Schizophyllum commune: An unexploited source for lignocellulose degrading enzymes

Abstract Lignocellulose represents the most abundant source of carbon in the Earth. Thus, fraction technology of the biomass turns up as an emerging technology for the development of biorefineries. Saccharification and fermentation processes require the formulation of enzymatic cocktails or the development of microorganisms (naturally or genetically modified) with the appropriate toolbox to produce a cost‐effective fermentation technology. Therefore, the search for microorganisms capable of developing effective cellulose hydrolysis represents one of the main challenges in this era. Schizophyllum commune is an edible agarical with a great capability to secrete a myriad of hydrolytic enzymes such as xylanases and endoglucanases that are expressed in a high range of substrates. In addition, a large number of protein‐coding genes for glycoside hydrolases, oxidoreductases like laccases (Lacs; EC 1.10.3.2), as well as some sequences encoding for lytic polysaccharide monooxygenases (LPMOs) and expansins‐like proteins demonstrate the potential of this fungus to be applied in different biotechnological process. In this review, we focus on the enzymatic toolbox of S. commune at the genetic, transcriptomic, and proteomic level, as well as the requirements to be employed for fermentable sugars production in biorefineries. At the end the trend of its use in patent registration is also reviewed.


| INTRODUC TI ON
In the past decade, the amount of research related to lignocellulosic ethanol (second generation ethanol) has increased extensively in the scientific community. Several bacteria and fungi species have been studied in terms of the intra and extracellular enzymatic complexes involved in the deconstruction of the polymeric components that make up the lignocellulosic biomass. On the other hand, every year a novel or modified pretreatment technology becomes available with the aim of improving yields during the saccharification of the lignocellulosic materials. However, we are still far away from producing economically competitive lignocellulosic bioethanol (Mohanram, Amat, Choudhary, Arora, & Nain, 2013), largely because the lack of microbial enzymatic cocktails that break down the recalcitrant lignocellulosic biomass in an efficient manner (Gupta, 2016). Since the amount of plant biomass has been estimated to be of 180 billions of tons only above the ground and near 40 millions tons in the ocean (Chen, 2014), the exploitation of these materials for production of biofuels and value-added products is a great alternative to reduce the fossil fuels dependence that as a society we have.
This review focuses on the unexploited and enormous biotechnological potential of the basidiomycete fungus Schizophyllum commune for the production of novel enzymes that could boost the biofuel and biomass derived product research. Also, this work summarizes the research that has been conducted in the last two decades and that supports the use of S. commune as a current aspirant for white, green and gray biotechnology applications.

| G ENER AL A S PEC TS OF SCHIZOPHYLLUM COMMUNE
Schizophyllum commune is an agarical mushroom-forming fungus, able to complete its life cycle in about 10 days and is one of the most commonly found fungi, whose distribution covers all continents with the exception of Antarctica .
This extensive repertoire of plant cell wall degrading and modifying enzymes makes S. commune an outstanding candidate for studies regarding the mechanism by which this fungus degrades biomass in order to exploit its potential and improve the efficiency of industrial processes such as the lignocellulosic ethanol production, bioconversion of agricultural by-products or biodegradation of xenobiotics and pollutants (Table 1).

| PROTEIN S INVOLVED IN CELLULOS E DECONS TRUC TION BY SCH IZOPHYLLUM COMMUNE
The subject of cellulose deconstruction by fungi leads us to think immediately of organisms like Trichoderma reesei, Neurospora crassa, and various Aspergillus species, considering the ascomycetes group, and mainly in Phanerochaete chrysosporium when referring to the basidiomycetes group, leaving out of study a significant group of basidiomycetes with the same or even greater potential to degrade cellulose.
One of these basidiomycetes is the "split gill" fungus S. commune, whose genome sequence was published in 2010 . Its genome revealed that it contains 240 candidate genes belonging to glycoside hydrolases from different families, almost 80 more GH genes than those reported for P. chrysosporium.
F I G U R E 1 Biotechnological applications of Schyzophyllum commune. Glycoside hydrolase (GH), carbohydrate esterase (CE), glycosyltransferase (GT), polysaccharide lyase (PL), lytic polysaccharide monooxygenase (AA9), laccase (AA1), peroxide-producing enzymes (AA3, and AA5) The study of the hydrolytic machinery in organisms like S. commune is attractive mainly for the lignocellulosic biofuels industry, since the number of published works is still growing year by year in this subject, but is not limited to this area. Something remarkable is that the amount and diversity of published work related with the S. commune ′s cellulolytic system is scarce (Table 2) when compared with published work (in this area) of fungi like T. reesei, N. crassa or P. chrysosporium, despite the fact that studies such as those carried out by Arboleda Valencia et al. (2011), Lee et al. (2014), Zhu et al., (2016) have demonstrated that S. commune has an important potential in the lignocellulose bioconversion field, even exhibiting cellulolytic and xylanolytic activities comparable with those obtained using an enzymatic commercial preparation of Trichoderma longibranchiatum.
The cellulose degradation mechanisms by ascomycetes and basidiomycetes have been revised by Glass, Schmoll, Cate, and Coradetti (2013) and Baldrian and Valásková (2008). However, although the role of the classic enzymes involved in cellulose deconstruction such as endoglucanases, cellobiohydrolases, cellobiose dehydrogenases and beta glucosidases is well documented in fungi, the role of the termed "amorphogenic proteins" or plant cell wall remodeling proteins (expansins and expansin-related proteins) in cellulose deconstruction is not well understood in both, ascomycetes and basidiomycetes. These amorphogenic proteins cause swelling of cellulose fibers and fragmentation of cellulose aggregations at the beginning of the enzymatic hydrolysis of cellulose before any detectable amount of reducing sugars is released (Gourlay et al., 2013).
From these latter proteins, the swollenin from T. reesei is the most studied, and after its discovering it was suggested as the C 1 factor of the cellulose enzymatic degradation mechanism originally proposed by Mandels and Reese (1999) and Reese, Siu, and Levinson (1950). Nevertheless, that hypothesis has been rejected by the work of Eibinger et al. (2016), who demonstrates that swollenin is not an amorphogenesis factor when acting on pure cellulose. Nonetheless, the possibility that one or more of these plant cell wall remodeling proteins may be acting as the C 1 factor is yet to be proven. Indeed, the genome of S. commune contains at least 17 expansin-related proteins, one of which has already been cloned and expressed in Pichia pastoris, showing a 23% increment in avicel hydrolysis when used as pretreatment before the addition of a cellulase mixture (Tovar-Herrera et al., 2015). However, the study of this type of proteins is relatively new in microbes, and there is a lot of information yet to be obtained from them.
Another group of proteins with great importance and also involved in biomass deconstruction is the group of enzymes known as lytic polysaccharide monooxygenases (LPMOs) classified in auxiliary activity families 9 (AA9), 10 (AA10), 11 (AA11), and 13 (AA13) in the CAZy database (Frandsen et al., 2016;Frommhagen et al., 2015;Hemsworth, Henrissat, Davies, & Walton, 2013;Silveira & Skaf, 2016;Vaaje-Kolstad et al., 2010). From these families, AA9 corresponds to fungal proteins involved in cellulose deconstruction (some of them are also active in hemicellulose), while AA10 belongs to a bacterial group of LMPOs active on cellulose and chitin, and AA11 and AA13 are fungal proteins active on chitin and starch, respectively. AA9 proteins have been studied in N. crassa (Tian et al., 2009), T. reesei (Tanghe et al., 2015), P. chrysosporium (Westereng et al., 2011), Chaetominium globosum (Kim et al., 2015) and Myceliophthora thermophile (Frommhagen et al., 2015), and have been reported to improve the release of glucose and oligosaccharides F I G U R E 2 Glycoside hydrolase (GH) genes present in the genome of Schizophyllum commune. Only those involved in plant cell wall deconstruction were considered
Three recent works have reported the presence of AA9 proteins in the secretomes of S. commune when cultured in avicel (one protein) (Sornlake et al., 2017), Jerusalem artichoke stalks (nine proteins) (Zhu et al., 2016), and Leucaena leucocephala wood chips (Singh et al., 2017)

| PROTEIN S INVOLVED IN HEMI CELLULOS E DECON S TRUC TI ON BY SCHIZOPHYLLUM COMMUNE
Hemicelluloses are heteropolysaccharides from the plant cell walls that constitute the second most abundant component of lignocellulosic biomass. Their complex structure is dependent on the source and mainly contains pentoses (xylose and arabinose), hexoses (glucose, galactose, and mannose) and, to a lesser extent, glucuronic and galacturonic acid. The bioconversion of hemicellulose to obtain ethanol or other value-added products, such as chemicals and biopolymers, is a well-researched topic. Through a pretreatment process, the hemicelluloses are degraded or broken down in the biomass, releasing fermentable sugars such as xylose, arabinose and glucose, and rendering the cellulose more accessible to cellulolytic enzymes (Lavarack, Griffin, & Rodman, 2002). Pretreatment of hemicelullose (usually chemically treated) is one of the most expensive steps of biomass processing, thus, studies to decrease the cost are of main interest from an economic point of view (Canam, Town, Iroba, Tabil, & Dumonceaux, 2013). The poor sustainability of the currently used acid/base-catalyzed processes has highlighted the need for a more environmentally friendly and mild pretreatment of the hemicellulosic biomass, such as biological ones, that also encompasses a high efficiency (Canam et al., 2013). Another disadvantage of chemical processes is that byproducts may potentially act as microbial inhibitors during the subsequent fermentation steps (Peng, Peng, Xu, & Sun, 2012). Therefore, enzymatic pretreatment and bioconversion have arisen as a suitable alternative that could be coupled to subsequent fermentation and might enhance the industrial processing of biomass. The main drawback of enzymatic processing is that high efficiency has not been achieved to date. New enzyme cocktails that can increase the yields of fermentable products and other valueadded chemicals are currently under study (Zhu et al., 2016).  TA B L E 4 Granted and applied patents related with S. commune

Technical and industrial fields of the patents (applied for or granted) Applicant(s) and year References
Selective and oriented enzyme production and preparation Preparation of glucoamylase TAX ADM Agency (Japan, 1984) Shimazaki and Sato (1984) Production of bilirubin-oxidase Takara Shuzo Co. Ltd (Japan, 1986; Matsui, Sato, and Nakajima (1986) and Susumu, Satou, and Takako (1984) Production of cholesterol oxidase and its use in modification of natural occurring spirostanes Toejepast Natuur Ondersoek, (Netherland, 1988 The reduced capacity of S. commune to degrade the lignin components from lignocellulose has been previously reported (Floudas et al., 2015;Horisawa et al., 2015;Zhu et al., 2016) in agreement with the lack of genes encoding class II peroxidases from the AA2 family . Interestingly, the main enzymatic activity detected in culture supernatants from S. commune grown in lignocellulosic substrates is hemicellulolytic (Zhu et al., 2016). Nevertheless, the production of xylanase activity in this fungus is under the regulatory control of cellulosic degradation byproducts (Haltrich & Steiner, 1994). Xylan or galactomannan do not induce xylanase or mannanase activities when provided as sole carbon source.
The analysis of the genome of S. commune has shown that noncellulosic polysaccharide-degrading enzymes are more abundant when compared to other model of lignocellulose decomposers . This fungus contains an extensive repertoire of xylan and pectin glycoside hydrolases as shown in Table 3, indicating a great potential for hemicellulose deconstruction. When compared with other basidiomycete fungi (the white-rot Phanerochaete chrysosporium and Ceriporiopsis subvermispora and the brown-rot Gloeophyllum trabeum), S. commune achieved the highest xylanase activity when growing on a lignocellulosic substrate (Zhu et al., 2016). Similarly, a crude enzymatic cocktail obtained from a solid-state fermentation of S. commune was more effective than a commercial enzyme cocktail from Trichoderma longibrachiatum in terms of reducing sugar release from pretreated lignocellulosic biomass (Zhu et al., 2016). In this case, while cellulolytic activities where similar, the level of xylanases was significantly higher in the S. commune enzymatic cocktail.

| LIGNIN DEGRADING ENZYMES AND ALTERNATIVE BIOTECHNOLOGICAL APPLICATIONS OF SCHIZOPHYLLUM COMMUNE
In addition to the cellulases and xylanases studied in S. commune (Table 1) In this context, there is an increase in the biotechnological significance of S. commnune in the last 15 years. Related to its genome, its enzymatic complexes and its biological versatility, more than The fields of innovation-patentability-biotechnology where the versatility of S. commune is currently being applied are summarized in Table 4.
In the field of lignocellulosic biomass, more than 1,100 patents (altogether applied and granted) related to the use of S. commune were reported in the last two decades (Gupta, 2016), including bio-fuel′s production and biomass derivatives. As stated before, it is recognized that economic utilization of widely distributed lignocellulosic biomass as a feedstock for the eco-sustainable production of biocarburants, biodiesel, molecular scaffolds, biomaterials, fuels, and chemicals with high-added value would represent a conceptual and methodological change in the strategic utilization of natural raw materials, allowing sustainable resources to be substituted for, and compete with, petroleum-based products.
In other research areas, S. commune has also been a subject of interest. For example, a nematicidal and bacteriostatic fumigant formulation has been prepared from an S. commune strain where the main bioactive component is β-bisabolol. This composition is environmentally friendly and shows a very wide spectrum of action (Kaiyin, 2017 bioprocessing and related processes (bio-pretreatment of agrowastes, biological saccharification, gelatin production, obtention of biofuels and bio-derivatives at the bio-refinery level, generation F I G U R E 3 Europe Patent Office patents . Distribution by technological application fields of enzymatic complexes for treatment of lignocellulosic materials and wastes, functional biofibers and bio-oligomers, solid fermentation, pith and lignin degradation, bio-oriented decomposition, etc.) the patents correspond to 22%, and, in the field of applied secondary metabolites, with great-added value, and utilization of enzymatic complexes (laccases, cellulases, xylanases, esterases, oxidases, production of glucans and polysaccharides with different molecular weights, ergothioneine, schizophyllan, glucosone, xylitols, trehalose, pantolactone, retinoids, organic acids, etc.), the patents number account for 31% of the total. It is noteworthy that the observed application-development trends will be maintained in the next 2-5 years, which supports the biotechnological versatility and applicability of this basidiomycete.

| CON CLUS IONS
Schizophyllum commune is a fungus that has a quite complete enzymatic set that can be used for diverse areas in the biotechnological field. Its genome description as well as the recently published works and patents related to this fungus, demonstrates part of the biotechnological potential that S. commune possess. This review is the first to concentrate most of the work that has been done with S. commune in the subject of plant biomass exploitation and the enzymes involved in its degradation, with a view to its future implementation in bio-refineries, pollutant degradation, formulation of enzymatic cocktails, bioconversion of agricultural by-products, as an example. Additionally, S. commune is a good source for hydrolytic, non-hydrolytic and oxidative enzymes which can help to understand the processes by which this fungus is capable of using the carbohydrates and phenolic compounds in the vast diversity of woods it can colonize, since classical genetics and genetic engineering techniques are available for S. commune.

ACK N OWLED G M ENTS
We are thankful to the National Council for Science and Technology (CONACyT) since OETH received a scholarship during the elaboration of this work. We also thank the financial support received from SEP-PRODEP-UAEMOR-PITC-381. RABG received a postdoctoral fellowship from the Quebec Government.

CO N FLI C T O F I NTE R E S T
Authors declare that there are no conflicts of interest.