The Diversity Challenge in Directed Protein Evolution

Abstract

Over the past decade, we have witnessed a bloom in the field of evolutive protein engineering which is fueled by advances in molecular biology techniques and high-throughput screening technology. Directed protein evolution is a powerful algorithm using iterative cycles of random mutagenesis and screening for tailoring protein properties to our needs in industrial applications and for elucidating proteins' structure function relationships. This review summarizes, categorizes and discusses advantages and disadvantages of random mutagenesis methods used for generating genetic diversity. These random mutagenesis methods have been classified into four main categories depending on the method employed for nucleotide substitutions: enzyme based methods (Category I), synthetic chemistry based methods (Category II), whole cell methods (Category III) and combined methods (Category I-II, I-III and II-III). The basic principle of each method is discussed and varied mutagenic conditions are summarized in Tables and compared (benchmarked) to each other in terms of: mutational bias, controllable mutation frequency, ability to generate consecutive nucleotide substitutions and subset diversity, dependency on gene length, technical simplicity/robustness and cost-effectiveness. The latter comparison shows how highly-biased and limited current diversity creating methods are. Based on these limitations, strategies for generating diverse mutant libraries are proposed and discussed (RaMuS-Flowchart; KISS principle). We hope that this review provides, especially for researchers just entering the field of directed evolution, a guide for developing successful directed evolution strategies by selecting complementary methods for generating diverse mutant libraries.

Enhancement of Cellulose Degradation Potential of Bacillus sp. Hcb-21 through Mutagenesis

“…Secondary and tertiary structures of protein also change when these forces are disturbed. Some researchers have also reported maximum production of cellulase at 40-50°C [29][30][31]. To increase the enzyme yield, mutant like E-11 produced in the present study seem extremely useful which can be successfully employed as a bioresource for various important biotechnological applications such as animal feed, textiles, pharmaceutical and chemical synthesis, clarification of fruit juices, manufacturing of bread and wine and renewable energy products, such as bio-ethanol after extensive R&D and scale-up/demonstration efforts.…”

Protein Engineering Methods and Applications

“…Rational design (Arnold, 1993) Site-directed mutagenesis (Arnold, 1993), (Antikainen & Martin, 2005) Evolutionary methods/directed evolution (Arnold, 1993) Random mutagenesis (Antikainen & Martin, 2005), (Wong et al, 2006), (Jackson et al, 2006), (Labrou, 2010) DNA shuffling (Antikainen & Martin, 2005), (Jackson et al, 2006) Molecular dynamics (Anthonsen et al, 1994) Homology modeling (Anthonsen et al, 1994) 'MolCraft'in vitro protein evolution systems (Shiba, 2004) Computational methods (computational protein design) (Jackson et al, 2006), (Van der Sloot et al, 2009), (Golynskiy & Seelig, 2010) Receptor-based QSAR methods (Lushington et al, 2007) NMR (Anthonsen et al, 1994) X-ray crystallography (Jackson et al, 2006) Peptidomimetics (Venkatesan & Kim, 2002) Phage display technology (Antikainen & Martin, 2005), (Sidhu & Koide, 2007), (Chaput et al, 2008) Cell surface display technology (Antikainen & Martin, 2005), (Gai & Wittrup, 2007), (Chaput et al, 2008) Flow cytometry / Cell sorting (Mattanovich & Borth, 2006 ) Cell-free translation systems (Shimizu et al, 2006) Designed divergent evolution (Yoshikuni & Keasling, 2007) Stimulus-responsive peptide systems (Chockalingam et al, 2007) Mechanical engineering of elastomeric proteins (Li, 2008) Engineering extracellular matrix variants (Carson & Barker, 2009) Traceless Staudinger ligation (Tam & Raines, 2009) De novo enzyme engineering (Golynskiy & Seelig, 2010) mRNA display …”

“…Their comparison was made according to a variety of parameters such as controllable mutation frequency, technical robustness, cost-effectiveness, etc. (Wong et al, 2006). "Cell-free translation systems" were also described as important tools for protein engineering and production.…”

To get what we aim for – progress in diversity generation methods

“…stabilities in presence of organic solvents, ionic liquids or detergents). Random mutagenesis methods have been grouped into three mutagenesis categories: (a) PCR-based; (b) chemical mutagens; and (c) whole cell methods [1,18]. Progress in the last 3 years is summarized by covering multicodon scanning mutagenesis (MCST), 2′-deoxyinosine 5′-triphosphate (dIT)P-PCR, prolonged overlap extension-PCR (POE-PCR), MegAnneal, tandem repeat insertion (TRINS), error-prone multiply-primed rolling circle amplification (epRCA), sequence saturation mutagenesis (SeSaM)-Tv-II and SeSaM-III [56][57][58][59][60][61][62][63] (Fig.…”