Tools for advanced and targeted genetic manipulation of the β-lactam antibiotic producer Acremonium chrysogenum.
Acremonium chrysogenum is the major producer of the β-lactam antibiotic cephalosporin C and therefore of great importance for the pharmaceutical industry. However, this filamentous fungus is known to reproduce solely by asexual means, shows only sporadic conidiospore production, and has gradual fragmentation of the vegetative mycelium into arthrospores. Due to these peculiar growth characteristics and life style, strain improvement by recombinant technologies is much more challenging than for other biotechnologically relevant fungi. Here, we describe several molecular tools for genetic engineering of A. chrysogenum, including a ΔAcku70 deletion strain for homologous recombination. No physiological or morphological changes occurred due to deletion of the ku70 gene or integration of the nat1 cassette in this recipient strain. We also used a xylose-inducible promoter from Sordaria macrospora (Smxyl) to demonstrate induction of the gfp reporter gene in A. chrysogenum. The Smxyl promoter was used for construction of a vector molecule to develop a one-step FLP/FRT recombination system in A. chrysogenum. This system was then used in the ΔAcku70 deletion strain to construct a marker-free recipient strain for targeted DNA insertion into genomic DNA. The applicability of our tools was demonstrated by construction of a marker-free transgenic strain, lacking any foreign genes.
Christian Doppler Laboratory for Fungal Biotechnology, Universitätsstraße 150, 44780 Bochum, Germany; Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, Universitätsstraße 150, 44780 Bochum, Germany.
S
S
Bloemendal
Christian Doppler Laboratory for Fungal Biotechnology, Universitätsstraße 150, 44780 Bochum, Germany; Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, Universitätsstraße 150, 44780 Bochum, Germany.
D
D
Löper
Christian Doppler Laboratory for Fungal Biotechnology, Universitätsstraße 150, 44780 Bochum, Germany; Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, Universitätsstraße 150, 44780 Bochum, Germany.
D
D
Terfehr
Christian Doppler Laboratory for Fungal Biotechnology, Universitätsstraße 150, 44780 Bochum, Germany; Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, Universitätsstraße 150, 44780 Bochum, Germany.
K
K
Kopke
Christian Doppler Laboratory for Fungal Biotechnology, Universitätsstraße 150, 44780 Bochum, Germany; Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, Universitätsstraße 150, 44780 Bochum, Germany.
J
J
Kluge
Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, Universitätsstraße 150, 44780 Bochum, Germany.
I
I
Teichert
Christian Doppler Laboratory for Fungal Biotechnology, Universitätsstraße 150, 44780 Bochum, Germany; Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, Universitätsstraße 150, 44780 Bochum, Germany. Electronic address: ulrich.kueck@rub.de.
U
U
Kück
D000164:Acremonium / Q000235:genetics* / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D017353:Gene Deletion; D005786:Gene Expression Regulation; D018390:Gene Targeting; D005818:Genetic Engineering; D011401:Promoter Regions, Genetic; D047090:beta-Lactams / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D047090:beta-Lactams
Acremonium chrysogenum; Cephalosporin C; FLP/FRT recombination system; Inducible promoter; Targeted insertion of foreign DNA