NOFO_DE-FOA-0003551_PERSEPHONE_Modification 01.pdf

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AR-311-10.24

FINANCIAL ASSISTANCE

NOTICE OF FUNDING OPPORTUNITY

ADVANCED RESEARCH PROJECTS AGENCY – ENERGY (ARPA-E)

U.S. DEPARTMENT OF ENERGY

PLANT ENGINEERING TO REVOLUTIONIZE SUSTAINABLE

ENERGY PRODUCTION AND HEIGHTEN OPPORTUNITIES FOR

NOVEL EFFICIENCY (PERSEPHONE)

Announcement Type: Initial Announcement Modification 01 Notice of Funding Opportunity No. DE-FOA-0003551

Assistance Listing Number 81.135

Mod. No. Date Description of Modifications*

01 2/6/2025 • Clarified the meaning of the Program Policy Factors in Section V.C.

*All modifications to the Notice of Funding Opportunity (NOFO) are highlighted in yellow in the body of the NOFO.

NOFOs are posted on ARPA-E eXCHANGE (https://arpa-e-foa.energy.gov/), Grants.gov (http://www.grants.gov/), and FedConnect (https://www.fedconnect.net/FedConnect/). Any modifications to the NOFO are also posted to these websites. You can receive an e-mail when a modification is posted by registering with FedConnect as an interested party for this NOFO. It is recommended that you register as soon as possible after release of the NOFO to ensure that you receive timely notice of any modifications or other announcements.

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TABLE OF CONTENTS

BASIC INFORMATION ......................................................................................................................................- 5 -

I. FUNDING OPPORTUNITY DESCRIPTION ...................................................................................................- 7 -

A. AGENCY OVERVIEW ........................................................................................................................................... - 7 - B. PROGRAM OVERVIEW ........................................................................................................................................ - 9 -

1. PROGRAM BACKGROUND ............................................................................................................................. - 9 -

2. TECHNICAL CHALLENGES ............................................................................................................................. - 12 -

3. EMERGING TECHNOLOGIES .......................................................................................................................... - 14 -

C. PROGRAM OBJECTIVES ................................................................................................................................ - 15 - D. TECHNICAL CATEGORIES OF INTEREST ............................................................................................................. - 16 - E. TECHNICAL PERFORMANCE TARGETS .................................................................................................................. - 17 - F. STATEMENT OF SUBSTANTIAL INVOLVEMENT ....................................................................................................... - 20 - G. FUNDING RESTRICTIONS............................................................................................................................... - 21 -

1. ALLOWABLE COSTS .................................................................................................................................... - 21 -

2. PRE-AWARD COSTS ................................................................................................................................... - 21 -

3. PATENT COSTS .......................................................................................................................................... - 21 -

4. CONSTRUCTION ......................................................................................................................................... - 22 -

5. FOREIGN TRAVEL ....................................................................................................................................... - 22 -

6. PERFORMANCE OF WORK IN THE UNITED STATES ............................................................................................ - 22 -

7. PURCHASE OF NEW EQUIPMENT .................................................................................................................. - 22 -

8. TECHNOLOGY TRANSFER AND OUTREACH ...................................................................................................... - 23 -

9. LOBBYING ................................................................................................................................................ - 23 -

10. CONFERENCE SPENDING ............................................................................................................................. - 24 -

11. INDEPENDENT RESEARCH AND DEVELOPMENT COSTS....................................................................................... - 24 -

12. BUY AMERICA REQUIREMENT FOR PUBLIC INFRASTRUCTURE PROJECTS .............................................................. - 25 -

13. REQUIREMENT FOR FINANCIAL PERSONNEL .................................................................................................... - 25 -

14. PARTICIPANTS, COLLABORATING ORGANIZATIONS, AND CURRENT AND PENDING SUPPORT ................................... - 25 -

II. ELIGIBILITY INFORMATION .................................................................................................................... - 27 -

A. ELIGIBLE APPLICANTS ....................................................................................................................................... - 27 -

1. INDIVIDUALS ............................................................................................................................................. - 27 -

2. DOMESTIC ENTITIES ................................................................................................................................... - 27 -

3. CONSORTIA .............................................................................................................................................. - 28 -

4. FOREIGN NATIONAL PARTICIPATION FROM COUNTRIES OF CONCERN ................................................................. - 29 -

5. FOREIGN PARTICIPATION CONSIDERATIONS ................................................................................................... - 29 -

B. COMPLIANCE AND RESPONSIVENESS ................................................................................................................... - 29 - C. LIMITATION ON NUMBER OF SUBMISSIONS ..................................................................................................... - 29 - D. COST SHARING ........................................................................................................................................... - 30 -

1. BASE COST SHARE REQUIREMENT ................................................................................................................ - 30 -

2. INCREASED COST SHARE REQUIREMENT ........................................................................................................ - 30 -

3. REDUCED COST SHARE REQUIREMENT .......................................................................................................... - 30 -

4. LEGAL RESPONSIBILITY ............................................................................................................................... - 31 -

5. COST SHARE ALLOCATION ........................................................................................................................... - 32 -

6. COST SHARE TYPES AND ALLOWABILITY ........................................................................................................ - 32 -

7. COST SHARE CONTRIBUTIONS BY FFRDCS AND GOGOS .................................................................................. - 33 -https://arpa-e.energy.gov/faqs mailto:ARPA-E-CO@hq.doe.gov mailto:ExchangeHelp@hq.doe.gov

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8. COST SHARE VERIFICATION ......................................................................................................................... - 33 -

III. APPLICATION CONTENTS AND FORMAT ............................................................................................ - 34 -

A. GENERAL APPLICATION CONTENT REQUIREMENTS ................................................................................................ - 34 -

1. MARKING OF CONFIDENTIAL INFORMATION ................................................................................................... - 34 -

2. EXPORT CONTROL INFORMATION ................................................................................................................. - 34 -

B. FULL APPLICATIONS ......................................................................................................................................... - 35 -

1. FIRST COMPONENT: TECHNICAL VOLUME ...................................................................................................... - 35 -

2. SECOND COMPONENT: SF-424 ................................................................................................................... - 35 -

3. THIRD COMPONENT: BUDGET JUSTIFICATION WORKBOOK/SF-424A ................................................................ - 36 -

4. FOURTH COMPONENT: SUMMARY FOR PUBLIC RELEASE .................................................................................. - 37 -

5. FIFTH COMPONENT: SUMMARY SLIDE ........................................................................................................... - 37 -

6. SIXTH COMPONENT: BUSINESS ASSURANCES & DISCLOSURES FORM .................................................................. - 37 -

C. REPLIES TO REVIEWER COMMENTS ................................................................................................................ - 37 -

IV. SUBMISSION REQUIREMENTS AND DEADLINES ................................................................................. - 39 -

A. UNIQUE ENTITY IDENTIFIER AND SAM REGISTRATION ........................................................................................... - 39 - B. USE OF ARPA-E EXCHANGE .......................................................................................................................... - 39 - C. REQUIRED DOCUMENTS CHECKLIST AND DEADLINES ......................................................................................... - 41 - D. COMPLIANCE ............................................................................................................................................. - 41 - E. INTERGOVERNMENTAL REVIEW ......................................................................................................................... - 42 -

V. APPLICATION REVIEW INFORMATION ................................................................................................... - 43 -

A. RESPONSIVENESS ............................................................................................................................................ - 43 -

1. TECHNICAL RESPONSIVENESS REVIEW ........................................................................................................... - 43 -

2. SUBMISSIONS SPECIFICALLY NOT OF INTEREST ................................................................................................. - 44 -

B. REVIEW CRITERIA ............................................................................................................................................ - 44 -

1. CRITERIA FOR FULL APPLICATIONS ................................................................................................................ - 44 -

2. CRITERIA FOR REPLIES TO REVIEWER COMMENTS ............................................................................................ - 45 -

C. PROGRAM POLICY FACTORS ......................................................................................................................... - 45 - D. REVIEW AND SELECTION PROCESS ................................................................................................................. - 46 -

1. FULL APPLICATIONS ................................................................................................................................... - 46 -

2. REPLY TO REVIEWER COMMENTS ................................................................................................................. - 47 -

3. PRE-SELECTION CLARIFICATIONS AND “DOWN-SELECT” PROCESS ...................................................................... - 47 -

4. SELECTION FOR AWARD NEGOTIATIONS ........................................................................................................ - 47 -

E. ARPA-E REVIEWERS ....................................................................................................................................... - 48 - F. ARPA-E SUPPORT CONTRACTORS ..................................................................................................................... - 48 - G. RISK REVIEW.............................................................................................................................................. - 48 -

VI. AWARD NOTICES AND AWARD TYPES ............................................................................................... - 50 -

A. AWARD NOTICES ............................................................................................................................................ - 50 -

1. REJECTED SUBMISSIONS ............................................................................................................................. - 50 -

2. FULL APPLICATION NOTIFICATIONS ............................................................................................................... - 50 -

B. PRE-AWARD COSTS ......................................................................................................................................... - 51 - C. RENEWAL AWARDS ..................................................................................................................................... - 51 - D. FUNDING AGREEMENT TYPES ....................................................................................................................... - 51 -https://arpa-e.energy.gov/faqs

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1. COOPERATIVE AGREEMENTS........................................................................................................................ - 52 -

2. FUNDING AGREEMENTS WITH FFRDCS/DOE LABS, GOGOS, AND FEDERAL INSTRUMENTALITIES .......................... - 52 -

3. OTHER TRANSACTIONS AUTHORITY .............................................................................................................. - 53 -

VII. POST-AWARD REQUIREMENTS AND ADMINISTRATION ..................................................................... - 54 -

A. NATIONAL POLICY REQUIREMENTS .................................................................................................................... - 54 -

1. NATIONAL POLICY ASSURANCES ................................................................................................................... - 54 -

2. ENVIRONMENTAL IMPACT QUESTIONNAIRE ................................................................................................... - 54 -

B. ADMINISTRATIVE REQUIREMENTS ...................................................................................................................... - 55 -

1. COST SHARE PAYMENTS ............................................................................................................................. - 55 -

2. INTELLECTUAL PROPERTY AND DATA MANAGEMENT PLANS ............................................................................. - 55 -

3. U.S. COMPETITIVENESS .............................................................................................................................. - 56 -

4. NONDISCLOSURE AND CONFIDENTIALITY AGREEMENTS REPRESENTATIONS .......................................................... - 57 -

5. INTERIM CONFLICT OF INTEREST POLICY FOR FINANCIAL ASSISTANCE .................................................................. - 58 -

6. COMPLIANCE AUDIT REQUIREMENT .............................................................................................................. - 58 -

7. RESEARCH SECURITY TRAINING REQUIREMENT ............................................................................................... - 58 -

8. FRAMEWORK FOR NUCLEIC ACID SYNTHESIS SCREENING .................................................................................. - 59 -

C. REPORTING ................................................................................................................................................ - 59 -

1. FRAUD, WASTE, AND ABUSE ....................................................................................................................... - 59 -

2. COMMERCIALIZATION PLAN AND SOFTWARE REPORTING ................................................................................. - 60 -

VIII. OTHER INFORMATION ...................................................................................................................... - 61 -

A. TITLE TO SUBJECT INVENTIONS .......................................................................................................................... - 61 - B. GOVERNMENT RIGHTS IN SUBJECT INVENTIONS ................................................................................................... - 62 -

1. GOVERNMENT USE LICENSE ........................................................................................................................ - 62 -

2. MARCH-IN RIGHTS .................................................................................................................................... - 62 -

C. RIGHTS IN TECHNICAL DATA ......................................................................................................................... - 62 - D. PROTECTED PERSONALLY IDENTIFIABLE INFORMATION ...................................................................................... - 63 -

IX. GLOSSARY ........................................................................................................................................ - 64 -https://arpa-e.energy.gov/faqs

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BASIC INFORMATION

KEY DATES:

Notice of Funding Opportunity (NOFO) Issue Date: January 17, 2025

Deadline for Full Application Questions to ARPA-E-CO@hq.doe.gov: 5 PM ET, February 21, 2025

Submission Deadline for Full Applications: 9:30 AM ET, March 4, 2025

Expected Reviewer Comment Release Date: 5 PM ET, March 27, 2025

Submission Deadline for Replies to Reviewer Comments: 5 PM ET, April 1, 2025

Anticipated Timeframe for Selection Notifications: May 2025

Anticipated Timeframe for Award: August 2025

Anticipated Period of Performance: August 2025 – August 2028

BASIC INFORMATION:

Total Amount to Be Awarded

Approximately $15 million, subject to the availability of appropriated funds to be shared between NOFOs DE-FOA-0003551 and DE-FOA-0003552.

Anticipated Number and Value of Awards

ARPA-E anticipates making approximately 5-7 awards between NOFOs DE-FOA- 0003551 and DE-FOA-0003552. ARPA-E may issue one, multiple, or no awards under this NOFO. The Federal share of awards under this NOFO may vary between $250,000 and $4 million.

Agency Contact Information

• Questions and answers (Q&As) about ARPA-E and this specific NOFO:

http://arpa-e.energy.gov/faq.

• Send other questions about the NOFO to: ARPA-E-CO@hq.doe.gov.

• Send questions about use of ARPA-E eXCHANGE to:

ExchangeHelp@hq.doe.gov.

Upon the issuance of a NOFO, only the Grants Officer via ARPA-E-CO@hq.doe.gov may communicate with Applicants. This “quiet period” remains in effect until ARPA-E’s public announcement of project selections. Emails sent to other email addresses will be disregarded.

The goal of the PERSEPHONE program is to develop disruptive new technologies for bioenergy crop genetic engineering. Bioenergy provides about 5% of domestic energy consumption and has the potential to provide 5-10% more. Agriculture also could revolutionize the energy sector in other ways, such as providing precursors for chemicals and materials that are currently derived from petroleum.

However, bioenergy crops may not maintain their current utility, much less achieve their potential, without transformative advances in tools to engineer them. Genetic engineering is an essential strategy to realize U.S. bioenergy potential and security.

The PERSEPHONE program will develop high-performance tools for bioenergy crop engineering, create novel genetic engineering modalities, and spur adoption by supporting innovative research on biocontainment. Specifically, PERSEPHONE aims to support the development of tools that could lead to an annual impact of the production of at least 1 quad of energy or mitigation of more than 60 MT of carbon dioxide equivalents (CO2e).

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The program will support projects in three areas:

• Technical Category A will develop robust, high-performing tools for the genetic engineering of bioenergy crops that overcome transformation genotype dependence, increase throughput 10x, and/or reduce cost and timeline by 4x and 2x, respectively.

• Technical Category B will support the creation of radically new technologies for plant genetic engineering that have a pathway to achieve metrics well beyond the targets established in Category A.

• Technical Category C will support adoption of genetically engineered bioenergy crops by developing technologies to reduce the risks of invasiveness to less than 0.1 percent and eliminate the flow of transgenes into the environment.

The program encourages the formation of multidisciplinary teams with expertise in relevant areas such as synthetic biology, bioengineering, plant genomics and genetics, materials, robotics, and machine learning. If PERSEPHONE is successful, the new genetic engineering tools will enable the rapid development and commercialization of bioenergy applications in areas as diverse as Sustainable Aviation Fuel (SAF), nitrogen management, and energy-efficient materials.

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I. FUNDING OPPORTUNITY DESCRIPTION

A. AGENCY OVERVIEW

The Advanced Research Projects Agency – Energy (ARPA-E), an organization within the Department of Energy (DOE), is chartered by Congress in the America COMPETES Act of 2007 (P.L. 110-69), as amended by the America COMPETES Reauthorization Act of 2010 (P.L. 111- 358), as further amended by the Energy Act of 2020 (P.L. 116-260):

“(A) to enhance the economic and energy security of the United States through the development of energy technologies that—

(i) reduce imports of energy from foreign sources;

(ii) reduce energy-related emissions, including greenhouse gases;

(iii) improve the energy efficiency of all economic sectors;

(iv) provide transformative solutions to improve the management, clean-up, and disposal of radioactive waste and spent nuclear fuel; and

(v) improve the resilience, reliability, and security of infrastructure to produce, deliver, and store energy; and

(B) to ensure that the United States maintains a technological lead in developing and deploying advanced energy technologies.”

ARPA-E issues this Notice of Funding Opportunity (NOFO) under its authorizing statute codified at 42 U.S.C. § 16538. The NOFO and any cooperative agreements or grants made under this NOFO are subject to 2 C.F.R. Part 200 as supplemented by 2 C.F.R. Part 910.

ARPA-E funds research on, and the development of, transformative science and technology solutions to address the energy and environmental missions of the Department. The agency focuses on technologies that can be meaningfully advanced with a modest investment over a defined period of time in order to catalyze the translation from scientific discovery to early-stage technology. For the latest news and information about ARPA-E, its programs and the research projects currently supported, see: http://arpa-e.energy.gov/.

ARPA-E funds transformational research. Existing energy technologies generally progress on established “learning curves” where refinements to a technology and the economies of scale that accrue as manufacturing and distribution develop drive improvements to the cost/performance metric in a gradual fashion. This continual improvement of a technology is important to its increased commercial deployment and is appropriately the focus of the private sector or the applied technology offices within DOE. In contrast, ARPA-E supports transformative research that has the potential to create fundamentally new learning curves.

ARPA-E technology projects typically start with cost/performance estimates well above the level of an incumbent technology. Given the high risk inherent in these projects, many will fail to progress, but some may succeed in generating a new learning curve with a projected cost/performance metric that is significantly better than that of the incumbent technology.

ARPA-E will provide support at the highest funding level only for submissions with significant https://arpa-e.energy.gov/faqs mailto:ARPA-E-CO@hq.doe.gov http://arpa-e.energy.gov/

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- 8 -technology risk, aggressive timetables, and careful management and mitigation of the associated risks.

ARPA-E funds technology with the potential to be disruptive in the marketplace. The mere creation of a new learning curve does not ensure market penetration. Rather, the ultimate value of a technology is determined by the marketplace, and impactful technologies ultimately become disruptive – that is, they are widely adopted and displace existing technologies from the marketplace or create entirely new markets. ARPA-E understands that definitive proof of market disruption takes time, particularly for energy technologies. Therefore, ARPA-E funds the development of technologies that, if technically successful, have clear disruptive potential, e.g., by demonstrating capability for manufacturing at competitive cost and deployment at scale.

ARPA-E funds applied research and development (R&D). The Office of Management and Budget defines “applied research” as an “original investigation undertaken in order to acquire new knowledge…directed primarily towards a specific practical aim or objective” and defines “experimental development” as “creative and systematic work, drawing on knowledge gained from research and practical experience, which is directed at producing new products or processes or improving existing products or processes.”1 Applicants interested in receiving financial assistance for basic research (defined by the Office of Management and Budget as “experimental or theoretical work undertaken primarily to acquire new knowledge of the underlying foundations of phenomena and observable facts”)1 should contact the DOE’s Office of Science (http://science.energy.gov/). Office of Science national scientific user facilities (http://science.energy.gov/user-facilities/) are open to all researchers, including ARPA-E Applicants and awardees. These facilities provide advanced tools of modern science including accelerators, colliders, supercomputers, light sources and neutron sources, as well as facilities for studying the nanoworld, the environment, and the atmosphere. Projects focused on early-stage R&D for the improvement of technology along defined roadmaps may be more appropriate for support through the DOE applied energy offices including: the Office of Energy Efficiency and Renewable Energy (http://www.eere.energy.gov/), the Office of Fossil Energy and Carbon Management (https://www.energy.gov/fecm/office-fossil-energy-and-carbon-management), the Office of Nuclear Energy (http://www.energy.gov/ne/office-nuclear-energy), and the Office of Electricity (https://www.energy.gov/oe/office-electricity).

ARPA-E encourages submissions stemming from ideas that still require proof-of-concept R&D efforts as well as those for which some proof-of-concept demonstration already exists.

Submissions can propose a project with the end deliverable being an extremely creative, but partial solution.

1 OMB Circular A-11 (https://www.whitehouse.gov/wp-content/uploads/2018/06/a11_web_toc.pdf), Section 84,

pg. 3.

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B. PROGRAM OVERVIEW

1. PROGRAM BACKGROUND

The goal of the Plant Engineering to Revolutionize Sustainable Energy Production and Heighten Opportunities for Novel Efficiency (PERSEPHONE) program is to develop disruptive new technologies for bioenergy crop genetic engineering.2 Bioenergy provides the U.S. with approximately 5 quadrillion British Thermal Units (quads)—about 5% of domestic energy consumption—and it has the potential to provide 6-11 additional quads through the cultivation of dedicated bioenergy crops.3 Beyond the production of biofuels, agriculture has the potential to revolutionize the energy sector by: providing precursors for chemicals and materials (e.g., polymers and building materials) that can complement petroleum-based products, capturing and utilizing atmospheric carbon dioxide, and accumulating critical minerals through phytomining. Moreover, bioenergy plays an important role in increasing energy security and reducing energy imports.4 However, bioenergy crops may not maintain their current utility, much less achieve their potential, without transformative advances in tools to engineer them.

Breeding and management practices have led to dramatic improvements in agriculture with crop yields increasing by 135% from 1961 to 2005.5 However, over the next decade U.S. yields are projected to increase more slowly (in the case of corn) or even decline (in the case of soybeans) due to changing patterns of temperature and precipitation.6 Looking ahead to the end of the century, corn yields across much of the U.S. may eventually decline by 10% or more.7 As a result, up to 5% of the U.S. energy supply is under threat unless new crops with increased resilience are developed. Meanwhile, demand for biofuels is increasing to meet the growing market for Sustainable Aviation Fuel (SAF). The Department of Energy’s SAF Grand Challenge has set a SAF production target of 35 billion gallons per year by 2050, which requires 60 billion

2 Bioenergy crops, as defined in this NOFO, include current bioenergy crops (e.g., corn, soybeans, and grain sorghum) as well as emerging bioenergy crops (e.g., miscanthus, poplar, biomass sorghum, sugarcane, and switchgrass). Other crops may be considered if they can be shown to meet the targets of the NOFO and be relevant to bioenergy.

3 U.S. DOE. 2024. "2023 Billion-Ton Report: An Assessment of U.S. Renewable Carbon Resources." U.S. Department of Energy Bioenergy Technologies Office. https://doi.org/10.2172/2441098.

4 US. DOE. 2013. “Replacing the Whole Barrel to Reduce U.S. Dependence on Oil.” U.S. Department of Energy Bioenergy Technologies Office. https://www.osti.gov/biblio/1220022/.

5 Jennifer A. Burney, Steven J. Davis, and David B. Lobell, “Greenhouse Gas Mitigation by Agricultural Intensification,” Proceedings of the National Academy of Sciences 107, no. 26 (June 15, 2010): 12052–57, https://doi.org/10.1073/pnas.0914216107.

6 Jayson Beckman, Maros Ivanic, and Noé J. Nava, “Estimating Market Implications From Corn and Soybean Yields Under Climate Change in the United States,” October 1, 2023, https://doi.org/10.32747/2023.8134358.ers.

7 Jonas Jägermeyr et al., “Climate Impacts on Global Agriculture Emerge Earlier in New Generation of Climate and Crop Models,” Nature Food 2, no. 11 (November 1, 2021): 873–85, https://doi.org/10.1038/s43016-021-00400-y.

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- 10 -gallons of ethanol to meet the target due to ethanol’s lower energy density.8,9 This is more than three times the combined production of ethanol and bio-based diesel in 2022, and will therefore require a step change in agricultural productivity and technology development.10

Beyond SAF, the potential 6-11 quad increase in available bioenergy depends upon rapid improvements in bioenergy crops other than corn, such as switchgrass, sorghum and miscanthus. Production of corn and soybeans is regionally concentrated with two-thirds of corn grown in seven Midwestern states.11 A greater diversity of bioenergy crops will expand the geographic range of production and impart greater resiliency to changing temperature and precipitation patterns.12 Many emerging bioenergy crops have favorable productivity or stress tolerance traits, including sorghum (drought tolerance), miscanthus (flood tolerance), oil seeds (cold tolerance), switchgrass (ability to cycle nutrients effectively), and poplar (high biomass productivity). These crops could add substantial genetic and geographic diversification to the U.S. bioenergy portfolio, but they have not been widely cultivated in the U.S. and are likely to require genetic engineering to meet their potential. Moreover, some of the greatest opportunities for bioenergy crops to impact energy involve traits that do not currently exist within the plant kingdom, such as nitrogen fixation and the accumulation of polymer precursors.13,14 Genetic engineering is an essential strategy to realize U.S. bioenergy potential and security.

Current tools for bioenergy crop genetic engineering suffer from severe limitations of speed, throughput, and generalizability. The PERSEPHONE program will develop high-performance tools for bioenergy crop engineering, create novel genetic engineering modalities, and spur adoption by supporting innovative research on biocontainment. These advances will enable the rapid innovation necessary for agriculture to enhance the economic and energy security of the U.S. and decrease American reliance on imported energy.

The downstream energy impact of the tools developed through the PERSEPHONE program can be illustrated through three main examples.

8 U.S. DOE, U.S. Department of Transportation, U.S. Department of Agriculture (USDA), and U.S. Environmental Protection Agency. 2022. SAF Grand Challenge Roadmap: Flight Plan for Sustainable Aviation Fuel.

https://www.energy.gov/sites/default/files/2022- 09/beto-saf-gc-roadmap-report-sept-2022.pdf.

9 United Nations International Civil Aviation Organization, “SAF Rules of Thumb,” n.d., https://www.icao.int/environmental-protection/Pages/SAF_RULESOFTHUMB.aspx.

10 United States Department of Agriculture Economic Research Service, U.S. Bioenergy Statistics, n.d., https://www.ers.usda.gov/data-products/u-s-bioenergy-statistics/.

11 Crop Acreage Data | Farm Service Agency.

12 Sarah Garland and Helen Anne Curry, “Turning Promise into Practice: Crop Biotechnology for Increasing Genetic Diversity and Climate Resilience,” PLOS Biology 20, no. 7 (July 26, 2022), https://doi.org/10.1371/journal.pbio.3001716.

13 Robert S. Allen et al., “Expression of 16 Nitrogenase Proteins Within the Plant Mitochondrial Matrix,” Frontiers in Plant Science 8 (March 3, 2017), https://doi.org/10.3389/fpls.2017.00287.

14 Chien-Yuan Lin et al., “In-Planta Production of the Biodegradable Polyester Precursor 2-Pyrone-4,6-Dicarboxylic Acid (PDC): Stacking Reduced Biomass Recalcitrance with Value-Added Co-Product,” Metabolic Engineering 66 (July 2021): 148–56, https://doi.org/10.1016/j.ymben.2021.04.011.

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• Production of SAF. The hydrotreated esters and fatty acids (HEFA) pathway, which uses vegetable oils and waste grease as a feedstock, is currently the dominant SAF production route and is expected to remain so into the 2030s.15 However, it is limited by the cost and availability of oil feedstocks. PERSEPHONE can help meet the demand for SAF by making it possible to engineer triacylglycerol (TAG) production into high-yield bioenergy crops like biomass sorghum. The model plant tobacco has been engineered to accumulate up to 30% TAG by dry weight in its leaves, and a similar accumulation in biomass sorghum would allow production of 10 billion gallons of SAF (equal to 1 quad of energy) on roughly 20 million (M) acres.16 Compared to current sorghum acreage (6.4M) and corn acreage (90M), this is ambitious but feasible.17 However, biomass sorghum is difficult to engineer today, necessitating the creation of new engineering tools.

• Advances in nitrogen management. Roughly 2% of global energy is devoted to ammonia synthesis, and approximately 70% of emissions from the production of corn for ethanol are due to the Haber-Bosch process or on-field N2O emissions.18,19 Recent progress in understanding how legumes form symbioses with nitrogen-fixing microbes strengthens the possibility of engineering this trait into bioenergy crops, and introducing in planta nitrogen fixation remains a lofty goal of plant engineering.20 Either path will likely require engineering large numbers of heterologous genes into crops with stringent regulatory control, a task which would be very difficult with present crop engineering technology.

• Production of wood for building materials. Wood can be transformed into a material with strength comparable to steel, but with less embodied energy by weight and negative emissions.21 However, the cost and scale of feedstock production will make it difficult to displace the roughly 46 megatons (MT) of steel used for construction in the U.S.22 Improved photosynthetic pathways could boost yield by 24%, but they have only been demonstrated in amenable cultivars.23 The PERSEPHONE program could enable researchers to rapidly engineer improved photosynthesis into diverse varieties of poplar, willow, and other short-rotation woody crops. If such an effort displaced 50% of U.S. crude steel, it would save 0.55 quads of energy per year. However, engineering such complex traits into diverse woody crops is not feasible with today’s tools.

15 RMI. 2024. “Sustainable Aviation Fuel Market Outlook.” Accessed November 2024. https://saf.rmi.org/.

16 Thomas Vanhercke et al., “Step Changes in Leaf Oil Accumulation via Iterative Metabolic Engineering,” Metabolic Engineering 39 (January 2017): 237–46, https://doi.org/10.1016/j.ymben.2016.12.007.

17 Aaron Ates and Olga Liefert, “Feed Outlook: April 2024.” United States Department of Agriculture Economic Research Service. https://www.ers.usda.gov/publications/pub-details/?pubid=108981.

18 IEA. 2021. “Ammonia Technology Roadmap.” https://www.iea.org/reports/ammonia-technology-roadmap/executive-summary.

19 Calculated using the 2024 GREET Model.

20 Min-Yao Jhu and Giles E. Oldroyd, “Dancing to a Different Tune, Can We Switch from Chemical to Biological Nitrogen Fixation for Sustainable Food Security?,” PLOS Biology 21, no. 3 (March 14, 2023), https://doi.org/10.1371/journal.pbio.3001982.

21 Jianwei Song et al., “Processing Bulk Natural Wood into a High-Performance Structural Material,” Nature 554,

no. 7691 (February 2018): 224–28, https://doi.org/10.1038/nature25476.

22 U.S. Geological Survey, Mineral Commodity Summaries, January 2023.

23 Paul F. South et al., “Synthetic Glycolate Metabolism Pathways Stimulate Crop Growth and Productivity in the Field,” Science 363, no. 6422 (January 4, 2019), https://doi.org/10.1126/science.aat9077.

https://arpa-e.energy.gov/faqs mailto:ARPA-E-CO@hq.doe.gov mailto:ExchangeHelp@hq.doe.gov https://saf.rmi.org/ https://www.iea.org/reports/ammonia-technology-roadmap/executive-summary https://www.iea.org/reports/ammonia-technology-roadmap/executive-summary https://www.iea.org/reports/ammonia-technology-roadmap/executive-summary

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By developing high-performing genetic engineering tools for bioenergy crops, the PERSEPHONE program will enable quad-scale energy and substantial greenhouse gas mitigation impacts in these diverse areas and beyond.

2. TECHNICAL CHALLENGES

Alongside breeding and management, plant genetic engineering is a complementary strategy that may further transform agriculture into a sustainable source of energy and materials. A typical plant genetic engineering workflow is illustrated in Figure 1 and involves the following steps:

0. Preparation of the transgene and vector;

1. Transformation of DNA into undifferentiated plant cells known as calli;

2. Regeneration of the cells into a plantlet via tissue culture;

3. Transplantation of the plantlet into soil in a greenhouse; and

4. Generation of explant and callus to iterate.

If the engineering targets have been reached, seeds can be harvested at the end of Step 3 and the workflow can cease there.

Figure 1. A typical plant genetic engineering workflow.

Challenges to plant engineering are particularly severe for bioenergy crops, as described in the recent DOE report, “Overcoming Barriers in Plant Transformation: A Focus on Bioenergy Crops.”24 Although a large majority of corn and soy grown in the U.S. is genetically modified, a

24 U.S. DOE. 2024. “Overcoming Barriers in Plant Transformation: A Focus on Bioenergy Crops,” DOE/SC-0215. U.S.

Department of Energy Office of Science. https://doi.org/10.2172/2335710.

https://arpa-e.energy.gov/faqs mailto:ARPA-E-CO@hq.doe.gov https://doi.org/10.2172/2335710

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- 13 -limited number of cultivars can be routinely transformed.25 The primary methods for plant transformation utilize disarmed Agrobacterium tumefaciens or biolistic DNA delivery.

Agrobacterium-based protocols have higher levels of transformation efficiency (up to 50% in optimized maize genotypes) but are effective on limited varieties of plants.26 Even when successful, the process of stably regenerating a transformed plant is lengthy and labor-intensive, especially for bioenergy crops such as sorghum, miscanthus, and poplar.

The challenges facing bioenergy crop engineering are summarized as follows:

• Low transformation efficiency;

• Lengthy and unreliable regeneration through tissue culture;

• Genotype-dependent techniques and recalcitrant varieties;

• Labor-intensive practices with low reproducibility;

• Regulatory uncertainty and biocontainment risk;

• Limitations on the size and complexity of genetic constructs;

• Excessive time, labor, and space to generate and maintain transformable material; and

• Difficulty controlling the genomic location and expression of trans-genes.

As a result, the rapid “Design-Build-Test-Learn” loops employed in other engineering fields to iterate and innovate remain out of reach in plant engineering.

On a national level, the consequences of this bottleneck are severe. Most labs lack the capacity to engineer relevant bioenergy crops, leaving them reliant on a dwindling number of centralized transformation facilities. U.S. laboratories deliver only 5,000-10,000 DNA designs into crops per year across all species, with only about 200 in bioenergy crops beyond corn and soybeans.27 Moreover, the cost of each design can range from $6,000 to $10,000.28 For comparison, the sorghum genome contains roughly 30,000 genes.29 The mismatch between current engineering capabilities and the scale of crop genomes makes it unlikely that the U.S.

will achieve transformative advances in bioenergy crops using currently available technology.

In addition to the challenges described above, advanced bioenergy crop engineering leads to unique regulatory and social considerations. Crops like sorghum and poplar have native relatives near likely areas of deployment, leading to concerns about the flow of transgenes into

25 USDA Economic Research Service. 2024. “Adoption of genetically engineered crops in the United States, 1996- 2024.” USDA ERS - Recent Trends in GE Adoption.

26 Yuji Ishida, Yukoh Hiei, and Toshihiko Komari, “Agrobacterium-Mediated Transformation of Maize,” Nature Protocols 2, no. 7 (June 21, 2007): 1614–21, https://doi.org/10.1038/nprot.2007.241.

27 U.S. DOE. 2024. “Overcoming Barriers in Plant Transformation: A Focus on Bioenergy Crops,” DOE/SC-0215. U.S.

Department of Energy Office of Science. https://doi.org/10.2172/2335710.

28 Wisconsin Crop Innovation Center Pricing. Accessed 12/2/24. https://cropinnovation.cals.wisc.edu/pricing-2/.

For each design, it is typical to produce 6-10 distinct transformed plant lines to compensate for random DNA integration. The price quoted is for one design.

29 Andrew H. Paterson et al., “The Sorghum Bicolor Genome and the Diversification of Grasses,” Nature 457, no.

7229 (January 2009): 551–56, https://doi.org/10.1038/nature07723.

https://arpa-e.energy.gov/faqs mailto:ARPA-E-CO@hq.doe.gov mailto:ExchangeHelp@hq.doe.gov https://www.ers.usda.gov/data-products/adoption-of-genetically-engineered-crops-in-the-united-states/recent-trends-in-ge-adoption/ https://cropinnovation.cals.wisc.edu/pricing-2/

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- 14 -native populations.30,31 The introduction of new bioenergy crops into an area also could raise concerns around invasiveness and ecosystem impacts.32 Widespread deployment of engineered bioenergy crops will require innovative biocontainment technologies to prevent environmental damage and facilitate regulatory compliance.

3. EMERGING TECHNOLOGIES

Recent advances both inside and outside the field of plant biotechnology have the potential to dramatically improve the ability of researchers to efficiently generate engineered plants. For example, expression of developmental regulatory genes can reduce the time and genotype-dependence of monocot transformation and regeneration.33 Similar genetic technologies have augmented biolistics-mediated, tissue culture-free editing through meristem induction.34 Meanwhile, advances in molecular genetics such as transposase-based gene insertion and “CRISPR-Combo” systems for simultaneous gene editing and activation have led to greater control and expanded possibilities for genome engineering; and rapid progress in fields such as drug delivery, nucleic acid medicine, machine learning, and robotics holds great promise for translation…