<>Second 5-Year Plan (2004-2008)
March 2004

Metabolic Engineering Working Group
Subcommittee on Biotechnology

The Metabolic Engineering Working Group (MEWG) was originally formed early in 1995 to foster one of the priority research areas (Manufacturing/ Bioprocessing) identified in the National Science and Technology Council (NSTC) Report: "Biotechnology for the 21st Century: New Horizons." After considerable deliberations, it was decided that the Metabolic Engineering of microbial, plant, and animal cells, as described in the Report, held the greatest promise of advancing the productivity of Bioprocessing. Further support for this research topic came from the recognition that Metabolic Engineering is expected to play a major role in the progress of the other three priority research areas mentioned in the Report, i.e. Agriculture, Environmental Biotechnology, and Marine Biotechnology and Aquaculture. With the recent issuance of the White House Memorandum (June 5, 2003) on "FY 2005 Interagency Research and Development Priorities", MEWG sees a continued opportunity for Metabolic Engineering to play a key role in meeting those priorities, and wishes to renew its original five-year plan.

MEWG GOALS

- Promote the Advancement of Metabolic Engineering

The MEWG has defined Metabolic Engineering (ME) as "the targeted and purposeful alteration of metabolic pathways found in an organism in order to better understand and utilize cellular pathways for chemical transformation, energy transduction, and supramolecular assembly." The MEWG believes that this emerging technology will have a significant effect on our nation's "quality-of-life" and international competitiveness. Accordingly, it should be the responsibility of the MEWG to promote and coordinate activities in this area.

- Coordinate Federal ME research activities for maximum productivity.

It is a goal of the MEWG to see that the current Federal expenditure for ME is coordinated for maximum efficiency and effectiveness. Key to this coordination is the dissemination of research program information among Federal Agencies with active ME programs.

- Identify and address gaps in ME research activities.

LIST OF MEWG AGENCIES

- Department of Agriculture (USDA)
- Department of Commerce (DOC)
- Department of Defense (DOD)
- Department of Energy (DOE)
- Environmental Protection Agency (EPA)
- National Aeronautics and Space Administration (NASA)
- National Institutes of Health (NIGMS/NIH)
- National Science Foundation (NSF)

ACCOMPLISHMENTS (1997-2003)

MEWG has generated a Metabolic Engineering Web Site (http://www.metabolicengineering.gov) that is providing a one-stop location for information on Federal Government activities in Metabolic Engineering.

MEWG has held four Interagency Conferences where results of funded ME projects have been shared with participating Grantees and Agency Representatives.

MEWG has issued five Interagency Announcements of Opportunity in Metabolic Engineering calling for research proposals that would centrally employ and/or significantly advance the techniques of ME. These proposals led to 45 research grants from the MEWG participating agencies. Grants from the first four competitions are listed on the MEWG web site.

While many of these grants are still in progress, examples of some particularly noteworthy outcomes include:

- A grant to Jay Keasling on the “Metabolic Engineering of Isoprenoid Production”, which was supported by the Office of Naval Research (ONR) and NSF, has led to the publication in Nature Biotechnology of “Engineering a Mevalonate Pathway in Escherichia coli for Production of Terpenoids”. The technology described in the paper has the potential for providing a microbial means of producing the anti-malarial drug Atremisinin, which is usually extracted from plants.

- A grant to Bernhard Palsson on the “in silico Analysis of the Escherichia coli Metabolic Genotype and the Construction of Selected Isogenic Strains” funded by ONR and NSF, and other grants to Palsson funded by NIH, have lead to numerous publications on this technology including a cover article in Nature Biotechnology. In silico, or computational methods to predict cellular responses to environmental stimuli based on the genome for a cell are being experimentally verified, and provide a powerful tool to understanding cellular behavior.

HOW THE PROPOSED ACTIVITY SUPPORTS THE MISSION OF EACH AGENCY

Metabolic engineering, being a broad, enabling technology, supports the missions of all of the current participating Federal Agencies in MEWG.

Department of Agriculture (USDA)

The Cooperative State Research, Education and Extension Service (CSREES) is the USDA agency that participates in the Interagency Metabolic Engineering Working Group. In the draft CSREES Strategic Plan, five goals are listed:

1. An agricultural production system that is highly competitive in the global economy.
2. A safe, secure food and fiber system.
3. Healthy, well-nourished population.
4. Greater harmony between agriculture and the environment.
5. Enhanced economic opportunity and quality of life for Americans.

These goals reflect the goals of the overall USDA strategic plan (enhancing economic opportunities for agricultural producers, supporting increased economic opportunities and improved quality of life in rural America, enhancing protection and safety of the nation’s agriculture and food supply, improving the nation’s nutrition and health, and protecting and enhancing the nation’s natural resource base and environment).

Metabolic Engineering (ME) can enhance competitiveness of the US agricultural system through the production of commercially useful products such as chemicals, biofuels, and biomolecules from agricultural commodities. Through modification of plants, animals, and microorganisms, ME can also result in new uses for existing crops and animals, added value to traditional agricultural products, and improved quality of agriculturally derived foods and materials. It is also possible through ME to produce plants with enhanced nutritional value or to modify plants and microorganisms for remediation of polluted environments.

The participation in MEWG has allowed CSREES to leverage funding for support of several research projects that address one or more of CSREES’ and USDA’s goals. Funding is supporting research on metabolic engineering of biofuels that may lead to maximized ethanol production as well as reduced costs. Another funded project involves production of flavor compounds in microbes that may eventually lead to improvements of metabolic function for processing of agricultural biomass and manufacture of bio-based industrial products. Funded metabolic engineering research projects in plants have the potential to produce fruits and vegetables with increased nutritional value and extended shelf-lives, to increase natural product-based disease and pest resistance, to enhance oil production in oilseeds, and to modify plants for production of pharmaceuticals and other economically important compounds. Thus, metabolic engineering, through both basic and applied research, is of vital importance for achieving the strategic goals of CSREES and USDA.

Department of Commerce (DOC)

The MEWG supports the DOC mission by advancing research and development of new commercial and industrial processes. As an emerging technology whose scientific basis is developing rapidly, ME is important to DOC’S National Institute of Standards and Technology (NIST) and especially its Biotechnology Division. NIST is especially interested in ME projects that support the development of biological and metabolic models, measurement methods and standards.

Department of Defense (DoD)

The DoD currently supports a broad range of research in the area of metabolic engineering through the Army Research Office (ARO) and other Army research activities, the Office of Naval Research (ONR), and the Defense Advanced Research Projects Agency (DARPA). The specific focus of the ARO, ONR, and DARPA efforts will be summarized and future directions in metabolic engineering research and technology development will be addressed.
The broad needs for the DoD that can be served through research efforts in metabolic engineering are summarized below. These science and technology targets will provide enhanced and expanded capabilities for the missions of the services and provide greatly expanded capabilities for the civilian sector.

• Materials
• Processes
• Devices
• Fabrication Schemes
• Information Processing

Current interests in metabolic engineering at ARO are focused on the characterization of biochemical pathways, inter- and intra-cellular signaling, and enzymatic mechanisms, and the genetic basis for manipulation of protein expression, structure and function, and cell fate, in systems with potential relevance to the Army. The goal is to develop a detailed understanding of how macromolecules and cells execute their designated functions and how they interact with other cells and macromolecules. With this information, it will be possible to design and engineer particular sub-cellular elements and metabolic pathways and cell systems to exhibit a set of specific functions and properties, according to Army needs, and to identify and non-invasively correct molecular deficiencies to optimize and maintain cognitive and physical performance under normal and extreme conditions. ARO currently supports research in several areas, including: how molecular transport, subcellular compartmentalization, and reaction sequences are involved in enzymatic regulation and superstructure formation; understanding and manipulating aminoacylation of tRNAs and genetic code expansion to produce new polymeric peptides containing non-natural amino acids; biologically based means for fabrication of functional nanostructures; systems engineering of cell differentiation processes; the role and regulation of classes of proteins differentially expressed in response to environmental or external stimuli; molecular genetics and genomics of human cognition, performance and function; and the design and implementation of unique biomolecular and cell based strategies for economically and environmentally favorable manufacturing, as well as the biodegradation of environmental pollutants.

One of the metabolic engineering foci at ONR, currently, is the microbial synthesis of energetic materials (EM) and EM precursors for the purposes of cost and environmental impact. Practically all such materials are non-natural products and their biosynthesis therefore requires the re-engineering of existing pathways and/or the assembly of new or hybrid pathways in one or more host organisms. An example of a simple EM precursor now under study is 1,2,4-butanetriol, which as its energetic trinitrate is used as a plasticizer in propellant and explosives formulations. More advanced EM targets, such as RDX, HMX and Cl20, involve high density fused ring cores with multiple nitramino (C-N(NO2)) substituents. While these are very difficult targets, they suggest worthwhile research goals such as the biosynthesis of highly electron withdrawing substituents on carbon (as in C-nitramino) or the assembly of strained heterocyclic rings. Clearly, a theoretical/experimental approach to the prediction of the true scope of enzyme reaction specificity, with energetic boundaries, would be particularly valuable in the design of pathways for EM biosynthesis. Other non-polymeric targets, besides EM, would include novel photonic/electronic/optical materials.

DARPA's metabolic engineering programs are driven by an interest in protecting human assets against biological threats and using biology to maintain human performance. The general concept of this thrust is to understand how nature controls the metabolic rate of cells and organisms (e.g., extremophiles, hibernation) and apply this understanding to problems of interest to DoD. Examples of current investments in metabolic engineering include efforts to develop technologies for engineering cells, tissues and organisms to survive in the battlefield environment so they can be used as sensors. Related basic research on biochemical circuit engineering in laboratory model organisms is also supported. In addition, DARPA is developing technologies that permit the long-term storage of cells including human blood. More complete descriptions of current DARPA programs and solicitations in these areas can be viewed at http://www.darpa.mil/dso.

Department of Energy (DOE)

The DOE is supporting research in metabolic engineering research, largely through the Offices of Science (SC), Energy Efficiency and Renewable Energy (EE), and Environmental Management (EM). The research falls in two main categories: 1) basic research, which involves the advancement of metabolic engineering fundamental knowledge and capabilities, and 2) applied research, which employs metabolic engineering techniques in development of target products. The basic research efforts of the Department reside within SC, whereas most of the applied research in this area is conducted within EE. In general, these research efforts are conducted by universities, national laboratories, and industry.
The Department's goals related to metabolic engineering research are to:

• Expand the level of knowledge and understanding of metabolic pathways and metabolic regulatory mechanisms related to the development of novel bio-based systems for the production, conservation, and conversion of energy.
• Apply metabolic engineering techniques to enhance and develop plants and microorganisms for use in the production of chemicals and fuels or for environmental remediation of waste sites.

Environmental Protection Agency (EPA)

The mission of the EPA is to protect human health and the environment from adverse effects of anthropogenic activity. Included in this mission are various elements for which metabolic engineering can play a useful role.

One prominent concern is the introduction of chemicals to the environment, which may have detrimental effects on humans and other biota. As mandated by statute and implemented by rule, the Agency routinely conducts evaluation of chemicals intended for use, currently in use, or determined to exist at significant levels in the environment. From these evaluations, the Agency may decide to implement management strategies designed to limit the potential for adverse effects.

The application of novel technologies such as the use of biotechnology as a substitute to conventional manufacturing and processing of raw materials into final products is consistent with the mission of the Agency. EPA implements this by supporting development of technologies which 1) use chemical substitutes that are less toxic; 2) produce more efficient activity resulting in decreased requirement for the chemical or; 3) develop engineering procedures which produce little or no toxic end products. Finally, consistent with the pollution prevention ethic is the reevaluation of chemical stewardship from one of "cradle to grave" to a more multigenerational philosophy in which a chemical may be utilized successively in different forms prior to final disposal. Metabolic engineering has a role to play by enabling the development of biological mechanisms for production or use that meet one or more of these criteria.

While it is generally accepted that chemical-based technologies have evolved to provide a higher standard of living for the general population, it is also recognized that the use of some chemicals, either through the chemical characteristics or the handling, synthesis or disposal, have produced negative effects on human health and/or the environment. Advances in technology allow scientists to better predict the potential for adverse effects from exposure to chemicals as well as mechanisms to diminish the negative effects of chemical production such as production of toxic byproducts and disposal of the chemical. The approach, which strives to identify synthetic pathways that are less polluting than existing pathways and that encourages the development of nontoxic chemical products, is referred to as "Green Chemistry". The use of metabolic engineering to evaluate the potential for increased risk from chemicals, by allowing the study of responsible metabolic pathways and by permitting modification of such pathways to reduce risk, is another way in which metabolic engineering firs within the EPA mission.

Finally, basic research, which utilizes methods of metabolic engineering, can provide longer-range approaches to assist EPA in its overall mission of protecting human health and the environment. The EPA supports extramural metabolic engineering research through the Technology for a Sustainable Environment (TSE) program, which awards grants in the area of pollution prevention. Since 1995, the TSE program has funded metabolic engineering research related to methanol conversion, solvent tolerance, biopolymer production and pesticide production-all focused on the elimination of pollution at the source.

National Aeronautics and Space Administration (NASA)

One of NASA’s strategic goals is to extend the duration & boundaries of human space flight to create new opportunities for exploration & discovery. To prepare for and hasten the journey, the NASA Office of Biological and Physical Research must address the following questions through its research:

• How can we assure the survival of humans traveling far from Earth?

• What technology must we create to enable the next explorers to go beyond where we have been?

NASA’s efforts in the area of metabolic engineering are on approaches and applications that will have a significant impact on the reduction of required mass, power, volume, crew time, and on increased safety and reliability, beyond the current baseline technologies. The targeted and purposeful alteration of metabolic pathways found in an organism may play a key role in the development of biological approaches and technologies that enable efficient use of spacecraft resources for long-duration space missions.

National Institutes of Health (NIGMS/NIH)

The NIGMS supports metabolic engineering research, usually in the form of grants to investigators in universities (R01s) or in small businesses (SBIRs). These grants support basic research in two general areas: 1) the development of microbial or plant-based metabolic routes to useful quantities of small molecules such as polyketides; and 2) the development of a much better understanding of the control architecture that integrates the genetic and catalytic processes in normal and aberrant cells.

National Science Foundation (NSF)

The mission of NSF is to:

- Promote the Progress of Science
- Advance the National Health, Prosperity, and Welfare
- Secure the National Defense
- Provide for Other Purposes

Support of ME research allows NSF to address specific goals within its mission. These include, but are not limited to: development of technologies integrating theoretical, computational, and experimental approaches to the study of metabolic processes; the targeted and purposeful alteration of metabolic pathways in living organisms in order to better understand and utilize these pathways for chemical transformation, energy transduction, and supramolecular assembly; providing a framework for studying the dynamics of interactions and interconversions of biological molecules in order to understand how organisms regulate specific physiological processes at the cellular and sub-cellular levels and the “cross-talk” between pathways; measurement and control of in vivo metabolic fluxes; metabolic control analysis of pathway groups or networks; and development of in vivo techniques to accomplish these goals.

Metabolic Engineering has been heavily supported in all five interagency competitions by three Directorates within NSF. There is a recognition at NSF that this Activity has been beneficial to NSF and that NSF would like to continue with this Activity.

Thus, whether a shared or unique focus, ME research has a broad based interest throughout the Federal research establishment.

PLAN

While the Interagency activities have been very successful, there remains much to be done.

MEWG proposes to continue to coordinate Federal ME research activities using the following mechanisms:

- Maintain an up-to-date web site
- Hold regular meetings of MEWG
- Sponsor annual Interagency Conferences on Metabolic Engineering

MEWG proposes to issue Interagency Announcements of Opportunities in Metabolic Engineering over the next five years calling for research proposals in Metabolic Engineering.

ANTICIPATED SYNERGIES OF THE MEWG EFFORTS

It is the intent of MEWG to provide a means for the participating Agencies to support projects in ME that would not normally be done by a single Agency. For example, a project at a government laboratory supported by one Agency could be complemented by a linkage to university participation in the project supported by another Agency. Three way linkages between industry, government laboratories, and academe are another possibility.

MEWG anticipates continued member participation in national and international conferences on Metabolic Engineering.

AVAILABILITY OF FUNDS TO SUPPORT THE INTERAGENCY ANNOUNCEMENT OF OPPORTUNITIES IN METABOLIC ENGINEERING

For the five Interagency Announcements already carried out, each Agency provided a letter of support for an Announcement indicating the amount of funding that might be available for proposals submitted in response to that Announcement. It is anticipated that this will continue with new Interagency Announcements. The actual amount each Agency commits to proposals from a particular Announcement, which can range from no support to support of multiple proposals, will depend on the quality and subject matter of the proposals that the Agency is willing to fund.

EVIDENCE OF THE TIMELINESS OF THE CONCEPT OF METABOLIC ENGINEERING AND THAT THE RELEVANT COMMUNITIES ARE READY TO RESPOND.

- All MEWG Agencies are interested in furthering the goals of MEWG.

- The recent issuance of a White House Memo (June 5, 2003) on "FY 2005 Interagency Research and Development Priorities" highlights the need for "Molecular-Level Understanding of Life Processes" where Metabolic Engineering has a far-reaching opportunity to contribute.