Enzyme activity measurements
L. squarrosulus was grown in simple synthetic medium with Reactive Black 5 (RB5), an azo dye that induces the production of ligninolytic enzymes. Our previous work on ligninolytic enzymes demonstrated that these enzymes were strongly stimulated in the presence of aromatic compounds and lignocellulose substrates. In particular, the recalcitrant dye RB5 stimulated higher titers of ligninolytic peroxidases. Lignin-degrading enzymes of this fungus, particularly versatile peroxidase, manganese peroxidase and laccase, were strongly induced in the presence of azo dye (Figure 1). [L1] Manganese oxidizing peroxidase activity increased to a maximum of 10 U/ml in induced medium compared to the uninduced medium in addition to laccase activity. The biomass-degrading enzyme activities of cellulase, xylanase, polygalacturonase, mannanase, α-glucosidase, β-glucosidase and xylan esterase were studied to obtain a deeper understanding of the regulation of these enzymes compared to ligninolytic enzymes. Only trivial α-glucosidase activity was observed in induced and uninduced media. The interference of the mannanase substrate locust bean gum with glucose in the culture supernatant impeded the detection of this enzyme.
An increase in cellulase activity was observed on day 7, with higher activity in uninduced medium than in induced medium. The same trend was observed with xylanase, acetyl esterase and polygalacturonase, whereas β-glucosidase activity was higher in induced medium. Although the maximum activity and duration varied for each enzyme, the performance of the uninduced medium was optimal for the production of biomass-degrading enzymes other than ligninolytic enzymes. Ligninolytic enzyme activity was higher in the induced medium, which was attributed to the complex aromatic nature of the azo dye. This evidently signified that the specific activity of ligninolytic enzymes was higher in the induced medium.
RNA extraction and sequencing
cDNA sequencing libraries were prepared from total RNA of late log phase cultures of L. squarrosulus grown in RB5-induced potato dextrose broth. Ten million reads of data were generated from an initial RNA concentration of 125 ng/ml with a mean fragment size of 447 bp. A total of 6,679,162 high-quality paired-end reads were obtained after removal of adaptor sequences and trimming of low-quality bases. The raw reads were aligned de novo through CLC assembly cells, and 25,244 contigs were obtained (Table 1).[L2]
Table 1: Summary of L. squarrosulus transcriptome assembly
Number of contigs
|
25, 244
|
Total size of contigs (bp)
|
2,31,81,074
|
Average Length (bp)
|
918
|
Length SD (bp)
|
856
|
GC content (%)
|
57.93
|
N50 contig length (bp)
|
1,340
|
N50 contig count
|
5,091
|
Functional annotation
Annotation of the sequences based on similarity was performed using BLAST2GO. BLAST2GO was used to determine the putative functions of the transcripts and categorize them by biological process, cellular component and molecular function. To further explore the function and significance of the transcripts, the sequences were analyzed through Function annotator. Functional annotator presented efficient interpretation of the reads on GO terms, enzyme groups, domain identification, subcellular localization, protein secretion, transmembrane proteins and on the taxonomic relationship at different levels. Sequence similarity with the NCBI nonredundant database yielded equivalent sequences with the lowest e-values.
The taxonomic information was furnished based on the proportion of similar sequences in L. squarrosulus close to Dichomitus squalens at the species level. The subsequent best hits were Trametes versicolor and Trametes cinnabarina, as depicted in Figure 2. Polyporaceae are a major group of white-rot fungi with L. squarrosulus, Dichomitus squalens and Trametes versicolor belonging to the core polyporoid clade, one of the major clades with rich catabolic ability under Polyporales. Analysis based on ITS, RNA polymerase II and nrLSU sequences supports the similarity of Lentinus to Dichomitus and Trametes at the genus level [24].
Gene Ontology terms for the transcripts were annotated through BLAST2GO and functional annotators. 3,365 GO terms were assigned to 10,494 transcripts. The most abundant GO term predicted by Function annotator was GO:0055114, specifying the oxidation–reduction process (biological process) with gene products marking to manganese peroxidase 3 precursor of Phlebia radiata (PEM3_PHLRA), laccase 1A of Trametes pubescens (AF414808.1, AF491761, AF414807.1), ligninase H2 of Phanerochaete chrysosporium(LIG4_PHACH), mannitol dehydrogenase (MTLD_BACP2), NADPH dependent D-xylose reductase (XYL1_CANBO), arabinitol dehydrogenase (ARD1_UROFA), arabinan endo-1,5-alpha-L-arabinosidase A (ABNA_EMENI), pyranose dehydrogenase (PDH3_LEUMG), α-fucosidase A (AFCA_ASPNC). This substantiates the potential of this fungus in biomass degradation through the production of diverse hydrolytic enzymes. Subsequently, the enriched GO terms were GO:0005524 (molecular function: ATP binding), GO:0008152 (cellular function: metabolic process) and GO:0016021 (cellular component: integral membrane components). Deeper classification of each of the GO terms predicted for the contigs was also visualized through a function annotator that presented fifteen levels of classification for each GO category.
Best matching hits of the putative proteins encoded by the transcripts against the NCBI nonredundant protein database were available for 16,779 transcripts that expressed similarity to chiefly Dichomitus and Trametes protein sequences.3,217 probable enzyme products likely to be produced by 12,379 transcripts were determined through PRIAM based on the ENZYME database. Although the most abundant hits were proteins involved in genome integrity and regulation, such as RNA-dependent RNA polymerase, RNA helicase, and protein kinases, there were significant representations of biomass-degrading enzymes, such as endo-1,3(4)-beta-glucanase, glucose oxidase, choline oxidase and a range of lignocellulose active enzymes, as depicted in Figure 3. This demonstrates that the expression of these enzymes by the fungus is not dependent on the presence of lignocellulose substrate in the culture medium.
Putative domain hits illustrated by functional annotators were based on the PFAM database. A total of 4952 unique conserved domains were identified against 11,585 transcripts. The most abundant domain hit was Major Facilitator Superfamily of secondary transporters (pfam07690), followed by Tymo_45kd_70kd (pfam03251), a kind of transposable element detected in Basidiomycetes. Similar transposable elements were also reported in Pleurotus ostreatus[25].
KEGG orthology designations were obtained for 3,327 transcripts. Transcripts encoding 20 putative cytochrome P450 polypeptides were present in the L. squarrosulus transcriptome. The probable pathway depiction for cytochrome P450 was metabolism of xenobiotics. In addition, mannosidase, α- and β-glucosidases, galactosidase, arabinofuranosidase, endoglucanase, pectin esterase, and polygalacturonase were identified, establishing the biodegradative potential of this fungus.
In addition to the representation of sequences involved in fungal internal metabolism, translation and transcription, there was considerable expression of a two-component signal transduction system coupled with the transcription factor SKN7. The SKN7 transcription factor is an important member of the two-component phosphorylation system that transfers signals to activate the promoters of genes in response to external stimuli and induces responses to oxidative stress, such as H2O2 [26]. In addition, multiple RTA1 domain-containing protein sequences were observed, again related to the stress response.
There was significant representation of contigs specifying laccase, as revealed through similarity searches to the nr database and by GO annotation. Laccase, a multicopper oxidase, catalyzes the oxidation of phenolic compounds using a molecular oxygen electron acceptor, as pointed out in the molecular function of the enzyme by GO as hydroquinone: oxygen oxidoreductase activity, with hydroquinone being a diphenol compound. Laccases are efficient in degrading the phenolic components of lignin and play a major role in lignin catabolic processes. Six transcripts of laccase were expressed in L. squarrosulus with 100% sequencecongruence to laccases of Trametes cinnabarina, Polyporus, and Lentinus tigrinus. Ten transcript sequences were found to encode proteins homologous to versatile peroxidase with significant protein similarity to versatile peroxidase protein isoforms of Pleurotus eryngii and of Trametes versicolor. Protein sequences of putative versatile peroxidases were subjected to multiple alignment through CLUSTALW with experimentally determined protein sequences of ligninolytic peroxidases in the Protein Data Bank (PDB). The phylogenetic tree rooted through the UPGMA method of the alignment is presented in Figure 4.
Two isoforms of manganese peroxidase were identified based on conserved domains and sequence similarity. However, no lignin peroxidase transcripts were observed in this species, as confirmed through biochemical analysis.
Peroxidases are more prominent in the lignin catabolic process due to their relatively high redox potential and hence require complete mineralization of lignin. The efficient function of ligninolytic machinery is indispensable without hydrogen peroxide, an electron acceptor for peroxidases. White-rot fungi produce H2O2 needed for lignin oxidation through diverse enzymes such as glyoxal oxidase, aryl-alcohol oxidase, aryl alcohol dehydrogenase and GMC oxidoreductases [27]. Glyoxal oxidase of this fungus exhibited 66% similarity in protein sequence to that of Phanerochaete chrysosporium. Multiple transcript copies of these enzymes were expressed by the fungus. A considerable depiction of H2O2-producing oxidases was made by the glucose-methanol-choline (GMC) superfamily encoding transcripts that supply the peroxide requisite for ligninolytic peroxidases.
Enhancement of the oxidoreduction process in the culture of this fungus was further validated by the ubiquitous presence of cytochrome P450 monooxygenases and oxidoreductases in the transcriptome [28, 29]. Cytochrome P450 enzymes are involved in the catalytic reaction of aromatic metabolism through diverse biochemical reactions. The enzymatic reaction of cleavage of β-O-4 linkages of lignin is enhanced by co-oxidants such as thiols, NAD+, glutathione, etc. Glutathione reductase enables regeneration of reduced glutathione and is expressed in cells exposed to oxidative stress [30]. Evidently, glutathione reductase was expressed in L. squarrosulus with considerable protein similarity to glutathione reductase of Dichomitus squalens. In addition, sequences of flavin-containing monooxygenases (FMOs) and dioxygenases were revealed in the transcriptome analysis. FMOs are proteins involved in degradation in a multitude of aromatics and have been reported in a number of fungal species, while dioxygenases are reportedly involved in ring cleavage of aromatics oxidation [31]. The reactive peroxides produced create a highly oxidative environment for enzymatic action. Fungi secrete svf1 protein in response to this oxidative stress for their survival, as evident through the sequences of oxidative stress survival svf1-like protein expressed by L. squarrosulus [32].
Plant polysaccharides are composed of cellulose, hemicellulose, pectin and lignin, which contribute to the bulk of the biomass. In addition to the expression of lignin-degrading enzymes in response to induction by aromatic compounds, polysaccharide-degrading enzymes also existed in the transcriptome, with regulation different from that of the control, mediuming a synergy in regulation of the former and the latter. Endoglucanase, cellobiohydrolase and β-glucosidase enzymes are important components of the cellulolytic machinery [33], and L. squarrosulus demonstrated the presence of mRNAs of these genes. Pectin lyase-like proteins belong to glycosyl hydrolase family 28, which acts by inverting the mechanism of α1,4 glycosydic linkage of polygalacturonates [34]. Sequences with pectin lyase-like protein activity belonging to pectin esterase and polygalacturonase in the transcriptome of this species state its role in the degradation of pectin. In addition to pectin lyases, transcripts of polysaccharide lyase classes of proteins were also expressed. In addition, there was a strong representation of substrate transporters, glycoside hydrolases, glycoside transferases, carbohydrate esterases and acetyl xylan esterases encompassing the major plant polysaccharide-degrading enzymes.
CAZyme annotation
The fungus undertaken in the current study was an extensive producer of a multitude of plant polysaccharide-degrading enzymes spanning across the families of carbohydrate-active enzymes. Fungal systems are reportedly affected by polysaccharide-degrading enzymes, and the enzymes acting on these polysaccharides in general are designated 156 families of glycoside hydrolases, 106 families of glycosyl transferases, 16 families of carbohydrate esterases and 29 families of polysaccharide lyases [35]. The transcriptome was enriched with transcripts of esterases of family carbohydrate esterases 10. Carbohydrate esterases deacetylate the conjugates of glucans and are binding components of polysaccharide-degrading machinery. Glycoside hydrolases of family 16 showed significant depiction in the transcriptome of L. squarrosulus. Additionally, the transcript sequences of six and seven hairpin glycosidases catalyzing O-glycosyl bonds were observed. This emphasizes the importance of this fungus in the catabolism of carbohydrates, primarily cellulose, hemicellulose and pectic polysaccharides. In this bracket, endoglucanases, β1, 3-1, and 4 glucanases, xyloglucanases, and xyloglucan:xyloglucosyl transferases are worth remarkable mention.
Chitinases of glycoside hydrolase family 18 were subsequently predominant and were supposed to be involved in cell wall remodeling and maintenance of the fungus. More than ten transcripts with GH3 and GH79 modules were identified in the transcriptome of L. squarrosulus. Similarly, ligninolytic enzymes that act in synchronization with glycoside hydrolases were also preponderant in the transcriptome, as revealed by CAZyme annotation belonging to the AA2 family. The other affluent CAZymes reported were GMC oxidoreductase (AA3), lytic polysaccharide monooxygenases cleaving cellulose chains (AA9) and lytic polysaccharide monooxygenases cleaving xylans (AA14). The overall distribution of CAZymes in the transcriptome based on dbCAN is illustrated in Figure 5-7.
CUPP assigned 508 transcripts to 6 families of polysaccharide lyases, 51 families of glycoside hydrolases, 23 families of glycoside transferases, 7 families of carbohydrate esterases and 10 families of auxiliary activities family. Of the enzymes characterized through CUPP based on the peptide signatures, chitinase (3.2.1.14) and chitin synthase (2.4.1.16) were predominant, followed by laccase (1.10.3.2). Ten transcript sequences were assigned to the laccase family, and seven transcripts encoded peptide signatures typical of versatile peroxidase (1.11.1.16). The top hits also included glyoxal oxidase (1.2.3.15) and glucan endo-1,3-beta-D-glucosidase (3.2.1.39). Prediction of CAZy families on peptide signatures revealed preponderance of glycosidehydrolases followed by auxiliary activities 3 family. Among the glycoside hydrolases, GH16 and GH18 were enriched in the transcript sequences of L. squarrosulus. GH16 comprises members that are active on β-1,4 and 1,3 glycosidic bonds, and GH18 members chitinase and endo-β-N-acetylglucosaminidase aid in the maintenance of the fungal cell wall, whereas GH5 activities include cellulases, endomannanases and xyloglucanases.
The auxiliary activities 2 family encoding lignin modifying peroxidases manganese peroxidase and versatile peroxidase were also abundant subsequent to glycoside hydrolases and auxiliary activities family 3.
Table 2:Nucleotide hits against mycoCLAP database.