Coupling structural characterization with secretomic analysis reveals mechanism of the disruption of cross-linked structure in bamboo culms by Echinodontium taxodii

Background: Overcoming the biomass recalcitrance is essential for efficient utilization of lignocellulosic biomass in industrial bio-refining. White-rot fungi can overcome the biomass recalcitrance and accelerate the conversion of lignocellulose to biofuels via a large number of special extracellular lignocellulolytic enzymes. Previous studies try to dissect the function of extracellular enzymes on biomass resistant cross-linked structures by secretome analysis, but the bio-alteration of cross-linked structures is ignored usually. A deeper and detailed understanding of relationship between secretome and bio-alteration of cross-linked structure in lignocellulosic biomass is still lack. Results: As an efficient wood-decaying fungus, Echinodontium taxodii could improve the conversion efficiency of lignocellulose to biofuels. This study coupled comparative analysis of fungal secretomes and 2D HSQC NMR analysis of lignocellulose fractions, aiming to elucidate the role of extracellular enzymes from Echinodontium taxodii 2538 in the disruption of resistant cross-linked structure of bamboo culms. Carboxylesterases, alcohol oxidases and Class-II peroxidases showed importance in the cleavage of cross-linked structures, including ester and ether linkages of lignin-carbohydrate complexes (LCCs) and inter-unit linkages of lignin, which contributed to biomass resistance removal and cellulose exposure during the early stage of fungal decay. Moreover, the rapid oxidation of Cα-OH was found to contribute to the lignin bio-depolymerization. Conclusions: These findings revealed the detailed mechanisms of biomass recalcitrance reduction by fungal pretreatment, and provide insight into efficient strategy of lignocellulose conversion. It will advance the development in design of carbohydrate MnP: peroxidase; LiP: lignin peroxidase; Lac: laccase; AAO: aryl-alcohol oxidase; GMC oxidoreductase: glucose-methanol-choline oxidoreductase; CAZymes: Carbohydrate-Active Enzymes; MWL: milled wood lignin; LigD: lignin dehydrogenase; S: syringyl; G: guaiacyl; H: p -hydroxyphenyl; PCA: p -coumarates; FA: ferulates; p CA: p -coumaric acid; Ac: acetic acid; GlcAE: esterified 4-O-methyl-α-D-glucuronic acid units; X1: β-D-xylopyranoside; Glc 1 : β-D-glucopyranoside; αX1: (1-4)-α-D-xylopyranoside; PhGlc: phenyl glycoside; Glc: glucose; Gal: galactose; Man: mannose; Ara: arabinose; SDS-PAGE: sodium dodecyl sulfate - polyacrylamide gel electrophoresis; LFQ: label-free quantification; LC-MS/MS: liquid chromatography-tandem mass


Background
Lignocellulosic biomass is one of the most abundant feedstock for the production of renewable fuels, biomaterials, and value-added chemicals [1][2][3]. The efficient release of sugars from biomass in industrial bio-refining is essential but restricted by complex cross-linked structures of lignocellulose, including the rigid lignin network coating the cellulose microfibril and lignin-carbohydrate complexes (LCCs), which cross-linked lignin and polysaccharide [4,5]. Certain basidiomycete so-called white-rot fungi evolved effective deconstruction strategy to disrupt resistant crosslinked structures of lignocellulose for overcoming the biomass recalcitrance. These fungi can mineralize lignin preferentially, and then the exposed cellulose can be easily hydrolyzed by extracellular glycoside hydrolases. In the previous studies, a series of white-rot fungi, especially selective lignin-degrading strain, have been used to accelerate the bioconversion of lignocellulose [6]. Selective white-rot fungi degraded complex substrates synergistically depending on a large number of special extracellular lignocellulolytic enzymes [7]. Dissecting the underlying mechanisms of resistant cross-linked structures degradation by the extracellular enzymes could advance the development in design of enzyme cocktail for efficient lignocellulose biorefinery [8].
Secretome provides a global insight of secreted protein during fungal deconstruction of lignocellulose [9]. Many studies have revealed crucial enzymes involved in biomass degradation through comparative secretomic analysis among the different growth stages or different substrates [10,11]. For example, Cai etal found that when Lentinula edodes was cultured with woody and non-woody 5 lignocellulosic biomass, several enzymes involved in lignin degradation were most abundant in glucose culture, while the abundance of cellulases and hemicellulases was higher in cellulose culture [12]. Kuuskeri et al. analyzed proteomes at two time points during the wood decay by Phlebia radiate, and found that the expression of specific oxidoreductases and peroxidases could be enhanced along with the release of lignin phenolic units on the early stage of decay [13]. However, due to the multiscale changes in lignocellulosic structure and complex interaction of extracellular enzymes, identifying the key factors responsible for overcoming biomass recalcitrance was still a challenge. A deeper and detailed understanding of relationship between secretome and bio-alteration of cross-linked structure in lignocellulosic biomass is still lack.
Echinodontium taxodii, an excellent white-rot fungus able to preferentially remove resistant lignin, has been utilized in the bio-refinery of woody and herbaceous feedstock [14,15]. Our recent study revealed that E. taxodii 2538 could enhance bamboo culms saccharification through increasing the medium pore volume of biomass and improving substrate accessibility, which was mainly attributed to the selective cleavage of cross-linkages in lignin units [16]. The disruption of LCCs may also play an important role in overcoming recalcitrance of bamboo, but there is still lack of unequivocal evidence that fungus degrades LCCs. As "woody grass", bamboo culm has different lignocellulosic structure from wood or grass, which contained higher lignin content than grasses and higher syringyl/guaiacyl (S/G) unit ratio than wood [17]. Although bamboo culm is one of the most abundant forest resource in Asia, few knowledge is known about the mechanism underlying bamboo cross-linked structure degradation by selective white-rot fungus.
The aim of this study was to elucidate the role of extracellular enzymes of E. taxodii 6 2538 in bamboo culms cross-linked structure disruption. The secretomes of E.
taxodii growing on bamboo for 20-days and 40-days were analyzed. Moreover, LCCs and lignin was isolated from bio-treated bamboo and characterized through twodimensional nuclear magnetic resonance (2D NMR) spectroscopic methods. Coupled with the analysis of fungal comparative secretomes and the structural characterization of biomass, the role of fungal extracellular enzymes on the biomass deconstruction was elucidated.

Results and Discussion
Dynamics of secretome of E. taxodii 2538 cultured in bamboo Lignocellulose degradation mainly relies on enzymatic reaction, especially extracellular enzymes [11]. The secretome of E. taxodii 2538 in bamboo biomass culture was focused on using iBAQ label-free quantification analysis. After sorted with N-terminal Sec-dependent secretion signal using SignalP 3.0, 132 secreted proteins were identified (Additional file 1: Table S1) in total. As shown in Fig. 1a, 122 proteins showed expression at day 20. The number of extracellular proteins decreased to 81 at day 40. According to biological functions, all secreted proteins were classified into oxidoreductases including lignolytic enzymes, glycoside hydrolases (GH), glycosyl transferases (GT), polysaccharide lyases (PL), esterases, proteases and peptidases, and other proteins [10]. Esterases and oxidoreductases achieved the most abundance at day 20, accounting for 33.41% and 33.38% abundance of total secretome. The glycoside hydrolases exhibited the highest diversity, with 62 and 45 proteins at day 20 and 40, respectively (Fig. 1b). In contrast, the relative abundance of glycoside hydrolases was up to 43.41% at day 40. The more number of extracellular enzymes and the higher diversity of secreted 7 proteins in early-stage showed potential relationship in response to the complexity of bamboo materials, compared with later-stage [11]. The changes in protein profile of E. taxodii 2538 reflected the response of fungus to changes in composition and structures of substrates.
The contributions and fold changes of individual proteins were estimated (Fig. 1).
Differences of individual protein expression at day 20 and day 40 were observed by relative abundance analysis of proteins. The fungal proteins including lignolytic enzymes, esterases and cellulases, were speculated to be important for cell wall deconstruction and analyzed at two time point.
Class-II peroxidases and laccase were believed to be responsible for the degradation of lignin [18]. Five manganese peroxidases (MnP), four lignin peroxidases (LiP) and one laccase (Lac) were identified in the secretomes of E. taxodii 2538. With high abundances, most of Class-II peroxidase were among top 10 expressed proteins at day 20. These results implied the importance of lignin degradation in lignocellulose conversion. As the cultivation time advanced to day 40, the protein profile was obviously different from day 20. Peroxidase and laccase exhibited completely different secretion patterns. The expression of most peroxidases at day 20 was significantly higher than that of day 40. The total abundance of the detected class-II peroxidase was reduced at day 40 (Fig. 1c). Only one LiP (Q53WT9) and two MnPs (Q70LM3 and A0A068PCH3) maintained high expression at day 40. Some class-II peroxidases including three lignin peroxidases (Q3L2T9, B2BF40 and A7U4S3) and two manganese peroxidases (B2BF37 and B1B554) were not detected at day 40. On the contrary, the expression of laccase (Q53WT9) was increased greatly at day 40.
The degradation ratio of lignin was higher on the early-stage of decay with E.
taxodii than on the later-stage (Additional file 2: Figure S1). Obviously, the expression of class-II peroxidases is consistent with the degradation of lignin.
Moreover, the secretomics analysis showed that the auxiliary enzymes (aryl-alcohol oxidase/dehydrogenase, copper radical oxidase, cellobiose dehydrogenase and glyoxal oxidase) were also upregulated significantly on the early stage of fungal decay (day 20), which are a class of important enzymes to generate H 2 O 2 for peroxidases [19]. These results indicated that Class-II peroxidases, instead of laccase, play an important role in the lignin degradation by E. taxodii 2538. Besides enzymes involved in lignin degradation, two alcohol oxidases were also identified in oxidoreductases and showed moderate expression (Fig. 1a). Alcohol oxidases were identified in many secretome of wood rot fungi [13,20,21]. The role of alcohol oxidases was defined as potential hydrogen peroxide donor, supplementing the function of auxiliary enzymes in H 2 O 2 -supply. However, the function of alcohol oxidases on substrate in cell wall deconstruction is unclear and to be confirmed.
It is worth noting that two carboxylesterases (A0A060SLH8 and B8PHY5) were detected. The two carboxylesterases showed very high abundance in the secretome for day 20 ( Fig. 1a and 1d). The LFQ of protein A0A060SLH8 achieved the highest expression among all secreted proteins. However, the expression level of carboxylesterase was significantly reduced at 40 days. Carboxylesterases represent a large class of proteins that catalyze the hydrolysis of esters, sulfates and amides.
Belonging to a subclass of carboxylesterases, ferulate esterase was thought to hydrolyze the ester bond between ferulic acids and hemicellulose, thereby White-rot fungal secretomes on various lignocelluloses have been investigated mostly concentrating on the model white-rot fungi [21][22][23][24]. With these data and the accumulating genomic knowledge on fungi, it is evident that each white-rot fungus expresses a unique array of CAZy and oxidoreductase enzymes and has an exquisite strategy for colonization and decay of wood.
As an efficient selective lignin-degradation white rot specie, E. taxodii 2538 was not fully investigated by omics approach before. Thus, we would like to perform an extensive and deep time point study on the secretome of fungus cultured on bamboo. The time point study in secretome analysis allowed us to observe dynamic changes in the abundances of E. taxodii expressed proteins, and to gain insight into the mechanism of cell wall deconstruction by enzymes.
Our results confirmed that the lignocellulolytic enzyme profile of E. taxodii is functional and composed of a variety of CAZy families including the auxiliary oxidoreductases, a set of Class-II peroxidases accompanied by H 2 O 2 producing enzymes, and a selection of hydrolases, esterases and lyases. All of these enzymes were necessary for complete degradation of the polymeric lignocellulose components. The expression profiles of multiple lignin-modifying enzymes (laccase and class-II peroxidases), together with H 2 O 2 producing oxidoreductases (AAO, GMCs), is more similar to the profiles of CAZymes expressed in P. chrysosporium [25,26]. Moreover, some unique features like high expression of certain class-II peroxidases and carboxylesterases but low expression of laccase at the early stage were different from many other Polyporales species [20]. In Phlebiopsis gigantea, ten class-II peroxidases genes were identified, but none of these were detected as secreted proteins in pine wood cultures [22]. The function of peroxidases and carboxylesterases in the disruption of lignin or LCCs was still unclear. Thus, the relationship between these enzymes and bio-alteration of cross-linked structure in bamboo culm needs further confirmation via the structural characterization.  Table S2) [27][28][29]. Table 1 summarized the relative abundance of lignin inter-unit linkages and aromatic units in the MWL samples.

2D-NMR analysis of bamboo LCCs
To confirm LCCs biodegradation by white-rot fungus, LCC samples isolated from raw and treated bamboo culm were analyzed by 2D NMR. Different from other LCC isolation, we applied a classic isolation method and obtained the Björkman LCC of bamboo samples. More carbohydrates exist in Björkman LCC, which composed with about 30% lignin and 70% carbohydrates (Additional file 2: Table S3). As a typical complex of lignin and carbohydrate, the isolated Björkman LCC samples were 13 suitable for exploration of LCC linkages. HSQC spectra of LCCs provided important structural information and were shown in Fig. 3 and 4. Currently, most studies on LCC structure focus on the woody plant [5]. Generally, the main types of LCC linkages in wood are believed to be phenyl glycoside bonds, esters and benzyl ethers. Bamboo as "woody grass" has different structure from herbaceous and woody plants, but studies on the structure and degradation of bamboo LCC are still limited. Especially for bamboo Björkman LCC, there was no report about its isolation and structural analysis. LCC linkages were identified and showed in Fig. 4. Table 2 summarized the relative abundance of major structures in the LCCs of raw and type. The signals of benzyl ether C2-linkages giving a cross-peak at 80-81/5. 1-4.9 ppm in LCCs disappeared rapidly at day 20 (Fig. 3). The signals of C1-linkages which giving a cross-peak at 80-81/4.5-4.7 ppm were not detected.
In summary, 2D-NMR analysis of the isolated LCC samples confirmed that the Most of lignin fragments in plant biomass are covalently linked to hemicellulose coating cellulose microfiber [32,37]. The cross-linked structure of lignin and 16 polysaccharides is partly responsible for the lignocellulose recalcitrance. Fungal esterase with ability to cleave ester bonds has been wildly used to improve saccharification of lignocellulose [38]. Recent studies speculated that the breakdown of LCCs during fungal treatment may play an important role in improving biomass digestibility [34, 38,39]. However, there is still lack of the unequivocal proof that fungus degrades LCCs. In this study, LCCs was isolated from fungus-

Isolation and HSQC analysis of MWL and LCC
The milled wood lignin (MWL) and lignin-carbohydrate complex (LCC) were isolation from raw and fungal treated bamboo (Additional file 2: Figure S2). The MWL and LCC samples from raw and treated bamboo culms were analyzed with heteronuclear single quantum coherence spectroscopy (HSQC). The isolated lignin (100 mg) was suspended in 0.75 ml of DMSO-d6 in the NMR tube. The experiments has been explained in detail in previous paper [16]. The brief parameters are: 1 H widths of 5,000 Hz in 2048 points with a recycle delay of 1.75 s; 13 C spectral widths of 25,000 Hz. Cetral DMSO peak (δ H /δ C 2.50/39.5) was used for reference. Correlation peaks of MWLs and LCCs were assigned according to the previous report (Additional file 2: Table S2 and S4). A semi-quantitative analysis of the integrals of the HSQC correlation peaks was performed using MestReNova (version 6.0.1).

Supplementary information
Additional file 1: Table S1. Detailed information of all secreted proteins identified in secretomes.
Additional file 2: Figure S1. The degradation ratio of lignin, cellulose and hemicellulose during fungal treatment of bamboo samples. Figure S2. The main isolation procedures of MWLs and LCCs from raw and treated bamboo. Table S2.
The assignments of 13 C-1 H peaks in HSQC spectrum from the isolated MWLs. Table   S3. Sugar and Lignin Analysis of MWL and LCC Preparations.

Consent for publication
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Availability of data and materials
All data generated or analyzed during this study are included in this published article and its additional information files.  All integral data was relative to methoxyl.  All integral data was relative to methoxyl. HSQC spectra of the isolated MWL samples from raw and treated bamboo. The up is the side Figure 3 HSQC spectra of the isolated LCC samples from raw and treated bamboo. The up is the side c 33 Figure 4 Main linkages identified in LCCs of bamboo.