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Additional file 5: Figure S4. Hydrolysis of un/pretreated wheat straw. The wild-type PC9 and the two transformants were used as a biological pretreatment of wheat straw for 19 and 35Â days. The pretreated and untreated straw samples were hydrolyzed by the commercial cellulase cocktail CellicÂŽ CTec2 (Novozymes), and the concentration of released soluble sugars was measured by the DNS method [84].

Additional file 2: Figure S2. Secreted cellulolytic activity of cre1-overexpressing or cre1-knockout transformants. Pleurotus ostreatus PC9 was genetically manipulated to either overexpress or knock out the cre1 gene. The fungi were grown on minimal medium, containing 2% MCC, for 7Â days. Secreted cellulolytic activity was measured by applying the supernatant fluids on phosphoric acid swollen cellulose and measuring the released soluble reducing sugars by the DNS method. The cre1-overexpressing transformant OE7 exhibited the lowest secreted cellulolytic activity and was thus selected for further analysis (termed herein OEcre1; red bar marked with black arrow). The cre1-knockout transformant showed the highest secreted cellulolytic activity and was thus selected for further analysis (termed herein KOcre1; green bar marked with black arrow).

Additional file 1: Figure S1. Genetic manipulation of cre1 overexpression (OEcre1) and knockout (KOcre1) transformants. a. Strategy for overexpression of cre1 in P. ostreatus (PC9). The β-tubulin promoter was fused to the cre1 coding sequence (CDS) and ligated to the carboxin-resistance-conferring cassette (Cbxr) to produce the TMS17 cassette. Small arrows indicate the location of primers (Additional file 6: Table S2) used for construction and detection of the construct. PCR verification of the integration was performed using primers TMS17DF and TMS17DR (Additional file 6: Table S2) b. Strategy for gene replacement of cre1 in P. ostreatus (Δku80, 20b). The hygromycin B-resistance-conferring cassette (Hygr) was fused to 2 kb of 5′ and 3′ genomic DNA flanking cre1 CDS to produce the TMS18 cassette. Wild-type cre1 genomic region is illustrated below. PCR verification of cre1 replacement was performed using primers CRE1DF and CRE1DR (Additional file 6: Table S2).

Additional file 7: Figure S5. cre1 gene expression analysis. PC9 and OEcre1 were grown for 7 days on minimal media containing either CMC or glucose as a carbon source. Culture biomass was ground under liquid nitrogen with mortar and pestle, and RNA was purified by RNeasy Plus Mini Kit (Qiagen) according to the manufacturer’s instructions. cDNA was synthesized using the qScript cDNA Synthesis Kit (Quanta BioSciences, Gaithersburg, MD, USA). Quantification of transcript abundance was determined on an ABI StepOnePlus Real-Time PCR Sequence Detection System and software (Applied Biosystems), using Power SYBR Green PCR Master Mix (Applied Biosystems), with an annealing temperature of 63 °C, according to the manufacturer’s default operating procedures. The endogenous internal control gene used was β-tubulin. Primers tubF543 (5′-GTGCGTAAGGAAGCTGAGGG-3′) and tubR777 (5′-TGTGGCATTGTACGGCTCAAC-3′) were used to amplify a 201-bp amplicon from β-tubulin. Primers cre1F_2214 (5′-GTGGATTGGGCGGGTCGA-3′) and cre1R_2623 (5′-CATCCGTTCCCATGAGCGAT-3′) were used to amplify a 201-bp amplicon from cre1. Target gene transcript abundance is expressed relative to the levels of β-tubulin according to their Ct values 2 [(Ct β−tubulin) − (Ct cre1)], in arbitrary units.

Additional file 6: Table S2. Oligonucleotides used in this study. The oligonucleotides that are used for either construction of cassettes or verification of the genomic integration of both OEcre1 or KOcre1 are detailed in the table. The location of the primer within the cassettes or in the genome is given in Additional file 1: Figure S1a and b.

Additional file 1: Figure S1. Purification profile of the different cellulosomes by Gel filtration chromatography. C. thermocellum growth media were centrifuged (10,900Â g, 7Â min), and the supernatant fluids were carefully removed from the pellet and concentrated 40 times using a Pellicon XL biomax 300 cassette (Millipore, Cat. No. PXB300C50). Concentrated samples were fractionated by size exclusion chromatography using a SuperdexS-200 prep grade 16/60 gel filtration column (GE Healthcare). (A) Chromatogram of glucose-derived cellulosomes (B) Chromatogram of CB-derived cellulosomes (C) Chromatogram of MCC-derived cellulosomes (D) Chromatogram of alSG-derived cellulosomes (E) Chromatogram of alCS-derived cellulosomes (F) Chromatogram of acCS-derived cellulosomes. Fractions containing the cellulosomes (according to SDS-PAGE analysis) are marked by black arrows. All cellulosomes-containing fractions were eluted immediately after void value due to the separation range of the column.

Additional file 5: Table S3. Soluble (non-cellulosomal) carbohydrate-active enzyme composition. Soluble (dockerin-lacking) carbohydrate-active enzyme compositions of the different cellulosomes were analyzed by label-free LCâ MS/MS mass spectrometry. The intensities were normalized by the intensity-based absolute quantification (iBAQ) method. The resultant iBAQ intensities were divided by the iBAQ intensity of ScaA in each sample, thereby generating a relative abundance index. Standard deviations of duplicate samples of CB- and MCC-derived cellulosomes and triplicates of glucose-, alSG-, alCS-, and acCS-derived cellulosomes were analyzed. Gene ID and CAZy annotation of the subunits are designated. Acronyms: GH, glycoside hydrolase; CBM, carbohydrate-binding module; CE, carbohydrate esterase; GT, glycosyl transferase.

Additional file 4: Table S2. Activities of C. thermocellum cellulosomal enzymes. Recombinant C. themocellum cellulosomal (type I dockerin-containing) enzymes were a kind gift of CelDezyner Ltd. (Rehovot, Israel). Activity assays were conducted in a final volume of 1 ml, containing 50 mM acetate buffer, 1% carboxymethyl cellulose (sodium salt, low viscosity CMC, BDH chemicals) and 7 nM enzyme. Samples were incubated with shaking for 3 h at 60 °C. Released soluble sugar (reducing ends) concentrations were analyzed by the dinitrosalicylic acid (DNS) method, as previously described [36]. Final soluble sugar concentrations were determined against a glucose calibration curve, and CMCase activities [µM reducing ends·µmol enzyme−1·min−1] were calculated. *A thermostable clone of Cel8A [66] was used.

Additional file 3: Table S1. Functional classes distribution of C. thermocellum DSM 1313 cellulosomal subunits. Cellulosomal subunits detected in the C. themocellum DSM 1313 proteome were sorted into five functional classes: endoglucanases, exoglucanases, hemicellulases, scaffoldins and others.

Additional file 1. Non-denaturing PAGE analysis of wild-type and mutant forms of Cel48S complexed with the monovalent scaffoldin Scaf¡T. Complexes were produced at different molar ratios and applied to non-denaturing polyacrylamide gels. For each gel, lanes 1 to 7 correspond to the respective enzyme/scaffoldin complexes at the following ratios: 0.4, 0.6, 0.8, 1.0, 1.2, 1.4 and 1.6. (Lane 4 signifies the calculated 1:1 ratio). Lane 8 corresponds to the specified enzyme alone at the presumed molar ratio. Red rectangle indicated the determined exact equimolar ratio.

Additional file 2. Comparative thermostability of wild-type β-glucosidase and BglA*. The enzymes were incubated at 66-72 °C for 1 h at pH 6.1. Residual activity was determined at a protein concentration of 1.05 µg/mL following incubation with 1 mM pNPG (Sigma-Aldrich, Israel) for 10 min at 60°C. Reactions were terminated by 1 M sodium carbonate and optical densities of the samples were measured at 405 nm (pNP released). Residual activities were calculated by dividing the activity of non-heated samples by that of the heated samples. The assay was performed at least twice in triplicate, error bars are indicated. Thermostable BglA* was generated by error-prone PCR as described by Anbar [24] using Gene-Morph II Random Mutagenesis Kit (Stratagene, La Jolla, CA). The thermostable BglA* was selected as it maintained the highest enzymatic activity after heat shock at 70 °C for 50 min (conditions in which the wild-type enzyme was no longer active). BglA* exhibited 127 % activity compared to the wild-type enzyme following heat shock at 66 °C for 75 min, and incubation with 1 mM pNPG for 45 min at 60 °C at 13 nM. Sequencing of the clone revealed 2 mutations: A17S and K268N.

Additional file 3. Thermostability of conventional versus thermostable designer cellulosomes and components. Non-denaturing PAGE (9 %) was employed for assessing total complex formation of the thermostable chimaeric scaffoldin and the wild-type or thermostable chimaeric enzymes into conventional designer cellulosomes (A) or thermostable designer cellulosomes (B), respectively, at the designated time periods and temperatures. In (C), the individual thermostable enzymes were incorporated into the thermostable scaffoldin. The stability of the trivalent scaffoldin either alone or in complex with each one of the thermostable dockerin-containing enzyme at various temperatures/times is shown. The tetravalent mesostable scaffoldin, bearing 3 thermostable cellulases was assayed similarly in (D). Pictograms denote composition of the designer cellulosomes and complementary free-enzyme cocktails. See Figures 1 and 4 and accompanying legends for descriptions of the pictograms.