The roles of the zinc finger transcription factors XlnR, ClrA and ClrB in the breakdown of lignocellulose by Aspergillus niger

Abstract

Genes encoding the key transcription factors (TF) XlnR, ClrA and ClrB were deleted from Aspergillus niger and the resulting strains were assessed for growth on glucose and wheat straw, transcription of genes encoding glycosyl hydrolases and saccharification activity. Growth of all mutant strains, based in straw on measurement of pH and assay of glucosamine, was impaired in relation to the wild-type (WT) strain although deletion of clrA had less effect than deletion of xlnR or clrB. Release of sugars from wheat straw was also lowered when culture filtrates from TF deletion strains were compared with WT culture filtrates. Transcript levels of cbhA, eglC and xynA were measured in all strains in glucose and wheat straw media in batch culture with and without pH control. Transcript levels from cbhA and eglC were lowered in all mutant strains compared to WT although the impact of deleting clrA was not pronounced with expression of eglC and had no effect on xynA. The impact on transcription was not related to changes in pH. In addition to impaired growth on wheat straw, the ΔxlnR strain was sensitive to oxidative stress and displayed cell wall defects in the glucose condition suggesting additional roles for XlnR. The characterisation of TFs, such as ClrB, provides new areas of improvement for industrial processes for production of second generation biofuels.

Keywords: Cell wall; Glysosyl-hydrolases; Regulation; Transcription factor; pH.

Fig. 1

pH measurements from glucose and wheat straw cultures. For both substrates the starting pH of the medium was pH 6.5. The results shown represent triplicates of three independent biological samples and the error bars indicate the standard error of the mean. A two-way ANOVA was performed for each condition in comparison to the WT strain. * represents the conditions for which the two-way ANOVA test result had a p-value < 0.05 which means that the difference observed is significant when compared to the WT

Fig. 2

Increase in the biomass content of the ΔxlnR, ΔclrB and ΔclrA cultures in glucose and wheat straw. The wheat straw cultures were inoculated with 1.5 g (wet weight) of mycelium from the corresponding glucose culture. The glucosamine content was measured from 1.5 g (wet weight) for the glucose culture and from the content of the full culture for the wheat straw cultures and the dry weight was calculated back using a standard curve of the glucosamine content against the dry weight. The results shown represent triplicates of three independent biological samples and the error bars indicate the standard error of the mean. * represents the conditions for which the t-test result had a p-value < 0.05 which means that the difference observed is significant when compared to the WT

Fig. 3

Measurement of sugars reducing ends released after incubation of equal volumes of the supernatants of the ΔxlnR, ΔclrB and ΔclrA wheat straw cultures with straw done by the DNS assay. A two-way ANOVA was performed for each condition in comparison to the WT strain. The results shown represent triplicates of three independent biological samples and the error bars indicate the standard error of the mean. * represents the conditions for which the two-way ANOVA test result had a p-value < 0.05 which means that the difference observed is significant when compared to the WT

Fig. 4

a Expression of the cbhA, eglC and xynA genes in A. niger grown in wheat straw media. The results shown represent triplicates of three independent biological samples and the error bars indicate the standard error of the mean. b Comparison of the expression of the cbhA, xynA and eglC genes in A. niger grown in wheat straw media under pH-controlled condition (pH 5.0 in fermenter) and no control of pH (flask). The results shown represent triplicates of two independent biological samples and the error bars indicate the standard error of the mean. For both experiments, the expression values represent the percentage of transcript level normalised against the level for actA. * represents the conditions for which the t-test result had a p-value < 0.05 which means that the difference observed is significant when compared to the WT

Fig. 5

Expression of the clrA and clrB genes in A. niger grown in wheat straw media. The results shown represent triplicates of three independent biological samples and the error bars indicate the standard error of the mean. * represents the conditions for which the t-test result had a p-value < 0.05 which means that the difference observed is significant when compared to the WT

Fig. 6

Cell wall analysis of sugars in the cell wall of the WT and ΔxlnR strains using HPLC

Fig. 7

Reactive oxygen species (ROS) accumulation and cell permeability using the dyes H2DCF-DA (a) and phloxine B (b). Phloxine B stains the cytosolic material due to a more permeable cell in the ΔxlnR and ΔclrB strains. H2DCF-DA emits a green fluorescence when it is oxidised by ROS in the cell. The fluorescence intensity was measured using the ImageJ software. The results shown represent 50 measurements of three independent biological samples and the error bars indicate the standard error of the mean. * represents the conditions for which the t-test result had a p-value < 0.05 which means that the difference observed is significant when compared to the WT. c Expression of the alternative oxidase gene (aox1) measured by qRT-PCR in the WT and the ΔxlnR and ΔclrB strains. The results shown represent triplicates of three independent biological samples and the error bars indicate the standard error of the mean