Abstract
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.
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- 1.
The argB and nicB auxotrophic mutants are also pyrG − and therefore the growth medium for these strains needs to be supplemented with uridine.
- 2.
A small sample of PCR fragments is routinely analyzed for purity and size. Optional is to confirm PCR product integrity by restriction analysis or sequencing.
- 3.
YE is added to a final concentration of 0.003 % to stimulate germination of A. niger. On MM + starch without YE, the wt strain also does not germinate very well.
- 4.
The split marker fragments are not purified from gel and template DNA (pyrG, hygB, Ble, argB, and nicB genes, respectively) used for amplification of the split marker might remain present in the next steps. We therefore include control transformations with both split markers separately. As no transformants are obtained in the transformation with only one flank (data not shown), the purification of the split marker fragment is not required, but is optional.
- 5.
Only the sporulating transformants on the phleomycin transformation plate (see Fig. 25.4) can grow on MM + phleomycin. The non-sporulating transformants do not grow, and are probably transient transformants, in which the split marker fragments have not integrated into the genome.
- 6.
The percentages of HR for the amyR gene are very high (41 % for wt, 98 % for Δku70). Usually we find 5–10 % HR for wt, and 80–100 % for Δku70 (Meyer et al. 2007).
References
Arentshorst M, Ram AFJ, Meyer V (2012) Using non-homologous end-joining-deficient strains for functional gene analyses in filamentous fungi. Methods Mol Biol 835:133–150
Bos CJ, Debets AJ, Swart K, Huybers A, Kobus G (1988) Genetic analysis and the construction of master strains for assignment of genes to six linkage groups in Aspergillus niger. Curr Genet 14:437–443
Buxton FP, Gwynne DI, Davies RW (1985) Transformation of Aspergillus niger using the argB gene of Aspergillus nidulans. Gene 37:207–214
Carvalho ND, Arentshorst M, Kwon MJ, Meyer V, Ram AFJ (2010) Expanding the ku70 toolbox for filamentous fungi: establishment of complementation vectors and recipient strains for advanced gene analyses. Appl Microbiol Biotechnol 87:1463–1473
De Ruiter-Jacobs YM, Broekhuijsen M, Unkles SE, Campbell EI, Kinghorn JR, Contreras R, Pouwels PH, van den Hondel CAMJJ (1989) A gene transfer system based on the homologous pyrG gene and efficient expression of bacterial genes in Aspergillus oryzae. Curr Genet 16:159–163
Fairhead C, Llorente B, Denis F, Soler M, Dujon B (1996) New vectors for combinatorial deletions in yeast chromosomes and for gap-repair cloning using ‘split-marker’ recombination. Yeast 12:1439–1457
Goswami RS (2012) Targeted gene replacement in fungi using a split-marker approach. Methods Mol Biol 835:255–269
Kuck U, Hoff B (2010) New tools for the genetic manipulation of filamentous fungi. Appl Microbiol Biotechnol 86:51–62
Lenouvel F, van de Vondervoort PJ, Visser J (2002) Disruption of the Aspergillus niger argB gene: a tool for transformation. Curr Genet 41:425–432
Meyer V (2008) Genetic engineering of filamentous fungi—progress, obstacles and future trends. Biotechnol Adv 26:177–185
Meyer V, Arentshorst M, El-Ghezal A, Drews AC, Kooistra R, van den Hondel CAMJJ, Ram AFJ (2007) Highly efficient gene targeting in the Aspergillus niger kusA mutant. J Biotechnol 128:770–775
Nielsen ML, Albertsen L, Lettier G, Nielsen JB, Mortensen UH (2006) Efficient PCR-based gene targeting with a recyclable marker for Aspergillus nidulans. Fungal Genet Biol 43:54–64
Ninomiya Y, Suzuki K, Ishii C, Inoue H (2004) Highly efficient gene replacements in Neurospora strains deficient for nonhomologous end-joining. Proc Natl Acad Sci U S A 101:12248–12253
Petersen KL, Lehmbeck J, Christensen T (1999) A new transcriptional activator for amylase genes in Aspergillus. Mol Gen Genet 262:668–676
Punt PJ, van den Hondel CAMJJ (1992) Transformation of filamentous fungi based on hygromycin B and phleomycin resistance markers. Methods Enzymol 216:447–457
Punt PJ, Oliver RP, Dingemanse MA, Pouwels PH, van den Hondel CAMJJ (1987) Transformation of Aspergillus based on the hygromycin B resistance marker from Escherichia coli. Gene 56:117–124
Van Hartingsveldt W, Mattern IE, van Zeijl CM, Pouwels PH, van den Hondel CAMJJ (1987) Development of a homologous transformation system for Aspergillus niger based on the pyrG gene. Mol Gen Genet 206:71–75
Verdoes JC, Punt PJ, van der Berg P, Debets F, Stouthamer AH, van den Hondel CAMJJ (1994) Characterization of an efficient gene cloning strategy for Aspergillus niger based on an autonomously replicating plasmid: cloning of the nicB gene of A. niger. Gene 146:159–165
Acknowledgments
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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Arentshorst, M., Niu, J., Ram, A.F.J. (2015). Efficient Generation of Aspergillus niger Knock Out Strains by Combining NHEJ Mutants and a Split Marker Approach. In: van den Berg, M., Maruthachalam, K. (eds) Genetic Transformation Systems in Fungi, Volume 1. Fungal Biology. Springer, Cham. https://doi.org/10.1007/978-3-319-10142-2_25
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DOI: https://doi.org/10.1007/978-3-319-10142-2_25
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