Development of novel genetic diversity-generation methods and their applications to directed protein evolution

Abstract

A method of mutagenic and unidirectional reassembly (MURA) that can generate libraries of DNA-shuffled and randomly truncated proteins was developed. The method involved fragmenting the template gene(s) randomly by DNase I and reassembling the small fragments with a unidirectional primer by PCR. The MURA products were treated with T4 DNA polymerase and subsequently with a restriction enzyme whose site was located on the region of the MURA primer. The N-terminal-truncated and DNA-shuffled library of a Serratia sp. phospholipase $A_1$ prepared by this method had an essentially random variation of truncated size and also showed point mutations associated with DNA shuffling. After high-throughput screening on triglyceride-emulsified plates, several mutants exhibiting absolute lipase activity (NPL variants) were obtained. The sequence analysis and the lipase activity assay on the NPL variants revealed that N-terminal truncations at a region beginning with amino acids 61 to 71, together with amino acid substitutions, resulted in the change of substrate specificity from a phospholipase to a lipase. We therefore suggest that the MURA method, which combines incremental truncation with DNA shuffling, can contribute to expanding the searchable sequence space in directed evolution experiments. A method termed as shuffled reassembly for simultaneous generation of homologous and non-homologous recombination from multiple parents (SHAREMP) was also developed by improving the MURA method. This method involved reassembling the small fragments of each gene group with an appropriate MURA primer, joining two overlapped-MURA libraries, and cloning them by a modified protocol of SHIPREC. The SHAREMP method could produce multiple crossovers without DNA sequence limitations, in which multiple homologous and non-homologous crossovers were found in applying the method to 4 bacterial metalloprotease genes (prtA, aprX, prtB, and prtC). The combination of DNA shuffling and sequence homolog...