Isolation and characterization of the Pichia stipitis transketolase gene and expression in a xylose-utilising Saccharomyces cerevisiae transformant.
The gene TKT from P. stipitis, encoding the enzyme transketolase (EC 2.2.1.1), was cloned from a genomic library by hybridization with a S. cerevisiae TKT1-gene-specific probe. The nucleotide sequence determined contains an open-reading frame of 2085 base pairs (bp) encoding a protein of 695 amino acids with a predicted molecular mass of 75113 Da. The TKT gene was actively expressed in S. cerevisiae when placed under the control of the homologous PDC1(-15) promoter and could complement a S. cerevisiae tkt deletion. The TKT protein was immunologically detectable using S. cerevisiae transketolase-specific antiserum. Overexpression of the P. stipitis TKT gene in a xylose-utilizing S. cerevisiae XYL1/XYL2 integrant led to a drastically extended generation time during growth on xylose minimal medium under aerobic conditions.
Metzger
M H
MH
Institut für Mikrobiologie, Heinrich-Heine-Universität, Düsseldorf, Germany.
Hollenberg
C P
CP
D001483:Base Sequence; D001709:Biotechnology; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics; D015870:Gene Expression; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D010843:Pichia / Q000201:enzymology* / Q000235:genetics*; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D014170:Transformation, Genetic; D014174:Transketolase / Q000235:genetics*; D014994:Xylose / Q000378:metabolism
D004271:DNA, Fungal; D014994:Xylose; D014174:Transketolase