TY - JOUR
T1 - Rapid thermostabilization of bacillus thuringiensis Serovar Konkukian 97-27 dehydroshikimate dehydratase through a structure-based enzyme design and whole cell activity assay
AU - Harrington, Lucas B.
AU - Jha, Ramesh K.
AU - Kern, Theresa L.
AU - Schmidt, Emily N.
AU - Canales, Gustavo M.
AU - Finney, Kellan B.
AU - Koppisch, Andrew T.
AU - Strauss, Charlie E.M.
AU - Fox, David T.
N1 - Funding Information:
D.T.F. was supported in this work by the Los Alamos National Laboratory under the U.S. Department of Energy, Laboratory Directed Research and Development grant [LDRD ER20100182ER] and Office of Energy Efficiency & Renewable Energy, Bioenergy Technologies Office Annual Operating Plan. L.H. was supported by the DOE Summer Undergraduate Laboratory Internships (SULI) program. R.K.J and C.E.M.S were supported by the Defense Threat Reduction Agency [CBCALL12-LS-6-0622]. Computational work was supported by the LANL Institutional Computing grant [W13_SynBio]. A.T.K. was supported in this work by the NAU College of Forestry, Natural Science and Engineering Dean’s office through faculty startup funding, and G.C. was supported by grant NIH-5R25GM056931, Initiative to Maximize Student Development in the Biomedical Sciences. We thank Drs. Ron Jacak and Brian Kuhlman (UNC Chapel Hill) for Rosetta point mutation scan (p_mut scan) protocol and Dr. Dung Vu (LANL) for assistance with the circular dichroism experiments.
Publisher Copyright:
© 2016 American Chemical Society.
PY - 2017/1/20
Y1 - 2017/1/20
N2 - 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.
AB - 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.
KW - Commodity chemicals
KW - Enzyme engineering
KW - Flow cytometry
KW - Shikimate pathway
KW - Thermostabilization
UR - http://www.scopus.com/inward/record.url?scp=85012838342&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85012838342&partnerID=8YFLogxK
U2 - 10.1021/acssynbio.6b00159
DO - 10.1021/acssynbio.6b00159
M3 - Article
C2 - 27548779
AN - SCOPUS:85012838342
SN - 2161-5063
VL - 6
SP - 120
EP - 129
JO - ACS Synthetic Biology
JF - ACS Synthetic Biology
IS - 1
ER -