Development of an efficient gene-targeting system in Aspergillus luchuensis by deletion of the non-homologous end joining system.

The industrial fungus Aspergillus luchuensis is used to produce a distilled spirit in Okinawa Island, Japan. Recently, the genome sequence of A. luchuensis RIB2604 (Aspergillus awamori NBRC 4314) was revealed and many functional genes are now expected to be analyzed. Gene targeting is necessary for analyzing the function of a gene; however, gene targeting frequencies in A. luchuensis are very low. To develop a highly efficient gene-targeting system for A. luchuensis, we disrupted A. luchuensis ligD (ALligD) encoding the human DNA ligase IV (ligIV) homologue using an Agrobacterium mediated gene transformation method. Deletion of ALligD dramatically improved homologous recombination efficiency (reached 100%) compared to that in the wild-type strain (0.8%), when 1000-bp homologous flanking regions were used. The ALligD disruptant showed no apparent defect in vegetative growth, and it exhibited increased sensitivity to phleomycin and high methyl methanesulphonate concentrations compared to the wild-type strain. Furthermore, using this ALligD disruptant, we disrupted ALpksP encoding an Aspergillus fumigatus polyketide synthase P (alb1/pksP) orthologue. The ALpksP disruptant displayed a decolourized conidial phenotype. This result indicated that ALpksP is a key factor for conidial black pigmentation in A. luchuensis. Our results indicate that the ALligD mutant is an efficient host for targeted gene disruption in A. luchuensis.

Takahashi T ，Mizutani O ，Shiraishi Y ，Yamada O ... -  《-》

A defect of LigD (human Lig4 homolog) for nonhomologous end joining significantly improves efficiency of gene-targeting in Aspergillus oryzae.

Mizutani O ，Kudo Y ，Saito A ，Matsuura T ，Inoue H ，Abe K ，Gomi K ... -  《-》

Efficient gene targeting in ligase IV-deficient Monascus ruber M7 by perturbing the non-homologous end joining pathway.

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.

He Y ，Shao Y ，Chen F 《Fungal Biology》

Agrobacterium tumefaciens-mediated transformation of Aspergillus fumigatus: an efficient tool for insertional mutagenesis and targeted gene disruption.

Sugui JA ，Chang YC ，Kwon-Chung KJ 《-》

Deletion of ligD significantly improves gene targeting frequency in the lignocellulolytic filamentous fungus Penicillium oxalicum.

To improve the gene targeting frequency (GTF) in the lignocellulolytic filamentous fungus Penicillium oxalicum HP7-1, the non-homologous end-joining (NHEJ) gene ligD was deleted. The obtained PoligD deletion mutant ΔPoligD showed no apparent defect in cellulase production, growth rate, and sensitivity towards osmotic stress and mutagen ethyl methanesulphonate (EMS), while increased sensitivity to high concentrations of methyl methanesulfonate (MMS). Deletion of PoligD gene resulted in significantly increased GTFs at three different loci in P. oxalicum, which are even higher than those in Poku70 deletion mutant. The GTF in ΔPoligD at PoargB (reached 97 %) and PoagaA (reached 90 %) loci increased 5.1- and 1.2-fold compared with that in wild-type strain (WT), while at the Podpp4 locus GTF was up to 27 % in ΔPoligD but close to 0 % in WT, with 0.5 kb homologous flanking regions. Furthermore, the argB and agaA nutritional selection in P. oxalicum was demonstrated and the PoargB and PoagaA genes could be used as selective markers in this fungus. Thus, the PoligD deletion mutant can be an important tool for the functional analysis of genes in P. oxalicum.

Qin X ，Li R ，Luo X ，Lin Y ，Feng JX ... -  《Fungal Biology》