title: Improved gene targeting frequency in marine-derived filamentous fungus Aspergillus glaucus by disrupting ligD.
abstract: Filamentous fungi from the marine environment have shown great potential as cell factories for the production of pharmacologically active metabolites, but extremely low frequency of homologous recombination brings difficulty to further molecular biology studies. To bypass this problem and develop a highly efficient gene targeting system in marine-derived filamentous fungus Aspergillus glaucus, LigD, a homolog of Neurospora crassa Mus-53 which is considered to play a significant role in nonhomologous end joining (NHEJ), was coloned and deleted, and frequency of targeted gene replacement (TGR) increased dramatically from <2% to 85% in comparison with that in the wild type, when containing 1000 bp of homologous flanking sequence. Such results strongly indicate that AgLigD is indeed involved in the repair of NHEJ in A. glaucus and functions in this pathway. Furthermore, the AgLigD-defective mutant has no discernible differences with wild type regarding sensitivity to mutagens and UV, growth characteristics and transformation frequency. The AgligD-deficient transformant, as the first NHEJ-defective mutant in the field of marine-derived filamentous fungus, will help in expediting studies of molecular biology of marine-derived microorganisms.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, People's Republic of China.
authors.forename: Zhe
authors.initials: Z
authors.lastname: Fang
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
authors.forename: Menghao
authors.initials: M
authors.lastname: Cai
authors.forename: Jian
authors.initials: J
authors.lastname: Zhang
authors.forename: Yuanxing
authors.initials: Y
authors.lastname: Zhang
authors.forename: Xiangshan
authors.initials: X
authors.lastname: Zhou
mesh_terms: D059001:Aquatic Organisms / Q000166:cytology / Q000235:genetics* / Q000528:radiation effects; D001230:Aspergillus / Q000166:cytology / Q000187:drug effects / Q000235:genetics* / Q000528:radiation effects; D059766:DNA End-Joining Repair / Q000187:drug effects / Q000528:radiation effects; D018390:Gene Targeting / Q000379:methods*; D005800:Genes, Fungal / Q000235:genetics*; D056426:Genetic Loci / Q000235:genetics; D006918:Hydroxyurea / Q000633:toxicity; D008741:Methyl Methanesulfonate / Q000633:toxicity; D009154:Mutation / Q000235:genetics*; D010641:Phenotype; D012689:Sequence Homology, Nucleic Acid; D014170:Transformation, Genetic / Q000187:drug effects / Q000528:radiation effects; D014466:Ultraviolet Rays
chemical_list: D008741:Methyl Methanesulfonate; D006918:Hydroxyurea