Potential of Selected Rumen Bacteria for Cellulose and Hemicellulose Degradation

210 M. ZOREC et al.: Cellulose and Hemicellulose Degradation by Rumen Bacteria, Food Technol. Biotechnol. 52 (2) 210–221 (2014)

ISSN 1330-9862 review (FTB-3544)

Ma{a Zorec, Ma{a Vodovnik and Romana Marin{ek-Logar* Department of Animal Science, Biotechnical Faculty, University of Ljubljana, SI-1230 Dom`ale, Slovenia Received: October 22, 2013 Accepted: May 8, 2014

Summary Herbivorous animals harbour potent cellulolytic and hemicellulolytic microorganisms that supply the host with nutrients acquired from degradation of ingested plant material. In addition to protozoa and fungi, rumen bacteria contribute a considerable part in the breakdown of recalcitrant (hemi)cellulosic biomass. The present review is focused on the enzymatic systems of three representative fibrolytic rumen bacteria, namely Ruminococcus flavefaciens, Prevotella bryantii and Pseudobutyrivibrio xylanivorans. R. flavefaciens is known for one of the most elaborated cellulosome architectures and might represent a promising candidate for the construction of designer cellulosomes. On the other hand, Prevotella bryantii and Pseudobutyrivibrio xylanivorans produce multiple free, but highly efficient xylanases. In addition, P. xylanivorans was also shown to have some probiotic traits, which makes it a promising candidate not only for biogas production, but also as an animal feed supple- ment. Genomic and proteomic analyses of cellulolytic and hemicellulolytic bacterial species aim to identify novel enzymes, which can then be cloned and expressed in adequate hosts to construct highly active recombinant hydrolytic microorganisms applicable for different biotechnological tasks.

Key words: rumen bacteria, Ruminococcus flavefaciens, Prevotella bryantii, Pseudobutyrivibrio xylanivorans, cellulosome, xylanase, probiotics

Introduction (in the cotton fibres) of the dry mass of plant tissue. It is Rumen represents one of the best studied anaerobic a homopolysaccharide consisting of 100 to 20 000 D-glu- ecosystems and constitutes a basis for the current under- copyranose residues linked by 1,4-b-glycosidic bonds. standing of microbial ecology in anaerobic digestive sys- Parallel glucan chains are tightly packed to form micro- tems, including human intestinal tract and anaerobic fibrils and larger macrofibrils. In most natural substrates biogas reactors. Due to the lack of (hemi)cellulolytic en- cellulose fibrils are dominated by highly ordered crystal- zymes, the digestion of plant biomass in herbivorous an- line areas, interrupted by less organized amorphous sec- imals strongly depends on rumen microbiota. The pri- tions. In plant cell walls, cellulose fibrils are embedded mary role of microorganisms in this complex food chain in a matrix of other polysaccharides: pectin (as homo- is the initiation of a plant biomass breakdown by cellu- galacturonan and rhamnogalacturonan) and hemicellulolytic and hemicellulolytic microorganisms. lose (xyloglucans, xylans, mannans and glucomannans, and 1,4-b- and 1,3-b-glucans). Among hemicellulosic The structure of plant cell wall substrates, xylan is the most abundant. The xylan struc- The major component of plant cell wall is cellulose, ture is more heterogeneous than cellulose and consists which comprises from 20 % (in certain grasses) up to 90 % of 1,4-linked b-D-xylopyranose backbone that bears many

*Corresponding author: Phone: +386 1 3203 849; E-mail: [email protected] M. ZOREC et al.: Cellulose and Hemicellulose Degradation by Rumen Bacteria, Food Technol. Biotechnol. 52 (2) 210–221 (2014) 211 different side chains. The backbone is often partially ac- zymes in extracellular multienzyme complexes, termed etylated and the type of substitution depends on botani- cellulosomes. These comprise a set of components with cal origin. Arabinoxylan (AX) is common type of xylan structural proteins (scaffoldins) binding different cata- found in grasses and cereals (e.g. oat spelt xylan). It car- lytic modules. The main interactions in the complex are ries L-arabinose side-chains that can be esterified by fe- the ones between the cohesion modules of the scaffol- rulic or coumaric acid. These enable cross-linking of xy- dins and dockerin module of the enzymes. Cellulosome lan backbone and enhance cell wall recalcitrance against structure varies among the species in variable number enzymatic attack. Hardwood xylan (e.g. birchwood xy- of the scaffoldins and diverse types of cohesin-binding lan) is glucuronoxylan (GX) with methylated or non-meth- sites that define the nature of the attached enzymes. ylated D-glucuronic acid side-chains. Most of the D-xy- Cellulosomes have so far mainly been described in some loses are acetylated. Softwood arabinoglucuronoxylan species from the bacterial order Clostridiales and in some (AGX) is more heavily substituted by methylated glucu- rumen anaerobic fungi (6). ronic acid than GX and additionally carries arabinofu- Degradation of a plant cell wall is usually conduct- ranose branches (e.g. beechwood xylan). Usually, AGX ed by the coordinated action of diverse members of the chains are shorter than GX (1). microbial community, acting as enzyme producers and/ or end-product utilizers. In nature, such communities Enzymes that degrade (hemi)cellulose – glycoside are found in different environments where intensive hydrolases plant biomass degradation takes place, rumen being one of the richest ones. Not only cellulolytic and xylanolytic Enzymes that catalyze the hydrolysis of glycosidic bacteria represent valuable sources of hydrolytic en- bonds and degrade polysaccharides into smaller struc- zymes and other metabolites, but some of them also ex- tural subunits are known as glycosidases or O-glycoside hibit promising probiotic traits. The characterization of hydrolases (GH). According to their primary structure the enzymes and their catalytic mechanisms, and under- (amino acid sequence similarity), the GHs are currently standing of the induction triggers promote their biotech- classified into 133 families. Some of these families are nological applications. Additionally, genomic analysis of further grouped in clans according to their 3D structure efficient cellulolytic and hemicellulolytic communities homologies (2). In addition to catalytic modules, many may identify genes coding for novel and/or more effi- GH also contain one or more carbohydrate-binding cient enzymes. Their subsequent cloning and expression modules (CBM). Typically, the CBMs and catalytic mod- in adequate hosts may lead to the production of recom- ules are connected by linker regions, allowing more flex- binant microorganism with optimized properties for the ibility to the catalytic part. The primary function of CBM degradation of recalcitrant plant biomass. is selective binding of the substrate and enzyme accumulation on its surface (3). The following article is focused on the cellulolytic and hemicellulolytic machinery of the three prominent Cellulases are glycoside hydrolases with the pri- bacterial strains from rumen that were comprehensively mary role of cleaving the cellulose chains into shorter studied in our research group (Chair of Microbiology cellodextrines with two to six units. According to their and Microbial Biotechnology, Biotechnical Faculty, Uni- mode of action, they are divided to endoglucanases and versity of Ljubljana, Slovenia) in collaboration with some exoglucanases (also cellobiohydrolases). Endoglucanases foreign institutes (see references): Ruminococcus flavefaci- randomly attack the internal glycosidic bonds in the ens, Prevotella ruminicola and Pseudobutyrivibrio xylanivo- amorphous regions of the cellulose chains and increase rans. the number of loose ends, which represent the substrates for exoglucanases. The latter are generally processive enzymes that bind to the substrate and then progress to Ruminococcus flavefaciens the end of the chain by releasing shorter saccharides. On the other hand, majority of the endoglucanases detach The first insight in R. flavefaciens cellulosomes from the substrate after each cleavage and then random- (strains FD-1 and 17) ly bind to another location (3). Due to xylan’s complex structure with diverse na- Ruminococcus flavefaciens is a Gram-positive, strictly ture of side-chains, degradation is also carried out by the anaerobic coccus-shaped bacterium, phylogenetically clas- synergistic action of many different enzymes. The enzyme sified in clostridial cluster IV (phylum Firmicutes). It is system usually consists of endoxylanases and b-xylosi- one of the most abundant cellulolytic bacteria in the gas- dases that cleave the polyxylopyranosyl backbone and trointestinal tract of the herbivores and currently the only rumen bacterium in which cellulosomes were experi- many different side-chain-removing enzymes like a-L-ara- mentally confirmed (7). Comparative nucleotide sequence binofuranosidases, a-glucuronidases, acetylxylan esterases, analysis in strains 17 and FD-1 revealed a sca gene clus- p -coumaric and ferulic acid esterases. Microbes usually ter encoding four scaffoldins, namely ScaA, ScaB, ScaC possess the whole set of enzymes, often producing mul- and ScaE. The arrangement of the cluster and spacer re- tiple forms of certain catalytic property with subtle dif- gions were well conserved within the species, whereas ferences in order to facilitate the solubilization of a vari- structural genes showed a considerable diversity, which ety of xylans (4,5). may be associated with the ability of individual strains Synergistic action of different enzymes with special- to degrade different types of (hemi)cellulosic substrates ized roles is needed for efficient degradation of natural (6,8). The core structural (enzyme-binding) proteins of R. cellulosic substrates. Some anaerobes developed more flavefaciens cellulosomes proved to be two glycosylated effective cellulolytic strategy by spatially combining en- scaffoldins, ScaA and ScaB. ScaB is the largest of the 212 M. ZOREC et al.: Cellulose and Hemicellulose Degradation by Rumen Bacteria, Food Technol. Biotechnol. 52 (2) 210–221 (2014) scaffoldins and provides a basis for anchoring up to Analysis of the 007C draft genome has revealed the seven ScaA subunits, resulting in a multi-scaffoldin com- same sca cluster arrangement as in other strains of R. plex. Each ScaA protein binds two (in strain FD-1) or flavefaciens: scaC-scaA-scaB-cttA-scaE (13). Since the amino three (in strain 17) enzyme subunits directly or via the acid sequences of the sca genes in strains 007C and 007S smallest scaffoldin, ScaC (9). Acting as an adapter pro- were nearly identical to scaffoldins of strain 17, the cel- tein, ScaC contributes to the variability of the cellulo- lulosome structure was predicted to be the same (Fig. 1). some structure and substrate specificity (10). ScaE is an anchoring scaffoldin, covalently bound to the cell wall. The 007C draft genome analysis also revealed sev- It has only one cohesin that binds to ScaB and anchors eral genes for putative cellulases, namely one for a gly- the whole complex to the cell (11). coside hydrolase GH48 and GH44 and a wide range of enzymes from families GH9 and GH5 (13). It is possible Interestingly, no CBMs similar to the previously de- that multiple putative endoglucanases from families GH5 scribed ones were found in R. flavefaciens. Instead, another protein with cellulose-binding capacity, CttA, was and GH9 evolved from gene duplication and point mu- suggested to play this role in strain 17. The newly de- tation accumulation in the two origin genes. Minor dif- scribed protein is encoded by gene cttA, which is located ferences in specificity and kinetic properties of these en- adjacent to previously described sca genes and is consid- zymes may offer the bacterium a competitive advantage ered part of the sca cluster. Its role was proposed after in its natural environment. In most 007C cellulases, dock- two regions of the recombinant protein were found to erin modules were also identified, confirming the en- strongly bind to microcrystalline cellulose. The CttA also zyme association in the cellulosome. As in strain FD-1, bears a ScaB-like dockerin which binds a cohesin of the substrate-binding modules were detected exclusively with cell-anchoring scaffoldin ScaE (12). the catalytic modules of GH9 and were mainly type CBM_3a (binding crystalline cellulose). Genomic and expression studies of R. flavefaciens Zymograms confirmed the existence of a wide range 007C and 007S of different endoglucanases in R. flavefaciens 007C and Further insight in the mechanisms of cellulose deg- 007S: four were produced constitutively, while at least radation was gained by studying mutants of R. flavefa- seven appeared to be induced exclusively when the strains ciens 007 (13). The first mutant, namely 007S, lost the were grown on insoluble cellulose (15). This is consistent ability to degrade cotton cellulose after being repeatedly with previous finding of Berg Miller et al. (16), who also subcultivated in cellobiose medium. After transferring reported cellulose-associated inducible transcription of 007S to the medium with cotton as a sole source of en- only part of the GH consortium, while the rest of the energy, the strain regained cotton degradation ability. The zymes appeared to be similarly expressed on cellobiose second mutant was named 007C (14). The mutants do and cellulose. Some recent findings argue that differen- not differ in the degradation of other substrates (cello- tial expression of cellulolytic enzymes may not be medi- biose, xylan, Avicel), and can therefore be used as a mo- ated by the substrate directly, but rather via transcrip- del for elucidating a mechanism of cotton cellulose deg- tional regulators that respond to differences in growth radation. rate on different carbon sources (17).

Fig. 1. Putative model of cellulosome organization in Ruminococcus flavefaciens 007 mutants. ScaB is the largest scaffoldin and carries several ScaA subunits. Different enzymes are attached to ScaA directly or via small scaffoldin ScaC. ScaE anchors the whole structure to the cell surface. CttA is cellulose-binding protein linked to the cell via ScaE (by M. Vodovnik) M. ZOREC et al.: Cellulose and Hemicellulose Degradation by Rumen Bacteria, Food Technol. Biotechnol. 52 (2) 210–221 (2014) 213

In the initial stage of growth of R. flavefaciens 007C role has not been identified yet. Interestingly, an open and 007S, the majority of the endoglucanases were cell- reading frame encoding a putative GH3 was found adja- -bound and their molecular mass corresponded mainly cent to the gene encoding Rub in R. flavefaciens, indicat- to cellulosomal GH9. In the later phases, additional smal- ing its possible association with cellulolytic system (13). ler endoglucanases were detected predominantly in the A novel mechanism of oxidative cellulose degradation in- supernatant fraction. Their molecular mass correspond- volving copper-dependent polysaccharide monooxygen- ed mainly to the enzymes from family GH5. It was pro- ases was recently described in fungi (22). These enzymes posed that GH9 carrying their own CBMs are the first to were recently reclassified as auxiliary activity family 9 attack intact crystalline cellulose. The released shorter (AA9, former GH61) and are able to cleave strongly packed chains were then hydrolyzed by other enzymes among cellulose microfibrils in the crystalline areas with their which are smaller, non-cellulosomal GH5 (13). strong oxidative action. It can be speculated that rubre- Proteomic analysis of R. flavefaciens 007C and 007S rythrin in R. flavefaciens may play a role in oxygen detox- extracellular fractions revealed two dominant celluloso- ification system as found in other anaerobic microorgan- mal enzymes – a GH9 (homologue of R. albus endoglu- isms (23). However, its involvement in similar oxidative canase Cel9B) and a GH48 (homologue of R. albus exo- reactions of recalcitrant cellulose breakdown is an inter- glucanase Cel48A) (18,19). The complementary action of esting area for future studies. the enzymes by the following scenario was suggested: after the GH9 endoglucanase cleaves cellulose chains in the amorphous regions, the GH48 exoglucanase binds to Yellow affinity substance – an auxiliary factor newly generated loose ends and travels along the chain, involved in cellulose degradation? processively cleaving off the saccharides. Among the most abundantly expressed proteins during growth on the cel- Several strains of R. flavefaciens have long been known lulose were not only a GH48 exoglucanase and some pu- to produce high amounts of a yellow affinity substance tative endoglucanases from family GH9, but also three (YAS) that strongly binds the cellulose (24). However, its products of sca gene cluster (ScaA, ScaB and CttA) and chemical structure and function still remain unknown. some ABC-transporters (18). In a study of Kope~ný and Hodrová (24), YAS was also found to enhance the attachment of cellulases to the sub- In further work, the extracellular protein profiles of strate. In our study, the majority of YAS was not associ- R. flavefaciens 007C and 007S were compared in order to ated directly with catalytic modules, but rather with the find protein candidates that may be crucial for the deg- three large, heavily glycosylated scaffoldins, namely ScaA, radation of highly crystalline cellulose such as cotton ScaB and CttA (13). This suggests its role in the attach- (13,18,20). In contrast to the previous study of Rincón et ment of the complex to the substrate, which was sup- al. (11), the analysis revealed no differences in relative ported by the fact that the addition of YAS to the washed amounts of CttA protein (12). However, two other pro- supernatant proteins of R. flavefaciens 007C increased the teins were highly upregulated in a cotton-degrading microcrystalline cellulose breakdown. strain 007C. The first of these was a type IV pili-related (T4P) protein, subsequently named Pil3. Its relative volume was more than 19-fold higher during growth on Potential for applications in biotechnology Avicel cellulose than on cellobiose. The analysis of genetic neighbourhood of the gene coding Pil3 revealed The study of R. flavefaciens cellulolytic system revealed the whole cluster of nine genes coding for different com- one of the most complex cellulosomal machineries with ponents of T4P biogenesis machinery. T4P are well stud- multimodular proteins and several putative auxiliary com- ied in Gram-negative species, where they play diverse ponents (type IV pili, YAS, etc.) that allow efficient cellu- roles, especially in cell adherence and motility (twitch- lose degradation in anaerobic environments. Newly gained ing motility, for example). On the other hand, not much knowledge may be usefully applied in many different is known about these structures in Gram-positive bacte- fields of biotechnology. Besides traditional applications ria. Only recently, analogues of a Gram-negative type IV in paper, food and textile industry, the biotechnological pilus gene cluster were found in some Gram-positive potential of cellulosomes is now becoming increasingly strains, particularly in the most ancient bacterial class recognized in bioenergy production (mainly biogas and Clostridia, which implies a common evolutionary origin biofuel production from plant biomass) (3). Together with (21). Since our study revealed a T4P-related protein to novel molecular biology tools, knowledge of efficient be highly upregulated during growth on cellulose exclu- cellulolytic systems can enable the construction of artifi- sively in cotton-degrading mutant 007C, it is possible cial cellulosomes with desired properties, such as in- that their role in R. flavefaciens is associated with the at- creased capacity for the degradation of lignocellulose, tachment to the highly crystalline regions of cellulose better stability in process conditions, etc. These chimeric (13). It is also possible that pili have a role in processive complexes are called 'designer cellulosomes' and are movement of the cells along the cellulose polymers and/ eventually meant to be expressed in industrial micro- or cellulose amorphogenesis (13). organisms. So far, solventogenic clostridia are the major The second protein that was exclusively upregulat- object for heterologous expression of cellulosomal pro- ed in R. flavefaciens 007C was a rubrerythrin-like protein teins and functional minicellulosome produced in other- (Rub) (13,18). This is a small protein (20 kDa) with an wise non-cellulolytic Clostridium acetobutylicum (25). Due N-terminal Fe2+ binding module and a C-terminus ho- to phylogenetic relatedness, the knowledge about rumi- mologous to rubredoxin. Although it is widely distrib- noccocal cellulolytic machinery may be of great appli- uted in anaerobic bacteria and archaea, its physiological cative value. 214 M. ZOREC et al.: Cellulose and Hemicellulose Degradation by Rumen Bacteria, Food Technol. Biotechnol. 52 (2) 210–221 (2014)

Prevotella bryantii xylanolytic system might be more efficient because native form of xylan bears more acetyl side-chains that Prevotella spp. are one of the dominant xylanolytic increase its solubility (36). The preference towards less bacterial genera detected in the rumen (26), P. bryantii substituted type of GX over AX was also detected, indi- being prevalent in liquid fraction of rumen samples (27). cating that side-chain decoration might hinder polymer Rumen isolates were formerly classified in one species, solubilization (36). On the other hand, among the pen- namely P. ruminicola. However, interstrain variability in tose sugars derived from xylan, uronic acid was utilized xylanase gene distribution, zymogram patterns (28) and with the greatest efficiency, exceeding xylose and arabi- 16S rDNA sequences (29) initiates full assessment of the nose levels (36). The preference of GX type of xylan is strains and eventually four species in the genus Prevo- probably a consequence of higher uronic acid content and tella were established (30). Rumen Prevotella strains are more potent side-chain-cleaving enzyme, a-glucuronidase, important in protein and peptide metabolism, some pos- recently found to be highly induced in xylan medium sess extracellular DNase activity and, nevertheless, they (39). are able to degrade several polysaccharides commonly present in rumen digesta like xylan, cellulose and starch Identification and characterization of P. bryantii B14 (30). Among xylanolytic strains, redefined P. ruminicola xylanolytic system and P. bryantii with type strain B14 were constituted. Strains of P. bryantii are strictly anaerobic Gram-negative In P. bryantii B14 four coding regions for xylanolytic bacteria belonging to the large Bacteroidetes phylum, and activity were identified and cloned (40), and each corre- are capable of xylan, soluble cellulose (carboxymethyl- sponds to a clearing band detected by zymogram analy- cellulose, CMC), starch and protein breakdown (30). sis (Fig. 2) (31). Briefly, zymograms were developed with denatured proteins on gels with incorporated xylan. The resulting gel with separated proteins was soaked in re- Xylanolytic activity of P. bryantii B14 naturating solutions overnight and then incubated at 38 As many rumen xylanolytic bacteria, strain B14 pos- °C. The xylanolytic activity of enzymes was detected as sesses multiple xylanase genes and has the highest xyla- clearing areas by Congo red staining (31). nolytic activity of all Prevotella spp. (31). The xylanolytic activity was clearly inducible with growth in xylan-containing medium and the activities were 20-times higher compared to the medium containing only glucose (32,33). Xylanase production was found to be primarily induced by medium (longer than five xylopyranose units) to large- -sized xylooligosaccharides (34). It was believed that oligosaccharides are generated by the action of P. bryantii extracellular xylanases or in the rumen by hydrolytic activity of other microorganisms. Monosaccharides including glucose, arabinose and xylose repressed xylanase production in ratios as little as 0.1 % (by mass per volume) when associated with xylan (35), pointing to catabolite repression of monosaccharides in P. bryantii B14. Xylanolytic activity of the strain B 4 was predomi- Fig. 2. Typical xylanogram of Prevotella bryantii B14. GH5 endo- 1 glucanases have additional xylanolytic activity, XynA and XynC nantly cell-bound and less than 5 % was detected in the are GH10 endoxylanases and XynB is GH43 exoxylanase/b-xy- culture supernatant (36). The CMCase was previously losidase. 1 – cell fraction, 2 – periplasmic fraction (by R. Ma- suspected to be cell-surface bound (37), but cell fraction- rin{ek-Logar) ation with osmotic shock released the majority of the CMCase and xylanase activities in the periplasmic fraction, the b-xylosidase and a-L-arabinofuranosidase remain- The first region corresponds to a previously de- ing cell-membrane associated (38). The polysaccharolytic scribed broad-specificity GH5 endoglucanase (41). The enzyme localization in the periplasm, where enzyme- zymograms revealed two proteins (of 82 and 88 kDa), -polymer interaction is obstructed by the outer membrane, being primarily CMCases with weak endoxylanolytic ac- was surprising at the time. It was assumed that periplas- tivity (31,33). Later, Matsushita et al. (42) found out that mic position protects the hydrolytic enzymes against en- GH5 endoglucanase is encoded in two overlapping read- vironmental detrimental factors and offers the bacterium ing frames and the obtained double band is a result of competitive fitness with segregation of the enzymes and reading frame shift. the degradation products from the extracellular matrix The other three xylanases lack CMCase activity. The (36). second clone exhibited b-xylosidase and a-L-arabinofu- Despite the apparent substrate inaccessibility, P. bry- ranosidase activity in addition to endoxylanase (40). In antii B14 was able to effectively utilize xylans (36). The this region two genes were found to be closely linked – majority of the produced xylooligosaccharides were rap- xynA, coding for GH10 endoxylanase, and xynB,anoxy- idly utilized and also larger water-soluble xylan frag- gen sensitive GH43 exoxylanase with associated b-xylo- ments were consumed to a high degree (36). Degrada- sidase and a-L-arabinofuranosidase activity (40). The XynB tion of less-accessible water-insoluble part of xylans was enzyme activities were severely affected by elevated the main limitation in utilization of isolated xylans in temperature and oxygen presence (40). The preference strain B14. It was suspected that in nature P. bryantii for GX over AX was detected and was most likely due M. ZOREC et al.: Cellulose and Hemicellulose Degradation by Rumen Bacteria, Food Technol. Biotechnol. 52 (2) 210–221 (2014) 215

to better solubility (40). The cloned XynB processively Transcriptomic analysis in P. bryantii B14 revealed homo- releases xylose from non-reducing ends of xylooligosac- logues of starch utilization system in Bacteroides thetaiota- charides and xylan, exhibiting exoxylanase action (40). It micron (46). In the predicted model the proteins of xylan cleaves also xylobiose, but no arabinose was released from utilization system (Xus) together with XynC are respon- native xylans although XynB degrades the artificial sub- sible for xylan binding, cleavage and transport of xylo- strate p-nitrophenylarabinofuranoside (32,40). On xyla- oligosaccharides across the outer membrane into the peri- nograms, the 33-kDa clearing band corresponds to xynB plasm (45). The presence of an N-terminal signal peptidase gene product and its activity was better detectable on II cleavage site in XynC indicates its position on the zymograms prepared with soluble xylan (less substituted outer leaflet of outer membrane (45) and explains the form with lower degree of polymerization) and addition enzyme low pH and oxygen tolerance in addition to re- of reducing agent to provide anaerobic conditions, con- sistance to proteolytic attack (31). The electrophoretic firming its free-end chain dependence and oxygen sensi- analysis of the partially purified 67-kDa enzyme (XynC) tivity also in native form (31). displayed other four to five non-xylanolytic proteins Cloned XynA enzyme was also thermolabile, but the (31), which is in accordance with the five genes identi- activity was not affected by oxygen (40), which repre- fied in the xus cluster (46). The predicted xylan-utiliza- sents an advantageous trait in biotechnological application system consists of XusA and XusC spanning the tions. With XynA enzyme xylan degradation products outer membrane most likely involved in oligosaccharide were mainly xylobiose and xylotriose, proving its endo- transport, XusB and XusD on the outer leaflet binding xylanolytic function (40). Again, cloned XynA exhibits the xylan, XynC with the catalytic action and XusE with higher activity on GX compared to AX as a substrate yet unknown function (45). The proteins seem to be strongly inter-linked and identification of high molecu- (32). On xylanograms P. bryantii B14 native XynA was presumably detected as a faint band at 41.5 kDa (31). lar xylanolytic complexes raised a speculation of xylano- some existence (47). The previously observed periplas- In AX breakdown XynB acts synergistically with matic location of XynC was most likely a result of firm XynA endoxylanases and up to 10-fold enhancement of association with the outer membrane and consequent re- reducing sugar release was determined (40). Synergism lease with periplasmic marker. Furthermore, membrane in xylan degradation is frequently recorded because the b association explains the formation of protein aggregates efficient action of exoxylanases/ -xylosidases depends upon isolation. On membrane disruption, the released on the availability of xylan loose ends generated by en- protein complexes are apparently bound along the re- doxylanases. maining xylan polymer, hence forming an inseparable The clone carrying xynC gene was found to code for aggregate. However, the aggregates retain xylanolytic another GH10 endoxylanase. On AX zymograms the activity (31). endoxylanase XynC was identified as clearing band at The comprehensive transcriptomic study discloses 67 kDa (31,33). XynC and the smallest, 33-kDa XynB many upregulated genes during growth of the strain B 4 were detected also in cultures grown in glucose-contain- 1 on AX (46). Most of them were annotated as coding for ing medium and therefore considered to be constitutive- proteins related to xylan metabolism and many were ly produced in low quantities (31). In xylan-grown cells designated as hypothetical proteins (46). The study re- the clearing bands were larger and more pronounced, in- vealed the major xylanolytic gene cluster with centrally dicating their inducibility (31). positioned xynR regulator gene, including previously The XynC enzyme was partially purified (31,43). identified xynC, xynB and xynA. Upstream to xynR, all Several methods were applied in enzyme isolation, most of the xus genes are located followed by xynC. This of them resulting in heavily aggregated states of peri- group contains six of the seven most highly induced plasmic extract (31). The isolation of native xylanolytic genes, indicating their crucial role in xylan metabolism enzymes from rumen bacteria is rarely reported, proba- (46). Regarding the highly conserved xylan utilization bly due to their high tendency to form aggregates. Fi- operon across xylanolytic Bacteroidetes (46), the hypothet- nally, successful HPLC separation of crude periplasmic ical model for signal transduction analogous to B. thetaio- proteins using DEAE-based compact porous discs (CIM tamicron mucin oligosaccharide induction was proposed DEAE-8) resulted in four peaks with xylanolytic activity (45). In strain B14 signal transduction is presumably ini- (31). The fraction including 67-kDa endoxylanase exhib- tiated by xylooligosaccharide transfer by transmembrane its the highest hydrolytic activity on soluble AX and protein XusC/D that triggers an unknown signalling showed good temperature stability with optimum at 39 cascade leading to activation of XynR and inducing the °C and pH=5.5 (31). Also with trypsin and pepsin con- transcription of xylan-utilization gene (45). centrations exceeding the physiological levels, XynC re- The elaborated xylanolytic system of P. bryantii B 4 tains its activity (31), indicating their possible applica- 1 was complemented with a characterization of a novel tion as feed additives for monogastric animals (35). GH5 endoxylanase (46) and three additional GH3 b-xy- A unique insertion of 160 amino acid residues in the losidases (48). Substrate affinity in multiple GH3 en- catalytic module of XynC was identified and no similar zymes revealed their unique role in the hydrolysis of sequence was retrieved in the database at the time (44). xylosidic linkages dependent on the type of substitution Due to its position next to the catalytic module, it was (48). The side-chain preference was not determined for suspected to be a novel type of substrate-binding mod- GH43 due to lack of appropriate substrates (36), but the ule (44). Later it was assigned to CBM family 4. production of several enzymes hydrolyzing the identical Recently, XynC was found to be part of xylan uti- chemical bond is substantiated by the heterogeneity of lization system located on the outer membrane (45). the substrate with side chains hindering the access. 216 M. ZOREC et al.: Cellulose and Hemicellulose Degradation by Rumen Bacteria, Food Technol. Biotechnol. 52 (2) 210–221 (2014)

Potential biotechnological applications of xylanases The active xylanolytic system consisted of at least seven electrophoretically distinct xylanases that were de- There are many areas of potential applications of xyla- tected on zymograms with AX (Fig. 3) (61,63). Multiple nases, especially cellulase-free enzymes, which are of clearing bands (up to 14 were detected in certain growth great value as they specifically remove xylan and leave phase) representing xylanolytic activity ranged from 30 cellulose intact. The enzymatic treatment is often a bet- to 146 kDa (61,64). Multiplicity together with high xy- ter alternative to chemical processing in regard to envi- lanolytic activity offers the bacterium a dominant posi- ronmental pollution and energy conservation. Xylanases tion in xylan breakdown process and not surprisingly, are routinely used in development of environment-friend- the butyrivibrios are among the most frequently isolated ly technologies in the pulp and paper industry (biobleach- bacteria from ruminants fed on forages (65). Multiple ing and biopulping) and for processing flour in bakery industry. Xylanases are also efficiently used as feed additives in non-ruminant livestock (poultry, pigs) as they release simple sugars from polymers that otherwise re- sist degradation by indigenous digestive enzymes. Ad- ditionally, xylanases reduce the viscosity of the intestinal content (e.g. arabinoxylan of wheat and rye) and by that improve the absorption of digested food, prevent colonization by pathogens and overall benefit the animal (49). Moreover, xylanases can be used to produce diverse xylooligosaccharides with prebiotic properties (50) and their application in functional foods industry is encouraged. Recently, the most interesting area where xylanases and xylanase-producing microorganisms are employed is the Fig. 3. The xylanogram of cell-associated xylanases in Pseudo- biofuel production, where they are used for the degra- butyrivibrio xylanivorans Mz5. Four major xylanases are indica- dation of lignocellulose material (51,52). ted (146, 100, 81 and 44 kDa) and the isolated Xyn11A (30 kDa). Lanes represent time-dependent emergence of the enzymes (10 Currently, mainly fungal xylanases are used in the hours to 9 days) (by M. Zorec) industrial applications, as fungi are considered more efficient producers than bacteria and archaea. However, occupational exposure to enzymes of fungal origin was forms of an enzyme could be a result of several distinct shown to trigger diverse allergic reactions (53,54). Bacte- genes, or proteins of a single gene subjected to differen- rial sources of potent xylanases are therefore gaining in- tial post-translational modifications (like glycosylation or creased interest and rumen microbiota can be efficiently proteolysis). In later growth phases, the detected multi- employed in that manner. plicity can also be a consequence of partial proteolysis or enzyme association with disintegrated extracellular Pseudobutyrivibrio xylanivorans polysaccharide (EPS) layer as reported for proteinases (66). The molecular masses of the four major enzymes identified as large clearing bands on xylanograms were One of the most xylanolytically active rumen bacte- 44, 81, 100 and 146 kDa (67). The majority of the xyla- ria are also the members of the Pseudobutyrivibrio-Buty- nolytic activity was cell-associated (61,63). The produc- rivibrio group. Historically, all Gram-negative staining tion of EPS in butyrivibrios is a common trait and the strains of rumen origin producing butyrate in glucose presence of a thick EPS layer was suspected also for medium were classified as Butyrivibrio fibrisolvens, later strain Mz5 as it formed mucoid colonies with emanating Butyrivibrio sp. considering the increasing phenotypic di- bubbles (61,68). The detected cell-bound activity was versity among the isolates (55–57). The full revision of therefore most likely a result of extracellular enzymes Butyrivibrio-like bacteria resulted in strain distribution retained in cell proximity because the glycocalyx pre- into four different phylogenetically coherent species, one vented their diffusion. In the later growth phases when of them newly described Pseudobutyrivibrio xylanivorans pH dropped below 5.8, the heteropolysaccharide matrix with type strain Mz5 (58), isolated in our lab. The spe- probably disintegrated and released the xylanolytic en- cies are classified to Lachnospiraceae family in the Firmi- zymes from the cell surface, when a decrease in cell-as- cutes phylum and their Gram-negative staining was found sociated xylanolytic activity with simultaneous increase to be a result of a very thin peptidoglycan layer (59). in the supernatant activity was observed (61,64). The electrophoretic equivalence of supernatant and cell-asso- Identification and characterization of ciated xylanases further supports these assumptions. Pseudobutyrivibrio xylanivorans Mz5 xylanolytic Strain Mz5 also possesses b-xylosidase and a-L-ara- system binofuranosidase activity that were mostly cell-bound On isolation, strainMz5 exhibited the highest xyla- (61). The peak of cell-associated xylanase activity was nolytic activity at least 1.6 times higher than other ru- detected in the mid-exponential phase at point where men xylanolytic species, including several Butyrivibrio- the 100- and 146-kDa xylanases were the most promi- -like strains (60,61). Based on the ability of strain Mz5 to nent (61,63). After the peak, xylanolytic activity declined grow on xylan as a sole carbohydrate source (62), we as- rapidly although the clearing bands on the xylanograms sumed that a wide range of enzymes capable of efficient intensified in brightness and in number (61). This dis- xylan degradation must be present. crepancy is due to method design as the majority of M. ZOREC et al.: Cellulose and Hemicellulose Degradation by Rumen Bacteria, Food Technol. Biotechnol. 52 (2) 210–221 (2014) 217 xylanases are inhibited by low pH that persists for the ule (CBM6) and a NodB-like module (78). Both non-cat- time of reducing sugar test, while xylanograms are de- alytic modules enhance xylan breakdown in a different veloped in the neutral pH that reactivates the enzymes manner. Binding subunit facilitates the activity towards to some extent. insoluble substrate with attaching and concentrating the The production of xylanolytic enzymes in strain Mz5 enzymes in proximity to xylan. Further on, NodB-like is highly inducible with AX and specific activity increased subunit removes acetyl side chains and exposes the xy- as much as 33-fold in comparison with glucose (60,61). lan backbone to xylanase attack. Similar module arrange- The AGX and GX xylan induced the xylanolytic activity, ment was found also in Clostridium thermocellum, Rumi- too, but the levels were approximately fivefold lower nococcus albus and soil bacterium Clostridium fimi (78). (61). On xylanograms two faint xylanolytic bands were The conservation level was particularly high in the cata- detected at 44 and 146 kDa in cells grown in glucose- lytic part and homologues of xyn11A of Mz5 were found -containing medium (61,63). The major part of xylanoly- also in other Pseudobutyrivibrio strains (75). High level of tic activity in glucose medium was detected in the culture similarity can be explained by the existence of common supernatant (61), which points to the fact that constitu- ancestral origin and subsequent horizontal gene transfer, tive enzymes are released to the extracellular space and which occurs frequently in the rumen (79). Nevertheless, by that expand the area of possible substrate contact. combining the catalytic module with binding and deace- These two xylanases are therefore considered to be con- tylating subunit offers a bacterium competitive advantage stitutively produced with the larger one being more abun- in xylan utilization in diverse environments. In certain dant as seen on xylanograms. The degradation products species, association of several complementary catalytic generated by the action of 44- and 146-kDa xylanases can modules with binding modules evolved in more elabo- then enter the cell and induce the synthesis of other en- rated multiprotein complexes such as cellulosomes. zymes. The linkage of the catalytic module to non-catalytic The partially purified 44-kDa enzyme efficiently de- proteins could contribute to several unsuccessful attempts graded AX and xylooligosaccharides to xylobiose and in isolation of other xylanases (67,69). The presence of xylose, but was inactive towards xylobiose and was thus CBM as pointed out earlier, additionally promotes na- considered to be exoxylanase without b-xylosidase activ- tive enzyme aggregation by accumulating them on the ity (67,69). The molecular mass of the protein indicated substrate residues. Additionally, membrane transporter homology to same size XynA xylanase found in Pseudo- proteins could be associated with enzyme complexes as butyrivibrio strain 49, which was classified into GH10 fa- it was revealed in Prevotella bryantii B14. By recent pro- mily (70). The presence of GH10 enzymes was suspected teomic analysis, several putative carbohydrate transport- also in Mz5 as they are widely distributed across the Bu- ers were identified in closely related rumen species, Bu- tyrivibrio-Pseudobutyrivibrio group (71). Additionally, an tyrivibrio proteoclasticus (80). Most of them were members identical gene sequence of related strain CF3 xynI2 gene of ABC transport system, some predicted to be involved encoding for GH10 xylanase was found (72). The exact in oligosaccharide transport (80). Like in Prevotella bryan- species designation of strain CF3 is pending, but its close tii B14, upregulation with xylan medium and clustering relatedness to the other strain of Pseudobutyrivibrio xyla- with other xylan utilization genes was identified (80). nivorans, DSM 10317 (58,73) relates strain CF3 to Mz5. Therefore, only partially isolated 81-kDa xylanase was Additionally, the smallest enzyme (30 kDa) was suc- obtained and the fraction exhibited a single clearing band cessfully purified (67,74). During growth in batch culture, on xylanogram with many other proteins still detected the enzyme emerged on xylanograms at the beginning on electrophoretograms (67). The 81-kDa enzyme is pre- of the stationary phase and persisted into the prolonged sumably a complete multimodular complex carrying a cultivation (61). The 30-kDa xylanase, XynT (later desig- 30-kDa xylanase subunit and thus displays almost iden- nated as Xyn11A) was classified into GH11 family and it tical degradation pattern to 30-kDa enzyme. The 81-kDa was the first family 11 xylanase found in the Butyrivibrio- xylanolytic activity on AX generates different xylooligo- -Pseudobutyrivibrio group (75). Since the activity of Xyn11A saccharides, xylopentaose being the largest (67). On the was at least threefold higher in the soluble fraction of other hand, the 30-kDa enzyme smallest product was xylan, the absence of the xylan-binding module was sus- tetraoligosaccharide, which might be a result of a minor pected. Xyn11A action on AX resulted in xylotetraose or active site alteration upon proteolytic cleavage. higher xylooligosaccharides placing the enzyme at the The putative mechanism for xylan degradation has beginning of the xylan breakdown process. The cleavage been proposed (67,72), starting by the action of constitu- site of Xyn11A was predicted to be near substitutions, tively synthesized 145-kDa endoxylanase, which releases because the activity towards less substituted types of smaller fragments of xylan and generates free ends of xylan was lower (76). Similar amino acid sequences to xylan. The 44-kDa exoxylanase binds to newly formed Xyn11A were also found in xylanases from strains of chain termini and produces wide range of different xylo- Clostridium, Bacillus and Ruminococcus (76). oligosaccharides including xylose. The enzymes are most On xylanograms the concurrent loss of visible bands likely detached from the cell to increase the chances for at 81 and 100 kDa with 30-kDa band emergence (61,63) coming into contact with xylan. Next, the xylooligosac- indicated that the smallest enzyme could be a proteo- charides are transported across the membrane by similar lytic fragment of the larger xylanases, retaining catalytic membrane transporters as found in B. proteoclasticus and function. When the xyn11A gene sequence was analyzed, induce the synthesis of other xylanolytic enzymes by a multimodular structure of the enzyme was revealed unknown mechanism. The induced xylanases are trans- (77,78). The gene consists of previously isolated catalytic ported across the membrane and trapped in the EPS module from GH11 family, followed by a binding mod- layer that retains the bulk of enzyme activity close to the 218 M. ZOREC et al.: Cellulose and Hemicellulose Degradation by Rumen Bacteria, Food Technol. Biotechnol. 52 (2) 210–221 (2014) cell surface. The attachment of the enzymes to the cell (90). Additionally, feeding mice with probiotic strain membrane is still unclear, but similar associations to lessened the colitis symptoms (91) and recently, an EPS membrane transporters as reported for B. proteoclasticus extract from the same strain has been shown to alleviate are not excluded. The amount of constitutively synthe- the atopic dermatitis lesions (92). Taken together, the sized xylanases also increases and joint performance of Pseudobutyrivibrio xylanivorans strain Mz5 is a promising all enzymes results in xylooligosaccharides of variable probiotic candidate having many beneficial characteris- lengths, some also substituted, and xylose. The products tics like butyrate, CLA and EPS production together with are then transported into the cell, where further degra- oxygen tolerance and an ability to attach to human epi- dation proceeds with the cell-associated b-xylosidase and thelial colorectal cells. arabinofuranosidase concluding with a variety of pen- toses and hexoses available for cell metabolism. Potential use of rumen bacteria in biogas production Potential use of Pseudobutyrivibrio xylanivorans Mz5 Recent studies of rumen-derived cellulolytic and xylanolytic bacteria are currently focused towards the bio- as a probiotic logical pretreatment of recalcitrant lignocellulosic sub- Probiotics are live microorganisms with beneficial strates in biogas production (93). Utilization of low-cost effects to the host in many ways: pathogen growth inhi- material such as waste plant residues is gaining atten- bition with production of organic acids and bacteriocins, tion in a sense of consolidated bioprocessing. Other pre- impeding pathogen adhesion and colonization by com- treatment methods like physical and chemical demand petitive exclusion, neutralizing the toxins and boosting certain energy input and have high environmental im- up the host immune system (81). Besides its use as a pact. Therefore, the biological pretreatment by appropri- source of enzymes, the strain Mz5 could potentially be ate microorganisms is preferred to improve the hydroly- applied as a probiotic due to several other advantageous sis rate of lignocellulose biomass, which is the rate-limit- traits. First, the strain Mz5 is a potent xylanolytic pro- ing step in biogas production (94). The promising results ducer and abundant rumen inhabitant. Therefore, buty- by utilization of cellulolytic R. flavefaciens 007C and xyla- rivibrios as rumen indigenous bacteria are promising nolytic Pseudobutyrivibrio xylanivorans Mz5 in substrate hosts for recombinant genes that could improve fibre pretreatment expand the area of biotechnological appli- degradation in rumen. Even though genetically modi- cation of fibrolytic rumen bacteria (93). fied strategies are heavily opposed by consumers, several solutions exist to minimize the risks and produce the microorganism safe enough to release (82). Then, it Conclusion produces high amounts of butyrate and lactate with concurrent acetate utilization from the medium (68,83). The Rumen is a rich source of fibrolytic enzymes and butyrate together with lactate induce decline in pH level, different microorganisms producing them, and thus rep- hence inhibiting the growth of many pathogens. In mono- resents a good source of productive bacteria for different gastric animals, butyrate was proved to have a primary biotechnological purposes. Ruminococcus flavefaciens car- role in metabolism and normal development of colono- ries one of the most elaborated architectures of the cellu- cytes and in having a detrimental effect on cancer cells losome and provides vast spectra of possibilities in con- (84). Mz5 also showed bacteriocin-like inhibition of some struction of designer cellulosomes. At the same time, it rumen bacteria and several strains of Salmonella enteri- represents an interesting pool of genes for genetic ma- tidis and E. coli isolated from poultry meat (85). The pro- nipulation of different host bacteria for high production duction of inhibitory substances additionally contributes of cellulases and hemicellulases. The xylanolytic Prevo- to competitive fitness of the bacterium in gastrointesti- tella and Pseudobutyrivibrio produce multiple forms of high- nal tract of animals. Another trait seems favourable at ly potent enzymes, but upscaling their isolation might the time, but is currently disputed about – beneficial ef- be difficult as they are linked to other non-catalytic ac- fects of the conjugated linoleic acid (CLA) in treating cessory proteins enhancing their function in vivo. The pro- obesity, cancer and preventing cardiovascular diseases in mising areas of application are as probiotics for mono- humans (86). Still, Butyrivibrio species are the principal gastric animals (especially pigs and poultry) and utiliza- CLA producers in the rumen from linoleic acid, and Mz5 tion in pretreatment bioprocesses for biogas production also produces the CLA-precursor trans-vaccenic acid (TVA) from lignocelluloses waste substrates originating mostly (87). CLA was detected only as an intermediate and TVA from agro-food industry. Due to the high applicative po- was the final product in biohydrogenation process (87). tentials of the three selected rumen bacteria, their ecol- Further on, adhesion test to human cell culture was adapt- ogy, physiology and biochemistry were thoroughly stud- ed for anaerobic bacteria (88). Cells of Mz5 successfully ied by our research group and our collaborators. attach to model Caco-2 cells and this finding additionally supports further in vivo studies. Nevertheless, oxygen tolerance was also detected as Mz5 retained viabil- References ity in partially oxygenated medium (89). Retaining the 1. A. Heredia, A. Jiménez, R. Guillén, Composition of plant viability upon oxygen exposure facilitates handling of cell walls, Z. Lebensm. Unters. Forsch. 200 (1995) 24–31. the cultures in different biotechnological applications. 2. 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