Biology Reference
In-Depth Information
Pathway
DNA
mRNA
Transcription
Translation
Promoter strength
RNA half-life
Secondary-
structures
RBS Strength
Promoter timing
Copy Number
Codon usage
protein half-life
protein optimization
Operon orgnization
Biosensors
Metabolites and Products
Allosteric regulation
O
O
CoA
O -
+ H 3 N
COO -
OO
FIGURE 11.1
Synthetic-biology-guided production of biofuels. In black are control points at which one can modify the biochemistry of the
host cell. In green are the mechanisms through which each of these control points can be modified as discussed.
PATHWAY DESIGN AND OPTIMIZATION
Initial Pathway Design and Validation
The first step in the design of any biofuel production pathway is deciding which fuel
compound will be produced using the pool of metabolic precursors available in the host
organism. Many potential fuel compounds or their synthetic intermediates occur naturally,
and their biosynthetic pathways have been determined. The suitability of biological
compounds for use as fuel replacements is reviewed elsewhere. 9 The next challenge is to
assemble a biochemical pathway capable of synthesizing the target molecule using known
enzymatic activities. 10 The genes that encode the necessary enzymes can often be drawn
from the vast library of publicly available sequences. Pathways to produce potentially any
molecule can be assembled from the rich diversity of enzymatic activities that have evolved
over billions of years. Entire biosynthetic pathways can be used as they are found in nature,
or can be assembled piecemeal by incorporating enzymes from different sources. This
diversity can be expanded yet further by engineering existing enzymes to generate
nonnatural products. 10 Biosynthetic pathways can also be constructed from catabolic
pathways engineered to run in the reverse direction than their usual physiological role. This
has been demonstrated using a reversed fatty-acid degradation pathway to produce butanol
in high titers in E. coli . 5
209
Construction of a biofuel pathway typically begins with selection of genes encoding
enzymes that comprise the individual pathway components. The full pathway is assembled
in one or more plasmids and expressed in the host organism of choice, which is often a
well-characterized and genetically tractable organism such as Escherichia coli or Saccharomyces
cerevisiae. Other organisms with different metabolic capabilities and toxicity tolerances that
may be better suited to producing the desired compound can certainly be used as hosts,
although they often present other challenges, most notably having fewer techniques for
genetic manipulations. 11 Production of the desired compound is usually assayed using an
appropriate chomatographic method. As it is unlikely that the initial design of the
pathway will produce high titer levels, production will be orders-of-magnitude lower than
is needed in an industrial process. Nonetheless, an important milestone will have been
achieved: proof that biological production of a fuel compound within a new host is
possible. The brevity with which the initial stage of pathway design and validation is
treated here is not intended to indicate its triviality or simplicity. It is a necessary first
step, but an equally important challenge lies in increasing titers and yields closer to
commercially viable levels.
 
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