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 encoding GTP cyclohydrolase II of the yeast Pichia guilliermondii was identified by transformation experiments. Detailed sequence analysis unveiled two potential ARS elements located 5' and 3' of the RIB1 open reading frame. The chromosomal fragment containing the ARS-like sequence 3' to the RIB1 structural gene, called PgARS, conferred high transformation frequencies of 10(4)-10(5) transformants/microg of DNA to a pUC19-derived plasmid in P. guilliermondii. The PgARS element also conferred autonomous replication to hybrid plasmids in this host. Based on this element a series of Escherichia coli shuttle vectors for efficient transformation of the flavinogenic yeast P. guilliermondii was developed.