Multi-omics analysis provides insights into lignocellulosic biomass degradation by Laetiporus sulphureus ATCC 52600

Background Wood-decay basidiomycetes are effective for the degradation of highly lignified and recalcitrant plant substrates. The degradation of lignocellulosic materials by brown-rot strains is carried out by carbohydrate-active enzymes and non-enzymatic Fenton mechanism. Differences in the lignocellulose catabolism among closely related brown rots are not completely understood. Here, a multi-omics approach provided a global understanding of the strategies employed by L. sulphureus ATCC 52600 for lignocellulose degradation. Results The genome of Laetiporus sulphureus ATCC 52600 was sequenced and phylogenomic analysis supported monophyletic clades for the Order Polyporales and classification of this species within the family Laetiporaceae. Additionally, the plasticity of its metabolism was revealed in growth analysis on mono- and disaccharides, and polysaccharides such as cellulose, hemicelluloses, and polygalacturonic acid. The response of this fungus to the presence of lignocellulosic substrates was analyzed by transcriptomics and proteomics and evidenced the occurrence of an integrated oxidative–hydrolytic metabolism. The transcriptomic profile in response to a short cultivation period on sugarcane bagasse revealed 125 upregulated transcripts, which included CAZymes (redox enzymes and hemicellulases) as well as non-CAZy redox enzymes and genes related to the synthesis of low-molecular-weight compounds. The exoproteome produced in response to extended cultivation time on Avicel, and steam-exploded sugarcane bagasse, sugarcane straw, and Eucalyptus revealed 112 proteins. Contrasting with the mainly oxidative profile observed in the transcriptome, the secretomes showed a diverse hydrolytic repertoire including constitutive cellulases and hemicellulases, in addition to 19 upregulated CAZymes. The secretome induced for 7 days on sugarcane bagasse, representative of the late response, was applied in the saccharification of hydrothermally pretreated grass (sugarcane straw) and softwood (pine) by supplementing a commercial cocktail. Conclusion This study shows the singularity of L. sulphureus ATCC 52600 compared to other Polyporales brown rots, regarding the presence of cellobiohydrolase and peroxidase class II. The multi-omics analysis reinforces the oxidative–hydrolytic metabolism involved in lignocellulose deconstruction, providing insights into the overall mechanisms as well as specific proteins of each step. Supplementary Information The online version contains supplementary material available at 10.1186/s13068-021-01945-7.


Background
Wood-decay basidiomycetes are essential for the carbon cycle because of their highly specialized biomass degradation. Their metabolic systems include carbohydrateactive enzymes (CAZymes), but also non-CAZymes and other associated non-enzymatic compounds. This ability allows them to be potentially used for the production of value-added biocompounds derived from lignocellulosic biomass [1][2][3].
Traditionally, wood-decay basidiomycetes have been classified as brown-rot or white-rot based on the capacity to degrade plant cell wall components. Accordingly, brown rots degrade cellulose and hemicellulose while only modifying lignin. These two decay modes have been distinguished based on the reduction or absence of some enzymes, such as ligninolytic peroxidases (PODs) class II (manganese-, lignin-and versatile-peroxidases), as well as enzymes involved in cellulose degradation such as cellobiohydrolase (CBH), lytic polysaccharide monooxygenase (LPMO) and cellobiose dehydrogenase (CDH) [3,4]. To compensate for the paucity of cellulolytic enzymes, some brown rots employ mechanisms for endoglucanase overproduction [5]. The lignocellulose degradation performed by brown-rot fungi involves chemical, biological and spatial relationships between fungal hyphae and the plant cell wall to perform a two-step mechanism: earlier lignocellulose oxidative (LOX) degradation mediated by Fenton reaction (H 2 O 2 + Fe 2+ → Fe 3+ + OH·) followed by a late hydrolytic mechanism. Key requirements for Fenton systems include mechanisms for extracellular peroxide production and iron reduction, involving extracellular fungal enzymes and metabolites, to generate reactive oxygen species (ROS) [6][7][8][9].
Most brown-rot agaricomycetes belong to the order Polyporales Gäum. Within this order, most brown-rot species belong to the "Antrodia clade", which includes the families Dacryobolaceae Jülich, Fomitopsidaceae Jülich, Laetiporaceae Jülich, and Sparassidaceae Herter, as well as a few unsolved groups [10]. L. sulphureus is considered a cosmopolitan species causing brown cubical heart rot in many deciduous and coniferous trees [11,12]. It is known to produce metabolites with antioxidant and antimicrobial properties [13,14] and natural dyes [15,16] in addition to the potential for bioremediation of treated wood [17] and decolorization of textile effluents [11]. During wood decay, L. sulphureus causes higher polysaccharide weight loss than lignin loss [18]. This fungus has superior potential to produce cellulolytic and hemicellulolytic enzymes in comparison to other representative brown rots [19] and the enzymatic repertoire secreted in the presence of carboxymethyl-cellulose (CMC) was analyzed by mass spectrometry [20]. However, the potential for lignocellulose degradation has not been explored at multi-omics level.
Omics approaches allow a deep understanding of the biology of an organism, including its behavior during growth on complex plant biomass [21]. In this work, genome sequencing followed by transcriptomic and proteomic analysis provided a global understanding of the strategies employed by L. sulphureus ATCC 52600 in the degradation of lignocellulosic by-products derived from sugarcane and Eucalyptus. In addition, a commercial enzymatic cocktail supplemented with the L. sulphureus secretome was evaluated for saccharification of hydrothermally pretreated grass (sugarcane straw) and softwood (pine).

Sequencing, annotation, and phylogenetic analysis of L. sulphureus ATCC 52600
The L. sulphureus ATCC 52600 genome sequence was assembled by a combination of paired-end (45,000,408 sequences) and mate-pair libraries (13,294,823 and 13,280,039 sequences), corresponding to 43.4 Mb ( Table 1).
Genomic features were similar to L. sulphureus var. sulphureus v1.0 [22]. Comparative analysis showed the strains sharing 8419 clusters of orthologous genes, with 7724 single-copy genes, which accounted for 60% and 56% of all coding sequences for the strains ATCC 52600 and var. sulphureus v1.0, respectively. The phylogenomic analysis considering whole-genome information strongly supported monophyletic clades for all families within the order Polyporales (Fig. 1). The strain ATCC 52600 clustered with L. sulphureus var. sulphureus v1.0 and Wolfiporia cocos in the family Laetiporaceae, which, in turn, appears as a sister clade of Fibroporiaceae (Fibroporia Conclusion: This study shows the singularity of L. sulphureus ATCC 52600 compared to other Polyporales brown rots, regarding the presence of cellobiohydrolase and peroxidase class II. The multi-omics analysis reinforces the oxidativehydrolytic metabolism involved in lignocellulose deconstruction, providing insights into the overall mechanisms as well as specific proteins of each step. Keywords: Basidiomycetes, Brown-rot, Genome, Transcriptome, Proteome, CAZymes, Fenton reaction, Sugarcane by-products radiculosa) and closely related to Dacryobolaceae (Postia placenta).

L. sulphureus ATCC 52600 displays a broad-range carbohydrate metabolism and slow glucose consumption
The presence of transporters for different carbohydrates such as glucose, mannose, and trehalose in the genome motivated further analysis of the L. sulphureus ATCC 52600 primary metabolism (Additional file 2: Table S1). The strain was able to grow on mono-, di-and polysaccharides (Additional file 1: Figure S2A), and faster colony growth was verified on pectin, followed by galacturonic acid, xylan, arabinose, and galactomannan (Additional file 1: Figure S2B). The growth analysis in liquid medium with glucose showed an extended lag period, with glucose consumption starting after 48 h of cultivation, which then decreased at a slow rate to around 40% at 168 h of cultivation (Additional file 1: Figure S2C). These data raised questions about the biological behavior of this basidiomycete growing on complex carbon sources in terms of protein expression and secretion, especially because of the presence of some non-canonical brownrot CAZymes such as CBH and AA2 peroxidase in the genome.
A set of genes encoding non-CAZy enzymes and proteins with a predicted function in the oxidative mechanism and Fenton reaction were also regulated, corresponding to 159 transcripts, out of which 62 were up and 97 downregulated ( Fig. 3a and Additional file 2: Table S2). The importance of this mechanism became clearer when the regulation of individual genes was analyzed, i.e., among the high upregulated transcripts (log2-fold change ≥ 2, n = 43), 67% were associated with oxidative mechanisms, including both AAs and non-CAZymes. The remaining CAZymes (33%) were mostly hydrolases including miscellaneous hemicellulases (acting on glucan, mannan, galactan), amylases, pectinase, and chitinases. Remarkably, the top upregulated transcripts included a series of AA oxidoreductases belonging to the families AA3_3, AA1_2, AA6, and AA7, as well as non-CAZy oxidoreductases, dehydrogenases, cytochrome, and enzymes involved in LMW metabolism. In turn, transcripts of predicted cellulose-or xylan-active enzymes were absent (Fig. 3b).

Proteins secreted by L. sulphureus ATCC 52600 during cultivation on pretreated plant biomass
The exoproteomes of L. sulphureus ATCC 52600 cultivated on lignocellulosic biomass were analyzed by tandem mass spectrometry. A total of 3328 spectra were identified, accounting for 112 proteins. This set of proteins was composed of 42 CAZymes, 8 peptidases/ proteases, 8 non-CAZy oxidoreductases, 7 esterases, 5 dehydrogenases, 32 miscellaneous proteins/domains (denominated "others"), and 10 hypothetical proteins of unknown function (Additional file 1: Figure S1C and Additional file 2: Table S3). Of note, most of the proteins identified were predicted with SP.

Performance of the L. sulphureus ATCC 52600 secretome on biomass conversion
Enzymatic activity profiles were evaluated on the L. sulphureus ATCC 52600 secretomes and most of the identified activities were found at higher levels on SCB. Activities were detected on arabinoxylan, β-glucan, starch, and xylan from beechwood (Additional file 1: Figure S3), corroborating the enzymes identified in the  Table S3). The secretome produced on SCB was then applied for supplementing commercial enzymatic cocktails in the saccharification of pretreated lignocellulosic biomass such as sugarcane straw (grass) and pine (softwood).
Glucan conversion obtained with sugarcane straw ranged from 40 to 55% by using the commercial cocktail at 85% or 100% enzyme load, respectively. Replacing 15% of the commercial cocktail with the secretome produced on SCB increased the glucan conversion by 7% (Fig. 5a). In turn, no differences were observed in the xylan conversion, which was around 30% (Fig. 5b). Saccharification of pine lignocellulose was noticeably less efficient for both glucan (12%) and xylan (around 8%) conversion, and unresponsive to the enzymatic supplementation with the L. sulphureus secretome.

L. sulphureus ATCC 52600 genome does not resemble typical brown-rot fungi
Genomic sequencing of filamentous fungi followed by transcriptomic and proteomic approaches has been widely employed to understand the strategies of microorganisms to degrade plant biomass [23][24][25][26][27][28][29]. Overall, the L. sulphureus ATCC 52600 genome revealed only subtle differences compared to the previously sequenced L. sulphureus var. sulphureus v1.0 [22], indicating that the strains might have undergone some changes in their ecological niches to shape their genomes to the environmental conditions. Our phylogenetic analysis, providing high resolution on the evolutionary history of organisms by considering whole-genome information [29], complements the previous phylogeny of the order Polyporales [10]. The phylogenetic tree (Fig. 1) strongly supports monophyletic clades for the families within the order Polyporales. L. sulphureus ATCC 52600 clusters with L. sulphureus var. sulphureus v1.0 and W. cocos giving further support to the existence of the family Laetiporaceae Jülich, as previously proposed [10] and currently present in Mycoguide, but retrieved as an invalid name in Myco-Bank and Index Fungorum.
The genomic CAZyme content in both L. sulphureus strains and the closely related brown-rot Polyporales shows a typical number of GHs, CEs, PLs, and GTs compared to W. cocos, P. placenta and F. radiculosa, whereas a lower number of CAZymes, particularly GHs, were observed in comparison with Fomitopsis pinicola (Fomitopsidaceae). In turn, L. sulphureus ATCC 52600 shows a higher AA content than in the other genomes [9]. Additionally, the genome presents several similarities with other brown-rot genomes associated with evolutionary reductions and losses in key enzymes involved in biomass Significance was analyzed using two-way ANOVA with Tukey's test relative to the reference reaction "85% Celluclast" (95% confidence interval) and is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001 breakdown, especially cellulases and lignin-modifying enzymes [3]. Accordingly, it presents a reduced number of genes coding CAZymes from the families GH1, GH3, GH5, GH7, GH10, AA9, and CE1 along with the absence of GH6, GH11, AA3_1, CBM1, and CE15 (Additional file 2: Table S1).
Considering these reductions or absences, other enzymes may also be necessary to achieve an effective breakdown of cellulose and hemicellulose, such as the AA9 and AA14 LPMOs. AA9s perform oxidative cleavage on cellulose and other glucans with great importance in lignocellulose degradation [30], presenting an average number of 3 genes in Polyporales genomes [3,26]. The recently established family AA14 also groups LPMOs that are widespread in fungi. Within the order Polyporales, there are 4.5 and 2.5 AA14 coding genes on average in white and brown-rot genomes, respectively [31]. This reduction pattern can also be observed for other gene reductions associated with the brown-rot lifestyle evolution. One characterized AA14 member from the whiterot Pycnoporus coccineus presents oxidative activity on xylans of xylan-coated cellulose fibers [31], and shares 48.8% identity with the L. sulphureus ATCC 52600 AA14 LPMO (Additional file 1: Figure S4).
Regarding the enzymes involved in the oxidative mechanism, AA3_1 CDHs are absent in L. sulphureus, as verified in P. placenta, W. cocos, and F. pinicola [32]. In turn, a large number of genes coding for AA3_2 (aryl alcohol oxidase and glucose 1-oxidase) and AA3_3 (alcohol oxidase) was identified, and the products H 2 O 2 (reduction of oxygen by oxidases) and hydroquinones (reduction of quinones) can support other enzymes that are important for lignocellulose deconstruction [33]. Similarly, AA5_1 glyoxal oxidases and AA6 benzoquinone reductase, which are also involved in Fenton reagents generation [34][35][36], were identified (Additional file 2: Table S1). Notably, the absence of CDH may also suggest the presence of other redox partners for the AA9 and AA14 LPMOs, such as AA3_2 flavoenzymes [37] and GMC oxidoreductases, among others [38], or the peroxide production might be driving LPMOs reaction [39].
The L. sulphureus genome also revealed some distinctions in the lignocellulolytic repertoire. For example, the well-known lack of cellulases in brown-rot fungi is generally attributed to a reduced number of GH6/GH7 CBHs [32,40,41], which are absent in brown-rot Polyporales [42]. Our sequencing, however, identified one putative GH7 CBH (g8442) in the L. sulphureus ATCC 52600 genome, in accordance with a GH7 CBH previously identified in the secretome of L. sulphureus growing on CMC [20]. Sequence analysis shows these enzymes sharing more than 90% identity, and the phylogeny using predicted and characterized fungal CBHs reveals 65% similarity with other fungal CBHs (Additional file 1: Figure S5). Additionally, analysis of 42 fungal genomes indicates that brown rots generally have a reduced number of GH45, in a 3:1 ratio in comparison with white rots [9]. Our initial search parameters identified one putative GH45 (g10751), coinciding with a GH45 (ID 174,393) previously identified in the L. sulphureus secretome [20]. These sequences share 92.5% identity; having an expansin domain predicted by InterPro v.78.1 [43], despite the previous classification as GH45 class C [20]. Expansins are closely related to GH45 endoglucanases and have been widely found in brown-rot strains [9], playing an important function in reducing biomass recalcitrance, consequently increasing the deconstruction of lignocellulose in synergism with cellulases [44].
Lignin degradation and the importance of different lignin-active enzymes in brown rots is a matter of debate, but it is widely recognized that brown rots present a reduced number of laccases and absence of PODs class II in comparison with white-rot strains [8,45,46]. L. sulphureus ATCC 52600 has AA1_1 and AA1_3 laccases, similar to F. pinicola, P. placenta, and W. cocos [47]. Additionally, 13 predicted PODs were identified in the L. sulphureus ATCC 52600 genome, and two of them with predicted AA2 domain. InterPro annotation classified one of them as an intracellular POD class I, while the other (g11846) was classified as a fungal ligninase/POD class II with a predicted SP. BLAST search retrieved 87% and 66% identity with PODs class II from L. sulphureus var. sulphureus v1.0 and W. cocos MD-104SS10 v1.0, respectively. POD class II has been reported as a single copy in P. placenta, W. cocos and F. pinicola genomes [32], and the P. placenta peroxidase (Ppl44056) was classified as a basal peroxidase, not closely related to LiP and MnP [48]. Laccases in Polyporales are multigenic [49] and have been characterized as functional enzymes in P. placenta and F. pinicola [50][51][52], playing a role in wood decay performed by P. placenta [50]. Significant lignolysis has been observed in Gloeophyllum trabeum (Gloeophyllales) and P. placenta without considering the involvement of PODs class II [6,53]. Nevertheless, the biological importance or the precise role of these PODs II found specifically in L. sulphureus and other closely related brown rots are uncertain since these enzymes have not been characterized to date.

Insights into the L. sulphureus ATCC 52600 biomass deconstruction mechanism
Several omics studies analyzing brown-rot fungi with significant taxonomic and niche distances such as W. cocos, F. radiculosa, P. placenta, G. trabeum, and Serpula lacrymans (Boletales), cultivated in different conditions show the common presence of a two-step mechanism involved in biomass deconstruction [8,25,48,[54][55][56][57][58][59][60]. The initial oxidoreductive step is estimated to persist for 48 h [8], which can be correlated with both the observed slow growth of P. placenta in cellulose and spruce [56] and the L. sulphureus growth and glucose consumption in liquid medium (Additional file 1: Figure S2C).
The transcriptome data of a short cultivation period reveal a series of upregulated genes related to the oxidative mechanism, probably induced by the recalcitrance of the non-pretreated sugarcane bagasse (Fig. 3 and Additional file 2: Table S2). The most upregulated transcripts include alcohol dehydrogenase, cytochrome P450, aldo/ keto reductases, and redox genes involved in the generation of hydrogen peroxide, while hydroquinone dehalogenase is involved in hydroquinone production that initiates Fenton reaction by carrying Fe 3+ [61]. Moreover, the presence of AA6 quinone reductases suggests that this enzyme takes part in the quinone redox cycle supporting Fenton chemistry, as previously observed in P. placenta [48], while also playing a role in the detoxification process [5]. Such observations are consistent with a biodegradative role of Fenton chemistry occurring during early cultivation of L. sulphureus on SCB, as verified in other brown-rot transcriptomes [9,62,63].
Regarding CAZymes (Fig. 3a and Additional file 2: Table S2), previous brown-rot transcriptomic studies similarly revealed a small set of cellulases and hemicellulases with predicted activity on glucans and mannans [8,48]. The upregulation of some cellulases and hemicellulases supports the existence of inducing mechanisms, which may depend on substrate exposure and availability but operating differently than reported for white-rot basidiomycetes or ascomycetes [56]. Additionally, the upregulation of two AA1 laccases indicates an ability to partially oxidize lignin. On the other hand, two other AA1 laccases, as well as two non-CAZy peroxidases were downregulated, so the importance of ligninases for this fungus remains unclear ( Fig. 3a and Additional file 2: Table S2). Transcripts of AA9 and AA14 LPMOs were upregulated, but not secreted, corroborating the concept of LPMOs being produced by fungi during early biomass degradation [62,64,65]. Our data indicate that L. sulphureus adopts mechanisms to integrate enzymatic and non-enzymatic systems at initial stages of brown-rot decay, as previously reported in G. trabeum [65]. Of note, the biological importance of LPMOs for brown-rot fungi remains unclear since their secretion has only been identified in G. trabeum growing on lignocellulose [48].
Furthermore, the growing of L. sulphureus on pectin and the upregulation of pectinases transcripts (Additional file 1: Figure S2A and B and Additional file 2: Table S2) support the mechanism in which pectin degradation also occurs in the early stage of degradation as previously observed in P. placenta and G. trabeum. Pectin removal by pectinases may facilitate the access of other enzymes to the plant cell wall components [8,56,57].
In contrast to the transcriptome, our secretome data (7-day cultivation) represents a late hydrolytic decay profile [56], which is supported by the absence of AAs in the secretome produced on Avicel. A core set of constitutive CAZymes was identified, comprising some GHs with predicted activity on cellulose and a wide diversity of GHs acting on glucans, xylan, mannans, trehalose, starch, and chitin (Additional file 2: Table S3). Apart from the xylan-active enzymes, the hemicellulase set is very similar to the profile observed in the transcriptome. This complete set of hemicellulases found in brown rots [56] allows the fungus to obtain energy sources from diverse substrates, providing an increase in survival capability under different environmental conditions.
Additionally, regulatory mechanisms may take place after the sensing and transport of inducers, resulting in the secretion of a repertoire of CAZymes targeted to substrate degradation. In that sense, differences in the enzymatic arsenal can be observed in the secretomes of L. sulphureus produced on grass and wood-derived substrates, which typically present different compositions [66,67]. The secretome produced on SCB showed the highest diversity of upregulated proteins probably due to substrate recalcitrance and pretreatment characteristics [68,69].
Endoglucanases are poorly secreted by L. sulphureus, apparently playing a minor role in cellulose degradation, despite the importance of processive endoglucanases in brown rots [70]. Two GH3 β-glucosidases are upregulated on SCB and notably the GH7 CBH undergoes upregulation exclusively on Avicel. These data, in addition to the basal secretion of some CAZymes commonly found in all substrates, show that CBH is inducible and it is not under carbon catabolite repression, as verified for the endoglucanase from G. trabeum [71] or cellulases from P. placenta [56]. However, the gene encoding GH7 CBH is not differentially expressed (transcriptomeearly stage), and the secretion of endoglucanases and β-glucosidases as well as oxidative agents, may compensate for that low expression in early stages [4,6].
In addition to the constitutive hemicellulases, a diversity of enzymes acting on glucan and mannan (mannosidases and α-galactosidases) were upregulated at both early and late response to biomass degradation (Additional file 2: Tables S2 and S3) corroborating a natural preference of brown rots for softwoods [1,3,7,59]. There is evidence that hemicellulose loss progresses faster than cellulose loss in coniferous wood decay performed by G. trabeum, F. pinicola, W. cocos and L. sulphureus [72,73]. Additionally, our secretome data show that L. sulphureus targets hemicellulose as part of the hydrolytic late response. Several enzymes active on xylan, the main hemicellulose in grasses [74], were secreted by L. sulphureus, i.e., one GH10 xylanase is upregulated on Avicel and SCB, while the production of another GH10 xylanase is constitutive. Also, one β-xylosidase is upregulated on SCB, while two α-L-arabinofuranosidases are widely secreted on the polymeric substrates (Additional file 2: Table S3). Moreover, transcripts of other arabinoxylan-degrading enzymes do not show early upregulation; rather, one GH30 xylanase and one GH51 arabinofuranosidase are downregulated (Additional file 2: Table S2). This result shows that L. sulphureus can adapt the metabolism to the degradation of a grass substrate, despite other brown rots from the Antrodia clade have been reported to be inefficient in the degradation of corn stalk [75].
Despite the ability of L. sulphureus to grow on xylan and galactomannan (Additional file 1: Figure S2A and B), in addition to the presence of some cellulases and several hemicellulases in the late-response secretome (Additional file 2: Table S3), biomass conversion was low, especially for softwood (Fig. 5). The lack of mannanases in the commercial cocktail [76] and the SCB secretome may be a possible explanation for the ineffective degradation of the pine biomass. Indeed, brown-rot fungi are known to grow and modify pine and other softwoods [73,77,78]; however, softwood is among the most recalcitrant lignocellulosic substrates for enzymatic processes, requiring severe pretreatment conditions as well as higher enzyme doses than hardwood or grass substrates [79]. Lignin content, larger amounts of extractive components, and smaller pore size are additional characteristics that may be further contributing to the poor degradation of this lignocellulose [80].
In Fig. 6, an overview of the L. sulphureus strategies for biomass deconstruction is proposed based on our multi-omics data. Our results are consistent with a temporal two-step oxidative-hydrolytic mechanism for the degradation of lignocellulose, while also demonstrating that this fungus does not resemble typical brownrot fungi in many aspects, thus contributing to the weak dichotomy between white-and brown-rot strains, as previously proposed [26]. Additional data applying biological approaches such as gene deletion and analysis of wood decay, as well as biochemical characterization of the enzymes would contribute to further address this question. Fig. 6 Biomass degradation mechanism from L. sulphureus ATCC 52600. Multi-omics analysis showing the range of CAZymes induced in response to sugarcane lignocellulose. The scheme represents the main CAZymes found in the transcriptome (in the basidium stem) and secretome analysis responsible for lignocellulose deconstruction and lignin modification by the oxidative mechanism, involving CAZymes, low-molecular-weight (LMW) compounds, and Fenton reaction. In parallel, monomers released from holocellulose are metabolized, unlike lignin, which remains partially degraded

Conclusions
Genome sequencing and analysis of expression and secretion patterns contributed to elucidate the mechanism involved in lignocellulose degradation by L. sulphureus ATCC 52600. In many aspects, this brown-rot fungus presents similarities with other model brown rots, while not resembling typical brown rots, especially due to the notable presence of cellobiohydrolase and POD class II. The transcriptomic analysis using highly recalcitrant biomass at a short cultivation period demonstrated the presence of early oxidative response, as well as other hallmarks of an early response such as the upregulation of pectinases and oxidative enzymes, including LPMOs. The late response was evaluated by proteomic analysis of secretomes produced on cellulose, and lignocellulose from grass (sugarcane bagasse and straw), and hardwood (Eucalyptus). Overall, the secretome profiles showed a common set of CAZymes in different conditions, with only subtle differences in the secretion of specific enzymes. Some cellulases displayed constitutive secretion while a more complex regulatory mechanism may be occurring for enzymes acting on xylan degradation. Another remarkable characteristic is the absence of AAs in the degradation of crystalline cellulose, but not in the degradation of lignocellulosic substrates.

Strain maintenance
L. sulphureus ATCC 52600 was purchased from Fundação André Tosello (CCT 4694). The strain was routinely maintained on solid media composed of 20 g/L malt extract and 2 g/L yeast extract and incubated for 7-10 days at 30 °C.

DNA extraction and sequencing
DNA extraction from mycelia was performed using phenol-chloroform, followed by RNAse treatment. High-quality DNA was obtained using the DNeasy Kit (Qiagen). Three Illumina libraries were constructed, a paired-end library with a 300-bp insert and two mate-pair libraries with 5-7 and 8-11 kb, according to the manufacturer's instructions. The libraries were sequenced on an Illumina HiSEq 2500 platform.

Phylogenetic analysis
The phylogenomic relationship of L. sulphureus ATCC 52600 and its closest described relatives of the family Laetiporaceae was determined based on orthologs singlecopy genes using FastOrtho tool (https:// github. com/ olson anl/ FastO rtho). The protein sequences of each 601 single-copy orthologous genes present in 31 basidiomycete genomes closely related to the family Laetiporaceae and L. sulphureus ATCC 52600 were aligned by Mafft v.7.299 [94] and the resulting individual alignments were concatenated to create a supermatrix using FASconCAT-G v.1.02 [95]. Evolutionary distance was inferred using maximum likelihood with RAxML v.8.2.0 [96], implementing PROTGAMMAWAG model and performing 1000 bootstrap replicates to evaluate the reliability of the reconstructed phylogenetic tree.

Cultivation conditions for transcriptome analysis
Pre-inoculum, consisting of 15 discs (8 mm diameter) of L. sulphureus ATCC 52600 pre-cultivated on agar plates, was inoculated into 100 mL of liquid medium and incubated under 180 rpm for 7 days at 30 °C. Mycelia were then filtered and washed with water and transferred to liquid medium containing 1.0 g of in natura sugarcane bagasse and 100 mL of medium pH 7.0 composed of 6 g/L (NH 4 ) 2 SO 4 , 1 g/L KH 2 PO 4 , 1 g/L KCl, and 1 g/L MgSO 4 . Cultivation was performed under 180 rpm for 24 h at 30 °C. Mycelia and substrate mixtures were collected by filtration, washed with sterile water, manually dried in filter paper, and stored at -80 °C before RNA extraction. Mycelium from the pre-inoculum was used as a standard before induction (T 0 ).

RNA extraction and sequencing
The mycelium was ground with liquid nitrogen and total RNA extraction was performed with mirVana ™ Total Isolation Kit (Thermo Fisher), according to the manufacturer's instructions. The resulting solution was treated with DNAse (DNA-Free RNA Kit, Zymo Research) and purified with RNeasy Kit (Qiagen), and quality was verified using RNAnano Bioanalyzer 2100 chip (Agilent). cDNA libraries were prepared according to the manufacturer's instructions and sequenced on the Illumina HiSEq 2500 platform.

Bioinformatics analysis of RNA-seq data
Reads were processed as described previously for the genome libraries and evaluation and filtration of the rRNAs were performed using SortmeRNA. The filtered data were mapped into the L. sulphureus ATCC 52600 reference genome sequenced in this work using the Tophat2 algorithm [97]. Differential gene expression analysis was based on counting data and performed with the Bioconductor DESeq2 package [98] using the R platform, by paired comparisons against the control condition. Transcripts showing differential expression (log2fold change ≥ 1 and ≤ − 1) relative to the non-induced condition (T0) were determined by applying p ≤ 0.05 as the threshold.

Carbohydrate metabolism and glucose consumption
Mycelia discs were excised from the border of the colony growing on potato dextrose agar plates and transferred to the center of minimal medium agar plates [99] supplemented with 1% (w/v) of the following substrates: glucose, arabinose, galacturonic acid, xylose, lactose, cellobiose, galactose, xylan from beechwood, pectin from citrus, and galactomannan from carob. Cultivation was performed in six replicates for 7 days at 30 °C, and growth rates were estimated from the daily measurement of the colony area using the software ImageJ 1.52a [100]. For cultivation in liquid medium, 15 mycelial discs were transferred into 250-mL Erlenmeyer flasks containing 100 mL of liquid minimal medium pH 5.5 supplemented with 1% (w/v) glucose for 7 days under static conditions at 30 °C. Cultivation was performed in triplicate and samples were taken at 6,12,24,48,72,96, and 120 h of cultivation. Residual glucose was measured by high-performance liquid chromatography (HPLC), as described below.

Cultivation conditions for proteomic analysis Pre-inoculum
L. sulphureus ATCC 52600 was grown on potato dextrose agar plates pH 5.5 at 25 °C. After 7 days of cultivation, 15 mycelium discs (8 mm diameter) were excised from the colony border and transferred to 250-mL Erlenmeyer flasks containing 50 mL of liquid medium composed of 0.5 g/L NH 4 CH 3 CO 2 , 0.5 g/L NaNO 3 , 0.5 g/L MgSO 4 , 0.2 g/L Na 2 HPO 4 , 0.8 g/L KH 2 PO 4 , 4.0 g/L yeast extract, and 10.0 g/L glucose. The pre-inoculum was incubated for 21 days under static conditions at 30 °C.

Cultivation
Pre-grown mycelia were removed by filtration, washed with distilled water, transferred to a 50-mL conical tube, and manually macerated with 2 g of glass beads. The macerated mycelia were then transferred to 250-mL Erlenmeyer flasks containing 50 mL minimal medium pH 5.5 supplemented with 1% (w/v) steam-exploded sugarcane bagasse (SCB), steam-exploded sugarcane straw (SCS), steam-exploded Eucalyptus residue (Eucalyptus grandis), Avicel ® PH-101 (Sigma), and glucose. Cultivation was performed in triplicate under static conditions for 7 days at 30 °C.
Spectra data were annotated based on the L. sulphureus ATCC 52600 genome. The adjusted conditions to validate protein identification were protein probability thresholds higher than 99% and at least 2 different peptides identifying a protein, each with 95% certainty. Once the parameters were defined, a 0.0% false discovery rate (FDR) was generated and spectrum count data were analyzed in a semi-quantitative method. Spectra counts are equivalent to the total number of standard spectra assigned to each protein and are commonly used to determine relative abundance [104]. As the spectra counting methodology was used for analysis, FDR was designated as one of the parameters to determine the reliability of the experimental data. FDR was defined as the expected correspondent percentage of each peptide spectrum [105]. Initially, a score was assigned to each peptide (primary analysis) performed with Mascot Distiller software. Subsequently, Mascot data were analyzed by Scaffold 4 Proteomic software attributing the number of spectra to the abundance and FDR to the reliability of the results. By using average spectra outputs from Scaffold 4, differentially secreted proteins were identified according to their spectra counting and quantitative values were applied to normalize the counts. The statistical analysis of the spectra was performed by the t-test (p ≤ 0.05) and fold change by category, using data from cultivation with glucose as standard.

Activity on different substrates
Enzymatic assays were performed using 50 μl of the following substrates: 5 mM 4-nitrophenyl β-D-cellobioside, 4-nitrophenyl β-D-xylopyranoside and 4-nitrophenyl β-D-glucopyranoside, and 0.5% (w/v) polygalacturonic acid, starch, CMC, xylan from beechwood (Sigma), wheat arabinoxylan, β-glucan and galactomannan (Megazyme). Assays were performed using 1 μg protein of the concentrated secretomes in 50 mM ammonium acetate buffer pH 5.5 for 4 h at 50 °C. Assays with the synthetic substrates were stopped with 100 μl of 1 M sodium bicarbonate and the released 4-nitrophenolate was measured at 405 nm. Reactions with polymeric substrates were stopped with 100 μl of 3,5-dinitrosalicylic acid (DNS) and the released reducing sugars were measured at 540 nm [106]. All assays were performed in triplicate. One enzyme unit (1 U; μmol/min) corresponds to the amount of enzyme that catalyzes the conversion of one micromole of substrate per minute under the assay conditions.

Enzymatic saccharification
Grass (sugarcane straw) and pine softwood (Pinus sp) lignocellulose were milled (1.0 cm length × 1.0 mm thickness), followed by hydrothermal pretreatment and composition characterization [107,108]. Saccharification reactions were performed in 1 mL working volume with substrate load at 2% (w/w) solids in 50 mM sodium acetate buffer pH 5.0 at 50 °C up to 48 h in a Thermomixer under 1000 rpm agitation. The L. sulphureus secretome produced on SCB (as described above) was evaluated by replacing 15% of protein load from commercial enzymatic cocktails. FPAse activity was previously assayed in the enzymatic cocktail [109,110] and the total protein load (equivalent to 15 FPU) was the combination 5:1 (w/w) Celluclast ® :glucosidase from Aspergillus niger (Merck) per gram of dry substrate (Celluclast ® at 150 mg protein/mL corresponding to 115 FPU/mL). Assays were performed in four replicates and the released sugars were measured by HPLC, as described below. Biomass conversion was calculated using glucan/xylan content in the biomass (pretreated/native) and the anhydrous correction factors of 1.13 for xylose and 1.1 for glucose [108,111,112].

HPLC analysis
Glucose, xylose, and cellobiose were quantified in a liquid chromatography system (Waters 515 Pump, Water 717 plus Injector/Sampler) coupled to an Aminex HPX87H (300 × 7.8 mm) column and equipped with a refractive index (RI) detector (Waters 410). Detector and column temperatures were set, respectively, to 40 and 45 °C; 50 mM H 2 SO 4 was used as a mobile phase at 0.6 ml/min flow rate; and 20 μl injection volume.
Additional file 1: Figure S1. Overview of L. sulphureus ATCC 52600 multi-omics analysis. Distribution of CAZymes and redox Non-CAZymes presented in the (A) genome and (B) transcriptome. (C) Categorization of all protein identified on the secretomes. Figure S2. Analysis of L. sulphureus ATCC 52600 growth on different carbohydrates. (A) Growth for 7 days on agar plates supplemented with different carbohydrates. (B) Growth rate estimated by colony area measurement. Values expressed relative to glucose. (C) Relative glucose consumption measured using HPLC during growth in liquid medium. Growth rates were analyzed using two-way ANOVA with Tukey's test, indicated as follows: *p<0.05, **p<0.01, ***p<0.001. Figure S3. Enzymatic activity profile of L. sulphureus ATCC 52600 secretomes. Enzymatic assays containing the different secretomes (Avicel; SCB: sugarcane bagasse; Eucalyptus: Eucalyptus grandis residue, SCS: sugarcane straw and glucose) were carried out in 50 mM sodium acetate buffer pH 5.5 for 240 min at 50 °C. Activities were analyzed assuming the secretome produced on SCB as control by two-way ANOVA with Tukey's test (95% confidence interval), indicated as follows: *p<0.05, **p<0.01, ***p<0.001. Reducing sugars were measured using the DNS method. Figure S4. Multiple AA14 sequence alignment. The alignment was generated by Clustal using two characterized lytic polysaccharide monooxygenases (LPMOs) from Pycnoporus coccineus CIRM-BRFM 310 (PcAA14A and PcAA14B) and two putative AA14 found in the L. sulphureus ATCC 52600 genome. Red boxes highlight the conserved amino acid residues constituting the histidine brace, a hallmark of LPMOs. Figure  S5. Multiple alignment of GH7 amino acid sequences. (A) Alignment