Optimisation of a host/vector system for heterologous gene expression by Hansenula polymorpha

Abstract

For the methylotrophic yeast, Hansenula polymorpha, expression vectors with different origins of replication have been constructed in order to analyse their influence on transformation and integration efficiency. The constructed plasmids are identical except for their origin of replication, which involve, respectively, that of the Saccharomyces cerevisiae 2-μm plasmid and a H. polymorpha ARS sequence (HARS2). A plasmid with no origin of replication served as a control. The plasmids also contained the α-galactosidase expression cassette, consisting of the Cyamopsis tetragonoloba α-galactosidase gene, the H. polymorpha methanol oxidase promoter and terminator, and the S. cerevisiae invertase signal sequence. The transformation frequencies of the expression vectors containing the 2-μm and the HARS2 origins of replication, and no origin of replication, were 2,50 and 15 per μg of DNA respectively, which demonstrates the negative effect of the 2-μm sequence on the transformation frequency. Autonomously replicating plasmids could be isolated from the transformants obtained with the plasmid containing either the 2-μm or the HARS2 sequence. Integration of the 2-μm based plasmid into the H. polymorpha genome could not be established using a standard procedure. This is in contrast with transformants containing a plasmid bearing the HARS2 sequence or else with no origin of replication, which shows that the 2-μm sequence negatively influences the integration of the expression vector into the H. polymorpha genome. Integration of expression plasmids occurred in 50% of the analysed integrants on the homologous methanol oxidase locus, and tandem integration was favoured. The level of specific mRNA, and the expression of the α-galactosidase protein by these integrants, was proportional to the number of integrated copies of the expression plasmid in the H. polymorpha genome.