Smelling in Multiple Dimensions. Agapakis and Tolaas. Current Opinion in Chemical Biology, 2012.
Smell is perhaps the most subjective of the human senses, making odors difficult to measure and define.
In everyday language, in the philosophy of aesthetics, and in the lab, this low opinion of odors means
that smells are often characterized simply along an axis of good or bad. Odors and the ways they are
perceived, however, are varied and incredibly complex, requiring an understanding of chemistry,
neuroscience, aesthetics, and social science. Science and art that engage the sense of smell have
the potential to expand our understanding of how biology and chemistry interact.
Natural strategies for the spatial optimization of metabolism in synthetic
biology. Agapakis, Boyle, and Silver.
Nature Chemical Biology, 2012.
Metabolism is a highly interconnected web of chemical reactions that power life.
Though the stoichiometry of metabolism is well understood, the multidimensional
aspects of metabolic regulation in time and space remain difficult to define,
model and engineer. Complex metabolic conversions can be performed by multiple
species working cooperatively and exchanging metabolites via structured networks
of organisms and resources. Within cells, metabolism is spatially regulated via
sequestration in subcellular compartments and through the assembly of multienzyme
complexes. Metabolic engineering and synthetic biology have had success in
engineering metabolism in the first and second dimensions, designing linear
metabolic pathways and channeling metabolic flux. More recently, engineering
of the third dimension has improved output of engineered pathways through isolation
and organization of multicell and multienzyme complexes. This review highlights
natural and synthetic examples of three-dimensional metabolism both inter- and
intracellularly, offering tools and perspectives for biological design.
A BioBrick compatible strategy for genetic modification of plants. Boyle, Burrill, Inniss, Agapakis et. al.,
Journal of Biological Engineering, 2012.
Plant biotechnology can be leveraged to produce food, fuel, medicine, and materials.
Standardized methods advocated by the synthetic biology community can accelerate the
plant design cycle, ultimately making plant engineering more widely accessible to
bioengineers who can contribute diverse creative input to the design process. This paper
presents work done largely by undergraduate students participating in the
2010 International Genetically Engineered Machines (iGEM) competition. Described here
is a framework for engineering the model plant Arabidopsis thaliana with standardized,
BioBrick compatible vectors and parts available through the Registry of Standard
Biological Parts (www.partsregistry.org). This system was used to engineer a
proof–of–concept plant that exogenously expresses the taste-inverting
protein miraculin. Our work is intended to encourage future iGEM teams and other synthetic biologists
to use plants as a genetic chassis. Our workflow simplifies the use of standardized
parts in plant systems, allowing the construction and expression of heterologous
genes in plants within the timeframe allotted for typical iGEM projects.
Towards a Synthetic Chloroplast. Agapakis, Niederholtmeyer, Noche et. al. PLoS ONE, 2011.
The evolution of eukaryotic cells is widely agreed to have proceeded through a series of
endosymbiotic events between larger cells and proteobacteria or cyanobacteria,
leading to the formation of mitochondria or chloroplasts, respectively. Engineered
endosymbiotic relationships between different species of cells are a valuable tool
for synthetic biology, where engineered pathways based on two species could take
advantage of the unique abilities of each mutualistic partner.
We explored the possibility of using the photosynthetic bacterium Synechococcus
elongatus PCC 7942 as a platform for studying evolutionary dynamics and for
designing two–species synthetic biological systems. We observed that
the cyanobacteria were relatively harmless to eukaryotic host cells compared
to Escherichia coli when injected into the embryos of zebrafish, Danio rerio,
or taken up by mammalian macrophages. In addition, when engineered with invasin from Yersinia pseudotuburculosis
and listeriolysin O from Listeria monocytogenes, S. elongatus was
able to invade cultured mammalian cells and divide inside macrophages. Our results show
that it is possible to engineer photosynthetic bacteria to invade the cytoplasm of mammalian
cells for further engineering and applications in synthetic biology. Engineered invasive
but non-pathogenic or immunogenic photosynthetic bacteria have great potential as synthetic biological devices.
A synthetic system links FeFe-hydrogenases to essential
E. coli sulfur metabolism. Barstow, Agapakis, Boyle et. al. Journal of Biological Engineering, 2011.
FeFe–hydrogenases are the most active class of H2-producing enzymes known in nature and
may have important applications in clean H2 energy production. Many potential uses are
currently complicated by a crucial weakness: the active sites of all known FeFe–hydrogenases
are irreversibly inactivated by O2. We have developed a synthetic metabolic pathway in
E. coli that links FeFe–hydrogenase activity to the production of the essential
amino acid cysteine. Our design includes a complementary host strain whose
endogenous redox pool is insulated from the synthetic metabolic pathway. Host
viability on a selective medium requires hydrogenase expression, and moderate O2
levels eliminate growth. This pathway forms the basis for a genetic selection for O2
tolerance. Genetically selected hydrogenases did not show improved stability in O2 and
in many cases had lost H2 production activity. The isolated mutations cluster significantly
on charged surface residues, suggesting the evolution of binding surfaces that may accelerate
hydrogenase electron transfer. Rational design can optimize a fully heterologous
three–component pathway to provide an essential metabolic flux while
remaining insulated from the endogenous redox pool. We have developed
a number of convenient in vivo assays to aid in the engineering of synthetic H2
metabolism. Our results also indicate a H2-independent redox activity in three different FeFe–hydrogenases,
with implications for the future directed evolution of H2-activating catalysts.
Modular electron transfer circuits for synthetic biology:
insulation of an engineered biohydrogen pathway. Agapakis and Silver, Bioengineered Bugs, 2010.
Electron transfer is central to a wide range of essential metabolic pathways,
from photosynthesis to fermentation. The evolutionary diversity and conservation of
proteins that transfer electrons makes these pathways a valuable platform for engineered
metabolic circuits in synthetic biology. Rational engineering of electron transfer
pathways containing hydrogenases has the potential to lead to industrial scale
production of hydrogen as an alternative source of clean fuel and experimental
assays for understanding the complex interactions of multiple electron transfer
proteins in vivo. We designed and implemented a synthetic hydrogen metabolism circuit
in Escherichia coli that creates an electron transfer pathway both
orthogonal to and integrated within existing metabolism. The design of such
modular electron transfer circuits allows for facile characterization of in
vivo system parameters with applications toward further engineering for alternative
energy production.
Insulation of a synthetic hydrogen metabolism circuit in bacteria. Agapakis, Ducat, Boyle et. al. Journal of Biological Engineering, 2010.
The engineering of metabolism holds tremendous promise for the production of desirable
metabolites, particularly alternative fuels and other highly reduced molecules.
Engineering approaches must redirect the transfer of chemical reducing equivalents,
preventing these electrons from being lost to general cellular metabolism.
This is especially the case for high energy electrons stored in iron–sulfur
clusters within proteins, which are readily transferred when two such clusters
are brought in close proximity. Iron sulfur proteins therefore require mechanisms
to ensure interaction between proper partners, analogous to many signal transduction
proteins. While there has been progress in the isolation of engineered metabolic
pathways in recent years, the design of insulated electron metabolism circuits in
vivo has not been pursued. Here we show that a synthetic hydrogen–producing
electron transfer circuit in Escherichia coli can be insulated from existing
cellular metabolism via multiple approaches, in many cases improving the function of
the pathway. Our circuit is composed of heterologously expressed FeFe–hydrogenase,
ferredoxin, and pyruvate-ferredoxin oxidoreductase (PFOR), allowing the production of
hydrogen gas to be coupled to the breakdown of glucose. We show that this synthetic
pathway can be insulated through the deletion of competing reactions, rational
engineering of protein interaction surfaces, direct protein fusion of interacting
partners, and co–localization of pathway components on heterologous protein scaffolds.
Through the construction and characterization of a synthetic metabolic circuit in vivo,
we demonstrate a novel system that allows for predictable engineering of an insulated
electron transfer pathway. The development of this system demonstrates working principles
for the optimization of engineered pathways for alternative energy production, as well as
for understanding how electron transfer between proteins is controlled.
Synthetic biology: exploring and exploiting genetic modularity
through the design of novel biological networks. Agapakis and Silver, Molecular Biosystems, 2009.
Synthetic biology has been used to describe many biological endeavors over the past
thirty years—from designing enzymes and in vitro systems, to manipulating existing metabolisms
and gene expression, to creating entirely synthetic replicating life forms. What
separates the current incarnation of synthetic biology from the recombinant DNA
technology or metabolic engineering of the past is an emphasis on principles from
engineering such as modularity, standardization, and rigorously predictive models.
As such, synthetic biology represents a new paradigm for learning about and using
biological molecules and data, with applications in basic science, biotechnology,
and medicine. This review covers the canonical examples as well as some recent advances
in synthetic biology in terms of what we know and what we can learn about the networks
underlying biology, and how this endeavor may shape our understanding of living systems.
