Enhanced enzyme performance by DNA shuffling

Abstract

In the quest for enhanced enzyme performance in non-natural applications, directed evolution emulates natural processes for the generation of diversity and selection of desirable traits. Classical methods for improving protein characteristics rely upon point mutation or cassette mutagenesis of a selected region within a desired sequence (i.e., focusing on a narrow sequence space). However, computer simulations of the evolution of linear sequences have demonstrated the importance of recombination of blocks of related sequences rather than sequence mutation alone. DNA shuffling includes in vivo and in vitro methods for recombination of nucleic acid sequences. In one format, DNA shuffling involves methods for in vitro homologous recombination of pools of related genes. For example, fragmentation of nucleic acid sequences that encode genes, can be followed by reassembly using the polymerase chain reaction (PCR). As the complete gene sequence can be shuffled, a wider sequence space can be accessed in searching for the desired, improved variant. Repeated cycles of recombination, optionally together with error-prone PCR to introduce point mutations, allow efficient molecular evolution of complex sequences in vitro, to provide biological molecules with improved properties.