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

Abstract

An Autonomously Replicating Sequence ele- ment adjacent to the RIB1 gene encoding GTP cyclohydrolase II of the yeast Pichia guilliermondii was identi®ed by transformation experiments. Detailed se- quence analysis unveiled two potential ARS elements located 5¢ and 3¢ of the RIB1 open reading frame. The chromosomal fragment containing the ARS-like se- quence 3¢ to the RIB1 structural gene, called PgARS, conferred high transformation frequencies of 104±105 transformants/lg 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 e cient transformation of the ¯avinogenic yeast P. guilliermondii was developed.