Efficient integrative transformation of Cephalosporium acremonium.

Abstract

A hybrid gene, IPNSp/HPTorf, was constructed by placing an 850 bp sequence of Cephalosporium acremonium DNA next to the 5' end of a bacterial open reading frame, HPTorf. The sequence was obtained as an 850 bp NcoI restriction fragment from the 5' non-coding region of the C. acremonium isopenicillin N synthetase (IPNS) gene. The HPTorf was obtained from a bacterial gene that coded for a hygromycin B phosphotransferase (HPT). Plasmids that contained IPNSp/HPTorf transformed C. acremonium to a stably maintained hygromycin B resistant phenotype. Southern analysis of total DNA from transformants demonstrated multiple integrations of the transforming DNA in the high molecular weight DNA of most transformants, but single integrations were observed in a few transformants. The number of transformants per microgram of DNA was about 100 times greater than for plasmids that contained the HPTorf without any juxtaposed eucaryotic promoter sequence. Plasmids with the promoterless HPTorf and plasmids with a truncated S. cerevisiae phosphoglycerate kinase promoter juxtaposed to the HPTorf transformed C. acremonium at equivalent low frequencies. Transformation of C. acremonium with linearized plasmid DNA produced at least 2-3 fold more transformants than the corresponding circular molecule. Several observations were made concerning protoplast formation and handling which made the transformation procedure more efficient and allowed a greater proportion of protoplasts to regenerate to viable walled cells. Plasmids were constructed that contained both the IPNSp/HPTorf and additional elements: fragments of C. acremonium ribosomal DNA (rDNA), or a fragment of C. acremonium mitochondrial DNA possessing activity as an autonomous replication sequence (ARS) in S. cerevisiae, or putative transcriptional termination/polyadenylation signals from the IPNS gene. These plasmids transformed C. acremonium at frequencies experimentally equivalent to those containing IPNSp/HPTorf without any of these additional elements.