Homologous recombination in the antibiotic producer Penicillium chrysogenum: strain ΔPcku70 shows up-regulation of genes from the HOG pathway

Abstract

In Penicillium chrysogenum, the industrial producer of the beta-lactam antibiotic penicillin, generating gene replacements for functional analyses is very inefficient. Here, we constructed a recipient strain that allows efficient disruption of any target gene via homologous recombination. Following isolation of the Pcku70 (syn. hdfA) gene encoding a conserved eukaryotic DNA-binding protein involved in non-homologous end joining (NHEJ), a Pcku70 knockout strain was constructed using a novel nourseothricin-resistance cassette as selectable marker. In detailed physiological tests, strain DeltaPcku70 showed no significant reduction in vegetative growth due to increased sensitivity to different mutagenic substances. Importantly, deletion of the Pcku70 gene had no effect on penicillin biosynthesis. However, strain DeltaPcku70 exhibits higher sensitivity to osmotic stress than the parent strain. This correlated well with comparative data from microarray analyses: Genes related to the stress response are significantly up-regulated in the Pcku70 deletion mutant. To demonstrate the applicability of strain DeltaPcku70, three genes related to beta-lactam antibiotic biosynthesis were efficiently disrupted, indicating that this strain shows a low frequency of NHEJ, thus promoting efficient homologous recombination. Furthermore, we discuss strategies to reactivate Pcku70 in strains successfully used for gene disruptions.

Evidence for Dicer-dependent RNA interference in the industrial penicillin producer Penicillium chrysogenum

“…P. chrysogenum producer strains are known to contain 6 to 14 copies of the clustered penicillin biosynthesis genes (Fierro et al, 2006). We have recently demonstrated that DPcku70 strains can be used to delete single gene copies from the penicillin cluster; however, a simultaneous deletion of all amplified gene copies was impossible (Hoff et al, 2009). Using an alternative RNAi approach, Ullán et al (2008) demonstrated recently the successful expressional downregulation of the multi-copy penicillin biosynthesis genes.…”

“…This plasmid served as template to generate a 3.8 kb PCR fragment to construct a DPclaeA knockout strain. Plasmid pKOPcVELA_Nat is a derivative of pKOPcVELA in which the phleomycin-resistance gene has been substituted by the nourseothricin-resistance cassette (Hoff et al, 2009). …”

“…Fragments of about 1 kb of the 59-and 39-flanking regions of Pcdcl2 were amplified using primers Pcdcl2-5s and Pcdcl2-5a and Pcdcl2-3s and Pcdcl2-3a, respectively, with genomic DNA from P2niaD18 as template. After subcloning into pDrive (Qiagen), the MluI-BamHI 59-fragment and the XhoI-XbaI 39-fragment were ligated into the corresponding restriction sites of plasmid pD-Phleo (Hoff et al, 2009), expressing the ble gene under control of the trpC promoter of A. nidulans. The Pcdcl2 knockout cassette was amplified by PCR using primers Pcdcl2-5s and Pcdcl2-3a with plasmid pKOPcDCL2 as template.…”

“…The Pcdcl2 knockout cassette was amplified by PCR using primers Pcdcl2-5s and Pcdcl2-3a with plasmid pKOPcDCL2 as template. The resulting 2.8 kb PCR fragment was used for transformation of DPcku70 (Hoff et al, 2009) to generate DPcdcl2.…”

“…In order to confirm a functional hairpin-induced silencing process in P. chrysogenum, a Pcdcl2 disruption strain was generated by homologous recombination in a DPcku70 background (Hoff et al, 2009). A gene-replacement cassette was generated containing the phleomycin-resistance marker flanked on both sites by approximately 1 kb of 59 and 39 genomic sequences from the Pcdcl2 locus (Fig.…”