21-05-2014, 12:19 PM
Genetic and metabolic engineering
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ABSTRACT
Recent advances in molecular biology techniques,
analytical methods and mathematical tools have led to a
growing interest in using metabolic engineering to
redirect metabolic fluxes for industrial and medical
purposes. Metabolic engineering is referred to as the
directed improvement of cellular properties through the
modification of specific biochemical reactions or the
introduction of new ones, with the use of recombinant
DNA technology (Stephanopoulos, 1999). This
multidisciplinary field draws principles from chemical
engineering, biochemistry, molecular and cell biology,
and computational sciences. The aim of this article is to
give an overview of the various strategies and tools
available for metabolic engineers and to review some of
the recent work that has been conducted in our
laboratories in the metabolic engineering area.
INTRODUCTION
Metabolic engineering is generally referred to as the
targeted and purposeful alteration of metabolic pathways
found in an organism in order to better understand and
utilize cellular pathways for chemical transformation,
energy transduction, and supramolecular assembly
(Lessard, 1996).
This multidisciplinary field draws
principles from chemical engineering, computational
sciences, biochemistry, and molecular biology. In essence,
metabolic engineering is the application of engineering
principles of design and analysis to the metabolic pathways
in order to achieve a particular goal. This goal may be to
increase process productivity, as in the case in production
of antibiotics, biosynthetic precursors or polymers, or to
extend metabolic capability by the addition of extrinsic
activities for chemical production or degradation.
Physiological studies
The concept of network rigidity, flexible and rigid nodes,
was introduced by Stephanopoulos and Vallino (1991).
The rigidity of a network or its resistance to variations in
metabolic change is due to control mechanisms established
to ensure balanced growth. For a engineering strategy to
be successful, a better understanding of the host cell is
necessary to determine the types of genetic modifications
needed to achieve the final goal. Some of the physiological
considerations that should be examined include the effects
of genetic manipulation on growth and possible effects on
“unrelated” systems. These negative effects of genetic
manipulation are often attributed to the metabolic burden.
In the case of the over-expression of phosphoenol pyruvate
forming enzymes, the heat-shock response and nitrogen
regulation were both inhibited (Liao et al., 1996a).
Inverse metabolic engineering
The classical approach of metabolic engineering, as
discussed above, requires detailed knowledge of the
enzyme kinetics, the system network, and intermediate
pools involved, and on such bases, a genetic manipulation
is proposed for some presumed benefits. In contrast, the
concept of inverse metabolic engineering is first to identify
the desired phenotype, then to determine environmental or
genetic conditions that confer this phenotype, and finally
to alter the phenotype of the selected host by genetic
manipulation (Bailey et al., 1996, Delgado and Liao,
1997). As in the case of expression of the oxygen binding
protein, VHb, in E. coli, the observed phenotype of high
heme cofactor levels in an obligate aerobe Vitreoscilla
under oxygen limitation suggested that synthesis of the
hemoglobin could improve growth of other organisms
under similar limitations (Bailey et al., 1996).