Directed Evolution of a Bond‐Forming Enzyme: Ultrahigh‐Throughput Screening of Microbial Transglutaminase Using Yeast Surface Display. Issue 57 (10th September 2018)

• Record Type: Journal Article
• Authors: Deweid, Lukas; Neureiter, Lara; Englert, Simon; Schneider, Hendrik; Deweid, Jakob; Yanakieva, Desislava; Sturm, Janna; Bitsch, Sebastian; Christmann, Andreas; Avrutina, Olga; Fuchsbauer, Hans‐Lothar; Kolmar, Harald
• Is Part Of: Chemistry. Volume 24:Issue 57(2018)
• Page Start: 15195
• Page End: 15200
• Publication Date: 2018-09-10
• Subjects: bioconjugation -- directed evolution -- enzyme catalysis -- FACS -- microbial transglutaminase -- protein engineering -- yeast surface display; Chemistry -- Periodicals; 540
• DOI: 10.1002/chem.201803485
• Languages: English
• ISSNs: 0947-6539

Abstract

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. Abstract : Fast‐track ligation : A novel ultrahigh‐throughput screen for microbial transglutaminase yields engineered biocatalysts with improved kinetics; the most potent mutant labeled monoclonal anti‐HER2 antibody trastuzumab with enhanced efficacy.