Directed Evolution of a Bond‐Forming Enzyme: Ultrahigh‐Throughput Screening of Microbial Transglutaminase Using Yeast Surface Display

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.

Recent progress in enzymatic protein labelling techniques and their applications

“…202–204 Concerning Q-tags, in addition to the classic and well-studied sequence of LLQG, 205 a Q-tag denoted as 7M48 (WALQRPH), 206 optimized from peptide sequences selected from phage-displayed libraries 202 was shown to have improved reaction rates; that was confirmed by engineering 7M48 into maltose binding protein (MBP) and analysing the labelling using propargylamine as the small molecule substrate. 147 Importantly, these tag sequences can be inserted at either the C-terminus, 207 N-terminus 208 or internal loops of the proteins of interest. 209…”

“…Construction of protein conjugates using MTG can be accomplished in one step using peptide or amine substrates appended to various cargos. Early examples included fluorophores, 207,210 the PEG polymers, 211 metal chelators, 212 or oligonucleotides. 204 Since 2013, two-step approaches consisting of initial protein modification with a bioorthogonal functional group, such as alkyne, 147 azide, 146,148 or tetrazine 92 have been widely explored.…”

Screening of yeast display libraries of enzymatically-cyclized peptides to discover macrocyclic peptide ligands

“…48 In the context of cyclization of short peptides, transglutaminase has been utilized either alone or in combination with orthogonal cyclization agents to construct cyclic and polycyclic constructs comprising 6 -12 residues. 37,49 Considerable efforts have been dedicated to the engineering of the enzyme, 50 the identification of its recognition sequence, and the optimization of the reaction conditions to increase the yield of ligation; 37,51 in particular, it has been recognized that appending the N-terminal dipeptide Ala-Leu significantly increases the cyclization yield. 37 In this study, we referred to the sequence ALQSGSRGGGKS as linear precursor to evaluate the efficiency of cyclization on the surface of yeast cells; this peptide, in fact, served as model substrate in prior work on transglutaminase-mediated cyclization.…”

Microbial Transglutaminase is Beneficial to Food Industries but a Caveat to Public Health

“…The most frequently used industrial mTG is secreted from Streptoverticillium mobaraense [3]. Novel mTGs are continuously described using ultrahigh-throughput screening and other biotechnological systems [10]. A recent new one, for example that exerts anti-phagocytic activity was reported in Streptococcus suis [11].…”