Rapid Thermostabilization of Bacillus thuringiensis Serovar Konkukian 97–27 Dehydroshikimate Dehydratase through a Structure-Based Enzyme Design and Whole Cell Activity Assay

详细信息    查看全文

• 作者：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
• 刊名：ACS Synthetic Biology
• 出版年：2017
• 出版时间：January 20, 2017
• 年：2017
• 卷：6
• 期：1
• 页码：120-129
• 全文大小：542K
• ISSN：2161-5063

文摘

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 (t_1/2³⁷ = 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 (t_1/2³⁷ = 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.