Cellulosome Systems in the Digestive Tract: Underexplored Enzymatic Machine for Lignocellulose Bioconversion

Abstract

Cellulosomes are sophisticated multi-enzyme complexes synthesized and secreted by anaerobic microorganisms, characterized by intricate structural components and highly organized modular assembly mechanisms. These complexes play a pivotal role in the efficient degradation of lignocellulosic biomass, significantly enhancing its bioconversion efficiency, and are thus regarded as invaluable enzymatic molecular machines. Cellulosomes are not only prevalent in anaerobic bacteria from soil and compost environments but are also integral to the digestive systems of herbivorous animals, primates and termites. The cellulosomes produced by digestive tract microbiota exhibit unique properties, providing novel enzymes and protein modules that are instrumental in biomass conversion and synthetic biology, thereby showcasing substantial application potential. Despite their promise, the isolation and cultivation of digestive tract microorganisms that produce cellulosomes present significant challenges. Additionally, the lack of comprehensive genetic and biochemical studies has impeded a thorough understanding of these cellulosomes, leaving them largely underexplored. This paper provides a comprehensive overview of the digestive tract cellulosome system, with a particular focus on the structural and functional attributes of cellulosomes in various animal digestive tracts. It also discusses the application prospects of digestive tract cellulosomes, highlighting their potential as a treasure in diverse fields.

1. Introduction

With population growth and accelerated industrialization, the progressive depletion of non-renewable resources and deteriorating environmental conditions have emerged as critical global challenges. In this context, renewable resources have garnered significant scientific interest owing to their renewability and environmental friendliness. Among them, lignocellulosic biomass (LCB) is a carbon-neutral feedstock derived from ubiquitous sources including agricultural waste (e.g., rice straw, wheat straw and corn straw) [1,2], forestry wastes [3,4] and cellulose-rich waste streams (e.g., paper) [5]. Global LCB production is estimated at ~200 billion metric tons annually [6], positioning it as a pivotal resource for sustainable biorefineries. Structurally, lignocellulose comprises three primary polymeric constituents: cellulose, hemicellulose and lignin [7]. This recalcitrant matrix can be biocatalytically converted into value-added products such as biofuels and platform chemicals through sequential processing involving pretreatment, saccharification and fermentation [8]. The saccharification process is a key step in biotransformation, which converts insoluble lignocellulosic substrates into soluble fermentable sugars [9,10]. The enzymatic machinery driving saccharification predominantly consists of polysaccharide-degrading hydrolases, including cellulases, hemicellulases and pectinases. Notably, aerobic fungi such as Trichoderma reesei have evolved specialized secretory pathways to produce extracellular enzyme cocktails, enabling efficient lignocellulose deconstruction [11]. A paradigm shift occurred in the 1980s with the discovery of cellulose-binding protein complexes in Clostridium thermocellum, an anaerobic thermophile exhibiting exceptional cellulolytic activity [12,13]. Subsequent characterization revealed these complexes to be multifunctional nanomachines, termed cellulosomes [14,15]. Architecturally, cellulosomes feature a modular scaffoldin subunit containing cohesin domains that orchestrate the assembly of dockerin-bearing catalytic modules [16]. However, the molecular weight and assembly complexity of cellulosomes in different species vary greatly, reflecting evolutionary adaptations to specific substrate niches [17]. This duality of conservation and diversification positions cellulosomes as evolutionarily optimized systems for lignocellulose valorization.

Comparative analyses demonstrate that cellulosome-mediated substrate conversion efficiency significantly surpasses that of free cellulase systems, prompting substantial research interest in its evolutionarily optimized lignocellulose deconstruction mechanisms [18]. To date, the most extensively characterized cellulosome-producing strains—C. thermocellum and Clostridium cellulolyticum—have predominantly been isolated from soil and composting systems [19]. Emerging evidence, however, reveals a broader phylogenetic distribution of cellulosome-producing microorganisms across diverse ecological niches, with notable prevalence in animal digestive ecosystems (

Table 1. Cellulosome systems in different digestive tract microorganisms.

Host	Strain	Scaffoldins/Cohesins	Dockerin-Containing Proteins/CAZymes	Genome Accession Number	Reference
Cow	Ruminococcus flavefaciens FD-1	17/27	223/154	ACOK00000000	[27,28]
Cow	Ruminococcus flavefaciens 17	11/21	180/123	AFNE00000000	[28,29]
Cow	Ruminococcus flavefaciens 007c	10/16	183/122	ATAX01000000	[28,30]
Cow	Ruminococcus albus 7	1/1	90/122	GCA_000179635.2	[28,31]
Cow	Ruminococcus albus 8	0/0	62/114	GCF_000178155.2	[28]
Sheep	Ruminococcus albus SY3	1/1	58/124	GCF_000586615.1	[28]
Human	Ruminococcus champanellensis 18P13	11/20	64/107	FP929052.1	[20,24]
Human and nonhuman primate	Ruminococcus primaciens, Ruminococcus hominiciens and Ruminococcus ruminiciens	-	-	-	[25]
Termite	Clostridium termitidis CT1112	5/5	22/355	AORV00000000	[26,32]
Sheep	Anaeromyces robustus	26/-	276/-	MCFG00000000	[33]
Goat	Neocallimastix californiae	55/-	422/-	MCOG00000000	[33]
Horse	Piromyces finnis	14/-	227/-	MCFH00000000	[33]

- not reported.

) [20,21]. For example, Ruminococcus flavefaciens and Ruminococcus albus in the digestive tract of ruminants (e.g., cattle, sheep, etc.) were found to be cellulosome-producing bacteria [22], which efficiently degrade lignocellulose, thereby promoting the digestion of feed. Recent studies have shown that the digestive tracts of humans [23,24] and non-human primates (NHPs) [25] also contain a variety of cellulosome-producing bacteria belonging to the genera Ruminococcus. Clostridium termitidis present in the gut of termites degrade lignocellulose into fermentable sugars for further digestion and absorption by the host termites [26]. These collective findings suggest that virtually all plant fiber-consuming animals harbor gut microbiota capable of cellulosome production, which synergistically enhances polysaccharide depolymerization to support host nutrition.

Structural and functional studies of cellulosomes have predominantly focused on their modular architecture and precisely coordinated assembly mechanisms in anaerobic bacteria isolated from environmental niches such as soil and compost systems [16]. However, due to technical challenges in isolating, culturing and genetically manipulating microorganisms from animal digestive tracts, their cellulosomes have yet to be thoroughly investigated, and more cellulosome-producing microorganisms await characterization [21,25]. Intriguingly, the observed lignocellulose degradation efficiency in animal gastrointestinal ecosystems demonstrates marked superiority over conventional soil/compost environments, highlighting the untapped potential of these digestive tract cellulosomes for biotechnological applications [34]. Although a large number of review articles on cellulosomes have been published, none of them focuses on the cellulosome system in the digestive tract. In this paper, the cellulosome system in the digestive tract is reviewed. The structure and application of the cellulosome are first summarized, then the distribution and characteristics of the cellulosome in the digestive tract of different animals are described in detail. Finally, the applications of the cellulosome in the digestive tract are prospected.

2. Cellulosome: Conservation and Diversity

Cellulosomes are assembled through high-affinity, non-covalent interactions between catalytic subunits and scaffoldins. Structurally, the catalytic subunit comprises a catalytic module and a small, non-catalytic module known as dockerin, while the scaffoldin consists of tandem, non-catalytic modules termed cohesins. A diverse array of enzymes, predominantly cellulases and hemicellulases, are assembled onto the scaffoldin via strong non-covalent interactions between the cohesin and dockerin, thereby forming the cellulosome. Moreover, some scaffoldins and enzymes may incorporate auxiliary modules, such as carbohydrate-binding modules (CBMs), which play a crucial role in substrate recognition and binding. Despite this conserved assembly mechanism, cellulosomes exhibit remarkable interspecies variation in molecular mass, architectural complexity, and specificity of cohesin–dockerin interactions, reflecting evolutionary adaptations to distinct ecological niches.

2.1. Fundamental Framework of Cellulosomes

The architectural framework of cellulosomes is defined by the modular assembly of dockerin-containing enzymatic subunits and cohesin-bearing scaffoldin proteins (Figure 1) [16,35]. Central to this assembly is the specific interaction between cohesin and dockerin domains, which can be classified into three distinct types based on sequence homology and structural analyses [16]. In most characterized cellulosome-producing bacteria, type I interactions mediate the binding of dockerin-bearing enzymes to primary scaffoldins containing tandem type I cohesins. Notably, certain primary scaffoldins incorporate a type II dockerin, enabling their hierarchical assembly with secondary scaffoldins harboring type II cohesins, thereby forming poly-cellulosome megacomplexes [16]. In contrast, type III interactions, predominantly observed in Ruminococcus species, demonstrate varying assembly patterns that can be further categorized into several groups [36]. Type II and type III assembly modules significantly diverge from type I in their overall structures and interaction modes, which ensures the specificity between the cohesin and the dockerin interaction types, thereby ensuring the rigor of hierarchical assembly.

The enzymatic repertoire of cellulosomes is predominantly composed of glycoside hydrolases (GHs), exhibiting remarkable functional diversity [37]. Through the spatial organization of complementary enzymes on the scaffoldin, cellulosomes achieve a proximity effect that amplifies synergistic interactions among catalytic subunits [16]. Further functional specialization is achieved through CBMs, which direct cellulosomes to lignocellulosic substrates, and S-layer homology (SLH) domains, which anchor cellulosomes to the cell wall [16]. This spatial arrangement facilitates rapid uptake of degradation products by the host cell, thereby mitigating product inhibition. Collectively, substrate targeting, enzyme–enzyme synergy, enzyme–cell coordination and proximity effects constitute the mechanistic basis for the exceptional catalytic efficiency of cellulosomes [16,38,39]. Additionally, auxiliary modules—including expansins, proteases, protease inhibitors and spore coat protein CotH—have been identified in certain cellulosomal systems, although their physiological roles remain largely unknown [40,41,42,43].

2.2. Species-Specific Diversity of Cellulosomes

The fundamental structure of cellulosomes remains consistent across different species, revealing a conserved basic framework. However, the diversity of cellulosomes is evident in the variations observed in assembly complexity and interaction modes among different species [17]. A striking example is Pseudobacteroides cellulosolvens (formerly Bacteroides cellulosolvens) [44], an anaerobic sludge isolate whose cellulosomes exhibit inverted interaction patterns compared to canonical systems. Specifically, its primary scaffoldin employs type II cohesins for enzyme recruitment, while type I cohesins are localized to the anchoring scaffoldin [45]. This bacterium also harbors an exceptionally complex cellulosomal system, comprising 31 scaffoldins and 212 dockerin-bearing enzymatic subunits [45], representing the most intricate cellulosome characterized to date. Similarly, ruminococcal cellulosomes utilize type III interactions, distinct from both type I and type II systems [28,36]. The species specificity of cohesin–dockerin interactions is remarkably stringent; for instance, type I cohesins from C. thermocellum exclusively recognize their cognate dockerins, preventing cross-species recognition and ensuring species-specific cellulosome assembly [46]. However, exceptions to this specificity rule have been documented for certain cellulosomal components [41,47].

The enzymatic composition of cellulosomes varies substantially across species, directly influencing their substrate degradation capabilities. For instance, cellulosomes from Clostridium clariflavum are enriched in hemicellulolytic enzymes compared to those of C. thermocellum, featuring diverse xylanases from GH11, GH30 and GH67 families [48]. Notably, GH30 xylanases constitute a major enzymatic component in C. clariflavum cellulosomes [49] but are minimally represented in C. thermocellum [50,51]. This divergence translates to distinct xylan degradation strategies: C. clariflavum efficiently converts xylan to xylose monomers, whereas C. thermocellum primarily generates xylo-oligosaccharides [48], and C. clariflavum exhibits higher activity than C. thermocellum in the breakdown of hemicellulose and unpretreated plant material [52]. Besides this comparison, however, the performance of different cellulosomes in lignocellulose bioconversion was seldomly compared experimentally for different species.

3. Applications of Cellulosome System in Lignocellulose Bioconversion and Biotechnology

The exceptional lignocellulose-degrading capacity of cellulosomes positions them as pivotal biocatalysts for LCB valorization. Their modular architecture facilitates heterologous expression and engineering, enabling diverse applications in synthetic biology and biotechnology.

3.1. Biomass Conversion

C. thermocellum, the most extensively studied cellulosome-producing bacterium, is the most efficient microorganism for cellulose degradation in nature, making it an ideal candidate strain for biomass conversion. This bacterium conducts intracellular glucose metabolism via an atypical glycolysis pathway and is capable of producing ethanol from cellulose [53]. As such, it is considered one of the most promising strains for the industrialization of lignocellulose conversion [54,55]. To harness this capability, Lynd et al. proposed consolidated bioprocessing (CBP), a strategy leveraging C. thermocellum for simultaneous lignocellulose degradation and ethanol production [56,57]. The CBP strategy faces a significant challenge in aligning the saccharification process with the ethanol fermentation production process. To address this, researchers have developed a strategy called consolidated bio-saccharification (CBS) that utilizes C. thermocellum and its cellulosomes for efficient lignocellulose saccharification [9,58]. Furthermore, research into hydrogen production from lignocellulose using C. thermocellum represents another important avenue of investigation [59].

Rumen microbiota also offers significant potential for biofuel production and fermented feed development [6]. The volatile fatty acids produced by rumen microbial fermentation serve as key precursors for biofuel synthesis, including alcohols, ketones and hydrocarbons. Co-cultivation of rumen microorganisms with complementary strains can enhance biofuel yields. Moreover, certain rumen bacteria and fungi exhibit robust metabolic activity, making them attractive candidates for fermented feed formulations [60].

3.2. Other Biotechnological Applications

Cellulosomes are widely used in synthetic biology and biotechnology. The concept of designer cellulosomes, pioneered by Bayer et al. [61], revolutionized their biotechnological utility. This approach involves engineering chimeric scaffoldins to assemble tailored multi-enzyme complexes with specific functionalities. For instance, displaying designer cellulosomes on Saccharomyces cerevisiae enables direct cellulose-to-ethanol conversion [62,63,64,65,66]. Beyond biofuels, designer cellulosomes can facilitate novel anabolic pathways to accelerate reaction rates and synthesize biofunctional materials [67,68,69].

Cellulosome components also find applications in diverse biotechnological fields. The cohesin–dockerin interaction enables integration with affinity systems (e.g., protein A, antibodies, lectins) for immunoassays, drug delivery, microarray technology and cell separation [16,70]. Notably, cohesin–dockerin-based biosensors [71] and CBM-fusion proteins for bioreactors [72], plant growth regulators [72] and protein purification [73] exemplify their versatility. The unique structural and functional properties of cellulosomes not only advance lignocellulose bioconversion but also expand their applicability across biotechnology.

4. Cellulosomes in Rumen Microbiota of Herbivores

Herbivores, particularly ruminants, exhibit remarkable lignocellulose degradation efficiency, attributable to the complex microbial ecosystems within their digestive tracts. Ruminants possess a specialized foregut (rumen) that maintains optimal temperature and pH conditions for anaerobic microbial communities (rumen microbiota). These microorganisms produce diverse carbohydrate-active enzymes (CAZymes), enabling the fermentation of plant biomass into volatile fatty acids [74,75]. Furthermore, rumination behavior—periodic regurgitation and re-chewing of feed—enhances substrate accessibility, thereby improving both conversion efficiency and digestibility [75,76]. This synergistic interplay between rumen microbiota and host physiology significantly enhances overall digestive efficiency.

Among rumen microbiota, bacteria are recognized as primary cellulose degraders, with dominant species belonging to the Bacteroidetes, Fibrobacteres and Firmicutes phyla [22]. These taxa employ distinct cellulolytic strategies: Bacteroidetes utilize free cellulases and polysaccharide utilization loci (PULs) to degrade cellulose and pectin [77]; Fibrobacteres, represented by Fibrobacter succinogenes, secrete cellulose-degrading enzymes via membrane vesicles or localize them on the cell surface [78]; and Firmicutes employ both free enzymes and cellulosomes for cellulose hydrolysis. Notably, two key cellulolytic species—R. albus and R. flavefaciens—belonging to Firmicutes produce cellulosomes that play a central role in ruminal cellulose degradation [22]. Additionally, anaerobic fungi capable of cellulosome production have been identified in the rumen, exhibiting structural distinctions from bacterial cellulosomes [33].

4.1. Cellulosomes of Rumen Ruminococcus

Comparative genomic analyses have revealed striking differences in the cellulosome systems of R. albus and R. flavefaciens [28]. R. flavefaciens possesses highly intricate cellulosomes comprising multiple scaffoldins and numerous dockerin-bearing enzymes, whereas R. albus lacks tandem-cohesin scaffoldins and exhibits a significantly reduced repertoire of dockerin-containing proteins.

4.1.1. Cellulosomes of R. flavefaciens

R. flavefaciens cellulosomes represent the most extensively characterized cellulosome system among digestive tract microorganisms. Prior to genome sequencing, numerous cellulosomal components, including scaffoldins and enzymes, were biochemically characterized [79,80,81,82,83,84,85,86,87]. Following the first genome report in 2009 [27], subsequent studies have comprehensively analyzed cellulosomes across various R. flavefaciens strains [28,88,89]. R. flavefaciens encodes over ten scaffoldins and more than 180 dockerin-bearing proteins [28,90], with cohesin–dockerin interactions classified into six distinct groups [88,91], resulting in a highly complex assembly (Figure 2A). Among these, ScaA and ScaB are the largest scaffoldins, containing three and nine cohesins, respectively, in R. flavefaciens FD-1. ScaA features a C-terminal dockerin that specifically binds to the cohesin of ScaB, while ScaB contains an X-dockerin at its C-terminus, enabling specific interaction with the cohesin of ScaE, which harbors a sortase motif for cell wall anchoring [82]. ScaC, an adaptor scaffoldin containing both dockerin and cohesin domains [83], is the most conserved scaffoldin across R. flavefaciens strains and is proposed as a genetic marker for metagenomic analyses [87,92]. Similarly, ScaH contains one dockerin and one cohesin, but its dockerin can bind to another ScaH molecule, suggesting a role as a spacer to reduce steric hindrance during cellulosome assembly [93]. Notably, R. flavefaciens strains 17 and 007c encode a unique protein with seven tandem dockerins, absent in strain FD-1, although its functional significance remains unexplored [28].

Within the six groups of cohesin–dockerin interactions, the structures of the cohesin–dockerin complexes in groups 1, 3, 4 and 5 have been solved (Figure 2B) [36,93,94,95,96,97], revealing the molecular basis of type III interactions and highlighting the diversity of cellulosome assembly in R. flavefaciens. Single-molecule force spectroscopy and steered molecular dynamics simulations of the interaction between ScaE cohesin and CttA X-Doc demonstrated exceptional mechanical strength, withstanding forces of 600–750 pN, making it one of the strongest biomolecular interactions known, comparable to half the strength of a covalent bond [98].

R. flavefaciens encodes over 200 dockerin-bearing proteins, with ~50% containing CAZyme modules, including GHs, polysaccharide lyases and carbohydrate esterases [88]. However, a significant proportion of these proteins contain domains/modules of unknown function [88], underscoring substantial knowledge gaps in ruminococcal cellulosome biology. To date, only a limited number of cellulosomal enzymes have been functionally characterized, including a papain-like cysteine peptidase [43], a GH5 endo-mannanase [99], a trimodular GH16 licheninase [100] and a GH5 endoglucanase [101]. High-throughput CBMome screening of 177 uncharacterized protein modules identified six novel CBM families [102], highlighting the potential of R. flavefaciens cellulosomes as a rich resource for discovering novel enzymes and functional modules.

4.1.2. Cellulosomes of R. albus

Early studies suggested that both R. albus and R. flavefaciens produce cellulosomes [103,104]; however, genomic analyses later revealed that R. albus strains lack scaffoldins necessary for cellulosome assembly, despite encoding dozens of dockerin-bearing proteins [104]. Unlike the diverse dockerin groups found in R. flavefaciens, R. albus dockerins are generally conserved and cannot be classified into distinct groups. Notably, R. albus encodes numerous CAZymes fused with a unique CBM37 module, absent in other species, which mediates enzyme attachment to both the bacterial cell surface and substrates [105,106]. Strains 7 and SY3 of R. albus encode a single-cohesin protein with a C-terminal dockerin, analogous to R. flavefaciens ScaC [28], though insufficient for cellulosome assembly. R. albus also produces multi-functional proteins similar to those in R. flavefaciens, but with a reduced repertoire of degradative enzymes [28]. Mutant analyses of R. albus defective in cellulose degradation identified deficiencies in two processive endocellulases, both containing CBM37 modules [107]. Subsequent transcriptomic studies of R. albus 7 grown on cellobiose and cellulose revealed upregulation of CBM37-containing enzymes on cellulose, rather than dockerin-bearing proteins [108], suggesting that dockerin-containing enzymes in R. albus do not play a central role in cellulose degradation. However, studies of a dockerin-containing endoglucanase EgV in R. albus F-40 demonstrated its localization on the cell surface [109], implying the presence of scaffoldin-like proteins or alternative mechanisms for dockerin-mediated attachment. Further research is needed to determine whether dockerin-bearing proteins form cellulosome-like complexes and to elucidate their functional roles.

4.2. Cellulosomes of Rumen Fungi

Rumen anaerobic fungi play a critical role in lignocellulose fermentation, producing both free enzymes and cellulosomes for substrate degradation. These fungi possess robust rhizoids, enabling their hyphae to penetrate plant cell walls, reducing internal tissue tension and facilitating cellulose degradation by other rumen microorganisms [110]. Most rumen fungi belong to the Neocallimastigomycota phylum and can produce free cellulases (non-cellulosomal), free cellulosomes (non-cell-surface-anchored) and cell-bound cellulosomes [111], potentially conferring a competitive advantage over bacterial enzyme systems.

Structurally, fungal cellulosomes differ from bacterial systems, utilizing non-catalytic dockerin domains (NCDDs) with distinct sequences to mediate assembly via interaction with scaffoldins [112]. Haitjema et al. identified 95 fungal scaffoldins capable of binding NCDDs and nearly 1600 dockerin domain proteins (DDPs) containing NCDDs in the cellulosomes of five anaerobic fungi, including Anaeromyces robustus, Neocallimastix californiae and Piromyces finnis [33]. While fungal scaffoldins likely orchestrate DDP assembly, the structural and biophysical basis of scaffoldin-NCDD interactions remains poorly characterized. The scaffoldin system in fungi is highly conserved, enabling extensive cross-species interactions, in contrast to the species-specific assembly of bacterial cellulosomes [113]. Enzymatically, fungal cellulosomes also differ from bacterial systems, containing GH3, GH6 and GH45 enzymes, which are rare in bacterial cellulosomes. Notably, GH3 enzymes exhibit β-glucosidase activity, enabling fungal cellulosomes to directly convert cellulose into fermentable monosaccharides, unlike Clostridium cellulosomes, which primarily produce low-molecular-weight oligosaccharides and disaccharides [114]. Additionally, fungal cellulosomes incorporate enzymes acquired through horizontal gene transfer from bacterial cellulosomes within the rumen microbiota [33,115,116].

5. Cellulosomes in the Intestinal Tract of Primates

Gut microbiota in primates play a pivotal role in host metabolism and immune regulation. Recent studies have identified cellulosome-producing bacteria in the gastrointestinal tracts of primates, including humans, highlighting their functional significance in digestive physiology, evolutionary adaptation and host–microbe symbiosis [25].

5.1. Cellulosomes in the Human Gut

Moraïs et al. [117] first reported that Ruminococcus champanellensis, a key species in the human gut microbiota, harbors genetic elements for cellulosome production and efficiently degrades microcrystalline cellulose (Avicel). The cellulosome architecture of R. champanellensis resembles that of R. flavefaciens [24,25], comprising 12 scaffoldins (Figure 3). Despite its structural complexity, the 20 cohesins on these scaffoldins exhibit specific recognition and binding to 64 dockerin-bearing proteins, ensuring structural stability in the complex intestinal environment [20]. This specificity is analogous to R. flavefaciens, where type III assembly modules display subtle variations among different members, resulting in diverse interaction patterns. Overall, cohesin–dockerin interactions can be classified into distinct groups based on structural and binding characteristics, providing molecular flexibility for cellulosome function and adaptation to diverse plant fiber structures.

The enzymatic repertoire of R. champanellensis cellulosomes exhibits remarkable diversity and complexity, including cellulases, xylanases, mannanases and other GHs, with up to 10 distinct cellulases [117]. These enzymes display varying modes of action and activity levels on cellulose substrates. For instance, comparative studies of cellulosome activity on carboxymethyl cellulose (CMC) and microcrystalline cellulose (Avicel) [118,119,120] have revealed unique functional roles in cellulose degradation. Genomic analyses of dockerin-bearing GHs in R. champanellensis identified multiple GH families, including GH5, GH8 and GH9 [117]. Despite their diversity, these enzymes share conserved catalytic domains in key amino acid sequences and three-dimensional structures, indicating a common catalytic mechanism. This structural adaptability enables cellulosomes to synergistically degrade complex polysaccharides in plant cell walls, enhancing the host’s energy harvest from diverse dietary fibers and improving adaptability to varying cellulose sources.

Recently, Moraïs et al. [25] expanded our understanding of human gut cellulose-degrading bacteria by identifying novel species, including Candidatus Ruminococcus primaciens, Ruminococcus hominiciens and Ruminococcus ruminiciens. These species are more prevalent in non-industrialized populations, correlating with dietary fiber intake and lifestyle factors. They acquire diet-adapted genes through horizontal gene transfer, such as the GH98 gene in R. hominiciens and the GH19 gene in R. primaciens, enabling them to occupy specific niches in fiber degradation and providing insights for modulating gut microbiota to improve host health.

Another notable species, Ruminococcus bromii, a dominant member of the human gut microbiota, plays a key role in starch degradation and energy release. Genomic analyses revealed four dockerin-bearing amylases and four cohesin-containing proteins, suggesting the existence of an amylosome, a cellulosome-like complex for starch hydrolysis [121]. This discovery extends the cellulosome paradigm, although the assembly and functional mechanisms of the amylosome remain to be fully elucidated.

It should be noted that while cellulosome-producing bacteria like R. champanellensis in the human gut can effectively hydrolyze cellulose to glucose [20], their abundance varies significantly depending on dietary habits and they are not consistently prevalent in the human digestive system [25]. Their roles in human food digestion and human healthy remain poorly understood and require further investigation.

5.2. Cellulosomes in the Gut of NHPs

Mammals lack endogenous GHs, polysaccharide lyases and carbohydrate esterases necessary for hydrolyzing β-1,4 glycosidic bonds in complex plant polysaccharides. Instead, their gastrointestinal microbiota degrades and ferment these structural polysaccharides, producing energy-rich short-chain fatty acids (SCFAs). For example, NHPs, which primarily consume plant-based diets [122], derive a significant portion of their daily energy from SCFAs [123]. With the rapid expansion of NHP gut microbiome datasets, particularly metagenomic and metatranscriptomic data, cellulosome-producing bacteria have been identified in NHP gastrointestinal tracts. For instance, metagenomic analyses of snub-nosed monkey Rhinopithecus bieti revealed a substantial abundance of lignocellulose-degrading bacteria, including key cellulosome producers such as C. thermocellum, C. cellulolyticum and R. flavefaciens [124]. Newman et al. demonstrated that dietary habits significantly influence the abundance of R. champanellensis in the gut microbiota of macaque Macaca fascicularis [125]. Houtkamp et al. identified fungal cellulosome-associated sequences, including GH48 enzymes, in the gut microbiome of zoo-housed gorillas [126]. Additionally, Li et al. detected genes encoding cohesin and dockerin modules of Ruminococcus in the metagenome of wild gibbons [127].

Recent work by Moraïs et al. [25] systematically investigated cellulosome-producing bacteria in human and NHP gut metagenomes, revealing that R. champanellensis and three novel species—R. primaciens, R. hominiciens and R. ruminiciens—are widely distributed in NHP guts and correlate with dietary patterns. Although these bacteria are present in both humans and NHPs, their evolutionary distribution differs: R. primaciens is associated with NHPs and ancient humans, while R. hominiciens is primarily linked to humans and apes. These species adapt to host ecosystems by acquiring genes from co-resident gut microbes, such as those for degrading specific plant polysaccharides. Phylogenetic analyses suggest that R. hominiciens may have originated from ruminant guts, whereas R. primaciens is closely related to ancestral human strains. The spread and adaptation of these bacteria across primates and ruminants reflect the dynamic exchange of microbial communities between distinct ecosystems.

6. Cellulosomes in Termite Guts

Termites, belonging to the Arthropoda, Insecta and Isoptera taxa, are hemimetabolous insects that play a critical role in carbon and nitrogen cycling due to their lignocellulose-degrading capabilities [128]. Their digestive system, comprising the foregut, midgut and highly specialized hindgut, harbors a diverse microbial community that facilitates the breakdown of complex polymers such as cellulose and lignin into digestible compounds, including SCFAs and monosaccharides [129,130,131]. However, cellulosomes were not identified in the termite gut microbiome until 2013, when genomic analysis of C. termitidis CT1112 revealed their presence [32,132].

C. termitidis CT1112, first isolated from the gut of the wood-feeding termite, Nasutitermes lujae [133], was originally classified within Clostridium cluster III [134]. Subsequent phylogenetic analyses led to the reclassification of cluster III members into four novel genera, with C. termitidis reassigned to Ruminiclostridium [135]. Further studies suggested that C. termitidis and Clostridium cellobioparum should be classified as subspecies of the same species, resulting in the current taxonomic designation of Ruminiclostridium cellobioparum subsp. termitidis [135]. For consistency with most published literature, this review retains the name Clostridium termitidis.

C. termitidis CT1112 can utilize cellulose, xylan, cellobiose and xylose as carbon sources, producing acetic acid, ethanol and lactic acid through mixed-acid fermentation [136]. Genomic analysis [32] identified 281 CAZymes, including 198 GHs distributed across 50 families. The genome also encodes twenty-two dockerin-bearing GHs, five cohesin-containing scaffoldins, and a ~20 kb gene cluster containing thirteen cellulosomal genes, resembling the simple cellulosome systems of well-characterized strains such as C. cellulolyticum [26]. This cluster includes scaffoldin genes followed by dockerin-bearing enzyme genes, suggesting a functional cellulosome. Additionally, C. termitidis produces non-cellulosomal cellulases, including exoglucanases (GH48, GH9), endoglucanases (GH5, GH8, GH9), xylanases (GH8, GH10, GH11, GH30) and mannanases (GH26), which synergize with the cellulosome to degrade cellulose into fermentable sugars [136]. Proteomic studies confirmed the active expression of cellulosomal components in C. termitidis CT1112, with all encoded dockerin- and cohesin-containing proteins detected in the proteome [136]. Transcriptomic and proteomic analyses further revealed substrate-dependent regulation of cellulosomal genes [137], mirroring the expression patterns observed in other cellulosome-producing bacteria [138,139].

Over the past two decades, significant advances have been made in understanding symbiotic digestion in termite guts [140]. Notably, termite gut microbiota exhibit diet-dependent community structures [141]. A comparative study of gut prokaryotic microbiomes from 11 higher termite genera, categorized into plant fiber- and soil-feeding groups, found higher cohesin/dockerin transcript levels in soil-feeding termites, suggesting more active cellulosome production in these species [142].

While the host’s role in mechanical grinding and enzymatic processing of digestive substrates is well-recognized, the mechanisms underlying lignocellulose degradation by cellulosomes remain poorly understood [132,143]. To date, few microorganisms have been isolated and characterized from termite guts. For C. termitidis CT1112, all cellulosome-related findings are based on omics studies, with no biophysical or biochemical investigations into its assembly or enzymatic properties. Comprehensive studies of termite gut cellulosomes are essential, as they will not only deepen our understanding of lignocellulose degradation in termite digestive systems but also unlock novel enzyme systems and protein components for advancing biomass conversion technologies.

7. Research and Application Prospect of Digestive Tract Cellulosomes

The application potential of digestive tract cellulosomes remains largely untapped, primarily due to limited knowledge about their unique properties and significant differences compared to cellulosomes from soil and compost environments. Further research is essential to fully elucidate their functional mechanisms and harness their biotechnological potential (Figure 4).

Key research priorities include:

• Enzymatic characterization of cellulosomal components, as most enzymes in digestive tract cellulosomes remain uncharacterized, and their roles in lignocellulose degradation have not been experimentally validated. Discovering novel enzymes and functional modules will enhance our understanding of these systems and provide new tools for lignocellulose biorefineries.
• Elucidating the structural organization and interaction mechanisms of cellulosomal components to understand how their arrangement contributes to functionality across different digestive tract environments.
• Exploring the regulation of cellulosome production in digestive tract microorganisms, as novel regulatory mechanisms are likely to be uncovered.
• Investigating the interactions between cellulosome-producing microorganisms and other gut microbes, which may reveal their roles in host health and functionality.
• Clarifying the contributions of cellulosome-producing microorganisms to greenhouse gas emissions (e.g., methane) and feed efficiency in livestock, which could inform strategies to reduce emissions and enhance productivity in the livestock industry.

Based on existing research, digestive tract cellulosomes are expected to have broad applications in multiple fields:

• Lignocellulose Biorefineries: The high efficiency of cellulosomes has already been leveraged for biofuel production and lignocellulose bio-saccharification [9,57]. Novel cellulosomal components from digestive tracts may enhance the activity and versatility of cellulosomes in biorefinery applications.
• Synthetic Biology and Biotechnology: The unique assembly of digestive tract cellulosomes offers novel modules for synthetic metabolic pathways, surface display systems, enzyme immobilization and biosensor development [61,67,68,69].
• Animal Nutrition: Cellulosomes have potential applications in improving feed utilization through cellulosome-producing bacterial additives or pre-treatment of lignocellulosic feeds [21,75].
• Environmental Sustainability: Digestive tract cellulosomes may mitigate greenhouse gas emissions from livestock [21] and enhance the composting of lignocellulosic agricultural waste [144]. Additionally, cellulosomal modules could be engineered to assemble plastic-degrading enzymes, improving the biotreatment of plastic waste and reducing pollution [145,146].

8. Conclusions

Cellulosomes are multi-enzyme complexes produced by microorganisms capable of cellulose degradation. They share a conserved structural framework, where dockerin-bearing catalytic subunits are assembled onto cohesin-containing scaffoldins through specific cohesin–dockerin interactions. However, cellulosomes exhibit significant interspecies variation in molecular weight, assembly patterns and catalytic composition, resulting in diverse structural architectures, substrate specificities and degradation efficiencies. This interplay of conservation and diversity reflects the evolutionary uniqueness and complexity of cellulosomes. Notably, cellulosomes from animal digestive tracts differ markedly from well-characterized systems such as those of C. thermocellum and C. cellulolyticum, exhibiting unique structural and functional features that warrant further investigation.

In ruminants, Ruminococcus species are the primary cellulose-degrading bacteria, utilizing cellulosomes to break down cellulose and enhance feed absorption. Similarly, cellulosome-producing bacteria have been identified in the digestive tracts of humans, NHPs, termites and other animals. These cellulosomes are highly adapted to their host environments and specific substrates, facilitating nutrient absorption and digestive efficiency. However, studies on digestive tract cellulosomes are limited due to the challenges of culturing these fastidious microorganisms and the lack of genetic tools. Key knowledge gaps include the assembly mechanisms, functional roles of cellulosomal components and synergistic interactions between cellulosomal and free enzymes. Future research must address these technical challenges to bridge the significant gaps in our understanding of digestive tract cellulosomes.

Cellulosomes have broad applications in biomass conversion, synthetic biology and biotechnology. Digestive tract cellulosomes, in particular, offer unique advantages: (1) They have unique characteristics, providing novel enzymes and functional modules for biomass conversion and synthetic biology. (2) They enhance cellulose degradation and nutrient absorption in hosts, potentially improving host health and physiological functions. (3) They may mitigate greenhouse gas emissions and improve feed efficiency in livestock. The underexplored potential of digestive tract cellulosomes underscores the need for in-depth studies to unlock their full biotechnological and ecological value. These enzymatic molecular machines are worthy of focused research to harness their unique properties for scientific and industrial applications.