Biology Reference
In-Depth Information
unwanted byproducts and reducing ethanol production.
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Multiple studies have focused
on engineering the XDH enzyme to have altered specificity towards NADP
1
, which would
match with the XR cofactor preference of NADPH.
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Similarly, the XR was altered to have
a preference for NADH, which led to a 40-fold increase in ethanol productivity over the
wild-type enzyme.
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COMBINATORIAL ASSEMBLY OF HOMOLOGOUS ENZYMES IN A PATHWAY
In the design of a pathway, there are often many different homologues of the desired
enzymes that conduct the specific chemistry required. Identifying the proper enzymes to be
used in the pathway can be challenging. Often, enzymes are chosen based on the highest
activity, but this is not always advantageous. Enzymes could have unknown properties, such
as side reactions and unknown regulation, which would reduce the overall productivity.
Also, balancing the enzyme activities in the pathway is difficult for rational design. Thus, a
library approach encompassing the automatic assembly of hundreds of genes was developed
(
Fig. 3.1B
).
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In this method, the fungal xylose utilization pathway, which consists of three
heterologously expressed enzymes (XR, XDH, and XKS) was constructed. A total of
20 homologues of XR, 22 XDHs, and 19 XKSs were cloned into the library. Combinatorial
assembly of these genes using the DNA assembler method resulted in a library of over
8000 xylose-utilizing pathways. After colony-size-based high-throughput screening, colonies
with the best utilization of xylose as a sole-carbon source were selected and subsequently
screened for ethanol production. This particular method is powerful, because it was able to
identify different combinations of the three types of enzymes in the pathway for different
media conditions and strains.
PROTEIN SCAFFOLDS AND SUBSTRATE CHANNELING
Industrial bioprocesses have taken advantage of immobilized enzymes to facilitate enzyme
reutilization and substrate
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enzyme diffusion for years. It is well known that by positioning
enzymes in a specific organized manner, more efficient mass transfer of the substrate will
occur. For example, cellulosomes are enzyme complexes that can degrade cellulose from plant
cell walls to fermentable sugars.
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This concept has also been developed into Consolidated
Bioprocessing or CBP,
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wherein cellulosomal degradation of biomass and ethanol
production can be simultaneously achieved. To this end, Wen et al. constructed a cellulosome
on the yeast surface.
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By docking three key cellullases including endoglucanase,
cellobiohydrolase, and a
-glucosidase onto a scaffolding displayed on the yeast surface, these
enzymes could efficiently break down the cellulose and convert the glucose to ethanol.
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β
Another interesting example is the enzyme tryptophan synthase, which contains a largely
hydrophobic tunnel connecting two active sites about 25 Å apart. In addition to protecting
the reactive intermediate, the tunnel allows for the chemical to be immediately channeled
from one active site to another. The same concept was applied to a pathway by Dueber
et al.
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The enzymes within the pathway were colocalized using a synthetic scaffold built
from protein
protein interaction domains, which specifically bind the corresponding
ligands of the metabolic enzymes. This created an easy access for the substrate to diffuse
from one enzyme active site to another. This technique prevents substrates from diffusing
away from the active site, and could make up for enzymes with low activity. Low-activity
enzymes can be expressed and coupled within the scaffold in multiple numbers. This
technique was applied to the mevalonate biosynthetic pathway and improved the titer by
22-fold compared to the nonscaffold wild-type control. The method was also shown to
lower the metabolic burden, thus improving cell growth.
APPLICATIONS OF PATHWAY ENGINEERING TOOLS
The tools and methods for pathway construction and optimization are varied. This is
significant because not every tool will work in each system, thus it is important to apply
