Industrial Biotransformations

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4 Optimization of Industrial Enzymes by Molecular Engineering
Site specific saturation mutagenesis is a method that can overcome the drawback of error
prone PCR mentioned above. This method generates all 19 amino acid exchanges virtually,
at a given position, by using customized degenerated oligonucleotides 5 , introducing
all possible codons. These degenerated oligonucleotides are usually incorporated into
the target gene by PCR reactions. This can be achieved by applying mega-primer PCR or overlap
extension PCR protocols [55, 56]. This technique was initially established to find ideal
amino acid exchanges at hot spot positions identified by other methods such as error prone
PCR. This approach is presently been extended to complete gene sequences, thereby generating
a complete saturation mutagenesis library containing all single mutants calculated by the
formulae given in Table 4.5 [54, 57, 58].
When evolving an enzyme by iterative rounds of random mutagenesis and screening,
the best performing variant identified in each generation constitutes the starting point
for the next round of mutagenesis (Fig. 4.2), thus many useful variants are discarded.
Furthermore, it is not obvious whether the selected variant will have the potential for
further improvement in the next generation or not. Deleterious mutations could be accumulated,
leading to a dead end for the in vitro evolution. The second evolution strategy based
on in vitro recombination techniques helps to overcome this problem. By recombining a pool
of better performing variants, libraries of chimeric genes can be produced, providing the possibility
of removing deleterious mutations and combining beneficial ones.
DNA shuffling was the first method described for in vitro recombination, and it immediately
proved to be a valuable tool for directed evolution of biocatalysts [59, 60]. Homologous
DNA sequences carrying mutations are recombined in vitro in a process consisting of random
gene fragmentation using DNase I and subsequent reassembly in a self-priming chain
extension method catalyzed by DNA polymerase. Recombination occurs by template switching:
a fragment originating from one gene anneals to a fragment from another gene.
DNA shuffling has been applied in a wide variety of experiments to improve single
gene encoded enzymes [61, 62] as well as operon encoded multi-gene pathways [63].
Furthermore, DNA shuffling has been used to improve viruses in terms of stability and
processing yields as well as changes in specificity (also known as tropism) for better application
in human gene therapy [64–66].
In addition to DNA shuffling, other efficient recombinative methods to generate variant
libraries followed including the PCR-based staggered extension process (StEP) [67] or
the assembly of designed oligonucleotides (ADO) [68]. Furthermore, many closely related
methods or “updates” have been described, as summarized in two recently published
review articles [69, 70] (see also Table 4.4).
All in vitro recombination methods described so far require relatively high levels of
DNA homology in the target sequences, otherwise the recombination events will only
occur in regions of homology or they will not occur at all. A different approach to create
libraries of fused gene fragments independent of sequence homology has been developed,
which was termed incremental truncation for the creation of hybrid enzymes (ITCHY)
[71]. Two parental genes are digested with exonuclease III in a tightly controlled manner
to generate truncated gene libraries with progressive 1 base pair deletions. The truncated
5) The genetic code is said to be degenerated
because more than one nucleotide triplet codes
for the same amino acid.