# Uncovering enzymatic tools promoting lignocellulose breakdown in the anaerobic bacterium Ruminiclostridium cellulolyticum

Nicolas Vita, Marion Holmière, Felipe Mejia-Otalvaro, Fabian Debard, Lison

Degeilh, Séverine Gagnot, David Crônier, Anouck Habrant, Florian Pion,

Yann Denis, et al.

► To cite this version:

Nicolas Vita, Marion Holmière, Felipe Mejia-Otalvaro, Fabian Debard, Lison Degeilh, et al. Uncovering enzymatic tools promoting lignocellulose breakdown in the anaerobic bacterium Ruminiclostridium cellulolyticum. Communications Biology, 2025, 9, pp.24. 10.1038/s42003-025-09287-y. hal-05441823

HAL Id: hal-05441823

https://hal.inrae.fr/hal-05441823v1

Submitted on 20 Jan 2026

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# Uncovering enzymatic tools promoting lignocellulose breakdown in the anaerobic bacterium *Ruminiclostridium cellulolyticum*

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Nicolas Vita<fnref n="1" />✉, Marion Holmière<fnref n="1" />, Felipe Mejia-Otalvaro², Fabian Debard<fnref n="1" />, Lison Degeilh³, Séverine Gagnot<fnref n="1" />, David Crônier⁴, Anouck Habrant⁵, Florian Pion⁵, Yann Denis⁶, Giuliano Sciara⁵, Craig Faulds³, Caroline Monteil⁷, Sébastien Santini⁸, Paul-Henri Ducrot⁵, Stéphanie Perret<fnref n="1" />, Véronique Aguié-Béghin⁴, Gaël Panis⁹,¹⁰, Eric Record³, Brigitte Chabbert⁴ & Henri-Pierre Fierobe<fnref n="1" />

Understanding the anaerobic deconstruction of recalcitrant lignocellulose remains challenging. Combining substrate composition and transcriptomic analyses, we shortlisted *Ruminiclostridium cellulolyticum* enzymes that modify lignocellulose and distinguished two members of the large SGNH hydrolase superfamily potentially enhancing lignocellulosic biomass degradation by acting on decorations of lignin and hemicelluloses but also on cross-links implicating lignin. Using genetic modifications, bioinformatics and biochemistry, we show they promote the plant cell wall ester-linked hydroxycinnamic acid derivatives release, a role never described for these proteins mainly synthesized by the restricted group of cellulolytic and cellulosome-producing bacteria. In addition to the recent observation of fungal limited lignin alterations in oxygen absence, this discovery is to the best of our knowledge, the first evidence of such anaerobic bacterial process that provides a better comprehension of the biogeochemical Earth's carbon cycle. Furthermore, a better knowledge of the anaerobic plant biomass degradation could help to design non-fossil resources based biotechnological applications, a cornerstone of bioeconomy development.

Plants have been the main environmental carbon sinks since the Carboni-ferous era, and currently hold ~82% of the estimated 550 billion tons of organic carbon on Earth<fnref n="1" />,². Much of this carbon is stored in Plant Cell Walls (PCW), making them the most abundant and renewable terrestrial reservoir of organic carbon. Most PCWs are essentially made up of lignocellulose, large and intertwined networks of cellulose microfibrils (20–50% of the vegetal biomass dry weight [VBDW])³,⁴ embedded into a matrix of hemi-celluloses (15–35% of VBDW)³,⁴, the whole being encased in a protective gangue of a branched polyaromatic polymer, lignin (15–30% of VBDW)³,⁴, a complex structure that grants this composite biomaterial high recalcitrance

to microbial degradation³,⁵. Nevertheless, (ligno)cellulolytic microorganisms occupying both aerobic and anaerobic biotopes, catalyze soil organic matter mineralization⁶ using efficient enzymatic and non-enzymatic degradative systems to access all plant biomass components⁷,⁸. These biological strategies can be leveraged to develop biotechnological applications for the sustainable production of chemicals and biofuels from easily accessible, low-priced and non-food-crop competing raw materials (forestry and industrial waste, agricultural residues)⁹⁻¹¹. For decades, lignocellulosic biomass has thus been considered as a credible answer to mitigate the consequences of fossil resource use and inescapable future depletion¹²⁻¹⁴.

<footnote n="1">¹CNRS, IMM, LCB-UMR7283, Aix-Marseille Université, Marseille, France. ²The Novo Nordisk Foundation Center for Biosustainability, Enzyme Engineering and Structural Biology group, Technical University of Denmark, Konges Lyngby, Denmark. ³INRAE, Biodiversité et Biotechnologie Fongiques, Aix-Marseille Université, Marseille, France. ⁴Université de Reims Champagne-Ardenne, INRAE, FARE, UMR A 614, Reims, France. ⁵INRAE, AgroParisTech, Institute Jean-Pierre Bourgin for Plant Sciences, Université Paris-Saclay, Versailles, France. ⁶CNRS, IMM, Plateforme Transcriptome, Aix-Marseille Université, Marseille, France. ⁷CNRS, CEA, UMR7265 Institut de Biosciences and Biotechnologies d'Aix-Marseille, Aix-Marseille Université, Saint-Paul-lez-Durance, France. ⁸CNRS, IMM, IGS-UMR7256, Aix-Marseille Université, Marseille, France. ⁹Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva, Geneva, Switzerland.
¹⁰Geneva Centre for Inflammation Research, Faculty of Medicine, University of Geneva, Geneva, Switzerland. ✉e-mail: nvita@imm.cnrs.fr</footnote>
Unlike cellulose and hemicelluloses, biodegradation by both aerobic and anaerobic cellulolytic microorganisms<sup>8,15,16</sup>, scarce information is available on lignin breakdown in anaerobic environments where dead plant matter accumulates, whereas it is extremely well-documented for both aerobic fungi and bacteria. The discovery of uncharacterized lignocellulose-active enzymes<sup>9</sup> or degradation processes, most particularly from anaerobes<sup>17</sup> and targeting or modifying lignin to at least favor the plant polysaccharide access, is then currently of prime importance to better understand the plant biomass degradation and to develop sustainable bioprocesses.

Lignin, built to be very difficult to break down by microorganisms<sup>18</sup>, confers to plants structural rigidity, water impermeability, and resistance to environmental factors, pathogens and other predators<sup>19</sup>. It is primarily composed of C-O and C-C inter-unit linked phenylpropanoid compounds, the monolignols (coniferyl alcohol (G), sinapyl alcohol (S), and p-coumaryl alcohol (H)) whose proportions show plant species and tissues specificities<sup>3,20-22</sup>. Other simple phenylpropanoid derivates, the hydroxycinnamic acids (HCA), are a key feature of grasses and other monocots, like in Wheat Straw (WS)<sup>20</sup>. In the latter abundant and therefore inexpensive source of lignocellulose (% of VBDW: ~16% lignin, ~38% cellulose and ~29% hemicelluloses)<sup>3,4</sup>, the two major cell wall-bound phenolic esters are the HCA derived from *p*-coumaric acid (*p*CA) and ferulic acid (FA)<sup>20</sup>, accounting, respectively, for 4 and 11% of total lignin content (expressed in molar contents considering H + G + S = 100%), respectively<sup>23</sup>. In this cultivated cereal residue, *p*CA is mainly acylating γ-OH of S-unit sidechains of lignin and decorates this aromatic polymer<sup>23-26</sup>, or, to a lower extent, participates in the *p*-coumaroylation of xylan<sup>27</sup>, whereas FA is linked to arabinosyl residues of arabinoxylan chains<sup>28</sup>, cross-links lignin-hemicelluloses, hemicelluloses-hemicelluloses and polysaccharide-protein via diferulate bounds<sup>23,25,28,29</sup>, or is ether-bridged in lignin carbohydrate complexes (LCC) present in herbaceous PCW<sup>30-32</sup>. Both HCAs' and lignin's characteristics contribute to high complexity, insolubility and stability of the lignocellulose. Cellulolytic fungi and bacteria are using a well-known class of enzymes, the cinnamoyl ester hydrolases (i.e., feruloyl esterases), to hydrolyze plant cell walls and more specifically HCAs linked to polysaccharides<sup>33-37</sup>. For lignin, aerobes employ non-selective depolymerization strategies based on specific arsenals of extracellular enzymes and accessory proteins (i.e., metalloproteins such as peroxidases, laccases...)<sup>21,22,38,39</sup> to generate free-oxygen- and/or phenolic- radicals that efficiently oxidize this polyaromatic biopolymer often uncontrollably generating a wide product variety. Because of the mechanism of this degradation process, the very existence of delignification in the absence of oxygen was even controversial until a recent publication describing a limited (compared to aerobic depolymerization yield) lignin deconstruction by anaerobic fungi from the *Neocallimastigomycota* genus<sup>40</sup>. However, no enzymatic actors involved in this process have been formally identified.

In this work, we demonstrate by substrate composition analysis that *Ruminiclostridium cellulolyticum*, a strict anaerobic cellulolytic and hemi-cellulolytic bacterium, can induce the release of FA and *p*CA from WS, this second HCA mainly decorating WS lignin<sup>23-26</sup>. Despite the absence of classical bacterial lignin degradation protein-encoding genes in *R. cellulolyticum* genome<sup>39,41</sup>, we defined by comparison of this strain's transcriptomes during growth on lignified and non-lignified substrates, a shortlist of potential actors with unknown function and possibly implicated in lignocellulosic assemblies processing and lignin modification. Focusing on three enzymes belonging to the very large SGNH hydrolase superfamily and recruited in large extracellular multienzymatic cellulolytic complexes called cellulosomes<sup>16,42</sup>, we applied biochemical and genetic approaches, allowing us to propose for two of them a role never reported for any SGNHs as acting on FA and *p*CA moieties of lignocellulosic assemblies (cinnamoyl ester hydrolases). Interestingly, bioinformatic analysis shows similar enzymes are found only in a limited variety of bacterial species and predominantly in anaerobic cellulolytic *clostridia*. Our data demonstrate, to the best of our knowledge, for the first time, that cellulosome-producing cellulolytic bacteria living in oxygen-free environments have developed specific and original extracellular mechanisms to deal with lignocellulosic

assemblies, at least to facilitate greater access to plant polysaccharides through lignin modification. Unsurprisingly, they drastically diverge from the enzymatic systems developed by aerobic organisms, which ultimately rely on reactive oxygen species (ROSs), very toxic for strict anaerobes.

## Results

### R. cellulolyticum anaerobically processes lignocellulosic assemblies

*R. cellulolyticum* isolated from decaying grass<sup>43</sup> and able to grow in pure culture on WS<sup>44</sup>, is an anaerobic hemicellulolytic and cellulolytic model bacterium that can degrade and utilize both types of plant polysaccharides<sup>42,44-46</sup> despite the lignin matrix surrounding and protecting them.

After 9-day cultures on hatched WS at 32 °C, difference spectra obtained from Infrared spectroscopy analysis<sup>47,48</sup> of the residual substrates show only a decreased signal in the bands mainly attributable to polysaccharides (1200–800 cm<sup>-1</sup>) compared with the non-inoculated WS-medium incubated under the same conditions (control) (Supplementary Fig. 1). However, the ¹H-¹³C HSQC NMR (2D-HSQC-NMR) analysis<sup>40,49</sup> of these residual substrates indicates that after growth, the relative proportions of aryl ether and tricin structures increase along with a slight reduction of S/G ratio and at a larger extent of the proportions of FA and *p*CA (Fig. 1A, B). Noteworthy, the decrease of the ratio between the FA and *p*CA quantities observable in the GC-MS compositional analysis of the corresponding extractives from alkaline hydrolyzed residual substrate<sup>20</sup> (Supplementary Fig. 2), confirms the loss of FA from WS after *R. cellulolyticum* growth. Moreover, it appears based on GC-MS compositional analysis of the culture supernatants<sup>47,51</sup> that FA and *p*CA, in higher amounts for this second HCA, accumulate in the culture medium, a phenomenon occurring in a more limited way for 4-OH benzoic acid (4-OH-B) (Fig. 1C and Supplementary Fig. 3).

For *p*CA, its accumulation in culture supernatants could be caused by its trimming mainly from the lignin decoration (WS *p*CA mostly acylates the γ-OH of S-unit sidechains)<sup>23-26</sup>, but also in smaller amounts from the xylan's *p*-coumaroylation<sup>27</sup>. For 4-OH-B, this compound, present as traces in WS lignin used in this study (Supplementary Fig. 2), could be directly released from the substrate or enzymatically produced from *p*CA<sup>52</sup>. The variation upon *R. cellulolyticum* growth of the S/G ratio, the proportion of WS aryl ether structure and the tricin content, a flavone known to be incorporated in the grass lignin polymers<sup>20,53</sup>, also supports a modification of lignocellulose structure. For FA, both its liberation in culture supernatants and its loss from WS may come from the removal of this HCA esterified on hemicelluloses<sup>28</sup> and the breaking of cross-links between lignin-hemicelluloses, hemicelluloses-hemicelluloses and polysaccharides-proteins<sup>23,25,28,29</sup>, and the ester- and ether-bridges present in LCC<sup>30-32</sup>. Altogether, our results reveal, to the best of our knowledge, the first time that an anaerobic cellulolytic bacterium can at least modify the lignocellulosic assemblies and, in a limited way, lignin to free polysaccharides usable for growth.

### Identification of *R. cellulolyticum* extracellular enzymes involved in anaerobic lignocellulosic assembly processing

*R. cellulolyticum* was anaerobically grown on cellobiose (Cb), a disaccharide, on crystalline cellulose (C), on a mix (Mix) of commercialized polysaccharides mimicking the overall PCW polysaccharidic composition [crystalline cellulose (~57%) + insoluble wheat arabinoxylan (~38%) + soluble xyloglucan (~5%)], or on hatched WS containing both lignin and polysaccharides. Following bacterial growths via metabolic profiling (MP, Supplementary Fig. 4A, B), subsequent transcriptome sequencings at the end of the exponential phase on each selected substrate (Supplementary Fig. 4C) were performed (Supplementary Fig. 5 and Supplementary data 1)<sup>40,54-56</sup> to identify *R. cellulolyticum* lignocellulosic assembly processing actors.

We first used Cb-, C-, and Mix-data as stencils to rule out from the WS-transcriptome, differentially expressed genes encoding enzymes with a
A

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¹H chemical shift

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¹H chemical shift

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¹H chemical shift

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¹³C chemical shift

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B

<table><thead><tr><th>Compound</th><th>Not inoculated</th><th>Inoculated</th><th>pSOS-2017</th></tr></thead><tbody><tr><td>A<sub>a</sub></td><td>67.63 ± 3.55</td><td>69.06 ± 6.43</td><td>68.97 ± 3.85</td></tr><tr><td>FA<sub>2</sub></td><td>17.51 ± 4.17</td><td>12.41 ± 0.57</td><td>13.98 ± 0.72</td></tr><tr><td>pCA<sub>2/6</sub></td><td>28.57 ± 2.53</td><td>22.97 ± 3.21</td><td>28.74 ± 1.21</td></tr><tr><td>T<sub>8</sub></td><td>21.78 ± 4.96</td><td>28.02 ± 4.35</td><td>22.91 ± 0.85</td></tr><tr><td>T<sub>6</sub></td><td>23.48 ± 4.29</td><td>30.24 ± 5.38</td><td>24.95 ± 2.71</td></tr><tr><td>G<sub>2</sub></td><td>100.00 ± 0.00</td><td>100.00 ± 0.00</td><td>100.00 ± 0.00</td></tr><tr><td>S<sub>2/6</sub></td><td>139.05 ± 4.27</td><td>134.09 ± 1.35</td><td>135.43 ± 5.38</td></tr><tr><td>S/G ratio</td><td>0.695 ± 0.021</td><td>0.670 ± 0.007</td><td>0.677 ± 0.027</td></tr></tbody></table>

C

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function related to the degradation of polysaccharidic fraction and exclusively extract those potentially related to the lignin presence in WS. Among the 3470 coding DNA sequence (CDS) annotated in *R. cellulolyticum* genome (~4.1 Mbp⁵⁷), 483 genes have a positive expression fold change (FC > 2 and adjusted *p* value (padj) <0.05) on WS compared to the other growth substrates (Fig. 2A). Since the early steps of microbial lignocellulose

deconstruction take place outside the cell²¹,²²,³⁸,³⁹, we curated the potential candidates inventory by only considering encoded proteins with a signal peptide⁵⁸ and consequently an extracellular localization (~70 candidates). Focusing on genes annotated as coding for hypothetical proteins or proteins hosting at least one domain with unknown or unorthodox function⁵⁷, we further shortened this list to 38 candidates by function prediction using
**Fig. 1 |** Impact of the growth of wild-type and overproducing SGNHh<sub>2017</sub>* R. cellulolyticum strains on hatched wheat straw (WS). A 2D-HSQC-NMR data acquired on residual insoluble substrates after anaerobic growth of wild-type (wt) R. cellulolyticum (Inoculated, middle panel) and the strain (pSOS-2017, right panel) hosting the vector allowing the overproduction of SGNHh<sub>2017</sub> without dockerin (SGNHh<sub>2017</sub>*). The corresponding data for the uninoculated control incubated in the same conditions are also depicted (Not inoculated, left panel). Sample preparation is described in the Materials and Methods section "Whole plant cell wall 2D-HSQC-NMR sample preparation and data acquisition". Structures of detected lignin subunits are color-coded to match their signals in NMR spectra (on the right of the panels). B Relative content of structures identified in A. calculated from contour volume integrals expressed in percentage, setting the signal attributable to

the G unit as 100% (mean and standard deviation calculated from biological triplicates for both Not inoculated and Inoculated conditions). C GC-MS compositional analysis of lyophilized culture supernatants after anaerobic growth on WS of wt R. cellulolyticum and the strain (pSOS-2017) hosting the vector allowing the overproduction of SGNHh<sub>2017</sub> without dockerin (SGNHh<sub>2017</sub>*). The structure of the detected compounds is depicted under the graph. The data represent µg of detected compound per g of dry total lyophilized samples (mean and standard deviation calculated from biological triplicates [with technical duplicates for the inoculated condition], the corresponding chromatograms are in Supplementary Fig. 3). Abbreviations: β-aryl ether unit (A); syringyl unit (S); guaiacyl unit (G); ferulic acid (FA); tricin unit (T); coumaric acid (pCA); 4-OH benzoic acid (4-OH-B). All error bars presented are standard errors of the mean.

BLASTP⁵⁹, the CAZy database¹⁵, InterProScan⁶⁰ and/or analysis of the genetic context of the corresponding encoding genes (Supplementary data 2 and Fig. 2B). In this selection, 30 encoded proteins are involved in hemi-celluloses and pectin hydrolysis, modification or decoration removal¹⁵,⁶¹, some of them possessing domain(s) with unknown functions. The identification of these enzymes is expected in this type of transcriptomic approach since WS contains minor additional polysaccharides (i.e., arabinan⁴) not included in the Mix we used in this study. We also identified the following proteins (Supplementary Data 2 and Fig. 2B): Ccel_1243, Ccel_2622, Ccel_2735, and Ccel_3134 (Protein Identification based on Old gene Locus Tag, PIDOLT), which are hypothetical proteins; Ccel_2642 and Ccel_3247 (PIDOLT), which have large unknown function domains; and Ccel_1101 (PIDOLT), which is a cellulosomal CotH homolog. The CotH protein family includes known non-CAZyme components of both fungal and bacterial cellulosomes without any defined function⁴²,⁶²,⁶³. However, in *Bacillus subtilis*, CotH is required for the assembly of several outer coat proteins and has a kinase activity⁶⁴⁻⁶⁶, an unexpected function for an extracellular *R. cellulolyticum* cellulosomal protein.

Moreover, expression pattern clustering comparing WS- and all other substrates- transcriptomic data highlighted a group of genes with a strong upregulation (cluster II, Fig. 2B), where it is most likely to find targets encoding proteins with functions related to the lignin fraction of WS lignocellulosic assemblies. Thus, the gene at locus CCEL_RS05325 encoding Ccel_1060 (PIDOLT) and annotated as "dockerin type I domain-containing protein", drew our attention since its FC ranged from 36 to 79 (Supplementary Data 2 and Fig. 2B). Besides, previous published proteomic data from our group showed that Ccel_1060 is only present in the cellulosomal fraction prepared from R. cellulolyticum grown on hatched WS and not from cultures on cellulose and oat spelt xylan⁴². Ccel_1060's catalytic domain (CD), linked to a typical C-terminal dockerin allowing its recruitment in cellulosomes, presents similarities with the SGNH hydrolases⁴² so called because all possess 4 conserved blocks of residues containing at least one strictly conserved key aminoacid for the catalysis⁶⁷: serine (S), glycine (G), asparagine (N) and histidine (H), respectively (Supplementary Fig. 6A-C). The SGNH hydrolase superfamily includes, at present >89,000 total members distributed across all domains of life with a wide variety of enzymatic esterase activities such as lipase, protease, acetyltransferase, thioesterase or carbohydrate esterase (CE)⁶⁸,⁶⁹. We consequently hereafter renamed Ccel_1060 as SGNHh<sub>1060</sub>, standing for SGNH hydrolase 1060.

In Blouzard et al., Ccel_2017, another SGNH hydrolase sharing SGNHh<sub>1060</sub> structural organization (Supplementary Fig. 6A) with a highly similar N-terminal CD (Supplementary Fig. 6B) and renamed herein SGNHh<sub>2017</sub> (PIDOLT), was detected only in WS cellulosomal fraction⁴², suggesting a potential role connected to the presence of lignin in WS. Consequently, SGNHh<sub>2017</sub> was included in this study despite its encoding gene (at locus CCEL_RS10195) has a light upregulation expression pattern belonging to the cluster I described in Fig. 2B (FC > 2 and ~1 comparing WS- respectively to [Mix- and C-] and Cb-data, Supplementary Data 2). We also mined the gene at locus CCEL_RS12090 (cluster I: FC ~ 4 to 6 comparing WS- to all other- data, Fig. 2B and Supplementary data 2) annotated in the genome as coding for a "GDSL-type esterase/lipase family protein", a

subclass of the SGNH hydrolase superfamily. The corresponding encoded protein Ccel_2395 (PIDOLT) renamed SGNHh<sub>2395</sub> in the rest of this document, has a CD (Supplementary Fig. 6A) displaying weak similarity with either SGNHh<sub>1060</sub> and SGNHh<sub>2017</sub> CDs (Supplementary Fig. 6C) and hosts a N-terminal dockerin. In CAZy, it is classified among the CE3 encompassing acylesterase and acetylxylan esterase¹⁵, in agreement with the function prediction proposed in the previous proteomic analysis, where it was shown to be absent in all tested cellulosomal fractions⁴².

Since 3 out of our 38 + 1 selected candidates (Fig. 2B and Supplementary Data 2) are cellulosomal SGNH hydrolases, we assumed that these enzymes may play important roles in R. cellulolyticum anaerobic lignocellulosic assembly processing. In addition, the three corresponding genes undergo different expression reprogramming between the mid- and the end-exponential phases of the growth on WS (Fig. 2C) that could be correlated to the evolution of the substrate structure and composition, which continuously change during the growth of R. cellulolyticum that degrade WS and use the generated degradation products. Highly similar SGNHh<sub>1060</sub> and SGNHh<sub>2017</sub>, and more divergent SGNHh<sub>2395</sub> might have different degradative activities on the various WS components, probably helping and intervening at different stages of lignocellulose degradation.

## SGNHh<sub>1060</sub> and SGNHh<sub>2017</sub> homologs help the lignocellulosic assembly remodeling in anaerobic cellulolytic bacteria producing cellulosomes

After heterologous overproduction and subsequent purification to homogeneity of the three selected SGNHs (Supplementary Fig. 7), their efficiency to cleave the *p*NP-acetate was shown (pNPA, Fig. 3A), thereby demonstrating their esterase activity⁷⁰. However, SGNHh<sub>1060</sub> also catalyzes the hydrolysis of *p*NP-butyrate (*p*NPB)⁷¹ and SGNHh<sub>2017</sub> is active towards *p*NPB⁷¹ and *p*NP-ferulate (*p*NPF)⁷² (Fig. 3A). Since it is well-known that the SGNH hydrolase superfamily gathers among others diverse carbohydrate-modifying enzymes from CE2, 3, 6, 12, and 17 families that mainly act on acetylated substrates (activities of CE2: 6-O-glycoside deacetylase, acetylmannan esterase, acylesterase, acetylxylan esterase; CE3: acylesterase, acetylxylan esterase; CE6: acetylxylan esterase; CE12: carboxylic ester hydrolase, pectin acylesterase, acetylxylan esterase, rhamnogalacturonan acylesterase; CE17: acetylmannan esterase)¹⁵,⁶⁸, SGNHh<sub>2395</sub>'s exclusive release of esterified acetyl from *p*NPA confirms this enzyme is a typical CE. This is in agreement with what was previously proposed by Blouzard et al.⁴², its CE3 CAZy classification¹⁵ and also with our substrate specificity and function predictions as "carbohydrate esterase" and "acetylxylan esterase" using dbCAN<sup>-73</sup> and ESTHER DB<sup>-74</sup> web servers.

In contrast, no such activity predictions were available or successful for SGNHh<sub>1060</sub> and SGNHh<sub>2017</sub> and both enzymes are also able to hydrolyze *p*-coumaric acid methyl ester (MpCA)⁷⁵⁻⁷⁷ with an additional SGNHh<sub>2017</sub> activity observed on ferulic acid methyl ester (MFA)⁷⁵⁻⁷⁷ (Fig. 3A). They also both display much weaker activities on methyl caffeate (MCA)⁷⁵⁻⁷⁷ and chlorogenic acid (CAC)⁷⁵⁻⁷⁷ but are inactive on methyl sinapate (MSA)⁷⁵⁻⁷⁷ (Fig. 3A). Thus, SGNHh<sub>1060</sub> and SGNHh<sub>2017</sub> additional activities on *p*-HCA ester derivates demonstrate they are at least not typical CE but are likely to be secreted cinnamoyl ester hydrolases³³⁻³⁷ with a broad range of substrate
<figure><img src="image_14.png" /><figcaption>**Fig. 2** | Comparative analysis of *R. cellulolyticum* transcriptomes from anaerobic cultures on more or less lignified substrates. A Venn diagram displaying the number of genes whose expression is upregulated at the end of the exponential phase (p-value [padj] <0.05 and Fold change [FC] > 2) between the three comparisons "WSvsCb", "WSvsC", and "WSvsMix" (Differentially Expressed Genes counts are available in Supplementary data 1) and the relationships between these comparisons. Cb, C, Mix and WS represent the transcriptomic data obtained from cultures on cellobiose, crystalline cellulose, Mix of selected polysaccharides and hatched wheat straw, respectively. The transcriptomic data from biological triplicates for each condition were analyzed and compared using DESeq²⁵⁶. B Heatmap showing the transcriptome dynamics at the end of *R. cellulolyticum* exponential growth phase on Cb, C, Mix and WS for the selected candidate genes coding for *R. cellulolyticum* actors potentially involved in the anaerobic lignocellulosic assembly handling (listed in Supplementary data 2). The blue to red scale represents the Log2FC. The</figcaption></figure>

identification of the products encoded by the selected genes is a Protein Identification based on Old gene Locus Tag (PIDOLT), and the presence of dockerin is indicated by an X. Encoded products with at least one domain with unorthodox or unknown function are in bold, and the three proteins further characterized in this study are in italics and underlined. The annotations are taken from the reference genome⁵⁷. The clusters based on expression pattern search are defined as follows: I/ genes with a slight upregulation profile, II/ genes with a strong upregulation profile and III/ genes with an intermediate upregulation profile. C Comparison "WSvsWSm" of the transcriptomes between the end- (WS) and the mid- (WSm) exponential phase of *R. cellulolyticum* grown on WS for the genes coding the 3 proteins further characterized in this study, combined with the transcriptome dynamics from the comparison "WSvsCb", "WSvsC", and "WSvsMix" described in (B).
A

<table><thead><tr><th rowspan="2">Substrate</th><th colspan="3">Specific activity (U.mg<sup>-1</sup>)</th></tr><tr><th>SGNHh<sub>1060</sub></th><th>SGNHh<sub>2017</sub></th><th>SGNHh<sub>2395</sub></th></tr></thead><tbody><tr><td>p-NPA</td><td>25.8 ± 0.742</td><td>15.8 ± 0.552</td><td>24.3 ± 0.948</td></tr><tr><td>p-NPB</td><td>55.3 ± 1.61</td><td>21.5 ± 1.64</td><td>0.480 ± 0.164</td></tr><tr><td>p-NPF</td><td>0.219 ± 0.042</td><td>1.96 ± 0.286</td><td>0.047 ± 0.026</td></tr><tr><td>MFA</td><td>n.d.</td><td>3.78 ± 0.237</td><td>0.037 ± 0.024</td></tr><tr><td>MCA</td><td>0.507 ± 0.029</td><td>0.333 ± 0.078</td><td>n.d.</td></tr><tr><td>MpCA</td><td>3.96 ± 0.222</td><td>3.30 ± 0.215</td><td>n.d.</td></tr><tr><td>MSA</td><td>n.d.</td><td>n.d.</td><td>n.d.</td></tr><tr><td>CAc</td><td>0.484 ± 0.037</td><td>0.219 ± 0.043</td><td>0.055 ± 0.016</td></tr></tbody></table>

<figure><img src="image_15.png" /><figcaption>**Fig. 3 | Specific activities of SGNHh<sub>1060</sub>, SGNHh<sub>2017</sub> and SGNHh<sub>2395</sub> on model esterase substrates and methyl-hydroxycinnamic acid derivatives.** A The three recombinant SGNHh activities were measured spectrophotometrically following the cleavage of the ester bond from pNP-acetate (pNPA), pNP-butyrate (pNPB) and pNP-ferulate (pNPF) ([Protein] = 44–53 nM, [Substrate] = 1 mM), or from methyl ferulic acid (MFA), methyl caffeic acid (MCA), methyl coumaric acid (MpCA), methyl sinapic acid (MSA) and chlorogenic acid (CAc) ([Protein] = 35–48 nM, [Substrate] = 100 µM). The experiments were performed in triplicate at 37 °C in 50 mM potassium phosphate, pH 7, with the addition of 1% Tween 80 in the buffer for pNPF assays. Negative controls were performed using the same experimental conditions without enzymes. Calculated specific activities are reported in mg of total protein. 1U = µmol pNP/min. n.d.: not detected. The structure of the substrates is represented below the table, and double arrows indicate the position of the cleaved bond. B, C The quantities (µM) of pCA (B) and FA (C) released according to time (hours) by the three recombinant SGNHh when individually applied on WS organosolv lignin are depicted ([Protein] = 1 µM, [Substrate] = 5 g L<sup>-1</sup>). The experiments were performed in triplicate at 37 °C in 50 mM potassium phosphate, pH 7. Negative controls were performed using the same experimental conditions without enzymes, and the presented data were corrected by subtraction of the quantities measured in the negative control from the quantities measured for each enzyme. All error bars presented are standard errors of the mean.</figcaption></figure>

specificity, a role never reported for any member of the SGNH hydrolase superfamily.

Furthermore, and in agreement with each tested SGNHhs' activity on p-HCA derivates (Fig. 3A), only pCA (Fig. 3B) and FA (Fig. 3C) are released upon incubation of purified SGNHh<sub>1060</sub> or SGNHh<sub>2017</sub> with WS organosolv lignin that contains a low amount of both esterified HCAs<sup>78–81</sup>. The specific activities determined for the release of pCA from organosolv lignin by SGNHh<sub>1060</sub> and SGNHh<sub>2017</sub> were 0.41 ± 0.01 and 0.50 ± 0.01 U/µmol, respectively. The specific activities obtained for the release of FA by SGNHh<sub>1060</sub> and SGNHh<sub>2017</sub> were 0.30 ± 0.06 and 0.99 ± 0.01 U/µmol, respectively. It is also noteworthy that more FA was removed from this polymeric substrate by SGNHh<sub>2017</sub> than by SGNHh<sub>1060</sub>, whereas as expected, no HCA was released upon incubation with SGNHh<sub>2395</sub>, thus confirming their observed substrates specificities (Fig. 3A). Moreover, the fact that no pCA and FA liberation was detected when purified SGNHh<sub>1060</sub>, SGNHh<sub>2017</sub> and SGNHh<sub>2395</sub> were applied on alkali- and Kraft-lignin, demonstrates that SGNHh<sub>1060</sub> and SGNHh<sub>2017</sub> only target esterified HCAs that are completely depleted during the production process of these two pure modified lignins<sup>82–84</sup>. Altogether, it suggests that when integrated into cellulosomes, both these enzymes may be responsible for releasing a significant amount of pCA and of FA from WS during R. cellulolyticum growth. Our data support their potential direct roles in modifying lignocellulosic assemblies by principally removing pCA that mainly acylates/decorates WS lignin<sup>23–26</sup> and to a lesser extent the xylan<sup>27</sup>, and FA that decorates arabinoxylan chains or by breaking diferulate bounds implicated in lignin-hemicelluloses and hemicelluloses-hemicelluloses linkages<sup>23,25,28,29</sup>, thus facilitating the access and degradation of fermentable PCW polysaccharides.

To further investigate this role, modified strains were generated by the single inactivation of the isolated genes coding for SGNHh<sub>1060</sub> (MTL1060 strain), SGNHh<sub>2017</sub> (MTL2017 strain) and SGNHh<sub>2395</sub> (MTL2395 strain) using the ClosTron technology<sup>85</sup>. They all displayed similar growth on Cb and WS compared with the wild-type (wt) R. cellulolyticum (Supplementary Fig. 8). Since R. cellulolyticum possesses, in addition to SGNHh<sub>2395</sub>, 12 others CE indexed in CAZy<sup>15</sup> (Supplementary data 1) including 7 secreted proteins (Ccel_0286, Ccel_0567, Ccel_1232, Ccel_1240, Ccel_1246, Ccel_2162 and Ccel_3463 [PIDOLT]), 5 of which are cellulosomal (Ccel_0286, Ccel_1232, Ccel_1240, Ccel_1246 and Ccel_2162 [PIDOLT]) and/or 4 of which are from CE2, 4 and 6 families acting on acetylated substrates (Ccel_0286, Ccel_0567, Ccel_1240 and Ccel_2162 [PIDOLT]), this functional redundancy could potentially rescue the SGNHh<sub>2395</sub> absence, hence the MTL2395 phenotype on WS. For MTL1060 and MTL2017, the lack of SGNHh<sub>1060</sub> could be compensated by SGNHh<sub>2017</sub> and vice versa due to their similarities and shared “cinnamoyl ester hydrolase” activity. However, we cannot exclude that other unidentified enzymes could functionally replace them.

Alternatively, SGNHh<sub>1060</sub>, SGNHh<sub>2017</sub>, or SGNHh<sub>2395</sub> variants without dockerin (to prevent the saturation of cellulosomes by the engineered SGNHhs<sup>86</sup>) were also individually overproduced in wt R. cellulolyticum where they were efficiently secreted in the free state (Supplementary Fig. 9). Compared with all other tested strains (Supplementary Fig. 10G–K), only the strain overproducing SGNHh<sub>2017</sub> showed an increased fermentation products excretion during growth on WS (Fig. 4A), surprisingly due to the release of formate in large quantity (Fig. 4C) but also a slightly higher production of acetate (Fig. 4B). Indeed, formate was not detected during the growth on Cb or Mix of this strain and of all other wt or modified strains (including the control transformed with pSOSzeroTm) regardless of the substrate (Supplementary Fig. 10 and 11). Furthermore, the strain over-producing SGNHh<sub>2017</sub> also displays a tendency to accumulate more pCA and FA in WS culture supernatant than wt R. cellulolyticum (Fig. 1C), whereas its growth induces a similar reduction of both the FA proportion and of the S/G ratio in the residual substrate, along with a comparable increase of the aryl ether structures (Fig. 1A, B). Noteworthy, the tricin structures and pCA proportions are not affected compared with the uninoculated control and contrary to what occurs for the wt strain (Fig. 1A, B). Since no significant expression FC (~1) is observed for the SGNHh<sub>2017</sub>
<figure><img src="image_16.png" /><figcaption>**Fig. 4 | Anaerobic growths of SGNHh<sub>1060</sub>*, SGNHh<sub>2017</sub>* or SGNHh<sub>2395</sub>* over-producing *R. cellulolyticum* strains on hatched wheat straw (WS).** The anaerobic growths of the wt *R. cellulolyticum* transformed with pSOSzeroTm (used as a control strain) or with the plasmids pSOS-1060, pSOS-2017 or pSOS-2395, allowing respectively the overproduction of SGNHh<sub>1060</sub>*, SGNHh<sub>2017</sub>* or SGNHh<sub>2395</sub>* were performed at 32 °C. Growth was followed by quantification by HPLC of the concentration of the total fermentation products excreted in the culture supernatants, the latter method being called Metabolic Profiling (MP) in this paper (reliability of</figcaption></figure>

this method is analyzed in Supplementary Fig. 4). The subsequent evolution over time of the concentration of the total fermentation products (Sum [lactate + acetate + formate + ethanol]) excreted in the culture supernatants is depicted (A) with zooms on the acetate (B) and formate excretion (C). The experiments were carried out in triplicate, and the mean and standard deviation are presented. More details on the quantification used to generate this figure are available in Supplementary Fig. 10. All error bars presented are standard errors of the mean.

encoding gene between WS- and Cb-conditions (Fig. 2B and Supplementary data 2), SGNHh<sub>2017</sub>* overproduction leads to its accumulation in larger quantities in the WS based culture supernatant (Supplementary Fig. 9). Its extracellular concentration increase could consequently strongly heighten the impact of SGNHh<sub>2017</sub> and SGNHh<sub>2017</sub>* combined activities on the lignocellulose network to further relax it and to promote better access to the PCW polysaccharides. This increased availability of fermentable sugars may have induced an excess carbon flux mainly directed toward the production of formate<sup>43</sup>. Considering the high similarity between SGNHh<sub>1060</sub> and SGNHh<sub>2017</sub> (Supplementary Fig. 6A, B), the lack of phenotype of the SGNHh<sub>1060</sub>* overproducing strain grown on WS compared to the control *R. cellulolyticum* strain is surprising (Fig. 4). However, the transcriptomic data show a high overexpression of the SGNHh<sub>1060</sub> encoding gene (FC ~ 66) between WS- and Cb-conditions (Fig. 2B and Supplementary data 2) that could be translated by a high secretion of SGNHh<sub>1060</sub> during the growth of wt *R. cellulolyticum* on WS. Thus, the quantity gain brought by the over-production of SGNHh<sub>1060</sub>* could be insufficient to induce a clear phenotype, in contrast to SGNHh<sub>2017</sub>* overproduction. Based on SGNHh<sub>2395</sub> CE activity, its overproduction should not have an effect on WS overall lignocellulose structure since its substrate is buried below the lignin matrix, and as a result, SGNHh<sub>2395</sub>* accumulation would not end up in a better deacetylation of carbohydrates whose access is then precluded, hence the observed phenotype.

Annotations from InterProScan⁶⁰ and NCBI Conserved Domain Database (CDD)⁷⁷ confirm SGNHh<sub>1060</sub>, SGNHh<sub>2017</sub> and SGNHh<sub>2395</sub> contain a “SGNH_hydrolase” catalytic domain (CD) from the Sequence Cluster cd00229⁸⁸. However, SGNHh<sub>2395</sub> CD is also classified in the cd01833 sub-family called “XynB_like”⁸⁹,⁹⁰ whereas no such sub-hierarchy is available for SGNHh<sub>1060</sub> and SGNHh<sub>2017</sub> CDs. To investigate the distribution of all three SGNH homologs among organisms, a BLASTP analysis⁵⁹ against the Refseq protein database using their CD sequence as query (Expect value threshold = 10<sup>-5</sup>) was performed. As expected, given its herein proposed function as typical CE, enzyme type known to be present in all life kingdoms, SGNHh<sub>2395</sub> homologs are widespread among the bacteria (>5000 hits) and to a lower extent in Dikarya (fungi, 805 hits) and archaea (11 hits). In contrast, only 90 hits for SGNHh<sub>2017</sub> corresponding to proteins exclusively secreted by bacteria could be found, including 36 out of the 38 hits detected for SGNHh<sub>1060</sub> (Supplementary data 3). The most similar homologs are appended with a dockerin domain that allows their recruitment in the

cellulosomes produced by the cellulolytic Gram-positive *clostridia* they are from, all of these microorganisms being close to *R. cellulolyticum* (*Ruminiclostrium*, *Anaerocolumna*, *Clostridium* and *Herbinix* genus, Fig. 5). Except for the cellulolytic and Gram-negative bacteria from the *Sorangium* genus homologs, the other detected proteins mainly come from marine Gram-negative *gammaproteobacteria* not reported to be cellulolytic. Yet, a number of them have been isolated from oil-contaminated waters or near oil wells, and are thought to have a hydrocarbon-related metabolism (Supplementary data 3 and Fig. 5).

Overall, the results of our biochemical, genetic, and bioinformatic approaches all trend toward the same direction and support the role of SGNHh<sub>1060</sub> and SGNHh<sub>2017</sub> in lignocellulosic assemblies remodeling, mainly restricted to cellulolytic anaerobic bacteria producing cellulosomes.

## Discussion

The microbial mineralization of dead plant matter in oxygen-deprived environments plays a significant role in the global carbon cycle⁶. The well-documented and characterized enzymatic systems deployed by anaerobes to degrade major plant polysaccharides, such as cellulose and hemicelluloses, are based on specific and efficient strategies⁷,⁸,¹⁵, often relying on cellulosomes¹⁶,⁶³. However, lignin hinders the activity of cellulolytic enzymes by precluding their access to PCW polysaccharides⁹¹, adsorbing them⁹², impeding their processive movement⁹³ or even inactivating them⁹⁴,⁹⁵. The way anaerobic microbes degrade or modify lignin to access the fermentable polysaccharides still remains elusive, and was even questioned until a recent study provided evidence that some anaerobic fungi can at least partly depolymerize lignin, but no enzymatic actor has been identified yet⁴⁰. Consequently, the investigation of the delignification of plant biomass by cellulolytic anaerobes is an emerging field that is critical for our understanding of a key mineralization step of Earth's carbon cycle occurring in oxygen-free environments.

Our study focuses on uncovering enzymatic tools promoting lignocellulose breakdown in anaerobic bacteria and on studying how they deal with lignin, whether they are modifying and/or “mildly” degrading it⁴⁰ in comparison to aerobic processes²¹,²²,³⁸,³⁹. It shows that the strict anaerobic and cellulolytic bacterium producing cellulosomes, *R. cellulolyticum*⁴²,⁸⁶, releases pCA and FA during its growth on WS, thereby supporting that this bacterium can at least partly remodel the lignocellulosic assemblies composing PCW to loosen or even open the intricate network of lignocellulose
<figure><img src="image_17.png" /><figcaption>**Fig. 5 | Occurrence of SGNHh<sub>2017</sub> and SGNHh<sub>1060</sub> homologs across bacteria.** A BlastP search⁵⁹ against the Refseq protein database using SGNHh<sub>2017</sub> and SGNHh<sub>1060</sub> catalytic domain sequence as queries and an Expect value threshold (E) of 10<sup>-5</sup> shows that homologs are only present in bacteria. Their characteristics (reported in Supplementary Data 3) are compiled in this heatmap. Data are ranked according to the average percentage identity of hits within a bacterial genus ("Average %id", 8th column, see below). The 1st column ("Genus") reports the bacterial genus associated with the aligned database sequences. The 2nd column ("Species (strains)") indicates the number of species and the number of strains (lowercase) per genus in which at least a hit was found. The 3rd column ("Gram") distinguishes the Gram-positive (+) and Gram-negative bacteria (-). The 4th column ("Cellulolytic") discriminates the cellulolytic (i.e., able to utilize cellulose as the sole carbon source) microorganisms (black square) from those for which this phenotype has not been reported (white square). Bacteria known to synthesize cellulosome and/or whose genome encodes cohesin-containing cellulosome scaffolding protein(s) (black square) are specified in the 5th column ("Cellulosome producer"). Strict anaerobes (black square) and aerobes or facultative anaerobes (white square) are discriminated in the 6th column ("Strict anaerobe"). The 7th column ("Hits") summarizes the number of different homologous proteins found per genus. The average percentage of identity of all proteins compared to SGNHh<sub>2017</sub> related to a single genus is indicated in the 8th column ("Average %id."), and symbolized by squares colored according to the scale shown at the bottom of the figure. The 9th column ("%with dockerin") summarizes the proportion of proteins displaying a dockerin module in a specific genus, which is symbolized by squares colored according to the scale shown at the bottom of the figure. It should be noted that all extracted proteins display a leader peptide except for one protein encoded by the genome of *Sorangium aterium* (WP_272098580.1) and one protein encoded by the genome of *Myxococcus fulvus* (WP_218035704.1), for which no leader peptide could be identified using the software SignalP 6.0⁵⁸.</figcaption></figure>

to grant a greater access to plant polysaccharides. Thus, we conducted a transcriptomic approach that allowed us to provide a shortlist of potential proteins involved in this process, including two homologous cellulosomal proteins (SGNHh<sub>1060</sub> and SGNHh<sub>2017</sub>) that were biochemically validated as esterases, active on *p*CA- and/or FA-ester derivatives and able to release these HCAs directly from polymeric substrates like WS organosolv lignin. Furthermore, in vivo overproduction and secretion of SGNHh<sub>2017</sub>* in *R. cellulolyticum* improved the growth of this modified strain on WS and increased consistently the release of *p*CA and FA from this complex lignified substrate. Moreover, its metabolism is reprogrammed toward the production of formate exclusively on this substrate presumably to deal with the carbon flux change resulting from a better access to the WS polysaccharides, this additional fermentation product possibly due to the activation of a Pyruvate Formate-Lyase pathway (encoding genes CCEL_RS11245 and CCEL_RS13060)⁵⁷.

Taken together, our results strongly suggest that SGNHh<sub>1060</sub> and SGNHh<sub>2017</sub> facilitate access to PCW polysaccharides by loosening the lignocellulose network via the removal of lignin decorations and by relaxing the lignin-hemicelluloses webs as was proposed for Glucuronoyl esterases (GE)⁹⁶,⁹⁷. These two enzymes, which are completely different from GEs and are produced specifically by anaerobic cellulolytic bacteria, may catalyze the cleavage of ester linkages leading to the release of both *p*CA, which is mainly an esterified lignin decoration in WS<sup>23-26</sup>(SGNHh<sub>1060</sub> and SGNHh<sub>2017</sub>), and FA (SGNHh<sub>2017</sub>), a compound implicated in lignin-hemicelluloses and hemicellulose boundaries<sup>23,25,28,29</sup>. This way, their combined actions should help to promote the disassembly of lignocellulose by breaking the LCC targeting ester bond in lignocellulosic biomass such as WS in O<sub>2</sub>-deprived environments, which seems to be particularly the case in cellulolytic anaerobes. This is consistent with the recent discovery that fungal wood decay can occur in anoxic conditions<sup>98</sup> and that the brown-rot fungus *Fomitopsis pinicola*, and possibly other brown-rot and white-rot fungi, have developed a specific lignocellulose degradation strategy to continue to colonize the anoxic core part of wood stumps and trunks. To do so, *F. pinicola* switches from ROS production (Fenton reaction and secretion of specific oxidases) to degrade lignin and access polysaccharides, towards the secretion of Glycoside Hydrolases and CE in anoxia to open the lignocellulosic material network without use of ROS, in order to degrade the parietal sugars and consequently penetrate further the wood. Then, our data confirm that CEs (1 CE3, 1 CE4, 1 CE6, 1 CE8 and 2 CEnc among the 38 + 1 targets identified in our transcriptomic approach, Fig. 2B and Supplementary data 2) are key for lignocellulosic biomass degradation in absence of oxygen but more specifically show that the esterase activity is essential for anaerobic cellulolytic bacteria producing cellulosomes to allow the unzipping of the lignin from the lignocellulose other components. Our study also highlights how SGNHh<sub>1060</sub> and SGNHh<sub>2017</sub>'s activity of secreted cinnamoyl ester hydrolases<sup>38-42</sup>, which was never reported for any members of the vast SGNH hydrolase superfamily that to date only includes lipases, proteases, and carbohydrate esterases<sup>67-69</sup>, could complement those of cellulases and hemicellulases in breaking down the complex lignocellulosic matrix by improving their access to the plant polysaccharidic fibers networks<sup>99</sup>. This could lead, for example, to an excess carbon flux and hence the metabolic reprogramming of the strain overproducing SGNHh<sub>2017</sub>*.

Noteworthily, despite that both SGNHh<sub>1060</sub> and SGNHh<sub>2017</sub> could participate to the anaerobic handling of the lignocellulosic assemblies (catalysis based on the formation of an oxyanion hole and no requirement of oxygen) to open the interwoven network of the substrate, our biochemical and genetic data combined with the different expression patterns of their encoding genes during growth on WS (Fig. 2B and Supplementary data 2) also suggest they do not have the same role and they intervene at different stages of the biomass degradation. Comparative genomic analyses indicate that the occurrence of enzymes highly similar to SGNHh<sub>1060</sub> and SGNHh<sub>2017</sub> seems to be restricted to a few mesophilic anaerobic bacteria producing cellulosomes. Moreover, despite the anaerobic fungus *Neocallimastix* strain MC-2 secretes *p*-coumaroyl esterase acting on *p*-coumaroyl-oligosaccharide esters and PAXX (0-[5-O-((E)-p-coumaroyl)-α-L-
arabinofuranosyl]-(1--3)-O--D-xylopyranosyl-(1- + 4)-D-
xylopyranose)¹⁰⁰, the genomes of the lignin deconstructing fungi from the
same genus⁴⁰ do not encode SGNHh-like enzymes. This suggests cellulolytic
anaerobes have developed different strategies to overcome the hurdle of
lignin, and it thus appears that their relationship with this polyaromatic
polymer is more complex than widely shared. For example, since cellulo-
somes from different species are recruiting proteins of still unknown
function⁴²,¹⁰¹ (as previously described for the herein studied R. cellulolyti-
cum's SGNHh₁₀₆₀ and SGNHh₂₀₁₇), these efficient cellulolytic nano-
machines are likely to directly act on all PCW lignocellulosic components.

Nevertheless, the promotion of the lignocellulosic assemblies utiliza-
tion by R. cellulolyticum is probably not restricted to SGNHh₁₀₆₀ and
SGNHh₂₀₁₇, and we are confident that some of the candidates listed in
Supplementary data 2 are potentially involved in anoxic lignin handling.
Together with the two identified SGNHhs, non-cellulosomal proteins with
unknown function could participate in this phenomenon (limited lignin
modification or deconstruction in the absence of oxygen) previously pro-
posed for the anaerobic fungi from the Neocallimastigomycota genus⁴⁰ and
also observed in the study (Fig. 1). Interestingly, a recent study showed that
R. cellulolyticum possesses an intracellular enzyme called XuaH that
removes *p*-hydroxycinnamoyl decorations from LCC derived from wheat
bran and WS¹⁰². Back in the context of our study, the complete removal of
aromatic moieties linking lignin to hemicelluloses does not seem to be
necessary for our model anaerobic cellulolytic bacterium to utilize plant
polysaccharide degradation products¹⁰². Thus, a “mild but necessary enzymatic delignification” that relaxes the lignocellulose network (by unhooking
the lignin matrix from the PCW polysaccharides to promote their better
access) and that produces LCC would be sufficient for R. cellulolyticum to
thrive on lignocellulosic biomass.

In addition, the discovery of enzymes from cellulolytic anaerobes⁹, such as SGNHh₁₀₆₀ and SGNHh₂₀₁₇ that are likely to participate to the degrafting of the lignin from the WS polysaccharidic fraction and to modify this polyaromatic polymer (decoration removal), could also contribute to develop biotechnological applications based on the conversion of all plant biomass components including the underused lignin¹⁸,¹⁰³,¹⁰⁴, a credible solution for transitioning away from fossil feedstock use towards a circular carbon Bioeconomy¹²⁻¹⁴,¹⁰⁵,¹⁰⁶. Indeed, as well as probably helping to overcome the lignin barrier and the effect of its degradation byproducts¹⁰⁷, SGNHh₁₀₆₀ and SGNHh₂₀₁₇ identified in this work could serve to extract aromatic synths still difficult to produce¹⁰⁸,¹⁰⁹ like pCA and FA that are valuable for industrial sectors like biofuels, chemicals, food and pharmaceuticals¹¹⁰,¹¹¹.

**Methods**

Cultivation conditions

Wild type (wt) or modified R. cellulolyticum H10 ATCC 35319 was cul-
tivated anaerobically at 32 °C in basal H10 medium (MOPS 18 g L⁻¹,
KH₂PO₄ 2.17 g L⁻¹, yeast extract 2 g L⁻¹, (NH₄)₂SO₄ 1.3 g L⁻¹, MgCl₂-6
H₂O 0.1 g L⁻¹; CaCl₂-2 H₂O 0.2 g L⁻¹, FeSO₄-7 H₂O 12.5 mg L⁻¹, cysteine
0.5 g L⁻¹, few drops of resazurine) supplemented with different carbohy-
drate sources:D- (+)-cellobiose (Cb) 4 g L⁻¹ (Sigma, St Louis, MO), Sig-
macell cellulose (C) 4 g L⁻¹ (Sigma) or hatched WS (chips ~ 4–6 mm)
5 g L⁻¹ ⁴²,⁴⁶,¹¹². We also worked with a mix (Mix) of polysaccharides:
2.3 g L⁻¹ Sigmacell cellulose (Sigma) + 1.5 g L⁻¹ insoluble wheat arabi-
noxylan WAXYi (Megazyme, Wicklow, Ireland) + 0.2 g L⁻¹ xyloglucan
(Megazyme). All insoluble substrates (C, WS or Mix) were added to the
media before sterilization, subsequently sterilized with the medium,
especially for WS, to avoid contamination of the cultures by other
microorganisms. For Cb, a sterile stock was used to supplement the ster-
ilized media. Modified strains of R. cellulolyticum MTL1060, MTL2017,
and MTL2395 were grown in basal H10 medium supplemented with
5 µg mL⁻¹ erythromycin, and R. cellulolyticum strains hosting the vectors
pSOS-1060, pSOS-2017 or pSOS295 were grown in basal H10 medium
supplemented with 5 µg mL⁻¹ thiamphenicol¹¹³.

*E. coli* strains (*E. coli* BL21 (DE3), T7 Express or NEB5α) were grown at 37 °C in Lysogenic Broth containing the appropriate antibiotic when necessary (50 mg L⁻¹ kanamycin, 100 mg L⁻¹ ampicillin, 32 mg L⁻¹ chloramphenicol).

Growth monitoring

Cell growth was measured by following the optical density at 600 nm (OD₆₀₀) for *E. coli* cultures. OD₄₅₀ was used for *R. cellulolyticum* cultures performed in basal H10 medium supplemented with soluble substrates⁴⁶,¹¹³. In the presence of insoluble substrates, we tested different methods to monitor cell growth: total protein quantification using the Folin assay⁴⁶ or measurements of *R. cellulolyticum* MP¹¹⁴. The second method seems more reliable than the Lowry quantification since the number of cells can be correlated with the quantification of fermentation products secreted in the culture supernatant (see Supplementary Fig. 4 and its legend for more explanation). In MP, 500 µL of culture was sampled at each culture time, cells were pelleted (10 min 14,000 × g). 200 µL of supernatant was then mixed with 50 µL of 25 mM H₂SO₄, and 25 µL were analyzed on a Dionex UltiMate 3000 (Thermo Fisher Scientific, Waltham, MA) equipped with an Aminex HPX-87H analytical column (Bio-Rad, Hercules, CA). The eluent was 5 mM H₂SO₄, and the flow rate was set at 0.6 mL min⁻¹. Injection of acetate, lactate, formate and ethanol standard solutions at known concentrations (0.5 mM–10 mM) was used to identify the fermentation products and quantify them after peak area integration using the software Chromeleon 7.2.10 (Thermo Fisher Scientific).

Whole PCW 2D-HSQC-NMR sample preparation and data
acquisition

Supernatant and residual insoluble WS from 400 mL after 9 days anaerobic cultures of R. cellulolyticum incubated at 32 °C or the control condition, where the same culture medium was incubated for the same period of time and at 32 °C, were separated by filtration on glass microfiber filters (Whatman GF/D, cutoff = 2.7 µm). Residual WS was further washed with 50 mL deionized water (MilliQ) and filtered again; the cleaning step was repeated three times. Both supernatants and washed residual WS were lyophilized for 24 h, and the lyophilized material was stored at room temperature until ¹H-¹³C HSQC NMR (2D-HSQC-NMR) and GC-MS analyses.

The preparation of 2D-HSQC-NMR samples is based on Kim and
Ralph method¹¹⁵. Washed residual WS (200 mg) was ground in zirconium
dioxide jars. Sixty mg of the ultrafine ground sample were placed in NMR
tubes before adding 0.6 mL of DMSO-d6/pyridine-d5 (4:1). The sample was
distributed along the length of the tube, and the tube was stored overnight at
20 °C. The tubes were then placed in an ultrasonic bath and sonicated until
the gel became apparently homogeneous. Acquisition was performed on a
600 MHz Bruker spectrometer equipped with TopSpin 3.1 software⁴⁹. The
HSQC NMR profile was obtained from the program (hsqcedetgpsisp2.2)
ns:42; TD F2:2102; TD F1:752; DS:16; D1:1 s; SW F2:14ppm; SW F1:162
ppm; NUS:50%; NusPoints:188. 2D profiles were calibrated on DMSO-d6:
1H: 2.50 ppm; 13 C: 39.5 ppm and assignment was based on the literature¹¹⁵.
Three biological replicates of R. cellulolyticum cultures and of the control
condition were analyzed, and the peak contour integration was performed
using TopSpin 4.3.0 Academic Software.

Fourier transform IR spectroscopy of the whole PCW

Two mg of washed WS samples prepared as described in the Materials and Methods section “Whole PCW 2D-HSQC-NMR sample preparation and data acquisition”, were ground in zirconium dioxide jars and pressed into KBr pellets. Spectra were acquired on an IR spectrophotometer in transmission mode (Nicolet 6700, Thermo Fisher Scientific), with wavenumbers ranging from 4000 to 400 cm⁻¹, and each sample underwent 16 scans at a resolution of 4 cm⁻¹⁴⁻⁷,⁴⁸. Spectra were corrected with baseline subtraction performed from 4000 to 400 cm⁻¹, followed by normalization to the area between 1900 and 800 cm⁻¹. Two biological replicates were analyzed, as well
as two replicates of the control condition described in the Materials and Methods section "Whole PCW 2D-HSQC-NMR sample preparation and data acquisition", were carried out.

GC-MS compositional analysis of R. cellulolyticum culture supernatant and post alkali hydrolysis of the washed residual WS

Three hundred mg of lyophilized culture supernatant prepared as described in the Material and Methods section “Whole PCW 2D-HSQC-NMR sample preparation and data acquisition”, were placed in a 12 mL vial with 2 mL of deionized water containing 0.1%v/v formic acid and with 50 µL of internal standard solution (o-coumaric acid, 0.489 mg mL⁻¹ in acetonitrile). The capped vial was shaken 1 h at room temperature. For the alkali hydrolysis, 30 mg of washed residual WS prepared as described in the Material and Methods section “Whole PCW 2D-HSQC-NMR sample preparation and data acquisition”, were placed in a 12 mL vial with 2 mL of deionized water containing NaOH 1 M and with 50 µL of internal standard solution (o-coumaric acid, 0.489 mg mL⁻¹ in acetonitrile). The capped vial was shaken 20 h at room temperature prior adding 0.4 mL of HCl 6 N, and the medium acidity was confirmed using pH indicator paper⁵⁰.

Then 2 mL ethyl acetate was added to both types of sample, and the capped vial was orbitally shaken at 1000 rpm for 1 h prior centrifugation (4800 rpm, 10 min, 5 °C). The organic supernatant was collected, dried and resuspended in 100 µL of ethyl acetate. Prior GC-MS, 20 µL of that solution was sylilated by adding 100 µL of BSTFA + 10 µL of anhydrous pyridine. Each sample was prepared in duplicate (for each of the two biological replicates and duplicates of the control condition described in the Materials and Methods section “Whole PCW 2D-HSQC-NMR sample preparation and data acquisition”), and blanks (0.1% formic acid in deionized water [MilliQ]) were prepared at the beginning and the end of the sequence. A calibration solution was prepared in acetonitrile containing 4-hydroxy benzoic acid (0.768 mg mL⁻¹), *p*-coumaric acid (0.778 mg mL⁻¹) and ferulic acid (0.873 mg mL⁻¹). Each calibration point was prepared in duplicate as follows: 5, 10, 15, 20, 60, 80 or 100 µL of this calibration solution were placed in a 12 mL vial and treated as described above for lyophilized culture supernatant. GC-MS analyses on 0.5 µL of all silylated samples were conducted using a 8860 Series GC system (Agilent) equipped with HP-5ms UI column (Agilent) using Hydrogen as mobile phase at a 1.25 ml min⁻¹ flow rate, and quantifications based on the calibration solution were performed using MassHunter Qualitative Analysis 10.0 (Agilent)⁴⁷,⁵¹.

RNA extraction, library preparation and sequencing (RNA-Seq)

*R. cellulolyticum* was grown in basal H10 medium supplemented with Cb, C, Mix or WS and cells were harvested at the end of the exponential phase of the growth with an additional sampling at the mid-exponential phase for WS (Supplementary Fig. 4C). Cell collection was performed by aerobic filtration on glass microfiber filters (Whatman GF/D, cutoff = 2.7 µm) and elution with 500 mL sterile deionized water (MilliQ)⁴⁹. Since the collection of the cells grown on insoluble substrates (C, Mix and WS) lasts 1 hour, cells collected from cultures on soluble substrates (Cb) were collected the same way and were kept at room temperature in water for 1 h in total in order to be exposed to the same oxidative stress time. After centrifugation at 4000 × g, 4 °C, 10 min, cells were washed with 40 mL of deionized water (MilliQ), centrifuged again at 8000 × g and 4 °C for 5 min, and the pellets were frozen in liquid nitrogen before storing at -80 °C until RNA extraction.

After cell pellets thawing and treatment at 37 °C for 20 min with ~1 g L⁻¹ lysozyme under vigorous agitation, total RNA extraction was performed using the Maxwell® 16 miRNA Tissue Kit (Promega, Madison, WI). Total RNAs were treated using the TURBO DNA-free™ kit (Invitrogen) following the supplier's protocol to get rid of any traces of DNA. The quality of the extracted total mRNA was verified using TapeStation microfluidic electrophoresis with RNA ScreenTape (Agilent Technologies, Santa Clara, CA), and samples were stored at -80 °C. Illumina libraries were produced from 250 ng of total RNA (quantified using a Qubit fluorometer and the Qubit RNA HS Assay Kit, both from Thermo Fisher

Scientific) and using the Zymo-Seq RiboFree® Total RNA Library Kit (Zymo Research, Irvine, CA) following the supplier protocol. The quality of the libraries was verified both by DNA quantification using the Qubit dsDNA BR Assay Kit measured with a Qubit fluorometer (Thermo Fisher Scientific) and microfluidic electrophoresis using a TapeStation with High Sensitivity D1000 ScreenTape (Agilent Technologies).

After libraries quality control using a Fragment Analyzer (Advanced Analytical, Cypress, CA), one run of paired-end (2 × 75 bp) RNA sequencing was performed by the TGML platform (Transcriptomics & Genomics Marseille Luminy, France) on an Illumina NextSeq 500 sequencer using NextSeq 500 High-Output Sequencing kit v2 (Illumina, San Diego, CA).

RNA-Seq data analysis

The fastq files from the RNA-Seq were downloaded from the TGML platform server, and the following data treatment was performed locally on an Ubuntu 20.04 OS.

Sequencing quality was verified through FastqQC (v0.12.1)¹¹⁶ before and after the adaptors trimming and the removal of reads with quality below 20 and length below 30 nucleotides performed using the Trimomatic software (v0.39; paramteters: -phred33 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:30 ILLUMINACLIP:TruSeq3-PE-2.fa:2:20:10)¹¹⁷. The read mapping on the *R. cellulolyticum* H10 nucleotidic reference sequence (GCF_000022065.1_ASM2206v1_genomic.fna)⁵⁷ was performed using the BWA MEM software (v0.7.17)¹¹⁸. Then reads assigned to the 3471 CDS features were counted using the htseq_count software (v2.0.3; mode 'intersection-strict')¹¹⁹ according to the GFF annotation file (updated version date: 30/03/2022).

Normalization and differential analysis data were carried out according to the DESeq2⁵⁶ R package with the default parameters (v1.34.0). This package has been launched through the SARTools R pipeline (v1.7.4)¹²⁰ which provides a helpful report to control the entire statistical analysis process. It contains graphs such as the Principal Component Analysis (PCA) that demonstrate a good level of correlation between biological replicates. We focused on the upregulated gene output files applying the following criteria on the differential analysis statistical values: adjusted *p* value (padj) < 0.05; expression Fold Change (FC) > 2.

The overlaps of the upregulated genes expressed in the different conditions were computed using the ggplot2 (v3.5.0)¹²¹ and ggvenn (v0.1.10) R packages. The clustering of the 38 + 1 selected targets and their visualization through a heatmap has been generated using the Bioconductor Complex Heatmap package (v2.10.0)¹²².

Bioinformatic analyses performed for identifying upregulated genes, their function and distribution across organisms

Products of the identified genes with upregulated expression were further analyzed using several bioinformatics tools to select targets potentially implicated in the anaerobic mild delignification process to be further biochemically characterized. Since the first step of lignocellulose degradation takes place outside the cell, we used SignalP 6.0⁵⁸ to predict the secreted proteins encoded in *R. cellulolyticum* reference genome⁵⁷: 268 secreted proteins with two types of signal peptides were found (198 SPI and 88 SPII). Looking for candidates implicated in lignocellulose degradation, including lignin, we first interrogated the CAZy database¹⁵ to determine if the identified targets contain annotated CAZy domains in combination with an InterProScan analysis⁶⁰ and a substrate prediction using the dbCAN-⁷³ and ESTHER DB-⁷⁴ web servers. To further determine if some of these genes encode secreted, totally unknown/hypothetical proteins or proteins bearing domains with unknown function, BLASTP searches were performed to identify homologous sequences of interest that might have similarities with domains of known or unknown function⁵⁹. Deeper function prediction was also performed by manual analysis of the genetic context available in the NCBI Gene database¹²³, of the genes encoding unknown/hypothetical proteins or proteins bearing domains with unknown function.

The distribution of the SGNH-selected candidate homologs in other organisms was analyzed using BLASTP⁵⁹ (Expect value threshold = 10⁻⁵,
analyses performed in November 2024) against the Refseq protein database using as query the sequence of their cd00029 domain as defined in InterPro (UniProt code for SGNHh<sub>1060</sub>: B8I9G0<sup>124</sup>, UniProt code for SGNHh<sub>2017</sub>: B8I3M0<sup>125</sup>, UniProt code for SGNHh<sub>2395</sub>: B8I5J2<sup>126</sup>).

Sequence comparisons and alignments were performed using MAFFT v7<sup>127</sup>.

## Construction of the expression plasmids of the SGNHh<sub>1060</sub>, SGNHh<sub>2017</sub> and SGNHh<sub>2395</sub>

Using genomic DNA from *R. cellulolyticum* as the template, the DNA regions encoding SGNHh<sub>1060</sub> and SGNHh<sub>2395</sub> without signal peptide were amplified using the forward and reverse primers listed in Supplementary table 1 and the NcoI-XhoI digested amplicons were cloned into the NcoI-XhoI linearized pET28b vector (Novagen, Madison, WI)<sup>128</sup>. The resulting plasmids pET28-1060 and pET28-2395 allow the production of C-terminal 6-histidine-tagged recombinant SGNHh<sub>1060</sub> and SGNHh<sub>2395</sub> without signal peptide, respectively. Ligation mixture was used to transform competent *E. coli* NEB5α (New England Biolabs, Ipswich, MA) and clones containing the insert were checked by sequencing. The plasmid pET28-2017 containing a synthetic gene adapted to *E. coli* codon bias that encodes SGNHh<sub>2017</sub> without a signal peptide with a C-terminal 6-histidine-tag was constructed by GenScript Biotech (Rijswijk, The Netherlands).

## Heterologous production and purification of the proteins of interest

*E. coli* BL21 (DE3) competent cells (New England Biolabs) transformed with the plasmids pET28-1060 or pET28-2395 were cultivated at 37 °C in 2.5 L conical Flask containing 1 L LB supplemented with 50 µg L<sup>-1</sup> kanamycin and under agitation at 150 rpm until the culture reached OD<sub>600</sub> 0.8–1. After cooling (1 h incubation at 4 °C), 20 µM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to the cultures that were subsequently incubated overnight at 15 °C under shaking at 150 rpm<sup>129</sup>. The following changes were applied to produce SGNHh<sub>2017</sub>: *E. coli* T7 Express competent cells (New England Biolabs) were transformed with pET28-2017, the culture volume was 400 mL of LB, 1 mM IPTG was used for the induction, which was performed 17 °C.

As previously described<sup>130</sup>, cells incubated for 16 h were harvested by centrifugation (4000 × g, 10 min, 4 °C) and resuspended in 100 mL of 30 mM Tris-HCl pH 8.0, 1 mM CaCl<sub>2</sub> supplemented with a few mg of DNase I. Cells were then disrupted using a French Press (1000 psi). After centrifugation at 15,000 × g, at 4 °C for 10 min, the supernatant (crude extract) was loaded on 2–4 mL of nickel-nitrilotriacetic acid resin (Thermo Fisher Scientific) equilibrated with 30 mM Tris-HCl, pH 8.0, 1 mM CaCl<sub>2</sub>, 5 mM imidazole. After washing with the same buffer, the proteins of interest were eluted using 30 mM Tris-HCl pH 8.0, 1 mM CaCl<sub>2</sub>, 100 mM imidazole. After concentration of the eluted fraction (1.5–2 mL) using a Vivaspin20 (cut-off 10 kDa, Sartorius, Göttingen, Germany), a second purification step was performed using a HiTrap Q HP fast flow column, 1 mL (Cytiva, Marlborough, MA) equilibrated with 30 mM Tris-HCl pH 8.0, 1 mM CaCl<sub>2</sub>. The elution step was performed by applying a linear gradient of 0–500 mM NaCl in 30 mM Tris-HCl pH 8.0, 1 mM CaCl<sub>2</sub>. Finally, proteins were dialyzed and concentrated in a Vivaspin20 (cut-off 10 kDa, Sartorius) against 10 mM Tris-HCl pH 8.0, 1 mM CaCl<sub>2</sub>. Purified protein concentration was evaluated by measuring the absorbance at 280 nm (A<sub>280</sub>) and the extinction coefficient determined using the Protparam tool<sup>131</sup> (21,952.05 M<sup>-1</sup> cm<sup>-1</sup> for SGNHh<sub>1060</sub>, 34,840 M<sup>-1</sup> cm<sup>-1</sup> for SGNHh<sub>2017</sub> and 39,942.5 M<sup>-1</sup> cm<sup>-1</sup> for SGNHh<sub>2395</sub>).

## Enzymatic activity of the proteins of interest on *p*-nitrophenyl and *p*-HCA derivates

*p*-nitrophenyl acetate (*p*NPA, Sigma, St Louis, MO) and *p*-nitrophenyl butyrate (*p*NPB, Sigma) were solubilized in ethanol, whereas *p*-nitrophenyl trans-ferulate (*p*NPF, Sigma) was solubilized in DMSO. The release of *p*-nitrophenol (*p*NP) at 400 nm was followed during incubation at 37 °C in 50 mM phosphate pH 7, with a substrate concentration of 1 mM<sup>70,71</sup>. For

*p*NPF esterase activities, 1% Tween 80 was added to the reaction<sup>72</sup>. All experiments were performed in triplicate using 44–53 nM of enzyme. *p*NP Extinction coefficients of 14,450 and 10,151 M<sup>-1</sup> cm<sup>-1</sup> at 400 nm were calculated from standard curves in 50 mM phosphate buffer pH 7 or Tween 80 supplemented buffer as needed, respectively. Negative controls were performed in the same experimental conditions without the addition of enzymes in order to take into account the eventual non-enzymatic hydrolysis of the substrates in the specific activity calculation. Specific activities are reported in mg of total protein. 1 U = µmol *p*NP min<sup>-1</sup>.

Esterase specific activities on ferulic acid methyl ester (MFA, Clin-iSciences, Nanterre, France)<sup>75-77</sup>, caffeic acid methyl ester (MCA, CliniSciences)<sup>75-77</sup>, *p*-coumaric acid methyl ester (*Mp*CA, CliniSciences)<sup>75-77</sup>, sinapic acid methyl ester (MSA, Interchim, Montluçon, France)<sup>75-77</sup> and chlorogenic acid (CAc, Sigma)<sup>75-77</sup> were also measured in triplicate at 37 °C in 50 mM phosphate pH 7, with 100 µM of substrate and using 35–48 nM of enzyme. All substrates (and products for the standard curves) were first resuspended in ethanol. The hydrolysis of each methyl ester derivative was followed by quantifying the appearance of the product of the reaction and the disappearance of the substrate using specific wavelengths for each substrate, where the difference between the absorbance of the substrate and that of the product is maximal. Extinction coefficients (Ec) for each substrate and product were calculated from standard curves measured at 37 °C in 50 mM phosphate buffer pH 7. The hydrolysis of MFA releasing the ferulic acid (FA, Sigma) was followed at 335 nm with Ec of 11,968 and 3550 M<sup>-1</sup> cm<sup>-1</sup> at 335 nm for MFA and FA, respectively. The hydrolysis of MCA releasing the caffeic acid (CA, Sigma) was followed at 335 nm with Ec of 11,294 and 4103 M<sup>-1</sup> cm<sup>-1</sup> at 335 nm for MCA and CA, respectively. The hydrolysis of *Mp*CA releasing the *p*-coumaric acid (*p*CA, Sigma, St Louis, MO) was followed at 320 nm with Ec of 12,249 and 4910 M<sup>-1</sup> cm<sup>-1</sup> at 320 nm for *Mp*CA and *p*CA, respectively. The hydrolysis of MSA releasing the sinapic acid (SA, Sigma) was followed at 335 nm with Ec of 12,851 and 4323 M<sup>-1</sup> cm<sup>-1</sup> at 335 nm for MSA and SA, respectively. The hydrolysis of CAc, releasing the caffeic acid (CA, Sigma) was followed at 335 nm with Ec of 12,617 and 4103 M<sup>-1</sup> cm<sup>-1</sup> at 335 nm for CAc and CA, respectively. Negative controls were performed in the same experimental conditions without the addition of enzymes in order to take into account the eventual non-enzymatic hydrolysis of the substrates in the specific activity calculation. Specific activities are reported in mg of total protein. 1 U = µmol of substrate transformed min<sup>-1</sup>.

## Enzymatic activity of the proteins of interest on polymeric substrates

FA and *p*CA releasing activities from alkali-lignin (Sigma), Kraft-lignin and WS organosolv lignin (Sigma) were also measured in triplicate at 37 °C in 50 mM phosphate pH 7, at a substrate concentration of 5 g L<sup>-1</sup> and using 1 µM of enzyme. Negative controls were performed using the same experimental conditions without enzymes. Sampling was performed over 24 hours and as follows: 500 µL of reaction mixture was centrifuged (10 min, 14,000 × g) and 200 µL of supernatant was mixed with 50 µL of 25 mM H<sub>2</sub>SO<sub>4</sub>. 25 µL were then analyzed by High-Pressure Liquid Chromatography (flow rate was 1 mL min<sup>-1</sup> at 30 °C) on a Dionex UltiMate 3000 (Thermo Fisher Scientific) equipped with a Zorbax XBD-C18 “eclipse” analytical column (Agilent Technologies) and coupled with a UV detection at 320 nm. The elution was performed with pure acetonitrile (Sigma) and 38.5 mM acetic acid + 16.5 mM sodium acetate (Sigma) as eluents A and B, respectively, using the multistep procedure as follows: isocratic separation (5 min 15% A + 85% B), separation gradient (15 min, 85-75% B), column wash (5 min, 75% B) and subsequent column equilibration (5 min, 85% B). Injection of CA, FA and *p*CA standard solutions at known concentrations (10–100 µM) was used to identify and quantify the released products using the software Chromeleon 7.2.10 (Thermo Fisher Scientific).

## Construction of *R. cellulolyticum* modified strains

Single inactivation of the isolated genes at loci CCEL_RS05325, CCEL_RS10195 or CCEL_RS12090, coding respectively for SGNHh<sub>1060</sub>, SGNHh<sub>2017</sub> and SGNHh<sub>2395</sub> was performed using the ClosTron
technology⁸⁵,¹¹³ to generate the *R. cellulolyticum* strains MTL1060, MTL2017 and MTL2395, respectively. The primers used for the construction and the verification by PCR of the intron insertion are listed in Supplementary table 1. Genomic DNA was extracted using the Nucleospin DNA Rapid Lyse kit (Macherey Nagel, Düren, Germany). For better lysis of *R. cellulolyticum* cells, the supplier protocol was modified as follows: lysis was performed in the recommended resuspension volume with proteinase K using FastPrep-24 mechanical lysis (2 × 40 s 6.5 m s⁻¹, Matrix B). The correct gene inactivation was then verified by whole-genome sequencing of these samples (Novogene, Cambridge, UK).

To overexpress the genes encoding the truncated forms of SGNHh₁₀₆₀* and SGNHh₂₀₁₇* without dockerin but hosting a C-terminal 6-histidine-tag and their own signal peptide, the corresponding DNA regions were amplified using *R. cellulolyticum* genomic DNA as the template and the primers listed in Supplementary table 1. The BamH1/Ehe1 digested amplicons were cloned into the BamH1/Ehe1 linearized pSOS956 vector¹¹³, allowing the expression of the corresponding gene under the control of the strong P*thl* promoter. For SGNHh₂₃₉₅*, a gene encoding the dockerin-free SGNHh₂₃₉₅ appended with a C-terminal 6-histidine-tag and its own signal peptide was synthesized by GenScript Biotech and cloned using the same methodology to generate the plasmid pSOS-2395. The resulting vectors were methylated and electro-transformed in *R. cellulolyticum*⁴⁶. Positive clones were selected on agar plates supplemented with 5 µg mL⁻¹ thiamphenicol anaerobically incubated at 32 °C, and verified by PCR using the forward and reverse primers listed in Supplementary table 1.

## PAGE and western blot analysis to control protein secretion by recombinant *R. cellulolyticum* strains

Modified strains producing SGNHh₁₀₆₀*, SGNHh₂₀₁₇* and SGNHh₂₃₉₅* were grown at 32 °C in basal H10 medium containing Cb (2 g L⁻¹). At the end of the exponential phase (OD₄₀ = 1), 1 mL of culture was centrifuged for 10 min at 14,000 × g and 4 °C, and the supernatant was mixed with 100 µL of a cold solution of 6.1 N trichloroacetic acid. The mix was incubated for 30 min at -20 °C and centrifuged for 10 min at 14,000 × g and 4 °C. The supernatant was discarded, and the pellet was washed with 500 µL of acetone (centrifugation for 10 min at 14,000 × g and 4 °C). The final pellet was resuspended in 15 µL of deionized water (MilliQ) and 15 µL of denaturing buffer, further analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue stained or probed by western blot using antibodies against 6-histidine-tag (Anti-His₆-Peroxidase, Roche, Basel, Switzerland)⁴⁶,¹³². For the western blot analysis, a sample prepared the same way from *R. cellulolyticum* transformed with the plasmid pSOSZeroTm (not expressing any SGNHh*) culture was used as a negative control, whereas 2 µg of purified C-terminal 6-histidine-tagged recombinant Cel5A was used as a positive control¹²⁹.

## Statistics and reproducibility

All experiments were at least performed in triplicate, except for the Fourier transform IR spectroscopy of whole PCW experiments, for which only duplicates were used. The number of biological replicates used for each experiment is indicated in the corresponding figure legends and/or in the corresponding Methods section. For all experiments, the corresponding mean and standard deviation were calculated (single point value and the mean are presented in the figures as individual data points with the corresponding error bars).

## RNA-seq data availability

RNA-seq data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus¹³³ and are publicly accessible through GEO Series accession number GSE290568. It contains the raw reads in FASTQ format for each tested substrate, growth phase and biological replicate (GSM8816752-GSM8816754 for cellobiose at the end of the exponential phase; GSM8816756-GSM8816757 for cellulose at the end of the exponential phase; GSM8816758-GSM8816760 for mix at the end of the exponential phase, GSM8816761-GSM8816763 for WS at the mid of the

exponential phase and GSM8816764-GSM8816766 for WS at the end of the exponential phase). It also contains the read counts grouped into a single matrix generated from this dataset (supplementary file "GSE290568_RNAseq_lignin_CDScount.csv.gz") and that were analyzed as described in the section "RNA-Seq data analysis" in the Methods to generate the Supplementary Data 1.

## Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

## Data and materials availability

All data are available in the main text, the Supplementary Figs. file, the Supplementary table and the Supplementary data. Raw data used to generate the figures from the main text and the Supplementary Figs. Files are gathered in the Excel files named "Supplementary data 4" and "Supplementary data 5" that are provided.

Received: 26 August 2025; Accepted: 17 November 2025;

Published online: 04 December 2025

## References

1. Bar-On, Y. M., Phillips, R. & Milo, R. The biomass distribution on Earth. *Proc. Natl. Acad. Sci. USA* **115**, 6506–6511 (2018).

2. Houghton, R. A., Hall, F. & Goetz, S. J. Importance of biomass in the global carbon cycle. *J. Geophys. Res.* **114**, 2009JG000935 (2009).

3. Chen, H. *Biotechnology of Lignocellulose: Theory and Practice*. (Springer Netherlands, Dordrecht, 2014).

4. Tufail, T. et al. Biochemical characterization of wheat straw cell wall with special reference to bioactive profile. *Int. J. Food Prop.* **21**, 1303–1310 (2018).

5. Zhang, X., Yang, W. & Blasiak, W. Modeling study of woody biomass: interactions of cellulose, hemicellulose, and lignin. *Energy Fuels* **25**, 4786–4795 (2011).

6. Schimel, J. P. & Schaeffer, S. M. Microbial control over carbon cycling in soil. *Front. Microbiol.* **3**, 31913 (2012).

7. Wilson, D. B. Three microbial strategies for plant cell wall degradation. *Ann. N. Y. Acad. Sci.* **1125**, 289–297 (2008).

8. Cragg, S. M. et al. Lignocellulose degradation mechanisms across the tree of life. *Curr. Opin. Chem. Biol.* **29**, 108–119 (2015).

9. Podolsky, I. A. et al. Harnessing Nature's Anaerobes for Biotechnology and Bioprocessing. *Annu. Rev. Chem. Biomol. Eng.* **10**, 105–128 (2019).

10. Heux, S., Meynial-Salles, I., O'Donohue, M. J. & Dumon, C. White biotechnology: state of the art strategies for the development of biocatalysts for biorefining. *Biotechnol. Adv.* **33**, 1653–1670 (2015).

11. Den Haan, R., Van Rensburg, E., Rose, S. H., Görgens, J. F. & Van Zyl, W. H. Progress and challenges in the engineering of non-cellulolytic microorganisms for consolidated bioprocessing. *Curr. Opin. Biotechnol.* **33**, 32–38 (2015).

12. Calvo-Flores, F. G. & Martin-Martinez, F. J. Biorefineries: achievements and challenges for a bio-based economy. *Front. Chem.* **10**, 973417 (2022).

13. Mori, R. Replacing all petroleum-based chemical products with natural biomass-based chemical products: a tutorial review. *RSC Sustain* **1**, 179–212 (2023).

14. European Commission. Joint Research Centre. *Biomass Production, Supply, Uses and Flows in the European Union: First Results from an Integrated Assessment*. (Publications Office, LU, 2018).

15. Cantarel, B. L. et al. The Carbohydrate-Active EnZymes database (CAZy): an expert resource for glycogenomics. *Nucleic Acids Res.* **37**, D233–D238 (2009).

16. Artzi, L., Bayer, E. A. & Moraïs, S. Cellulosomes: bacterial nanomachines for dismantling plant polysaccharides. *Nat. Rev. Microbiol.* **15**, 83–95 (2017).
17. Lankiewicz, T. S., Lillington, S. P. & O'Malley, M. A. Enzyme discovery in anaerobic fungi (neocallimastigomycetes) enables lignocellulosic biorefinery innovation. *Microbiol. Mol. Biol. Rev.* **86**, e00041-22 (2022).

18. Vigh, M. A trashed treasure: lignin could become a large and renewable source of organic compounds for the chemical industry to replace fossil fuel-based chemicals. *EMBO Rep.* **24**, e57103 (2023).

19. Vanholme, R., Demedts, B., Morreel, K., Ralph, J. & Boerjan, W. Lignin biosynthesis and structure. *Plant Physiol.* **153**, 895-905 (2010).

20. Peracchi, L. M., Panahabadi, R., Barros-Rios, J., Bartley, L. E. & Sanguinet, K. A. Grass lignin: biosynthesis, biological roles, and industrial applications. *Front. Plant Sci.* **15**, 1343097 (2024).

21. Bugg, T. D. H., Ahmad, M., Hardiman, E. M. & Rahmanpour, R. Pathways for degradation of lignin in bacteria and fungi. *Nat. Prod. Rep.* **28**, 1883 (2011).

22. Cui, L. et al. Lignin biodegradation and its valorization. *Fermentation* **8**, 366 (2022).

23. Del Rio, J. C. et al. Structural characterization of wheat straw lignin as revealed by analytical pyrolysis, 2D-NMR, and reductive cleavage methods. *J. Agric. Food Chem.* **60**, 5922-5935 (2012).

24. Wang, X. et al. Recent advances in transforming agricultural biorefinery lignins into value-added products. *J. Agric. Food Res.* **12**, 100545 (2023).

25. Chen, M., Li, Y., Lu, F., Luterbacher, J. S. & Ralph, J. Lignin hydrogenolysis: phenolic monomers from lignin and associated phenolates across plant clades. *ACS Sustain. Chem. Eng.* **11**, 10001-10017 (2023).

26. Del Rio, J. C. et al. Differences in the chemical structure of the lignins from sugarcane bagasse and straw. *Biomass. Bioenergy* **81**, 322-338 (2015).

27. Terrett, O. M. & Dupree, P. Covalent interactions between lignin and hemicelluloses in plant secondary cell walls. *Curr. Opin. Biotechnol.* **56**, 97-104 (2019).

28. Oliveira, D. M. et al. Feruloyl esterases: Biocatalysts to overcome biomass recalcitrance and for the production of bioactive compounds. *Bioresour. Technol.* **278**, 408-423 (2019).

29. Vismeh, R. et al. Profiling of diferulates (plant cell wall cross-linkers) using ultrahigh-performance liquid chromatography-tandem mass spectrometry. *Analyst* **138**, 6683 (2013).

30. You, T.-T., Zhang, L.-M., Zhou, S.-K. & Xu, F. Structural elucidation of lignin-carbohydrate complex (LCC) preparations and lignin from Arundo donax Linn. *Ind. Crops Products* **71**, 65-74 (2015).

31. Yoo, C. G., Meng, X., Pu, Y. & Ragauskas, A. J. The critical role of lignin in lignocellulosic biomass conversion and recent pretreatment strategies: a comprehensive review. *Bioresour. Technol.* **301**, 122784 (2020).

32. Tarasov, D., Leitch, M. & Fatehi, P. Lignin-carbohydrate complexes: properties, applications, analyses, and methods of extraction: a review. *Biotechnol. Biofuels* **11**, 269 (2018).

33. Borneman, W. S., Hartley, R. D., Morrison, W. H., Akin, D. E. & Ljungdahl, L. G. Feruloyl and p-coumaroyl esterase from anaerobic fungi in relation to plant cell wall degradation. *Appl. Microbiol. Biotechnol.* **33**, 345-351 (1990).

34. Wong, D. W. S., Chan, V. J. & Liao, H. Metagenomic discovery of feruloyl esterases from rumen microflora. *Appl. Microbiol. Biotechnol.* **103**, 8449-8457 (2019).

35. Qi, M. et al. Isolation and characterization of a ferulic acid esterase (Fae1A) from the rumen fungus Anaeromyces mucronatus: A ferulic acid esterase in rumen fungus. *J. Appl. Microbiol.* **110**, 1341-1350 (2011).

36. Donaghy, J. A., Bronnenmeier, K., Soto-Kelly, P. F. & McKay, A. M. Purification and characterization of an extracellular feruloyl esterase from the thermophilic anaerobe Clostridium stercorarium. *J. Appl. Microbiol.* **88**, 458-466 (2000).

37. Goldstone, D. C. et al. Structural and functional characterization of a promiscuous feruloyl esterase (Est1E) from the rumen bacterium *Butyrvibrio proteoclasticus*. *Proteins* **78**, 1457-1469 (2010).

38. Bugg, T. D. H. The chemical logic of enzymatic lignin degradation. *Chem. Commun.* **60**, 804-814 (2024).

39. Grgas, D. et al. The bacterial degradation of lignin—a review. *Water* **15**, 1272 (2023).

40. Lankiewicz, T. S. et al. Lignin deconstruction by anaerobic fungi. *Nat. Microbiol.* **8**, 596-610 (2023).

41. Bugg, T. D. H., Williamson, J. J. & Rashid, G. M. M. Bacterial enzymes for lignin depolymerisation: new biocatalysts for generation of renewable chemicals from biomass. *Curr. Opin. Chem. Biol.* **55**, 26-33 (2020).

42. Blouzard, J. et al. Modulation of cellulosome composition in *Clostridium cellulolyticum*: Adaptation to the polysaccharide environment revealed by proteomic and carbohydrate-active enzyme analyses. *Proteomics* **10**, 541-554 (2010).

43. Petitdemange, E., Caillet, F., Giallo, J. & Gaudin, C. Clostridium cellulolyticum sp. nov., a Cellulolytic, mesophilic: species from decayed grass. *Int. J. Syst. Bacteriol.* **34**, 155-159 (1984).

44. Celik, H. et al. A two-component system (XydS/R) controls the expression of genes encoding CBM6-containing proteins in response to straw in Clostridium cellulolyticum. *PLoS One* **8**, e56063 (2013).

45. Liu, N. et al. Selfish uptake versus extracellular arabinoxylan degradation in the primary degrader Ruminiclostridium cellulolyticum, a new string to its bow. *Biotechnol. Biofuels* **15**, 127 (2022).

46. Fosses, A. et al. A seven-gene cluster in Ruminiclostridium cellulolyticum is essential for signalization, uptake and catabolism of the degradation products of cellulose hydrolysis. *Biotechnol. Biofuels* **10**, 250 (2017).

47. Malric-Garajova, S. et al. Modification of a marine pine kraft lignin sample by enzymatic treatment with a Pycnoporus cinnabarinus Laccase. *Molecules* **28**, 4873 (2023).

48. Chabbert, B. et al. Action of lytic polysaccharide monooxygenase on plant tissue is governed by cellular type. *Sci. Rep.* **7**, 17792 (2017).

49. Auxenfans, T., Crônier, D., Chabbert, B. & Paës, G. Understanding the structural and chemical changes of plant biomass following steam explosion pretreatment. *Biotechnol. Biofuels* **10**, 36 (2017).

50. Daou, M. et al. Fungal treatment for the valorization of technical soda lignin. *JoF* **7**, 39 (2021).

51. Majira, A. et al. Enhancing the antioxidant activity of technical lignins by combining solvent fractionation and ionic-liquid treatment. *ChemSusChem* **12**, 4799-4809 (2019).

52. Konzock, O., Tous-Mohedano, M., Cibin, I., Chen, Y. & Norbeck, J. Cinnamic acid and p-coumaric acid are metabolized to 4-hydroxybenzoic acid by Yarrowia lipolytica. *AMB Expr.* **13**, 84 (2023).

53. Lan, W. et al. Elucidating tricin-lignin structures: assigning correlations in HSQC spectra of monocot lignins. *Polymers* **10**, 916 (2018).

54. Zhang, L., Sun, L., Zhang, B. & Chen, L. Identification of differentially expressed genes (DEGs) relevant to prognosis of ovarian cancer by use of integrated bioinformatics analysis and validation by immunohistochemistry assay. *Med Sci. Monit.* **25**, 9902-9912 (2019).

55. Ardissoone, S., Kint, N., Petrignani, B., Panis, G. & Viollier, P. H. Secretion relieves translational co-repression by a specialized flagellin paralog. *Dev. Cell* **55**, 500-513.e4 (2020).

56. Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. *Genome Biol.* **15**, 550 (2014).

57. Index of /genomes/all/GCF/000/022/065/GCF_000022065.1_ASML2206v1. https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/022/065/GCF_000022065.1_ASML2206v1/.
58. Teufel, F. et al. SignalP 6.0 predicts all five types of signal peptides using protein language models. *Nat. Biotechnol.* **40**, 1023–1025 (2022).

59. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. *J. Mol. Biol.* **215**, 403–410 (1990).

60. Jones, P. et al. InterProScan 5: genome-scale protein function classification. *Bioinformatics* **30**, 1236–1240 (2014).

61. Østby, H. & Várnai, A. Hemicellulolytic enzymes in lignocellulose processing. *Essays Biochem.* **67**, 533–550 (2023).

62. Brown, J. L. et al. Co-cultivation of the anaerobic fungus Caecomyces churrovis with Methanobacterium bryantii enhances transcription of carbohydrate binding modules, dockerins, and pyruvate formate lyases on specific substrates. *Biotechnol. Biofuels* **14**, 234 (2021).

63. Haitjema, C. H. et al. A parts list for fungal cellulosomes revealed by comparative genomics. *Nat. Microbiol.* **2**, 17087 (2017).

64. Nguyen, K. B. et al. Phosphorylation of spore coat proteins by a family of atypical protein kinases. *Proc. Natl. Acad. Sci. USA* **113**, E3482-91 (2016).

65. Freitas, C. et al. A protein phosphorylation module patterns the *Bacillus subtilis* spore outer coat. *Mol. Microbiol.* **114**, 934–951 (2020).

66. Zilhão, R. et al. Assembly requirements and role of CotH during spore coat formation in *Bacillus subtilis*. *J. Bacteriol.* **181**, 2631–2633 (1999).

67. Mølgaard, A., Kauppinen, S. & Larsen, S. Rhamnogalacturonan acetylesterase elucidates the structure and function of a new family of hydrolases. *Structure* **8**, 373–383 (2000).

68. Anderson, A. C., Stangherlin, S., Pimentel, K. N., Weadge, J. T. & Clarke, A. J. The SGNH hydrolase family: a template for carbohydrate diversity. *Glycobiology* **32**, 826–848 (2022).

69. Akoh, C. C., Lee, G.-C., Liaw, Y.-C., Huang, T.-H. & Shaw, J.-F. GDSL family of serine esterases/lipases. *Prog. Lipid Res.* **43**, 534–552 (2004).

70. Correia, M. A. S. et al. Crystal structure of a cellulosomal family 3 carbohydrate esterase from Clostridium thermocellum provides insights into the mechanism of substrate recognition. *J. Mol. Biol.* **379**, 64–72 (2008).

71. Li, Z. et al. Structure-guided protein engineering increases enzymatic activities of the SGNH family esterases. *Biotechnol. Biofuels* **13**, 107 (2020).

72. Xu, Z., He, H., Zhang, S., Guo, T. & Kong, J. Characterization of feruloyl esterases produced by the four lactobacillus species: L. amylovorus, L. acidophilus, L. farciminis and L. fermentum, isolated from ensiled corn stover. *Front. Microbiol.* **8**, 941 (2017).

73. Zheng, J. et al. dbCAN3: automated carbohydrate-active enzyme and substrate annotation. *Nucleic Acids Res.* **51**, W115–W121 (2023).

74. Chatonnet, A., Perochon, M., Velluet, E. & Marchot, P. The ESTHER database on alpha/beta hydrolase fold proteins - an overview of recent developments. *Chem. Biol. Interact.* **383**, 110671 (2023).

75. Dilokpimol, A. et al. Screening of novel fungal Carbohydrate Esterase family 1 enzymes identifies three novel dual feruloyl/acetyl xylan esterases. *FEBS Lett.* **596**, 1932–1943 (2022).

76. Li, X. et al. Identification of a novel feruloyl esterase by functional screening of a soil metagenomic library. *Appl. Biochem. Biotechnol.* **187**, 424–437 (2019).

77. Nieter, A. et al. A chlorogenic acid esterase with a unique substrate specificity from Ustilago maydis. *Appl. Environ. Microbiol.* **81**, 1679–1688 (2015).

78. Xu, F., Sun, J.-X., Sun, R., Fowler, P. & Baird, M. S. Comparative study of organosolv lignins from wheat straw. *Ind. Crops Products* **23**, 180–193 (2006).

79. Li, H.-L. et al. Optimizing extraction and structural characterization of organosolv lignin from wheat straw. *Cellul. Chem. Technol.* **51**, 433–455 (2017).

80. Saberikhah, E., Mohammadi-Rovshandeh, J. & Mamaghani, M. Spectroscopic comparison of organosolv lignins isolated from wheat straw. *Cellul. Chem. Technol.* **47**, 409–418 (2013).

81. Sun, R.-C., Lawther, J. M. & Banks, W. B. Isolation and characterization of organosolv lignins from wheat straw. *Wood Fiber Sci.* **30**, 56–63 (1998).

82. Mun, J.-S., Pe, J. A. & Mun, S.-P. Chemical characterization of kraft lignin prepared from mixed hardwoods. *Molecules* **26**, 4861 (2021).

83. Lu, M. et al. Fractionation of lignin from corncob with high-yield p-coumaric acid production in deep eutectic solvents followed by pyrolysis to produce monophenols. *Int. J. Biol. Macromol.* **285**, 138331 (2025).

84. Qian, S. et al. Effects of combined enzymatic hydrolysis and fed-batch operation on efficient improvement of ferulic acid and p-coumaric acid production from pretreated corn straws. *Bioresour. Technol.* **366**, 128176 (2022).

85. Kuehne, S. A. & Minton, N. P. ClosTron-mediated engineering of Clostridium. *Bioengineered* **3**, 247–254 (2012).

86. Ravachol, J. et al. Combining free and aggregated cellulolytic systems in the cellulosome-producing bacterium Ruminiclostridium cellulolyticum. *Biotechnol. Biofuels* **8**, 114 (2015).

87. Wang, J. et al. The conserved domain database in 2023. *Nucleic Acids Res.* **51**, D384–D388 (2023).

88. CDD Conserved Protein Domain Family: SGNH_hydrolase. https://www.ncbi.nlm.nih.gov/Structure/cdd/cd00229 (2023).

89. CDD Conserved Protein Domain Family: XynB_like. https://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?h=1&uid=238871.

90. Zhang, J.-X., Martin, J. & Flint, H. J. Identification of non-catalytic conserved regions in xylanases encoded by the xynB and xynD genes of the cellulolytic rumen anaerobe Ruminococcus flavefaciens. *Mole. Gen. Genet.* **245**, 260–264 (1994).

91. Li, H., Pu, Y., Kumar, R., Ragauskas, A. J. & Wyman, C. E. Investigation of lignin deposition on cellulose during hydrothermal pretreatment, its effect on cellulose hydrolysis, and underlying mechanisms. *Biotechnol. Bioeng.* **111**, 485–492 (2014).

92. Djajadi, D. T., Pihlajaniemi, V., Rahikainen, J., Kruus, K. & Meyer, A. S. Cellulases adsorb reversibly on biomass lignin. *Biotechnol. Bioeng.* **115**, 2869–2880 (2018).

93. Haviland, Z. K. et al. Lignin impairs Cel7A degradation of in vitro lignified cellulose by impeding enzyme movement and not by acting as a sink. *Biotechnol. Biofuels* **17**, 7 (2024).

94. Ximenes, E., Kim, Y., Mosier, N., Dien, B. & Ladisch, M. Deactivation of cellulases by phenols. *Enzym. Microb. Technol.* **48**, 54–60 (2011).

95. Zhai, R., Hu, J. & Jin, M. Towards efficient enzymatic saccharification of pretreated lignocellulose: Enzyme inhibition by lignin-derived phenolics and recent trends in mitigation strategies. *Biotechnol. Adv.* **61**, 108044 (2022).

96. Martins, P. A., Bourmaud, C. L., Luterbacher, J. S. & Agger, J. W. Glucuronoyl esterases improve cellulose hydrolysis by lignocellulose degrading enzymes and enhance lignin extraction. *Int. J. Biol. Macromolecules* **314**, 144218 (2025).

97. Pentari, C. et al. Exploring the synergy between fungal CE15 glucuronoyl esterases and xylanases for lignocellulose saccharification. *Biotechnol. Biofuels Bioprod.* **18**, 38 (2025).

98. Röllig, R. et al. Wood decay under anoxia by the brown-rot fungus Fomitopsis pinicola. *Nat. Commun.* **16**, 7352 (2025).

99. Phienluphon, A. et al. Boosting sugarcane trash decomposition: synergistic action and proximity effect of xylanase and feruloyl esterase co-displayed on the cell surface of *Pichia pastoris* (*Komagataella phaffi*). *Sustain. Energy Fuels* **8**, 873–883 (2024).
100. Borneman, W. S., Ljungdahl, L. G., Hartley, R. D. & Akin, D. E. Isolation and characterization of p-coumaroyl esterase from the anaerobic fungus Neocallimastix strain MC-2. *Appl. Environ. Microbiol.* **57**, 2337–2344 (1991).

101. Minor, C. M. et al. A genomic analysis reveals the diversity of cellulosome displaying bacteria. *Front. Microbiol.* **15**, 1473396 (2024).

102. Liu, N. et al. Intracellular removal of acetyl, feruloyl and p-coumaroyl decorations on arabinoxylo-oligosaccharides imported from lignocellulosic biomass degradation by Ruminiclostridium cellulolyticum. *Microbiol. Cell Fact.* **23**, 151 (2024).

103. Sethupathy, S. et al. Lignin valorization: status, challenges and opportunities. *Bioresour. Technol.* **347**, 126696 (2022).

104. Abu-Omar, M. M. et al. Guidelines for performing lignin-first biorefining. *Energy Environ. Sci.* **14**, 262–292 (2021).

105. Vennestrom, P. N. R., Osmundsen, C. M., Christensen, C. H. & Taarning, E. Beyond petrochemicals: the renewable chemicals industry. *Angew. Chem. Int. Ed.* **50**, 10502–10509 (2011).

106. Christensen, C. H., Rass-Hansen, J., Marsden, C. C., Taarning, E. & Egeblad, K. The renewable chemicals industry. *ChemSusChem* **1**, 283–289 (2008).

107. Ujor, V. C. & Okonkwo, C. C. Microbial detoxification of lignocellulosic biomass hydrolysates: Biochemical and molecular aspects, challenges, exploits and future perspectives. *Front. Bioeng. Biotechnol.* **10**, 1061667 (2022).

108. Chen, M. et al. High value valorization of lignin as environmental benign antimicrobial. *Mater. Today Bio* **18**, 100520 (2023).

109. Chauhan, P. S. et al. Next generation applications of lignin derived commodity products, their life cycle, techno-economics and societal analysis. *Int. J. Biol. Macromol.* **197**, 179–200 (2022).

110. De Oliveira Silva, E. & Batista, R. Ferulic acid and naturally occurring compounds bearing a feruloyl moiety: a review on their structures, occurrence, and potential health benefits. *Comp. Rev. Food Sci. Food Safe* **16**, 580–616 (2017).

111. Pei, K., Ou, J., Huang, J. & Ou, S. p -Coumaric acid and its conjugates: dietary sources, pharmacokinetic properties and biological activities. *J. Sci. Food Agric.* **96**, 2952–2962 (2016).

112. Giallo, J., Gaudin, C., Belaich, J. P., Petitdemange, E. & Caillet-Mangin, F. Metabolism of glucose and cellobiose by cellulolytic mesophilic Clostridium sp. strain H10. *Appl. Environ. Microbiol.* **45**, 843–849 (1983).

113. Kampik, C. et al. Handling several sugars at a time: a case study of xyloglucan utilization by *Ruminiclostridium cellulolyticum*. *mBio* **12**, e02206-21 (2021).

114. Borne, R. et al. Engineering of a new Escherichia coli strain efficiently metabolizing cellobiose with promising perspectives for plant biomass-based application design. *Metab. Eng. Commun.* **12**, e00157 (2021).

115. Kim, H. & Ralph, J. Solution-state 2D NMR of ball-milled plant cell wall gels in DMSO-d6/pyridine-d5. *Org. Biomol. Chem.* **8**, 576–591 (2010).

116. Babraham Bioinformatics - FastQC a quality control tool for high throughput sequence data. https://www.bioinformatics.babraham.ac.uk/projects/fastqc/.

117. Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. *Bioinformatics* **30**, 2114–2120 (2014).

118. Li, H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. Preprint at https://doi.org/10.48550/ARXIV.1303.3997 (2013).

119. Anders, S., Pyl, P. T. & Huber, W. HTSeq—a Python framework to work with high-throughput sequencing data. *Bioinformatics* **31**, 166–169 (2015).

120. Varet, H., Brillet-Guéguen, L., Coppée, J.-Y. & Dillies, M.-A. SARTools: a DESeq2- and EdgeR-based R pipeline for comprehensive differential analysis of RNA-seq data. *PLoS One* **11**, e0157022 (2016).

121. Wickham, H. *Ggplot2*. (Springer International Publishing, Cham, https://doi.org/10.1007/978-3-319-24277-4_2016).

122. Gu, Z., Eils, R. & Schlesner, M. Complex heatmaps reveal patterns and correlations in multidimensional genomic data. *Bioinformatics* **32**, 2847–2849 (2016).

123. Home - Gene - NCBI. https://www.ncbi.nlm.nih.gov/gene.

124. cellulase (B8I9G0) - protein - InterPro. https://www.ebi.ac.uk/ interpro/protein/UniProt/B8I9G0/.

125. cellulase (B8I3M0) - protein - InterPro. https://www.ebi.ac.uk/ interpro/protein/UniProt/B8I3M0/.

126. cellulase (B8I5J2) - protein - InterPro. https://www.ebi.ac.uk/ interpro/protein/UniProt/B8I5J2/.

127. Katoh, K. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. *Nucleic Acids Res.* **30**, 3059–3066 (2002).

128. Borne, R. et al. Catalytic subunit exchanges in the cellulosomes produced by *Ruminiclostridium cellulolyticum* suggest unexpected dynamics and adaptability of their enzymatic composition. *FEBS J.* **287**, 2544–2559 (2020).

129. Borne, R., Bayer, E. A., Pagès, S., Perret, S. & Fierobe, H. Unraveling enzyme discrimination during cellulosome assembly independent of cohesin - dockerin affinity. *FEBS J.* **280**, 5764–5779 (2013).

130. Ravachol, J., Borne, R., Tardif, C., De Philip, P. & Fierobe, H.-P. Characterization of all family-9 glycoside hydrolases synthesized by the cellulosome-producing bacterium clostridium cellulolyticum. *J. Biol. Chem.* **289**, 7335–7348 (2014).

131. Expasy - ProtParam. https://web.expasy.org/protparam/.

132. Ferdinand, P.-H. et al. Are cellulosome scaffolding protein CipC and CBM3-containing protein HycP, involved in adherence of clostridium cellulolyticum to cellulose? *PLoS One* **8**, e69360 (2013).

133. Barrett, T. et al. NCBI GEO: archive for functional genomics data sets-update. *Nucleic Acids Res.* **41**, D991–D995 (2013).

## Acknowledgements

We thank the TGML platform (Transcriptomics & Genomics Marseille Luminy, France) and the URCATech PLANET analytic platform (Université de Reims Champagne Ardenne, France) for performing the Illumina RNA libraries sequencing and the NMR analysis, respectively. We also thank Dr. Wanassa Beroual and Dr. Emanuele Biondi for their assistance during the testing and development phases of the RNA-seq experiments; Dr. Patrick Viollier, Laboratory for hosting the 1st author during his 3-months research visit as part of EMBO Short-Term Fellowship in order to acquire new skills in RNA sequencing and data analysis; Dr. Frédéric Carrière for his help, advice and expertise on esterase activity; Romain Borne for his help with the pET28-1060 plasmid construction; and François Gaudard-Schmitter for sample preparation and IR spectra acquisition. This work was supported by a Ph. D. fellowship from the Ministère de la Recherche et de l'Enseignement Supérieur granted to MH by the École Doctorale Sciences du vivant ED 658 (Aix-Marseille Université, France). This research was supported by an EMBO Short-Term Fellowship grant Number 8089 (NV), an Institut Microbiologie, Bioénergies et Biotechnologie (IM2B) Nouveaux Entrants grant (NV), and an Agence Nationale de la Recherche grant ANR-21-CE43-0018 (C.F., E.R., F.P., G.S., H.P.F., L.D., M.H., N.V., P.H.D., S.G., and S.P.).

## Author contributions

Conceptualization: H.P.F. and N.V. Methodology: B.C., C.F., E.R., G.S., H.P.F., N.V., P.H.D., S.P., and V.A.B. Validation: B.C., D.C., F.P., H.P.F., M.H., N.V., S.G., and S.P. Formal analysis: B.C., D.C., F.P., H.P.F., M.H., N.V., and S.G. Investigation: A.H., B.C., D.C., F.D., F.M.O., F.P., G.P., H.P.F., L.D., M.H., N.V., S.G., and Y.D. Data curation: C.M., F.M.O., H.P.F., M.H., N.V., S.G., and S.S. Writing—original draft: H.P.F., M.H., and N.V. wrote the paper with input from all authors. Writing—review & editing: all authors contributed. Visualization: H.P.F., M.H., N.V., and S.G. Supervision and project administration: H.P.F. and N.V. Funding acquisition: E.R., G.S., H.P.F., and N.V.
**Competing interests**

The authors declare no competing interests.

**Additional information**

**Supplementary information** The online version contains supplementary material available at https://doi.org/10.1038/s42003-025-09287-y.

**Correspondence** and requests for materials should be addressed to Nicolas Vita.

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