A new vector for efficient gene targeting to the pyrG locus in Aspergillus niger
© Arentshorst et al.; licensee BioMed Central. 2015
Received: 13 February 2015
Accepted: 26 February 2015
Published: 14 March 2015
The possibility for efficient gene targeting for the controlled integration of DNA constructs is an important tool in fungal genetics.
In this study, we report a new targeting vector based on the pyrG marker in Aspergillus niger. The DNA sequence to be targeted is surrounded by two fragments of the pyrG gene to allow homologous recombination of the recombinant DNA at the pyrG locus. The 5’ end of the targeting cassette contains a non-functional truncated pyrG open reading frame (first 112 bases deleted) and the 3’ untranslated region (3’ UTR). At the 3’ end, the targeting cassette consists of the 3’ flanking region of the pyrG gene. A unique NotI site between the flanks allows the insertion of a gene of interest. The linearized targeting cassette is transformed to the A. niger pyrG mutant strain AB4.1 or a derivative thereof. By using a constitutively expressed luciferase reporter gene (mluc) as an example, it is shown that the targeting system is efficient as 4 out of 6 (67%) AB4.1 transformants and 51 out of 66 (77%) MA169.4 (ku70 − ) transformants contained the reporter gene at the pyrG locus. A luciferase (lux) activity assay, performed with independently obtained transformants in which the mluc reporter was integrated at the pyrG locus, showed comparable and reproducible lux activities.
The new pyrG targeting vector is an important improvement to the existing method for gene targeting in A. niger. Although the vector is specific for A. niger, the presented design and approach is easily applicable for constructing integration vectors for other fungi.
However, there are certain drawbacks of using the pyrG* method. Transformation frequencies are on average 10 to 20 times lower compared to regular pyrG transformations, often resulting in only one or two transformants per transformation. In addition, the percentage of successful integration of the reporter construct, varying between 10 to 50%, is rather low, probably due to a recombination or repair event that restores the mutation in the pyrG gene in AB4.1. Another disadvantage of the pyrG* system is that after targeted integration, two pyrG repeats are present around the construct, making the loss of the reporter construct via a loop out event possible (Figure 1). Finally, despite the well established positive effect on gene targeting efficiencies in ku70 mutants [7,8], we noticed that deletion of the ku70 gene did not improve transformation or targeted integration frequencies of the pyrG* plasmid. The reason for this is not known and systematic studies to analyse homologous integration using circular (pyrG*) or linear DNA fragments in wild type or ku70 strains to clarify this have not been reported. These drawbacks make the construction of A. niger strains with targeted integration, such as of reporter constructs, a time consuming and laborious exercise.
Strains used in this study
derivative of N400
UV mutant of N402
cspA1, pyrG378, kusA::DR-amdS-DR
ku70 disruption in AB4.1
cspA1, PgpdA-mluc-TtrpC on pyrG locus
PgpdA-mluc-TtrpC on pyrG locus in AB4.1
cspA1, kusA::DR-amdS-DR, PgpdA-mluc-TtrpC on pyrG locus
PgpdA-mluc-TtrpC on pyrG locus in MA169.4
Primers used in this study
CGGAATTCGG CGCGCCCGGC TGACGTTACC ACCACT*
pyrG-3’ UTR PCR (Figure 2A)
AAGGAAAAAA GCGGCCGCAG TCAGACCTAA TGCCTCGGG
pyrG-3’ UTR PCR (Figure 2A)
AAGGAAAAAA GCGGCCGCCG TCGCGTGATA AGGGTTG
3’ pyrG PCR (Figure 2A)
CGAGCTCGGC GCGCCTCGGG TCAATTTCCT CTGTTG
3’ pyrG PCR (Figure 2a)
ATTTGCGGCC GCAGAACGCC GAGAAGAACT GG
PgpdA-mluc PCR (Figure 2A)
AAGGAAAAAA GCGGCCGCTC TAGAAAGAAG GATTACCTC
PgpdA-mluc PCR (Figure 2A)
In the pyrG** system, the reporter construct has to integrate at the pyrG locus via homologous integration. In order to test the performance of the pyrG** system in a ku70 −background, strain MA169.4 (pyrG − , ku70 − ) was transformed with 10 μg of linear DNA, isolated by digestion of plasmid pMA349 with AscI. In total 66 primary transformants were obtained and purified. These transformants were analysed for their luciferase activity in a lux assay (data not shown), resulting in 51 out of 66 (77%) strains that show lux activity. Southern blot analysis (data not shown) of 15 selected strains showed that 13 strains, which showed comparable lux activities in the lux assay, contain the lux reporter construct at the pyrG locus. In the other two strains, that were negative in the lux assay, the reporter construct was not present. No ectopic integrations were detected in these 15 transformants.
In the experiments described above, the results indicate that the pyrG** targeting system is both useful in wild-type and ku70 − strains, even though the transformation frequency in the wild-type strain is much lower. It is likely that this lower frequency of transformation is due to the ectopic integration of the cassette, which does not result in transformants as this integration does not reconstitute the pyrG gene.
The new targeting system has recently been successfully used in two independent studies in our laboratory. In the first study (A-M Burggraaf-van Welzen, unpublished results) 4 different ku70 − strains and 2 different reporter genes were used. Out of 28 transformants analysed, 23 transformants contained the reporter construct at the pyrG locus (82%). In the second study (J. Niu, unpublished results), a Δku70 strain was transformed with 6 different reporter constructs. Out of 122 primary transformants analysed, 105 transformants contained the reporter construct (86%). Southern analysis of 24 transformants (four of each construct) confirmed integration at the pyrG locus in all transformants analysed. These studies further confirm the efficiency and ease at which transformants with targeted integrations are obtained.
The described method to obtain transformants with targeted integration is not restricted to pyrG mutants, but can also be used for other auxotrophic markers. Prerequisite is that the mutation in the auxotrophic marker is determined to allow design of the targeting cassette.
Availability of supporting data
The data set supporting the results of this article is included within the article. Plasmids and plasmids maps and DNA sequences are deposited at Fungal Genetics Stock Centre.
We thank Anne-Marie Burggraaf-van Welzen, Jiachen Cui and Jing Niu for sharing unpublished results. We thank Peter Punt and Cees van den Hondel for helpful discussions. The plasmids pMA334 and pMA349 are deposited at Fungal Genetics Stock Centre (www.fgsc.net) and we thank the FGSC for making them available.
- van Hartingsveldt W, Mattern IE, van Zeijl CM, Pouwels PH, van den Hondel CAM. Development of a homologous transformation system for Aspergillus niger based on the pyrG gene. Mol Gen Genet. 1987;206(1):71–5.View ArticlePubMedGoogle Scholar
- Osmani AH, Oakley BR, Osmani SA. Identification and analysis of essential Aspergillus nidulans genes using the heterokaryon rescue technique. Nat Protoc. 2006;1:2517–26.View ArticlePubMedGoogle Scholar
- d’Enfert C. Selection of multiple disruption events in Aspergillus fumigatus using the orotidine-5’-decarboxylase gene, pyrG, as a unique transformation marker. Curr Genet. 1996;30:76–82.View ArticlePubMedGoogle Scholar
- Boeke JD, LaCroute F, Fink GR. A positive selection for mutants lacking orotidine-5’-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet. 1984;197:345–6.View ArticlePubMedGoogle Scholar
- Arentshorst M, Ram AFJ, Meyer V. Using non-homologous end-joining-deficient strains for functional gene analyses in filamentous fungi. Methods Mol Biol. 2012;835:133–50.View ArticlePubMedGoogle Scholar
- de Ruiter-Jacobs YM, Broekhuijsen M, Unkles SE, Campbell EI, Kinghorn JR, Contreras R, et al. A gene transfer system based on the homologous pyrG gene and efficient expression of bacterial genes in Aspergillus oryzae. Curr Genet. 1989;6:159–63.View ArticleGoogle Scholar
- Meyer V, Arentshorst M, El-Ghezal A, Drews AC, Kooistra R, van den Hondel CAM, et al. Highly efficient gene targeting in the Aspergillus niger kusA mutant. J Biotechnol. 2007;128:770–5.View ArticlePubMedGoogle Scholar
- Carvalho NDSP, Arentshorst M, Kwon MJ, Meyer V, Ram AFJ. Expanding the ku70 toolbox for filamentous fungi: establishment of complementation vectors and recipient strains for advanced gene analyses. Appl Microbiol Biotechnol. 2010;87:1463–73.View ArticlePubMed CentralPubMedGoogle Scholar
- van Gorcom RFM, van den Hondel CAM. Expression analysis vectors for Aspergillus niger. Nucleic Acids Res. 1988;16:9052.View ArticlePubMed CentralPubMedGoogle Scholar
- Damveld RA, van Kuyk PA, Arentshorst M, Klis FM, van den Hondel CAM, Ram AFJ. The Aspergillus niger MADS-box transcription factor RlmA is required for cell wall reinforcement in response to cell wall stress. Fungal Genet Biol. 2005;42:165–77.View ArticlePubMedGoogle Scholar
- Carvalho NDSP, Arentshorst M, Kooistra R, Stam H, Sagt CM, van den Hondel CAM, et al. Effects of a defective ERAD pathway on growth and heterologous protein production in Aspergillus niger. Appl Microbiol Biotechnol. 2011;89:357–73.View ArticlePubMed CentralPubMedGoogle Scholar
- Meyer V, Wanka F, van Gent J, Arentshorst M, van den Hondel CAM, Ram AFJ. Fungal gene expression on demand: an inducible, tunable, and metabolism-independent expression system for Aspergillus niger. Appl Environ Microbiol. 2011;77:2975–83.View ArticlePubMed CentralPubMedGoogle Scholar
- Carvalho NDSP, Arentshorst M, Weenink XO, Punt PJ, van den Hondel CAM, Ram AFJ. Functional YFP tagging of the essential GDP-mannose transporter reveals an important role for the secretion related small GTPase SrgC protein in maintenance of Golgi bodies in Aspergillus niger. Fungal Biol. 2011;115:253–64.View ArticlePubMedGoogle Scholar
- Bos CJ, Debets AJ, Swart K, Huybers A, Kobus G, Slakhorst SM. Genetic analysis and the construction of master strains for assignment of genes to six linkage groups in Aspergillus niger. Curr Genet. 1988;14:437–43.View ArticlePubMedGoogle Scholar
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