title: RNA-silencing in Penicillium chrysogenum and Acremonium chrysogenum: validation studies using beta-lactam genes expression.
abstract: In this work we report the development and validation of a new RNA interference vector (pJL43-RNAi) containing a double-stranded RNA expression cassette for gene silencing in the filamentous fungi Penicillium chrysogenum and Acremonium chrysogenum. Classical targeted gene disruption in these fungi is very laborious and inefficient due to the low frequency of homologous recombination. The RNAi vector has been validated by testing the attenuation of two different genes of the beta-lactam pathway; pcbC in P. chrysogenum and cefEF in A. chrysogenum. Quantification of mRNA transcript levels and antibiotic production showed knockdown of pcbC and cefEF genes in randomly isolated transformants of P. chrysogenum and A. chrysogenum, respectively. The process is efficient; 15 to 20% of the selected transformants were found to be knockdown mutants showing reduced penicillin or cephalosporin production. This new RNAi vector opens the way for exploring gene function in the genomes of P. chrysogenum and A. chrysogenum.
authors.affiliation: Institute of Biotechnology (INBIOTEC), Avda. Real no. 1, 24006 León, Spain.
authors.forename: Ricardo V
authors.initials: RV
authors.lastname: Ullán
authors.forename: Ramiro P
authors.initials: RP
authors.lastname: Godio
authors.forename: Fernando
authors.initials: F
authors.lastname: Teijeira
authors.forename: Inmaculada
authors.initials: I
authors.lastname: Vaca
authors.forename: Carlos
authors.initials: C
authors.lastname: García-Estrada
authors.forename: Raúl
authors.initials: R
authors.lastname: Feltrer
authors.forename: Katarina
authors.initials: K
authors.lastname: Kosalkova
authors.forename: Juan F
authors.initials: JF
authors.lastname: Martín
mesh_terms: D000164:Acremonium / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D002511:Cephalosporins / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D005821:Genetic Techniques; D009154:Mutation; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism; D010406:Penicillins / Q000378:metabolism*; D010408:Penicillium chrysogenum / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic; D047090:beta-Lactams / Q000378:metabolism
chemical_list: D002511:Cephalosporins; D005656:Fungal Proteins; D010406:Penicillins; D012330:RNA, Double-Stranded; D047090:beta-Lactams; D010088:Oxidoreductases; C038370:isopenicillin N synthetase