Identification and Application of Novel Autonomously Replicating Sequences (ARSs) for Promoter-Cloning and Co-Transformation in Candida utilis

Abstract

In order to develop practical recombinant DNA techniques in the industrially important yeast Candida utilis, at least six plasmids harboring autonomously replicating sequences (ARSs) were isolated from a C. utilis genomic library. Two ARSs were subjected to detailed analysis. Sequences of 1.9 and 1.8 kb were found to be necessary to exert ARS activity in a plasmid as assessed by transformation efficiency and mitotic stability. Both fragments were found to be rich in AT content (69.5% and 70.8% respectively), and to contain an 11-bp ARS consensus sequences (10 and 13 motifs with one base difference respectively). Using the ARS-containing plasmid as a promoter-cloning vector, several DNA fragments having promoter activities were cloned and characterized. Co-transformation of C. utilis with an integrating DNA fragment and a replicating plasmid yielded plasmid-free transformants harboring the fragment integrated into the C. utilis genome.