Figure 5. Distribution of selected directed evolution studies by mutagenesis method.

Random mutagenesis: experiments that mutate the whole gene in the absence of guiding criteria; Saturation mutagenesis: iterative and/or simultaneous application of saturation mutagenesis; Gene shuffling: use of recombinatorial methods to generate diversity; Step-wise method application: cases where random mutagenesis, saturation mutagenesis or gene shuffling were used iteratively; Saturation mutagenesis of ‘hot spots’: probing of positions known to affect function, identified through stochastic methods; Combination of improved variants: recombination of variants through site-directed mutagenesis or gene shuffling.