Site saturation mutagenesis: Methods and applications in protein engineering
122
Citation
66
Reference
10
Related Paper
Citation Trend
Keywords:
Saturated mutagenesis
Protein Engineering
Thermostability
Site-directed mutagenesis
Protein design
Directed mutagenesis
This chapter treats exclusively directed evolution of proteins, the primary focus being on recent methodology developments. During the past 25 years, numerous gene mutagenesis methods have been developed. It is difficult even for experts to make the optimal choice, because comparative studies focusing on relative efficiency are rare. A few select studies in directed evolution are reviewed in the chapter, because they allow conclusions regarding the relative merits of different mutagenesis methods and strategies. In a landmark study, single-gene DNA shuffling was applied to turn Escherichia coli β-galactosidase (BGAL) into a β-fucosidase, thereby changing the substrate scope. The enzyme hydrolyzes β-galactosyl linkages such as the β (1,4)-linkage in lactose. In principle, epPCR and DNA shuffling are independent of structural data, whereas saturation mutagenesis generally needs such knowledge to make a decision regarding the randomization sites. Alternatively, saturation mutagenesis can be applied systematically at every single amino acid position, as was reported in the thermostabilization of a xylanase. Subsequent to the first directed evolution study regarding the enantioselectivity of enzymes, which involved four cycles of epPCR in the hydrolytic kinetic resolution of a chiral ester catalyzed by a lipase, numerous academic and industrial studies have appeared that contribute to the generalization of this new approach to asymmetric catalysis. Subsequent investigations addressed the reasons for the apparent efficacy of iterative combinatorial active-site saturation testing as a form of iterative saturation mutagenesis (ISM).
Cite
Citations (2)
Thermostability
Saturated mutagenesis
Protein Engineering
Site-directed mutagenesis
Directed mutagenesis
Cite
Citations (25)
Terpenes represent about half of known natural products, with terpene synthases catalyzing reactions to increase the complexity of substrates and generate cyclizations of the linear diphosphate substrates, therefore forming rings and stereocenters. With their diverse functionality, terpene synthases may be highly evolvable, with the ability to accept a wide range of non-natural compounds and with high product selectivity. Our hypothesis is that directed evolution of terpene synthases can be used to increase selectivity of the synthase on a specific substrate. In the first part of the work presented herein, three natural terpene synthases, Cop2, BcBOT2, and SSCG_02150, were tested for activity against the natural substrate and a non-natural substrate, called Surrogate 1, and the relative activities on both the natural and non-natural substrates were compared. In the second part of this work, a terpene synthase variant of BcBOT2 that has been evolved for thermostability, was used for directed evolution for increased activity and selectivity on the non-natural substrate referred to as Surrogate 2. Mutations for this evolution were introduced using random mutagenesis, with error prone polymerase chain reactions, and using site-specific saturation mutagenesis, in which an NNK library is designed with a specific active site amino acid targeted for mutation. The mutant enzymes were then screened and selected for enhancement of the desired functionality. Two neutral mutants, 19B7 W367F and 19B7 W118Q, were found to maintain activity on Surrogate 2, as measured by the screen.
Saturated mutagenesis
Terpene
Protein Engineering
Thermostability
Natural product
Cite
Citations (0)
Directed evolution is a new developed approach to protein modification engineering.Cytochrome P450BM-3 from B.Megaterium was evolved by random mutagenesis and Site-saturation mutagenesis.Some P450BM-3 mutants were obtained by high through-put screening of the combination with the active agar plates with some amount of substrate indole and 96-well microtile plates of catalytic activities according to colourimetric product formation of blue pigment indigo that the P450BM-3 mutants are able to produce.
Saturated mutagenesis
Site-directed mutagenesis
Directed mutagenesis
Protein Engineering
Cite
Citations (0)
Esterase
Cite
Citations (47)
Saturated mutagenesis
Thermostability
Directed mutagenesis
Site-directed mutagenesis
Protein Engineering
Cite
Citations (216)
Abstract Directed evolution has emerged as a powerful method for engineering the catalytic profile of enzymes. It is based on repetitive cycles of random gene mutagenesis and expression coupled with efficient high‐throughput screening or selection for a given catalytic property such as thermostability, substrate acceptance, and/or enantioselectivity. In the 1990s, directed evolution was established on a broad front by applying standard mutagenesis methods such as error‐prone polymerase chain reaction (epPCR), saturation mutagenesis, and DNA shuffling. The current challenge is to devise optimal strategies for probing protein sequence space, thereby allowing for fast directed evolution. This article covers the progress of the last few years.
DNA shuffling
Saturated mutagenesis
Thermostability
Protein Engineering
Directed Molecular Evolution
Site-directed mutagenesis
Shuffling
Directed mutagenesis
Sequence space
Cite
Citations (0)
Abstract Ionic liquids (ILs) are attractive (co‐)solvents for biocatalysis. However, in high concentration (>10 % IL), enzymes usually show decreased activity. No general principles have been discovered to improve IL resistance of enzymes by protein engineering. We present a systematic study to elucidate general engineering principles by site saturation mutagenesis on the complete gene bsla . Screening in presence of four [BMIM]‐based ILs revealed two unexpected lessons on directed evolution: 1) resistance improvement was obtainable at 50–69 % of all amino acid positions, thus explaining the success of small sized random mutant libraries; 2) 6–13 % of substitutions led to improved resistance. Among these, 66–95 % were substitutions by chemically different amino acids (e.g., aromatic to polar/aliphatic/charged amino acids), thus indicating that mutagenesis methods introducing such changes should, at least for lipases like BSLA, be favored to improve IL resistance.
Saturated mutagenesis
Protein Engineering
Biocatalysis
Cite
Citations (47)
Abstract The most thoroughly studied enzyme in directed evolution is the lipase from Pseudomonas aeruginosa (PAL) as a catalyst in the hydrolytic kinetic resolution of 2‐methyldecanoic acid p ‐nitrophenyl ester. Seminal studies utilized epPCR, saturation mutagenesis and DNA shuffling or combinations thereof. With current emphasis on efficacy in laboratory evolution, however, we recently applied our previously developed method, iterative saturation mutagenesis (ISM), to the same catalytic system, discovering that this approach is much more efficient than the original strategies. Herein, we consider PAL once more, this time testing ISM as a means to broaden the substrate scope of this lipase by studying bulky substrates of the type 2‐phenylalkanoic acid esters as substrates that are not accepted by the WT. Highly active and enantioselective ( E up to 436) mutants were evolved, a process that required only small mutant libraries and thus a minimum of screening effort. A theoretical investigation using molecular dynamics simulations and docking experiments revealed the source of enhanced activity and stereoselectivity.
Saturated mutagenesis
Saturation (graph theory)
Enzyme Kinetics
Cite
Citations (30)
The traditional approaches used in directed evolution, including mutator strains, error-prone polymerase chain reaction, gene insertion and deletion, saturation mutagenesis, DNA shuffling and related recombinant gene mutagenesis, circular mutation and domain swapping, as well as solid-phase gene synthesis techniques, are presented critically in this chapter. Computational tools and approaches, as well as machine learning algorithms established in rational enzyme design, are also described. As outlined in Section 3.18 of this chapter, the techniques in directed evolution and rational design no longer develop on separate tracks but have merged recently with the construction of powerful techniques such as Focused Rational Iterative Site-specific Mutagenesis (FRISM).
Rational design
Saturated mutagenesis
Shuffling
DNA shuffling
Directed mutagenesis
Site-directed mutagenesis
Protein Engineering
Synthetic Biology
Cite
Citations (0)