Isolation, identification, degradation mechanism and exploration of active enzymes in the ochratoxin A degrading strain Acinetobacter pittii AP19.

Ochratoxin A (OTA) is a polyketide mycotoxin that commonly contaminates agricultural products and causes significant economic losses. In this study, the efficient OTA-degrading strain AP19 was isolated from vineyard soil and was identified as Acinetobacter pittii. Compared with growth in nutrient broth supplemented with OTA (OTA-NB), strain AP19 grew faster in nutrient broth (NB), but the ability of the resulting cell lysates to remove OTA was weaker. After cultivation in NB, the cell lysate of strain AP19 was able to remove 100% of 1 mg/L OTA within 18 h. The cell lysate fraction > 30 kDa degraded 100% of OTA within 12 h, while the fractions < 30 kDa were practically unable to degrade OTA. Further anion exchange chromatography of the > 30 kDa fraction yielded two peaks exhibiting significant OTA degradation activity. The degradation product was identified as OTα. Amino acid metabolism exhibited major transcriptional trends in the response of AP19 to OTA. The dacC gene encoding carboxypeptidase was identified as one of the contributors to OTA degradation. Soil samples inoculated with strain AP19 showed significant OTA degradation. These results provide significant insights into the discovery of novel functions in A. pittii, as well as its potential as an OTA decomposer.

LaeA regulates morphological development and ochratoxin A biosynthesis in Aspergillus niger.

The global regulator LaeA and its orthologs govern the morphogenetic development and secondary metabolism of several filamentous ascomycetes. In Aspergillus niger, it has been shown that an LaeA ortholog (AnLaeA) regulates the production of citric acid and secondary metabolites. In this work, we constructed AnlaeA disruption and overexpression strains to investigate the roles of AnLaeA in morphological development and ochratoxin A (OTA) biosynthesis in A. niger. Phenotypic observation, chemical analysis, and gene expression analysis indicated that AnLaeA acts as a negative regulator of conidial morphogenesis and positively regulates gene expression of the OTA cluster in A. niger grown in CYA medium. However, it was observed that the upregulation of gene expression of the OTA cluster does not necessarily increase OTA production. Our results contribute to a better understanding of the AnlaeA regulatory mechanism and suggest the AnlaeA gene as a potential target for developing control strategies for A. niger infection and OTA biosynthesis.

Deletion and Overexpression of the AnOTAbzip Gene, a Positive Regulator of Ochratoxin A Biosynthesis in Aspergillus niger.

The ochratoxin A (OTA) biosynthetic gene cluster includes a bZIP transcription factor (TF) gene (OTAbzip) that has been identified in different fungal species. However, most previous studies identified the OTAbzip gene in ochratoxigenic fungi using bioinformatics methods, while few studies focused on deleting the gene, let alone overexpressing it, to characterize the function of the OTAbZIP TF. Here, we characterized the AnOTAbZIP TF in an ochratoxigenic isolate of Aspergillus niger by deleting and overexpressing the AnOTAbzip gene and examining the role of AnOTAbZIP in morphological development, OTA biosynthesis, and pathogenicity. Chemical and gene expression analyses revealed that AnOTAbZIP positively regulates OTA biosynthesis, since the loss of OTA production and the downregulation of the OTA biosynthetic genes were observed in the ΔAnOTAbzip strain, compared with the wild-type (WT) and OE::AnOTAbzip strains. In terms of pathogenicity, the ΔAnOTAbzip strain produced a greater lesion on grape berries, especially with respect to the OE::AnOTAbzip strain, rather than WT. Finally, the ΔAnOTAbzip strain was also more tolerant to oxidative stress with respect to the OE::AnOTAbzip and WT strains in that order. These new findings improve our understanding of the AnOTAbZIP regulatory mechanism and help develop strategies to attenuate plant pathogenicity and reduce OTA biosynthesis of A. niger.

Deacetoxycephalosporin C synthase (expandase): Research progress and application potential.

Cephalosporins play an indispensable role against bacterial infections. Deacetyloxycephalosporin C synthase (DAOCS), also called expandase, is a key enzyme in cephalosporin biosynthesis that epoxides penicillin to form the hexavalent thiazide ring of cephalosporin. DAOCS in fungus Acremonium chrysogenum was identified as a bifunctional enzyme with both ring expansion and hydroxylation, whereas two separate enzymes in bacteria catalyze these two reactions. In this review, we briefly summarize its source and function, improvement of the conversion rate of penicillin to deacetyloxycephalosporin C through enzyme modification, crystallography features, the prediction of the active site, and application perspective.

Improving citric acid production of an industrial Aspergillus niger CGMCC 10142: identification and overexpression of a high-affinity glucose transporter with different promoters.

BACKGROUND: Glucose transporters play an important role in the fermentation of citric acid. In this study, a high-affinity glucose transporter (HGT1) was identified and overexpressed in the industrial strain A. niger CGMCC 10142. HGT1-overexpressing strains using the PglaA and Paox1 promoters were constructed to verify the glucose transporter functions. RESULT: As hypothesized, the HGT1-overexpressing strains showed higher citric acid production and lower residual sugar contents. The best-performing strain A. niger 20-15 exhibited a reduction of the total sugar content and residual reducing sugars by 16.5 and 44.7%, while the final citric acid production was significantly increased to 174.1 g/L, representing a 7.3% increase compared to A. niger CGMCC 10142. Measurement of the mRNA expression levels of relevant genes at different time-points during the fermentation indicated that in addition to HGT1, citrate synthase and glucokinase were also expressed at higher levels in the overexpression strains. CONCLUSION: The results indicate that HGT1 overexpression resolved the metabolic bottleneck caused by insufficient sugar transport and thereby improved the sugar utilization rate. This study demonstrates the usefulness of the high-affinity glucose transporter HGT1 for improving the citric acid fermentation process of Aspergillus niger CGMCC 10142.

Enhancement of Acid Protease Activity of Aspergillus oryzae Using Atmospheric and Room Temperature Plasma.

Atmospheric and room temperature plasma (ARTP) system is a novel and efficient mutagenesis protocol for microbial breeding. In this study, ARTP was employed to treat spores of Aspergillus oryzae strain 3.042 for selection of high acid protease producers. With an irradiation time of 150 s at the lethal rate of 90%, 19 mutants with higher acid protease activity were initially selected based on different mutant colony morphology and ratio of the clarification halo of protease activity to the colony diameter. Measurements of the acid protease activity revealed that mutant strain B-2 is characterized by a steady hereditary stability with increased acid protease, neutral protease and total protease activities of 54.7, 17.3, and 8.5%, respectively, and decreased alkaline protease activity of 8.1%. In summary, the identified mutant strain B-2 exhibits great potential for the enhancement of the insufficient acid protease activity during the middle and later stages of soy sauce fermentation.

Efficient Coproduction of Mannanase and Cellulase by the Transformation of a Codon-Optimized Endomannanase Gene from Aspergillus niger into Trichoderma reesei.

Cellulase and mannanase are both important enzyme additives in animal feeds. Expressing the two enzymes simultaneously within one microbial host could potentially lead to cost reductions in the feeding of animals. For this purpose, we codon-optimized the Aspergillus niger Man5A gene to the codon-usage bias of Trichoderma reesei. By comparing the free energies and the local structures of the nucleotide sequences, one optimized sequence was finally selected and transformed into the T. reesei pyridine-auxotrophic strain TU-6. The codon-optimized gene was expressed to a higher level than the original one. Further expressing the codon-optimized gene in a mutated T. reesei strain through fed-batch cultivation resulted in coproduction of cellulase and mannanase up to 1376 U·mL-1 and 1204 U·mL-1, respectively.

Insights into the roles of non-catalytic residues in the active site of a GH10 xylanase with activity on cellulose.

Bifunctional glycoside hydrolases have potential for cost-savings in enzymatic decomposition of plant cell wall polysaccharides for biofuels and bio-based chemicals. The N-terminal GH10 domain of a bifunctional multimodular enzyme CbXyn10C/Cel48B from Caldicellulosiruptor bescii is an enzyme able to degrade xylan and cellulose simultaneously. However, the molecular mechanism underlying its substrate promiscuity has not been elucidated. Herein, we discovered that the binding cleft of CbXyn10C would have at least six sugar-binding subsites by using isothermal titration calorimetry analysis of the inactive E140Q/E248Q mutant with xylo- and cello-oligosaccharides. This was confirmed by determining the catalytic efficiency of the wild-type enzyme on these oligosaccharides. The free form and complex structures of CbXyn10C with xylose- or glucose-configured oligosaccharide ligands were further obtained by crystallographic analysis and molecular modeling and docking. CbXyn10C was found to have a typical (ß/α)8-TIM barrel fold and "salad-bowl" shape of GH10 enzymes. In complex structures with xylo-oligosaccharides, seven sugar-binding subsites were found, and many residues responsible for substrate interactions were identified. Site-directed mutagenesis indicated that 6 and 10 amino acid residues were key residues for xylan and cellulose hydrolysis, respectively. The most important residues are centered on subsites -2 and -1 near the cleavage site, whereas residues playing moderate roles could be located at more distal regions of the binding cleft. Manipulating the residues interacting with substrates in the distal regions directly or indirectly improved the activity of CbXyn10C on xylan and cellulose. Most of the key residues for cellulase activity are conserved across GH10 xylanases. Revisiting randomly selected GH10 enzymes revealed unreported cellulase activity, indicating that the dual function may be a more common phenomenon than has been expected.

Overexpressing key component genes of the secretion pathway for enhanced secretion of an Aspergillus niger glucose oxidase in Trichoderma reesei.

Vast interest exists in developing T. reesei for production of heterologous proteins. Although rich genomic and transcriptomic information has been uncovered for the T. reesei secretion pathway, little is known about whether engineering its key components could enhance expression of a heterologous gene. In this study, snc1, a v-SNARE gene, was first selected for overexpression in T. reesei. In engineered T. reesei with additional copies of snc1, the Aspergillus niger glucose oxidase (AnGOD) was produced to a significantly higher level (2.2-fold of the parental strain). hac1 and bip1, two more component genes in the secretion pathway, were further tested for overexpression and found to be also beneficial for AnGOD secretion. The overexpression of one component gene more or less affected the expression of the other two genes, suggesting a complex regulating mechanism. Our study demonstrates the potential of engineering the secretion pathway for enhancing heterologous gene production in T. reesei.

Revisiting overexpression of a heterologous ß-glucosidase in Trichoderma reesei: fusion expression of the Neosartorya fischeri Bgl3A to cbh1 enhances the overall as well as individual cellulase activities.

BACKGROUND: The filamentous fungus Trichoderma reesei has the capacity to secret large amounts of cellulase and is widely used in a variety of industries. However, the T. reesei cellulase is weak in ß-glucosidase activity, which results in accumulation of cellobiose inhibiting the endo- and exo-cellulases. By expressing an exogenous ß-glucosidase gene, the recombinant T. reesei cellulase is expected to degrade cellulose into glucose more efficiently. RESULTS: The thermophilic ß-glucosidase NfBgl3A from Neosartorya fischeri is chosen for overexpression in T. reesei due to its robust activity. In vitro, the Pichia pastoris-expressed NfBgl3A aided the T. reesei cellulase in releasing much more glucose with significantly lower amounts of cellobiose from crystalline cellulose. The NfBgl3A gene was hence fused to the cbh1 structural gene and assembled between the strong cbh1 promoter and cbh1 terminator to obtain pRS-NfBgl3A by using the DNA assembler method. pRS-NfBgl3A was transformed into the T. reesei uridine auxotroph strain TU-6. Six positive transformants showed ß-glucosidase activities of 2.3-69.7 U/mL (up to 175-fold higher than that of wild-type). The largely different ß-glucosidase activities in the transformants may be ascribed to the gene copy numbers of NfBgl3A or its integration loci. The T. reesei-expressed NfBgl3A showed highly similar biochemical properties to that expressed in P. pastoris. As expected, overexpression of NfBgl3A enhanced the overall cellulase activity of T. reesei. The CBHI activity in all transformants increased, possibly due to the extra copies of cbh1 gene introduced, while the endoglucanase activity in three transformants also largely increased, which was not observed in any other studies overexpressing a ß-glucosidase. NfBgl3A had significant transglycosylation activity, generating sophorose, a potent cellulase inducer, and other oligosaccharides from glucose and cellobiose. CONCLUSIONS: We report herein the successful overexpression of a thermophilic N. fischeri ß-glucosidase in T. reesei. In the same time, the fusion of NfBgl3A to the cbh1 gene introduced extra copies of the cellobiohydrolase 1 gene. As a result, we observed improved ß-glucosidase and cellobiohydrolase activity as well as the overall cellulase activity. In addition, the endoglucanase activity also increased in some of the transformants. Our results may shed light on design of more robust T. reesei cellulases.

Identification of the C-Terminal GH5 Domain from CbCel9B/Man5A as the First Glycoside Hydrolase with Thermal Activation Property from a Multimodular Bifunctional Enzyme.

Caldicellulosiruptor bescii encodes at least six unique multimodular glycoside hydrolases crucial for plant cell wall polysaccharides degradation, with each having two catalytic domains separated by two to three carbohydrate binding modules. Among the six enzymes, three have one N- or C-terminal GH5 domain with identical amino acid sequences. Despite a few reports on some of these multimodular enzymes, little is known about how the conserved GH5 domains behave, which are believed to be important due to the gene duplication. We thus cloned a representative GH5 domain from the C-terminus of a multimodular protein, i.e. the bifunctional cellulase/mannanase CbCel9B/Man5A which has been reported, and expressed it in Escherichia coli. Without any appending CBMs, the recombinant CbMan5A was still able to hydrolyze a variety of mannan substrates with different backbone linkages or side-chain decorations. While CbMan5A displayed the same pH optimum as CbCel9B/Man5A, it had an increased optimal temperature (90°C) and moreover, was activated by heating at 70°C and 80°C, a property not ever reported for the full-length protein. The turnover numbers of CbMan5A on mannan substrates were, however, lower than those of CbCel9B/Man5A. These data suggested that evolution of CbMan5A and the other domains into a single polypeptide is not a simple assembly; rather, the behavior of one module may be affected by the other ones in the full-length enzyme. The differential scanning calorimetry analysis further indicated that heating CbMan5A was not a simple transition state process. To the best knowledge of the authors, CbMan5A is the first glycoside hydrolase with thermal activation property identified from a multimodular bifunctional enzyme.

Implication of a galactomannan-binding GH2 ß-mannosidase in mannan utilization by Caldicellulosiruptor bescii.

Many glycoside hydrolases involved in deconstruction of cellulose and xylan from the excellent plant cell wall polysaccharides-degrader Caldicellulosiruptor bescii have been cloned and analyzed. However, far less is known about the enzymatic breakdown of mannan, an important component of hemicellulose. We herein cloned, expressed and purified the first ß-mannosidase CbMan2A from C. bescii. CbMan2A is thermophilic, with an optimal temperature of 80 °C. CbMan2A hydrolyzes mannooligosaccharides with degrees of polymerization from 2 to 6 mainly into mannose and shows strong synergy with CbMan5A, an endo-mannanase from the same bacterium, in releasing mannose from ß-1,4-mannan. Thus CbMan2A forms the missing link in enzymatic conversion of mannan into the ready-to-use mannose by C. bescii. Based on these observations, a model illustrating how CbMan2A may assist C. bescii in mannan utilization is presented. In addition, CbMan2A appeared to bind to insoluble galactomannan in a pH-dependent fashion. Although the relation of this feature to mannan utilization remains elusive, CbMan2A represents an excellent model for investigation of the binding of GH2 ß-mannosidases to galactomannan.

The N-Terminal GH10 Domain of a Multimodular Protein from Caldicellulosiruptor bescii Is a Versatile Xylanase/ß-Glucanase That Can Degrade Crystalline Cellulose.

The genome of the thermophilic bacterium Caldicellulosiruptor bescii encodes three multimodular enzymes with identical C-terminal domain organizations containing two consecutive CBM3b modules and one glycoside hydrolase (GH) family 48 (GH48) catalytic module. However, the three proteins differ much in their N termini. Among these proteins, CelA (or C. bescii Cel9A [CbCel9A]/Cel48A) with a GH9/CBM3c binary partner in the N terminus has been shown to use a novel strategy to degrade crystalline cellulose, which leads to its outstanding cellulose-cleaving activity. Here we show that C. bescii Xyn10C (CbXyn10C), the N-terminal GH10 domain from CbXyn10C/Cel48B, can also degrade crystalline cellulose, in addition to heterogeneous xylans and barley ß-glucan. The data from substrate competition assays, mutational studies, molecular modeling, and docking point analyses point to the existence of only one catalytic center in the bifunctional xylanase/ß-glucanase. The specific activities of the recombinant CbXyn10C on Avicel and filter paper were comparable to those of GH9/CBM3c of the robust CelA expressed in Escherichia coli. Appending one or two cellulose-binding CBM3bs enhanced the activities of CbXyn10C in degrading crystalline celluloses, which were again comparable to those of the GH9/CBM3c-CBM3b-CBM3b truncation mutant of CelA. Since CbXyn10C/Cel48B and CelA have similar domain organizations and high sequence homology, the endocellulase activity observed in CbXyn10C leads us to speculate that CbXyn10C/Cel48B may use the same strategy that CelA uses to hydrolyze crystalline cellulose, thus helping the excellent crystalline cellulose degrader C. bescii acquire energy from the environment. In addition, we also demonstrate that CbXyn10C may be an interesting candidate enzyme for biotechnology due to its versatility in hydrolyzing multiple substrates with different glycosidic linkages.

A new α-galactosidase from thermoacidophilic Alicyclobacillus sp. A4 with wide acceptor specificity for transglycosylation.

An α-galactosidase gene (gal36A4) of glycosyl hydrolase family 36 was identified in the genome of Alicyclobacillus sp. A4. It contains an ORF of 2,187 bp and encodes a polypeptide of 728 amino acids with a calculated molecular mass of 82.6 kDa. Deduced Gal36A4 shows the typical GH36 organization of three domains--the N-terminal ß-sheets, the catalytic (ß/α)8-barrels, and the C-terminal antiparallel ß-sheet. The gene product was produced in Escherichia coli and showed both hydrolysis and transglycosylation activities. The optimal pH for hydrolysis activity was 6.0, and a stable pH range of 5.0-11.0 was found. The enzyme had a temperature optimum of 60 °C. It is specific for α-1,6-glycosidic linkages and had a K m value of 1.45 mM toward pNPGal. When using melibiose as both donor and acceptor of galactose, Gal36A4 showed the transfer ratio of 23.25 % at 96 h. With respect to acceptor specificity, all tested monosaccharides, disaccharides, and oligosaccharides except for D-xylose and L-arabinose were good acceptors for transglycosylation. Thus, Gal36A4 may find diverse applications in industrial fields, especially in the food industry.

A C-terminal proline-rich sequence simultaneously broadens the optimal temperature and pH ranges and improves the catalytic efficiency of glycosyl hydrolase family 10 ruminal xylanases.

Efficient degradation of plant polysaccharides in rumen requires xylanolytic enzymes with a high catalytic capacity. In this study, a full-length xylanase gene (xynA) was retrieved from the sheep rumen. The deduced XynA sequence contains a putative signal peptide, a catalytic motif of glycoside hydrolase family 10 (GH10), and an extra C-terminal proline-rich sequence without a homolog. To determine its function, both mature XynA and its C terminus-truncated mutant, XynA-Tr, were expressed in Escherichia coli. The C-terminal oligopeptide had significant effects on the function and structure of XynA. Compared with XynA-Tr, XynA exhibited improved specific activity (12-fold) and catalytic efficiency (14-fold), a higher temperature optimum (50°C versus 45°C), and broader ranges of temperature and pH optima (pH 5.0 to 7.5 and 40 to 60°C versus pH 5.5 to 6.5 and 40 to 50°C). Moreover, XynA released more xylose than XynA-Tr when using beech wood xylan and wheat arabinoxylan as the substrate. The underlying mechanisms responsible for these changes were analyzed by substrate binding assay, circular dichroism (CD) spectroscopy, isothermal titration calorimetry (ITC), and xylooligosaccharide hydrolysis. XynA had no ability to bind to any of the tested soluble and insoluble polysaccharides. However, it contained more α helices and had a greater affinity and catalytic efficiency toward xylooligosaccharides, which benefited complete substrate degradation. Similar results were obtained when the C-terminal sequence was fused to another GH10 xylanase from sheep rumen. This study reveals an engineering strategy to improve the catalytic performance of enzymes.

A thermophilic endo-1,4-ß-glucanase from Talaromyces emersonii CBS394.64 with broad substrate specificity and great application potentials.

Thermophilic cellulases are of significant interest to the efficient conversion of plant cell wall polysaccharides into simple sugars. In this study, a thermophilic and thermostable endo-1,4-ß-glucanase, TeEgl5A, was identified in the thermophilic fungus Talaromyces emersonii CBS394.64 and functionally expressed in Pichia pastoris. Purified recombinant TeEgl5A exhibits optimal activity at pH 4.5 and 90 °C. It is highly stable at 70 °C and over a broad pH range of 1.0-10.0, and shows strong resistance to most metal ions, sodium dodecyl sulfate (SDS), and proteases. TeEgl5A has broad substrate specificity and exhibits high activity on substrates containing ß-1,4-glycosidic bonds and ß-1,3-glycosidic bonds (barley ß-glucan, laminarin, lichenan, CMC-Na, carob bean gum, and birchwood xylan). Under simulated mashing conditions, addition of 60 U TeEgl5A reduced more viscosity (10.0 vs.7.6 %) than 80 U of Ultraflo XL from Novozymes. These properties make TeEgl5A a good candidate for extensive application in the detergent, textile, feed, and food industries.

Identification and molecular characterization of an Akirin2 homolog in Chinese loach (Paramisgurnus dabryanus).

Akirin is a nuclear factor involved in innate immune responses of arthropods and mammals. In this study we have cloned an Akirin2 gene, pdakirin2, from freshwater Chinese loach (Paramisgurnus dabryanus) and characterized its biological functions. Phylogenetic analysis revealed deduced PdAkirin2 had high sequence identities to Akirin2 homologs from fish and mammals (70-91%), it contained two conserved nuclear localization signals (NLSs) with verified sub-cellular localization. Quantitative real-time (qRT)-PCR analysis indicated that PdAkirin2 was present in a wide range of loach tissues and showed up-regulation with challenges of Aeromonas hydrophila NJ-1, LPS and poly I:C. PdAkirin2 as an immune factor had significant effects on the expression of cytokines (TNFα, IFN-α, IFN-γ, IL-4 and IL-1ß) and transcription factor NF-κB. This study provides insights into the potential role of PdAkirin2 in the innate immune system.

Identification, expression and bioactivity of Paramisgurnus dabryanus ß-defensin that might be involved in immune defense against bacterial infection.

ß-defensins are a large family of multi-disulfide-bonded peptides with broad-spectrum antimicrobial activities that contribute to innate host defense in many organisms, but little information is available about ß-defensins produced by freshwater fish lacking scales. We therefore cloned and identified a ß-defensin gene from Chinese loach (Paramisgurnus dabryanus) by designing degenerate primers and using thermal asymmetric interlaced PCR. This gene is the first defensin gene ever identified in a non-scaled freshwater fish. Annotation of the protein domain architecture showed that the putative Chinese loach ß-defensin contains the signature motif of six conserved cysteines within the mature peptide, an aspect similar to ß-defensins of other marine fish. We also used quantitative real-time PCR to investigate the expression pattern of the Chinese loach ß-defensin gene, mRNA of which could be observed in various tissues. After challenge with the pathogenic bacterium Aeromonas hydrophila, ß-defensin expression was induced in the eye, gill, skin, and spleen of the adult loach. The bioactivity of the recombinant P. dabryanus ß-defensin was examined against pathogenic bacteria, and the results suggest that this class 2 ß-defensin has potential applications for treatment of bacterial infections.

Comparative quantitative analysis of gene expression profiles of glycoside hydrolase family 10 xylanases in the sheep rumen during a feeding cycle.

Xylanase is a crucial hydrolytic enzyme that degrades plant polysaccharides in the rumen. To date, there is no information on the genetic composition and expression characteristics of ruminal xylanase during feeding cycles of ruminants. Here, the major xylanase of the glycoside hydrolase family 10 (GH 10) from the rumen of small-tail Han sheep was investigated during a feeding cycle. We identified 44 distinct GH 10 xylanase gene fragments at both the genomic and transcriptional levels. Comparison of their relative abundance showed that results from the evaluation of functional genes at the transcriptional level are more reliable indicators for understanding fluctuations in xylanase levels. The expression patterns of six xylanase genes, detected at all time points of the feeding cycle, were investigated; we observed a complex trend of gene expression over 24 h, revealing the dynamic expression of xylanases in the rumen. Further correlation analysis indicated that the rumen is a dynamic ecosystem where the transcript profiles of xylanase genes are closely related to ruminal conditions, especially rumen pH and bacterial population. Given the huge diversity and changing composition of enzymes over the entire rumen, this research provides valuable information for understanding the role of functional genes in the digestion of plant material.