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Intein-mediated assembly of tunable scaffoldins for facile synthesis of designer cellulosomes

Abstract

In this study, extended artificial scaffoldins possessing multiple cohesin modules were created in vivo by employing split-intein-mediated protein ligation. Artificial scaffoldins having one Clostridium thermocellum cohesin (Coht), one carbohydrate binding module (CBM) from Clostridium cellulolyticum scaffolding protein CipC, and one to five cohesins (Cohc) derived from CipC, were assembled. These scaffoldins were used to assemble cellulosomal enzyme complexes for investigating the interplay among endoglucanase, exoglucanase, and scaffoldin-borne CBM, on the hydrolysis of a model microcrystalline cellulose substrate, Avicel. The cellulosomal complexes were assembled in vitro by incubating recombinant C. thermocellum endoglucanase (At) and C. cellulolyticum exoglucanase (Ec), with the various artificial scaffoldins. Under a fixed total cellulase concentration, improved hydrolysis is noted by recruiting both Ec and At on the same scaffoldin, for all scaffoldins tested, compared with free cellulases. The improvement is more profound with scaffoldins having a higher Cohc/Coht ratio (i.e., increased Ec/At ratio). Furthermore, among scaffoldins having the same Cohc/Coht ratio, highest rates of Avicel hydrolysis are noted when Coht, and hence an endoglucanase, is situated next to the CBM and not flanked by Cohc. These results point to the importance of using scaffoldins with sufficiently high numbers of cohesin units to achieve an optimal exo-/endo-glucanase ratio to create efficient designer cellulosomes. Furthermore, intein-trans-splicing is proven here to be an effective method for assembling complex scaffoldins and more intricate cellulosomes.

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Funding

This work was supported in part by the USDA NIFA AFRI grant (2010-65504-20349), NIFA multi-state project HAW05034-R, the Hawaii Community Foundation (44272, 11ADVC-49237), and by a Specific Cooperative Agreement (SCA no.58-5320-3-022) with the Daniel K. Inouye U.S. Pacific Agricultural Research Center.

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Correspondence to Wei Wen Su.

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The authors declare that they have no conflict of interest.

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This work does not contain any study with human participants or animals.

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Han, Z., Su, W.W. Intein-mediated assembly of tunable scaffoldins for facile synthesis of designer cellulosomes. Appl Microbiol Biotechnol 102, 1331–1342 (2018). https://doi.org/10.1007/s00253-017-8701-y

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  • DOI: https://doi.org/10.1007/s00253-017-8701-y

Keywords

  • Scaffoldin
  • Cellulosome
  • Intein
  • Self-assembly
  • Cellulose hydrolysis