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Anal Biochem. 2013 Nov 15;442(2):241-8. doi: 10.1016/j.ab.2013.07.041. Epub 2013 Aug 6.

An assay for functional xylose transporters in Saccharomyces cerevisiae.

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1
State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China.

Abstract

It has been considered that more efficient uptake of xylose could promote increased xylose metabolic capacity of several microorganisms. In this study, an assay to screen xylose transporters was established in the Saccharomyces cerevisiae strain, which expresses the xylosidase gene of Bacillus pumilus intracellularly. The absorbed xylose analog p-nitrophenyl-β-d-xylopyranoside (pNPX) rapidly hydrolyzed to p-nitrophenol (pNP), which displayed a yellow tint when exposed to xylosidase in vivo. The xylose transporter activities of the strain were computed using the pNP production rate, which was detected extracellularly. This method could be used for both high-throughput screening and smaller scale investigations. AraEp, which is a pentose transporter of Corynebacterium glutamicum, was expressed in S. cerevisiae and exhibited better transport capacity than the endogenous transporters Hxt7p and Gal2p. Moreover, a mutant of AraEp with 103% greater transport capacity was screened out, and the computer simulation suggested that transmembrane domain 5 was an important factor for the transport capacity of AraEp in S. cerevisiae.

KEYWORDS:

AraEp; Budding yeast; Xylose absorption; Xylosidase; pNPX

PMID:
23928049
DOI:
10.1016/j.ab.2013.07.041
[Indexed for MEDLINE]
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