Fiedler Markus R M, Gensheimer Tarek, Kubisch Christin, Meyer Vera
Institute of Biotechnology, Department of Applied and Molecular Microbiology, Technische Universität Berlin, Gustav-Meyer-Allee 25, 13355, Berlin, Germany.
BMC Microbiol. 2017 Mar 8;17(1):57. doi: 10.1186/s12866-017-0960-3.
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.
对于黑曲霉而言，有一系列广泛的营养缺陷型和显性抗性标记可用。然而，只有少数标记能实现对目标基因在选定基因组位点的靶向修饰，这阻碍了功能基因组学研究。因此，我们旨在通过构建一个具有特征化hisB位点的组氨酸营养缺陷型菌株，来扩展黑曲霉中可用的标记集，以实现目标基因的整合和缺失。
通过使用可反向选择的pyrG标记破坏黑曲霉hisB基因，建立了一个组氨酸营养缺陷型菌株。去除该标记后，获得了一个hisB - 、pyrG - 菌株，该菌株用作进一步研究的受体菌株。我们在此表明，来自构巢曲霉和黑曲霉的hisB直系同源基因均可用于在该受体菌株中恢复组氨酸原养型。构巢曲霉的hisB基因适用于在黑曲霉的不同位点进行高效基因靶向，而黑曲霉的hisB基因则允许将Tet-on驱动的荧光素酶报告构建体高效整合到内源性无功能的hisB位点。随后对荧光素酶活性的分析表明，hisB位点在非诱导条件下调控严格，与pyrG整合位点相比，其荧光素酶表达水平更高。
综上所述，我们在此为黑曲霉提供了一种替代选择标记hisB，它具有高效的同源整合率以及与成熟的pyrG选择标记相比更优的高表达水平。
