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Spore-Based Designer Enzyme Cascade Biocatalysts

  • Author(s): Chen, Long
  • Advisor(s): Ge, Xin
  • et al.

Despite with high sufficiency and intrinsic specificity, industrial applications of biocatalysts are still restrained due to relatively high cost of production, limited stability and reusability, and expensive co-factor regeneration. This dissertation aims to develop a Bacillus subtilis spore-based biocatalysis platform to address these issues. Different from all previous spore display studies, anchoring scaffoldins based on cellulolsome were designed and constructed to display cascade enzymes in stoichiometrically controllable manner, assemble multimeric enzyme complex, and recruit large number of synergic enzymes. We found among B. subtilis outer coat proteins, CotG mediated a high expression level of cohesin domains. Functional display of dockerin tagged xylose reductase and phosphite dehydrogenase on spore surface at optimized stoichiometry achieved efficient regeneration of NADP(H). Because coat proteins do not need to translocate across membrane during of sporulation, co-expression of spore surface anchoring scaffoldins carrying multiple cohesin domains and dockerin tagged β-galactosidase (β-gal) in the mother cell compartment, resulted in self-assembly of tetrameric β-gal in its active form with a high display density of > 3 x 104 per spore. Spore display dramatically increased transgalactosylation yields in water/organic emulsions, enhanced thermostability and reusability, and exhibited long-term storage stability at ambient temperature for more than 60 days. We further assembled recombinant cellulosomes containing primary and secondary scaffoldins with 1-5 copies of type I/II cohesion domain that combinatorically can dock 1-25 cellulase molecules. The quantitative correlation between cellulosome size and its cellulolytic activity was studied for the first time. Overall this dissertation suggests that spore-based biocatalysts incorporating multiple synergic enzymes hold a great potential in a wide range of biocatalysis applications.

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