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Abstract
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he yeast Saccharomyces cerevisiae has 20 genes that encode Hexose Transporter
proteins, including HXT1-HXT17, GAL2, SNF3 and RGT2. Two of these genes
(SNF3 and RGT2) act as glucose sensors while the HXT1-HXT17 genes function
in direct transportation of glucose. Earlier research has shown that alcohol
fermentation can be augmented by increasing the expression of these genes, resulting in
increasing ethanol production. The aim of this study was the identification and isolation
of the Restores Glucose Transport 2 (RGT2) gene from Saccharomyces cerevisiae
genome. Specific primers were employed in PCR so as to clone RGT2 into a vector
under a suitable expression promoter for recombinant yeast. After gene amplification,
ligation was achieved between the amplified fragments and pGEM-T vector and the
recombinant colonies were identified by the blue-white screening method. Candidate
recombinant plasmids were sequenced. The nucleotide sequence of the open reading
frame was found to be 2292 bp long with a deduced amino acid of 763 residues. The
estimated molecular mass and the predicted isoelectric point of the deduced polypeptide
were 83.173 kDa and 5.68 respectively. The deduced protein sequence showed a high
similarity to RGT2 sequences in the NCBI database, especially with P301 strain of
Saccharomyces cerevisiae (100 % similarity). Finally, the RGT2 gene was cloned into
the pGBKT7 expression vector which is suitable for protein expression in yeast via the
restriction sites NcoI and PstI. A phylogenic study of the RGT2 gene and other hexose
transporter families showed that this gene has the most similarity with SNF3. Therefore,
by isolation, cloning and sequence identification and transformation of this gene into
yeast, ethanol production via alcohol fermentation can be increased.
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