title: Efficient gene targeting in ligase IV-deficient Monascus ruber M7 by perturbing the non-homologous end joining pathway.
abstract: Inactivating the non-homologous end joining (NHEJ) pathway is a well established method to increase gene replacement frequency (GRF) in filamentous fungi because NHEJ is predominant for the repair of DNA double strand breaks (DSBs), while gene targeting is based on homologous recombination (HR). DNA ligase IV, a component of the NHEJ system, is strictly required for the NHEJ in Saccharomyces cerevisiae and Neurospora crassa. To enhance the GRF in Monascus ruber M7, we deleted the Mrlig4 gene encoding a homolog of N. crassa DNA ligase IV. The obtained mutant (MrΔlig4) showed no apparent defects in vegetative growth, colony phenotype, microscopic morphology, spore yield, and production of Monascus pigments and citrinin compared with the wild-type strain (M. ruber M7). Gene targeting of ku70 and triA genes revealed that GRF in the MrΔlig4 strain increased four-fold compared with that in the wild-type strain, reached 68 % and 85 %, respectively. Thus, the MrΔlig4 strain is a promising host for efficient genetic manipulation. In addition, the MrΔlig4 strain is more sensitive than M. ruber M7 to a DNA-damaging agent, methyl methanesulfonate.
authors.affiliation: Key Laboratory of Environment Correlative Dietology, Huazhong Agricultural University, Ministry of Education, Wuhan 430070, Hubei Province, PR China; College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei Province, PR China.
authors.forename: Yi
authors.initials: Y
authors.lastname: He
authors.affiliation: Key Laboratory of Environment Correlative Dietology, Huazhong Agricultural University, Ministry of Education, Wuhan 430070, Hubei Province, PR China; College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei Province, PR China.
authors.forename: Yanchun
authors.initials: Y
authors.lastname: Shao
authors.affiliation: Key Laboratory of Environment Correlative Dietology, Huazhong Agricultural University, Ministry of Education, Wuhan 430070, Hubei Province, PR China; Hubei Collaborative Innovation Center for Industrial Fermentation, Wuhan 430068, Hubei Province, PR China; College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei Province, PR China; The Food College of SHIHEZI University, Shihezi, 832003, Xinjiang Province, PR China. Electronic address: chenfs@mail.hzau.edu.cn.
authors.forename: Fusheng
authors.initials: F
authors.lastname: Chen
mesh_terms: D002953:Citrinin / Q000378:metabolism; D059766:DNA End-Joining Repair; D011088:DNA Ligases / Q000235:genetics / Q000378:metabolism*; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D017353:Gene Deletion; D018390:Gene Targeting / Q000379:methods*; D005827:Genetics, Microbial / Q000379:methods*; D008969:Molecular Sequence Data; D032741:Monascus / Q000166:cytology / Q000235:genetics* / Q000254:growth & development; D010860:Pigments, Biological / Q000378:metabolism; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA; D013172:Spores, Fungal / Q000254:growth & development
chemical_list: D004271:DNA, Fungal; D010860:Pigments, Biological; D002953:Citrinin; D011088:DNA Ligases
keywords: DNA ligase IV; Gene replacement frequency; Genetic manipulation; Homologous recombination; Mutagen sensitivity