Directed evolution of antibiotic resistant and DNA modifying enzymes

Since the fIrst site directed mutagenesis in the mid 1980s, protein engineering has become a very powerful means for generating enzyme variants that illuminate the relationship between enzyme sequence, structure and function. This is having an increasing impact on our ability to improve the utility of enzymes for industrial applications. The new approach to protein engineering, directed evolution that mimics Darwinian evolution, can explore a sequence space very effectively in a short space of time if there is a good screen/selection available for the enzyme. We applied directed evolution on Xenobiotic acetyl transferase from Agrobacterium tumeJacians (AtXAT), which confers low level resistance to the antibiotic chloramphenicol. Three rounds of mutagenesis resulted in XAT variants with 100-fold higher resistance toward the antibiotic. Analysis of the clones showed the constitutive nature of XAT gene expression as a result of mutations in the promoter region. The mutagenesis and selection cycles were reapplied to the ORF of AtXAT, and mutants were isolated demonstrating a 20-fold higher chloramphenicol resistance. Analysis of the mutants showed one key mutation out of five amino acid changing mutations that resulted in higher stability of the enzyme. The enhanced stability was a result of a salt-bridge formation on the surface ofthe protein. We also applied directed evolution to M.HhaI and M.HaeIII C5-methyltrasferase to alter the DNA sequence specifIcity. C5-methyl transferases are very useful tools for molecular biology and provide a good system to understand nucleic acid-protein interactions. Using DNA shuffling and an algorithm known as SCHEMA we created a library of mutants and chimeras for the two above-mentioned enzymes, but a more robust screening method is needed to identify mutants/hybrids with altered specifIcity.EThOS - Electronic Theses Online ServiceGBUnited Kingdo