RESUMO
Saturation mutagenesis constitutes a powerful method in the directed evolution of enzymes. Traditional protocols of whole plasmid amplification such as Stratagene's QuikChange sometimes fail when the templates are difficult to amplify. In order to overcome such restrictions, we have devised a simple two-primer, two-stage polymerase chain reaction (PCR) method which constitutes an improvement over existing protocols. In the first stage of the PCR, both the mutagenic primer and the antiprimer that are not complementary anneal to the template. In the second stage, the amplified sequence is used as a megaprimer. Sites composed of one or more residues can be randomized in a single PCR reaction, irrespective of their location in the gene sequence.The method has been applied to several enzymes successfully, including P450-BM3 from Bacillus megaterium, the lipases from Pseudomonas aeruginosa and Candida antarctica and the epoxide hydrolase from Aspergillus niger. Here, we show that megaprimer size as well as the direction and design of the antiprimer are determining factors in the amplification of the plasmid. Comparison of the results with the performances of previous protocols reveals the efficiency of the improved method.
Assuntos
Evolução Molecular Direcionada/métodos , Biblioteca Gênica , Mutagênese , Reação em Cadeia da Polimerase/métodos , Aspergillus niger/enzimologia , Aspergillus niger/genética , Bacillus megaterium/enzimologia , Bacillus megaterium/genética , Proteínas de Bactérias/genética , Candida/enzimologia , Candida/genética , Sistema Enzimático do Citocromo P-450/genética , Epóxido Hidrolases/genética , Lipase/genética , NADPH-Ferri-Hemoproteína Redutase/genética , Plasmídeos , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genéticaRESUMO
Saturation mutagenesis probes define sections of the vast protein sequence space. However, even if randomization is limited this way, the combinatorial numbers problem is severe. Because diversity is created at the codon level, codon redundancy is a crucial factor determining the necessary effort for library screening. Additionally, due to the probabilistic nature of the sampling process, oversampling is required to ensure library completeness as well as a high probability to encounter all unique variants. Our trick employs a special mixture of three primers, creating a degeneracy of 22 unique codons coding for the 20 canonical amino acids. Therefore, codon redundancy and subsequent screening effort is significantly reduced, and a balanced distribution of codon per amino acid is achieved, as demonstrated exemplarily for a library of cyclohexanone monooxygenase. We show that this strategy is suitable for any saturation mutagenesis methodology to generate less-redundant libraries.
Assuntos
Códon , Mutagênese Insercional/métodos , Proteínas/genética , Aminoácidos/genética , Técnicas de Química Combinatória/métodos , Primers do DNA/genética , Biblioteca Gênica , Código Genético , Oxigenases/genéticaRESUMO
A current challenge in synthetic organic chemistry is the development of methods that allow the regio- and stereoselective oxidative C-H activation of natural or synthetic compounds with formation of the corresponding alcohols. Cytochrome P450 enzymes enable C-H activation at non-activated positions, but the simultaneous control of both regio- and stereoselectivity is problematic. Here, we demonstrate that directed evolution using iterative saturation mutagenesis provides a means to solve synthetic problems of this kind. Using P450 BM3(F87A) as the starting enzyme and testosterone as the substrate, which results in a 1:1 mixture of the 2ß- and 15ß-alcohols, mutants were obtained that are 96-97% selective for either of the two regioisomers, each with complete diastereoselectivity. The mutants can be used for selective oxidative hydroxylation of other steroids without performing additional mutagenesis experiments. Molecular dynamics simulations and docking experiments shed light on the origin of regio- and stereoselectivity.