Biology Reference
In-Depth Information
prohibitively inefficient and time-consuming. To simplify the process, a computational
method for predicting the translation rate of mRNA was developed by Salis et al.
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The
translation initiation of mRNA is comprised of several different molecular interactions.
The Gibbs free energy of initiation can be measured and quantified, and consequently the
total Gibbs free energy of the reaction can be determined. An expression of the translation
efficiency was modeled on the basis of the
G. This computational tool was successfully
applied to predict the reliable operation of a synthetic AND gate by balancing the
expression of the proteins in the circuit. In principle, this method can also be used to
predict the optimal RBS sequences of bottleneck enzymes to balance the metabolic flux.
Δ
Strategies for Optimizing a Metabolic Pathway on the Protein Level
Changing gene expression can greatly optimize the pathway; however, in many
circumstances the inherent limitations associated with activities and specificities of the
desired enzymes in the pathway cannot be overcome by optimizing the gene expression.
Thus tools for engineering the pathway on the protein level are also needed.
INCREASING ENZYME ACTIVITY OR IMPROVING SUBSTRATE SPECIFICITY
Leonard et al.
64
engineered a diterpenoid biosynthetic pathway for levopimaradiene production.
Two rate-limiting enzymes, geranylgeranyl disphosphate synthase (GGPPS) and levopimaradiene
synthase (LPS), were identified as bottlenecks. These enzymes were engineered independently for
higher activity. The mutant enzymes with the highest in vitro activity were selected and
subsequently cloned into the full pathway. The pathways with the engineered GGPPS and LPS
enzymes increased production and also increased the selectivity toward the desired product. This
resulted in a 2600-fold increase in levopimaradiene production.
Another interesting example of protein engineering in pathway optimization is altering
substrate specificity. Zhang et al.
65
constructed a pathway to produce 3-methyl-1-pentanol,
a nonnatural alcohol, by engineering a promiscuous enzyme to have altered specificity
towards the desired substrate, (
S
)-2-keto-methylhexanoate. KivD, the promiscuous key
enzyme, needed to be engineered to reduce the formation of byproducts, thus driving the
carbon flux toward the desired alcohol. The mutant exhibited a specificity towards (
S
)-2-
keto-methylhexanoate 40-fold higher than its natural substrate. The wild-type enzyme
specificity towards (
S
)-2-keto-methylhexanoate was only four-fold higher than the natural
substrate. Another enzyme in the system also needed engineering: LeuA required a higher
activity toward (
S
)-2-keto-3-methylvalerate to reduce the bottleneck. The combination of the
mutated KivD and LeuA produced 685.7 mg/L of (
S
)-3-methyl-1-pentanol, whereas the
control consisting of the two wild-type enzymes produced no detectable amounts of the
desired product. By introduction of these mutants, it was possible to expand the
E. coli
metabolism to produce C5 and C8 alcohols.
52
ALTERING COFACTOR SPECIFICITY
Many pathways involved in the synthesis of value-added compounds employ
oxidation-reduction reactions catalyzed by enzymes using cofactors NAD(P)
1
and
NAD(P)H. These cofactors are just as critical in natural metabolism as in the
heterologous pathways. Thus, the competition for the cofactors between the enzymes
requiring them can be a major limiting factor.
66
Engineering of cofactor recycling systems
has proven to be beneficial: by altering the cofactor specificity of the enzymes within the
pathway, an internal regeneration mechanism can be incorporated. A particular project
which has received much attention in this area is the conversion of xylose to ethanol.
Two of the major enzymes in the pathway, thexylosereductase(XR)andxylitol
dehydrogenase (XDH), have an imbalance in their cofactor usage, as XR prefers NADPH
and XDH prefers NAD
1
. A number of studies analyzing this pathway have shown that the
redox imbalance generated during the xylose assimilation limits cell growth, producing
