Several studies have demonstrated that the saprophytic, parasitic and/or plant pathogenic nature of fungi makes them an important reservoir of carbohydrate-active enzymes (CAZymes) which are responsible for the biosynthesis, breakdown or modification of polysaccharides and their derivatives which make up plant cell walls (Ospina-Giraldo et al., 2010; Zhao et al., 2014; Brouwer et al., 2014). CAZymes may be grouped into five functional classes: glycoside hydrolases (GHs), glycosyl transferases (GTs), polysaccharide lyases (PLs), carbohydrate esterases (CEs) and the latest addition to the CAZymes database, enzymes possessing auxiliary activities (AA) (Lombard et al., 2014; http://www.cazy.org).
Glycoside hydrolases cleave the glycosidic bond between carbohydrates or between carbohydrates and a non-carbohydrate substituent such as protein or lipids while GTs are involved in the biosynthesis of di-, oligo- and polysaccharides by catalysing the transfer of sugar moieties and forming glycosidic bonds (Lombard et al., 2014). Glycoside hydrolases such as cellulases and xylanases have been extensively studied in fungi for industrial exploitation as discussed at length in Chapter 1. Polysaccharide lyases degrade pectin and glycosaminoglycans while CEs catalyse the de-O or de-N-acylation of substituted sugars (Zhao et al., 2013). Auxiliary activity enzymes are redox enzymes that include lytic polysaccharide mono-oxygenases and ligninolytic enzymes (Veneault-Fourrey et al., 2014).
CAZymes can make-up 1-5% of an organisms coding sequences (Lombard et al., 2014).
Recent studies have taken to analysing the collection of CAZymes encoded in an organism’s
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genome, referred to as the “CAZome”. The information garnered from CAZome analysis has been used to investigate the metabolic potential of organisms, in particular, fungal plant pathogens and parasites, as CAZymes involved in cell-wall degradation are seen as pathogenicity factors (Ospina-Giraldo et al., 2010; Zhao et al., 2013; Brouwer et al., 2014).
With the explosion in genome sequencing efforts over the past decade, there has been a concurrent surge in the prediction of protein functions by computational means (Dassa et al., 2012; Häkkinen et al., 2012). The functional annotation of genes relies on the existence of current functional information via homology searches (Levasseur et al., 2014). This is not a straightforward task, as when it comes to CAZymes, they share low sequence identity and thus prediction of enzymatic activity cannot be based on their amino acid sequence alone (Henrissat and Davies, 1997). The CAZy Database provides a tremendous amount of information specifically on enzymes involved in the building and breakdown of complex carbohydrates and glycoconjugates (Cantarel et al., 2009). The CAZymes in the database are classified based on both their amino acid sequence and three-dimensional structure, thus allowing for differentiation of enzymes that possess different functions and belong to the same family, or of enzymes possessing the same function yet belonging to different families (Busk and Lange, 2013). A typical example is that of enzymes possessing endoglucanase (EG) activity; they have representatives in 17 different GH families (Juturu and Wu, 2014).
Functional prediction of GHs could be performed by aligning amino acid sequences of a GH family and deducing phylogenetic relationships, however, apart from the low sequence identity of proteins within a family, enzymes with the same function may have developed as a result of convergent evolution in different ancestors and would thus make the prediction unreliable; skewing the correlation between enzyme activity and phylogeny (Henrissat and Davies, 1997; Busk and Lange, 2013). A much more specific and sensitive approach for the
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annotation of CAZymes is proposed by Park et al. (2010) which involves similarity searches against nonredundant sequences in the CAZy database much like the algorithm used by the basic local sequence alignment tool (BLAST) together with annotating CAZy family and protein family (Pfam) domains since CAZymes are usually modular proteins.
With plant biomass being an attractive renewable feedstock for the production of biofuel and the polysaccharide degradative ability of fungi, it is clear to see the need for CAZome analysis of saprophyts such as white- and brown-rot fungi, as well as species isolated from unique ecological niches. A brief overview of the CAZyme genes encoded in the genome of important CAZyme producers such as thermophiles Thielavia terrestris, Myceliopthora thermophila and the paradigm cellulase producer Trichoderma reesei, can be found in Table 3.1. The study of the CAZyme repertoire of plant biomass degrading fungi will greatly aid in the development of industrial enzyme cocktails for efficient degradation of plant biomass (Benoit et al., 2015).
Table 3.1: Distribution and comparison of the number of putative CAZyme genes between important fungal CAZyme producers
Species GH GT PL CE AA CBM Total Reference
Thielavia terrestris 212 91 4 28 58 80 473 http://www.cazy.org Myceliophthora
thermophila
195 87 8 28 50 47 415 http://www.cazy.org Penicillium
chrysogenum
225 103 9 22 22 51 432 http://www.cazy.org Aspergillus
nidulans
264 92 21 31 33 44 485 http://www.cazy.org Trichoderma
reesei
174 94 4 19 NR 41 332 Xie et al. (2014);
Payne et al. (2015)
NR: Not reported; GH: Glycoside hydrolase; GT: Glycosyl transferase; PL: Polysaccharide lyase; Carbohydrate esterase; AA: Auxillary activity enzymes; CBM: Carbohydrate binding domain
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Table 3.1 reveals a trend that in most organisms, GHs are the most highly represented class of CAZymes. However, not all GHs are involved polysaccharide degradation as will be discussed later in this chapter. There has been about 30 CAZymes that have been characterized from T. reesei, however, with CAZome analysis, there has been predictions of 44 uncharacterized enzymes that are involved in the degradation of biomass (Häkkinen et al., 2012). Thus, CAZome analysis is powerful bioinformatic method that reveals information on an organism’s metabolic potential and nutritional lifestyle.
Phialophora alba, a thermophilic ascomycete fungus isolated from Eucalyptus woodchips and has been shown to possess highly active thermostable xylanases (Mosina, 2013). It was found that maximum endoglucanase activity is produced on Day 4 with two isozymes being produced with pH optima at 4 and 9 with temperature optim at 60°C (Dweba, 2013;
Mbandlwa, 2013) Research and information on Phialophora species in general is scarce. To our knowledge, P. alba is only the second Phialophora sp. known to produce thermophilic CAZymes, and this is the first known report on the genome of P. alba. Thielavia terrestris and Myceliopthora thermophila are among the most well studied thermophiles with regard to their cellulolytic activity and thermostable enzymes and will thus be featured prominently in this discussion along with other well known CAZyme producers (Berka et al., 2011).
The aim of this phase of study was to data-mine the genome of P. alba and identify genes that encode EGs and other CAZymes of interest and those that could be utilised in industrial applications.