Schematic diagram outlining the general approach adopted to alter the amino acid (aa) sequence by site-directed mutagenesis. The DNA fragment encoding the wild-type (unaltered) protein of interest is cloned into a plasmid vector. An appropriate oligonucleotide (oligo) is designed, the ends of which are complementary to the nucleotide (nt) sequence of the gene, while the central portion of the oligo contains an altered nt sequence corresponding to the desired change in the protein (marked with a star). The dsDNA is denatured to make the template strand available for hybridization with the mutagenic oligo (Step 1). The addition of dNTPs, DNA polymerase, and DNA ligase enables DNA synthesis, forming closed circular molecules (Step 2). The mutated plasmid is selected, and the desired change is verified (by sequencing). The mutant plasmid is selected and transformed into a final host strain (Step 3), where large amounts of the mutant protein can be produced using standard recombinant methods.
design approaches to enzyme engineering are not particularly suited to generating enzymes in a random, selection-driven manner . In direct contrast to rational design, the technique of directed evolution requires minimal knowledge of the enzyme mechanism and of how the enzyme structure relates to its function [26-28,52,56]. Directed evolution approaches involve either random mutagenesis of the gene encoding the enzyme or recombination of gene fragments encoding homologs of the enzyme in question, followed by stringent selection and screening processes for identifying enzymes with desired properties. In this respect, the directed evolution approach mimics Darwinian evolution. In contrast to the natural evolution of species, which can take millions of years, directed evolution approaches can be performed in any modern molecular biology laboratory in a matter of weeks or months, and with an unlimited number of parents [33,37,38].
Directed evolution is essentially a two-step procedure (Figure 2.3):
1. Generation of initial diversity, followed by cloning this library into an appropriate expression vector and host organism
2. Screening and selection of the library of mutants created in step 1 to obtain an enzyme with the desired altered trait(s)
General approach to directed evolution
1. Target gene(s)
2. Library of recombinant/ mutated genes
3. Library of expressed recombinant/ mutated proteins
4. Protein(s) with desired properties
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