2018
    Co-Authors: Fabio Luis Da Silva Faria Oliveira, Lisbeth Olsson, David Moreno, Cecilia Geijer
    Abstract:

    Candida Intermedia is a non-conventional yeast species with a natural ability to produce ethanol from xylose, making it an attractive non-GMO alternative for lignocellulosic biomass conversion in biorefineries and/or gene donor to Saccharomyces cerevisiae to improve its xylose fermentation capacity. We have de novo genome sequenced the C. Intermedia strain CBS 141442, previously isolated in our lab, which allows us to study the yeast at a genomic and molecular level. The aim of this project was to develop a molecular toolbox for C. Intermedia to enable also targeted genome editing and subsequent mutant phenotyping. C. Intermedia is a haploid yeast belonging to the CTG clade of fungal species, and thus requires drug-resistant markers adapted for the alternative codon usage of these organisms. Transformation of linearized plasmid containing the CaNAT1 marker flanked by the TEF1 promoter and terminator from Ashbya gossypii [1] resulted in hundreds of Nourseothricin-resistant transformants. We then constructed an ADE2-deletion cassette, where the CaNAT1 marker was fused to the upstream and downstream sequences (1000bp) of CiADE2. Transformations resulted in less than 1% of ade2 mutants with the characteristic red pigmentation, which indicates that the non-homologous end joining pathway (NHEJ) is dominant over the homologous recombination (HR) pathway in this yeast. Using the cell cycle inhibitor hydroxyurea to arrest cells in the S-phase has been shown to improve the HR/NHEJ ratio in other yeasts [2], and increased the ADE2 deletion efficiency to 4% in C. Intermedia. To further improve the targeted deletion rate, we applied the "split-marker” strategy previously developed for Saccharomyces cerevisiae [3]. Here, the selectable marker gene is truncated in two different fragments, and the gene is not functional until homologous recombination takes place between the two overlapping parts of the fragments. The truncated marker gene fragments were flanked by homologous sequences (1000 bp) upstream and downstream of the target gene using fusion PCR, thereby avoiding a tedious cloning step. This approach increased the targeted gene disruption of ADE2 to 56%. As proof of concept, the method was also used to delete KU70, the xylose reductase gene XYL1_2 as well as a large gene cluster in C. Intermedia, with allele-specific HR efficiencies between 87 and 100%. The split-marker approach for targeted gene-disruptions will pave the way for high throughput genetic analysis in C. Intermedia as well as in other yeasts where NHEJ is the predominant form of recombination.

Cecilia Geijer - One of the best experts on this subject based on the ideXlab platform.

  • Genomic and transcriptomic analysis of Candida Intermedia reveals the genetic determinants for its xylose-converting capacity.
    Biotechnology for Biofuels, 2020
    Co-Authors: Cecilia Geijer, Antonio D Moreno, Simon Stenberg, S Mazurkewich, Fábio Faria-oliveira, Lisbeth Olsson
    Abstract:

    Background: An economically viable production of biofuels and biochemicals from lignocellulose requires microorganisms that can readily convert both the cellulosic and hemicellulosic fractions into product. The yeast Candida Intermedia displays a high capacity for uptake and conversion of several lignocellulosic sugars including the abundant pentose d-xylose, an underutilized carbon source since most industrially relevant microorganisms cannot naturally ferment it. Thus, C. Intermedia constitutes an important source of knowledge and genetic information that could be transferred to industrial microorganisms such as Saccharomyces cerevisiae to improve their capacity to ferment lignocellulose-derived xylose. Results: To understand the genetic determinants that underlie the metabolic properties of C. Intermedia, we sequenced the genomes of both the in-house-isolated strain CBS 141442 and the reference strain PYCC 4715. De novo genome assembly and subsequent analysis revealed C. Intermedia to be a haploid species belonging to the CTG clade of ascomycetous yeasts. The two strains have highly similar genome sizes and number of protein-encoding genes, but they differ on the chromosomal level due to numerous translocations of large and small genomic segments. The transcriptional profiles for CBS 141442 grown in medium with either high or low concentrations of glucose and xylose were determined through RNA-sequencing analysis, revealing distinct clusters of co-regulated genes in response to different specific growth rates, carbon sources and osmotic stress. Analysis of the genomic and transcriptomic data also identified multiple xylose reductases, one of which displayed dual NADH/NADPH co-factor specificity that likely plays an important role for co-factor recycling during xylose fermentation. Conclusions: In the present study, we performed the first genomic and transcriptomic analysis of C. Intermedia and identified several novel genes for conversion of xylose. Together the results provide insights into the mechanisms underlying saccharide utilization in C. Intermedia and reveal potential target genes to aid in xylose fermentation in S. cerevisiae.

  • genomic and transcriptomic analysis of Candida Intermedia reveals the genetic determinants for its xylose converting capacity
    Biotechnology for Biofuels, 2020
    Co-Authors: Cecilia Geijer, Antonio D Moreno, Fabio Fariaoliveira, Simon Stenberg, S Mazurkewich, Lisbeth Olsson
    Abstract:

    An economically viable production of biofuels and biochemicals from lignocellulose requires microorganisms that can readily convert both the cellulosic and hemicellulosic fractions into product. The yeast Candida Intermedia displays a high capacity for uptake and conversion of several lignocellulosic sugars including the abundant pentose d-xylose, an underutilized carbon source since most industrially relevant microorganisms cannot naturally ferment it. Thus, C. Intermedia constitutes an important source of knowledge and genetic information that could be transferred to industrial microorganisms such as Saccharomyces cerevisiae to improve their capacity to ferment lignocellulose-derived xylose. To understand the genetic determinants that underlie the metabolic properties of C. Intermedia, we sequenced the genomes of both the in-house-isolated strain CBS 141442 and the reference strain PYCC 4715. De novo genome assembly and subsequent analysis revealed C. Intermedia to be a haploid species belonging to the CTG clade of ascomycetous yeasts. The two strains have highly similar genome sizes and number of protein-encoding genes, but they differ on the chromosomal level due to numerous translocations of large and small genomic segments. The transcriptional profiles for CBS 141442 grown in medium with either high or low concentrations of glucose and xylose were determined through RNA-sequencing analysis, revealing distinct clusters of co-regulated genes in response to different specific growth rates, carbon sources and osmotic stress. Analysis of the genomic and transcriptomic data also identified multiple xylose reductases, one of which displayed dual NADH/NADPH co-factor specificity that likely plays an important role for co-factor recycling during xylose fermentation. In the present study, we performed the first genomic and transcriptomic analysis of C. Intermedia and identified several novel genes for conversion of xylose. Together the results provide insights into the mechanisms underlying saccharide utilization in C. Intermedia and reveal potential target genes to aid in xylose fermentation in S. cerevisiae.

  • evolutionary engineered Candida Intermedia exhibits improved xylose utilization and robustness to lignocellulose derived inhibitors and ethanol
    Applied Microbiology and Biotechnology, 2019
    Co-Authors: Antonio D Moreno, Lisbeth Olsson, Antonella Carbone, Rosita Pavone, Cecilia Geijer
    Abstract:

    The development of robust microorganisms that can efficiently ferment both glucose and xylose represents one of the major challenges in achieving a cost-effective lignocellulosic bioethanol production. Candida Intermedia is a non-conventional, xylose-utilizing yeast species with a high-capacity xylose transport system. The natural ability of C. Intermedia to produce ethanol from xylose makes it attractive as a non-GMO alternative for lignocellulosic biomass conversion in biorefineries. We have evaluated the fermentation capacity and the tolerance to lignocellulose-derived inhibitors and the end product, ethanol, of the C. Intermedia strain CBS 141442 isolated from steam-exploded wheat straw hydrolysate. In a mixed sugar fermentation medium, C. Intermedia CBS 141442 co-fermented glucose and xylose, although with a preference for glucose over xylose. The strain was clearly more sensitive to inhibitors and ethanol when consuming xylose than glucose. C. Intermedia CBS 141442 was also subjected to evolutionary engineering with the aim of increasing its tolerance to inhibitors and ethanol, and thus improving its fermentation capacity under harsh conditions. The resulting evolved population was able to ferment a 50% (v/v) steam-exploded wheat straw hydrolysate (which was completely inhibitory to the parental strain), improving the sugar consumption and the final ethanol concentration. The evolved population also exhibited a better tolerance to ethanol when growing in a xylose medium supplemented with 35.5 g/L ethanol. These results highlight the potential of C. Intermedia CBS 141442 to become a robust yeast for the conversion of lignocellulose to ethanol.

  • genomic and transcriptomic analysis of Candida Intermedia reveals genes for utilization of biotechnologically important carbon sources
    35th International Specialised Symposium on Yeasts Antalya, 2019
    Co-Authors: Fabio Luis Da Silva Faria Oliveira, Lisbeth Olsson, Cecilia Geijer
    Abstract:

    A future biobased society relies on efficient industrial microorganisms that can convert all sugars from agricultural, forestry and industrial waste streams into fuels, chemicals and materials. To be able to tailor-make such potent cell factories, we need a far better understanding of the proteins responsible for the assimilation of biotechnologically important carbon sources including pentoses, disaccharides and oligomers. The yeast Candida Intermedia, known for its superior growth on xylose owing to its efficient uptake and conversion systems, can also utilize a range of other important carbon sources such as cellobiose, galactose and lactose. The aim of this project was to identify the genomic determinants for the utilization of these mono- and disaccharides in our in-house isolated C. Intermedia strain CBS 141442. Genome sequencing and transcriptional (RNA seq) data analysis during growth in defined medium supplemented with glucose, xylose, galactose, lactose or cellobiose, revealed numerous distinct clusters of coregulated genes. By scanning the CBS 141442 genome for genes encoding Major Facilitator Superfamily (MFS) sugar transporters, and the RNA-seq dataset for the corresponding transcriptional profiles, we identified several novel genes encoding putative xylose transporters and multiple Lac12-like transporters likely involved in the uptake of disaccharides in C. Intermedia. We also found that the yeast possesses no less than three genes encoding aldose reductases with different transcriptional profiles, and heterologous expression of the genes in Saccharomyces cerevisiae showed that the aldose reductases have different substrate and co-factor specificities, suggesting diverse physiological roles. Taken together, the results of this study provide insights into the mechanisms underlying carbohydrate metabolism in C. Intermedia, and reveals several genes with potential future applications in cell factory development.

  • split marker recombination for efficient targeted gene deletions in Candida Intermedia
    Non-conventional Yeasts: from Basic Research to Application Rzeszow, 2018
    Co-Authors: Fabio Luis Da Silva Faria Oliveira, Lisbeth Olsson, David Moreno, Cecilia Geijer
    Abstract:

    Candida Intermedia is a non-conventional yeast species with a natural ability to produce ethanol from xylose, making it an attractive non-GMO alternative for lignocellulosic biomass conversion in biorefineries and/or gene donor to Saccharomyces cerevisiae to improve its xylose fermentation capacity. We have de novo genome sequenced the C. Intermedia strain CBS 141442, previously isolated in our lab, which allows us to study the yeast at a genomic and molecular level. The aim of this project was to develop a molecular toolbox for C. Intermedia to enable also targeted genome editing and subsequent mutant phenotyping. C. Intermedia is a haploid yeast belonging to the CTG clade of fungal species, and thus requires drug-resistant markers adapted for the alternative codon usage of these organisms. Transformation of linearized plasmid containing the CaNAT1 marker flanked by the TEF1 promoter and terminator from Ashbya gossypii [1] resulted in hundreds of Nourseothricin-resistant transformants. We then constructed an ADE2-deletion cassette, where the CaNAT1 marker was fused to the upstream and downstream sequences (1000bp) of CiADE2. Transformations resulted in less than 1% of ade2 mutants with the characteristic red pigmentation, which indicates that the non-homologous end joining pathway (NHEJ) is dominant over the homologous recombination (HR) pathway in this yeast. Using the cell cycle inhibitor hydroxyurea to arrest cells in the S-phase has been shown to improve the HR/NHEJ ratio in other yeasts [2], and increased the ADE2 deletion efficiency to 4% in C. Intermedia. To further improve the targeted deletion rate, we applied the "split-marker” strategy previously developed for Saccharomyces cerevisiae [3]. Here, the selectable marker gene is truncated in two different fragments, and the gene is not functional until homologous recombination takes place between the two overlapping parts of the fragments. The truncated marker gene fragments were flanked by homologous sequences (1000 bp) upstream and downstream of the target gene using fusion PCR, thereby avoiding a tedious cloning step. This approach increased the targeted gene disruption of ADE2 to 56%. As proof of concept, the method was also used to delete KU70, the xylose reductase gene XYL1_2 as well as a large gene cluster in C. Intermedia, with allele-specific HR efficiencies between 87 and 100%. The split-marker approach for targeted gene-disruptions will pave the way for high throughput genetic analysis in C. Intermedia as well as in other yeasts where NHEJ is the predominant form of recombination.

Isabel Spencermartins - One of the best experts on this subject based on the ideXlab platform.

  • the expression in saccharomyces cerevisiae of a glucose xylose symporter from Candida Intermedia is affected by the presence of a glucose xylose facilitator
    Microbiology, 2008
    Co-Authors: Maria Jose Leandro, Isabel Spencermartins, Paula Goncalves
    Abstract:

    Two glucose/xylose transporter genes from Candida Intermedia were recently cloned and characterized: GXF1, which encodes a glucose/xylose facilitator; and GXS1, which encodes a glucose/xylose proton symporter. Here we report the functional expression of these transporters in Saccharomyces cerevisiae. While Gxf1p seems to be fully functional in S. cerevisiae, the symporter Gxs1p exhibits very low glucose/xylose transport activity, which could not be ascribed to insufficient production of the protein or incorrect subcellular localization. In addition, co-expression of glucose/xylose facilitators with Gxs1p strongly reduced GXS1 mRNA levels, and consequently symport activity, in glucose-grown, but not in ethanol-grown, cells. The observed decrease in GXS1 transcript levels seems to be related to an enhanced glucose influx mediated by glucose facilitator protein(s), and not to a specific interaction between Gxs1p and other transporters. We found GXS1 mRNA levels to be severely reduced as a result of glucose addition, and we show that this effect takes place at the level of GXS1 mRNA stability. Our results suggest that a decrease in mRNAs encoding high-affinity/active sugar transport systems may be a widespread and conserved mechanism in yeasts, limiting expression of these proteins whenever their activity is dispensable.

  • two glucose xylose transporter genes from the yeast Candida Intermedia first molecular characterization of a yeast xylose h symporter
    Biochemical Journal, 2006
    Co-Authors: Maria Jose Leandro, Paula Goncalves, Isabel Spencermartins
    Abstract:

    Candida Intermedia PYCC 4715 was previously shown to grow well on xylose and to transport this sugar by two different transport systems: high-capacity and low-affinity facilitated diffusion and a high-affinity xylose–proton symporter, both of which accept glucose as a substrate. Here we report the isolation of genes encoding both transporters, designated GXF1 (glucose/xylose facilitator 1) and GXS1 (glucose/xylose symporter 1) respectively. Although GXF1 was isolated by functional complementation of an HXT-null (where Hxt refers to hexose transporters) Saccharomyces cerevisiae strain, isolation of the GXS1 cDNA required partial purification and micro-sequencing of the transporter, identified by its relative abundance in cells grown on low xylose concentrations. Both genes were expressed in S. cerevisiae and the kinetic parameters of glucose and xylose transport were determined. Gxs1 is the first yeast xylose/glucose–H+ symporter to be characterized at the molecular level. Comparison of its amino acid sequence with available sequence data revealed the existence of a family of putative monosaccharide–H+ symporters encompassing proteins from several yeasts and filamentous fungi.

  • high capacity xylose transport in Candida Intermedia pycc 4715
    Fems Yeast Research, 2003
    Co-Authors: Mark Gardonyi, Mans Osterberg, Carla Rodrigues, Isabel Spencermartins, Barbel Hahnhagerdal
    Abstract:

    Xylose-utilising yeasts were screened to identify strains with high xylose transport capacity. Among the fastest-growing strains in xylose medium, Candida Intermedia PYCC 4715 showed the highest xylose transport capacity. Maximal specific growth rate was the same in glucose and xylose media (μmax=0.5 h−1, 30°C). Xylose transport showed biphasic kinetics when cells were grown in either xylose- or glucose-limited culture. The high-affinity xylose/proton symport system (Km=0.2 mM, Vmax=7.5 mmol h−1 g−1) was more repressed by glucose than by xylose. The less specific low-affinity transport system (K=50 mM, Vmax=11 mmol h−1 g−1) appeared to operate through a facilitated-diffusion mechanism and was expressed constitutively. Inhibition experiments showed that glucose is a substrate of both xylose transport systems.

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