Industrial Biotransformations

4.2
Learning from Nature
Nature itself appears to provide a solution to the apparent dilemma of the frequent
incompatibilities of enzyme properties and organic chemistry: natural evolution produces
enzyme variants by mutation and subsequently tests activity by selecting the “fittest”
variant. This process can be mimicked in a test-tube using modern molecular biological
methods of site directed or random mutagenesis and subsequent analysis of the
enzyme activity. This collection of (molecular) biological methods has been termed “molecular
engineering”, which provides a powerful tool for the development of biocatalysts
with novel properties.
Before beginning an enzyme optimization by means of molecular engineering, three
requirements are necessary: (a) the enzyme coding gene must be known and isolated
from gene-libraries or amplified directly from genomic DNA using PCR; (b) the identified
gene must be cloned into an appropriate expression vector for production of the
enzymatically active biocatalyst; and (c) the analysis of enzymatic activity must be established
to identify changes in biocatalyst properties. In the age of complete genome [9–13]
and environmental genome sequencing [14] projects, the availability of gene sequences
do not usually constitute a major drawback to molecular engineering approaches. However,
having the gene in ones hands does not as a consequence mean that the catalytically
active enzyme is actually available. Therefore, one essential requirement is the availability
of powerful expression systems based on homologous or heterologous microbial
hosts, as described in the next section.
4.3
Enzyme Production Using Bacterial Expression Hosts
An efficient bacterial overexpression system consists of a vector harboring the gene (or
genes) of interest under the control of a promoter that might be regulated by trans-acting
elements (so-called regulatory proteins) leading to a modulated gene expression in the
prokaryotic host cell. These expression vectors are naturally occurring extra-chromosomal
DNA, so-called plasmids, usually carrying resistance genes enabling the cells to survive
in toxic environments or to cope with antibiotic attacks from other microorganisms.
One of the first and best-studied “general purpose” cloning vectors is pBR322 1 .
This plasmid, isolated by Bolivar and Rodriguez et al. [15], consists of 4361 base pairs.
As shown in Fig. 4.1(A), pBR322 contains two antibiotic resistance genes (ampicillin and
tetracycline), an origin of replication for Escherichia coli as the host cell and unique
1) Plasmid names usually start with a “p”, which is
the abbreviation for plasmid. The subsequent letters
and/or numbers are abbreviations chosen by
the scientist who created or isolated the plasmid.
In the case of pBR322, the letters represent the
4.3 Enzyme Production Using Bacterial Expression Hosts
initials from the molecular biology scientists
Bolivar and Rodriguez, whereas the “322” is a
serial number.
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