Comparative analysis of functional diversity of rumen microbiome in bison and beef heifers.

IMPORTANCE: Ruminants play a key role in the conversion of cellulolytic plant material into high-quality meat and milk protein for humans. The rumen microbiome is the driver of this conversion, yet there is little information on how gene expression within the microbiome impacts the efficiency of this conversion process. The current study investigates gene expression in the rumen microbiome of beef heifers and bison and how transplantation of ruminal contents from bison to heifers alters gene expression. Understanding interactions between the host and the rumen microbiome is the key to developing informed approaches to rumen programming that will enhance production efficiency in ruminants.

Analysis of Complex Carbohydrate Composition in Plant Cell Wall Using Fourier Transform Mid-Infrared Spectroscopy.

Fourier transform mid-infrared spectroscopy (FTIR) is a powerful tool for compositional analysis of plant cell walls. The infrared spectrum generates a fingerprint of a sample with absorption peaks corresponding to the frequency of vibrations between the bonds of the atoms making up the material. Here we describe a method focused on the use of FTIR in combination with principal component analysis (PCA) to characterize the composition of the plant cell wall. The FTIR method described here facilitates high-throughput identification of the major compositional differences across a large set of samples in a low-cost and non-destructive manner.

Mechanistic insights into the digestion of complex dietary fibre by the rumen microbiota using combinatorial high-resolution glycomics and transcriptomic analyses.

There is a knowledge gap regarding the factors that impede the ruminal digestion of plant cell walls or if rumen microbiota possess the functional activities to overcome these constraints. Innovative experimental methods were adopted to provide a high-resolution understanding of plant cell wall chemistries, identify higher-order structures that resist microbial digestion, and determine how they interact with the functional activities of the rumen microbiota. We characterized the total tract indigestible residue (TTIR) from cattle fed a low-quality straw diet using two comparative glycomic approaches: ELISA-based glycome profiling and total cell wall glycosidic linkage analysis. We successfully detected numerous and diverse cell wall glycan epitopes in barley straw (BS) and TTIR and determined their relative abundance pre- and post-total tract digestion. Of these, xyloglucans and heteroxylans were of higher abundance in TTIR. To determine if the rumen microbiota can further saccharify the residual plant polysaccharides within TTIR, rumen microbiota from cattle fed a diet containing BS were incubated with BS and TTIR ex vivo in batch cultures. Transcripts coding for carbohydrate-active enzymes (CAZymes) were identified and characterized for their contribution to cell wall digestion based on glycomic analyses, comparative gene expression profiles, and associated CAZyme families. High-resolution phylogenetic fingerprinting of these sequences encoded CAZymes with activities predicted to cleave the primary linkages within heteroxylan and arabinan. This experimental platform provides unprecedented precision in the understanding of forage structure and digestibility, which can be extended to other feed-host systems and inform next-generation solutions to improve the performance of ruminants fed low-quality forages.

New recombinant fibrolytic enzymes for improved in vitro ruminal fiber degradability of barley straw1.

This study used a high-throughput in vitro microassay, in vitro batch culture, and the Rumen Simulation Technique (RUSITEC) to screen recombinant fibrolytic enzymes for their ability to increase the ruminal fiber degradability of barley straw. Eleven different recombinant enzymes in combination with a crude mixture of rumen enzymes (50% recombinant enzyme:50% crude mixture of rumen enzymes) were compared with the crude mixture of rumen enzymes alone. In the microassay, all treatments were applied at 15 mg of protein load per gram barley straw glucan. Based on the microassay results, 1 recombinant endoglucanase [EGL7A, from the glycoside hydrolase (GH) family 7], 2 recombinant xylanases (XYL10A and XYL10C, from GH10), and a recombinant enzyme mixture were selected and compared with a crude mixture of fibrolytic enzymes from Aspergillus aculeatus for their ability to hydrolyze barley straw. For batch culture, enzymes were applied to barley straw at 2 dosages (100 and 500 µg of protein/g of substrate DM). All enzymes increased (P < 0.05) DM disappearance and total VFA production, but the mixture of recombinant enzymes was not superior to the use of a single recombinant enzyme. Based on positive results (P < 0.05) for total DM disappearance and VFA production in batch culture, 3 enzymes (EGL7A, XYL10A, and XYL10C) were selected and applied to barley straw at 500 µg of protein per gram for further assessment in RUSITECs fed a concentrate:barley straw diet (300:700 g/kg DM). In RUSITECs, the recombinant enzyme XYL10A increased (P < 0.05) barley straw DM, NDF, and ADF disappearance, whereas EGL7A and XYL10C had no effect. The enzymes selected based on the high-throughput in vitro microassay consistently increased barley straw degradation in ruminal batch culture, but not in the semicontinuous culture RUSITEC system.

Saccharification efficiencies of multi-enzyme complexes produced by aerobic fungi.

In the present study, we have characterized high molecular weight multi-enzyme complexes in two commercial enzymes produced by Trichoderma reesei (Spezyme CP) and Penicillium funiculosum (Accellerase XC). We successfully identified 146-1000 kDa complexes using Blue native polyacrylamide gel electrophoresis (BN-PAGE) to fractionate the protein profile in both preparations. Identified complexes dissociated into lower molecular weight constituents when loaded on SDS PAGE. Unfolding of the secondary structure of multi-enzyme complexes with trimethylamine (pH >10) suggested that they were not a result of unspecific protein aggregation. Cellulase (CMCase) profiles of extracts of BN-PAGE fractionated protein bands confirmed cellulase activity within the multi-enzyme complexes. A microassay was used to identify protein bands that promoted high levels of glucose release from barley straw. Those with high saccharification yield were subjected to LC-MS analysis to identify the principal enzymatic activities responsible. The results suggest that secretion of proteins by aerobic fungi leads to the formation of high molecular weight multi-enzyme complexes that display activity against carboxymethyl cellulose and barley straw.

Identification of novel enzymes to enhance the ruminal digestion of barley straw.

Crude enzyme extracts typically contain a broad spectrum of enzyme activities, most of which are redundant to those naturally produced by the rumen microbiome. Identification of enzyme activities that are synergistic to those produced by the rumen microbiome could enable formulation of enzyme cocktails that improve fiber digestion in ruminants. Compared to untreated barley straw, Viscozyme® increased gas production, dry matter digestion (P < 0.01) and volatile fatty acid production (P < 0.001) in ruminal batch cultures. Fractionation of Viscozyme® by Blue Native PAGE and analyses using a microassay and mass-spectrometry revealed a GH74 endoglucanase, GH71 α-1,3-glucanase, GH5 mannanase, GH7 cellobiohydrolase, GH28 pectinase, and esterases from Viscozyme® contributed to enhanced saccharification of barley straw by rumen mix enzymes. Grouping of these identified activities with their carbohydrate active enzymes (CAZy) counterparts enabled selection of similar CAZymes for downstream production and screening. Mining of these specific activities from other biological systems could lead to high value enzyme formulations for ruminants.

Analysis of Complex Carbohydrate Composition in Plant Cell Wall Using Fourier Transformed Mid-Infrared Spectroscopy (FT-IR).

Fourier transformed mid-infrared spectroscopy (FTIR) is a powerful tool for compositional analysis of plant cell walls (Acebes et al., Front Plant Sci 5:303, 2014; Badhan et al., Biotechnol Biofuels 7:1-15, 2014; Badhan et al., BioMed Res Int 2015: 562952, 2015; Roach et al., Plant Physiol 156:1351-1363, 2011). The infrared spectrum generates a fingerprint of a sample with absorption peaks corresponding to the frequency of vibrations between the bonds of the atoms making up the material. Here, we describe a method focused on the use of FTIR in combination with principal component analysis (PCA) to characterize the composition of the plant cell wall. This method has been successfully used to study complex enzyme saccharification processes like rumen digestion to identify recalcitrant moieties in low-quality forage which resist rumen digestion (Badhan et al., BioMed Res Int 2015: 562952, 2015), as well as to characterize cell wall mutant lines or transgenic lines expressing exogenous hydrolases (Badhan et al., Biotechnol Biofuels 7:1-15, 2014; Roach et al., Plant Physiol 156:1351-1363, 2011). The FTIR method described here facilitates high-throughput identification of the major compositional differences across a large set of samples in a low cost and nondestructive manner.

Improvement in Saccharification Yield of Mixed Rumen Enzymes by Identification of Recalcitrant Cell Wall Constituents Using Enzyme Fingerprinting.

Identification of recalcitrant factors that limit digestion of forages and the development of enzymatic approaches that improve hydrolysis could play a key role in improving the efficiency of meat and milk production in ruminants. Enzyme fingerprinting of barley silage fed to heifers and total tract indigestible fibre residue (TIFR) collected from feces was used to identify cell wall components resistant to total tract digestion. Enzyme fingerprinting results identified acetyl xylan esterases as key to the enhanced ruminal digestion. FTIR analysis also suggested cross-link cell wall polymers as principal components of indigested fiber residues in feces. Based on structural information from enzymatic fingerprinting and FTIR, enzyme pretreatment to enhance glucose yield from barley straw and alfalfa hay upon exposure to mixed rumen-enzymes was developed. Prehydrolysis effects of recombinant fungal fibrolytic hydrolases were analyzed using microassay in combination with statistical experimental design. Recombinant hemicellulases and auxiliary enzymes initiated degradation of plant structural polysaccharides upon application and improved the in vitro saccharification of alfalfa and barley straw by mixed rumen enzymes. The validation results showed that microassay in combination with statistical experimental design can be successfully used to predict effective enzyme pretreatments that can enhance plant cell wall digestion by mixed rumen enzymes.

Formulation of enzyme blends to maximize the hydrolysis of alkaline peroxide pretreated alfalfa hay and barley straw by rumen enzymes and commercial cellulases.

BACKGROUND: Efficient conversion of lignocellulosic biomass to fermentable sugars requires the synergistic action of multiple enzymes; consequently enzyme mixtures must be properly formulated for effective hydrolysis. The nature of an optimal enzyme blends depends on the type of pretreatment employed as well the characteristics of the substrate. In this study, statistical experimental design was used to develop mixtures of recombinant glycosyl hydrolases from thermophilic and anaerobic fungi that enhanced the digestion of alkaline peroxide treated alfalfa hay and barley straw by mixed rumen enzymes as well as commercial cellulases (Accelerase 1500, A1500; Accelerase XC, AXC). RESULTS: Combinations of feruloyl and acetyl xylan esterases (FAE1a; AXE16A_ASPNG), endoglucanase GH7 (EGL7A_THITE) and polygalacturonase (PGA28A_ASPNG) with rumen enzymes improved straw digestion. Inclusion of pectinase (PGA28A_ASPNG), endoxylanase (XYN11A_THITE), feruloyl esterase (FAE1a) and ß-glucosidase (E-BGLUC) with A1500 or endoglucanase GH7 (EGL7A_THITE) and ß-xylosidase (E-BXSRB) with AXC increased glucose release from alfalfa hay. Glucose yield from straw was improved when FAE1a and endoglucanase GH7 (EGL7A_THITE) were added to A1500, while FAE1a and AXE16A_ASPNG enhanced the activity of AXC on straw. Xylose release from alfalfa hay was augmented by supplementing A1500 with E-BGLUC, or AXC with EGL7A_THITE and XYN11A_THITE. Adding arabinofuranosidase (ABF54B_ASPNG) and esterases (AXE16A_ASPNG; AXE16B_ASPNG) to A1500, or FAE1a and AXE16A_ASPNG to AXC enhanced xylose release from barley straw, a response confirmed in a scaled up assay. CONCLUSION: The efficacy of commercial enzyme mixtures as well as mixed enzymes from the rumen was improved through formulation with synergetic recombinant enzymes. This approach reliably identified supplemental enzymes that enhanced sugar release from alkaline pretreated alfalfa hay and barley straw.

Expression of a fungal ferulic acid esterase in alfalfa modifies cell wall digestibility.

BACKGROUND: Alfalfa (Medicago sativa) is an important forage crop in North America owing to its high biomass production, perennial nature and ability to fix nitrogen. Feruloyl esterase (EC 3.1.1.73) hydrolyzes ester linkages in plant cell walls and has the potential to further improve alfalfa as biomass for biofuel production. RESULTS: In this study, faeB [GenBank:AJ309807] was synthesized at GenScript and sub-cloned into a novel pEACH vector containing different signaling peptides to target type B ferulic acid esterase (FAEB) proteins to the apoplast, chloroplast, endoplasmic reticulum and vacuole. Four constructs harboring faeB were transiently expressed in Nicotiana leaves, with FAEB accumulating at high levels in all target sites, except chloroplast. Stable transformed lines of alfalfa were subsequently obtained using Agrobacterium tumefaciens (LBA4404). Out of 136 transgenic plants regenerated, 18 independent lines exhibited FAEB activity. Subsequent in vitro digestibility and Fourier transformed infrared spectroscopy (FTIR) analysis of FAEB-expressing lines showed that they possessed modified cell wall morphology and composition with a reduction in ester linkages and elevated lignin content. Consequently, they were more recalcitrant to digestion by mixed ruminal microorganisms. Interestingly, delignification by alkaline peroxide treatment followed by exposure to a commercial cellulase mixture resulted in higher glucose release from transgenic lines as compared to the control line. CONCLUSION: Modifying cell wall crosslinking has the potential to lower recalcitrance of holocellulose, but also exhibited unintended consequences on alfalfa cell wall digestibility due to elevated lignin content. The combination of efficient delignification treatment (alkaline peroxide) and transgenic esterase activity complement each other towards efficient and effective digestion of transgenic lines.

Development of cellulosic secondary walls in flax fibers requires beta-galactosidase.

Bast (phloem) fibers, tension wood fibers, and other cells with gelatinous-type secondary walls are rich in crystalline cellulose. In developing bast fibers of flax (Linum usitatissimum), a galactan-enriched matrix (Gn-layer) is gradually modified into a mature cellulosic gelatinous-layer (G-layer), which ultimately comprises most of the secondary cell wall. Previous studies have correlated this maturation process with expression of a putative ß-galactosidase. Here, we demonstrate that ß-galactosidase activity is in fact necessary for the dynamic remodeling of polysaccharides that occurs during normal secondary wall development in flax fibers. We found that developing stems of transgenic (LuBGAL-RNAi) flax with reduced ß-galactosidase activity had lower concentrations of free Gal and had significant reductions in the thickness of mature cellulosic G-layers compared with controls. Conversely, Gn-layers, labeled intensively by the galactan-specific LM5 antibody, were greatly expanded in LuBGAL-RNAi transgenic plants. Gross morphology and stem anatomy, including the thickness of bast fiber walls, were otherwise unaffected by silencing of ß-galactosidase transcripts. These results demonstrate a specific requirement for ß-galactosidase in hydrolysis of galactans during formation of cellulosic G-layers. Transgenic lines with reduced ß-galactosidase activity also had biochemical and spectroscopic properties consistent with a reduction in cellulose crystallinity. We further demonstrated that the tensile strength of normal flax stems is dependent on ß-galactosidase-mediated development of the phloem fiber G-layer. Thus, the mechanical strength that typifies flax stems is dependent on a thick, cellulosic G-layer, which itself depends on ß-galactosidase activity within the precursor Gn-layer. These observations demonstrate a novel role for matrix polysaccharides in cellulose deposition; the relevance of these observations to the development of cell walls in other species is also discussed.