In This Issue
Fall Issue of The Bridge on the Convergence of Engineering and the Life Sciences
October 1, 2013 Volume 43 Issue 3

Applications of Synthetic Biology to Enhance Life

Tuesday, September 24, 2013

Author: Jay D. Keasling and J. Craig Venter

For centuries a principal goal of science has been, first, to understand life at its most basic level and, second, to learn to control it. René Descartes (1596–1650), a pioneer of optics most often associated with “I think, therefore I am,” looked forward in his Discourse on the Method (1637) to a day when humans would become “masters and possessors of nature.”

Modern science has revealed that life ultimately consists of DNA-driven biological machines: all living cells run on DNA “software,” which directs hundreds of thousands of protein “robots.” Researchers have been digitizing life for decades, since they first figured out how to read the software of life by sequencing DNA. It is now possible to go in the other direction by starting with computerized digital code, designing a new form of life, chemically synthesizing its DNA, and then “booting it up” to produce the actual organism (Venter 2013).

Before genetic engineering, breeding and crosses were used to alter the makeup of pets, livestock, and crops, but it took many (human) generations to produce a better hunting dog and a high-yielding rice. Genetic engineering promised the ability to leapfrog over the timescales of breeding. And in the four decades since its initial development genetic engineering has had profound impacts, in the development of drugs (e.g., human insulin, human growth hormone), production of chemicals (e.g., 1,3-propanediol, amino acids, indigo dye), and generation of crops with advanced traits (e.g., Roundup-ready corn and soybeans).

Indeed, many of the most pressing problems facing humankind could be solved through the engineering of biology: synthesis of chemicals and fuels from sunlight and carbon dioxide, creation of life-saving drugs, production of food to feed the masses, and cleanup of the environment. But genetic engineering still takes far too long, is far too costly, and is far too unpredictable. Engineering even the simplest microorganism to produce some of the simplest organic chemicals can take a decade or more. And engineering a complicated organism like a plant can take much longer, not to mention the approval process, in part because the engineering is so unpredictable.

Synthetic Biology
Definition and Development

Synthetic biology—the application of engineering principles and designs to biology—promises to decrease development times (and thus cost) and to increase the reliability of genetic modification. It builds on advances in molecular and systems biology and seeks to transform biology in the same way that synthesis transformed chemistry and integrated circuit design transformed computing.

The element that distinguishes synthetic biology from traditional molecular biology is the focus on design and construction. Just as engineers now design integrated circuits based on the known physical properties of materials and then fabricate functioning circuits and entire processors (with relatively high reliability), synthetic biologists will soon design and build cells. Synthetic biology is to biology what synthetic chemistry is to chemistry. Just as synthetic chemists develop models for chemical reactions and apply those reactions to a myriad of chemicals to test their generality, one can imagine developing rules for biology to build new biological systems that are not found in the natural world and as such learn more about how biological systems function and enable the examination of new functions.

Progress toward synthetic biology has been made practical by developments in DNA sequencing and synthesis. Sequencing has increased understanding of the components and organization of natural biological systems and synthesis has enabled the testing of designs of new, synthetic biological parts. Furthermore, advances in long DNA synthesis make it possible to construct complicated genetic circuits and metabolic pathways. With these advances, synthetic biologists have made tremendous progress on the construction of genetic circuits and even entire chromosomes.

Benefits

There are at least two reasons to do synthetic biology. The first is to learn: a functional system reconstituted from its basic parts can yield a deeper understanding of fundamental design principles. Using synthetic biology, researchers can test models of how biology works by building systems and measuring differences between expectation and observation. Well-characterized genetic controls and wires enable the biologist to more easily perturb an existing biological system to study its inner workings.

One such example is work from the Lim laboratory on understanding how scaffolding complexes flexibly control the flow of information in yeast mitogen-activated protein kinase (MAPK) pathways (Bashor et al. 2008; Reményi et al. 2005). The understanding of these mechanisms has not only shed light on how normal responses occur and how they may have evolved but also allowed the engineering of rationally altered MAPK pathways with custom-designed nonnatural input/output response behaviors as well as precisely tuned response dynamics.

Similarly, reconstituting a biological function in a naïve host can be one of the best ways to understand all the components necessary to make it function. An example is demonstrated in recent work from the Voigt laboratory, where all native regulation was removed and the entire 23.5 kb segment of chromosomal DNA controlling nitrogen fixation in Klebsiella oxytoca, consisting of 20 genes in 7 operons, was removed and replaced with a completely redesigned, functioning, synthetic segment controlled exclusively by T7 promoters and ribosome binding sites of differing strengths (Temme et al. 2012).

The second reason to do synthetic biology is to solve some important problems or improve life or the environment. Advances in synthetic biology will accelerate the development of lower-cost, bio-based, sustainable production methods for food and feed ingredients, chemicals, and biofuels in the next 25 years. Increasingly, rational design of living organisms will become a hallmark of future bio-based production methods. Future decades will witness the introduction of semisynthetic cells (containing one or more synthetic chromosomes driving metabolic functions) and wholly synthetic cells into bio-based manufacturing processes.

Recent Accomplishments and Future Opportunities
Health

As many as 60 percent of successful drugs are of natural origin (Cragg et al. 1997), including some of the most potent anticancer, antibacterial, and antifungal drugs. Many of these are produced in small amounts in their native hosts, making them expensive to harvest. Organic chemistry methods are widely used to synthesize many pharmaceuticals (whether of natural origin or not) and to functionalize many pharmaceutically relevant natural products in use today. With considerable effort, functionalities can be introduced into molecules with precision. More recently, enzymes have been used for in vitro combinatorial functionalization of complex molecules. The next logical step in the synthesis of chemotherapeutics is the use of enzymes for combinatorial synthesis inside the cell. This would make it possible to produce drug candidates from inexpensive starting materials and avoid purification of the enzymes, as might be necessary for in vitro synthesis.

The production of a drug in a heterologous host generally involves the introduction of several genes in a biosynthetic cascade. During the past decade, metabolic engineering has started to change how drugs are developed from natural products in fundamentally new and practically useful ways. For example, the recognition that secondary metabolite biosynthetic pathways in bacteria and fungi are intimately linked at the genetic level greatly simplifies the cloning and sequence analysis of biosynthetic, regulatory, and self-resistance genes associated with a target natural product. The ability to produce these compounds in reagent quantities in biologically friendly heterologous hosts simplifies protein engineering and metabolic engineering programs.

Production of the antimalarial drug artemisinin is an example of the engineering of microorganisms to produce a needed drug. Artemisinin, which is extracted from Artemisia annua L. (family Asteraceae; commonly known as sweet wormwood), is highly effective against multidrug-resistant Plasmodium spp. but is in short supply and unaffordable to most malaria sufferers (Enserink 2005). Recently, Saccharomyces cerevisiae was engineered to produce high titers of artemisinic acid using an amorphadiene synthase, a novel cytochrome P450 monooxygenase, and several accessory enzymes from A. annua that perform a three-step oxidation of amorpha-4,11-diene to artemisinic acid (Figure 1) (Chang et al. 2007; Martin et al. 2003; Newman et al. 2006; Paddon et al. 2013; Ro et al. 2006; Westfall et al. 2012). Sanofi has licensed the yeast that produces artemisinic acid and developed a chemical process to convert the acid to artemisinin. This microbial production process will reduce the time to produce artemisinin and stabilize the supply and prices.

Figure 1

The artemisinin example is only the beginning. We foresee the day when nearly any natural product, and many nonnatural products, are produced in recombinant microbes, greatly expanding the numbers and types of drugs available.

Fuels

Alternative transportation fuels are in high demand due to concerns about climate change, the global petroleum supply, and energy security (Kerr 2007; Stephanopoulos 2007).

Advanced Biofuels from Sugar

The most widely used biofuels are ethanol generated from starch (corn) or sugar cane and biodiesel produced from vegetable oil or animal fats (Fortman et al. 2008).

Ethanol and carbon dioxide are terminal products of fermentative metabolism; therefore, it stands to reason that evolutionary selective pressure has forced cells to achieve high metabolic flux through this pathway and near-theoretical product yield. Saccharomyces cerevisiae produces ethanol anaerobically at 96 percent of theoretical maximum yield, but ethanol is not an ideal fuel molecule: its energy content is only about 70 percent, and because of its corrosivity and high hygroscopicity it is not compatible with the existing fuel infrastructure for distribution and storage.

Biodiesel has similar problems: it cannot be transported in pipelines because its cloud and pour points are higher than those for petroleum diesel (petrodiesel), and its energy content is approximately
11 percent lower than that of petrodiesel.

Furthermore, both ethanol and biodiesel are currently produced from limited agricultural resources, even though there is a large, untapped resource of plant biomass (lignocellulose) that could be used as a renewable source of carbon-neutral, liquid fuels (Blanch et al. 2008). Microbial production of transportation fuels from renewable lignocellulose has several advantages. First, the production is not reliant on agricultural resources commonly used for food, such as corn, sugar cane, soybean, and palm oil. Second, lignocellulose is the most abundant biopolymer on earth. Third, new biosynthetic pathways can be engineered to produce fossil fuel replacements, including short-chain, branched-chain, and cyclic alcohols, alkanes, alkenes, esters, and aromatics.

Table 1

There has been much progress recently in engineering microorganisms to produce gasoline, diesel, and jet fuel molecules (Steen et al. 2010; Schirmer et al. 2010; Chou et al. 2012) (Table 1), but these fuels have not been produced at scales anywhere near that of ethanol. Furthermore, achievement of the near-theoretical yields and high productivity from sugar required for improved economics is challenged by the fact that these products are secondary, not primary, energy-generating metabolites and can be inhibitory to cell function. Recent advances in synthetic biology and metabolic engineering will make it possible to overcome these hurdles and engineer microorganisms for the cost-effective production of biofuels from cellulosic biomass.

Algae

Production of biofuels from photoauxotrophic algae holds significant long-term promise because of their ability to produce lipid molecules that can be converted for blending with petroleum-derived crude that can be refined to form fuels, high areal lipid productivity, and use of marginal inputs (nonarable land, brackish water or sea water, and carbon dioxide). The highest reported sustained biomass productivities of ~25 grams biomass/m2/day are about one-third of estimated practical theoretical maximum productivity of ~75 grams biomass/m2/day (Eroglu et al. 2011). Oleaginous phototrophic cells typically show less accumulation of lipids (30–40 percent of dry cell weight) under nutrient limitation relative to heterotrophic cells, which have been reported to accumulate up to 70–80 percent of dry cell weight under similar conditions.

Recent research by Synthetic Genomics–ExxonMobil and others suggests that the development of economical algal biofuel processes will not be achieved through modestly engineered native cells. The relative absence of methods to target transgenes into specific chromosome locations means that exhaustive rounds of sequential gene introduction and subsequent screening and/or selection are needed to obtain engineered cells that successfully and stably express multiple desired transgenes. Growth rates averaging one or at best two doublings per day significantly lengthen this process. More significant metabolic engineering is necessary, and can be achieved only through the use of semi- or fully synthetic algae cells.

Three major improvements are required for economical fuel production: improved photosynthetic efficiency in mass culture; greater channeling of fixed carbon into higher fuel value lipids; and development of robust, “domesticated” algal cell lines that persist and compete in lower-cost open or semiopen environments. The development of synthetic chromosome technology for algae will accelerate the development of algal strains with coordinated, predictable, and stable expression of desired metabolic modifications.

Chemicals

Many petrochemicals are accessible through microbial metabolic pathways. Although there is an increasing market pull for renewable products, their production must be cost competitive with the incumbent petrochemicals to achieve market penetration. Therefore, the overall objectives for bio-based chemicals are both to create new metabolism to enable the synthesis of new products and to push the performance of resulting biocatalysts to their theoretical limits in terms of yield, titer, and productivity to achieve favorable economics.

The best example of engineered bio-based chemicals production is 1,3-propanediol (PDO), which remains the industry benchmark for development of a nonnatural commodity chemical. In a traditional genetic engineering tour de force, DuPont and Genencor engineered Escherichia coli to produce PDO, which was piloted and commercialized by Tate and Lyle (2007). The successful microbial catalyst had over two dozen genetic changes and required a decade of effort and over $100 million to complete the genetic engineering.

Table 2

Since the development of PDO, several other commodity chemicals are approaching commercialization (Table 2). For these products and others on the horizon, the low price points and margins of most commodity chemicals fail to support the development cost and timelines required of traditional genetic engineering approaches and demand more modern approaches to strain development.

Food and Feed Applications

The challenge of feeding a growing population with healthier alternatives in an environmentally sustainable manner is significant. The United Nations’ Food and Agriculture Organization estimates that the worldwide food supply will need to increase by 70 percent by 2050 (FAO 2009). Successful efforts will provide healthier foods, which will reduce the prevalence of chronic diseases, such as cardiovascular disease and diabetes, that impair quality of life and create a significant economic burden on the global healthcare system (WHO 2003).

These goals must be achieved, however, in the face of several long-term constraints. First, the availability of cultivatable land is limited and will be further restricted by competing demand from a growing population and climate change, which may result in a reduction in cultivated land area by 2050.

Second, the growing demand for animal protein throughout the world, a trend correlated with economic development, will intensify the need for primary food sources. The production of 1 kilogram of animal protein requires 10 kg of vegetable or microbial protein and 15,000 liters of water (Millstone and Lang 2008). A similar situation exists with respect to fish supply, where a 30-year lack of growth in wild fish harvest has driven a growing demand for farmed fish.

Third, the availability of essential nutrients, such as nitrogen, phosphorus, and nonbrackish water, will be limited and increasingly costly (Déry and Anderson 2007; Fry and Haden 2006). This constraint will severely limit the world’s ability to achieve higher crop yields through chemically intensive agronomic practices, which have been used in developed countries to improve yield.

It is estimated that the combined effects of climate change, land degradation, cropland losses, water scarcity, and species infestations may cause projected yields to be 5–25 percent short of demand by 2050 (Sheeran 2009). The production of foods that are healthier, on marginal land with fewer chemical inputs and yields that can support a growing population, will be enabled by the application of synthetic genomic technologies to develop improved or novel plant, animal, and microbial food sources, as described in the following sections.

Terrestrial Crops

The introduction of genetically engineered crops in the 1990s highlighted both the yield improvement promise of this technology and environmental and public acceptance concerns that will have to be addressed before its broader-scale adoption.

Initial plant genetic engineering efforts involved the introduction of single genes to inhibit insect damage (e.g., Bt toxin) or facilitate the use of broad-spectrum herbicides (e.g., glyphosate resistance) for a limited number of high-acreage crops, corn, soybean, and rapeseed canola. Synthetic genomics technologies (e.g., for synthetic chromosomes) are needed to consolidate the larger number of simultaneous genetic changes, or “stacked traits,” required either to combine beneficial traits or to provide for multigenetic traits and introduce them with a single transformation event.

Major food-producing crops will be “semisynthetic”: they will contain at least one synthetic chromosome that encodes genes driving multiple beneficial traits such as enhanced photosynthetic efficiency, drought resistance, salt tolerance, and production of beneficial micronutrients. These synthetic chromosomes will be introduced into a variety of genetic backgrounds that are better suited for growth in broader geographical regions.

Conventional breeding techniques will be a thing of the past, replaced by molecular breeding techniques in which plant variants are crossed based on knowledge of differences in their genomic sequences. Molecular breeding will be used in a variety of cereal grain and vegetable crops, such as sorghum, pearl millet, quinoa, potatoes, tomatoes, and beans.

Meat-Producing Animals

Animals and fish used as food sources are exclusively the product of natural or conventional breeding. Recent advances enabling the introduction of genes in multiple animal species and the cloning of whole organisms have generated speculation about the potential of using transgenic animals as food sources. Two developments of note include reports of a transgenic pig modified to have a healthier, omega-3 fatty acid–rich profile (Lai et al. 2006) and a transgenic Chinook salmon modified to grow twice as fast as its natural counterpart (Marris 2010).

Analogous to the situation described for terrestrial crops, bioengineered animals that exhibit faster growth rates, improve yield on costly feed ingredients, and provide healthier sources of meat protein will be readily available in 2050. Genetic engineering efforts are likely to focus on reducing the amount of saturated fat, with a commensurate increase in the amount of monounsaturated and polyunsaturated fat, and partially replacing cholesterol with plant sterols.

Microbial Food Sources

A number of commonly used food ingredients are produced using natural, classically improved, and/or genetically modified microorganisms. Ingredients produced by the latter, through microbial fermentation, include amino acids, acidulants (e.g., citric acid), texturizing ingredients (e.g., xanthan gum), enzymes, and colorants. A fungal microorganism, Fusarium venenatum, is the source of a vegetarian, but expensive, protein source, Quorn™, which is increasingly available in packaged food products (Wiebe 2004).

Cultivated, highly productive photosynthetic microalgae will become a major new food and feed ingredient source. These simple plants will be semi- or wholly synthetic, designed to produce macro- and micronutrients with significantly higher productivity and areal yield than natural counterparts. A single-cell “algal bean” will provide a renewable and sustainable supply of oil, flour, and protein for use as food and feed ingredients.

Why microalgae? They are simple, single-cell plants that can achieve significantly higher areal productivities on marginal land with nonpotable water and limited nutrient inputs. Current oil and protein yields of natural microalgae already are severalfold higher than those achieved in terrestrial crops, and can theoretically go much higher with appropriate engineering, as shown in Figure 2. Economic modeling indicates that achievement of the yields shown in Figure 2 can result in production costs comparable to those for traditional crops. Furthermore, microalgae are inherently excellent sources of beneficial nutrients such as long-chain polyunsaturated omega-3 fatty acids, carotenoids, antioxidants, and certain vitamins.

Figure 2

Development of algae crops, however, will require significant engineering to further increase yield necessary for cost reduction, tailor nutritional, taste, and texture profile for consumption by animals and humans, and provide measures for appropriate biocontainment.

Ethical, Legal, and Social Implications of Synthetic Biology

Synthetic biology combines methods for the chemical synthesis of DNA with computational techniques for DNA design. As discussed above, these new techniques have the potential to accelerate scientific and technological progress in a variety of areas. But they also present challenges:

  • Synthetic biology can be “dual-use”—in addition to useful advances for society, it provides those with nefarious intent new ways to cause harm.
  • Improvements in the speed and cost of DNA synthesis are opening the field to new participants (e.g., engineers and computer scientists) who must be trained to work safely in the lab.
  • New products made using these new techniques must be reviewed and reviewable by a regulatory system that was designed for earlier generations of genetic engineering.
  • The ability to modify life in new ways will challenge many and offend some.
  • Finally, and just as important from a policy perspective, the controversies that have surrounded the use of genetic engineering for the last several decades remain and perhaps even dominate the policy debate surrounding synthetic biology.

Biosecurity

Biosecurity was the first societal concern related to synthetic biology to reach the attention of policymakers, beginning with the synthesis in 2002 of an infectious poliovirus constructed in the laboratory directly from nucleic acids by Eckard Wimmer and colleagues (Cello et al. 2002). While this work was built on methods slowly developed over the prior 30 years, the paper demonstrated for the first time in a post–September 11 world the feasibility of synthesizing a complete human pathogen using only the published DNA sequence and mail-ordered raw materials. Wimmer and his coworkers required about a year to synthesize the approximately 7500 bp poliovirus. In 2004, a team at the Venter Institute (JCVI) synthesized a similar size virus (phiX-174, which infects bacteria rather than humans) in about two weeks. The biosecurity implications of the new technology reached the attention of policymakers in both Congress and the administration.

 In 2004, in response to these developments, the National Institutes of Health (NIH) established the National Scientific Advisory Board for Biosecurity (NSABB) to provide advice about dual-use biological research (i.e., research with legitimate scientific purpose that may be misused to pose a biologic threat to public health and/or national security). Synthetic biology falls within NSABB’s purview.

In 2010, the US Department of Health and Human Services (HHS) published Screening Framework Guidance for Providers of Synthetic Double-Stranded DNA, “to minimize the risk that unauthorized individuals or individuals with malicious intent will obtain ‘toxins and agents of concern’ through the use of nucleic acid synthesis technologies” (HHS 2010, 3). It is now standard practice for suppliers of synthesized DNA (e.g., SGI-DNA, a subsidiary of Synthetic Genomics, Inc.) to screen orders to determine whether they contain dangerous “sequences of concern” and to make sure customers are legitimate research users. However, the 2010 HHS guidance for screening synthetic nucleotides applies only to providers of synthetic double-stranded DNA.

Biosafety

Commercial products that might cause harm to the environment or human health are subject to a long list of federal laws and regulations. The US oversight and regulatory framework for products developed using genetic engineering—including synthetic biology—stems from the 1986 Coordinated Framework for Regulation of Biotechnology (OSTP 1986). The Coordinated Framework assigned primary responsibility for regulating the products of biotechnology to three agencies: the Food and Drug Administration (FDA), the Animal and Plant Health Inspection Service (APHIS) of the US Department of Agriculture (USDA), and the Environmental Protection Agency (EPA), using an array of laws in place at the time. The key laws are the Food, Drug, and Cosmetic Act (for food products and animal biotechnology), the Plant Pest Act (for plant biotechnology), and the Toxic Substances Control Act (for microbes).

The public likely expects that any living organisms modified by synthetic biology and intended for market will first be reviewed for possible adverse effects to the environment or human health. Although the Coordinated Framework has been in place for more than 25 years, it is still controversial. Known harms to the environment or human health from introduced, genetically engineered products have been minimal, but some view the current system as too lax, others as too burdensome.

Ethical Issues

Ethical issues related to synthetic biology were reviewed by the Presidential Commission for the Study of Bioethical Issues (PCSBI) in response to a request by President Obama after the JCVI announcement of the creation of the first self-replicating synthetic genome in a bacterial cell. The resulting report, New Directions: The Ethics of Synthetic Biology and Emerging Technologies (PCSBI 2010), presents 18 recommendations; the major recommendation is that the “Federal government start to coordinate and oversee what all Federal agencies are doing in the field of synthetic biology.”1 The report’s authors “do not recommend that additional agencies or oversight bodies need to be created to oversee synthetic biology,” but “do recommend that the government stay current on the advances with the science and remain forward looking about the potential benefits and risks to the public.”2

Required Advances in Synthetic Biology Technology

Just as a new building must have both a good design and robust, sound, and efficient methods for construction, so do new bio-based processes require more complete knowledge of cellular design and cost-effective and efficient cell construction methods. Major gaps in knowledge of gene structure-function relationships and of the hierarchical regulatory networks that dictate temporal metabolic activity, coupled with several limitations in the ability to accurately synthesize and assemble large strands of DNA “software,” have hampered the development of complex engineered cells.

Further advances in synthetic biology technologies are needed to achieve greater penetration of bio-based manufacturing processes. First, the cost of synthesizing and assembling DNA must drop by two orders of magnitude to facilitate large-scale engineering of microbial, plant, and animal systems. DNA synthesis and assembly costs are projected to decrease significantly during this decade. Low cost must be accompanied by high accuracy in systems where even one error in the genome may be lethal (Gibson et al. 2010). Second, more robust methods of introducing and activating synthetic chromosomes in higher organisms must be developed. Third, knowledge of the biological conversion of genotype (genes present) to phenotype (trait[s]) must increase exponentially in order to inform rational engineering or breeding of biological systems. It is not enough to have the tools to build an organism. What is it that needs to be built? Fourth, tools to diagnose and troubleshoot engineered cells must continue to improve, much as tools to troubleshoot errors in complex software have evolved.

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FOOTNOTES

1 Quoted from the report website, “Synthetic Biology FAQs: What are the major findings of the synthetic biology report?” (www.bioethics.gov/node/353).

2 On a historical note, the earliest look at the ethical and societal implications of synthetic biology was by Cho and colleagues (1999). Interestingly, the societal concerns they identify are similar to those identified above, though the relative emphasis of concerns differs somewhat from today’s policy discussions.

About the Author:Jay D. Keasling (NAE) is a professor in the Departments of Chemical Engineering and Bioengineering at the University of California, Berkeley; director of the Physical Biosciences Division at Lawrence Berkeley National Laboratory and of the Synthetic Biology Engineering Research Center; and CEO of the Joint BioEnergy Institute. J. Craig Venter (NAS) is founder, chairman, and CEO of the J. Craig Venter Institute and Synthetic Genomics Inc., both in La Jolla, California.