Biology Reference
In-Depth Information
BOX 11.1 BIOSENSORS
One technology that is becoming increasingly important in the synthetic biology toolkit is biosensors.
This class of molecules, which can be either naturally occurring or specifically engineered, can
include protein or RNA. The output of a biosensor can include increased transcription whose activities
change depending on the biochemical state of the cell or the presence of a specific molecule, such
as a biofuel or a biofuel precursor. Of particular relevance are transcription factors that respond to
precursors, intermediates, or products in the synthetic pathway. This type of biosensor has been
used for a variety of applications. The clearest use of this technology is for directed evolution.
Biosensors that are responsive to the product or key intermediate in a pathway can be coupled to the
transcription of a screenable marker such as GFP, 78 or a selectable marker such as
-lactamase, to
isolate the highest producing strains out of a pool of mutants. 79 When a suitable biosensor does not
exist, it may be possible to generate a synthetic one using directed evolution. Cirino and coworkers
demonstrated that there is some plasticity in the molecular recognition of the arabinose responsive
regulator AraC. Placing GFP under the control of P BAD promoter allowed FACS screening of a library
of AraC mutants. Using both a positive and negative screen they were able to identify mutants which
were activated by D-arabinose, the sugar with the inverse stereochemistry of the natural L-ligand
which also exhibited the same tight control and low background associated with the natural protein. 80
β
controlling expression of those genes using a chemically inducible promoter. Furthermore,
growth retardation associated with overexpression of one of the rate-limiting proteins was
not seen, despite a higher protein expression level. These improvements were attributed to a
better coordination of lycopene biosynthesis with the metabolic state of the cell, resulting in
better diversion of flux away from acetate into lycopene. Finally, because production is
coupled to glycolytic excess, the extra carbon is channeled into lycopene rather than the
toxic overflow product acetate, reducing the production of this waste by 66%.
215
A naturally occurring biosensor of an intermediate in the synthesis of the biodiesel fatty acid
ethyl esters was recently used to drive expression of genes that further modify the
intermediate into the final biofuel product. In E. coli, the transcription factor FadR responds
to fatty acyl-CoAs and free fatty acids 5 by relieving inhibition of downstream genes. The
expression of an ethanol production pathway and a wax ester synthase gene responsible for
esterifying the fatty acids with ethanol were placed under control of FadR. These genes were
not transcribed until sufficient levels of fatty acyl-CoA had accumulated, preventing not only
wasteful expression of downstream proteins before they had substrates to act upon, but also
accumulation of the toxin ethanol to high levels. In addition, this type of regulation allows
the cell to coordinate the levels of the reactants; the transcriptional output of the biosensor
increases with increasing concentrations of acyl-CoA, which in turn drives faster production
of ethanol and the wax ester synthase. With this technology it was possible to push FAEE
levels up three-fold to 1.6 g/L and to 28% of the theoretical maximum yield. 54a
A recent study that demonstrated the production of a precursor to the important drug Taxol
in E. coli is another example of optimization of a pathway by combining copy number
modulation with promoter strength. Ajikumar et al. 57 split their pathway into two parts
(referred to as the
), and varied copy
number and promoter strength of each module independently. By measuring production
levels, they mapped out a production landscape as a function of expression level of both the
upstream and downstream pathways. Tellingly, production levels did not monotonically
increase with increasing expression of either half of the pathway, but rather exhibited
localized peaks that indicate improved balancing of the two halves of the pathway.
'
upstream module
'
and the
'
downstream module
'
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