Efficient cloning system for construction of gene silencing vectors in Aspergillus niger.
An approach based on Gateway recombination technology to efficiently construct silencing vectors was developed for use in the biotechnologically important fungus Aspergillus niger. The transcription activator of xylanolytic and cellulolytic genes XlnR of A. niger was chosen as target for gene silencing. Silencing was based on the expression vector pXLNRir that was constructed and used in co-transformation. From all the strains isolated (N = 77), nine showed poor xylan-degrading activities in two semi-quantitative plate assays testing different activities for xylan degradation. Upon induction on D-xylose, transcript levels of xlnR were decreased in the xlnR-silenced strains, compared to a wild-type background. Under these conditions, the transcript levels of xyrA and xynB (two genes regulated by XlnR) were also decreased for these xlnR-silenced strains. These results indicate that the newly developed system for rapid generation of silencing vectors is an effective tool for A. niger, and this can be used to generate strains with a tailored spectrum of enzyme activities or product formation by silencing specific genes encoding, e.g., regulators such as XlnR.
Oliveira
José Miguel
JM
Laboratory of Microbiology, Wageningen University, Wageningen, The Netherlands.
van der Veen
Douwe
D
de Graaff
Leo H
LH
Qin
Ling
L
D001234:Aspergillus niger / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D050542:D-Xylulose Reductase / Q000235:genetics / Q000378:metabolism; D043364:Endo-1,4-beta Xylanases / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D020868:Gene Silencing; D005822:Genetic Vectors / Q000235:genetics*; D014158:Transcription, Genetic; D014990:Xylans / Q000378:metabolism
D005656:Fungal Proteins; D014990:Xylans; D050542:D-Xylulose Reductase; D043364:Endo-1,4-beta Xylanases