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.
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.
The ability to rationally design biological systems holds tremendous promise for applications in medicine, manufacturing, energy, and the environment. As biological complexity and evolution can pose threats to the ease and stability of an engineering approach, emerging principles of biological design have urged abstraction and standardization of biological modules with defined functions. However, the power of biology as a design substrate lies first and foremost in the rich diversity and complexity of evolved biological systems. Instead of flattening and eliminating such diversity, can we instead employ our ever-deepening understanding of processes that drive diversity and evolutionary change as tools for synthetic biology design? This dissertation explores several such design principles and platforms for synthetic biology—protein domains that transfer high-energy electrons, cyanobacteria, plants, and cheese serve as physical platforms, while gene recombination, cellular cooperation, and personalization emerge as conceptual platforms. A more integrated and biological approach to synthetic biology has potential to lead to robust designs with multiple future applications.
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.
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 pestis 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.
We were fascinated by the similarities between cheese and human microbiodiversity and curious about the historic origin of cheese microflora. Given the physicality of cheesemaking, we speculated on the human origins of many of the unique cheese flavors. To explore this hypothesis and to foreground the microbiology of our food and bodies, we sought out to make cheeses with starter cultures isolated from the human body. Swabs from hands, feet, noses, and armpits were inoculated into fresh, pasteurized, organic whole milk and incubated overnight at 37° Celsius. The milk curds were then strained and pressed, yelding unique smelling fresh cheeses. Eight cheeses were produced in total for further study, with bacterial origins from the bodies of the Synthetic Aesthetics team.
Comparative smell-omics of the cheeses analyzed with headspace technology. The largest difference in odor and bacterial diversity was between Armpit-3 (pink) and Foot-5 (blue). Armpit-3 had the most pleasing, cheese-like smell, and also contains the largest number of ketones previously identified as being involved in cheese odor. For more information, read my thesis chapter on the Synthetic Aesthetics project (PDF).
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.