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Ku70 and ku80 null mutants improve the gene targeting frequency in Monascus ruber M7
Yi He1, Qingpei Liu, Yanchun Shao
1College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei Province, People's Republic of China.
Abstract:
Normally, gene targeting by homologous recombination occurs rarely during a transformation process since non-homologous recombination is predominant in filamentous fungi. In our previous researches, the average gene replacement frequency (GRF) in Monascus ruber M7 was as low as 15 %. To develop a highly efficient gene targeting system for M. ruber M7, two M. ruber M7 null mutants of ku70 (MrΔku70) and ku80 (MrΔku80) were constructed which had no apparent defects in the development including vegetative growth, colony phenotype, microscopic morphology and spore yield compared with M. ruber M7. In addition, the production of some significant secondary metabolites such as pigments and citrinin had no differences between the two disruptants and the wild-type strain. Further results revealed that the GRFs of triA (encoding a putative acetyltransferase) were 42.2 % and 61.5 % in the MrΔku70 and MrΔku80 strains, respectively, while it was only about 20 % in M. ruber M7. Furthermore, GRFs of these two disruptants at other loci (the pigE, fmdS genes in MrΔku70 and the ku70 gene in MrΔku80) were investigated, and the results indicated that GRFs in the MrΔku70 strain and the MrΔku80 strain were doubled and tripled compared with that in M. ruber M7, respectively. Therefore, the ku70 and ku80 null mutants of M. ruber M7, especially the ku80-deleted strain, will be excellent hosts for efficient gene targeting.
Insights
Gene targeting efficiency in filamentous fungi was significantly improved by creating ku70 and ku80 null mutants. The ku80-deleted strain of Monascus ruber M7 shows the highest gene replacement frequency, making it an excellent host for genetic engineering.
Area of Science:
- Molecular Biology
- Mycology
- Genetic Engineering
Background:
- Homologous recombination is inefficient in filamentous fungi like Monascus ruber M7 due to predominant non-homologous recombination.
- Previous studies reported low average gene replacement frequencies (GRF) of approximately 15% in M. ruber M7.
Purpose of the Study:
- To develop a highly efficient gene targeting system for M. ruber M7.
- To construct ku70 and ku80 null mutants to enhance gene replacement frequency.
Main Methods:
- Construction of ku70 (MrΔku70) and ku80 (MrΔku80) null mutants in M. ruber M7.
- Assessment of developmental characteristics and secondary metabolite production in the mutants.
- Measurement of gene replacement frequencies (GRFs) for various genes in wild-type and mutant strains.
Main Results:
- The MrΔku70 and MrΔku80 mutants exhibited no significant defects in growth, morphology, or spore yield compared to the wild-type.
- Secondary metabolite production (pigments, citrinin) remained unchanged in the disruptants.
- GRFs for the triA gene were 42.2% in MrΔku70 and 61.5% in MrΔku80, significantly higher than the ~20% in wild-type M. ruber M7.
- GRFs at other loci were doubled in MrΔku70 and tripled in MrΔku80 compared to the wild-type.
Conclusions:
- The ku70 and ku80 null mutants of M. ruber M7 are viable and do not impair essential developmental or metabolic functions.
- Deletion of ku70 and ku80 genes substantially enhances gene targeting efficiency via homologous recombination in M. ruber M7.
- The ku80-deleted strain is particularly promising as a superior host for efficient gene targeting in M. ruber M7.
