TKL2, A 2ND TRANSKETOLASE GENE OF SACCHAROMYCES-CEREVISIAE - CLONING,SEQUENCE AND DELETION ANALYSIS OF THE GENE

Transketolase activity is indispensable for the generation of erythros e 4-phosphate and therefore necessary for the biosynthesis of the arom atic amino acids. Yeast mutants with a deletion of the transketolase g ene, TKL1, can grow without aromatic amino acid supplement indicating an additional source of erythrose 4-phosphate in the cells. Here we de scribe the cloning of TKL2, a gene coding for a second transketolase e nzyme in Saccharomyces cerevisiae. The deduced protein sequence of TKL 2 demonstrates 71% identitity with TKL1 [Sundstrom, M., Lindqvist, Y., Schneider, G., Hellman, U. & Ronne, H. (1993) J. Biol. Chem., in the press]. Double mutants for both genes, TKL1 and TKL2, are auxotrophic for aromatic amino acids, indicating a complete block in the transketo lase activity. Deletion of TKL2 alone does not lead to a significant p henotype, and transketolase activity is not reduced in these mutants. Overexpression of TKL2 on a multi-copy plasmid in a tkl1 background sh owed that TKL2 is functionally expressed: transketolase enzyme activit y was detectable in the transformants and the protein reacts with anti -transketolase serum in Western blot analysis. In addition, transforma tion of the tkl1 tkl2 double mutant with the TKL2 plasmid can compensa te the growth defect on a medium without aromatic amino acids.