commentary
Engineering synergy in biotechnology Jens Nielsen, Martin Fussenegger, Jay Keasling, Sang Yup Lee, James C Liao, Kristala Prather & Bernhard Palsson
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© 2014 Nature America, Inc. All rights reserved.
Biotechnology is a central focus in efforts to provide sustainable solutions for the provision of fuels, chemicals and materials. On the basis of a recent open discussion, we summarize the development of this field, highlighting the distinct but complementary approaches provided by metabolic engineering and synthetic biology for the creation of efficient cell factories to convert biomass and other feedstocks to desired chemicals.
S
ince the industrial revolution, progress in manufacturing has been defined in terms of making more things available more consistently, cheaply and quickly. However, change has too often come at the overuse of limited resources or environmental costs. Thus, society in general, and scientists in particular, have sought to improve the efficiency of various processes by discovering alternate paths to reach similar goals. Since the 1980s, scientists have made tremendous advances along one such path: the development of new, sustainable bioprocesses for production of fuels, chemicals and materials. These bioprocesses in turn depend upon the design and construction of efficient cell factories that can ensure robust conversion of a raw material to the product of interest. In May 2013, at the Copenhagen Bioscience Conference on Cell Factories and Biosustainability in Favreholm, Denmark, we involved all of the participants (>130) in an open discussion on the potential contributions and complementarity of metabolic engineering and synthetic biology in providing enabling solutions for the field and society at large. The objective of this discussion was to identify commonalities between the two research fields and also how synergy and complementarity can be exploited for further advancing the design and construction of new cell factories. Here we summarize the outcome of this discussion. Manipulating microorganisms for human benefit is no recent matter: traditionally, species that naturally produced a desired molecule were identified and then improved through classical strain engineering on the basis of
mutagenesis and screening. This has been a very efficient approach and has resulted in low-cost production processes for many different chemicals, including penicillin1, citric acid, glutamate and lysine2, to name a few. With the advent of molecular biology and the associated portfolio of restriction enzymes, engineering plasmids for stable high-level expression, recyclable markers and other tools in the late 1980s, chemical engineers realized that biological systems could be programmed to produce chemicals with higher precision and cost efficiency than could be achieved by classical chemical synthesis protocols3,4. Indeed, these engineers anticipated that using and refining molecular biology tools could open the door to design not only individual reactions but also entire gene networks and whole organisms, resulting in coining the phrase ‘cell factories’. The subsequent collaboration between molecular biologists and chemical engineers led to the distinct field of metabolic engineering that brought engineering concepts to the design and construction of cell factories5–8; the particular strength of chemical engineers in mathematical modeling originally shaped the field on a trajectory that continues today. At this time, the activities of the early metabolic engineers focused on reprogramming one gene at a time. In addition to improved chemical production, this reprogramming led to some of the inaugural genetic circuits now popularized by the field of synthetic biology, perhaps best exemplified by the toggle switch, which was first designed by metabolic engineers9, refined and reinvented in prokaryotes by synthetic biologists10 and
nature chemical biology | VOL 10 | MAY 2014 | www.nature.com/naturechemicalbiology
later replicated in mammalian cells11. Since those days, metabolic engineering has been transformed further by advanced methods for genome engineering, including genome sequencing projects and intensive functional genomic research, as well as by improved methods to interrogate and understand cellular metabolism, including systems biology as a mechanism to provide detailed information about global metabolic interaction networks (Box 1). Following the initial definition of metabolic engineering as a discipline in the early 1990s3,4, the first international conference on metabolic engineering was held in 1996 in Danvers, Massachusetts, USA, and in 1998 the journal Metabolic Engineering was established. The Metabolic Engineering conference has grown into a lively international conference series organized every two years and recently resulted in the foundation of the International Metabolic Engineering Society (IMES; http://www.aiche.org/ sbe/community/imes/about), which currently has more than 500 members, with Metabolic Engineering signed on as a society journal. The society and the associated conference have a very active industrial involvement, and they are an excellent example of how basic engineering science can be translated into the design and implementation of new industrial processes. Indeed, metabolic engineering has driven the development of several new industrial processes. Some prominent examples are outlined below. • Dupont has launched a process for production of 1,3-propanediol using a metabolically engineered Escherichia coli12. 1,3-propanediol is used as one of the key chemicals in the production of 319
commentary Box 1 | Definitions of metabolic engineering and synthetic biology. Metabolic engineering The research field is broadly defined as ‘the development of methods and concepts for analysis of metabolic networks, typically with the objective of finding targets for engineering of cell factories’. The improvement of cellular properties may involve a range of different strategies, i.e., improvement of substrate range, production of new chemicals, improvement of yield and productivity and improvement of cellular robustness (improved tolerance towards toxic compounds and so on).
npg
© 2014 Nature America, Inc. All rights reserved.
Synthetic biology The research field is broadly defined as ‘the design and construction of new biological systems (e.g., genetic control systems, metabolic pathways, chromosomes, cells) that do not exist in nature through the assembly of well-characterized, standardized, reusable components’. These constructed systems may be useful for engineering metabolism or a host of other biotechnology projects.
the polymer Sorona, which is used for the manufacturing of fabrics, carpets and a wide range of plastic-based materials. The process was developed through the close collaboration of Dupont, Genencor and Tate&Lyle. • Gevo and Butamax, a Dupont-BP joint venture, have both developed a process for production of isobutanol using metabolically engineered yeast13. Isobutanol can be used as blend-in gasoline and it can also be used for synthesis of jet fuels and other chemicals. There have been several studies on optimizing the isobutanol pathway in E. coli14. • Amyris has developed a process for production of artemisinin using a metabolically engineered yeast15. This followed several years of development in one of our research groups (J.K.), and Sanofi launched the process in 2013 for production of 70 million malaria cures. • Genomatica has developed a process for production of 1,4-butanediol using a metabolically engineered E. coli16. 1,4-butanediol has a world market exceeding 1 million tons and is used for the synthesis of solvents and polymers. These and many other examples provide exciting evidence that metabolic engineering projects can provide meaningful and economic solutions to chemical challenges. As molecular biology and engineering became more closely intertwined, new questions arose regarding the properties and behaviors of biological systems. In particular, electrical engineers and computer scientists began to explore whether the kinds of logical principles that define computer science could be applied to biology, with these investigations coalescing into the field of synthetic biology17. Because the field grew more out of a philosophical concept rather than a preexisting scientific topic, the research 320
spans a broad range but is unified in its use of core engineering concepts, such as mathematical models to describe (bio) physical and (bio)chemical systems, and the resulting ability to use those same models for design, i.e., to predict successful system modifications to achieve a predetermined goal. Indeed, much of the work done under the umbrella of synthetic biology is not about the analysis or modification of metabolic pathways or whole biological systems but focuses on construction of generic ‘parts’ and ‘devices’ that oscillate, switch or sense or perform the biological equivalent of computation. Synthetic biology has also made its presence known in terms of associations such as the International Association Synthetic Biology (IASB; http://www.ia-sb. eu/go/synthetic-biology/), conferences such as the BioBricks Foundation Synthetic Biology Conference (Sbx.0) Series, the very successful International Genetically Engineered Machine (iGEM; http://www. igem.org/) student competition series and journals such as ACS Synthetic Biology, launched in 2012. Though synthetic biology did not begin with a mandate to manipulate cells for practical purposes, the obvious conceptual and practical synergies with metabolic engineering have led to a close coupling of the two fields. At the conference, we discussed how these synergies could be further exploited to maximize progress in biotechnology, and this resulted in three messages. First, we should take full advantage of our common engineering origins, frameworks and language. Clearly, both metabolic engineering and synthetic biology feature a focus on predictable design and hence have a common approach to studying and engineering biological systems. In both fields, there is a vision of being able to predict and design biological systems; in metabolic engineering, there is primarily a focus on metabolism of cell
factories, and in synthetic biology the focus is on an even broader context involving regulatory circuits and biomolecules. This does not, however, mean that metabolic engineering only involves engineering the metabolic network, but there is a focus on achieving the objective of overproducing a desired product through the optimization of the metabolic network, which is intertwined with gene regulatory and signaling networks. Second, we should appreciate the distinct approaches of the two fields. Metabolic engineering often involves a top-down approach where the metabolic network of a cell factory is retrofitted to overproduce a certain metabolite18. This may involve altering the regulation of carbon fluxes such that the carbon source is preferentially directed toward the product of interest and as little as possible carbon is lost in byproducts and to cellular growth. An analogy of this process is the retrofitting or reengineering of an existing chemical plant to produce new products by changing the connections between the different reactors and unit operations. In contrast, synthetic biology tends to take a bottom-up approach by reconstructing new biological parts that can find broad biological use18 or specifically meet predefined objectives. Though many examples focus on a single gene or small group of genes, increasingly synthetic biologists may aim to create a completely synthetic biological pathway. Though it has been possible to construct a completely synthetic genome19, translation of this result to the design of new efficient cell factories from scratch will depend on both increased knowledge of biological systems and the identification of a desired pathway for which it would be easier to start fresh than retrofit an existing cell. Working across fields with these specific interests in mind can often lead to overlap in which new biological parts are being used in combination with reengineering of a cell factory18. The potential contribution of parts from synthetic biology to metabolic engineering is illustrated with the construction of devices (to accomplish a particular goal) from well-characterized, standardized components. These devices derived from synthetic biology would find use in controlling biosynthetic pathways in a microorganism retrofitted to produce a desired product. For example, the synthetic biologist would use a computeraided design (CAD) program to design a genetic control circuit from a database of characteristics of biological components generated by a biofab20. She might desire to query for the following components
nature chemical biology | VOL 10 | MAY 2014 | www.nature.com/naturechemicalbiology
commentary Systems biology Synthetic biology
Mathematical models
Computational design for forward engineering of genetic circuits, biological systems or cells
S A
Metabolic engineering
B
Computational analysis of metabolic networks for their reengineering
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© 2014 Nature America, Inc. All rights reserved.
Biological system
Figure 1 | Illustration of how systems biology impacts both synthetic biology and metabolic engineering. The objective of systems biology is to develop predictive mathematical models for biological systems, and these models can be used for design of completely synthetic cells (forward engineering) and for modifying existing cells through metabolic engineering (reengineering). At the same time, the use of computational methods (as in other engineering disciplines) is at the core of both synthetic biology and metabolic engineering.
the time needed for tuning devices and increase their reliability. Of the relevant synthetic biology parts that are needed for advancing metabolic engineering are biosensors that can be used for highthroughput fluorescence-activated cell sorting screening of improved producers and promoters with high-precision expression and that can be controlled in response to environmental conditions. Third, we should build on existing knowledge to advance design and construction faster. Metabolic engineers think in terms of a host organism being defined in large part by its underlying metabolism and so seek to develop increasingly complete mathematical models of metabolic pathways, whereas synthetic biologists enjoy the challenge of understanding why a designed part is not working according to known principles. By integrating these strengths, we can more quickly move from conceiving a phenotype to actually constructing a
to build the circuit: a complex protein consisting of a DNA binding element and a metabolite binding element, a promoter-operator sequence or a ribosome binding site and so on. Once designed, the CAD program would output the DNA sequences, which would be ordered from a DNA synthesis company. When it arrived, the synthetic biologist would clone the DNA encoding the control system in front of a metabolic pathway that either produces or uses the chemical that the circuit measures and actuates in response. Because the circuit had been designed from well-characterized, standardized components, it would meet performance criteria in the retrofitted host with little tuning of its characteristics. In a less-thanideal world, the synthetic biologist may not have a CAD program but could still design biological systems from well-characterized components using mathematical models and other design tools. The use of wellcharacterized components would reduce
Table 1 | Overlap and differences between metabolic engineering and synthetic biology. Metabolic engineering Common themes Common tools
Synthetic biology Non-natural behavior
Molecular biology, mathematical modeling
Working domain
Chemistry, enzymes, genes, pathways, biological networks, cells
Genes, expression circuits, cells
Unique domains
Chemical and biochemical networks
Logic parts and domains
Objective
Engineering a cellular phenotype
Engineering of biological parts
Specialized tools
Flux quantification and network analysis
Synthesis of biological components
Applications
Industrial biotechnology
Diverse
nature chemical biology | VOL 10 | MAY 2014 | www.nature.com/naturechemicalbiology
working phenotype. At the moment, though a pathway can be built in silico in a few minutes, it can and usually does take much longer to improve its function within the host to the point that it can support a commercial process17. Indeed, several commercial examples, such as those mentioned above, have shown that designing a cell factory is a monumental task where the cost to build a new cellular phenotype is in the range of hundreds of millions of US dollars. Improving these processes will take focused effort and a realistic expectation of the challenges in translating from concept to practice. In parallel, both groups can also contribute to unraveling cell function from an engineering perspective, seeking to enumerate design principles governing cellular functions akin to chemical engineering where first principles of reaction kinetics, thermodynamics and transport phenomena can be used to design reactors de novo. With advancement in our understanding of cellular function, in particular through systems biological research where predictive mathematical models are being developed21–24, we anticipate the design of completely new biological components that can be used for metabolic engineering purposes (Fig. 1). Our discussions resulted in the following conclusion. The two fields of metabolic engineering and synthetic biology differ in their foundational objectives: Metabolic engineering was established to enable and develop the biological production of drugs, chemicals, fuels and materials, i.e., a defined practical endpoint. Synthetic biology was established with the objective of building biological parts, modules and systems to understand and manipulate biological systems, i.e., a fundamental scientific focus. The two disciplines clearly overlap, but there are elements of each that remain distinct (Table 1). We expect that metabolic engineering will increasingly adopt strategies from synthetic biology, e.g., for gene synthesis and fine control of gene expression, whereas many synthetic biologists are taking an increased interest in an objective-driven strategy of engineering circuits and whole-cell metabolism, a characteristic of metabolic engineering. Similarly, companies focused on industrial biotechnology have embraced metabolic engineering, and many have begun to exploit tools from synthetic biology. A multifaceted approach building on the strengths of each field may therefore serve as one of the pillars underlying a modern, sustainable society. ◾ Jens Nielsen, Jay Keasling, Sang Yup Lee and Bernhard Palsson are at the Novo Nordisk Foundation 321
commentary ETH Zurich, Department of Biosystems Science and Engineering, Basel, Switzerland. James C. Liao is at the Department of Chemical and Biomolecular Engineering, University of California–Los Angeles, Los Angeles, California, USA. Kristala Prather is at the Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. Kristala Prather is also at the Synthetic Biology Engineering Research Center (SynBERC), Cambridge, Massachusetts, USA. e-mail: [email protected] References 1. Thykaer, J. & Nielsen, J. Metab. Eng. 5, 56–69 (2003). 2. de Graaf, A.A., Eggeling, L. & Sahm, H. Adv. Biochem. Eng. Biotechnol. 73, 9–29 (2001). 3. Bailey, J.E. Science 252, 1668–1675 (1991). 4. Stephanopoulos, G. & Vallino, J.J. Science 252, 1675–1681 (1991). 5. Stephanopoulos, G., Aristidou, A. & Nielsen, J. Metabolic Engineering: Principles and Methodologies. (Academic Press, 1998) 6. Nielsen, J. Appl. Microbiol. Biotechnol. 55, 263–283 (2001).
7. Stephanopoulos, G., Alper, H. & Moxley, J. Nat. Biotechnol. 22, 1261–1267 (2004). 8. Keasling, J.D. Science 330, 1355–1358 (2010). 9. Chen, W., Kallio, P.T. & Bailey, J.E. Gene 130, 15–22 (1993). 10. Gardner, T.S., Cantor, C.R. & Collins, J.J. Nature 403, 339–342 (2000). 11. Kramer, B.P. et al. Nat. Biotechnol. 22, 867–870 (2004). 12. Nakamura, C.E. & Whited, G.M. Metabolic engineering for microbial production of 1,3-propanediol. Curr. Opin. Biotechnol. 14, 454–459 (2003). 13. Hong, K.-K. & Nielsen, J. Cell. Mol. Life Sci. 69, 2671–2690 (2012). 14. Atsumi, S., Hanai, T. & Liao, J.C. Nature 451, 86–89 (2008). 15. Paddon, C.J. et al. Nature 496, 528–532 (2013). 16. Yim, H. et al. Nat. Chem. Biol. 7, 445–452 (2011). 17. Stephanopoulos, G. ACS Synth. Biol. 1, 514–525 (2012). 18. Nielsen, J. & Keasling, J.D. Nat. Biotechnol. 29, 693–695 (2011). 19. Gibson, D.G. et al. Science 329, 52–56 (2010). 20. Holtz, W.J. & Keasling, J.D. Cell 140, 19–23 (2010). 21. Feist, A.M. & Palsson, B.O. Nat. Biotechnol. 26, 659–667 (2008). 22. Lee, S.Y., Lee, D.-Y. & Kim, T.Y. Trends Biotechnol. 23, 349–358 (2005). 23. Tyo, K.E.J., Kocharin, K. & Nielsen, J. Curr. Opin. Microbiol. 13, 255–262 (2010). 24. Lee, J.W. et al. Nat. Chem. Biol. 8, 536–546 (2012).
Competing financial interests The authors declare no competing financial interests.
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© 2014 Nature America, Inc. All rights reserved.
Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark. Jens Nielsen is also at the Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden, and the Science for Life Laboratory, Royal Institute of Technology, Solna, Sweden. Jay Keasling is also at the Joint Bioenergy Institute, Emeryville, California, USA, the Department of Chemical and Biomolecular Engineering and the Department of Bioengineering, University of California–Berkeley, Berkeley, California, USA, the Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA, and the Synthetic Biology Engineering Research Center (SynBERC), Berkeley, California, USA. Sang Yup Lee is also at the Department of Chemical and Biomolecular Engineering, KAIST, Daejeon, Korea. Bernhard Palsson is also at the Department of Bioengineering, University of California–San Diego, La Jolla, California, USA. Martin Fussenegger is at
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nature chemical biology | VOL 10 | MAY 2014 | www.nature.com/naturechemicalbiology
Engineering synergy in biotechnology Jens Nielsen, Martin Fussenegger, Jay Keasling, Sang Yup Lee, James C Liao, Kristala Prather & Bernhard Palsson
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© 2014 Nature America, Inc. All rights reserved.
Biotechnology is a central focus in efforts to provide sustainable solutions for the provision of fuels, chemicals and materials. On the basis of a recent open discussion, we summarize the development of this field, highlighting the distinct but complementary approaches provided by metabolic engineering and synthetic biology for the creation of efficient cell factories to convert biomass and other feedstocks to desired chemicals.
S
ince the industrial revolution, progress in manufacturing has been defined in terms of making more things available more consistently, cheaply and quickly. However, change has too often come at the overuse of limited resources or environmental costs. Thus, society in general, and scientists in particular, have sought to improve the efficiency of various processes by discovering alternate paths to reach similar goals. Since the 1980s, scientists have made tremendous advances along one such path: the development of new, sustainable bioprocesses for production of fuels, chemicals and materials. These bioprocesses in turn depend upon the design and construction of efficient cell factories that can ensure robust conversion of a raw material to the product of interest. In May 2013, at the Copenhagen Bioscience Conference on Cell Factories and Biosustainability in Favreholm, Denmark, we involved all of the participants (>130) in an open discussion on the potential contributions and complementarity of metabolic engineering and synthetic biology in providing enabling solutions for the field and society at large. The objective of this discussion was to identify commonalities between the two research fields and also how synergy and complementarity can be exploited for further advancing the design and construction of new cell factories. Here we summarize the outcome of this discussion. Manipulating microorganisms for human benefit is no recent matter: traditionally, species that naturally produced a desired molecule were identified and then improved through classical strain engineering on the basis of
mutagenesis and screening. This has been a very efficient approach and has resulted in low-cost production processes for many different chemicals, including penicillin1, citric acid, glutamate and lysine2, to name a few. With the advent of molecular biology and the associated portfolio of restriction enzymes, engineering plasmids for stable high-level expression, recyclable markers and other tools in the late 1980s, chemical engineers realized that biological systems could be programmed to produce chemicals with higher precision and cost efficiency than could be achieved by classical chemical synthesis protocols3,4. Indeed, these engineers anticipated that using and refining molecular biology tools could open the door to design not only individual reactions but also entire gene networks and whole organisms, resulting in coining the phrase ‘cell factories’. The subsequent collaboration between molecular biologists and chemical engineers led to the distinct field of metabolic engineering that brought engineering concepts to the design and construction of cell factories5–8; the particular strength of chemical engineers in mathematical modeling originally shaped the field on a trajectory that continues today. At this time, the activities of the early metabolic engineers focused on reprogramming one gene at a time. In addition to improved chemical production, this reprogramming led to some of the inaugural genetic circuits now popularized by the field of synthetic biology, perhaps best exemplified by the toggle switch, which was first designed by metabolic engineers9, refined and reinvented in prokaryotes by synthetic biologists10 and
nature chemical biology | VOL 10 | MAY 2014 | www.nature.com/naturechemicalbiology
later replicated in mammalian cells11. Since those days, metabolic engineering has been transformed further by advanced methods for genome engineering, including genome sequencing projects and intensive functional genomic research, as well as by improved methods to interrogate and understand cellular metabolism, including systems biology as a mechanism to provide detailed information about global metabolic interaction networks (Box 1). Following the initial definition of metabolic engineering as a discipline in the early 1990s3,4, the first international conference on metabolic engineering was held in 1996 in Danvers, Massachusetts, USA, and in 1998 the journal Metabolic Engineering was established. The Metabolic Engineering conference has grown into a lively international conference series organized every two years and recently resulted in the foundation of the International Metabolic Engineering Society (IMES; http://www.aiche.org/ sbe/community/imes/about), which currently has more than 500 members, with Metabolic Engineering signed on as a society journal. The society and the associated conference have a very active industrial involvement, and they are an excellent example of how basic engineering science can be translated into the design and implementation of new industrial processes. Indeed, metabolic engineering has driven the development of several new industrial processes. Some prominent examples are outlined below. • Dupont has launched a process for production of 1,3-propanediol using a metabolically engineered Escherichia coli12. 1,3-propanediol is used as one of the key chemicals in the production of 319
commentary Box 1 | Definitions of metabolic engineering and synthetic biology. Metabolic engineering The research field is broadly defined as ‘the development of methods and concepts for analysis of metabolic networks, typically with the objective of finding targets for engineering of cell factories’. The improvement of cellular properties may involve a range of different strategies, i.e., improvement of substrate range, production of new chemicals, improvement of yield and productivity and improvement of cellular robustness (improved tolerance towards toxic compounds and so on).
npg
© 2014 Nature America, Inc. All rights reserved.
Synthetic biology The research field is broadly defined as ‘the design and construction of new biological systems (e.g., genetic control systems, metabolic pathways, chromosomes, cells) that do not exist in nature through the assembly of well-characterized, standardized, reusable components’. These constructed systems may be useful for engineering metabolism or a host of other biotechnology projects.
the polymer Sorona, which is used for the manufacturing of fabrics, carpets and a wide range of plastic-based materials. The process was developed through the close collaboration of Dupont, Genencor and Tate&Lyle. • Gevo and Butamax, a Dupont-BP joint venture, have both developed a process for production of isobutanol using metabolically engineered yeast13. Isobutanol can be used as blend-in gasoline and it can also be used for synthesis of jet fuels and other chemicals. There have been several studies on optimizing the isobutanol pathway in E. coli14. • Amyris has developed a process for production of artemisinin using a metabolically engineered yeast15. This followed several years of development in one of our research groups (J.K.), and Sanofi launched the process in 2013 for production of 70 million malaria cures. • Genomatica has developed a process for production of 1,4-butanediol using a metabolically engineered E. coli16. 1,4-butanediol has a world market exceeding 1 million tons and is used for the synthesis of solvents and polymers. These and many other examples provide exciting evidence that metabolic engineering projects can provide meaningful and economic solutions to chemical challenges. As molecular biology and engineering became more closely intertwined, new questions arose regarding the properties and behaviors of biological systems. In particular, electrical engineers and computer scientists began to explore whether the kinds of logical principles that define computer science could be applied to biology, with these investigations coalescing into the field of synthetic biology17. Because the field grew more out of a philosophical concept rather than a preexisting scientific topic, the research 320
spans a broad range but is unified in its use of core engineering concepts, such as mathematical models to describe (bio) physical and (bio)chemical systems, and the resulting ability to use those same models for design, i.e., to predict successful system modifications to achieve a predetermined goal. Indeed, much of the work done under the umbrella of synthetic biology is not about the analysis or modification of metabolic pathways or whole biological systems but focuses on construction of generic ‘parts’ and ‘devices’ that oscillate, switch or sense or perform the biological equivalent of computation. Synthetic biology has also made its presence known in terms of associations such as the International Association Synthetic Biology (IASB; http://www.ia-sb. eu/go/synthetic-biology/), conferences such as the BioBricks Foundation Synthetic Biology Conference (Sbx.0) Series, the very successful International Genetically Engineered Machine (iGEM; http://www. igem.org/) student competition series and journals such as ACS Synthetic Biology, launched in 2012. Though synthetic biology did not begin with a mandate to manipulate cells for practical purposes, the obvious conceptual and practical synergies with metabolic engineering have led to a close coupling of the two fields. At the conference, we discussed how these synergies could be further exploited to maximize progress in biotechnology, and this resulted in three messages. First, we should take full advantage of our common engineering origins, frameworks and language. Clearly, both metabolic engineering and synthetic biology feature a focus on predictable design and hence have a common approach to studying and engineering biological systems. In both fields, there is a vision of being able to predict and design biological systems; in metabolic engineering, there is primarily a focus on metabolism of cell
factories, and in synthetic biology the focus is on an even broader context involving regulatory circuits and biomolecules. This does not, however, mean that metabolic engineering only involves engineering the metabolic network, but there is a focus on achieving the objective of overproducing a desired product through the optimization of the metabolic network, which is intertwined with gene regulatory and signaling networks. Second, we should appreciate the distinct approaches of the two fields. Metabolic engineering often involves a top-down approach where the metabolic network of a cell factory is retrofitted to overproduce a certain metabolite18. This may involve altering the regulation of carbon fluxes such that the carbon source is preferentially directed toward the product of interest and as little as possible carbon is lost in byproducts and to cellular growth. An analogy of this process is the retrofitting or reengineering of an existing chemical plant to produce new products by changing the connections between the different reactors and unit operations. In contrast, synthetic biology tends to take a bottom-up approach by reconstructing new biological parts that can find broad biological use18 or specifically meet predefined objectives. Though many examples focus on a single gene or small group of genes, increasingly synthetic biologists may aim to create a completely synthetic biological pathway. Though it has been possible to construct a completely synthetic genome19, translation of this result to the design of new efficient cell factories from scratch will depend on both increased knowledge of biological systems and the identification of a desired pathway for which it would be easier to start fresh than retrofit an existing cell. Working across fields with these specific interests in mind can often lead to overlap in which new biological parts are being used in combination with reengineering of a cell factory18. The potential contribution of parts from synthetic biology to metabolic engineering is illustrated with the construction of devices (to accomplish a particular goal) from well-characterized, standardized components. These devices derived from synthetic biology would find use in controlling biosynthetic pathways in a microorganism retrofitted to produce a desired product. For example, the synthetic biologist would use a computeraided design (CAD) program to design a genetic control circuit from a database of characteristics of biological components generated by a biofab20. She might desire to query for the following components
nature chemical biology | VOL 10 | MAY 2014 | www.nature.com/naturechemicalbiology
commentary Systems biology Synthetic biology
Mathematical models
Computational design for forward engineering of genetic circuits, biological systems or cells
S A
Metabolic engineering
B
Computational analysis of metabolic networks for their reengineering
npg
© 2014 Nature America, Inc. All rights reserved.
Biological system
Figure 1 | Illustration of how systems biology impacts both synthetic biology and metabolic engineering. The objective of systems biology is to develop predictive mathematical models for biological systems, and these models can be used for design of completely synthetic cells (forward engineering) and for modifying existing cells through metabolic engineering (reengineering). At the same time, the use of computational methods (as in other engineering disciplines) is at the core of both synthetic biology and metabolic engineering.
the time needed for tuning devices and increase their reliability. Of the relevant synthetic biology parts that are needed for advancing metabolic engineering are biosensors that can be used for highthroughput fluorescence-activated cell sorting screening of improved producers and promoters with high-precision expression and that can be controlled in response to environmental conditions. Third, we should build on existing knowledge to advance design and construction faster. Metabolic engineers think in terms of a host organism being defined in large part by its underlying metabolism and so seek to develop increasingly complete mathematical models of metabolic pathways, whereas synthetic biologists enjoy the challenge of understanding why a designed part is not working according to known principles. By integrating these strengths, we can more quickly move from conceiving a phenotype to actually constructing a
to build the circuit: a complex protein consisting of a DNA binding element and a metabolite binding element, a promoter-operator sequence or a ribosome binding site and so on. Once designed, the CAD program would output the DNA sequences, which would be ordered from a DNA synthesis company. When it arrived, the synthetic biologist would clone the DNA encoding the control system in front of a metabolic pathway that either produces or uses the chemical that the circuit measures and actuates in response. Because the circuit had been designed from well-characterized, standardized components, it would meet performance criteria in the retrofitted host with little tuning of its characteristics. In a less-thanideal world, the synthetic biologist may not have a CAD program but could still design biological systems from well-characterized components using mathematical models and other design tools. The use of wellcharacterized components would reduce
Table 1 | Overlap and differences between metabolic engineering and synthetic biology. Metabolic engineering Common themes Common tools
Synthetic biology Non-natural behavior
Molecular biology, mathematical modeling
Working domain
Chemistry, enzymes, genes, pathways, biological networks, cells
Genes, expression circuits, cells
Unique domains
Chemical and biochemical networks
Logic parts and domains
Objective
Engineering a cellular phenotype
Engineering of biological parts
Specialized tools
Flux quantification and network analysis
Synthesis of biological components
Applications
Industrial biotechnology
Diverse
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working phenotype. At the moment, though a pathway can be built in silico in a few minutes, it can and usually does take much longer to improve its function within the host to the point that it can support a commercial process17. Indeed, several commercial examples, such as those mentioned above, have shown that designing a cell factory is a monumental task where the cost to build a new cellular phenotype is in the range of hundreds of millions of US dollars. Improving these processes will take focused effort and a realistic expectation of the challenges in translating from concept to practice. In parallel, both groups can also contribute to unraveling cell function from an engineering perspective, seeking to enumerate design principles governing cellular functions akin to chemical engineering where first principles of reaction kinetics, thermodynamics and transport phenomena can be used to design reactors de novo. With advancement in our understanding of cellular function, in particular through systems biological research where predictive mathematical models are being developed21–24, we anticipate the design of completely new biological components that can be used for metabolic engineering purposes (Fig. 1). Our discussions resulted in the following conclusion. The two fields of metabolic engineering and synthetic biology differ in their foundational objectives: Metabolic engineering was established to enable and develop the biological production of drugs, chemicals, fuels and materials, i.e., a defined practical endpoint. Synthetic biology was established with the objective of building biological parts, modules and systems to understand and manipulate biological systems, i.e., a fundamental scientific focus. The two disciplines clearly overlap, but there are elements of each that remain distinct (Table 1). We expect that metabolic engineering will increasingly adopt strategies from synthetic biology, e.g., for gene synthesis and fine control of gene expression, whereas many synthetic biologists are taking an increased interest in an objective-driven strategy of engineering circuits and whole-cell metabolism, a characteristic of metabolic engineering. Similarly, companies focused on industrial biotechnology have embraced metabolic engineering, and many have begun to exploit tools from synthetic biology. A multifaceted approach building on the strengths of each field may therefore serve as one of the pillars underlying a modern, sustainable society. ◾ Jens Nielsen, Jay Keasling, Sang Yup Lee and Bernhard Palsson are at the Novo Nordisk Foundation 321
commentary ETH Zurich, Department of Biosystems Science and Engineering, Basel, Switzerland. James C. Liao is at the Department of Chemical and Biomolecular Engineering, University of California–Los Angeles, Los Angeles, California, USA. Kristala Prather is at the Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. Kristala Prather is also at the Synthetic Biology Engineering Research Center (SynBERC), Cambridge, Massachusetts, USA. e-mail: [email protected] References 1. Thykaer, J. & Nielsen, J. Metab. Eng. 5, 56–69 (2003). 2. de Graaf, A.A., Eggeling, L. & Sahm, H. Adv. Biochem. Eng. Biotechnol. 73, 9–29 (2001). 3. Bailey, J.E. Science 252, 1668–1675 (1991). 4. Stephanopoulos, G. & Vallino, J.J. Science 252, 1675–1681 (1991). 5. Stephanopoulos, G., Aristidou, A. & Nielsen, J. Metabolic Engineering: Principles and Methodologies. (Academic Press, 1998) 6. Nielsen, J. Appl. Microbiol. Biotechnol. 55, 263–283 (2001).
7. Stephanopoulos, G., Alper, H. & Moxley, J. Nat. Biotechnol. 22, 1261–1267 (2004). 8. Keasling, J.D. Science 330, 1355–1358 (2010). 9. Chen, W., Kallio, P.T. & Bailey, J.E. Gene 130, 15–22 (1993). 10. Gardner, T.S., Cantor, C.R. & Collins, J.J. Nature 403, 339–342 (2000). 11. Kramer, B.P. et al. Nat. Biotechnol. 22, 867–870 (2004). 12. Nakamura, C.E. & Whited, G.M. Metabolic engineering for microbial production of 1,3-propanediol. Curr. Opin. Biotechnol. 14, 454–459 (2003). 13. Hong, K.-K. & Nielsen, J. Cell. Mol. Life Sci. 69, 2671–2690 (2012). 14. Atsumi, S., Hanai, T. & Liao, J.C. Nature 451, 86–89 (2008). 15. Paddon, C.J. et al. Nature 496, 528–532 (2013). 16. Yim, H. et al. Nat. Chem. Biol. 7, 445–452 (2011). 17. Stephanopoulos, G. ACS Synth. Biol. 1, 514–525 (2012). 18. Nielsen, J. & Keasling, J.D. Nat. Biotechnol. 29, 693–695 (2011). 19. Gibson, D.G. et al. Science 329, 52–56 (2010). 20. Holtz, W.J. & Keasling, J.D. Cell 140, 19–23 (2010). 21. Feist, A.M. & Palsson, B.O. Nat. Biotechnol. 26, 659–667 (2008). 22. Lee, S.Y., Lee, D.-Y. & Kim, T.Y. Trends Biotechnol. 23, 349–358 (2005). 23. Tyo, K.E.J., Kocharin, K. & Nielsen, J. Curr. Opin. Microbiol. 13, 255–262 (2010). 24. Lee, J.W. et al. Nat. Chem. Biol. 8, 536–546 (2012).
Competing financial interests The authors declare no competing financial interests.
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Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark. Jens Nielsen is also at the Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden, and the Science for Life Laboratory, Royal Institute of Technology, Solna, Sweden. Jay Keasling is also at the Joint Bioenergy Institute, Emeryville, California, USA, the Department of Chemical and Biomolecular Engineering and the Department of Bioengineering, University of California–Berkeley, Berkeley, California, USA, the Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA, and the Synthetic Biology Engineering Research Center (SynBERC), Berkeley, California, USA. Sang Yup Lee is also at the Department of Chemical and Biomolecular Engineering, KAIST, Daejeon, Korea. Bernhard Palsson is also at the Department of Bioengineering, University of California–San Diego, La Jolla, California, USA. Martin Fussenegger is at
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