The non-catalytic amino acid Asp446 is essential for enzyme activity of the modular endocellulase Cel9 from Myxobacter sp. AL-1

The modular endocellulase Cel9 of the bicistronic operon cel9-cel48 of Myxobacter sp. AL-1 shares not only amino acid sequence similarity but also biochemical properties similar to those of Thermobifida fusca endo/exocellulase E4. Amino acid alignments of a T. fusca E4 cellulase subfamily of family 9 cellulases revealed that Asp(446) of Myxobacter sp. AL-1 Cel9, a putatively noncatalytic residue, is highly conserved in one of the catalytic domains of this subfamily. Directed mutagenesis of residue aspartate (Asp(446)) to alanine generated a Cel9 mutant that lost more than 99% of its activity, suggesting that Asp(446) plays an essential structural role in Cel9 during cellulose degradation. Owing to its high degree of conservation and essential role, we propose that Asp(446) of Myxobacter sp. AL-1 Cel9 is a good landmark that distinguishes members of the E4 subfamily of family 9 cellulases.     

<Emphasis Type="Italic">Thermobifida fusca</Emphasis> exoglucanase Cel6B is incompatible with the cellulosomal mode in contrast to endoglucanase Cel6A

Cellulosomes are efficient cellulose-degradation systems produced by selected anaerobic bacteria. This multi-enzyme complex is assembled from a group of cellulases attached to a protein scaffold termed scaffoldin, mediated by a high-affinity protein–protein interaction between the enzyme-borne dockerin module and the cohesin module of the scaffoldin. The enzymatic complex is attached as a whole to the cellulosic substrate via a cellulose-binding module (CBM) on the scaffoldin subunit. In previous works, we have employed a synthetic biology approach to convert several of the free cellulases of the aerobic bacterium, Thermobifida fusca, into the cellulosomal mode by replacing each of the enzymes’ CBM with a dockerin. Here we show that although family six enzymes are not a part of any known cellulosomal system, the two family six enzymes of the T. fusca system (endoglucanase Cel6A and exoglucanase Cel6B) can be converted to work as cellulosomal enzymes. Indeed, the chimaeric dockerin-containing family six endoglucanase worked well as a cellulosomal enzyme, and proved to be more efficient than the parent enzyme when present in designer cellulosomes. In stark contrast, the chimaeric family six exoglucanase was markedly less efficient than the wild-type enzyme when mixed with other T. fusca cellulases, thus indicating its incompatibility with the cellulosomal mode of action.     

Temporal secretion of a multicellulolytic system in Myxobacter sp. AL-1. Molecular cloning and heterologous expression of cel9 encoding a modular endocellulase clustered in an operon with cel48, an exocellobiohydrolase gene.

The Gram-negative soil micro-organism Myxobacter sp. AL-1 possesses at least five extracellular cellulases, the production of which is regulated by the growth cycle. We cloned the complete gene for one of these cellulases, termed cel9, which encoded a 67-kDa modular family 9 endoglycohydrolase, which was produced during the stationary phase of growth and was strongly enhanced by avicel. The predicted product of cel9 matches the structural architecture of family 9 cellulases such as Thermonospora fusca endo/exocellulase E4. Cel9 protein was synthesized in Escherichia coli from a multicopy plasmid and in Bacillus subtilis from the isopropyl thiogalactoside-inducible Pspac promoter and was purified from the culture medium. Thermal stability, optimum pH and temperature dependence of Cel9 were similar when expressed from either source, and were indistinguishable from related cellulases produced by thermophilic bacteria. Downstream from cel9 was found a partial ORF, designated cel48, the deduced product of which was highly similar to bacterial exocellobiohydrolases and processive endoglucanases belonging to family 48 of the glycosyl hydrolases. The cel9 and cel48 genes appear to be arranged as part of an operon.     

Heterologous co-production of <Emphasis Type="Italic">Thermobifida fusca</Emphasis> Cel9A with other cellulases in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The processive endoglucanase Cel9A of the moderately thermophilic actinomycete Thermobifida fusca was functionally produced in Saccharomyces cerevisiae. Recombinant Cel9A displayed activity on both soluble (carboxymethylcellulose) and insoluble (Avicel) cellulose substrates confirming its processive endoglucanase activity. High-performance anionic exchange chromatography analyses of soluble sugars released from Avicel revealed a cellobiose/glucose ratio of 2.5 ± 0.1. Growth by the recombinant strain on amorphous cellulose was possible due to the sufficient amount of glucose cleaved from the cellulose chain. This is the first confirmed report of S. cerevisiae growing on a cellulosic substrate as sole carbohydrate source while only expressing one recombinant gene. To improve the cellulolytic capability of S. cerevisiae and to investigate the level of synergy among cellulases produced by a recombinant host, the cel9A gene was co-expressed with four cellulase-coding genes of Trichoderma reesei: two endoglucanases cel5A (egII) and cel7B (egI), and two cellobiohydrolases cel6A (cbhII) and cel7A (cbhI). Synergy, especially between the Cel9A and the two cellobiohydrolases, resulted in a higher cellulolytic capability of the recombinant host.     

Molecular Cloning of Novel Cellulase Genes <Emphasis Type="Italic">cel</Emphasis>9A and <Emphasis Type="Italic">cel</Emphasis>12A from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> GXN151 and Synergism of Their Encoded Polypeptides

A thermophilic bacterial strain GXN151 which could degrade Avicel efficiently was isolated and identified as Bacillus licheniformis. A genomic library of GXN151 was constructed and two novel endoglucanase genes designated cel9A and cel12A were isolated by screening the library on carboxylmethyl cellulase indicator plates. The analysis of amino acid sequences deduced from the genes indicated that Cel9A consisted of a catalytic domain belonging to glycosyl hydrolase family 9, a linker domain, and a carbohydrate binding module family 3 from N-terminal to C-terminal; Cel12A had only one catalytic domain belonging to glycosyl hydrolase family 12. The combinations of Cel9A and Cel12A produced by the recombinant E. coli exhibited synergistic action against substrates of carboxylmethyl cellulose as well as Avicel.     

Simultaneous release of recombinant cellulases introduced by coexpressing colicin E7 lysis in <Emphasis Type="Italic">Escherichia coli</Emphasis>

With the increasingly serious global environment problem caused by slather use of fossil fuels, numerous efforts have been made in developing renewable alternatives. Converting lignocellulose to bioenergy has accomplished by cellulases but the production is limited, therefore the recombinant expression platforms in E. coli have been established. Since E. coli is partly restricted by its inability to secrete enzymes to extracellular membrane, we constructed a synthetic circuit to coexpress cellulases and colicin E7 lysis to solve this problem. The pBAD-RBS-lysis-TT (BBa_K117010) sequence from iGEM was added to form a chimeric plasmid based on pET system, which harboring cellulases from Bacillus subtilis or Thermobifida fusca. The presence of arabinose for the promoter pBAD, leading to induce lysis and further breakdown of the host membrane to release cellulase to extracellular membrane. The extracellular activities increase to 48.1 and 55.5% under lysis gene functioned on Cel5 and CD1, respectively. This is the first attempt to show that cellulases have successfully expressed and released to extracellular membrane without cumbersome steps. Finally, this synthetic circuit simplifies and achieves the simultaneous release and heterologous expression of the cellulases as well as obtain a long-term stable enzyme in E. coli system.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C,and <Superscript>15</Superscript>N backbone chemical shift assignments of 4E-BP1<Subscript>44–87</Subscript> and 4E-BP1<Subscript>44–87</Subscript> bound to eIF4E

The eukaryotic translational initiation factor 4G (eIF4G) interacts with the cap-binding protein eIF4E through a consensus binding motif, Y(X)₄LΦ (where X is any amino acid and Φ is a hydrophobic residue). 4E binding proteins (4E-BPs), which also contain a Y(X)₄LΦ motif, regulate the eIF4E/eIF4G interaction. The non- or minimally-phosphorylated form of 4E-BP1 binds eIF4E, preventing eIF4E from interacting with eIF4G, thus inhibiting translation initiation. 4EGI-1, a small molecule inhibitor of the eIF4E/eIF4G interaction that is under investigation as a novel anti-cancer drug, has a dual activity; it disrupts the eIF4E/eIF4G interaction and stabilizes the binding of 4E-BP1 to eIF4E. Here, we report the complete backbone NMR resonance assignment of an unliganded 4E-BP1 fragment (4E-BP1_44–87). We also report the near complete backbone assignment of the same fragment in complex to eIF4E/m⁷GTP (excluding the assignment of the last C-terminus residue, D87). The chemical shift data constitute a prerequisite to understanding the mechanism of action of translation initiation inhibitors, including 4EGI-1, that modulate the eIF4E/4E-BP1 interaction.     

Nucleotide and Deduced Amino Acid Sequences of a New Subtilisin from an Alkaliphilic <Emphasis Type="Italic">Bacillus</Emphasis> Isolate

The gene for a new subtilisin from the alkaliphilic Bacillus sp. KSM-LD1 was cloned and sequenced. The open reading frame of the gene encoded a 97 amino-acid prepro-peptide plus a 307 amino-acid mature enzyme that contained a possible catalytic triad of residues, Asp³², His⁶⁶, and Ser²²⁴. The deduced amino acid sequence of the mature enzyme (LD1) showed approximately 65% identity to those of subtilisins SprC and SprD from alkaliphilic Bacillus sp. LG12. The amino acid sequence identities of LD1 to those of previously reported true subtilisins and high-alkaline proteases were below 60%. LD1 was characteristically stable during incubation with surfactants and chemical oxidants. Interestingly, an oxidizable Met residue is located next to the catalytic Ser²²⁴ of the enzyme as in the cases of the oxidation-susceptible subtilisins reported to date.Received: 19 November 2002 / Accepted: 19 December 2002     

Interisland Evolution of <Emphasis Type="Italic">Trimeresurus flavoviridis</Emphasis> Venom Phospholipase A<Subscript>2</Subscript> Isozymes

Abstract Trimeresurus flavoviridis snakes inhabit the southwestern islands of Japan. A phospholipase A₂ (PLA₂), named PL-Y, was isolated from Okinawa T. flavoviridis venom and its amino acid sequence was determined from both protein and cDNA. PL-Y was unable to induce edema. In contrast, PLA-B, a PLA₂ from Tokunoshima T. flavoviridis venom, which is different at only three positions from PL-Y, is known to induce edema. A new PLA₂, named PLA-B′, which is similar to PLA-B, was cloned from Amami-Oshima T. flavoviridis venom gland. Three T. flavoviridis venom basic [Asp⁴⁹]PLA₂ isozymes, PL-Y (Okinawa), PLA-B (Tokunoshima), and PLA-B′ (Amami-Oshima), are identical in the N-terminal half but have one to four amino acid substitutions in the β1-sheet and its vicinity. Such interisland sequence diversities among them are due to isolation in the different environments over 1 to 2 million years and appear to have been brought about by natural selection for point mutation in their genes. Otherwise, a major PLA₂, named PLA2, ubiquitously exists in the venoms of T. flavoviridis snakes from the three islands with one to three synonymous substitutions in their cDNAs. It is assumed that the PLA2 gene is a prototype among T. flavoviridis venom PLA₂ isozyme genes and has hardly undergone nonsynonymous mutation as a principal toxic component. Phylogenetic analysis based on the amino acid sequences revealed that T. flavoviridis PLA₂ isozymes are clearly separated into three groups, PLA2 type, basic [Asp⁴⁹]PLA₂ type, and [Lys⁴⁹]PLA₂ type. Basic [Asp⁴⁹]PLA₂-type isozymes may manifest their own particular toxic functions different from those of the isozymes of the PLA2 type and [Lys⁴⁹]PLA₂ type.     

Structural elements of the C-terminal domain of subunit E (E<Subscript>133-222</Subscript>) from the <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> V<Subscript>1</Subscript>V<Subscript>O</Subscript> ATPase determined by solution NMR spectroscopy

Subunit E of the vacuolar ATPase (V-ATPase) contains an N-terminal extended α helix (Rishikesan et al. J Bioenerg Biomembr 43:187–193, 2011) and a globular C-terminal part that is predicted to consist of a mixture of α-helices and β-sheets (Grüber et al. Biochem Biophys Res Comm 298:383–391, 2002). Here we describe the production, purification and 2D structure of the C-terminal segment E_133-222 of subunit E from Saccharamyces cerevisiae V-ATPase in solution based on the secondary structure calculation from NMR spectroscopy studies. E_133-222 consists of four β-strands, formed by the amino acids from K136-V139, E170-V173, G186-V189, D195-E198 and two α-helices, composed of the residues from R144-A164 and T202-I218. The sheets and helices are arranged as β1:α1:β2:β3:β4:α2, which are connected by flexible loop regions. These new structural details of subunit E are discussed in the light of the structural arrangements of this subunit inside the V₁- and V₁V_O ATPase.     

Reversibility and binding kinetics of Thermobifida fusca cellulases studied through fluorescence recovery after photobleaching microscopy

Cellulases are enzymes capable of depolymerizing cellulose. Understanding their interactions with cellulose can improve biomass saccharification and enzyme recycling in biofuel production. This paper presents a study on binding and binding reversibility of Thermobifida fusca cellulases Cel5A, Cel6B, and Cel9A bound onto Bacterial Microcrystalline Cellulose. Cellulase binding was assessed through fluorescence recovery after photobleaching (FRAP) at 23, 34, and 45 °C. It was found that cellulase binding is only partially reversible. For processive cellulases Cel6B and Cel9A, an increase in temperature resulted in a decrease of the fraction of cellulases reversibly bound, while for endocellulase Cel5A this fraction remained constant. Kinetic parameters were obtained by fitting the FRAP curves to a binding-dominated model. The unbinding rate constants obtained for all temperatures were highest for Cel5A and lowest for Cel9A. The results presented demonstrate the usefulness of FRAP to access the fast binding kinetics characteristic of cellulases operating at their optimal temperature.     

Protective Effect of Enzymatic Extracts from Microalgae Against DNA Damage Induced by H<Subscript>2</Subscript>O<Subscript>2</Subscript>

The enzymatic extracts from seven species of microalgae (Pediastrum duplex, Dactylococcopsis fascicularis, Halochlorococcum porphyrae, Oltmannsiellopsis unicellularis, Achnanthes longipes, Navicula sp. and Amphora coffeaeformis) collected from three habitats (freshwater, tidal pool, and coastal benthic) at Jeju Island in Korea were investigated for their antioxidant activity. Of the extracts tested, the AMG 300 L (an exo 1, 4-α-d-glucosidase) extract of P. duplex, the Viscozyme extract of Navicula sp., and the Celluclast extract of A. longipes provided the most potential as antioxidants. Meanwhile, the Termamyl extract of P. duplex in an H₂O₂ scavenging assay exhibited an approximate 60% scavenging effect. In this study, we report that the DNA damage inhibitory effects of P. duplex (Termamyl extract) and D. fascicularis (Kojizyme extract) were nearly 80% and 69% respectively at a concentration of 100 μg/ml. Thus, it is suggested that the microalgae tested in this study yield promising DNA damage inhibitory properties on mouse lymphoma L 5178 cells that are treated with H₂O₂. Therefore, microalgae such as P. duplex may be an excellent source of naturally occurring antioxidant compounds with potent DNA damage inhibition potential.     

Effect of Prostaglandins E<Subscript>1</Subscript> and F<Subscript>1α</Subscript> on Biosynthesis of Collagen

THE prostaglandins (PG) are possible mediators of inflammation. Prostaglandins E and F are present in inflammatory exudates^1–3 and could be related to the increase of collagen biosynthesis associated with inflammation. Vane and his colleagues^4–6 recently observed that indomethacin, aspirin and sodium salicylate potently block the biosynthesis of prostaglandins. These anti-inflammatory drugs are also inhibitors of collagen biosynthesis^7,8. Morphological studies⁹ have revealed increased deposition of collagen or collagen-related elements in organ cultures of chick embryo skin containing prostaglandins E₁ and B₁. We report here results which indicate stimulation of collagen biosynthesis by prostaglandins E₁ and F_1α evaluated by hydroxylation of proline and lysine and glycosylation of hydroxylysine in 10 day chick embryo tibiae.     

The <Emphasis Type="Italic">b</Emphasis><Subscript>arg36</Subscript> contributes to efficient coupling in F<Subscript>1</Subscript>F<Subscript>O</Subscript> ATP synthase in <Emphasis Type="Italic">Escherichia coli</Emphasis>

In Escherichia coli, the F₁F_O ATP synthase b subunits house a conserved arginine in the tether domain at position 36 where the subunit emerges from the membrane. Previous experiments showed that substitution of isoleucine or glutamate result in a loss of enzyme activity. Double mutants have been constructed in an attempt to achieve an intragenic suppressor of the b _arg36→ile and the b _arg36→glu mutations. The b _arg36→ile mutation could not be suppressed. In contrast, the phenotypic defect resulting from the b _arg36→glu mutation was largely suppressed in the b _{arg36→glu,glu39→arg} double mutant. E. coli expressing the b _{arg36→glu,glu39→arg} subunit grew well on succinate-based medium. F₁F_O ATP synthase complexes were more efficiently assembled and ATP driven proton pumping activity was improved. The evidence suggests that efficient coupling in F₁F_O ATP synthase is dependent upon a basic amino acid located at the base of the peripheral stalk.     

Synergistic activity of Paenibacillus sp. BP-23 cellobiohydrolase Cel48C in association with the contiguous endoglucanase Cel9B and with endo- or exo-acting glucanases from Thermobifida fusca

Cellobiohydrolase Cel48C from Paenibacillus sp. BP-23, an enzyme displaying limited activity on most cellulosic substrates, was assayed for activity in the presence of other bacterial endo- or exocellulases. Significant enhanced activity was observed when Cel48C was incubated in the presence of Paenibacillus sp. BP-23 endoglucanase Cel9B or Thermobifida fusca cellulases Cel6A and Cel6B, indicating that Cel48C acts synergistically with them. Maximum synergism rates on bacterial microcrystalline cellulose or filter paper were obtained with a mixture of Paenibacillus cellulases Cel9B and Cel48C, accompanied by T. fusca exocellulase Cel6B. Synergism was also observed in cell extracts from recombinant clone E. coli pUCel9-Cel48 expressing the two contiguous Paenibacillus cellulases Cel9B and Cel48C. The enhanced cellulolytic activity displayed by the cellulase mixtures assayed could be used as an efficient tool for biotechnological applications like pulp and paper manufacturing.     

Proteomic identification of CBM37-containing cellulases produced by the rumen cellulolytic bacterium <Emphasis Type="Italic">Ruminococcus albus</Emphasis> 20 and their putative involvement in bacterial adhesion to cellulose

The objective of this study was to identify and characterize other proteins than fimbrial proteins potentially involved in R. albus 20 adhesion to cellulose using an adhesion-related antiserum preparation (i.e. anti-Adh serum). From protein fractions of R. albus 20 grown on cellulose, the serum recognized at least 10 cellulose-binding proteins (CBPs), among which homologs of glycoside hydrolases (family 5, 9 and 48) of R. albus 8 (i.e. Cel5G, Cel9B and Cel48A) were identified by a proteomic approach. In strain 20, Cel9B and Cel48A were identified as two major CBPs and as bacterial cell-associated proteins. The anti-Adh serum was also shown to target the C-terminal family 37 carbohydrate-binding module (CBM37) of Cel9B and Cel48A, indicating that this module, unique to R. albus, may play a significant role in bacterial adhesion to cellulose as suggested previously for R. albus 8. Overall, our results support the hypothesis of an adhesion mechanism involving the CBM37 of Cel9B and Cel48A. This adhesion mechanism may not be restricted to these two enzymes but may also involve other CBM37-containing proteins such as Cel5G and the other uncharacterised proteins recognized by the anti-Adh serum. The EMBL accession numbers for the sequences reported in this paper are FM872295 for Cel9B and FM872296 for Cel48A.     

Processivity, Substrate Binding, and Mechanism of Cellulose Hydrolysis by Thermobifida fusca Cel9A

Thermobifida fusca Cel9A-90 is a processive endoglucanase consisting of a family 9 catalytic domain (CD), a family 3c cellulose binding module (CBM3c), a fibronectin III-like domain, and a family 2 CBM. This enzyme has the highest activity of any individual T. fusca enzyme on crystalline substrates, particularly bacterial cellulose (BC). Mutations were introduced into the CD or the CBM3c of Cel9A-68 using site-directed mutagenesis. The mutant enzymes were expressed in Escherichia coli; purified; and tested for activity on four substrates, ligand binding, and processivity. The results show that H125 and Y206 play an important role in activity by forming a hydrogen bonding network with the catalytic base, D58; another important supporting residue, D55; and Glc(−1) O1. R378, a residue interacting with Glc(+1), plays an important role in processivity. Several enzymes with mutations in the subsites Glc(−2) to Glc(−4) had less than 15% activity on BC and markedly reduced processivity. Mutant enzymes with severalfold-higher activity on carboxymethyl cellulose (CMC) were found in the subsites from Glc(−2) to Glc(−4). The CBM3c mutant enzymes, Y520A, R557A/E559A, and R563A, had decreased activity on BC but had wild-type or improved processivity. Mutation of D513, a conserved residue at the end of the CBM, increased activity on crystalline cellulose. Previous work showed that deletion of the CBM3c abolished crystalline activity and processivity. This study shows that it is residues in the catalytic cleft that control processivity while the CBM3c is important for loose binding of the enzyme to the crystalline cellulose substrate.     

Statistical optimization of cellulases production by <Emphasis Type="Italic">Penicillium</Emphasis><Emphasis Type="Italic">chrysogenum</Emphasis> QML-2 under solid-state fermentation and primary application to chitosan hydrolysis

Solid-state fermentation conditions for cellulases production by a newly isolated Penicillium chrysogenum QML-2 were investigated using statistical methods. At first, significant variables for cellulases production including (NH₄)₂SO₄, initial pH and inoculum size were screened by using Plackett-Burman Design. Then the optimal regions of the significant variables were investigated by using the method of steepest ascent. Finally, central composite design and response surface analysis were adopted to determine the optimal values of the significant variables and investigate the combined effects of each variable’s pair on cellulases production. The results showed that the optimal ranges of (NH₄)₂SO₄ concentration, initial pH and inoculum size for three types of cellulases activities were 1.97–2.15 g, pH 4.32–4.41 and 13.3–13.7% (v/w), respectively. Using the mixture of corn stover powder and wheat bran (CSP/WB, 1/1) as carbon source, the optimization resulted in 370.15, 101.76 and 321.56 U/g for maximal endoglucanase activity, filter paper activity and β-glucosidase activity, respectively. Compared with maximum values of cellulases activities (endoglucanase activity 85.21 U/g, filter paper activity 16.62 U/g and β-glucosidase activity 67.68 U/g) obtained under unoptimized conditions, the optimization resulted in 3.34, 5.12 and 3.75 folds improvement for endoglucanase activity, filter paper activity and β-glucosidase activity, respectively. For chitosan hydrolysis, the crude cellulases had the optimal temperature of 55°C, pH of 4.4 and exhibited Michaelis constant (K _m) value of 8.34 mg/ml and maximum velocity (V _max) of 2.21 μmol glucosamine/min by 1 ml of the crude cellulases.     

Characterization of All Family-9 Glycoside Hydrolases Synthesized by the Cellulosome-producing Bacterium Clostridium cellulolyticum

The genome of Clostridium cellulolyticum encodes 13 GH9 enzymes that display seven distinct domain organizations. All but one contain a dockerin module and were formerly detected in the cellulosomes, but only three of them were previously studied (Cel9E, Cel9G, and Cel9M). In this study, the 10 uncharacterized GH9 enzymes were overproduced in Escherichia coli and purified, and their activity pattern was investigated in the free state or in cellulosome chimeras with key cellulosomal cellulases. The newly purified GH9 enzymes, including those that share similar organization, all exhibited distinct activity patterns, various binding capacities on cellulosic substrates, and different synergies with pivotal cellulases in mini-cellulosomes. Furthermore, one enzyme (Cel9X) was characterized as the first genuine endoxyloglucanase belonging to this family, with no activity on soluble and insoluble celluloses. Another GH9 enzyme (Cel9V), whose sequence is 78% identical to the cellulosomal cellulase Cel9E, was found inactive in the free and complexed states on all tested substrates. The sole noncellulosomal GH9 (Cel9W) is a cellulase displaying a broad substrate specificity, whose engineered form bearing a dockerin can act synergistically in minicomplexes. Finally, incorporation of all GH9 cellulases in trivalent cellulosome chimera containing Cel48F and Cel9G generated a mixture of heterogeneous mini-cellulosomes that exhibit more activity on crystalline cellulose than the best homogeneous tri-functional complex. Altogether, our data emphasize the importance of GH9 diversity in bacterial cellulosomes, confirm that Cel9G is the most synergistic GH9 with the major endoprocessive cellulase Cel48F, but also identify Cel9U as an important cellulosomal component during cellulose depolymerization.