CLONING AND SEQUENCING OF THE URA3 GENE OF KLUYVEROMYCES-MARXIANUS CBS-6556

The URA3 gene, coding for orotidine-5'-phosphate decarboxylase, from K luyveromyces marxianus CBS 6556, was isolated from a genomic DNA libra ry. The K. marxianus URA3 gene encodes a protein of 267 amino acids wi th a calculated molecular weight of 29.3 kDa. Comparison of the K. mar xianus protein with the corresponding enzymes of Saccharomyces cerevis iae and Kluyveromyces lactis showed amino acid sequence identities of 81% and 88%, respectively. Using contour-clamped homogenous electric f ield gel electrophoresis, the genomic copy was found to be located on chromosome VI. We have used the cloned gene for the construction of a K. marxianus leu2 mutant. This mutant contains no heterologous sequenc es, which is essential to make it acceptable for application in the fo od industry.