Biology Reference
In-Depth Information
57
g/ml for a
protein with three site mutations.
114
Although not attempted, the use of this strain in a cell-
free system could further improve the production of proteins containing unnatural amino
acids.
μ
g/ml for a protein with one unnatural amino acid site mutation, and 68
μ
Small Molecule Metabolites
Similar to CFPS, cell-free metabolic engineering for overproducing small molecule
metabolites occurs via either a bottom-up or top-down approach. Since the original
discovery that cell extracts could convert sugar into ethanol,
115
there have been relatively
few top-down examples of metabolic engineering. Efforts primarily by the Swartz and Panke
laboratories have developed the field; the former for activating energy pathways for fueling
cell-free protein synthesis reactions,
116
and the latter for making desired small molecule
metabolites. In one example of this approach, the Panke group constructed an enzyme
catalytic system by fine-tuning a catalytic pathway with information from real-time analysis
of concentrations of the pathway intermediates.
117
In contrast to extract-based systems, the bottom-up approach exploits the organization of
synthetic enzymatic pathways from purified components, sometimes to facilitate a process
or reaction that does not occur in nature.
9,118
In one instance, a 13-step synthetic enzymatic
pathway using starch and water produced hydrogen at yields far higher than the
theoretical yields of biological hydrogen fermentations (illustrated in
Fig. 15.4
).
9
Indeed, because carbon flux is not directed towards cell growth, cell-free systems can
achieve higher theoretical yields than natural biological processes found in living
organisms.
116
Unfortunately, the high cost of purifying stable, standalone enzymes, and
cofactor regeneration costs, currently limit synthetic enzymatic pathways to laboratory
scale research.
289
FIGURE 15.4
A cell-free synthetic enzymatic pathway for converting starch and water into hydrogen and carbon dioxide: this artificial
pathway has been reported to produce hydrogen at yields greater than the theoretical limits for industrial anaerobic
fermentations (12 H
2
per glucose compared to 4 H
2
per glucose).
9
Molecular graphics images were produced using the
UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San
Francisco (supported by NIH P41 RR-01081).
