3.1. Sequence Agnostic Random Mutagenesis

Random mutagenesis techniques that do not require any structural information and allow for mutation at any position within the protein-coding region of a gene can be considered “sequence agnostic”. Sequence (and structure) agnostic mutagenesis approaches are the methods of choice for library generation when no structural data is available. The mutant libraries generated in this way can be used for directed evolution, in combination with high-throughput screening or selection techniques (Section 4). Methods for random mutagenesis include error prone PCR (epPCR) [66], DNA shuffling [67], in vivo mutagenesis using mutator strains [68], and external mutagens [69] (Table 2).

Overview of random mutagenesis methods.

A powerful and versatile yet straightforward technique, epPCR is the most common method for creating mutant libraries of a single gene. In epPCR, conditions are chosen to allow for a relatively high mutation rate by the DNA polymerase (i.e., low fidelity of replication). This can typically be achieved by adjusting the concentration of DNA polymerase and MgCl₂, adding MnCl₂, and adjusting the ratio of dNTPs, or by using an engineered DNA polymerase mutant with reduced fidelity [66]. As it is the most common mutagenesis technique, it comes as no surprise that epPCR has been applied to engineer novel GBPs. In one example from 2007, Yabe et al. cloned an earthworm galactose-binding lectin, EW29Ch, as the starting point for directed evolution wherein variants from successive generations were selected from mutant libraries generated by epPCR. This approach produced an engineered GBP specific for α2,6-sialic acid, a ligand not recognized by the parent protein [70].

In other examples, epPCR can also be combined with other mutagenesis techniques, such as DNA shuffling. DNA shuffling involves recombination of a population of homologous genes that have diverged either naturally or through laboratory mutagenesis of a parent (e.g., by epPCR). In this technique, random fragmentation of genes in a library (e.g., by DNase I digestion) is followed by PCR-based reassembly of overlapping fragments with sufficient homology, which effectively recombines mutations within the gene library [71]. Examples of DNA shuffling in combination with epPCR are seen in protein engineering efforts that have introduced mutations to the CBM of cyclodextrin glucanotransferase from Bacillus sp., and the glycan-binding regions of N-oligosaccharyltransferase from Campylobacter jejuni resulting in increased specificity and efficiency of those enzymes [72,73].

Alternative to the in vitro mutagenesis methods described above, one can perform in vivo mutagenesis on a target gene. These in vivo methods involve manipulating the DNA replication and repair machinery of the organism in which the target gene is cloned. For example, in mutator strains like E. coli XL1-red, which is deficient in three of the primary DNA repair pathways (carrying mutations mutS, mutD, and mutT), imperfect replication of DNA results in the accumulation of mutations in the cloned gene (along with the vector) [68]. In an example from 2011, Mendonça and Marana used in vivo mutagenesis in E.coli XL1-Red to alter the specificity of a β-glycosidase, SfβGly, from Spodoptera frugiperda [74]. Mutants from a library generated in the mutator strain were screened for their specificity towards fucosides vs. glucosides, and several variants were identified that differed from the parent enzyme in their substrate preference. Given that glycosidases can serve as scaffolds for GBPs through their catalytic inactivation (Section 2.4), this can be a useful strategy for engineering novel GBPs. The advantage of mutator strains is that their use involves simple protocols, generally involving transformation of the mutator strain by a plasmid followed by propagation and plasmid recovery. However, mutator strains get progressively sick as they divide due to the deficiencies in their DNA repair mechanisms, and consequently this mutagenesis method often requires frequent re-transformations. Other in vivo mutagenesis methods use external mutagens such as UV radiation or mutagenic chemicals (e.g., ethyl methanesulfonate) which can avoid some of the challenges of maintaining mutator strains.

Regardless of the sequence agnostic random mutagenesis techniques used, one disadvantage is that the produced libraries only cover a small fraction of the possible mutations and require considerable effort to screen. Rational and semi-rational mutagenesis can be more efficient at producing effective mutations based on the structural and functional information when it is available.