DNA SHUFFLING OF A FAMILY OF GENES FROM DIVERSE SPECIES ACCELERATES DIRECTED EVOLUTION

DNA shuffling is a powerful process for directed evolution, which gene rates diversity by recombination(1,2), combining useful mutations from individual genes. Libraries of chimaeric genes can be generated by ra ndom frag;mentation of a pool of related genes, followed by reassembly of the fragments in a self-priming polymerase reaction. Template swit ching causes crossovers in areas of sequence homology. Our previous st udies used single genes and random point mutations as the source of di versity(3-6). An alternative source of diversity is naturally occurrin g homologous genes, which provide 'functional diversity.' To evaluate whether natural diversity could accelerate the evolution process, we c ompared the efficiency of obtaining moxalactamase activity from four c ephalosporinase genes evolved separately with that from a mixed pool o f the four genes. A single cycle of shuffling yielded eightfold improv ements from the four separately evolved genes, versus a 270- to 540-fo ld improvement from the four genes shuffled together, a 50-fold increa se per cycle of shuffling. The best clone contained eight segments fro m three of the four genes as well as 33 amino-acid point mutations, Mo lecular breeding by shuffling can efficiently mix sequences from diffe rent species, unlike traditional breeding techniques, The power of fam ily shuffling may arise from sparse sampling of a larger portion of se quence space.