Page 38 - Cascade_Biocatalysis_Integrating_Stereoselective_and_Environmentally_Friendly_Reactions

14 1 Directed Evolution of Ligninolytic Oxidoreductases

(ii) The K for H O was enhanced up to 15-fold while the catalytic efficiency
m 2 2
was maintained. We are currently working on evolving oxidative stability
(i.e., resistance to H O ) using the R4 mutant as the initial variant. All
2
2
peroxidases are inhibited by catalytic concentrations of H O (so-called sui-
2
2
cide inactivation), a mechanism-based phenomena that has provoked great
PCR 1
PCR 1 product

Parent gene Linearized
plasmid
PCR 2 product
(a) PCR 2 In vivo recombination
Mutagenic library 1

Parent gene
(b) Linearized plasmid
Mutagenic library 2 In vivo recombination
Primer

Linearized
plasmid
Annealing
Parent genes and Denaturing and Mutagenic
(c) extension annealing (with the library In vivo recombination
introduction of mutations)
Parent genes
DNAse I

Random Random PCR Linearized
(d) fragmentation assembly amplification plasmid In vivo recombination
Focused PCR insert
mutagenic PCR (with point mutations)

Parent gene Linearized
plasmid
High fidelity
(e) High fidelity PCR PCR inserts In vivo recombination

P1 G1 T1
P2 G2 T2
P3 G3 T3
Gene expression Linearized plasmid
(f) cassettes In vivo recombination

Figure 1.5 Different genetic methods for library creation in S. cerevisiae: (a) IVOE;
(b) IvAM; (c) StEP + in vivo DNA shuffling; (d) CLERY; (e) MORPHING; and (f) DNA
assembler.