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Biphenyl Dioxygenases:
Functional Versatilities
and Directed Evolution
2004-20622
이재학
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
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Directed evolution
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DNA Shuffling
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DNA Shuffling
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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.
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Random priming recombination
Random priming recombination
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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.
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Heteroduplex recombination
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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).
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Staggered extension process(StEP)
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Directed evolution
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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.
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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.
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