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402 Metabolic Engineering

divided into seven categories, depending on the approach tems for improved performance for heterologous protein
taken or of the aim: production. A major problem encountered with this strat-
egy is, however, that the secretory capacity in a given ex-
Heterologous protein production pression system is strongly protein specific. Thus, for one

Extension of substrate range protein it may be a certain step in the secretion pathway

Pathways leading to new products that is limiting the production, e.g., folding, formation of

Pathways for degradation of xenobiotics disulfide bridges, or glycosylation, whereas other proteins

Engineering of cellular physiology for process may easily be processed through the pathway and secreted

improvement at high rates. Even when working with generic expression
Elimination or reduction of by-product formation systems it is therefore normally necessary to further opti-

Improvement of yield or productivity mize the expression system for the specific protein to be

produced, and there have been several attempts to engineer
Belowfollowsashortdiscussionofthedifferentcategories the glycosylation pathway or the secretion pathway.
with some presentation of a few examples.
B. Extension of Substrate Range
A. Heterologous Protein Production
Biotech processes are attractive for replacement of many
The first breakthrough in genetic engineering paved the classical chemical processes, since they potentially may
way for a completely new route for production of phar- apply agricultural waste products as raw materials (typi-
maceutical proteins like human growth hormone (hGH) cal as carbon source), and thereby sustainable industrial
and human insulin; it also opened the possibility to pro- processes may be obtained for the production of fuels,
duce many other pharmaceuticals. The first products (hu- chemicals, and materials. Often the industrial strain ap-
man insulin and hGH) were produced in recombinant plied for a given process has a narrow substrate spectrum,
E. coli, but soon followed the exploitation of other ex- and it is therefore necessary to extend the substrate range.
pression systems like S. cerevisiae (introduced for pro- Here it is relevant to consider two different strategies:
duction of human insulin), insect cells, and mammalian
cells (Chinese hamster ovary cells and hybridoma cells). Introduction of a gene encoding a membrane-bound
Today there are more than 55 protein drugs, largely recom- protein that transports the substrate into the cell in
binant proteins and monoclonal antibodies that are often addition to genes encoding the necessary pathway that
referred to as biotech drugs, and the 20 top-selling drugs is responsible for channeling the substrate into the
represents sales of more than 16 bio US$. The choice of central carbon metabolism
expression system depends upon many factors: Introduction of a gene encoding a secreted protein that
converts the substrate to compounds that can be
1. the desirability of posttranslational modification and directly assimilated and metabolized by the host
secretion organism
2. the stability of the protein in question
3. the projected dose of protein per patient (which Expression of specific permeases is difficult, but the first
determines whether the cost of the drug becomes strategy is still the preferred one for engineering cells to
critical) take up monosaccharides since often these compounds can
be taken up by nonspecific permeases. Thus, one can focus
Thus for proteins used in large doses, like human insulin, it on engineering the pathway converting the monosaccha-
is important that the production costs are kept low, which ride or disaccharide into the central carbon metabolism.
requires an expression system with a high productivity, The second strategy is typically applied for di-, oligo-, and
i.e., E. coli or S. cerevisiae. For very complex molecules polysaccharides, which may be difficult (or impossible)
like tissue plasminogen activator (tPA) and erythropoi- to transport into the cell. Here it is necessary to ensure
etin, it is not, however, possible to obtain sufficiently ac- efficient secretion of an enzyme (often of heterologous
tive compounds in microbial systems, and here a higher nature) that may degrade the substrate. Besides ensuring
eukaryotic expression system is required. proper secretion of the hydrolytic enzyme, it is important
When a certain expression system has been imple- to ensure that the hydrolysis rate is sufficiently high to
mented for the production of one product, it is often de- ensure supply of mono- or disaccharides for growth. In
sirable to use this expression system within the company the literature there are many examples of extension of the
to produce other products as well. There have therefore substrate range and among the most prominent example
been several attempts to engineer generic expression sys- is engineering of S. cerevisiae such that it can metabolize