Error-Prone PCR and Effective Generation of Gene Variant Libraries for Directed Evolution

Abstract

Any single-enzyme directed evolution strategy has two fundamental requirements: the need to efficiently introduce variation into a gene of interest and the need to create an effective library from those variants. Generation of a maximally diverse gene library is particularly important when employing nontargeted mutagenesis strategies such as error-prone PCR (epPCR), which seek to explore very large areas of sequence space. Here we present comprehensive protocols and tips for using epPCR to generate gene variants that exhibit a relatively balanced spectrum of mutations and for capturing as much diversity as possible through effective cloning of those variants. The detailed library preparation methods that we describe are generally applicable to any directed evolution strategy that uses restriction enzymes to clone gene variants into an expression plasmid.

Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently

“…Refinement of these methods has allowed greater control over the mutation bias, rate of mutations 530,535–537 and the development of alternative methodologies like Mutagenic Plasmid Amplification, 538 replication, 539 error-prone rolling circle 540 and indel 541–543 mutagenesis. Typically, for reasons indicated above, the epPCR mutation rate is tuned to produce a small number of mutations per gene copy (although orthogonal replication in vivo may improve this 544 ), since entirely random epPCR produces multiple stop codons (3 in every 64 mutations) and a large proportion of non-functional, truncated or insoluble proteins.…”

Control of metabolite efflux in microbial cell factories: current advances and future prospects

“…In the past, and partly because of the enormous number of possible sequences [374][375][376] this was done rather empirically, using methods such as error-prone PCR (ePCR) [377][378][379] to introduce mutations. Although showing the utility of the general directed evolution strategy, this had three highly undesirable consequences: (i) there was no control over which mutations were made, (ii) the search could only be local, as high mutation rates necessarily introduced stop codons [379,380], and (iii) the reliance on selection of local 'winners' as starting points for the next generation inevitably meant that search was soon trapped in local minima from which it was impossible to escape (as was evident from many published studies showing a lack of further improvement after 3 or so generations, despite quite poor k cat values) [324].…”

Functional Site Discovery in a Sulfur Metabolism Enzyme by Using Directed Evolution

“…To introduce random mutations,w eu sed the GeneMorph II system, which uses a unique error-prone PCR enzyme blend to achievee quivalent rates of mutation at A'sa nd T's vs. G's and C's. [13] In the initial stage of this study,w ev aried the amount of input DNA template to generally allow mutagenesisa to nly as ingle site per round of selection. The PAPR mutant library was generated in the pBR322 plasmide ncoding the rop gene, restricting the plasmidc opyn umber to~15-20 per cell.…”