RNA-sequencing reveals the complexities of the transcriptional response to lignocellulosic biofuel substrates in Aspergillus niger

Abstract

Background: Saprobic fungi are the predominant industrial sources of Carbohydrate Active enZymes (CAZymes) used for the saccharification of lignocellulose during the production of second generation biofuels. The production of more effective enzyme cocktails is a key objective for efficient biofuel production. To achieve this objective, it is crucial to understand the response of fungi to lignocellulose substrates. Our previous study used RNA-seq to identify the genes induced in Aspergillus niger in response to wheat straw, a biofuel feedstock, and showed that the range of genes induced was greater than previously seen with simple inducers.

Results: In this work we used RNA-seq to identify the genes induced in A. niger in response to short rotation coppice willow and compared this with the response to wheat straw from our previous study, at the same time-point. The response to willow showed a large increase in expression of genes encoding CAZymes. Genes encoding the major activities required to saccharify lignocellulose were induced on willow such as endoglucanases, cellobiohydrolases and xylanases. The transcriptome response to willow had many similarities with the response to straw with some significant differences in the expression levels of individual genes which are discussed in relation to differences in substrate composition or other factors. Differences in transcript levels include higher levels on wheat straw from genes encoding enzymes classified as members of GH62 (an arabinofuranosidase) and CE1 (a feruloyl esterase) CAZy families whereas two genes encoding endoglucanases classified as members of the GH5 family had higher transcript levels when exposed to willow. There were changes in the cocktail of enzymes secreted by A. niger when cultured with willow or straw. Assays for particular enzymes as well as saccharification assays were used to compare the enzyme activities of the cocktails. Wheat straw induced an enzyme cocktail that saccharified wheat straw to a greater extent than willow. Genes not encoding CAZymes were also induced on willow such as hydrophobins as well as genes of unknown function. Several genes were identified as promising targets for future study.

Conclusions: By comparing this first study of the global transcriptional response of a fungus to willow with the response to straw, we have shown that the inducing lignocellulosic substrate has a marked effect upon the range of transcripts and enzymes expressed by A. niger. The use by industry of complex substrates such as wheat straw or willow could benefit efficient biofuel production.

Keywords: Aspergillus; Biofuels; RNA-seq; Transcriptome; Wheat straw; Willow.

Figure 1

Percentage of transcripts belonging to (A) CAZy families and (B) particular enzyme activities. Expression levels were measured following 24 h of culturing A. niger in media containing ball-milled willow as the only carbon source. CAZy families representing more than 2% of total CAZy expression are indicated. The percentage values are the RPKM values for (A) particular CAZy families expressed as a proportion of the total RPKM value of all the families in the GH, CE, PL and AA classes or (B) particular enzyme activities expressed as a proportion of the total RPKM value of all the activities analysed. Classifications for CAZy are according to CAZy.org. Genes were assigned to particular enzyme activities as described in the Methods section and Additional file 7.

Figure 2

Expression per (A) CAZy family and (B) selected enzyme activities as a percentage of total gene expression. For (A), only those families with total RPKM values of 100 or more upon at least one of the substrates are shown. Gene expression was measured following 24 h culturing of A. niger in the presence of willow or wheat straw as the only carbon source. The families and activities are presented in order of decreasing expression from the willow cultures. The error bars represent standard error. Genes were assigned to particular enzyme activities as described in Methods section and Additional file 7. A ‘*’ symbol is used to indicate whether the differences between willow and straw cultures were statistically significant (unequal variances t-test, p <0.05).

Figure 3

Specific activity towards para -nitrophenyl (pNP) substrates from supernatants from A. niger cultured with either wheat straw or willow. A ‘*’ symbol is used to indicate whether the differences between willow and straw cultures were statistically significant (Student’s t-test, p < 0.05). Abbreviations are: pNP-β-cel: 4-Nitrophenyl-β-D-cellobioside; pNP-α-ara: 4-Nitrophenyl-α-L-arabinofuranoside; pNP-β-glu; 4-Nitrophenyl-β-D-glucopyranoside and pNP-β-xyl: 4-Nitrophenyl-β-D-xylopyranoside. The error bars represent standard errors.

Figure 4

Saccharification of lignocellulosic substrates by supernatants (S/Ns) from A. niger cultured with either wheat straw or willow. An equal amount of protein from concentrated S/Ns from A. niger cultured with either straw or willow was used to saccharify straw or willow substrates. The glucose and reducing end group equivalents were quantified with the GODPOD and DNS assays respectively. The glucose or reducing end groups released by equal amounts of protein from the different culture S/Ns in a 24 h period are expressed per mg of the lignocellulosic substrate in the saccharification assay. The results are the mean of assays from S/Ns from 3 independent cultures with either lignocellulosic substrate and the error bars represent standard errors.