Jay Keasling

Lawrence Berkeley National Laboratory – Joint BioEnergy Institute, Berkeley

Synthetic Biology for Synthetic Fuels

Today, carbon-rich fossil fuels, primarily oil, coal and natural gas, provide 85% of the energy consumed in the United States. As world demand increases, oil reserves may become rapidly depleted. Fossil fuel use increases CO2 emissions and raises the risk of global warming. The high-energy content of liquid hydrocarbon fuels makes them the preferred energy source for all modes of transportation. In the US alone, transportation consumes around 13.8 million barrels of oil per day and generates over 0.5 gigatons of carbon per year. This release of greenhouse gases has spurred research into alternative, non-fossil energy sources. Among the options (nuclear, concentrated solar thermal, geothermal, hydroelectric, wind, solar and biomass), only biomass has the potential to provide a high-energy-content transportation fuel. Biomass is a renewable resource that can be converted into carbon-neutral transportation fuels.

Currently, biofuels such as ethanol are produced largely from grains, but there is a large, untapped resource (estimated at more than a billion tons per year) of plant biomass that could be utilized as a renewable, domestic source of liquid fuels. Well-established processes convert the starch content of the grain into sugars that can be fermented to ethanol. The energy efficiency of starch-based biofuels is however not optimal, while plant cell walls (lignocellulose) represents a huge untapped source of energy. Plant-derived biomass contains cellulose, which is more difficult to convert to sugars, hemicellulose, which contains a diversity of carbohydrates that have to be efficiently degraded by microorganisms to fuels, and lignin, which is recalcitrant to degradation and prevents cost-effective fermentation.  The development of cost-effective and energy-efficient processes to transform lignocellulosic biomass into fuels is hampered by significant roadblocks, including the lack of specifically developed energy crops, the difficulty in separating biomass components, low activity of enzymes used to deconstruct biomass, and the inhibitory effect of fuels and processing byproducts on organisms responsible for producing fuels from biomass monomers.

We are engineering the metabolism of platform hosts (Escherichia coli and Saccharomyces cerevisiae) for production of advanced biofuels.  Unlike ethanol, these biofuels will have the full fuel value of petroleum-based biofuels, will be transportable using existing infrastructure, and can be used in existing automobiles and airplanes.  These biofuels will be produced from natural biosynthetic pathways that exist in plants and a variety of microorganisms.  Large-scale production of these fuels will reduce our dependence on petroleum and reduce the amount of carbon dioxide released into the atmosphere, while allowing us to take advantage of our current transportation infrastructure. 


Sunil Chandran

Amyris Inc.

Advances in the industrialization of synthetic biology

Malaria, caused by the Plasmodium falciparum parasite, is estimated to affect about 350-500 million people annually, resulting in more than a million deaths, with the highest mortality occurring in children under the age of five. Unfortunately, P. falciparum is now resistant to most drug treatments except for artemisinin-based combination therapies (ACTs).  Artemisinin is currently extracted from the plant Artemisia annua, but an additional source is needed to meet anticipated demand, stabilize the market, lower costs and increase availability of ACTs. We have developed a fermentation process for the biosynthetic production of artemisinin precursors, with subsequent chemical conversion to artemisinin. The synthetic biology underlying the development of the S. cerevisiae strains for the production of artemisinin precursors amorphadiene and artemisinic acid will be described, along with process development to produce significant quantities of amorphadiene. This route, which involves the fermentative production of artemisinin precursors followed by chemical conversion to artemisinin will result in a stable, scalable, alternative source of artemisinin that can be incorporated into ACT’s.

The artemisinin project demonstrated the challenges of engineering strains through multiple iterations of strain design, testing, analysis and re-design.  This iterative process is dramatically enhanced by the ability to construct thousands of strains in a short time frame.  To rapidly construct thousands of yeast strains per week, standardized and modular molecular biology systems for DNA construction have been designed in several labs.  One such system, termed RYSE for Rapid Yeast Strain Engineering, was developed at Amyris.  RYSE is based on standardized end junctions that allow the joining of multiple DNA fragments.  Standardized ends allow the generic use and archiving of DNA fragments, termed RYSE-Associated Bits or “RABits.”  RABits, which may be thought of as “parts,” serve as the functional unit of construction in RYSE.


Peter Facchini

Department of Biological Sciences, University of Calgary

How plants make morphine – can we teach this trick to a microorganism?

Among the vast catalogue of plant natural products are ~2500 known benzylisoquinoline alkaloids (BIAs) that include the narcotic analgesic morphine and other pharmaceuticals produced in opium poppy. Genes corresponding to most of the enzymes involved in the biosynthesis of morphine and others functioning in branch pathways leading to the antimicrobial sanguinarine, the vasodilator papaverine and the potential anticancer drug noscapine have been identified. The first committed step in BIA biosynthesis, norcoclaurine synthase, is catalyzed by a unique member of the pathogenesis-related (PR)10 protein family. All other known enzymes belong to a variety of families including cytochromes P450, O- and N-methyltransferases, FAD-linked oxidoreductases, 2-oxoglutarate-dependent dioxygenases, acyl-CoA-dependent acyltransferases and three types of NADPH-dependent reductases. Biochemical genomics and cell biology applications have rapidly accelerated our understanding of BIA metabolism in opium poppy. Tapping into the enzyme variants responsible for the vast BIA diversity beyond opium poppy requires genetic resources for exotic plant species and the development of a common functional genomics platform. Next-generation sequencing has been used to generate deep transcriptome resources for 20 BIA-producing species from four plant families. Yeast strains are being engineered using available and newly discovered biosynthetic genes to produce a variety of BIAs, including those capable of de novo codeine and morphine biosynthesis. These strains allow the discovery of novel BIA biosynthetic genes using plug-and-play synthetic biology based on candidate genes available in our deep transcriptome databases, and complement our plant functional genomics tools.

Ron Weiss

Department of Biological Engineering, Massachusetts Institute of Technology

Synthetic biology: from parts to modules to therapeutic systems

Synthetic biology is revolutionizing how we conceptualize and approach the engineering of biological systems. Recent advances in the field are allowing us to expand beyond the construction and analysis of small gene networks towards the implementation of complex multicellular systems with a variety of applications. In this talk I will describe our integrated computational / experimental approach to engineering complex behavior in living systems ranging from bacteria to stem cells. In our research, we appropriate design principles from electrical engineering and other established fields. These principles include abstraction, standardization, modularity, and computer aided design. But we also spend considerable effort towards understanding what makes synthetic biology different from all other existing engineering disciplines and discovering new design and construction rules that are effective for this unique discipline. We will briefly describe the implementation of genetic circuits and modules with finely-tuned digital and analog behavior and the use of artificial cell-cell communication to coordinate the behavior of cell populations. The first system to be presented is an RNAi-based logic circuit that can detect and destroy specific cancer cells based on their microRNA expression profiles. We will also discuss preliminary experimental results for obtaining precise spatiotemporal control over stem cell differentiation for tissue engineering applications. We will conclude by discussing the design and preliminary results for creating an artificial tissue homeostasis system where genetically engineered stem cells maintain indefinitely a desired level of pancreatic beta cells despite attacks by the autoimmune response, relevant for diabetes.

Pamela A. Silver

Department of Systems Biology, Harvard Medical School and The Wyss Institute for Biologically Inspired Engineering, Harvard University

Designing Biological Systems for Health and Sustainability

Biology presents us with an array of design principles.  From studies of both simple and more complex systems, we understand some of the fundamentals of how Nature works. We are interested in using the foundations of biology to engineer cells in a logical and predictable way to perform certain functions. By necessity, the predictable engineering of biology requires knowledge of quantitative behavior of individual cells and communities and the ability to construct reliable models.  By building and analyzing synthetic systems, we learn more about the fundamentals of biological design as well as engineer useful living devices with myriad applications.  For example, we are interested in building cells that can perform specific tasks, such as remembering past events and thus acting as a biological computer. Moreover, we design cells with predictable biological properties that serve as cell-based sensors, factories for generating useful commodities and improved centers for carbon fixation.  We have recently constructed synthetic protein/RNA structures to increase the efficiency of biological reactions. In doing so, we have made new findings about how cells interact with and impact on their environment.

Radhakrishnan Mahadevan

Chemical Engineering & Applied Chemistry, University of Toronto

Model-based Design of Metabolism

Bioprocess development for biofuels and biochemicals typically requires several rounds of metabolic engineering to meet process targets including product yield, titer and productivity, all of which impact the process economics. Recent advances in experimental and computational technologies have enabled the detailed characterization of biological systems. In particular, the molecular components of these systems including the list of genes, proteins they encode, and compounds that interact with these proteins can be determined. Similar advances in computational modeling techniques have allowed the development of genome-scale models of metabolism in several organisms.  In this talk, the use of such models for metabolic engineering will be presented. Model refinement through the incorporation of a fundamental physical constraint that accounts for membrane area will be described. In the first part, a rational approach based on bi-level optimization to enhance bioprocess productivity by forcing co-utilization of substrates will be shown.  Experimental results from the application of this approach to enforce substrate co-utilization in Escherichia coli will be discussed. In the next part of the talk, a novel nested nonlinear optimization method for metabolic engineering resulting in over hundred different strain design strategies for biochemicals production will be presented.  Finally, we will also examine the role of the redundant pathways from a design perspective and present computational results on how these pathways is valuable for robust design.

Matthew Scott

Department of Applied Mathematics, University of Waterloo

Indirect regulation of bacterial gene expression imposed by growth and division

In bacteria, the rate of cell proliferation and the level of gene expression are intimately connected. Uncovering these relations is important both for understanding the physiological functions of endogenous genetic circuits and for designing robust synthetic systems. I will discuss a phenomenological study that reveals intrinsic constraints governing the allocation of resources towards protein synthesis and other aspects of cell growth. The use of such empirical relations, analogous to phenomenological laws, may facilitate our understanding and manipulation of complex biological systems before underlying regulatory circuits are fully elucidated.


Mads Kaern

Ottawa Institute of Systems Biology, Faculty of Medicine, University of Ottawa

A rapid in vitro DNA assembly method for genetic network engineering

We describe the development of a DNA assembly method that enables rapid in vitro fusion of multiple smaller DNA fragments into a single molecule with a defined sequence. The method addresses two problems in DNA synthesis and genetic network engineering - the creation of double-sanded DNA molecules from multiple smaller fragments; and the time-consuming, stepwise assembly of genetic network parts into larger functional modules. Here, we demonstrate the use of the method in three applications where the desired DNA product is obtained in a matter of hours rather than days. We first show how the method can be applied to assemble six DNA fragments that adhere to standards established by the BioBrick Foundation using standardized primer sets, restriction endonucleases and DNA ligation. We then show how a similar DNA product can be created without the use of restriction enzymes or ligation, allowing for ''scarless'' assembly of DNA fragments. Finally, we show how a slight modification of the method enables the assembly of a larger number of fragments.

Joel Bader

John Hopkins University School of Medicine

The Saccharomyces Cerevisiae 2.0 Project

Richard Feynman's blackboard read, "What I cannot create, I do not understand."  Synthetic biology has the goal of understanding biology by creating it.  We specifically aim to create a living yeast cell whose DNA traces back to an oligo synthesizer, and before that to a computer program, rather than a parent cell.  The Saccharomyces cerevisiae 2.0 (Sc2.0) project is an international collaboration to achieve this aim.  We have developed computer-aided design / computer-aided manufacture (CAD/CAM) software to design new versions of genes and genomes, to convert the electronic designs to physical DNA, and to replace native DNA with our synthetic version.  The synthetic DNA has been designed to eliminate transposons and other elements of questionable fitness benefit; we have also introduced recombination sites to permit rapid genome scrambling and evolution.  This international collaborative effort includes the Boeke and Chandrasegaran labs (also at Johns Hopkins), the Dai lab (Tinsghua University, China), and BGI (Shenzhen, China). References: Dymond et al. 2011 Nature 477: 471

Nathan Hillson

Lawrence Berkeley National Laboratory – Joint BioEnergy Institute, Berkeley

DNA assembly design software and automation devices

The production of clean renewable biofuels from cellulosic starting material requires concerted feedstock engineering, deconstruction of plant matter into simple sugars, and microbial fermentation of the sugars into biofuel. These three efforts share significant molecular biological challenges, including the construction of large enzymatic libraries (e.g. vast collections of glycosyl transferases, cellulases, and efflux pumps), the generation of combinatorial libraries (e.g. multi-functional enzyme domain fusions; variations in copy number, promoter and ribosomal binding site strength), and the concurrent assembly of multiple biological parts (e.g. the incorporation of an entire metabolic pathway into a single target vector). With these challenges in mind, we have developed two on-line software tools, j5 and DeviceEditor (http://j5.jbei.org), that automate the design of sequence agnostic, scar-less, multi-part assembly methodologies and translates them to robotics-driven protocols. Given a target library to construct, the software provides automated oligo, direct synthesis, and cost-optimal assembly process design, and integrates with liquid-handling robotic platforms to set up the PCR and multi-part assembly reactions. This work reduces the time, effort and cost of large scale cloning and assembly tasks, as well as enables research scales otherwise unfeasible without the assistance of computer-aided design tools and robotics. We are also pursuing the development of microfluidic devices that couple DNA library construction with functional assessment, a compelling process integration that is anticipated for the next round of scale-enabling foundational technologies.