A novel homologous dominant selection marker for genetic transformation of Penicillium chrysogenum: overexpression of squalene epoxidase-encoding ergA.
Genetic engineering requires genetic selection markers. For generation of biosafe strains in industrial applications, homologous dominant selection markers allowing "self-cloning" are best suited but scarce. Here we describe a novel homologous dominant genetic selection system for the filamentous fungus Penicillium chrysogenum based on overexpression of the P. chrysogenum squalene epoxidase-encoding ergA gene, which confers resistance against terbinafine. Terbinafine (TRB) is a potent antifungal drug used in therapy of fungal infections. Overexpression of ergA was driven by the P. chrysogenum endoxylanase xylP promoter that is highly inducible by xylose. The suitability of the novel selection marker cassette for genetic manipulation was proven by its use for targeted deletion of the transcription factor nosA in P. chrysogenum. NosA-deficiency did not affect growth rates on solid or in liquid media, conidiation in light or darkness, and resistance to hydrogen peroxide. However, NosA-deficiency significantly decreased penicillin productivity. As TRB inhibits the growth of a variety of fungal species, this novel selection marker is expected to be suitable for genetic engineering of diverse fungal species.
Sigl
Claudia
C
SANDOZ GmbH, Biochemiestrasse 10, 6252 Kundl, Austria.
Handler
Monika
M
Sprenger
Georg
G
Kürnsteiner
Hubert
H
Zadra
Ivo
I
D000935:Antifungal Agents / Q000494:pharmacology; D015139:Blotting, Southern; D025141:Drug Resistance, Fungal; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D005819:Genetic Markers / Q000235:genetics*; D005947:Glucose / Q000378:metabolism; D009281:Naphthalenes / Q000494:pharmacology; D010404:Penicillin V / Q000378:metabolism; D010408:Penicillium chrysogenum / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D050603:Squalene Monooxygenase / Q000235:genetics* / Q000378:metabolism; D000077291:Terbinafine; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic / Q000235:genetics*; D014994:Xylose / Q000378:metabolism
D000935:Antifungal Agents; D005656:Fungal Proteins; D005819:Genetic Markers; D009281:Naphthalenes; D014157:Transcription Factors; D014994:Xylose; D050603:Squalene Monooxygenase; D000077291:Terbinafine; D005947:Glucose; D010404:Penicillin V