Homologous transformation of Cephalosporium acremonium with the nitrate reductase-encoding gene (niaD)

We report the development of a homologous transformation system for Cephalosporium acremonium using the niaD gene of the nitrate assimilation (NA) pathway. Mutants in the NA pathway were selected on the basis of chlorate resistance by conventional means. Screening procedures were developed to differentiate between nitrate reductase apoprotein structural gene mutants (niaD) and molybdenum cofactor gene mutants (cnx) as wt C. acremonium, unlike most filamentous fungi, fails to grow on minimal medium with hypoxanthine as a sole source of nitrogen. Phage clones carrying the niaD gene were isolated from a C. acremonium library constructed in lambda EMBL3 using the A. nidulans niaD gene as a heterologous probe. An 8.6-kb EcoRI fragment was subcloned into pUC18, and designated pSTA700. pSTA700 was able to transform stable niaD mutants to NA at a frequency of up to 40 transformants per microgram DNA. Transformants were easily visible since the background growth was low and no abortives were observed. Gene replacements, single copy homologous integration and complex multiple integrations were observed. The niaD system was used to introduce unselected markers for hygromycin B resistance and benomyl resistance into C. acremonium by cotransformation.