Industrial Biotransformations

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4 Optimization of Industrial Enzymes by Molecular Engineering
4.4.3
Random Mutagenesis Methods
The simple concept of laboratory evolution – random mutagenesis and screening or
selection of the “fittest” – should be planned carefully by the experimenter right from the
beginning of the project. The first problem the scientist is confronted with is implementing
the huge sequence space of proteins: using the 20 building blocks of proteins, the
different amino acids encoded by the DNA, an enormous number of novel combinations
can be generated. For example, an enzyme consisting of 300 amino acid residues can
theoretically exist in 20 300 possible linear combinations of all 20 amino acids at each position.
This infinite number of combinations cannot be generated by any one scientist – at
least nor by nature itself, because the mass of such a library would exceed the mass of
the whole universe – which makes de novo enzyme design by completely random approaches
impossible. Therefore, two different strategies using good starting points in the
“fitness landscape” of protein-sequence space have been developed: random point mutations
and in vitro recombination.
The insertion of random point mutations into DNA has been used to generate mutant
strains since industrial biocatalysis began. UV and X-ray radiation or mutagenic chemicals
such as nitrous acid, formic acid or hydrazine have been used to generate production
strains in industry as a way of so-called “classical strain improvement”: random mutagenesis
of the whole genome and subsequent screening for better performing variants.
Today, this is still an option as a way of improving production strains without creating socalled
genetically manipulated organisms (GMOs). Many successful examples have been
published or patented; however, the accumulation of deleterious mutations in the genome
of the target organism makes the process difficult and unpredictable. Therefore, the
Biotech Company Maxygen (Redwood City, CA, USA) and its subsidiary Codexis (Redwood
City, CA, USA) have recently introduced a new strategy to speed up classical strain
improvement using whole genome shuffling. Here, the genomes of mutants created by
classical strain improvement are recombined by protoplast fusions, creating a pool of
recombinants, which is screened for better production strains. This technique has proved
to be useful for the rapid improvement in the production of the macrolite antibiotic tylosin
from Streptomyces fradiae [39] as well as changing the pH tolerance of a Lactobacillus
strain of commercial interest, so that it will grow under acidic conditions [40].
Nevertheless, most directed evolution strategies use in vitro mutagenesis methods
applied to defined target genes. According to this, the most commonly used method is
error prone PCR (epPCR), a technique that is as simple as regular PCR. The Taq-polymerase
4 , an enzyme without in vitro proof-reading activity, is used to amplify the target gene
under “suboptimal” conditions. This increases the naturally occurring error rate from
0.1–2 × 10 –4 [41, 42] to 1–5 × 10 –3 base substitutions by: (a) increasing the concentration of
MgCl 2, (b) addition of MnCl 2, (c) using an unbalanced concentration of nucleotides, or
(d) using a mixture of triphosphate nucleoside analogues [43–47]. In addition, companies
such as Statagene (La Jolla, CA, USA) or BD Biosciences – Clontech (Palo Alto, CA,
4) Abbreviation for the hyperthermophilic bacterium
Thermus aquaticus