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240 10 Perspectives on Multienzyme Process Technology

10.5.1
Recombinant DNA Technology

The development of recombinant DNA (rDNA) technology enables several possi-
bilities for the real exploitation of biocatalysts in the full sense of the word. First it
has provided a cheap way to produce a given biocatalyst. The desired enzyme (or
enzymes) can now be overexpressed meaning that it represents a much bigger frac-
tion of the available protein in the cell. This not only reduces the required scale of
the fermentation but for isolated enzyme applications also reduces the downstream
burden prior to biocatalysis. Secondly a synthetically interesting enzyme found in
nature may be expressed in a poor host for production (e.g., the host may be
pathogenic or may grow only under conditions far from those used for application).
Such a situation can be overcome by genetic engineering through cloning into a
new host organism. Typical hosts in an industrial setting are, for example, Bacillus
subtilis, Escherichia coli,or Pichia pastoris as they are fast growing (overcoming the
risk of contamination) and the genetics are well understood, although others are
used, depending on the application. In some cases, protein secretion is possible.
These developments have revolutionized the biotechnology industry because they
have provided biocatalysts at a reasonable cost. The ability to grow cells to a high
titer based on sophisticated fed-batch feeding profiles has also had a major impact.
Recombinant DNA technology also enables an alteration of the properties of the
biocatalyst. For cells this can involve alteration of pathways (blocking nonproductive
routes) and increasing flux or even creating de novo pathways [30, 31]. Today this
is a hugely exciting area of industrial biotechnology that will develop into entirely
new routes to chemicals. Whether it is to be carried out inside or outside the cell is
still an open question. In some cases, compartmentalization is useful and in other
cases it is not. For enzyme options too, purification, immobilization, and protein
engineering to improve specific activity under the required condition must be con-
sidered. For example, in a two enzyme scheme, an enzyme may be engineered to
work under a compromised condition for both or at the optimum for one or alterna-
tively the other enzyme, depending on the relative costs [32]. The ability to swap the
amino acids either in the active site or even at remote positions of the protein has
been found capable of altering and controlling substrate repertoire, stability, activity
(reaction rate), and selectivity. Today, synthetic chemists routinely use so called
‘‘directed evolution’’ combined with rational strategies based on structure–function
relationships to engineer proteins [33]. For the future, this will be applied to many
more processes at a full scale. Examples already exist but it is clear this will
develop enormously in the near future. Most complex is that improvements in both
the biocatalyst(s) and the process need to go hand-in-hand. Consequently process
engineers have an important role here in integrating the targets required for a
cost-effective process together with the possibilities provided by the ‘‘biocatalyst
engineers.’’