ISOLATION AND CHARACTERIZATION OF THE PICHIA-STIPITIS TRANSKETOLASE GENE AND EXPRESSION IN A XYLOSE UTILIZING 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 determine d contains an open-reading frame of 2085 base pairs (bp) encoding a pr otein of 695 amino acids with a predicted molecular mass of 75 113 Da. The TKT gene was actively expressed in S. cerevisiae when placed unde r the control of the homologous PDC1(- 15) promoter and could compleme nt a S. cerevisiae tkt deletion. The TKT protein was immunologically d etectable using S. cerevisiae transketolase-specific antiserum. Overex pression of the P. stipitis TKT gene in a xylose-utilizing S. cerevisi ae XYL1/XYL2 integrant led to a drastically extended generation time d uring growth on xylose minimal medium under aerobic conditions.