Xylose isomerase from polycentric fungus Orpinomyces: gene sequencing, cloning, and expression in Saccharomyces cerevisiae for bioconversion of xylose to ethanol.
The cDNA sequence of the gene for xylose isomerase from the rumen fungus Orpinomyces was elucidated by rapid amplification of cDNA ends. The 1,314-nucleotide gene was cloned and expressed constitutively in Saccharomyces cerevisiae. The deduced polypeptide sequence encoded a protein of 437 amino acids which showed the highest similarity to the family II xylose isomerases. Further, characterization revealed that the recombinant enzyme was a homodimer with a subunit of molecular mass 49 kDa. Cell extract of the recombinant strain exhibited high specific xylose isomerase activity. The pH optimum of the enzyme was 7.5, while the low temperature optimum at 37 degrees C was the property that differed significantly from the majority of the reported thermophilic xylose isomerases. In addition to the xylose isomerase gene, the overexpression of the S. cerevisiae endogenous xylulokinase gene and the Pichia stipitis SUT1 gene for sugar transporter in the recombinant yeast facilitated the efficient production of ethanol from xylose.
Madhavan
Anjali
A
Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz Khas, 110016 Delhi, India.
Tamalampudi
Sriappareddy
S
Ushida
Kazunari
K
Kanai
Daisuke
D
Katahira
Satoshi
S
Srivastava
Aradhana
A
Fukuda
Hideki
H
Bisaria
Virendra S
VS
Kondo
Akihiko
A
D019747:Aldose-Ketose Isomerases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001711:Biotransformation; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D019281:Dimerization; D004795:Enzyme Stability; D000431:Ethanol / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism; D020085:Neocallimastigales / Q000201:enzymology* / Q000235:genetics*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013696:Temperature; D014994:Xylose / Q000378:metabolism*
D004271:DNA, Fungal; D005656:Fungal Proteins; D009004:Monosaccharide Transport Proteins; C118625:SUT1 protein, Pichia stipitis; D000431:Ethanol; D014994:Xylose; D017853:Phosphotransferases (Alcohol Group Acceptor); C032700:xylulokinase; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase