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Background Renewable energy has become a field of high interest over the past decade, and production of biofuels from cellulosic substrates has a particularly high potential as an alternative source of energy. Industrial deconstruction of biomass, however, is an onerous, exothermic process, the cost of which could be decreased significantly by use of hyperthermophilic enzymes. An efficient way of breaking down cellulosic substrates can also be achieved by highly efficient enzymatic complexes called cellulosomes. The modular architecture of these multi-enzyme complexes results in substrate targeting and proximity-based synergy among the resident enzymes. However, cellulosomes have not been observed in hyperthermophilic bacteria. Results Here, we report the design and function of a novel hyperthermostable “designer cellulosome” system, which is stable and active at 75 °C. Enzymes from Caldicellulosiruptor bescii , a highly cellulolytic hyperthermophilic anaerobic bacterium, were selected and successfully converted to the cellulosomal mode by grafting onto them divergent dockerin modules that can be inserted in a precise manner into a thermostable chimaeric scaffoldin by virtue of their matching cohesins. Three pairs of cohesins and dockerins, selected from thermophilic microbes, were examined for their stability at extreme temperatures and were determined stable at 75 °C for at least 72 h. The resultant hyperthermostable cellulosome complex exhibited the highest levels of enzymatic activity on microcrystalline cellulose at 75 °C, compared to those of previously reported designer cellulosome systems and the native cellulosome from Clostridium thermocellum . Conclusion The functional hyperthermophilic platform fulfills the appropriate physico-chemical properties required for exothermic processes. This system can thus be adapted for other types of thermostable enzyme systems and could serve as a basis for a variety of cellulolytic and non-cellulolytic industrial objectives at high temperatures. Electronic supplementary material The online version of this article (10.1186/s13068-019-1386-y) contains supplementary material, which is available to authorized users.

Supplementary Information Additional Materials and Methods Strains, Media, and Growth Conditions Caldicellulosiruptor bescii and Escherichia coli strains used in this study are listed in Table S1. All C. bescii strains were grown anaerobically at 65°C on solid or in liquid low osmolarity defined (LOD) medium (1), as previously described, with .5 % w/v D(+)-Cellobiose (Acros Organics, NJ, U.S.A., Code: 108465000, Lot: A0384025) as the sole carbon source, final pH 6.8, for routine growth and transformation experiments (2). For growth of uracil auxotrophic strains based off of JWCB029 (ΔpyrFA ldh::ISCbe4 Δcbe1 ΔcelA), the LOD medium contained 40 µM uracil. This concentration of uracil does not support growth of C. bescii as sole carbon source. E. coli strain DH5α was used for plasmid DNA construction and preparation using standard techniques as described (3). E. coli cells were cultured in LB broth supplemented with apramycin (50 μg/mL) and plasmid DNA was isolated using a Qiagen Mini-prep kit (Qiagen, Hilden, Germany). Chromosomal DNA from Caldicellulosiruptor strains was extracted using the Quick-gDNA™ MiniPrep (Zymo Research, Irvine, CA, U.S.A.) as previously described (4). Construction and Transformation of CelA and CelA Derivative Expression Vectors Plasmids in this study were generated using Q5 High-Fidelity DNA polymerase (New England BioLabs, Ipswich, MA, U.S.A.), restriction enzymes (New England BioLabs, Ipswich, MA, U.S.A.), and Fast-link TM DNA Ligase (Epicentre Technologies, Madison, WI, U.S.A.) according to the manufacturer's instructions. For the construction of pJYW008 (Figure S1, Table S1), a 10.64 kb DNA fragment was synthesized with the reverse primer JY017 and forward primer JY018 using pDCW173 (4) as a template. After amplification, the PCR product was ligated via blunt-end ligation. The 9.96 kb (amplified using DCB151 and DCB152) and 10.62 kb (amplified using DCB152 and DCB153) PCR amplified DNA fragments, using pDCW173 as a template, were synthesized for the construction of pDCYB037 and pDCYB038 (Table S1), respectively. These two linear DNA fragments were digested with SphI and ligated to construct pDCYB037 and pDCYB038, respectively. The C. bescii CelA gene sequence (Cbes_1867; GenBank accession number Z86105) was codon optimized for expression in E. coli and cloned into a pET28b(+) vector using NcoI and XhoI sites (GenScript, Piscataway, NJ, U.S.A.). The sequence for a 6x histidine tag was placed at the C-terminus to facilitate protein purification. It was referred as pDCYB017 (Table S1). For the construction of pDCYB018 (Table S1), a 7.81 kb DNA fragment was synthesized with the reverse primer DCB068 and forward primer DCB069 using pDCYB017 as a template. After amplification, the PCR product was ligated via blunt-end ligation. The 7.78 kb (amplified using DCB155 and DCB157) and 7.11 kb (amplified using DCB157 and DCB256) PCR amplified DNA fragments, using pDCYB017 as a template, were synthesized for the construction of pDCYB075 and pDCYB076 (Table S1), respectively. These two linear DNA fra...

BackgroundThermophilic microorganisms and their enzymes offer several advantages for industrial application over their mesophilic counterparts. For example, a hyperthermophilic anaerobe, Caldicellulosiruptor bescii, was recently isolated from hot springs in Kamchatka, Siberia, and shown to have very high cellulolytic activity. Additionally, it is one of a few microorganisms being considered as viable candidates for consolidated bioprocessing applications. Moreover, C. bescii is capable of deconstructing plant biomass without enzymatic or chemical pretreatment. This ability is accomplished by the production and secretion of free, multi-modular and multi-functional enzymes, one of which, CbCel9A/Cel48A also known as CelA, is able to outperform enzymes found in commercial enzyme preparations. Furthermore, the complete C. bescii exoproteome is extremely thermostable and highly active at elevated temperatures, unlike commercial fungal cellulases. Therefore, understanding the functional diversity of enzymes in the C. bescii exoproteome and how inter-molecular synergy between them confers C. bescii with its high cellulolytic activity is an important endeavor to enable the production of more efficient biomass degrading enzyme formulations and in turn, better cellulolytic industrial microorganisms.ResultsTo advance the understanding of the C. bescii exoproteome we have expressed, purified, and tested four of the primary enzymes found in the exoproteome and we have found that the combination of three or four of the most highly expressed enzymes exhibit synergistic activity. We also demonstrated that discrete combinations of these enzymes mimic and even  improve upon the activity of the whole C. bescii exoproteome, even though some of the enzymes lack significant activity on their own.ConclusionsWe have demonstrated that it is possible to replicate the cellulolytic activity of the native C. bescii exoproteome utilizing a minimal gene set, and that these minimal gene sets are more active than the whole exoproteome. In the future, this may lead to more simplified and efficient cellulolytic enzyme preparations or yield improvements when these enzymes are expressed in microorganisms engineered for consolidated bioprocessing.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1014-2) contains supplementary material, which is available to authorized users.

Biomass deconstruction remains integral for enabling second-generation biofuel production at scale. However, several steps necessary to achieve significant solubilization of biomass, notably harsh pretreatment conditions, impose economic barriers to commercialization. By employing hyperthermostable cellulase machinery, biomass deconstruction can be made more efficient, leading to milder pretreatment conditions and ultimately lower production costs. The hyperthermophilic bacterium Caldicellulosiruptor bescii produces extremely active hyperthermostable cellulases, including the hyperactive multifunctional cellulase CbCel9A/Cel48A. Recombinant CbCel9A/Cel48A components have been previously produced in Escherichia coli and integrated into synthetic hyperthermophilic designer cellulosome complexes. Since then, glycosylation has been shown to be vital for the high activity and stability of CbCel9A/Cel48A. Here, we studied the impact of glycosylation on a hyperthermostable designer cellulosome system in which two of the cellulosomal components, the scaffoldin and the GH9 domain of CbCel9A/Cel48A, were glycosylated as a consequence of employing Ca. bescii as an expression host. Inclusion of the glycosylated components yielded an active cellulosome system that exhibited long-term stability at 75°C. The resulting glycosylated designer cellulosomes showed significantly greater synergistic activity compared to the enzymatic components alone, as well as higher thermostability than the analogous nonglycosylated designer cellulosomes. These results indicate that glycosylation can be used as an essential engineering tool to improve the properties of designer cellulosomes. Additionally, Ca. bescii was shown to be an attractive candidate for production of glycosylated designer cellulosome components, which may further promote the viability of this bacterium both as a cellulase expression host and as a potential consolidated bioprocessing platform organism.