Revisiting the Lipase from <i>Pseudomonas aeruginosa</i>: Directed Evolution of Substrate Acceptance and Enantioselectivity Using Iterative Saturation Mutagenesis

Prasad, Shreenath; Bocola, Marco; Reetz, Manfred T.

doi:10.1002/cphc.201100031

Cited by 29 publications

(20 citation statements)

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“…aturation mutagenesis (also called oligonucleotide-directed randomization) is a protein-engineering technique that has been used widely and successfully to improve protein properties such as catalytic activity, enantioselectivity, thermostability, and binding affinity (3,12,14,16). We use the term "activity" for the protein's property under optimization, but the methodology developed below is aimed at any desirable protein feature that may be influenced by mutation.…”

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confidence: 99%

When Second Best Is Good Enough: Another Probabilistic Look at Saturation Mutagenesis

Nov

2012

Appl Environ Microbiol

102

107

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We developed new criteria for determining the library size in a saturation mutagenesis experiment. When the number of all possible distinct variants is large, any of the top-performing variants (e.g., any of the top three) is likely to meet the design requirements, so the probability that the library contains at least one of them is a sensible criterion for determining the library size. By using a criterion of this type, one may significantly reduce the library size and thus save costs and labor while minimally compromising the quality of the best variant discovered. We present the probabilistic tools underlying these criteria and use them to compare the efficiencies of four randomization schemes: NNN, which uses all 64 codons; NNB, which uses 48 codons; NNK, which uses 32 codons; and MAX, which assigns equal probabilities to each of the 20 amino acids. MAX was found to be the most efficient randomization scheme and NNN the least efficient. TopLib, a computer program for carrying out the related calculations, is available through a user-friendly Web server. Saturation mutagenesis (also called oligonucleotide-directed randomization) is a protein-engineering technique that has been used widely and successfully to improve protein properties such as catalytic activity, enantioselectivity, thermostability, and binding affinity (3,12,14,16). We use the term "activity" for the protein's property under optimization, but the methodology developed below is aimed at any desirable protein feature that may be influenced by mutation.In saturation mutagenesis, one or more positions along the protein sequence are identified as likely to accommodate beneficial mutations and are then randomized, i.e., the amino acids at these positions are replaced by random ones. The randomization originates at the DNA level, typically via degenerate primers containing a mixture of sequences at the chosen codons. To decrease the chances of introducing a premature stop codon, reduced codon sets are often used: NNB, NNS, and NNK codons (where N ϭ A/C/G/T, B ϭ C/G/T, S ϭ C/G, and K ϭ G/T) are popular choices that still encode all 20 amino acids, but the use of codon sets encoding fewer amino acids has been also advocated (9, 15). More sophisticated randomization schemes, such as MAX (6, 7), result in equal probabilities for all 20 amino acids (or for some predetermined subset thereof) without encoding stop codons. Either way, a large number of random variants, which together constitute a library, are produced and then screened in an attempt to discover a highly active variant among them. Clearly, the larger the library, the higher the probability of exploring more distinct variants. We shall use the term "variant space" to denote the set of all possible distinct variants in a given experiment; this space is determined by the number of positions randomized and the randomization scheme.The probabilistic literature on saturation mutagenesis (1, 5, 8, 11) focuses mainly on two mathematical quantities: the first is the expected percentage of variant spa...

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confidence: 99%

When Second Best Is Good Enough: Another Probabilistic Look at Saturation Mutagenesis

Nov

2012

Appl Environ Microbiol

102

107

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“…The results from each CAST library can then be combined pairwise by multiple mutations or by iterative strategies and re-randomized as explained above. The impressive results obtained with enzyme specificity and enantio-selectivity [32,34] highlight the suitability of the method to evolve new functions for biocatalysts. Lipases are a good example of the powerful application of CAST.…”

Section: Methodsmentioning

confidence: 99%

“…Saturation mutagenesis has played a key role in engineering several lipases both for thermal stability and enantio-selectivity, with at least 20 research papers published in the last 5 years. Among groups involved in lipases engineering, Reetz and co-workers achieved relevant results [29,32,34] by applying SSM, ISM and CAST for enhancing enantio-selectivity and B-FIT for tuning thermal stability properties. The SSM approach was applied to Pseudomonas aeruginosa lipase, a well-known catalyst applied to hydrolysis of carboxylic acid esters and transesterification of primary and secondary alcohols, with the aim of redesigning the substrate recognition pocket to enable catalysis on more bulky substrates, such as benzoic acid esters.…”

Section: Recent Successful Applicationsmentioning

confidence: 99%

“…Further works on the same enzyme by ISM highlighted the enormous potentiality of iterative saturation versus other methods such as error prone PCR, shuffling and even the previous SSM, in particular for enhancing the stereospecificity of reactions. In fact, a more recent paper reports, on the same enzyme, the gain of function for the bulky 2-phenylalkanoic acid esters that are not recognized by the WT and the selection of variants with enantio-selectivity of E = 436, achieved with only small mutant libraries and thus a minimum of screening effort [34]. …”

Section: Recent Successful Applicationsmentioning

confidence: 99%

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Improvement of Biocatalysts for Industrial and Environmental Purposes by Saturation Mutagenesis

Valetti

Gilardi

2013

Biomolecules

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Laboratory evolution techniques are becoming increasingly widespread among protein engineers for the development of novel and designed biocatalysts. The palette of different approaches ranges from complete randomized strategies to rational and structure-guided mutagenesis, with a wide variety of costs, impacts, drawbacks and relevance to biotechnology. A technique that convincingly compromises the extremes of fully randomized vs. rational mutagenesis, with a high benefit/cost ratio, is saturation mutagenesis. Here we will present and discuss this approach in its many facets, also tackling the issue of randomization, statistical evaluation of library completeness and throughput efficiency of screening methods. Successful recent applications covering different classes of enzymes will be presented referring to the literature and to research lines pursued in our group. The focus is put on saturation mutagenesis as a tool for designing novel biocatalysts specifically relevant to production of fine chemicals for improving bulk enzymes for industry and engineering technical enzymes involved in treatment of waste, detoxification and production of clean energy from renewable sources.

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“…A recent survey regarding the location of mutations that improve enzyme properties shows that to manipulate the enantioselectivity of enzymes, closer mutations are more effective than distant mutations (7). With the availability of structural information, the methodology of CASTing (combinatorial activesite saturation test) used in an iterative manner has been particularly successful in tailoring enzyme enantioselectivity (8)(9)(10)(11).…”

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confidence: 99%

Exploring the Enantioselective Mechanism of Halohydrin Dehalogenase from Agrobacterium radiobacter AD1 by Iterative Saturation Mutagenesis

Guo

Chen

Zheng

et al. 2015

Appl Environ Microbiol

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Halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) shows great potential in producing valuable chiral epoxides and ␤-substituted alcohols. The wild-type (WT) enzyme displays a high R-enantiopreference toward most aromatic substrates, whereas no S-selective HheC has been reported to date. To obtain more enantioselective enzymes, seven noncatalytic active-site residues were subjected to iterative saturation mutagenesis (ISM). After two rounds of screening aspects of both activity and enantioselectivity (E), three outstanding mutants (Thr134Val/Leu142Met, Leu142Phe/Asn176His, and Pro84Val/ Phe86Pro/Thr134Ala/Asn176Ala mutants) with divergent enantioselectivity were obtained. The two double mutants displayed approximately 2-fold improvement in R-enantioselectivity toward 2-chloro-1-phenylethanol (2-CPE) without a significant loss of enzyme activity compared with the WT enzyme. Strikingly, the Pro84Val/Phe86Pro/Thr134Ala/Asn176Ala mutant showed an inverted enantioselectivity (from an E R of 65 [WT] to an E S of 101) and approximately 100-fold-enhanced catalytic efficiency toward (S)-2-CPE. Molecular dynamic simulation and docking analysis revealed that the phenyl side chain of (S)-2-CPE bound at a different location than that of its R-counterpart; those mutations generated extra connections for the binding of the favored enantiomer, while the eliminated connections reduced binding of the nonfavored enantiomer, all of which could contribute to the observed inverted enantiopreference.T he application of biocatalysts in the production of optically pure compounds has attracted much attention during the past few decades. Enantioselectivity (E) is one of the key parameters that define the usefulness of enzymes in the corresponding industrial synthesis of fine chemicals. Thus, identification of enzymes with high enantioselectivity for a desired transformation is important. In nature, biocatalysts with high enantioselectivity for specific industrial applications are not readily available. Directed evolution has become a routine approach for developing such novel biocatalysts, and a plethora of successful examples have been reported (1-6). A recent survey regarding the location of mutations that improve enzyme properties shows that to manipulate the enantioselectivity of enzymes, closer mutations are more effective than distant mutations (7). With the availability of structural information, the methodology of CASTing (combinatorial activesite saturation test) used in an iterative manner has been particularly successful in tailoring enzyme enantioselectivity (8-11).Microbial halohydrin dehalogenases attract a great deal of attention for their ability to produce optically pure epoxides and halohydrins (12, 13). The enzymes which are involved in the biodegradation pathway of halopropanols catalyze the intramolecular nucleophilic displacement of a halogen by a vicinal hydroxyl group in halohydrins, producing epoxide and HCl. Halohydrin dehalogenase HheC has been studied extensively because of its high enzymatic act...

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Revisiting the Lipase from Pseudomonas aeruginosa: Directed Evolution of Substrate Acceptance and Enantioselectivity Using Iterative Saturation Mutagenesis

Cited by 29 publications

References 58 publications

When Second Best Is Good Enough: Another Probabilistic Look at Saturation Mutagenesis

When Second Best Is Good Enough: Another Probabilistic Look at Saturation Mutagenesis

Improvement of Biocatalysts for Industrial and Environmental Purposes by Saturation Mutagenesis

Exploring the Enantioselective Mechanism of Halohydrin Dehalogenase from Agrobacterium radiobacter AD1 by Iterative Saturation Mutagenesis

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