Biphenyl Dioxygenases: Functional Versatilities and Directed Evolution

1. Biphenyl Dioxygenases: • Functional Versatilities • and Directed Evolution 2004-20622 이재학

2. Directed evolution • Natural enzymes are usually not so well suited for biotechnology applications, because of the distinct conditions and different demands • Biocatalysis applications depend on methods to tailor nature's catalysts or redesigning them anew • Laboratory evolution methods are now used widely to fine-tune the selectivity & activity of enzymes • Requirements for directed evolution • 1. Desired function must be physically possible • 2. The function must be biologically/evolutionary feasible • 3. Generation of ‘large’ variant libraries • 4. Rapid screen or selection for desired function

3. Directed evolution

4. DNA Shuffling

5. DNA Shuffling

6. Random priming recombination • A simple and efficient method for in vitro mutagenesis and recombination of polynucleotide sequences. • The method involves priming template polynucleotide(s) with random-sequence primers and extending to generate a pool of short DNA fragments which contain a controllable level of point mutations. • The fragments are reassembled during cycles of denaturation, annealing and further enzyme- catalyzed DNA polymerization to produce a library of full-length sequences. • Screening or selecting the expressed gene products leads to new variants with improved functions, as demonstrated by the recombination of genes encoding different thermostable subtilisins in order to obtain enzymes more stable than either parent.

7. Random priming recombination Random priming recombination

8. Heteroduplex recombination • The optimized insert heteroduplex recombination protocol efficiently generates chimeric DNA libraries with sequence components from two parent sequence. • The Multiple homologous sequences can be recombined and ‘shuffled’ by this approach, simply by repeating the heteroduplex formation and transformation steps with additional sequences. • This method should be effective for recombining relatively large target sequences.

9. Heteroduplex recombination

10. Staggered extension process(StEP) • StEP recombination. Only one primer and single strands from two parent genes (templates) are shown. • (A) Denatured template genes are primed with one defined primer. • (B) Short fragments are produced by brief Polymerase-catalyzed primer extension. • (C) Through another cycle of StEP, fragments randomly prime the templates (template switching) and extend further. • (D) This process is repeated until full-length genes are produced resulting in a genepool of recombined parent genes (E).

11. Staggered extension process(StEP)

12. Directed evolution

13. Directed evolution of biphenyl dioxygenases • Directed evolution of biphenyl dioxygenases revealed that they can be grouped onto families that are similar in size and amino acid sequence. Enzyme belonging to the same family have evolved from a common ancestor to acquire a new catabolic function through various genetic events, such as gene transfer, recombination, duplication, multiple point mutation, deletion, and integration. Thus, we could learn how new degradation abilities appeared through a long historcal period. • The deduced amino acid sequences of such evolved large subunits showed only a few amino acid changes from the original enzymes.

14. Conclusion • Biphenyl dioxygenase is an interesting enzyme that provides a good model system for molecular evolutionary engineering. • It demonstrates that evolved biphenyl dioxygenases can be used for the degradation of PCBs and other environmental pollutants, including dioxins and chlorinated ethenes. • The use of evolved biphenyl dioxygenases is effective for the synthesis of high-value organic molecules.