Efficient Generation of Aspergillus niger Knock Out Strains by Combining NHEJ Mutants and a Split Marker Approach
To generate gene deletion mutants in Aspergillus niger, we combined the use of nonhomologous end-joining (NHEJ) mutants (ku70 mutant) and the split marker approach. The combination of both tools resulted in efficient PCR amplification because of the reduced length of the PCR fragments and efficient homologous recombination frequencies. A set of five selection markers, two dominant selection markers (hph; hygromycin B resistance and BLE; phleomycin resistance) and three auxotrophic markers (pyrG, argB, and nicB) were successfully used in a split marker approach to obtain amyR knock outs with high efficiency. AmyR encodes a transcription factor that is required for the expression of starch degrading enzymes and disruption of amyR results in the inability to grow on starch. The strategy to generate the gene deletion constructs is such that with one set of four gene-specific primers, a gene deletion mutant can be generated with either one of the five selection markers. The strategy is based on fusion PCR and omits the necessity for cloning the disruption cassettes. This accelerates the process of generating gene deletion cassettes which can now be accomplished within eight hours. The split marker approach can also be used to make gene deletions in a wild-type background instead of a Δku70 background. In this chapter, we present protocols and considerations that we used to generate gene knock out constructs by fusion PCR and to obtain and verify gene knock outs with any of the five marker genes using the split marker approach. The method is easily transferable to other filamentous fungi.
Keywordsku70 Gene targeting Homologous recombination Nicotinamide auxotrophy Uracil auxotrophy Arginin auxotrophy Phleomycin resistance Hygromycin resistance
Jing Niu was supported by a grant from the China Scholarship Council. The research group of A.F.J. Ram is part of the Kluyver Centre for Genomics of Industrial Fermentation which is supported by the Netherlands Genomics Initiative.
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