A novel directed evolution approach for co-evolution of β-glucosidase activity and organic acid tolerance

Directed evolution is a potent tool for protein engineering; however, Error-prone PCR and DNA Shuffling often lead to a high frequency of negative and reverse mutations, especially in the case of large genes. This study introduces two innovative techniques to tackle these challenges: Segmental error-prone PCR (SEP) and Directed DNA shuffling (DDS). SEP involves averagely dividing large genes into small fragments, independently and randomly mutagenizing them in vitro, and reassembling them as well as other unmutated fragments in Saccharomyces cerevisiae. DDS selectively amplifies mutated fragments of positive variants from SEP and reassembles them in S. cerevisiae to produce complete genes with cumulative positive mutations. We have used these two techniques to simultaneously improve the activity of β-glucosidase and its tolerance to organic acids, which validates the effectiveness and feasibility of the approach.