Identification of an ARS element and development of a high efficiency transformation system for Pichia guilliermondii

An Autonomously Replicating Sequence element adjacent to the RIB1 gene enco ding GTP cyclohydrolase II of the yeast Pichia guilliermondii was identifie d by transformation experiments. Detailed sequence analysis unveiled two po tential ARS elements located 5' and 3' of the RIB1 open reading frame. The chromosomal fragment containing the ARS-like sequence 3' to the RIB1 struct ural gene, called PgARS conferred high transformation frequencies of 10(4)- 10(5) transformants/mu g of DNA to a pUC19-derived plasmid in P. guilliermo ndii, The PgARS element also conferred autonomous replication to hybrid pla smids in this host. Based on this element a series of Escherichia coli shut tle vectors for efficient transformation of the flavinogenic yeast P. guill iermondii was developed.