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Article

Strategies for Efficient Expression of Heterologous Monosaccharide Transporters in Saccharomyces cerevisiae

1
Center of Biological Sciences, Department of Biochemistry, Federal University of Santa Catarina, Florianópolis 88040-900, SC, Brazil
2
Laboratório Nacional de Energia e Geologia, I.P., Unidade de Bioenergia, Estrada do Paço do Lumiar 22, 1649-038 Lisboa, Portugal
3
ITQB NOVA, Instituto de Tecnologia Química e Biológica António Xavier, Av. da República, 2780-157 Oeiras, Portugal
4
Discovery, R&D, Chr. Hansen A/S, 2970 Hørsholm, Denmark
*
Author to whom correspondence should be addressed.
Academic Editors: Margarida Casal, Sandra Paiva and Isabel Soares-Silva
J. Fungi 2022, 8(1), 84; https://doi.org/10.3390/jof8010084
Received: 30 November 2021 / Revised: 9 January 2022 / Accepted: 12 January 2022 / Published: 15 January 2022
(This article belongs to the Special Issue Fungi Nutrient Transportation)
In previous work, we developed a Saccharomyces cerevisiae strain (DLG-K1) lacking the main monosaccharide transporters (hxt-null) and displaying high xylose reductase, xylitol dehydrogenase and xylulokinase activities. This strain proved to be a useful chassis strain to study new glucose/xylose transporters, as SsXUT1 from Scheffersomyces stipitis. Proteins with high amino acid sequence similarity (78–80%) to SsXUT1 were identified from Spathaspora passalidarum and Spathaspora arborariae genomes. The characterization of these putative transporter genes (SpXUT1 and SaXUT1, respectively) was performed in the same chassis strain. Surprisingly, the cloned genes could not restore the ability to grow in several monosaccharides tested (including glucose and xylose), but after being grown in maltose, the uptake of 14C-glucose and 14C-xylose was detected. While SsXUT1 lacks lysine residues with high ubiquitinylation potential in its N-terminal domain and displays only one in its C-terminal domain, both SpXUT1 and SaXUT1 transporters have several such residues in their C-terminal domains. A truncated version of SpXUT1 gene, deprived of the respective 3′-end, was cloned in DLG-K1 and allowed growth and fermentation in glucose or xylose. In another approach, two arrestins known to be involved in the ubiquitinylation and endocytosis of sugar transporters (ROD1 and ROG3) were knocked out, but only the rog3 mutant allowed a significant improvement of growth and fermentation in glucose when either of the XUT permeases were expressed. Therefore, for the efficient heterologous expression of monosaccharide (e.g., glucose/xylose) transporters in S. cerevisiae, we propose either the removal of lysines involved in ubiquitinylation and endocytosis or the use of chassis strains hampered in the specific mechanism of membrane protein turnover. View Full-Text
Keywords: xylose; hxt-null; ubiquitinylation; lysine; truncated permease; endocytosis; membrane protein turnover; XUT1; ROD1; ROG3 xylose; hxt-null; ubiquitinylation; lysine; truncated permease; endocytosis; membrane protein turnover; XUT1; ROD1; ROG3
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MDPI and ACS Style

Knychala, M.M.; dos Santos, A.A.; Kretzer, L.G.; Gelsleichter, F.; Leandro, M.J.; Fonseca, C.; Stambuk, B.U. Strategies for Efficient Expression of Heterologous Monosaccharide Transporters in Saccharomyces cerevisiae. J. Fungi 2022, 8, 84. https://doi.org/10.3390/jof8010084

AMA Style

Knychala MM, dos Santos AA, Kretzer LG, Gelsleichter F, Leandro MJ, Fonseca C, Stambuk BU. Strategies for Efficient Expression of Heterologous Monosaccharide Transporters in Saccharomyces cerevisiae. Journal of Fungi. 2022; 8(1):84. https://doi.org/10.3390/jof8010084

Chicago/Turabian Style

Knychala, Marilia M., Angela A. dos Santos, Leonardo G. Kretzer, Fernanda Gelsleichter, Maria José Leandro, César Fonseca, and Boris U. Stambuk. 2022. "Strategies for Efficient Expression of Heterologous Monosaccharide Transporters in Saccharomyces cerevisiae" Journal of Fungi 8, no. 1: 84. https://doi.org/10.3390/jof8010084

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