- Methodology article
- Open Access
HisB as novel selection marker for gene targeting approaches in Aspergillus niger
© The Author(s). 2017
- Received: 8 October 2016
- Accepted: 17 February 2017
- Published: 8 March 2017
For Aspergillus niger, a broad set of auxotrophic and dominant resistance markers is available. However, only few offer targeted modification of a gene of interest into or at a genomic locus of choice, which hampers functional genomics studies. We thus aimed to extend the available set by generating a histidine auxotrophic strain with a characterized hisB locus for targeted gene integration and deletion in A. niger.
A histidine-auxotrophic strain was established via disruption of the A. niger hisB gene by using the counterselectable pyrG marker. After curing, a hisB - , pyrG - strain was obtained, which served as recipient strain for further studies. We show here that both hisB orthologs from A. nidulans and A. niger can be used to reestablish histidine prototrophy in this recipient strain. Whereas the hisB gene from A. nidulans was suitable for efficient gene targeting at different loci in A. niger, the hisB gene from A. niger allowed efficient integration of a Tet-on driven luciferase reporter construct at the endogenous non-functional hisB locus. Subsequent analysis of the luciferase activity revealed that the hisB locus is tight under non-inducing conditions and allows even higher luciferase expression levels compared to the pyrG integration locus.
Taken together, we provide here an alternative selection marker for A. niger, hisB, which allows efficient homologous integration rates as well as high expression levels which compare favorably to the well-established pyrG selection marker.
- Aspergillus niger
- Gene expression
- Selection marker
The filamentous fungus Aspergillus niger is an industrially exploited cell factory with a broad product portfolio including primary metabolites, proteins and enzymes . Recent data proved that A. niger can also serve as a suitable host for secondary metabolite production [2, 3]. Additionaly, A. niger is a model system used to study fundamental molecular and cellular processes. Various selection systems are available for transformation of A. niger, including nutritional (pyrG, trpC, amdS, niaD, sC, agaA and argB) and antibiotic resistance (hph, ble) markers [4–11]. Recently this set was expanded by two new nutritional markers (nicB and adeA) which can be used for gene deletion in A. niger . However, in order to study the function and interplay of several genes, or to construct/re-engineer a complete metabolic pathway in A. niger, it is of advantage having a range of selection markers at hand to choose the best one suited for a given approach. The number of nutritional markers for A. niger is limited to seven, as recently published  and although marker recycling using the Cre/loxP system has been established for Aspergilli [13, 14], they often suffer from poor recombination events ranging from 5 to 20%. So far, within the set of nutritional selection markers available for A. niger, only the pyrG and agaA loci [4, 15, 16] meet the need of a well characterized locus for efficient homologous integration of single copy expression cassettes. In fungi, a transcriptionally active, non-protein encoding locus that is targeted by exogenous expression cassettes at high frequency has been an important molecular technique underlying transformation, mutant complementation, and functional genomic approaches to study gene function . Even the highly efficient CRISPR/Cas system which has been applied recently for filamentous fungi [17–21], depends on well characterized loci for genomic integration of the genes of interest.
In order to establish an alternative auxotrophic selection marker for A. niger, which can also be used for both gene targeting or insertion of an expression cassette into a well characterised locus, we chose the histidine biosynthesis pathway as a target. This pathway was intensively studied in Salmonella typhimurium, Escherichia coli and Corynebacterium glutamicum (for reviews see [22, 23]) and in A. nidulans . It generates histidine in ten reaction steps catalysed by seven enzymes in a branched pathway out of phosphoribosyl pyrophosphate supplied via the pentose phosphate pathway. As it was shown that deletion of hisB in A. nidulans and A. fumigatus results in histidine auxotrophy [25, 26], we selected hisB, which catalyses the sixth step in the histidine biosynthesis pathway and disrupted it via a direct targeting approach in A. niger. Subsequent integration of the well-established Tet-on system  using luciferase as reporter gene enabled us to evaluate gene expression characteristics at the hisB locus in comparison to the widely used pyrG locus. We could furthermore demonstrate that genome editing using the hisB orthologue of A. nidulans is feasable.
Strains, growth conditions and molecular techniques
A. niger strains used in this work
kusA::DR-amdS-DR, pyrG − (AB4.1 derivative)
kusA::DR-amdS-DR, pyrG + , hisB::ThisB-AopyrG-ThisB (MA169.4 derivative)
kusA::DR-amdS-DR, pyrG - , hisB - (MF40.6 derivative)
kusA::DR-amdS-DR, hisB - pyrG + (MF41.3 derivative)
kusA::DR-amdS-DR, pyrG + , olvA::AnidhisB, (MF42.2 derivative)
kusA::DR-amdS-DR, pyrG + , olvA + , AnidhisB + (transformed with pSE1.6, MA42.2 derivative)
MA169.4 pyrG +
kusA::DR-amdS-DR, AopyrG + , olvA + (transformed with pAW34, MA169.4 derivative)
kusA::DR-amdS-DR, Tet-on (single copy) (MF42.2 derivative)
kusA::DR-amdS-DR, Tet - on -mluc (single copy) (MF42.2 derivative)
pyrG + , Tet - on (single copy) (AB4.1 derivative)
pyrG + , Tet - on -mluc (single copy) (AB4.1 derivative)
To obtain pyrG - strains via counterselection, 2 x 107 spores were plated on MM plates containing 75 mg/ml 5-Fluoroorotic acid (FOA), 10 mM uridine, 10 mM proline and 10 mM histidine. Plates were incubated at 30°C for 1-2 weeks until single colonies were visible. FOA-resistant mutants were purified on MM + FOA plates once and tested for their uridine auxothrophy on MM plates containing 10 mM histidine or 10 mM histidine and 10 mM uridine, respectively.
Construction of a hisB disruption vector
To construct a hisB disruption plasmid we used an approach which was published recently . In brief, 533 bp and 500 bp sequences of the hisB coding and 3’ sequence were amplified via PCR using primers listed in Additional file 1: Table S1. Both fragments were inserted via Gibson cloning into the BsrGI linearized plasmid pAO4-13 carrying the A. oryzae pyrG gene  giving rise to the counter-selectable hisB disruption plasmid pMF22.1.
Construction of an olvA deletion cassette
The plasmid pAW34 (, kindly provided by Arthur Ram) containing the AopyrG gene flanked by the 5’ and 3’ region of olvA was used as a backbone. The A. nidulans hisB (AN6536) gene was amplified using primers listed in Additional file 1: Table S1 and cloned into the XhoI/HindIII linearized pAW34 via Gibson cloning giving rise to plasmid pSE1.6.
Construction of luciferase reporter constructs
The A. niger pyrG* gene within Tet-on plasmids pVG2.2 (containing the empty Tet-on system, ) and pVG4.1 (containing a codon optimized version of the luciferase mluc under control of the Tet-on system, ) was replaced by a 2291 bp fragment amplified by fusion PCR, containing the full length hisB gene without a functional start codon flanked by 5’ and 3’ region of the hisB gene, giving rise to pTG1.2 and pTG2.15, respectively (Additional file 2: Figure S2).
Measurement of the luciferase activity
Ninety six well microtiter plate assays were performed as described earlier  with slight modifications. In brief, 5 x 104 spores were inoculated in 200 μl CM medium  supplemented 1.4 mM luciferin and 0, 5 (A) or 20 μg/ml (B) doxycycline in a microtiter plate and incubated at 30°C in a Victor3 (Perkin Elmer). OD and luminescence were measured every 30 min.
To evaluate whether the A. nidulans hisB gene (AN6536) could be used as selection marker to complement the histidine auxotrophy, the genomic and protein sequences were compared using BlastN and BlastP, revealing a high conservation (90.2%) on the protein level, whereas the nucleotide sequence conservation was considerably lower (74.1%), which was thought to be crucial for the correct integration of the deletion cassette into the region of choice instead of complementing A. niger hisB gene at its endogenous locus. For an easy read-out of the transformation and gene replacement efficiency, the olvA gene was chosen. This gene encodes a hydrolase involved in DHN-melanin synthesis in A. niger and its deletion results in an incomplete melanin biosynthesis and thus green spore formation .
Homologous recombination efficiency of individual transformations, as assessed by phenotypic spore color screening using A. oryzae pyrG and A. nidulans hisB as selection markers to delete the olvA gene in kusA - recipient strains MF42.2 and MA169.4
No. of transformants
No. of positive mutants
Homologous integration efficiency [%]
MF42.2 (hisB - , pyrG + )
MA169.4 (pyrG - )
89 ± 1.9
While the luminescence activity of the strains carrying the reporter construct integrated into the pyrG or hisB locus are comparable (induction with 5 μg/ml doxycycline), the values of the TG2.3 are 3 times higher when using 20 μg/ml doxycycline, possibly reflecting a higher transcriptional activity at the hisB locus under the conditions used. It is notable that neither the vector control (TG1.14, 20 μg/ml doxycycline) nor the non-induced luciferase constructs (TG2.3 and VG8.27) showed any luciferase activity during the experiment, clearly demonstrating that the system is tight at the hisB locus in the absence of the inducer.
In summary, we report here a straight forward approach to rationally generate auxotrophic markers in the filamentous fungus A. niger which was employed to create a histidine auxotrophic strain which can be used as a recipient isolate for endogenous deletion of genes using the A. nidulans orthologue hisB. In addition, we characterized the hisB* locus for functionality to integrate expression constructs, which revealed an expression level for the luciferase reporter with a higher performance and tighter characteristics under non-induced conditions compared to the well-used pyrG locus. The tools described here significantly increase the tractability of A. niger at the molecular level and suggest hisB could be used for similar applications in other model or pathogenic filamentous fungi.
We are grateful to Susanne Engelhardt for her excellent technical assistance.
This work was financially supported by the Marie Curie Career Integration Grant (CIG303864) to VM.
Availability of data and materials
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.
MF and VM designed the study and drafted the manuscript. MF, TG and CK performed the experiments. All authors contributed to the writing and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
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