Environmental Engineering Reference
In-Depth Information
2.7. Pathway Engineering
Metabolic pathway engineering integrates the approaches and technologies described
above and has the fundamental goals of modifying biosynthetic pathways, assessing the
physiological outcomes of the genetic modifications, and using the resulting information to
improve further the pathways in question [42]. The optimization of entire metabolic networks,
rather than individual enzymes, is often necessary because kinetic control is frequently
distributed throughout a pathway rather than concentrated in a single reaction. When levels of
a particular enzyme are altered, the fluxes not only of its direct product(s) and substrates are
altered, but also of metabolites in related pathways linked to the pathway of interest through
regulatory and common-substrate relationships. As a result, multiple points of intervention,
frequently requiring fairly small changes in enzyme activity, may be required to achieve the
desired metabolic changes [42, 43].
Pathway engineering is primarily directed toward one of several distinct goals. The first
of these is the elucidation of pathways of interest, involving both identification of component
reactions as well as reaction and/or transport bottlenecks [42]. Numerous mathematical tools
are being brought to bear on this goal, in combination with genomic and microarray-
generated expression data, and have had great success in elucidating the structures of
metabolic pathways and distribution of kinetic control within them [42]. Two recent
breakthroughs in this area are found in the development of a new kinetics format, termed
linear log kinetics, which has proven remarkably accurate in describing intracellular kinetic
behavior of metabolic networks, and the second is the development of a conceptual and
experimental framework known as FANCY for elucidation of gene function by analysis of
the metabolome, or total metabolite composition of a cell [43]. Additional promising in silico
metabolic pathway modeling approaches interpret and predict cellular functions within the
extremes of allowable possibilities, followed by the use of biochemical rationales to select the
most reasonable behaviors [43]. This quantitative analysis of pathways leads to the
understanding necessary, in turn, to target specific promising genetic modifications [42].
Another primary goal of pathway engineering, enabled by the first, is the modification of
a pathway such that preferred substrates can be used, where preferred substrates are either
less expensive, more widely available, or more environmentally friendly than the
conventional substrates. Increased attention is being given to the use of renewable resources
for the synthesis of specialty and commodity chemicals by so-called green processes. In
addition, biomass- derived substrates are among the most widely available renewable
resources-those generated from agriculture and municipal, agricultural, and forest wastes,
among others.
The majority of inexpensive biomass is composed of lignocellulose. However, this
contains a significant proportion of less-readily fermented five-carbon sugars in combination
with the readily utilized six-carbon sugars. The great promise of pathway engineering for
facilitating utilization of renewable agricultural materials is revealed by the development of
metabolic pathways for the use of biomass-derived mixed sugars for the production of
ethanol, described further in Chapter IV [43].
A third goal of pathway engineering is the development of pathways for the synthesis of
novel chemical structures, particularly antibiotics, carotenoids, and polyhydroxyalkanoates,
the latter of which are described further in Chapter III.
These efforts involve futuristic approaches, such as combining genes from different
pathways and/or different organisms; inserting genes into a pathway or deleting genes from a
