Abstract
Approaches that depend on directed evolution require reliable methods to generate DNA diversity so that mutant libraries can focus on specific target regions. We took advantage of the high frequency of homologous DNA recombination in Saccharomyces cerevisiae to develop a strategy for domain mutagenesis aimed at introducing and in vivo recombining random mutations in defined segments of DNA. Mutagenic Organized Recombination Process by Homologous IN vivo Grouping (MORPHING) is a one-pot random mutagenic method for short protein regions that harnesses the in vivo recombination apparatus of yeast. Using this approach, libraries can be prepared with different mutational loads in DNA segments of less than 30 amino acids so that they can be assembled into the remaining unaltered DNA regions in vivo with high fidelity. As a proof of concept, we present two eukaryotic-ligninolytic enzyme case studies: i) the enhancement of the oxidative stability of a H2O2-sensitive versatile peroxidase by independent evolution of three distinct protein segments (Leu28-Gly57, Leu149-Ala174 and Ile199-Leu268); and ii) the heterologous functional expression of an unspecific peroxygenase by exclusive evolution of its native 43-residue signal sequence.
Highlights
In the past two decades directed evolution strategies have had a huge impact on protein engineering and synthetic biology [1,2,3,4,5]
MORPHING is a method of generating DNA diversity based on the high frequency of homologous recombination of S. cerevisiae
Like many other ligninolytic oxidoreductases, unspecific peroxygenase (UPO) is not readily expressed in heterologous hosts so that it can be tailored by directed evolution and we recently addressed this problem by subjecting the whole UPO gene to several rounds of random mutagenesis, recombination and screening in S. cerevisiae
Summary
In the past two decades directed evolution strategies have had a huge impact on protein engineering and synthetic biology [1,2,3,4,5]. Combining directed evolution with new computational and hybrid approaches has allowed researchers to design ‘‘smart’’ mutant libraries to address bottlenecks in enzyme functionality, helping to maintain the balance between activity and stability, or even creating novel catalytic activities [6,7]. Despite the wide array of focused evolution methods, there remains a need for consistent domain mutagenesis/recombination strategies targeting specific protein subsets for random mutagenesis and recombination, while conserving the remaining protein regions. This kind of focused-indiscriminate approach has received little attention in the literature [19,20], it can effectively unmask structural determinants of specific enzymatic attributes, which can be optimized using the aforementioned methods
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