Faculty Opinions recommendation of Co-expression of a mammalian accessory trafficking protein enables functional expression of the rat MCT1 monocarboxylate transporter in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae the activity for the lactate–proton symporter is dependent on JEN1 gene expression. Pichia pastoris was transformed with an integrative plasmid containing the JEN1 gene. After 24 h of methanol induction, Northern and Western blotting analyses indicated the expression of JEN1 in the transformants. Lactate permease activity was obtained in P. pastoris cells with a Vmax of 2.1 nmol·s−1·mg of dry weight−1. Reconstitution of the lactate permease activity was achieved by fusing plasma membranes of P. pastoris methanol-induced cells with Escherichia coli liposomes containing cytochrome c oxidase, as proton-motive force. These assays in reconstituted heterologous P. pastoris membrane vesicles demonstrate that S. cerevisiae Jen1p is a functional lactate transporter. Moreover, a S. cerevisiae strain deleted in the JEN1 gene was transformed with a centromeric plasmid containing JEN1 under the control of the glyceraldehyde-3-phosphate dehydrogenase constitutive promotor. Constitutive JEN1 expression and lactic acid uptake were observed in cells grown on either glucose and/or acetic acid. The highest Vmax (0.84 nmol·s−1·mg of dry weight−1) was obtained in acetic acid-grown cells. Thus overexpression of the S. cerevisiae JEN1 gene in both S. cerevisiae and P. pastoris cells resulted in increased activity of lactate transport when compared with the data previously reported in lactic acid-grown cells of native S. cerevisiae strains. Jen1p is the only S. cerevisiae secondary porter characterized so far by heterologous expression in P. pastoris at both the cell and the membrane-vesicle levels.

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Functional expression of xylose isomerase in flocculating industrial Saccharomyces cerevisiae strain for bioethanol production

Journal of Bioscience and Bioengineering ◽

10.1016/j.jbiosc.2015.10.013 ◽

2016 ◽

Vol 121 (6) ◽

pp. 685-691 ◽

Cited By ~ 9

Author(s):

Yun-Cheng Li ◽

Guo-Ying Li ◽

Min Gou ◽

Zi-Yuan Xia ◽

Yue-Qin Tang ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Xylose Isomerase ◽

Functional Expression ◽

Bioethanol Production

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Functional Expression and Characterization of Schizosaccharomyces pombe Avt3p as a Vacuolar Amino Acid Exporter in Saccharomyces cerevisiae

PLoS ONE ◽

10.1371/journal.pone.0130542 ◽

2015 ◽

Vol 10 (6) ◽

pp. e0130542 ◽

Cited By ~ 8

Author(s):

Soracom Chardwiriyapreecha ◽

Kunio Manabe ◽

Tomoko Iwaki ◽

Miyuki Kawano-Kawada ◽

Takayuki Sekito ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Schizosaccharomyces Pombe ◽

Functional Expression

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Functional Expression of a Bacterial Xylose Isomerase in Saccharomyces cerevisiae

Applied and Environmental Microbiology ◽

10.1128/aem.02522-08 ◽

2009 ◽

Vol 75 (8) ◽

pp. 2304-2311 ◽

Cited By ~ 174

Author(s):

Dawid Brat ◽

Eckhard Boles ◽

Beate Wiedemann

Keyword(s):

Saccharomyces Cerevisiae ◽

Heterologous Expression ◽

Xylose Fermentation ◽

Thermus Thermophilus ◽

Xylose Isomerase ◽

Functional Expression ◽

Yeast Cells ◽

Highly Active ◽

Excellent Starting Point ◽

Starting Point

ABSTRACT In industrial fermentation processes, the yeast Saccharomyces cerevisiae is commonly used for ethanol production. However, it lacks the ability to ferment pentose sugars like d-xylose and l-arabinose. Heterologous expression of a xylose isomerase (XI) would enable yeast cells to metabolize xylose. However, many attempts to express a prokaryotic XI with high activity in S. cerevisiae have failed so far. We have screened nucleic acid databases for sequences encoding putative XIs and finally were able to clone and successfully express a highly active new kind of XI from the anaerobic bacterium Clostridium phytofermentans in S. cerevisiae. Heterologous expression of this enzyme confers on the yeast cells the ability to metabolize d-xylose and to use it as the sole carbon and energy source. The new enzyme has low sequence similarities to the XIs from Piromyces sp. strain E2 and Thermus thermophilus, which were the only two XIs previously functionally expressed in S. cerevisiae. The activity and kinetic parameters of the new enzyme are comparable to those of the Piromyces XI. Importantly, the new enzyme is far less inhibited by xylitol, which accrues as a side product during xylose fermentation. Furthermore, expression of the gene could be improved by adapting its codon usage to that of the highly expressed glycolytic genes of S. cerevisiae. Expression of the bacterial XI in an industrially employed yeast strain enabled it to grow on xylose and to ferment xylose to ethanol. Thus, our findings provide an excellent starting point for further improvement of xylose fermentation in industrial yeast strains.

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