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.
Keywords: commodity chemicals; enzyme engineering; flow cytometry; shikimate pathway; thermostabilization.