A NOVEL-APPROACH TO EXPRESS A HETEROLOGOUS GENE ON KLUYVEROMYCES-LACTIS LINEAR KILLER PLASMIDS - EXPRESSION OF THE BACTERIAL APH GENE FROM A CYTOPLASMIC PROMOTER FRAGMENT WITHOUT IN-PHASE FUSION TO THE PLASMIDOPEN READING FRAME

Using the cytoplasmically localized killer plasmids pGKL1 and pGKL2 of Kluyveromyces lactis a new linear hybrid plasmid (pWKL1) was construc ted. It consists of pGKL1 into which, in addition to the previously de veloped cytoplasmically expressible LEU2 selection marker, a phosphot ransferase encoding bacterial antibiotic resistance gene has been inte grated. In the hybrid plasmid pWKL1 this gene was preceded by a putati ve transcriptional signal from pGKL2, i.e., the upstream conserved seq uence of ORF5 (UCS5). All foreign genes, so far integrated and express ed in linear plasmids, required in addition to a cytoplasmic promoter (UCS) an in phase fusion to the corresponding open reading frame. In t his study the UCS5, not containing the translational start of ORF5, an d the aph coding region were intentionally not combined in phase, in o rder to check whether expression can occur under these conditions. Klu yveromyces lactis transformants carrying pWKL1 were highly resistant a gainst the antibiotic geneticine (G418) and exhibited elevated levels of APH activity. Expression of the aph gene was due to the presence of the UCS5 containing fragment (110 bp). Thus, an in phase fusion was n ot required for expression. This is the first report that, concomitant with a selectable marker gene, foreign DNA can be integrated in a lin ear plasmid via homologous recombination and more importantly, that in phase fusions are not required generally for expression of heterologo us genes in linear plasmids. (C) 1994 Academic Press, Inc.