CLONING AND SEQUENCE-ANALYSIS OF THE PICHIA-PASTORIS TRP1, IPP1 AND HIS3 GENES

The Pichia pastoris TRP1 and HIS3 genes were cloned by complementation of the Saccharomyces cerevisiae trp1 and his3 mutants, respectively, and their nucleotide sequence was determined. The P. pastoris TRP1 gen e includes an open reading frame (ORF) of 714 nucleotides correspondin g to a polypeptide of 237 amino acids whose sequence shares about 40% identity with that of TRP1 encoding proteins in other yeast species. D NA sequencing showed that an ORF of 858 nucleotides, encoding a protei n of 285 amino acids with high homology to inorganic pyrophosphatases (IPP1), is located downstream of the P, pastoris TRP1 gene. Both genes converge in this chromosomal region, showing a genetic organization a nalogous to that found in the Kluyveromyces lactis genome. The P. past oris HIS3 gene possesses an ORF of 675 nucleotides, encoding a polypep tide of 224 amino acids which shows 74.1% identity to the homologous S . cerevisiae protein. The hexameric consensus GCN4 binding sequence (T GACTC), characteristic of many amino acid biosynthetic penes, is prese nt in the promoter region. The TRP1 and IPP1 sequences were deposited in the EMBL databank under Accession Number AJ001000. The Accession Nu mber of the HIS3 gene is U69170. (C) 1998 John Wiley B Sons, Ltd.