Expression of Aspergillus niger CAZymes is determined by compositional changes in wheat straw generated by hydrothermal or ionic liquid pretreatments

Lignocellulose, which is composed of polysaccharides and lignin, is an abundant plant raw material that is used to make liquid biofuels [1]. The polysaccharides are a source of simple sugars that can be released enzymatically by saccharification for subsequent fermentation to biofuels [2]. One barrier to cost-efficient production of biofuels is the inefficient saccharification step, which requires excessive amounts of costly enzymes produced by industry using fungi [3]. As part of an approach to improve efficiency, lignocellulosic substrates are routinely pretreated to render the lignocellulose more amenable to saccharification by enzymes [4]. Production of more efficient and cheaper enzyme mixtures would be aided by improved knowledge gained from studying the responses of nature’s lignocellulose-degrading fungi to pretreated lignocellulosic feedstocks.

Pretreated lignocelluloses offer the opportunity to study how fungi respond and adapt to major changes in the composition and structure of lignocellulose. Pretreatments consist of a physical and/or chemical process that has the aim of opening up the structure of the lignocellulose to improve the efficiency of saccharification [5]. Hydrothermal pretreatments, which are commonly used, tend to remove some hemicelluloses from the lignocellulose as well as break linkages between the polysaccharides and lignin [6]. The use of ionic liquids is an emerging pretreatment technology which facilitates the solubilisation of lignocellulose and, crucially, regenerates insoluble but now highly saccharifiable polymers [7]. In our study we pretreated wheat straw, which is a major agricultural waste residue with potential for use to produce bioethanol [8].

Previously, various untreated lignocelluloses as well as individual polysaccharides have been used to study the transcriptional response of the ascomycetes Aspergillus niger [9–13], Aspergillus nidulans [14], Trichoderma reesei [15–18], Neurospora crassa [14, 19, 20] and Myceliophthora thermophila [21]. Transcriptional responses of mixed cultures of ascomycete fungi with untreated lignocellulose have also been studied [22]. A limited number of studies have used pretreated substrates, namely where A. niger was exposed to steam-exploded bagasse [10] or where T. reesei was exposed to steam-exploded bagasse, wheat straw or spruce [15]. The most prominent feature of these studies was the induction of many genes encoding Carbohydrate-Active enZymes (CAZy, as found in the CAZy database, http://www.cazy.org/ [23]) that saccharify lignocellulose (for reviews, see Daly et al. [24] and Glass et al. [25]).

There are several major gaps in our understanding of the fungal response to pretreated lignocellulose that previous studies could not adequately address. Firstly, as fungi were exposed only to steam explosion-based pretreatment [10, 15], the effect of the majority of pretreatment-induced changes in lignocellulose composition and structure is so far unknown. Secondly, previous studies had limited temporal resolution making it difficult to delineate the temporal aspects of the response to the substrates. Kolbusz et al. [21] coined the phrase ‘cryptic CAZy’ to refer to those genes encoding biomass-degrading CAZymes that have not been shown to be induced in response to lignocelluloses. Their expression may just be temporally limited rather than being irrelevant to lignocellulose degradation. Thirdly, concentrations of sugars present in cultures with recalcitrant untreated feedstocks can be relatively low, such as in Delmas et al. [9]. Highly saccharifiable, pretreated substrates facilitate the study of the response to a different range of concentrations of those same sugars. Furthermore, the sensing of ratios of sugar concentrations rather than just concentrations of individual sugars is a recently established paradigm for fungi [26] and a broad time course facilitates exposure to many more different ratios of these sugars.

A. niger, a saprobic ascomycete filamentous fungus, is both a model organism and used industrially for the production of organic acids and enzymes [27, 28]. A. niger possesses a large repertoire of CAZymes that are active towards plant polysaccharides [13, 27]. A. niger has regulatory mechanisms mediated by transcription factors such as XlnR and AraR that allow the fungus to respond to the presence of polysaccharides in its environment [29, 30]. Both broad spectrum and specific inducers mediate this regulation. For example, xylose not only induces genes encoding xylanolytic enzymes but also endoglucanases via XlnR [29], whereas specific aromatic compounds induce the gene encoding the feruloyl esterase FaeB [31], which generally functions to cleave linkages that involve the inducing aromatics. Previously, we have shown a role for carbon starvation in the induction of CAZy genes during the early response of A. niger to wheat straw [9, 11].

In this study, we investigated the temporal responses of A. niger to untreated and pretreated wheat straw by measuring genome-wide transcript abundance, specific proteins and secreted enzyme activities. To our knowledge, this is the first study providing an extensive time course of the fungal responses to untreated and pretreated lignocelluloses, as well as the first in reporting the fungal response to ionic liquid-pretreated lignocellulose. We identify uncharacterised CAZy genes, regulators and transporters relevant to lignocellulose degradation by relating gene expression with exposure to the untreated and pretreated straw.