Biology Reference
In-Depth Information
concentration of bicarbonate inside the microcompartment enables a higher flux than
RubisCO might otherwise achieve. Other compartments, such as the Pdu and Eut
(1,2-propanediol and ethanolomine bacterial microcompartments, respectively) are thought
to benefit their hosts by blocking the diffusional loss of toxic intermediates produced by the
pathway into the cytoplasm. The protein shell of the microcompartment may also act as a
regulation point by controlling the entry and efflux of precursors and products, enabling
metabolite-level regulation on these pathways. The details of protein targeting into the
microcompartments are becoming clear,
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and promising applications for harnessing
microcompartments for biofuel production are as yet unexplored.
CENTRAL DOGMA ENGINEERING: HOW MANY ENZYMES AND AT WHAT TIME?
The ideal starting point in optimizing a biofuel production pathway is with biochemically
characterized enzymes that are known to be capable of providing flux at a level needed for
high production rates in vivo. The next step is to design a genetic system that expresses the
optimal level of each enzyme at the correct time. Unfortunately, just as it is difficult to
perform biochemical measurements of pathway enzymes, it is also extremely difficult to
know a priori how much enzyme is needed in vivo for an optimum pathway, and to
precisely engineer those protein levels. The use of in vitro assays, such as cell-free
approaches that enable each pathway component to be titrated individually, can inform
optimization.
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One goal of synthetic biology is to be able to engineer protein expression systems that
robustly produce enzymes at a predetermined level, and at precise times. A simplistic
viewpoint that approximates flux as a product of enzyme concentration, substrate
concentration, and catalytic rate would assume that more enzymes would produce higher
flux. Following this logic, the optimal solution for maximum flux might be to engineer the
highest possible enzyme concentrations. Unfortunately, enzyme activities are often
feedback-regulated by metabolites, and high concentrations of intermediates can be toxic to
the cell.
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Furthermore, expressing a high level of pathway protein may be detrimental for
other reasons, such as squandering transcription and translation machinery generating
superfluous levels of protein.
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It has become clear that enzyme levels within a biofuel
pathway must be carefully balanced. Metabolic control theory
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predicts that the best
approach to maximizing productivity is to modify each component of a synthetic pathway
in a way that increases fluxes but maintains intermediate concentrations near their
physiological levels. Considered an extremely difficult undertaking when first articulated,
advances in synthetic biology and protein engineering, which enable the careful tuning of
enzyme levels and catalytic activities, may make this approach more tractable, if still
challenging.
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From Gene to mRNA: Modulating Transcription of Pathway Genes
The concentration of a protein can be expected to increase as the concentration of the
mRNA transcripts that encode the protein increases, although this is not always a simple
relationship (as reviewed in
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), and there is little to no correlation between mRNA levels
and protein levels at the single-cell level.
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Fortunately for the bioengineer, factors that
determine mRNA levels, such as DNA copy number and promoter strength, usually have
predictable effects on protein expression, and can be readily modulated using
straightforward genetic methods.
Typically when demonstrating biosynthesis of a fuel, production pathways are encoded on
plasmids. Plasmid copy number can be easily modulated by using plasmids with various
origins of replication that confer low- or high-copy numbers. This can considerably simplify
efforts to manipulate gene copy number and resulting protein concentrations, which can
identify enzymes that are expressed at insufficient levels. This approach was used to
