Abstract
Engineering proteins for designer functions and biotechnological applications almost invariably requires (or at least benefits from) multiple mutations to non-contiguous residues. Several methods for multiple site-directed mutagenesis exist, but there remains a need for fast and simple methods to efficiently introduce such mutations – particularly for generating large, high quality libraries for directed evolution. Here, we present Darwin Assembly, which can deliver high quality libraries of >108 transformants, targeting multiple (>10) distal sites with minimal wild-type contamination (<0.25% of total population) and which takes a single working day from purified plasmid to library transformation. We demonstrate its efficacy with whole gene codon reassignment of chloramphenicol acetyl transferase, mutating 19 codons in a single reaction in KOD DNA polymerase and generating high quality, multiple-site libraries in T7 RNA polymerase and Tgo DNA polymerase. Darwin Assembly uses commercially available enzymes, can be readily automated, and offers a cost-effective route to highly complex and customizable library generation.
Highlights
Biology, through natural enzymes, has explored and exploited a vast repertoire of chemical reactions, including many that can be harnessed for the manufacture of clinically, biotechnologically and culturally relevant molecules [1]
Expression and functional optimization, or more radical engineering is often required to generate the desired enzymatic activity, whether boosting an existing activity or changing enzyme function altogether. Those needs have led to the flourishing of directed evolution and protein engineering and it has repeatedly proven possible to enhance a number of protein properties, including expression, folding [2], thermostability [3], substrate specificity [4,5,6] and catalytic efficiency [7,8]
Mutations can be incorporated into the primers to change the sequence in the PCR product, but this method is limited to the number of mutations that can be incorporated into a single iPCR primer pair
Summary
Through natural enzymes, has explored and exploited a vast repertoire of chemical reactions, including many that can be harnessed for the manufacture of clinically, biotechnologically and culturally relevant molecules [1]. Sequential cycles of individual site mutagenesis and screening can be effective in navigating from natural to engineered catalyst [4,5,7,12,13] but such an approach requires a sequence landscape where improvement can be detected in each of the intermediates and pre-defines the evolutionary path followed [14,15]. Such iterative approaches cannot uncover phenotypes requiring epistatic mutations as multiple mutations are never made in the same round [16,17,18]. Mutations can be incorporated into the primers to change the sequence in the PCR product, but this method is limited to the number of mutations that can be incorporated into a single iPCR primer pair
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