Iterative Saturation Mutagenesis: A Powerful Approach to Engineer Proteins by Systematically Simulating Darwinian Evolution

Abstract

Iterative saturation mutagenesis (ISM) is a widely applicable and powerful strategy for the efficient directed evolution of enzymes. First, one or more amino acid positions from the chosen enzyme are assigned to multi-residue sites (i.e., groups of amino acids or "multisites"). Then, the residues in each multisite are mutated with a user-defined randomization scheme to all canonical amino acids or a reduced amino acid alphabet. Subsequently, the genes of chosen variants (usually the best but not necessarily) are used as templates for saturation mutagenesis at other multisites, and the process is repeated until the desired degree of biocatalyst improvement has been achieved. Addressing multisites iteratively results in a so-called ISM scheme or tree with various upward branches or pathways. The systematic character of ISM simulates in vitro the natural process of Darwinian evolution: variation (library creation), selection (library screening), and amplification (template chosen for the next round of randomization). However, the main feature of ISM that distinguishes it from other directed evolution methods is the systematic probing of a defined segment of the protein sequence space, as it has been shown that ISM is much more efficient in terms of biocatalyst optimization than random methods such as error-prone PCR. In addition, ISM trees have also shed light on the emergence of epistasis, thereby rationally improving the strategies for evolving better enzymes. ISM was developed to improve catalytic properties such as rate, substrate scope, stereo- and regioselectivity using the Combinatorial Active-site Saturation Test (CAST), as well as chemical and thermal stability employing the B-Factor Iterative Test (B-FIT). However, ISM can also be invoked to manipulate such protein properties as binding affinity among other possibilities, including protein-protein interactions. Herein, we provide general guidelines for ISM, using CAST as the case study in the quest to enhance the activity and regioselectivity of the monooxygenase P450BM3 toward testosterone.

Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently

“…In addition to these more conventional methods, new approaches are continually being developed to improve efficiency and to reduce the number of steps in the workflow, for example Mutagenic Oligonucleotide-Directed PCR Amplification (MOD-PCR), 559 Overlap Extension PCR (OE-PCR), 560–564 Sequence Saturation Mutagenesis, 565–571 Iterative Saturation Mutagenesis, 557,572–579 and a variety of transposon-based methods. 580–583 However, a common issue with site-directed mutagenesis methods is the large number of steps involved and the limited number of positions that can be efficiently targeted at a time.…”

Economical analysis of saturation mutagenesis experiments

“…More recently, SM has been used to engineer promoters 9 , transcriptional enhancers 10 , ribosome binding sites 11 , cis -regulatory elements and trans -acting factors 12 , and protein-coding genes using emergent genome engineering tools 13 . Consequently, SM, especially when applied iteratively 3 4 5 14 15 , has become an effective tool in protein engineering 2 4 5 , and holds great potential in the directed evolution of metabolic pathways 16 17 18 and genomes 19 20 21 .…”

A mix-and-read drop-based in vitro two-hybrid method for screening high-affinity peptide binders

“…In this work, we screened and sorted 10 6 drops in just half an hour, which was sufficient to assay a library size of 10 5 molecules. With the state-of-art microfluidic techniques, we should be able to increase the library size to 10 6 , which may be suitable for screening a target-focused library where a few specific changes are made by site-directed mutagenesis 50 51 . To screen an even larger library that is comparable to other methods (Table S1), it may be possible to improve the sorting speed or load multiple DNA templates into each drop, followed by successive rounds of screening for further enrichments.…”