Abstract
In Penicillium chrysogenum, the industrial producer of the β-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 ΔPcku70 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 ΔPcku70 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 ΔPcku70, three genes related to β-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.
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Acknowledgements
We thank Kerstin Kalkreuter and Stefanie Mertens for their excellent technical assistance and Eva Szczypka and Gabriele Frenßen-Schenkel for the artwork. Furthermore, we thank Drs. Hubert Kürnsteiner, Ernst Friedlin, Rudolf Mitterbauer, Heiko Eichhorn and Thomas Specht for helpful discussion. This work was funded by the Sandoz GmbH (Kundl, Austria) and the Christian Doppler Society (Vienna, Austria).
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Fig. 1S
Comparison of the Penicillium chrysogenum (Pc) Pcku70 protein with homologues of Aspergillus fumigatus (Af; XM_748769), N. crassa (Nc; BAD16622), Sordaria macrospora (Sm; AM117808) and human (Hs; P12956) (GIF 226 kb)
Fig. 2S
Phenotype of strains P2niaD18 and ΔPcku70 grown on minimal media supplemented with 0.4 M up to 1.2 M NaCl or KCl (GIF 143 kb)
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Hoff, B., Kamerewerd, J., Sigl, C. et al. Homologous recombination in the antibiotic producer Penicillium chrysogenum: strain ΔPcku70 shows up-regulation of genes from the HOG pathway. Appl Microbiol Biotechnol 85, 1081–1094 (2010). https://doi.org/10.1007/s00253-009-2168-4
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DOI: https://doi.org/10.1007/s00253-009-2168-4