[Molecular evolution of AdoMet synthetase by DNA recombination with a novel separate-mixing method]

We describe a new approach to in vitro DNA recombination termed Separate-Mixing method in this study. The reaction process of this method consists of two stages: at the first stage the reaction was implemented in two parallel teams, which generated random recombination by template-switching of growing polynucleotides from primers in the presence of unidirectional single-stranded DNA fragments used as templates, and then both teams were mixed together for further extension and recombination of DNA sequences at the second stage. Because of the particular strategy, the reaction process was also accompanied by the other two processes of DNA shuffling and StEP simultaneously. Two AdoMet synthetase genes sam2 from Saccharomyces cerevisiae and metK from Escherichia coli, which have only 56% homology on the DNA level were used for recombination with Separate-Mixing method. DNA recombination was available after a single round of reaction. With sequencing of 10 randomly selected recombinants, no unshuffled parental clone was found, and also no unexpected insertion, deletion or rearrangement was detected. An evolved gene sam' was obtained after screen and selection, which could obviously increase the accumulation of AdoMet in S. cerevisiae.