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 journalArticle

3 Citations (Scopus)

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 (t1/237 = 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 (t1/237 = 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

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Hydro-Lyases
Bacillus thuringiensis
Bacilli
Assays
Catalyst activity
Enzymes
Sorting
Escherichia coli
Mutation
Proteins
Green Fluorescent Proteins
Libraries
Half-Life
Serogroup
Rate constants
Flow Cytometry
Hot Temperature
Metals
Fluorescence
Cells

Keywords

  • commodity chemicals
  • enzyme engineering
  • flow cytometry
  • shikimate pathway
  • thermostabilization

ASJC Scopus subject areas

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

Cite this

Rapid Thermostabilization of Bacillus thuringiensis Serovar Konkukian 97-27 Dehydroshikimate Dehydratase through a Structure-Based Enzyme Design and Whole Cell Activity Assay. / Harrington, Lucas B.; Jha, Ramesh K.; Kern, Theresa L.; Schmidt, Emily N.; Canales, Gustavo M.; Finney, Kellan B.; Koppisch, Andrew T.; Strauss, Charlie E M; Fox, David T.

In: ACS Synthetic Biology, Vol. 6, No. 1, 20.01.2017, p. 120-129.

Research output: Contribution to journalArticle

Harrington, Lucas B. ; Jha, Ramesh K. ; Kern, Theresa L. ; Schmidt, Emily N. ; Canales, Gustavo M. ; Finney, Kellan B. ; Koppisch, Andrew T. ; Strauss, Charlie E M ; Fox, David T. / Rapid Thermostabilization of Bacillus thuringiensis Serovar Konkukian 97-27 Dehydroshikimate Dehydratase through a Structure-Based Enzyme Design and Whole Cell Activity Assay. In: ACS Synthetic Biology. 2017 ; Vol. 6, No. 1. pp. 120-129.
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AU - Kern, Theresa L.

AU - Schmidt, Emily N.

AU - Canales, Gustavo M.

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