Improved xylose uptake in Saccharomyces cerevisiae due to directed evolution of galactose permease Gal2 for sugar co-consumption.

Reznicek, O; Facey, S J; de Waal, P P; Teunissen, A W R H; de Bont, J A M; Nijland, J G; Driessen, A J M; Hauer, B

Reznicek, O; Facey, S J; de Waal, P P; Teunissen, A W R H; de Bont, J A M; Nijland, J G; Driessen, A J M; Hauer, B.

Afiliación

Reznicek O; Institute of Technical Biochemistry, University of Stuttgart, Stuttgart, Germany.
Facey SJ; Institute of Technical Biochemistry, University of Stuttgart, Stuttgart, Germany.
de Waal PP; DSM, AX Delft, The Netherlands.
Teunissen AW; DSM, AX Delft, The Netherlands.
de Bont JA; Jandebontbioconsultancy, Wageningen, The Netherlands.
Nijland JG; Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology, Zernike Institute for Advanced Materials and Kluyver Centre for Genomics of Industrial Fermentation, University of Groningen, Groningen, The Netherlands.
Driessen AJ; Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology, Zernike Institute for Advanced Materials and Kluyver Centre for Genomics of Industrial Fermentation, University of Groningen, Groningen, The Netherlands.
Hauer B; Institute of Technical Biochemistry, University of Stuttgart, Stuttgart, Germany.

J Appl Microbiol ; 119(1): 99-111, 2015 Jul.

Article en En | MEDLINE | ID: mdl-25882005

RESUMEN

AIMS: Saccharomyces cerevisiae does not express any xylose-specific transporters. To enhance the xylose uptake of S. cerevisiae, directed evolution of the Gal2 transporter was performed. METHODS AND RESULTS: Three rounds of error-prone PCR were used to generate mutants with improved xylose-transport characteristics. After developing a fast and reliable high-throughput screening assay based on flow cytometry, eight mutants were obtained showing an improved uptake of xylose compared to wild-type Gal2 out of 41 200 single yeast cells. Gal2 variant 2·1 harbouring five amino acid substitutions showed an increased affinity towards xylose with a faster overall sugar metabolism of glucose and xylose. Another Gal2 variant 3·1 carrying an additional amino acid substitution revealed an impaired growth on glucose but not on xylose. CONCLUSIONS: Random mutagenesis of the S. cerevisiae Gal2 led to an increased xylose uptake capacity and decreased glucose affinity, allowing improved co-consumption. SIGNIFICANCE AND IMPACT OF THE STUDY: Random mutagenesis is a powerful tool to evolve sugar transporters like Gal2 towards co-consumption of new substrates. Using a high-throughput screening system based on flow-through cytometry, various mutants were identified with improved xylose-transport characteristics. The Gal2 variants in this work are a promising starting point for further engineering to improve xylose uptake from mixed sugars in biomass.

Asunto(s)

Proteínas de Transporte de Monosacáridos/genética; Proteínas de Saccharomyces cerevisiae/genética; Saccharomyces cerevisiae/enzimología; Xilosa/metabolismo; Transporte Biológico; Evolución Molecular Dirigida; Glucosa/metabolismo; Ensayos Analíticos de Alto Rendimiento; Proteínas de Transporte de Monosacáridos/metabolismo; Mutagénesis; Saccharomyces cerevisiae/genética; Saccharomyces cerevisiae/metabolismo; Proteínas de Saccharomyces cerevisiae/metabolismo

Palabras clave

Gal2; biofuels; biotechnology; sugar co-consumption; yeasts

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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Saccharomyces cerevisiae / Xilosa / Proteínas de Transporte de Monosacáridos / Proteínas de Saccharomyces cerevisiae Tipo de estudio: Prognostic_studies Idioma: En Revista: J Appl Microbiol Asunto de la revista: MICROBIOLOGIA Año: 2015 Tipo del documento: Article País de afiliación: Alemania Pais de publicación: Reino Unido

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