Efficient cloning system for construction of gene silencing vectors in Aspergillus niger

Abstract

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.

Fig. 1

Construction of expression vectors for post-transcriptional gene silencing in A. niger. The arrow indicates the recombination reaction of pFIRD1 destination vector with two molecules of entry clone containing a partial coding sequence. ccdB-Cm ^R ccdB gene-chloramphenicol resistance gene; S spacer region

Fig. 2

Semi-quantitative assays for analysis of xylan-degrading activities. a With AZCL xylan for detection of endoxylanase activity. b With MUX for detection of β-xylosidase and xylanase activities. C Wild-type strain (NW219::pGW635); numerals designate each silenced strain (NW219::pGW635::pXLNRir)

Fig. 3

mRNA levels for xlnR, xyrA and xynB in silenced and non-silenced strains, compared to wild type. C Wild-type strain (NW219::pGW635); numerals designate each strain from co-transformation (NW219::pGW635::pXLNRir). Error bars represent 95% confidence intervals (1.962 × SE)

Fig. 4

Genome integrity of pXLNRir. Genome integrity was assessed for both silenced (a and b) and non-silenced strains (c and d). a, c PCR-amplified 5′-region of pXLNRir. b, d PCR-amplified 3′-region of pXLNRir. Each triangle represents the expected fragment for intact pXLNRir. M Molecular weight marker; numerals designate each strain from co-transformation (NW219::pGW635::pXLNRir); C wild-type strain (NW219::pGW635)

Fig. 5

Inverted repeats of pXLNRir. PCR-amplified portion of inverted repeat of pXLNRir from genomic DNA of non-silenced and silenced strains. Triangle Expected fragment for intact inverted repeat region. M Molecular weight marker; P silencing plasmid pXLNRir; C wild-type strain (NW219::pGW635); numerals designate each non-silenced and silenced strain (NW219::pGW635::pXLNRir)