Rapid thermostabilization of bacillus thuringiensis Serovar Konkukian 97-27 dehydroshikimate dehydratase through a structure-based enzyme design and whole cell activity assay

Lucas B. Harrington, Ramesh K. Jha, Theresa L. Kern, Emily N. Schmidt, Gustavo M. Canales, Kellan B. Finney, Andrew T. Koppisch, Charlie E.M. Strauss, David T. Fox

Research output: Contribution to journalArticlepeer-review

6 Scopus citations

Abstract

Thermostabilization of an enzyme with complete retention of catalytic efficiency was demonstrated on recombinant 3-dehydroshikimate dehydratase (DHSase or wtAsbF) from Bacillus thuringiensis serovar konkukian 97-27 (hereafter, B. thuringiensis 97-27). The wtAsbF is relatively unstable at 37 °C, in vitro (t1/2 37 = 15 min), in the absence of divalent metal. We adopted a structure-based design to identify stabilizing mutations and created a combinatorial library based upon predicted mutations at specific locations on the enzyme surface. A diversified asbF library (∼2000 variants) was expressed in E. coli harboring a green fluorescent protein (GFP) reporter system linked to the product of wtAsbF activity (3, 4- dihydroxybenzoate, DHB). Mutations detrimental to DHSase function were rapidly eliminated using a high throughput fluorescence activated cell sorting (FACS) approach. After a single sorting round and heat screen at 50 °C, a triple AsbF mutant (Mut1), T61N, H135Y, and H257P, was isolated and characterized. The half-life of Mut1 at 37 °C was >10-fold higher than the wtAsbF (t1/2 37 = 169 min). Further, the second-order rate constants for both wtAsbF and Mut1 were approximately equal (9.9 × 105 M-1 s-1, 7.8 × 105 M-1 s-1, respectively), thus demonstrating protein thermostability did not come at the expense of enzyme thermophilicity. In addition, in vivo overexpression of Mut1 in E. coli resulted in a ∼60-fold increase in functional enzyme when compared to the wild-type enzyme under the identical expression conditions. Finally, overexpression of the thermostable AsbF resulted in an approximate 80-120% increase in DHB accumulation in the media relative to the wild-type enzyme.

Original languageEnglish (US)
Pages (from-to)120-129
Number of pages10
JournalACS Synthetic Biology
Volume6
Issue number1
DOIs
StatePublished - Jan 20 2017

Keywords

  • Commodity chemicals
  • Enzyme engineering
  • Flow cytometry
  • Shikimate pathway
  • Thermostabilization

ASJC Scopus subject areas

  • Biomedical Engineering
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)

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