Developing efficient technologies to enhance the conversion of low-quality forages into available energy within the rumen is pivotal for sustainable dairy and beef production . Potentially, such technologies can reduce feed costs and improve the competitiveness of cattle management, while reducing the environmental footprint of meat and milk production . Supplementing ruminant diets with exogenous enzymes has the potential to enhance feed efficiency [7, 8]. However, current commercial enzymes have been tailored for bio-refinery and bioprocessing applications and as such are not optimized for the physiological conditions of the rumen [23, 24, 25]. Likewise, commercial enzymes often contain redundant enzyme activities already encoded within the rumen microbiota. Understanding the rate-limiting steps in ruminal fiber digestion from the perspective of plant cell wall chemistry and endogenous catalytic potential is critical for the successful design of technologies based on enzyme addition, programming of the microbiome, and breeding plant cell walls that are more amenable to digestion by microorganisms within the ruminant digestive tract. Here we present an informed experimental platform (Fig. 8) that couples high-resolution cell wall structural analysis with targeted meta-transcriptomic analysis of rumen microbiota so as to expand the present understanding of the factors that limit plant cell wall degradation.
Straw residues that escape ruminal total tract digestion (TTIR) can provide valuable insight into nutrient utilization by rumen and lower-tract microbes and into plant cell wall components that confer recalcitrance to the hydrolysis of straw. Presented here is the first report using ELISA-based glycome profiling to study the progression of fiber utilization and recalcitrance during total tract digestion of fiber in ruminants. This methodology has been extensively used to characterize plant cell walls including those of genetically modified plants, and microbially and chemically treated plant biomass [9, 10, 26]. Glycome profiling provided us with an unprecedented ability to decipher the architecture of native barley straw and its residual fraction that is recalcitrant to digestion in cattle (TTIR) as it can detect diverse structural features within plant cell wall polysaccharide classes (Fig. 1). For example, a comprehensive suite of more than 25 mAbs that recognize different substructures, including each of the major side-chains of xyloglucans was applied, while another set of approximately 30 mAbs that recognize diverse xylan epitopes including Ara, GlcA and MeGlcA substitutions as well as various lengths of backbone residues  were used to assay xylans (Fig. 1). This not only allowed us to identify recalcitrant polymers with high precision, but also to differentiate polysaccharide subclasses that were removed during passage through the ruminant digestive tract or that exhibit high recalcitrance and were enriched in the TTIR. For instance, 4-O-Me-GlcA xylan epitopes that are lignin bound, as well as those xylans loosely associated with the cell walls in barley straw were removed during passage through the digestive tract (Fig. 1). Alternatively, 2-Ara-substituted xylans, as well as xylan epitopes recognized by the Xylan-2 and Xylan-3 groups of antibodies, were enriched in TTIR, suggesting that these epitopes directly contribute to recalcitrance. Relative proportions of xyloglucan and xylan epitopes in TTIR remained largely unchanged from the proportions of these epitopes in native barley straw (Fig. 1). However, it was clear that glycome profiling alone could not fully describe the glycan composition of TTIR. For example, there was a significant fraction of glycans that were loosely associated with the TTIR (AO extract), yet, with the exception of de-esterified HG epitopes (DP > 4), there was no antibody binding to this extracted fraction from TTIR. This lack of antibody binding may be attributed to the fact that short-chain oligosaccharides released during digestion fail to bind to ELISA plates . Alternatively, while the antibody collection used here recognizes a large and diverse set of plant cell wall glycan epitopes, there may be other epitopes native to barley straw or generated during passage through the ruminant digestive tract that are not recognized by the current antibody library. Therefore, to confirm glycome profiling results we quantified individual glycosidic linkages using partially methylated acetyl alditol derivatization, which measures all glycan linkages present. The linkage data showed that the TTIR was enriched in HX, XG, and AB when compared with barley straw at the whole cell wall level (Fig. 2, Table 1), suggesting that these components were more recalcitrant than other components within this biomass. Fractionation of the walls showed that the EDTA + carbonate extract was strongly enriched in HX and XG (Fig. 2, Table 1), which was in contrast to the results from glycome profiling, where the equivalent extract (AO) only showed enhanced HG content (Fig. 1). This may reflect the composition of the large fraction of cell wall glycans that are loosely associated with TTIR, but which were not assayable in the glycome profiling ELISAs for the reasons mentioned above. Consistent abundance of HX and XG within the whole cell wall and the EDTA/carbonate and KOH extracts of TTIR suggest that these epitopes contribute to the recalcitrance of barley straw within the ruminant digestive tract. Apart from cell wall composition, linkage analysis also provided useful insight into the predominant bonds within identified recalcitrant polysaccharides. The results presented in this study could prove to be a valuable resource for crop breeders to develop feed stocks that exhibit increased digestibility in ruminants.
Identification of predominant recalcitrant bonds coupled to transcriptomic and differential expression analyses, provided two complementary approaches to identify candidate linkages to target for improved plant cell wall digestion. To identify potential enzymes tailored for the digestion of recalcitrant linkages, TTIR and barley straw were used as substrates to culture rumen microbial communities in batch cultures. Incubation with TTIR provided an unique experimental approach that enabled the rumen microbial digestion of recalcitrant plant cell walls to be studied. This is the first such attempt where recalcitrant residues like TTIR have been used to study rumen community composition and CAZyme expression. Nutritional value and effective fermentation of TTIR was confirmed by ELISA results and through comparison of total gas production using barley straw and TTIR as substrates in in vitro cultures (Additional File 8). Firmicutes (Lachnospiraceae, Ruminococcaceae, Clostridiaceae), Bacteroidetes (Prevotellaceae, Bacteroidaceae and unclassified Bacteroidiales), Actinobacteria, Fibrobacteres, Proteobacteria and Spirochaetae were found to be dominant bacterial phyla at the transcriptome level (Table 2). These results are in agreement with previously reported metatranscriptomic analysis, such as Dai et al. , that reported Firmicutes, Bacteroidetes, Spirochaetes, Proteobacteria, Actinobacteria and Fibrobacteres are the most abundant bacterial phyla. Likewise, Comtet-Marre et al.  found that bacteria represented 77.5% of rumen ssu rRNA reads with Firmicutes, Bacteroidetes, Fibrobacteres, Proteobacteria, Spirochaetae and Lentisphaerae as predominant phyla. Although the present study found a similar core microbiome, there was variation in the abundance of taxonomic families as compared to previous literature [28, 29]. This variation may be attributed to the shorter incubation time (48 h), and absence of host effects in closed in vitro batch culture system as compared to in vivo animal-based studies. Shorter incubation times favor rapidly growing bacteria over the slower growing fungal and protozoal members of ruminal microbial communities. In contrast to the host environment where fermentation by-products like VFA and ammonia are absorbed across the intestinal epithelium, closed batch culture systems accumulate by-products, which can alter the fermentation process towards certain types of microbial communities. Furthermore, host genetics have also been reported to influence microbial populations , as a region on chromosome 6 in cattle has recently been shown to be associated with Actinobacteria, Euryarchaeota and Fibrobacteres densities . While whole animal experiments can not specifically study the digestion of TTIR, in vitro batch cultures provided us with the level of control over experimental variables required to investigate those factors that limit plant cell wall digestion.
Gene expression analyses of rumen microbial communities cultured on barley straw and TTIR demonstrated substrate-specific modes of digestion, as evidenced by the expression levels of CAZy family transcripts. At the family level, higher expression of putative endo-glucanases GH5, GH9, and GH8 on TTIR indicated an abundance of metabolizable cellulose within TTIR, as confirmed in the linkage analyses. Furthermore, GH48 cellobiohydrolases were the dominant exo-glucanases expressed during the digestion of TTIR by rumen microbes. In contrast, GH6 and GH48 were the predominant families associated with the microbial digestion of barley straw (Additional File 6B). Both GH6 and GH48 are known to efficiently hydrolyze the crystalline regions of cellulose [28, 32]; while cellobiohydrolases of family GH48 act on the reducing ends of cellulose chains, and GH6 attack non-reducing ends, generating cellobiose . The high expression levels of these exo-glucanases suggest there is a greater abundance of amorphous cellulose at the reducing ends of micro-fibrils in TTIR as compared to barley straw, as result of its exposure to CAZymes during passage through the ruminant digestive tract. The level of expression of GH43 hemicellulases in both TTIR and barley straw was higher than other hemicellulose-targeting activities (Additional File 6E). Among the 501 bacteria in the Hungate catalogue, the GH43 family was reported as the most abundant hemicellulase . Metagenomic [35–37] and metatransciptomic [28, 29, 38–40] studies have suggested that members of the GH43 family are the principal hemicellulases within the rumen. GH43 encode a range of debranching enzymes, including arabinofuranosidases, xylanases, galactanases, arabinanases and β-xylosidases, which aid in the degradation of arabinoxylan . Indeed, glycomics revealed that arabinoxylan was enriched in TTIR, highlighting its resistance to ruminal digestion. Additionally, abundance of branched linkages like 3,4-Xylp, t-GlcAp, 2,3,4-Xylp, t-Araf in TTIR also confirm high resistance of heteroxylan to degradation by rumen microbiota (Fig. 2). Metatranscriptomic analyses reflected the efforts of the rumen microbiota to degrade this polysaccharide, with the GH10 family being the dominant family of xylanases (Additional File 6D). This family has been previously reported to digest heteroxylan and release xylotriose with a methylglucuronic acid (MeGA) moiety at the non reducing end which is hydrolyzed by members of the GH5 family of xylanases . The GH5 xylanases have been shown to be active against glucuronoxylan and MeGA substituents on the xylan backbone . Pectin polysaccharides differed between barley straw and TTIR (Figs. 1 and 2). Accordingly, expression levels of pectin-targeting CAZy families differed between rumen microbiota incubated with TTIR vs barley straw (Additional File 6F). Extracted rumen microbial communities cultured on barley straw expressed more GH78s, a family that contains α-l-rhamnosidases that act on rhamnogalacturonan and arabinogalactan-protein linkages . In contrast, with TTIR there was a higher expression of GH28, a family that encodes polygalacturonases and rhamnogalacturonases that hydrolyse the α-1,4 galacturonate linkage in HG and RG-I respectively; and GH53s, which encode for β-1,4-galactanases .
Metatranscriptomics of barley straw and TTIR cultures provided a broad picture of the enzymatic potential of the rumen in terms of CAZy families. However, to gain more insight into the enzymatic processes of ruminal digestion, differential expression analyses, a more nuanced approach with in-depth analyses was used. Based on differential expression, several GH families putatively involved in cellulose, hemicellulose, and pectin digestion were identified (Fig. 5). Informed by glycomics, the relative contribution of dominant DE transcripts was determined for candidate CAZyme families with a focus on those activities important for digestion of recalcitrant plant cell walls (Fig. 6). Since candidate transcripts were expressed by the microbial community in the presence of TTIR, it is highly likely that these transcripts coded for enzymes targeted at inaccessible or non-hydrolyzable linkages within recalcitrant polymers of TTIR. As identified transcripts represent uncharacterized microbial genes, phylogenetic analysis using SACCHARIS was employed in an attempt to postulate enzyme function and specificity for DE transcripts from the selected families GH5, GH11, GH43, and CE15 important for XG, HX, and AB saccharification. No significant difference was seen between barley straw and TTIR in the relative contribution of GH5 and GH9 family DE transcripts (Fig. 6), suggesting that rumen microbes may lack specialized xyloglucanases for the utilization of XG, despite its abundance within TTIR. Thus, xyloglucanases may represent a rate-limiting enzyme activity in the rumen, and exogenous supplementation of xyloglucanases may promote straw digestion within this environment. Although no xyloglucanases were identified, substrate-specific gene expression targeted towards HX and AB digestion was evident from phylogenetic trees of GH11, GH43 and CE15 DE transcripts, whereby the expression of specialized xylanases and arabinanases were induced within the rumen microbial community for the digestion of TTIR. Interestingly, GH11 transcripts that were overexpressed in TTIR cultures clustered distantly from the barley straw overexpressed GH11 transcripts, and some of the early diverging sequences, are GH11-like, and may represent members of a new family (Fig. 7A). The divergence of TTIR-overexpressed transcripts also suggests that changes in expression levels is a result of changes of the predominant bacterial species, differing between TTIR and barley straw microbes for utilizing recalcitrant HX. Furthermore, the TTIR-overexpressed GH11 transcript TR83509|c0_g1 was observed to contain an unique arrangement with four tandem GH11-like domains (Fig. 7A), possibly the first report of a novel repetitive xylanase architecture. The phylogenetic analyses of the overexpressed GH43 transcripts revealed that both TTIR (TR712918|c0_g1) and barley straw (TR941931|c2_g1 and TR300921|c2_g2) clustered together with endo-α-1,5-l-arabinanases (Fig. 7B), suggesting differential regulation of arabinanase activity in rumen microbiota cultured on barley straw vs TTIR. The recalcitrance of arabinan to microbial digestion was also reflected by linkage analysis and glycome profiling (Figs. 1 and 2; Table 1). Transcripts TR400300|c0_g2 and TR451123|c0_g1 were overexpressed in incubations with barley straw and clustered with arabinofuranosidases, refective of the cross-linked nature of HX in barley straw (Fig. 7B). Likewise, the three overexpressed CE15 transcripts with TTIR were embedded among acetyl xylan esterase activity, further reflecting the recalcitrant nature of HX. The results shown in this study also identified 5-linked Araf within arabinan, 3- linked Araf within arabinoxylan and Xyl-3AC and Xyl-2AC within acetyl-xylan as linkages that resisted microbial digestion (Fig. 7). These results suggest that rumen microorganisms are quite efficient in utilizing loosely bound pectin and hemicellulose, but are limited in their ability to digest cross-linked core pectin and hemicellulose.
Previous metatranscriptomic studies have been mostly limited to GH family levels with limited or absent information regarding substrate specificity of identified transcripts [28, 29, 38–40]. In this study, transcript-level SACCHARIS analyses in combination with glycome profiling and linkage analysis suggested that substrate-specific CAZyme transcript expression is inducible. The TTIR-overexpressed transcripts and putative activities may identify those CAZymes with the greatest potential to improve the rate and extent of recalcitrant plant cell wall residues by the rumen microbial consortia. Identified target CAZymes of rumen origin are likely to stand a better chance of success when applied as additive enzymes as they are optimized for the complex physiological conditions within the rumen. Future recombinant production and characterization of these candidate CAZymes and their screening is needed to ascertain their synergy to the natural compliment of CAZymes produced by rumen microbiota. In addition, the barley straw overexpressed transcripts and their closely clustered CAZymes signifies important enzyme activities for effective barley straw digestion and hold high intellectual value for their wide application to industrial sectors such as bioenergy, and green chemistry. Here we have shown that integrating high-resolution analytical methods, such as cell wall structural characterization, in vitro batch culture, RNA-sequencing and DE analysis (Fig. 8), can be successfully used to identify recalcitrant polymers and resistant linkages within feeds, as well as inform the biological processes involved in ruminal digestion of polysaccharides.