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Directed Evolution of a Bond‐Forming Enzyme: Ultrahigh‐Throughput Screening of Microbial Transglutaminase Using Yeast Surface Display
Author(s) -
Deweid Lukas,
Neureiter Lara,
Englert Simon,
Schneider Hendrik,
Deweid Jakob,
Yanakieva Desislava,
Sturm Janna,
Bitsch Sebastian,
Christmann Andreas,
Avrutina Olga,
Fuchsbauer HansLothar,
Kolmar Harald
Publication year - 2018
Publication title -
chemistry – a european journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.687
H-Index - 242
eISSN - 1521-3765
pISSN - 0947-6539
DOI - 10.1002/chem.201803485
Subject(s) - tissue transglutaminase , yeast , directed evolution , enzyme , high throughput screening , substrate (aquarium) , biochemistry , chemistry , cell sorting , recombinant dna , substrate specificity , cell , biology , gene , mutant , ecology
Abstract Microbial transglutaminase from Streptomyces mobaraensis (mTG) has emerged as a useful biotechnological tool due to its ability to crosslink a side chain of glutamine and primary amines. To date, the substrate specificity of mTG is not fully understood, which poses an obvious challenge when mTG is used to address novel targets. To that end, a viable strategy providing an access to tailor‐made transglutaminases is required. This work reports an ultrahigh‐throughput screening approach based on yeast surface display and fluorescence‐activated cell sorting (FACS) that enabled the evolution of microbial transglutaminase towards enhanced activity. Five rounds of FACS screening followed by recombinant expression of the most potent variants in E. coli yielded variants that possessed, compared to the wild type enzyme, improved enzymatic performance and labeling behavior upon conjugation with an engineered therapeutic anti‐HER2 antibody. This robust and generally applicable platform enables tailoring of the catalytic efficiency of mTG.