Microbes Environ. Vol. 19, No. 3, 203–210, 2004 http://wwwsoc.nii.ac.jp/jsme2/ Minireview

Fiber Digestion by Rumen

AKIO TAKENAKA1*, KIYOSHI TAJIMA1, MAKOTO MITSUMORI1 and HIROSHI KAJIKAWA1

1 Rumen Microbiology Laboratory, National Institute of Livestock and Grassland Science, Tsukuba, Ibaraki 305–0901, Japan

(Received May 22, 2004—Accepted July 5, 2004)

The study of fibrolytic enzymes of rumen ciliate protozoa has lagged behind that of rumen bacteria and fungi. However, molecular biology has enhanced the investigation of rumen protozoa, and significant information has been accumulated in the last decade. The cellulolytic activities of cell-free extracts from each species of rumen ciliate protozoa have been summarized. Large Ophyroscolecidae such as Epidinium, Polyplastron, and Eudiplo- dinium have higher levels of endoglucanase and xylanase activity. On the other hand, Entodinium spp. have only weak activity to degrade plant fibers. The molecular weight of the main endoglucanase appears to be less than 40k Daltons, and the endoglucanases have the ability to bind cellulose. A total of eight fibrolytic enzyme genes have been cloned from cDNA libraries of Epidinium caudatum and Polyplastron multivesuculatum, and have been registered in GenBank. Four of these 8 clones have high homology with glycoside hydrolase family 5 endo- glucanase (GHF5) genes. The remaining four have homology with xylanase (GHF10 and GHF11) genes. The guanine (G)cytocine (C) content (mol%) of protozoal genes is about 30% and the codon usage is specific and different from that of the endoglucanase or xylanase genes originating from other microorganisms. The codon for Lys in the protozoal ORF is mainly (about 90%) AAA, unlike the Lys codons in rumen bacteria (25%) and the fungal gene (27%). The phylogenetic tree for family 5 endoglucanases shows that the endoglucanases of four are closely related to each other and form a cluster. On the other hand, the xylanases of these ciliates appear more closely related to xylanases from gram-positive bacteria such as Ruminococcus and Clostridium than to other xylanases of eukaryotic origin.

Key words: rumen microorganism, cellulase, xylanase, fibrolytic enzyme gene

Ruminants are herbivorous mammals that possess a spe- Information on the fibrolytic enzymes from rumen ciliate cial organ called the rumen, within which the fermentation protozoa has been particularly limited because it has not of their food occurs. Ruminants are able to digest plant been possible to culture rumen ciliates in vitro in axenic cul- polysaccharides including cellulose and xylan through tures. Despite these limitations, the effect of rumen proto- the activities of specifically adapted rumen zoa on ruminant physiology has been investigated11,21). Pre- microorganisms1,4,23). Rumen microorganisms are briefly liminary work on the role of rumen protozoa has compared classified into three categories, bacteria, fungi, and proto- the metabolism between faunated and defaunated (or unfau- zoa, each of which appears to have the ability to degrade fi- nated; i.e., ciliate-free) ruminants, though there remains bers. Although rumen bacteria and fungi have been well in- some controversy regarding animals and feeding vestigated, rumen protozoa have received little attention. conditions24). The next stage of the research investigated the digestion of fibers by rumen ciliates, using an enzymatic * Corresponding author; E-mail: [email protected], Tel: 81–29– analysis of cell-free extracts acting on purified fiber com- 838–8660, Fax: 81–29–838–8606 pounds or synthesized substrates. Mono-faunated animals 204 TAKENAKA et al.

(which have only one species of rumen ciliate) have occa- sumed to be of importance in the metabolism and nutrition sionally been used as a source of rumen ciliates. Finally, of ruminants9). These protozoa are principally ciliates, and molecular biology has been adapted to investigate the prop- are roughly classified into two types: the Entodiniomor- erties of rumen ciliates not only to establish a systematic phids (Oligotrichs) and the Holotrichs. These rumen ciliate classification using rRNA sequences but also to conduct protozoa infect via another host and only by direct contact. genetic analysis of fiber-degrading enzymes. In this article, Consequently, an unfaunated ruminant, that is, one with no we examine the enzymatic activities found in the cell-free ciliates in the rumen, can be obtained by isolating the calf extract of each species of rumen ciliate protozoa and the from the dam soon after birth. A mono-faunated ruminant, fibrolytic enzyme genes cloned from cDNA libraries of which has only one species of ciliate, can be established by two major fibrolytic ciliates, Epidinium caudatum and inoculating one species of ciliate into an unfaunated animal. Polyplastron multivesiculatum. The rumen ciliate protozoa have been classified on the basis of their morphology14,23), and ciliates could be sorted by size using nylon mesh with a specific lattice size. Fibrolytic en- Fibrolytic enzyme activity of rumen ciliates zyme activities toward the cell-free extract of each species Rumen protozoa were first observed in 1843 and as- of rumen ciliates have been analyzed.

Table 1. Cellulase activity of rumen ciliate protozoaa

Carboxymethylcellulase Production Micro- Protozoan Growth Food Loss of Azure b c of reducing viscosity crystalline cellulase conditions Source material cellulase Diplodinium pentacanthum RND6ND21 Diploplastron affine R31233620 CGW5442ND30 CG 43NDND46 Entodinium bursa CGWND0 ND0 E. caudatum R2NDND8 CGR0 0 ND0 E. furca bilobum CGW2 NDND0 E. longinucleatum CGWNDNDND0 E. simplex CGW0 NDND0 Entodinium spp. R ND 0–6 ND 0–5 Epidinium ecaudatum caudatum R 100 100 100 100 C GW 41 114 ND 47 Eremoplastron bovis R 43 42 122 36 Eudiplodinium maggii R64367923 CGW6 NDND24 C G 76 67 ND 114 Ophryoscolex caudatus R 116 54 80 67 C GW 72 105 ND 85 Polyplastron multivesiculatum CGW5545ND45 C G 90 42 ND 54 Mixed small protozoa R 11 3–4 ND 5–6 Mixed large protozoa R 40 ND ND ND a Data were taken from reference number 2. Results expressed as activity/mg protein relative to that of Epidinium caudatum taken as 100. b R: rumen-grown, C: cultured in vitro. c G: grown on dried grass alone, GW: grown on dried grass and ground wheat, GR: grown on dried grass and rice starch. ND: not determined. Fiber Digestion by Rumen Protozoa 205

Fig. 1. Activity of fibrolytic enzymes from rumen protozoa. Das: Dasytricha ruminantium, Epi: Epidinium caudatum, Poly: Polyplastron multivesiculatum, Ent: Entodinium spp., PNPG: p-nitrophenyl--D-glucoside, PNPC: p-nitrophenyl--D-cellobioside, PNPX: p-nitrophenyl-- D-xyloside.

Coleman2) reported the cellulolytic activities of cell-free endoglucanase and xylanase activity, whereas Dasytricha extracts from many species of rumen ciliates toward car- had no cellulase activity but had strong xylanase activity. boxymethylcellulose (CMC), microcrystalline cellulose, Dasytricha also had relatively high levels of activity against and azure cellulose as substrates (Table 1). The ciliates used p-nitrophenyl--D-glucoside and p-nitrophenyl--D-cello- in that experiment were obtained directly from mono-fau- bioside, though these activities might come from symbiotic nated sheep, or after cultivation in vitro with dried grass or bacteria, given that they decreased after cultivation with an- grass and concentrates. Diploplastron, Epidinium Eudiplo- tibiotics (data not shown). Entodinium spp. had low levels dinium, Ophryoscolex, and Polyplastron had higher levels of of activity against all of the substrates used in this experi- cellulase activity, whereas Entodinium spp. had trace levels. ment (Fig. 1). The gel filtration of the crude cell-free frac- Ciliates grown in vitro on grass alone tended to have more tion revealed that Epidinium and Polyplastron had 3 to 5 enzymatic activity than those grown on starch and grass. peaks of cellulase and xylanase between 15 to 100 k dal- Mixed species of protozoa were also assayed, and the frac- tons, and the lower molecular weight fibrolytic enzyme had tion of large protozoa was four-times more active against cellulose-binding activity18,19). In contrast, gel filtration of CMC than that of small protozoa. the cell extract of Ruminococcus albus showed that cellulas- Ushida and Jouany22) reported gas production from es of this microbe were enormous, a result which supported starch, xylan, and cellulose by rumen ciliates after 24 hours the hypothesis that R. albus13,15) and R. flavefacience7) have culture in vitro with antibiotics from mono-faunated sheep. cellulosomes, a macromolecule for the fiber-digesting com- Polyplastron and Eudiplodinium species could ferment cel- plex, reported in thermophilic cellulolytic bacteria12). lulose and xylan well, whereas there was no gas production In conclusion, based on all of these observations, large from cellulose or xylan by Entodinium or spp.. Ophryoscolecidae have comparatively strong fibrolytic Epidinium, which had strong cellulolytic activity toward activity and might play an important role in the digestion of soluble substrates such as CMC and hydroxyethylcellulose, fiber in the rumen. was less active against crystalline cellulose and xylan. Our group also analyzed the enzymatic activities of 4 ru- men ciliate species isolated from mono-faunated calves18) (Fig. 1). Epidinium and Polyplastron had higher livels of 206 TAKENAKA et al.

been registered, including 8 fibrolytic enzyme genes Molecular cloning of fibrolytic enzyme genes from isolated from cDNA libraries of two rumen ciliate protozoa cDNA of rumen ciliates species, Epidinium caudatum17) and Polyplastron Molecular biology is a valuable tool for the investigation multivesiculatum5,6,20) (Table 2). Since the 1990s, fiber-de- of unculturable microorganisms, and has been used in at- grading enzyme genes have been aggressively cloned from tempts to examine rumen ciliate protozoa. 18S ribosomal many kinds of microbes including rumen microbes. A clas- DNA sequences registered in the DDBJ/EMBL/GenBank sification for glycoside hydrolases based on amino acid se- databases have been used for the phylogenetic classification quence similarities has been developed10). There is a direct of rumen ciliates25,26,27). Some functional genes have also relationship between the sequence and the three-dimension-

Table 2. Fibrolytic enzyme genes isolated from cDNA libraries of the rumen ciliate protozoa registered in GenBank

Size of gene Species of protozoa Name of gene GC% a b Accession No. References (kb) Substrate GHF Epidinium caudatum epi2 1.64 30.1 CMC 5 AB104616 epi3 1.25 31.0 CMC 5 AB011273 16 celA 2.03 30.5 MUC 5 AB104617 xynA 1.73 33.0 AZCL-xylan 10 AB104618 Polyplastron multivesiculatum celA 2.64 31.1 MUC 5 AB104619 polyX 0.77 34.1 AZCL-xylan 11 AB011274 xynA 0.77 33.6 oat spelts xylan 11 AJ009528 4 xynB 1.76 32.2 oat spelts xylan 10 AJ252150 5 a Substrates for screening: CMC: Carboxymethyl cellulose, MUC: 4-metylumbelliferyl--D-cellobioside, AZCL-xylan: azurine-crosslinked xylan b Glycosyl Hydrolase Family Number

Fig. 2. The estimated structures of fibrolytic enzyme genes isolated from rumen protozoa. Fiber Digestion by Rumen Protozoa 207

Fig. 3 Substrate specificity of cellulases produced by the recombinant cloned from rumen ciliate cDNA libraries. Enzymatic activity is measured as the amount of reducing sugar liberated, and the values are relative to CMC activity as 100%. CMC: Carboxymethyl cellulose, Xylan: Xylan from ort spelts, -Glucan: -Glucan from barley, N.D.: Not detectable (0.0005 units/mg protein or less).

Fig. 4. Phylogenetic tree for family 5 endoglucanases. Numbers mean the frequency within 1000 bootstraps, and the length of the line indicates genetic distance. The accession number of each gene is indicated in parentheses. The scale bar indicates the percentage of amino acid substi- tutions. ( : originated from rumen protozoa, : originated from rumen fungi, : originated from rumen bacteria.) 208 TAKENAKA et al.

Fig. 5. Phylogenetic tree for family 10 and 11 xylanases. Numbers mean the frequency within 1000 bootstraps, and the length of the line indi- cates genetic distance. The accession number of each gene is indicated in parentheses. The scale bar indicates the percentage of amino acid substitutions. ( : originated from rumen protozoa, : originated from rumen fungi, : originated from rumen bacteria.)

al structure3). Recently, glycoside hydrolases were classified Properties of the fibrolytic enzyme gene from into 93 families, some of which are grouped into 14 clans rumen ciliates that represent larger families (http://afmb.cnrs-mrs.fr/ CAZY/GH.html). Four cellulase genes were classified as Codon usage in the fibrolytic enzyme genes of rumen cil- family 5 endoglucanase genes based on an analysis of ho- iate protozoa is presented in the universal codon usage mology, and xylanase genes were classified as family 10 or table8). The characteristics of protozoal codon usage were family 11 xylanase genes. The structures of these genes are classified into three groups. The first group is specific for indicated in Fig. 2. Two genes cloned as exoglucanase ciliates such as Lys, Tyr, His, and Asn. Codon usage of this genes using 4-methylumbelliferyl--D-cellobioside as a group is such that the 3rd position tends to be A or T instead substrate have two domains of the family 5 endoglucanases. of C or G. More than 80% of the codons for Lys are AAA, The substrate specificities of the protein (CelA) expressed those for Tyr are TAT, those for His are CAT, and those for by these genes are different from those of endoglucanases Asn are AAT in fibrolytic genes from rumen protozoa16). produced from epi2 or epi3, which have one domain of fam- The second group is similar to fungi and contains the ily 5 endoglucanases (Fig. 3). We are now investigating codons for Gln, Glu, Asp, and Ser. The substitutions of whether this specific structure affects the substrate specific- CAA for Gln, GAA for Glu, and GAT for Asp represented ity of the enzyme. Two family 10 xylanase genes have a the majority of codon usage in fibrolytic enzyme genes of carbohydrate-binding module. rumen protozoa and fungi. Protozoal codon usage of Ser is also similar to that of fungi, because AGT is more frequent Fiber Digestion by Rumen Protozoa 209 in ciliates and fungi than in other microorganisms. The last V. Aurilia, Y. Shoham, E.A. Bayer and H.J. Flint. 2001. Cellulo- group is Cys, in that codon usage for Cys is similar to that of somal Scaffoldin-like Proteins from Ruminococcus flavefaciens. J. Bacteriol. 183: 1945–1953. Ruminococcus spp., Butyrivibrio spp., and E. coli. These 8) Eschenlauer, S.C.P., N.R. McEwan, R.E. Calza, R.J. Wallace, R. properties of codon usage indicate that fibrolytic enzyme Onodera and C.J. Newbold. 1998. Phylogenetic position and genes cloned from cDNA libraries originated from rumen codon usage of two centrin genes from the rumen ciliate protozo- ciliate protozoa16). an. FEMS Microbiol. Lett. 166: 147–154. 9) Gruby, D. and O. Delafond. 1843. 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