Aminoacyl-tRNA Synthetases: Investigations of Specificity for Application in ProxiMAX / Synthetic Biology.

ProxiMAX randomization technology (Colibra) is a defined saturation mutagenesis process that delivers precision control of both identity and relative ratio of amino acids at specified locations within a protein library. The process is non-degenerate, meaning that encoded DNA libraries are as small as is physically possible. Since no constraints are imposed by the genetic code, ProxiMAX can encode all 20 or any desired subsets of amino acids with ease. Moreover, its use of a maximum of 20 codons in saturated positions, without sequence restraints, means that many codons remain available to encode additional, unnatural amino acids (UAAs). Incorporation of UAAs is particularly relevant in expanding engineered protein repertoires. Ultimately, we aim to combine ProxiMAX with an in vitro transcription/translation system to design and express synthetic protein libraries containing multiple UAAs simultaneously. We have employed ProxiMAX randomisation to encode 18 natural amino acids (excluding Cys andMet) in various, putative amino acid-binding locations of an E. coli alanyl-tRNA synthetase. Two variant libraries, each lacking any amino acid editing function, have been created. Our poster will describe the synthesis of those libraries, each encoding >107 novel variant proteins, their composition, quality and our progress towards screening and deconvolution of the libraries to discover novel synthetases, with an initial focus on specificity for D-amino acids.