Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

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.

Harnessing glycoenzyme engineering for synthesis of bioactive oligosaccharides

“…Semi-rational libraries (approx. 10 2 -10 4 variants) can be constructed using for example the NNK or NDT degenerate codons using subsequently different approaches such as (c) iterative saturation mutagenesis (ISM) [22], (d ) combinatorial active site saturation testing (CASTing) [23], and using molecular biology methods such as: (e) OmniChange [24], or ( f ) the recently described Darwin assembly method [25]. The resulting smaller libraries can then be screened using low-or medium-throughput screening assays such as liquid assays in microtitre plates coupled with analytical detection or screening on a solid medium for the isolation of interesting mutants.…”

XNA Synthesis and Reverse Transcription by Engineered Thermophilic Polymerases

“…Among thermostable B-family polymerases, engineering efforts have focused on DNAPs from Thermococcus gorgonarius Liu et al, 2018;, Thermococcus kodakarensis (Larsen et al, 2016), and 9°N (Gardner & Jack, 1999), with mutations being portable between homologous enzymes, at least for some chemistries (Dunn, Otto, Fenton, & Chaput, 2016). Here we demonstrate a comparison of HNA synthesis between a published HNA polymerase from T. gorgonarius [T.6G12 : Tgo DNAP V93Q, D141A, E143A, A485L, V590A, E609K, I610M, K659Q, E664Q, Q665P, R668K, D699Q, K671H, K674R, T686R, A681S, L704P, E730G) and the same mutations ported into T. kodakariensis DNAP (K.6G12: KOD DNAP V93E, D141A, E143A, A485L, V590A, E609K, I610M, K659Q, E664Q, Q665P, R668K, D699Q, K671H, K674R, T686R, A681S, L704P, E730G)] using Darwin Assembly (Cozens & Pinheiro, 2018).…”

Clarifying the Difference between Iterative Saturation Mutagenesis as a Rational Guide in Directed Evolution and OmniChange as a Gene Mutagenesis Technique

“…The subsequently introduced term CAST is simply a convenient acronym that can be used to distinguish it from SM at remote sites for other purposes such as increasing protein robustness . Importantly, when applying the CAST strategy , SSM and CSM can be performed by using any gene mutagenesis technique, including those based mainly on PCR, such as the popular QuikChange, MegaPrimer, overlap‐extension PCR and others,– or, in principle, methods based on either gene assembly such as Gibson, ADO and others, or gene synthesis such as Twist, Sloning and others . OmniChange is yet another alternative when opting for the CAST strategy.…”