Abstract
Directed evolution requires the creation of genetic diversity and subsequent screening or selection for improved variants. For DNA mutagenesis, conventional site-directed methods implicitly utilize the Boolean AND operator (creating all mutations simultaneously), producing a combinatorial explosion in the number of genetic variants as the number of mutations increases. We introduce GeneORator, a novel strategy for creating DNA libraries based on the Boolean logical OR operator. Here, a single library is divided into many subsets, each containing different combinations of the desired mutations. Consequently, the effect of adding more mutations on the number of genetic combinations is additive (Boolean OR logic) and not exponential (AND logic). We demonstrate this strategy with large-scale mutagenesis studies, using monoamine oxidase-N (Aspergillus niger) as the exemplar target. First, we mutated every residue in the secondary structure-containing regions (276 out of a total 495 amino acids) to screen for improvements in kcat. Second, combinatorial OR-type libraries permitted screening of diverse mutation combinations in the enzyme active site to detect activity toward novel substrates. In both examples, OR-type libraries effectively reduced the number of variants searched up to 1010-fold, dramatically reducing the screening effort required to discover variants with improved and/or novel activity. Importantly, this approach enables the screening of a greater diversity of mutation combinations, accessing a larger area of a protein’s sequence space. OR-type libraries can be applied to any biological engineering objective requiring DNA mutagenesis, and the approach has wide ranging applications in, for example, enzyme engineering, antibody engineering, and synthetic biology.
Highlights
N atural evolution is based on the random creation of genetic diversity and subsequent selection of a desired fitness.[1]
Numerous studies have utilized asymmetric PCR for the purposes of site-directed mutagenesis.[39−43] In this twostep process, the first step consists of an asymmetric PCR that generates a single-stranded DNA product, created by using an unequal concentration of DNA oligonucleotide primers
In this study we demonstrate a methodology to create a novel type of variant library, whereby multiple discrete DNA regions can be mutated in an OR-type fashion
Summary
N atural evolution is based on the random creation of genetic diversity and subsequent selection of a desired fitness.[1]. Genetic diversity is generated for a target gene, enabling the discovery, selection and isolation of variants encoding an improvement in the desired fitness (e.g., increased activity). Research Article base codons used and the level of diversity at individual residues Even with these methods, the library size quickly becomes too large to test experimentally when looking to mutate multiple amino acids (typically four or more). The effect on library size of mutating multiple residues in this way is additive and not multiplicative/exponential We demonstrate that this strategy can be employed to reduce the library size significantly (often by many orders of magnitude), as well as decreasing its complexity, enabling the mutation of a larger number of regions in the same library. This has the highly desirable effect of significantly reducing the overall size of the library, while still testing all the desired codons and mutations
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