Biology Reference
In-Depth Information
efforts to optimize the translation level of an individual gene may affect expression of the
other pathway genes in ways that are difficult or impossible to predict. This relationship
between mRNA secondary structure and its effect upon both mRNA stability and translation
has been exploited to create E. coli mutants with varying protein concentrations. To
demonstrate this principle, a combinatorial library was generated in which different hairpins
were introduced into the intergenic regions between the three genes responsible for
producing mevalonate atoB , HMGS , and tHMGR . This secondary structure led to expression
differences for each gene in different mutants. With this strategy it was possible to identify
levels of these proteins that lead to the production of seven-fold more mevalonate than the
starting strain. 61 A very recent, more comprehensive model of gene expression takes into
account the kinetics of mRNA folding and degradation, and uses RNA devices (aptazymes
and ribozymes) to tune these parameters directly. RNA devices were placed within the
5 0 -untranslated region of mRNA to modulate stability of the transcript. This enabled reliable
static and dynamic control over the expression of the downstream gene over a wide range. 62
HOST ENGINEERING
The organism that hosts the biofuel production pathway will, of course, heavily
influence the behavior of the biofuel production pathway. In addition to expressing and
maintaining the biofuel synthesis pathway, the host organism supplies the metabolic
precursors and enzyme cofactors. However, no organism has evolved to generate chemicals
on a commodity scale for our consumption. Organisms have evolved to address goals (such
as self-replication) that are often at odds with engineering efforts; thus, the engineer must
consider the host physiology and biochemistry if high titers are to be obtained. This section
will review approaches to modifying production hosts that result in improved titers of final
products. For the sake of brevity, we will consider only rational approaches rather than
methods that employ mutagenesis or random, undirected perturbations that are then
screened for improved titers. The reader should consider that we are still quite ignorant of
many details of cellular physiology and metabolism, so undirected approaches often
produce completely unexpected targets for improvements. 63
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KNOCKING OUT COMPETING PATHWAYS
The starting materials of biofuels are typically precursors to building blocks that will
ultimately become components of the cell (fatty acids, amino acids), or secondary
metabolites involved in other processes (isoprenoids). An engineered biofuel pathway will
compete with native pathways for these resources. Removing or attenuating competing
pathways through genetic manipulations have been repeatedly demonstrated to improve
product titers. For instance, in anaerobic conditions in the absence of an electron acceptor,
E. coli will use pyruvate as an electron acceptor in mixed acid fermentation, generating
lactate, acetate, and succinate. Pathways used to generate higher alcohols, such as butanol,
use pyruvate as a carbon source. The deletion of key enzymes required for mixed acid
fermentation relieves competition of the biofuel for pyruvate and acetyl-CoA, increasing
yield of the desired final product. 28,64 Furthermore, as these pathways compete for the
electron carrier NADH, deletion of the pathways also increases the availability of NADH for
alcohol production.
Other deletions that have no direct effect on precursor pools may improve titer by
eliminating the generation of substrates that might act as a competitive inhibitor of an
enzyme along the biofuel production pathway. The product of ilvD generates a compound
that acts as a competitive inhibitor for one of the enzymes involved in producing butanol
from a 2-keto acid. Deletion of the ilvD resulted in a three-fold production improvement. 28
Gene deletions may also increase product yield by eliminating pathways that catabolize
biofuel precursors that are present in excess. Steen et al. improved the yield of fatty acids in
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