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Biological and Biotechnology Sciences

INTRODUCTION

The U.S. Army Combat Capabilities Development Command (DEVCOM) Army Research Laboratory’s (ARL’s) biological and biotechnology sciences research competency is focused on “Investigating the fundamental sciences of biology, biological systems, and biomaterials, with the goal of enabling transformational Army capabilities.”¹ As an enabling competency, it provides knowledge and advanced materials not reachable from other routes to partnering ARL competencies and to organizations in an effort to develop game-changing Army capabilities. The competency is immersed in a rapidly expanding field, encompassing foundational biological research as well as the growing fields of engineering with biology and engineering biological systems. It touches many other fields, making use of new techniques as well as contributing new capabilities.²

Within the research competency are three core competencies: biology in military environments, biosynthesis and biomaterials, and synthetic biology tools. The biology in military environments core competency focuses on discovering, understanding, and controlling microbiomes that are believed to be relevant to military applications (what the competency describes as “military material”). On the discovery end, this includes bioprospecting from assets, bioinformatics on what the core competency calls “unknown-unknowns,” single isolate and community omics, and genome mining. On the controlling end, activities include the characterization and measurement of polymer degradation and metal corrosion by microbes (single cell or community).³

The biosynthesis and biomaterials core competency focuses on bio-derived materials—specifically their production, characterization, and processing for operational use. Their research looks at biological routes to inorganics (e.g., magnetosomes), bio-polymer synthesis and processing (e.g., melanin, bacterial cellulose, and silk), and peptide discovery and development (e.g., protein catalyzed capture agents, bacterial displays, and peptide resins).⁴

The synthetic biology tools core competency focuses on developing novel genetic engineering capabilities to harness military-relevant chassis. This includes onboarding novel chassis, the design and development of genetic parts and circuits, and genetic engineering using traditional benchtop design and assembly approaches. The core competency also focuses on high-throughput genetic engineering

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¹ V. Martindale and J. Sumner, 2024, “Biological and Biotechnology Sciences Technical Advisory Board—Story of the Competency,” U.S. Army Combat Capabilities Development Command (DEVCOM) Army Research Laboratory (ARL) presentation to the committee, July 9.

² Passages from V. Martindale and J. Sumner, 2024, “Biological and Biotechnology Sciences Technical Advisory Board—Story of the Competency,” DEVCOM ARL presentation to the committee, July 9.

³ Passages from B. Adams, 2024, “Synthetic Biology and Biology in Military Environments,” DEVCOM ARL presentation to the committee, July 9.

⁴ Passages from M.B. Coppock, 2024, “Biosynthesis and Materials Overview,” DEVCOM ARL presentation to the committee, July 9.

strategies—high-throughput approaches to facilitate deoxyribonucleic acid (DNA) introduction, assembly, and screening steps of the genetic engineering processes.⁵

On June 9–11, 2024, the Panel on Assessment of Biological and Biotechnology Sciences visited the Adelphi Laboratory Center in Adelphi, Maryland. During this visit, the panel viewed podium and poster presentations, held discussions about the competency facilities, and spoke to the scientific researchers within the competency and within these three core competencies. Below is the summary of the panel’s findings as they relate to the assessment criteria. As part of the assessment, the Army Research Laboratory Technical Assessment Board (ARLTAB) and its panels were asked by ARL to provide suggestions of specific people and organizations relevant to the work it is doing, or could be doing, that it could connect with. The fruits of this brainstorming activity are captured in the pages of the following chapter. It is important to note, however, that while certain individuals or organizations are mentioned, the ARLTAB and its panels are in no way being prescriptive about connecting to these outside entities and understand there may be other exemplars of equal value in the research community. ARL should therefore use its best judgement as to whether these ideas could be helpful.

TECHNICAL QUALITY OF THE CORE COMPETENCY

Biology in Military Environments Core Competency

Accomplishments and Advancements

The review of the biology in military environments core competency included a focus on ARL’s extramurally funded and intramural projects, both of which demonstrate ARL’s prioritization of fundamental biology research in the development of tools for applicative use. As part of this review, presentations where also shown from ARL’s extramural exploratory biology research thrust, which is discussed below.

The intramural team’s progress since its very recent establishment (inception around 2018) is highly impressive and commendable. Under excellent leadership, the biological and biotechnology sciences competency has grown from a few people with a vision and empty laboratories to a strong portfolio supported by state-of-the-art equipment. (1) maintaining broad awareness of ongoing research, (2) investing in cutting-edge exploration, and (3) preventing technological surprise.⁶ The research presented during the assessment is making headway toward these goals. Notably, good progress is being made on standardization of multiple phyla cloning and genome manipulation, and this work is encouraged to continue.

It is also commendable that ARL appreciates that the state of the art is to consider complex, natural communities with the challenges associated with their study. It was noted during the review that there was a strong commitment to a similar goal running through the synthetic biology tools core competency, which looks at novel chassis and genetic circuits and parts. A drive toward efficiency and improved outcomes requires collaboration across these core competencies so that truly interchangeable protocols and parts are developed.

Another overall strength is the clear understanding by ARL researchers that cutting-edge research considers complex biological processes, including the function of complex microbial communities, despite challenges associated with working with natural communities. There is, however, a need for improvement in the core competency’s research relating to microbial bioprospecting, which is discussed in the next section, “Research Portfolio Opportunities.”

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⁵ Passages from B. Adams, 2024, “Synthetic Biology and Biology in Military Environments,” DEVCOM ARL presentation to the committee, July 9.

⁶ V. Martindale, 2024, “BBS in Context: The Big Picture,” DEVCOM ARL presentation to the committee, July 9.

Furthermore, initial ARL seed investments into plastics degradation have positioned it to be a leader in polyurethane degradation. The intramural ARL researchers have rightfully recognized a dearth in standardized methods to characterize and screen for polyurethane degradation. Substrate complexity and variability in methods has led to a number of false positives in the field where enzymes and strains actually consume polymer additives rather than the underlying backbone or only work on one of many plastics subtypes. Current intramural efforts at ARL to develop standardized, and more importantly, high-throughput, assays for plastics degradation have the potential to set the standard for the field and accelerate discovery and engineering of relevant platforms for polyurethane degradation. This work is pioneering, potentially standard setting, and will accelerate the development of chassis and proteins of interest—it needs to be prioritized. Historical models for this are the National Renewable Energy Laboratory’s standard assays for biomass characterization and degradation⁷ and the Idaho Research Laboratory’s well-characterized feedstock library.⁸

Other ARL successes include the extramural efforts on novel chassis for polyurethane degradation that may lead to novel enzymes and relevant degradation rates at scale. Continued growth and success in these areas promise to tackle a global challenge, while providing potentially enabling capabilities for the Army. It should be noted that the Department of Energy (DOE) has recently invested significant resources into plastics degradation, and thus some interagency coordination and communication is warranted to reduce the potential of duplicated efforts. That said, ARL’s focus on polyurethane is a unique niche from more “popular” work on polyethylene terephthalate (PET), nylon, polyethylene, and polypropylene and represents a gap where ARL can lead.

The competency goals with respect to plastics degradation are aligned with the state of the art in the field and reflect an understanding of the underlying science and research conducted elsewhere, and ARL’s research methods associated with plastic degradation are consistent with standards in the field.

Research Portfolio Opportunities

Plastics degradation is a relatively nascent field with many open areas. There is a missed opportunity for more discovery-based work that can identify novel or unknown enzyme classes from polyurethane degrading isolates, which can be developed by the intramural researchers and also establish the extramural branch of ARL as a leader in this space. Potential strategies that can enable this include proteomics to identify secreted enzymes that degrade plastics, materials characterization to confirm the extent and types of chemical degradation, and expanded data science/bioinformatics support to mine and manage generated data sets.

In addition, current ARL efforts focused on rational cutinase engineering are duplicative. While applied here to polyurethane degradation, the enzymatic activity being optimized in this case is similar to that in other polymers such as PET. Here, the literature is extensive, with recent examples leveraging artificial intelligence (AI) to enhance activity by several orders of magnitude to industrially relevant rates, rather than the 2-fold observed in the presented ARL research. Moreover, it should be noted that cutinase/polyesterase activity is more or less a “solved” problem (although there are still unknown aspects of both of these enzyme groups and activities, including chitin LPMOs [lytic polysaccharide monooxygenases]). The challenge for polyurethane is the carbamate bond where discovery is still needed. ARL may consider conversations with leaders, such as those noted in the publications below (e.g., researchers such as Craig Criddle), who may help them define future directions:

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⁷ National Renewable Energy Laboratory Bioenergy, “Biomass Compositional Analysis Laboratory Procedures,” https://www.nrel.gov/bioenergy/biomass-compositional-analysis.html, accessed October 15, 2024.

⁸ Idaho National Laboratory, “Bioenergy Feedstock Library,” Biomass Feedstock National User Facility, https://bioenergylibrary.inl.gov/Home/Home.aspx, accessed October 15, 2024.

• “Machine Learning-Aided Engineering of Hydrolases for PET Depolymerization” by Hongyuan Lu and colleagues.⁹
• “Surface Engineering of a Cutinase from Thermobifida cellulosilytica for Improved Polyester Hydrolysis” by Enrique Herrero Acero and colleagues.¹⁰
• “Revisiting the Activity of Two Poly(Vinyl Chloride)- and Polyethylene-Degrading Enzymes” by Anton A. Stepnov and colleagues.¹¹
• “Characterization of Biodegradation of Plastics in Insect Larvae” by Wei-Min Wu and Craig S. Criddle.¹²

Another finding connected to the above discussion is that an overarching observation relevant to many of the projects presented during the panel’s assessment was that modern computational biology, bioinformatics, and AI approaches are not yet being harnessed at the level they could be to support the core competency’s projects. It is imperative that these approaches be incorporated into the core competency work.

On a different note, in broadly viewing the entire core competency portfolio, the panel found that the extramural research might benefit from expansion into research themes that align with ongoing research in the field, and from bringing additional research laboratories into the extramural research portfolio (if they are not already doing so with more recent project acquisitions). In line with this observation, one suggested approach going forward is to increase conference attendance—especially at high-caliber conferences such as the Gordon Research Conferences (GRCs)—as it will allow scientists and managers in ARL to stay abreast of cutting-edge advances in their fields and connect to other leading-edge researchers. This is a high priority for networking with scientists who may quickly add new technology and relevance that could be of interest to ARL and inform its portfolio acquisitions and collaborations.

This suggestion extends to intramural researchers as well. Much of ARL’s work is related to microbial ecology, and thus it was surprising that ARL seemed absent from the recent International Symposium on Microbial Ecology (ISME) meeting, which is the largest nonprofit meeting of its kind. It is important that both ARL program managers and scientists have the ability to attend such conferences.

Other ways of meeting potential new collaborators are through scanning public databases for currently funded grants or perhaps hosting workshops. ARL may also consider the creation of an internship program for intramural scientists to spend some time in external laboratories and, if feasible, reciprocal arrangements for external researchers (potentially extramural ARL collaborators) to spend time at ARL intramural laboratories, potentially taking advantage of ARL’s scientific instrumentation.

Another observation is that there is substantial technical overlap between the research being conducted in-house and in other government laboratories, both within the Department of Defense (DoD) and other agencies. For example, the National Aeronautics and Space Administration (NASA) has an interest that is similar to the core competency in life in extreme environments, slow growth, etc. While the motivation is very different, the technical aspect of the ARL program would benefit from joint programs that utilize the expertise and infrastructure of other agencies. ARL may want to consider reaching out to other agencies or facilities with similar research interests to explore collaborations and joint projects. This

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⁹ H. Lu, D.J. Diaz, N.J. Czarnecki, C. Zhu, W. Kim, R. Shroff, D.J. Acosta, et al., 2022, “Machine Learning-Aided Engineering of Hydrolases for PET Depolymerization,” Nature 604:662–667.

¹⁰ E.H. Acero, D. Ribitsch, A. Dellacher, S. Zitzenbacher, A. Marold, G. Steinkellner, K. Gruber, et al., 2013, “Surface Engineering of a Cutinase from Thermobifida Cellulosilytica for Improved Polyester Hydrolysis,” Biotechnology and Bioengineering 110:2581–2590.

¹¹ A.A. Stepnov, E. Lopez-Tavera, R. Klauer, C.L. Lincoln, R.R. Chowreddy, G.T. Beckham, V.G.H. Eijsink, et al., 2024, “Revisiting the Activity of Two Poly(Vinyl Chloride)- and Polyethylene-Degrading Enzymes,” Nature Communications 15:8501.

¹² W.-M. Wu and C.S. Criddle, 2021, “Chapter Five—Characterization of Biodegradation of Plastics in Insect Larvae,” in Methods in Enzymology, Vol. 648.

could be initiated through a workshop. The additional advantage to this approach is that other federal agencies have closed facilities and a similar approach to controlled unclassified information (CUI) when needed. Workshops with scientists from industry and academia may also be useful.

Current work funded by federal agencies is public information, and researching these research project descriptions could also help ARL understand what work is already being done, to avoid duplication, but also to potentially identify new academic collaborators or industry laboratory partners in particular areas. Some search tools that can help the competency determine the work going on in other agencies include the National Institutes of Health (NIH) “RePORT”¹³ and the National Science Foundation (NSF)-funded awards search tools.¹⁴ DOE grants are also searchable in the Portfolio Award and Management System.¹⁵

ARL may also consider initiating calls for ideas that actively look for research groups that are focused on one field or have an interdisciplinary focus and bring them together. This may help ARL to get the latest advances that are not public yet. A call for ideas, should, however, be done with some discernment, as one concern is that this approach could easily be biased in the favor of more established faculty and thus miss some innovative ideas from other voices.

Another finding was that ARL could be more intentional about developing opportunities for its researchers to collaborate more closely. First, there appears to be a disconnect between the research carried out by ARL’s intramural researchers and the research funded extramurally. The nature of the disconnect between these two entities (intramural and extramural) is understood, because there are the logistical obstacles associated with combining these groups. However, the current disconnect seems to be a major limitation in expanding the research areas that align with ARL’s scientific goals, and there is a loss of opportunity that could be addressed by overlapping collaborative research. ARL can thus focus on better integration of the core competency’s intramural and extramural research efforts by providing a platform for each entity to network more directly with the other. Perhaps symposia, reciprocal internships, joint posters at meetings, or other opportunities could provide the missing overlap for these two groups by promoting increased communication and collaboration. It would likewise benefit ARL’s intramural researchers to visit outside laboratories to gain exposure to additional areas of expertise and expand their skillsets for carrying out their research.

Furthermore, there appears to be a disconnect between research being carried out by different intramural groups. Many areas of research across different core competencies could benefit from increased communication and collaboration. For example, the state-of-the-art microfluidics approaches and expertise in the synthetic biology tools core competency (specifically its work on DNA ENTRAp (project name: Custom Microfluidic Cartridges for Army Science: DNA ENTRAP–A Microfluidic Platform for Enhanced DNA Transfer) could be employed in research concerning microbial growth. Likewise, molecular dynamics simulations and modeling can be incorporated to provide powerful insight into complex biological systems. ARL can provide more opportunities within ARL for communication between intramural researchers across core competencies and also competency programs. This could be achieved similarly as the above suggestion with the incorporation of annual symposia or mini-conferences held specifically within and for ARL intramural researchers.

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¹³ National Institutes of Health, “Welcome to Research Portfolio Online Reporting Tools (RePORT),” https://report.nih.gov, accessed October 15, 2024.

¹⁴ National Science Foundation, “Awards Simple Search,” https://www.nsf.gov/awardsearch, accessed October 15, 2024.

¹⁵ Department of Energy, “Portfolio Analysis and Management System (PAMS),” https://www.energy.gov/science/portfolio-analysis-and-management-system-pams, accessed October 15, 2024.

Opportunities Identified for Individual Projects

This section provides both opportunities identified for and also commentary about some of the individual projects that were shown during the review.

• ARO Microbiology: Slow Microbial Growth. The extramurally funded research shown through this presentation could be put in two categories: (1) challenging metabolism studied in laboratories and (2) field-environment experiments including dry environments and permafrost. The former of these is basic laboratory science in academic laboratory programs (e.g., Bacterial Longevity through researchers at the University of Washington; the “Unknome” (database of important genes of unknown function) through researchers at Michigan State University and Mg++ dependence of supercoiling with researchers at Yale University) represent a portfolio of excellent investigators and scientific approaches.

  In the second group of field-based approaches, the experimental capabilities described were excellent, in particular given that several of these research groups are working in the field under extreme conditions. One concern was that while data presented (such as deuterium isotope ratio mass spectrometry of lipids of organisms in permafrost tunnel) suggested metabolic pathways were active in the microorganisms within these extreme cold temperatures, it was unclear whether these data could be attributed to growth or to metabolically active but non-dividing organisms. Additionally, it should be noted that microbial metabolism in gypsum halite crusts has long been known,¹⁶ but the work on iron utilization could be of interest if magnetite is present. ARL interest in the effects of melting of the permafrost is timely. The following article gives an idea who funds microbial work for permafrost.¹⁷ A unique niche would be to advance the basic science in the field. Characterizing in the native ecosystem has not been addressed. It seems that this area in particular is of broad interest and would benefit from interagency coordination, for example, the use of NASA satellite data and other ground studies and NIH for its interest in the release of disease vectors. NSF, DOE, and the U.S. Geological Survey may have an interest as well.

  The idea of using bacteria to sequester precious elements as a future direction is valuable. Note some of this is happening already (e.g., uranium oxidation; lanthanide recycling,^18,19 and other work from Martinez-Gomez laboratory). Thus, there may be an opportunity to reach out to others working on similar research for additional extramural partners here.

• Breakdown of Polymeric Materials Using Synthetic Biology Approaches. The research topic for this project, which focused on the isolation of new plastic degrading enzymes is of current interest across broad scientific communities. The approach carried out by the intramural researchers to isolate new organisms capable of degrading plastic is logical and thorough and appeared to be successful in identifying and confirming both bacterial and fungal species capable of degrading polyurethane plastics. Downstream methods for characterizing these organisms involved sequencing and bioinformatic analysis of the genomes but focused specifically on identifying enzymes that have already been established to play a role in plastic degradation. Thus, despite the strong workflow, a critical issue within

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¹⁶ L.J. Rothschild, L.J. Giver, M.R. White, and R.L. Mancinelli, 1994, “Metabolic Activity of Microorganisms in Evaporites,” Journal of Phycology 30:431–438.

¹⁷ M.P. Waldrop, C.L. Chabot, S. Liebner, S. Holm, M.W. Snyder, M. Dillon, S.R. Dudgeon, et al., 2023, “Permafrost Microbial Communities and Functional Genes Are Structured by Latitudinal and Soil Geochemical Gradients,” The ISME Journal 17:1224–1235.

¹⁸ T. Rogiers, R. van Houdt, A. Williamson, N. Leys, N. Boon, and K. Mijnendonckx, 2022, “Molecular Mechanisms Underlying Bacterial Uranium Resistance,” Frontiers in Microbiology 13:822197.

¹⁹ N.M. Good, C.S. Kang-Yun, M.Z. Su, A.M. Zytnick, C.C. Barber, H.N. Vu, J.M. Grace, et al., 2023, “Scalable and Consolidated Microbial Platform for Rare Earth Element Leaching and Recovery from Waste Sources,” Environmental Science and Technology 58:570–579.

• this project was the repeated isolation of cutinases, as described in a broader conversation above (the writings below provide a few additional thoughts on directions ARL may consider moving it).

  This result means there is significant room for improvement on the downstream applications for identifying plastic-degrading enzymes. While the use of sequencing and subsequent bioinformatics could be of high value for identifying novel enzymes capable of plastic degradation, the application was limited to focus on already discovered enzymes. This research has the potential to be cutting-edge if methods to identify novel plastic-degrading enzymes are employed. High-throughput screens can rule out additional cutinase expressors before the step of genome sequencing. Isolations can be designed that do not yield positive hits for cutinase activity. For example, in bacteria there are genetic methods such as transposon sequencing (Tn-seq) that could be employed to identify all genes that contribute to plastic degradation in the genome.²⁰ There is significant potential in this project to identify new and diverse enzymes and metabolic pathways that could be implemented for downstream plastic-degrading functions. One potential way to explore these possibilities is to leverage the expertise of bacterial and fungal geneticists and molecular biologists to provide additional expertise on methods for identifying and characterizing novel enzymes. The study of enzyme variants using AI (e.g., Alpha Fold in this case) may not lead to new insights, as team members acknowledged during their presentation (although there are differing opinions on the potential of AI to discover novel enzyme activities). In the pursuit of improved amidases and cutinases, they may want to consider approaches such as deep mutational scanning or in vitro evolution.

Exploratory Biology Research Thrust

The research portfolio focusing on the extramural exploratory biology research thrust is exciting, impressive, and expansive. Elements of the research explore fundamental outstanding questions in biology that could have significant impact on both expanding our understanding of basic biology as well as contributing to the development of new biological tools for applicative use. The exploratory biology portfolio covered diverse biological topics aiming to establish techniques to identify novel signals, integrate computational predictions in complex systems, dissect interactions between prokaryotes and larger-order organisms, and explore the composition and function of microbial communities. The range of approaches being applied to the signal generation, propagation, and detection work presented were impressive and the continuation of this thrust is encouraged.

There is a large opportunity to expand the portfolio on microbial community work. For example, one current major focus revolves around identifying metabolic interactions among two to three species within a microbial community (e.g., secondary metabolite modification within simplified soil community), yet networks dissecting relationships between few species has been ongoing for decades. The current challenge in dissecting metabolic networks of large communities is the ability to do this in more complex communities containing much greater biodiversity. For example, cutting-edge research on complex metabolic interactions focuses on communities that are either more representative of natural communities or of pre-existing environmental biofilms, which remains challenging as these systems are often unculturable. There is also room for expansion into exploring the spatial organization of complex microbial communities. Studying the spatial organization of complex communities is highly challenging, and cutting-edge research in this arena still focuses on small microbial communities consisting of one to two species. There is significant opportunity for this area of study to expand the understanding of how metabolic interactions are achieved in communities more reflective of those found in natural environments. Understanding the spatial distribution of individual cells and how individuals localize

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²⁰ M.N. Price, K.M. Wetmore, R.J. Waters, M. Callaghan, J. Ray, H. Liu, J.V. Kuehl, et al., 2018, “Mutant Phenotypes for Thousands of Bacterial Genes of Unknown Function,” Nature 557:503–509.

throughout communities will provide insight into both the physical and chemical interactions taking place and how these interactions contribute to biofilm growth, stagnation, or death. In fact, spatial distribution in microbial communities was the subject of two of the plenaries at the recent ISME meeting, suggesting that a broad swath of the research community considers this a key question.

There is also opportunity to expand into the study of diverse biological species. The current portfolio contains projects on a few different species, but it is largely limited to bacteria and does not appear to consider the broad diversity that exists in nature including archaea, fungi, algae, protists, and phages. It is important for the microbial community researchers to recognize that archaea and phage are part of complex communities, and bioinformatics tools can now follow these members of complex communities. Similarly, protists should not be discounted as they can play a vital role in the function of microbial communities.²¹

For a comprehensive study of complex microbes, more research focusing on these and other diverse species need to be incorporated.

Another beneficial aspect of the exploratory research portfolio included the possibility for allocating resources for the expansion of fundamental biology in the current portfolio into translational applications for future use. The potential for continuation of ongoing basic research provides a significant opportunity for applications and real-world outcomes. This being said, the exploratory biology area is enormous and cannot be covered comprehensively by ARL, so judicious choice of areas and collaborations are key.

Biosynthesis and Biomaterials Core Competency

Achievements and Advancements

The ARL biosynthesis and biomaterials core competency research team highlighted several projects that are at par with other research institutions nationally and internationally and show a strong understanding of research done elsewhere. In addition, all of the PSQs relevant to the core competency are cutting edge state-of-the-art questions that the field is actively trying to address. The paragraphs below describe some of the successful research efforts ARL has been developing.

Building on the initiation of adapting biological self-assembly mechanisms of proteins and DNA to develop novel biomaterials by the researchers David Tirrell (California Institute of Technology) and Ned Seeman (New York University), there has been significant interest in applying dynamic properties of biomolecules into materials systems and identifying their applications. Such studies have primarily focused on biomedical applications but there is significant interest in the development of novel functional biomaterials beyond health-related fields. The work shown through the ARL presentations “Biosynthesis and Materials Overview and Controlling Biomolecular Assembly” and “Expanding the Chemistry of Biology to Access Novel Biomaterials” did an excellent job of building on well-established approaches for the development of melanin, magnetosome, and silk applications. Specifically, this work has done a terrific job of illustrating biology’s role in templating materials in ways that traditional, chemically based approaches have not been able to achieve.

Building on early discoveries from Peter Shultz in the 1980s, there has been significant interest and excitement around the use of bio-orthogonal chemistries.²² In 2022, the Nobel Prize was awarded to Carolyn Bertozzi at Stanford University²³ highlighting the continued progress in this area. The research

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²¹ S. Guo, W. Xiong, X. Hang, Z. Gao, Z. Jiao, H. Liu, Y. Mo, et al., 2021, “Protists as Main Indicators and Determinants of Plant Performance,” Microbiome 9:64.

²² C.C. Liu and P.G. Schultz, 2010, “Adding New Chemistries to the Genetic Code,” Annual Review of Biochemistry 79:413–444.

²³ ACS Publications, “Collection: Bioorthogonal and Click Chemistry Curated by Prof. Carolyn R. Bertozzi, 2022 Winner of the Nobel Prize in Chemistry,” https://pubs.acs.org/page/vi/bioorthogonal-click-chemistry, accessed October 15, 2024.

shown through the presentations “Controlling Biomolecular Assembly” and “Expanding the Chemistry of Biology to Access Novel Biomaterials” on novel biosynthesis of non-peptide linkages and unnatural amino acids in cell free systems represents pioneering cutting-edge work in this field. As biology and biotechnology continues to evolve, these fields provide equal opportunities for innovation across nations and institutions. Securing pioneering technologies in biotechnology is becoming increasingly crucial, as it offers a competitive advantage and ensures leadership in a rapidly advancing global scientific landscape. Leveraging these emerging technologies will be essential for maintaining a strategic edge in both research and application.

The research presented on capture agents, through the poster “Peptide Resins for Heavy Metal Water Filtration,” which looked at single amino acids, or a few with the same amino acids and existing peptides, represents solid work building from concepts initiated in the early 1990s by Kit Lam (University of California, Davis) through one-bead-one-compound approaches for peptide discovery and applications. This work streamlined well-established and existing systems to allow for fast following approaches to bring these capabilities to the broader specialized needs and ecosystem of ARL. This a strong incremental advance in support of the core competency’s scientific goals.

The assessment criteria asked for commentary on how the competency demonstrates and reflects a broad understanding of the underlying science and research conducted elsewhere. Thus, it should be noted that work presented around traditional self-assembling biomaterials, novel chemistry, and protein capture is well-connected to leading laboratories in the field and building from state-of-the-art scientific endeavors. The laboratories selected are excellent nodes into the broader, cutting-edge scientific community, and the internal pipelines for discovery represent well-thought-out deployment of established tools and techniques to enable rapid response.

The capabilities around high-performance computing demonstrates a strong understanding and capability in well-established legacy systems. These systems are being utilized to the best of their abilities and are an important foundational infrastructure necessary for building next-generation data science capabilities. The research methods around self-assembling biomaterials, novel chemistry, and protein capture are sound and represent a thorough understanding of the quantitative metrics, design of experiments, and traditional modeling approaches. Such work reflects high-performing teams with excellent capabilities to bring state-of-the-art biotechnology into the ARL ecosystem.

Concerning their fungal research, the core competency team has rightly chosen melanins as one of their focused application efforts, as these polymers present a plethora of useful properties, ranging from protection from ultraviolet radiation, enzymatic lysis, temperature extremes, and oxidative damage. The presentations showed the impressive progress the core competency has made on isolation of fungal melanin and its conversion into useful materials, such as graphitic carbon. The team has made incredible progress toward its goals. They are working toward the state of art in the synthetic biology area and are state of art as far as production of melanin goes.

Research Portfolio Opportunities

The assessment criteria asked for commentary on the soundness of the research methods and methodologies used by the competency. While successes in this area are listed in the previous section, it is important to note that the methods used to evaluate biomanufacturing lacked industry standard quantitative assessments that are critical in understanding gaps in the transition path from current methods to commercial viability. An excellent summary of the key metrics to benchmark was recently published by one of the leaders in the field, Jens Nielsen.²⁴ All future work on biomanufacturing will need to develop a clear data science strategy with key quantitative metrics meticulously recorded for changes around environment and genetic impact on the titer (g/L), rate (g/L/hr), and yield (g/g). Additional factors around purity of products, cell density, genetics, environment, and additional metadata will need to be

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²⁴ O. Konzock and J. Nielsen, 2024, “TRYing to Evaluate Production Costs in Microbial Biotechnology,” Trends in Biotechnology (11):1339–1347, https://doi.org/10.1016/j.tibtech.2024.04.007.

recorded. Increasing collaborations with external biomanufacturing partners could further enhance alignment with industry standards, including scalability and purity.

More broadly speaking on this topic of data science, all efforts within the core competency would benefit from a clear modern data strategy. A modern data science strategy is a framework and approach to managing data that puts “AI at the center.” This implies that efforts to benchmark, collect, and process data should focus on making such data accessible to transformer-based AI methodologies. Unique to this space are practices for metadata collection that connect scientific efforts traditionally considered to be totally independent, because AI can help to identify novel and non-obvious relationships that may catalyze new areas for scientific exploration, support existing scientific efforts and/or potentially catalyze cross-cutting collaborations with other core competencies and competencies. The development of a modern data strategy could be informed by connecting with leaders in the data science field, such as Ilias Tagkopoulos, who is director of NSF’s and the U.S. Department of Agriculture’s AI Institute for Next Generation Food Systems. Furthermore, NSF has invested in 25 AI centers, many of which are built for biological systems. The leadership for the coordinating organization of these 25 AI centers is Steven Brown.²⁵ He could be a highly valuable contact for coordination to discuss potential synergies and learnings that can be adapted based on the >$250 million public investment in this space.

Since the invention of transformers in ~2017, advances in AI are rapidly transforming the field of biology and the broader world. Modern organizations are being built with an “AI in the center” as the framework in order to deploy a coherent data strategy enabling construction of the necessary data assets to leverage transformer-based AI methodologies. For example, private industry over the past 6 months has deployed more than $2 billion in building “AI in the center biotechnology” companies such as Xaira, EvolutionaryScale, Generate, Isomorphic Labs, and Profluent as examples. Just recently, Flagship Ventures raised $3.6 billion to deploy into the AI driven biotechnology sector (“Companies using AI or ML for healthcare research and drug discovery are on track to raise more venture money in 2024 than in any previous year apart from 2021.”²⁶). In addition, the following articles are relevant:

• “New AI Drug Discovery Powerhouse Xaira Rises with $1B in Funding”²⁷
• “EvolutionaryScale Lands $142 mln to Advance AI in Biology”²⁸
• Generate:Biomedicines Announces Close of $273M Series C Financing to Advance Its Generative AI Pipeline of Preclinical and Clinical Protein Therapeutics”²⁹
• “Isomorphic Labs Kicks Off 2024 with Two Pharmaceutical Collaborations”³⁰
• “Profluent Secures $35M Funding Led by Spark Capital to Advance AI-First Protein Design”³¹

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²⁵ AI Institutes Virtual Organization, “AI Institutes Virtual Organization,” https://aiinstitutes.org/about-aivo, accessed October 15, 2024

²⁶ M. Senior, 2024, “Biotech Financing: Darkest Before the Dawn,” Nature Biotechnology 42:1331–1338.

²⁷ A. Armstrong, 2024, “New AI Drug Discovery Powerhouse Xaira Rises with $1B in Funding,” Fierce Biotech, April 24, https://www.fiercebiotech.com/biotech/new-ai-drug-discovery-powerhouse-xaira-rises-1bfunding.

²⁸ A. Tong and K. Hu, 2024, “EvolutionaryScale Lands $142 mln to Advance AI in Biology,” Reuters, June 25, https://www.reuters.com/technology/evolutionaryscale-lands-142-mln-advance-ai-biology-2024-06-25.

²⁹ Generate: Biomedicines, 2023, “Generate: Biomedicines Announces Close of $273M Series C Financing to Advance Its Generative AI Pipeline of Preclinical and Clinical Protein Therapeutics,” Press Release, updated September 14, https://generatebiomedicines.com/news/series-c-financing-announcement.

³⁰ Isomorphic Labs News, 2024, “Isomorphic Labs Kicks Off 2024 with Two Pharmaceutical Collaborations,” updated July 1, https://www.isomorphiclabs.com/articles/isomorphic-labs-kicks-off-2024-with-two-pharmaceuticalcollaborations.

³¹ Synbiobeta News, 2024, “Profluent Secures $35M Funding Led by Spark Capital to Advance AI-First Protein Design,” updated March 21, https://www.synbiobeta.com/read/profluent-secures-35m-funding-led-by-spark-capitalto-advance-ai-first-protein-design.

• “Flagship Pioneering Raises $3.6 Billion to Fuel Breakthrough Innovations That Transform Human Health and Sustainability”³²

Current projects within the core competency either did not utilize AI or considered legacy ML approaches as an afterthought. ARL could consider adding dedicated intramural staff and external efforts to build programming with AI in the center. This will benefit both the biological and biotechnology sciences competency and ARL more broadly.

Furthermore, all of the projects in the biosynthesis and biomaterials core competency would benefit from closer integration with the synthetic biology tools core competency. This will increase throughput and standardization of data generation to improve interoperability and robustness. For projects such as Biomanufactured Cellulose as a Versatile Feedstock for Military Applications, the integration with the synthetic biology capability is likely to be essential for success given the inherent limitations with the natural organism evolutionary constraints.

Switching to other areas of the assessment criteria focused on identifying opportunities for the core competency, there is a major opportunity to double-down in the area of extending the toolkit of biology and novel biomaterials, and there can be a greater diversification of biomaterials being studied by the core competency. This is a rapidly developing field being accelerated through synthetic biology and AI capabilities. Specifically, scientists now have the ability to create component parts, and it will be important to understand the utility of these parts, as well as what products and materials can be made that were not previously accessible.

Supramolecular polymers are promising areas of research in the biomaterials field and, broadly speaking, the integration of covalent and supramolecular polymers will benefit not just biomaterials but also the critical field of sustainable materials, including biodegradation and recycling. Supramolecular polymers introduce the great opportunity of non-covalent bonding among monomers and thus their easy reuse.³³ They also introduce opportunities for dynamic materials for robotic applications,^34,35 bioactivity toward cell signaling, and generally for rapid stimulus-responsive properties given their highly dynamic nature. For all the potential applications of these non-covalent polymers, they could be used as bulk materials, nanostructures, or coatings. Great examples in nature are cell cytoskeletons and muscle with synergistic functionalities from well-organized supramolecular polymers, such as peptides and structured proteins. Diversifying the biomaterials portfolio with supramolecular polymers appears to align with the core competency’s scientific goals. Attending GRC, such as the Peptide Materials GRC and the Supramolecular Chemistry GRC, will be helpful in understanding and developing this new opportunity.

The work across the core competency, especially the melanin and magnetosome work, highlight the importance of biology in the field of materials through providing a templating mechanism not traditionally accessible through chemistry-based approaches. There are opportunities to now understand more broadly what the limitations of templating are using cellular systems by connecting with the synthetic biology capabilities in ARL to control biological morphologies and compositions. Furthermore, additional mechanisms of templating could be explored, such as those recently demonstrated by the Baker

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³² Flagship Pioneering News, 2024, “Flagship Pioneering Raises $3.6 Billion to Fuel Breakthrough Innovations That Transform Human Health and Sustainability,” updated July 1, https://www.flagshippioneering.com/news/press-release/flagship-pioneering-raises-3-6-billion-to-fuel-breakthrough-innovations-that-transform-human-health-and-sustainability.

³³ T. Aida, E.W. Meijer, and S. Stupp, 2012, “Functional Supramolecular Polymers,” Science 335:813–817.

³⁴ C. Li, A. Iscen, H. Sai, K. Sato, N.A. Sather, S.M. Chin, Z. Álvarez, L.C. Palmer, G.C. Schatz, and S.I. Stupp, 2020, “Supramolecular-Covalent Hybrid Polymers for Light-Activated Mechanical Actuation,” Nature Materials 19:900–909.

³⁵ S.D. Cezan, C. Li, J. Kupferberg, L. Dordevic, A. Aggarwal, L.C. Palmer, M.O. de la Cruz, and S.I. Stupp, 2024, “Fast Photoactuation Driven by Supramolecular Polymers Integrated into Covalent Networks,” Advanced Functional Materials, https://doi.org/10.1002/adfm.202400386.

laboratory.³⁶ Attending meetings such as the Peptide Materials GRC and Supramolecular Chemistry GRC could also provide exposure to a diverse set of leaders in the field that are moving beyond legacy approaches. This will be critical to bring cutting-edge capabilities into the ARL that work to engineer the system for homogeneity, controlled growth, etc.

There is also an opportunity to expand into more recently developed approaches enabled by the advent of synthetic biology and AI in the area of protein capture agents. Over the past 5 years, significant advances have been made in the development of “mini-binders” and “nanocages,” pioneered by the Baker and King laboratories at the University of Washington.³⁷ These concepts have moved past the conceptual stage with examples now commercially deployed and many other under commercial development. There is a critical opportunity to expand the capture agents to move beyond traditional methods and adopt modern AI-driven approaches. In the development of new protein capture agents, there would be a significant benefit of connecting with the Baker laboratory to understand new opportunities around modern approaches for designing protein binders. By leveraging these methods, there will be potential to address the challenges identified, create more capabilities for manufacturing viability, as well as better determine efficacy in complex biological environments. The core competency could also explore the broader community represented by the RosettaCommons, which is the foremost-leading scientific community around AI-driven biotechnology. RosettaCommons engages with more than 100 laboratories from across the world.³⁸ Connecting with RosettaCommons may help ARL to further understand recent advances and capabilities in protein and peptide science. Another potential resource to explore could be DeepMind, developers of AlphaFold.³⁹

Concerning the core competency’s biomanufacturing focus, in the development of dynamic functional protein-based materials using recombinant protein technology, there will be expected challenges or significant efforts needed in biomanufacturing. ARL’s intramural efforts on biomanufacturing can bring the appropriate synergies and reduce the amount of future efforts in terms of the design-build-test learn concept. Innovation in biomanufacturing is currently being invested through a collaboration between NSF’s and DOE’s Bioenergy Technology Office, which is being funded by the Agile BioFoundry. It could be highly beneficial to connect with groups such as these that are working on protein production to understand advanced technologies in the area.

In reference to the core competency’s research on fungi, templating melanin production on fungal hyphae to produce unique materials is a strength of the core competency, and these choices are resulting in polymers not possible with chemical approaches. The team has bonded graphitic carbon to multiple polymeric surfaces. Melanins are polymeric natural products either derived from the tyrosine L-dopa pathway (most organisms) or the polyketide dihydroxynaphthalene (DHN) melanin pathway (only in fungi). This latter pathway has more diversity in nature and is amenable to molecular genetics to produce variations in melanin polymers and melanin colors. There is an obvious need for improved production of biomaterials obtained from fungi. Thus, as previously mentioned, there is a need for better linkage between synthetic biology approaches in the synthetic biology tools core competency with materials science research in the biosynthesis and biomaterials core competency.

The panel was asked by ARL to provide suggestions for other useful biomaterials for future consideration. Other fungal biomaterials to consider are hydrophobins, which are only synthesized by fungi. They are excellent surfactants and are hydrophobic and used in nano technology. For pertinent reviews, ARL may consider the following articles:

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³⁶ A. Saragovi, H. Pyles, P. Kwon, N. Hanikel, F.A. Dávila-Hernández, A.K. Bera, A. Kang, et al., 2024, “Controlling Semiconductor Growth with Structured De Novo Protein Interfaces,” bioRxiv, https://doi.org/10.1101/2024.06.24.600095.

³⁷ Baker Laboratory, “Publications,” https://www.bakerlab.org/publications, accessed October 15, 2024.

³⁸ Rosetta Commons, “Rosetta Commons,” https://www.rosettacommons.org/about, accessed October 15, 2024.

³⁹ AlphaFold, “Overview,” https://deepmind.google/technologies/alphafold, accessed October 15, 2024.

• “Fungan Hydrophobins and Their Self-Assembly into Functional Nanomaterials”⁴⁰
• “Apergillus Hydrophobins: Physicochemical Properties, Biochemical Properties, and Functions in Solid Polymer Degradation”⁴¹
• “A Cryo-Electron Microscopy Support Film Formed by 2D Crystals of Hydrophobin HFBI”⁴²

Most fungi contain numerous hydrophobin genes, and Luciano-Rosario and colleagues illustrate a technology to recycle a marker gene to delete a 7-gene hydrophobin family.⁴³

The assessment criteria also asked for promising areas of research or novel approaches that the competency could consider pursuing, and some of these areas have already been described above. An excellent area for the core competency to explore is food biotechnology, which has taken off over the past 10 years, with >$55 billion in private and public sector investment.⁴⁴

After this initial flurry of investments in a new sector, it is now becoming clear where the state of the art in the field of food biotechnology lies. Academic domain experts, such as Bruce German (director of Foods for Health Institute), as well as industry leaders, such as Harold Schmitz (The March Group, former chief scientific officer of Mars, Inc.), and Victor Friedberg (founder of New Epoch, S2G, FoodShot), are critical leaders to discuss areas of opportunity in this field. Furthermore, new companies such as Meati (founder Justin Whiteley) and Better Meat⁴⁵ that have recently developed new strains of filamentous fungi, scaled manufacturing, and launched successful commercial food products may be able to discuss needed capabilities given ARL’s interest in fungi. Additionally, recent organizations have been launched to bring food into the modern biotechnology world through the Periodic Table of Foods⁴⁶ and discussion with those leaders (e.g., Selena Ahmed⁴⁷) to understand the potential of modern food biotechnology could be transformative.

As previously mentioned, other exciting areas of research include biological systems that can create novel component parts through leveraging abiotic chemistry approaches. Exploration could be initiated to understand the scope of abiotic chemistries that can be developed to create component parts and on understanding what materials and products can be made using these novel component parts. There is an opportunity to integrate the synthetic biology tools core competency with the biosynthesis and biomaterials core competency to work toward this goal.

Opportunities Identified for Individual Projects

• Manipulating Melanin Content and Hyphal Branching in Dematiaceous Fungi (Poster). This presentation tested the effects of different growth medium components, temperature, lighting conditions, and inhibitors of DOPA (3,4-dihydroxyphenylalanine) and DHN (1,8-

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⁴⁰ V. Lo, J.I-C. Lai, and M. Sunde, 2019, “Fungan Hydrophobins and Their Self-Assembly into Functional Nanomaterials,” Advances in Experimental Medicine and Biology 1174:161–185.

⁴¹ T. Tanaka, Y. Terauchi, A. Yoshimi, and K. Abe, 2022, “Apergillus Hydrophobins: Physicochemical Properties, Biochemical Properties, and Functions in Solid Polymer Degradation,” Microorganisms 10:1498.

⁴² H. Fan, B. Wang, Y. Zhang, Y. Zhu, B. Song, H. Xu, Y. Zhai, M. Qiao, and F. Sun, 2021, “A Cryo-Electron Microscopy Support Film Formed by 2D Crystals of Hydrophobin HFBI,” Nature Communications 12:7257.

⁴³ D. Luciano-Rosario, J.L. Eagan, N. Arylal, E.G. Dominguez, C.M. Hull, and N.P. Keller, 2022, “The Hydrophobin Gene Family Confers a Fitness Trade-Off Between Spore Dispersal and Host Colonization in Penicillium expansum,” mBio 13:e0275422.

⁴⁴ AgFunder, “AgFunder Global AgriFoodTech Investment Report 2024,” https://agfunder.com/research/agfunder-global-agrifoodtech-investment-report-2024, accessed October 15, 2024.

⁴⁵ The Better Meat Co., “The Better Meat Co.,” https://www.bettermeat.co, accessed October 23, 2024.

⁴⁶ The Periodic Table of Food Initiative, “Mapping Food Quality to Improve Human and Planetary Health,” https://foodperiodictable.org, accessed October 15, 2024.

⁴⁷ The Periodic Table of Food Initiative, “Selena Ahmed,” https://foodperiodictable.org/bio/selena-ahmed, accessed October 15, 2024.

• dihydroxynaphthalene) melanin synthesis on melanin production in Curvularia lunata. The team also tested char yield and morphology of the melanized fungal cells and the final graphitic carbon. This project would benefit from additional testing of growth parameters and collaboration with the synthetic biology tools core competency team to speed achievement of increased melanin production. For example, it became clear during conversations that the impact of the nitrogen source or level was not tested, and it is known that nitrogen availability controls melanin production in some fungal species.⁴⁸

Synthetic Biology Tools Core Competency

Accomplishments and Advancements

ARL has developed and presented a broad portfolio of synthetic biology research across extramural and intramural programs that rightly addresses highly scientifically relevant and high-impact questions in the field. There is a growing recognition within the field that microbiomes and diverse non-model chassis will be pivotal to providing scalable solutions to emerging challenges (both grand civilian societal ones and military), and thus ARL’s focus in these areas is aptly chosen.

More importantly, accomplishments from the competency have begun to mature into scalable bioproduction efforts (e.g., magnetosomes). ARL’s recent sprint to onboard 10 novel organisms in 10 weeks should be lauded as among the first in field. The success of this core competency can be attributed in part to a robust extramural program that currently funds multiple recognized leaders in synthetic biology research via the Army Center for Synthetic Biology. They are doing innovative foundational work in genetic tool development, microbiome engineering, and biomaterials. The investments in the Army Center for Synthetic Biology are well spent and promise to lead the field. Accomplishments of note here are the culturomics platform within the Center for Harnessing Microbiota from Military Environments (CHARMME), which uses the latest in automation and ML to isolate, classify, and characterize environmental bacterial and fungal soil species.⁴⁹ Similarly, the Predictive Materials Design (PreMaDe) project, which is an extramural collaboration that includes contributors from Stanford University, Northwestern University, University of Texas at Austin, Massachusetts Institute of Technology, Johns Hopkins University, and University of Illinois Urbana-Champaign whose goal is to “develop new experimental and computational tools that enable the scalable synthesis, assembly, and characterization of rationally designed biological materials with control over final material properties”⁵⁰ and is leading the way for cell-free synthesis—that is, more portable and robust synthesis—of functional materials.

Generally speaking, the competency has an understanding of science being performed in the field at large and the researchers are addressing cutting-edge questions and seeking out novel niches—for example, their microbiome work is focused on material synthesis rather than more common small molecule chemicals. Additionally, they are using research methods and methodologies that are both sound and appropriate.

ARL’s synthetic biology approaches utilizing fungi appear to be a relatively recent research direction for ARL. As mentioned in other core competency writings of this chapter, the speed with which the ARL research teams have pivoted to work with filamentous fungi is impressive. They have made significant progress in a relatively short period of time. Their establishment of high-throughput methods is state of the art.

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⁴⁸ S.R. Pomberio-Sponchiado, G.S. Sousa, J.C.R. Andrade, H.F. Lisboa, and R.C.R. Gonçlaves, 2017, “Production of Melanin Pigment by Fungi and Its Biotechnological Applications,” in Melanin, M. Blumenberg, ed., IntechOpen, https://doi.org/10.5772/67375.

⁴⁹ S. McElhinny, 2024, “U.S. Army Center for Synthetic Biology,” DEVCOM ARL presentation to the committee, July 9.

⁵⁰ Ibid.

ARL’s decision to utilize fungi for some of its synthetic biology approaches is commendable. The wide range of metabolic capabilities and growth habits of these organisms are amenable to synthetic and downstream biomaterials applications. Such organisms have been, until recently, relatively understudied, but are now recognized as an emerging technological powerhouse, as illustrated by several new industries centered around fungal proclivities. Fungi should provide a rich source of interesting polymers and other compounds for production of biomaterials. The next section provides identified opportunities for how ARL may further develop this research thrust.

Research Portfolio Opportunities

In situ engineered microbes present a risk to the environments within which they are released, potentially proliferating beyond their intended region of function and disrupting ecosystems. Uncontained engineered microbes also present a tactical risk where U.S. intellectual property and competitive advantage may be “stolen” by foreign actors that can gather a physical sample and analyze the underlying genomes. Biocontainment work would mitigate these risks and maintain U.S. tactical advantage by developing solutions to constrain engineered systems to a given operating area and include self-destruct fail-safes (kill switches) to destroy the engineered microbes and/or their modified genomes to prevent uncontrolled access to U.S. technology.⁵¹ If work is not being performed in this area, there is a clear opportunity for ARL to expand its efforts here. ARL can also consider enhancing its consideration of containment and other control interactions with the electromagnetic spectrum (e.g., antennas, receivers) via biological/biohybrid metamaterials and supramolecular assembled biomaterials.

The assessment criteria asked for commentary on opportunities within the competency where improved direction, increased focus, connections with other research lines, or other changes could better and more quickly address the competency objectives. At a high level, it is understood that ARL is building a biofoundry for non-model chassis microbes or a (distributed) facility that combines biology, automation, AI, synthetic biology, materials science, and chemistry to rapidly prototype and test engineered biological systems. However, the design of this biofoundry may need more intentionality. Biofoundries exploit automation and computer-aided design (CAD) to rapidly iterate through the design-build-test-learn cycle to develop practical biological systems that meet a given need. Significant investment has been made in developing technologies to “build” novel biological systems (e.g., automation, part libraries, microfluidic transformation platform). However, corresponding investments are needed for the “design,” “test,” and “learn” components. For example, a unified data framework/architecture is needed to enable the creation of CAD tools to inform design of genetic circuits and AI algorithms that can mine generated data sets for insight for future designs. Similarly, there is an opportunity to align “build” methodologies for species that meet specific scientific needs, creating a single pipeline that can onboard a novel isolate, and facilitate complex circuit design (as opposed to the current implementation where pipeline elements have been optimized for different classes of species). It is anticipated that this will require an intentional strategy with an infrastructure investment in data repository systems or a partnership with another agency or institution (e.g., NSF and/or DoD) to generate or adopt best practices and databases for creation, storage, and access of research data and metadata.

As mentioned, the inclusion of filamentous fungi as target organisms for synthetic biology is a ripe and timely research direction. The core competency has made significant progress on this relatively new research area and is poised to make important contributions to the field, as fast followers. However, there are some concerns that the core competency has not considered some technologies that will speed up its progress in the long run. Considering the short time since the fungal research program was initiated, there is room for skill acquisition among the researchers. It appears that the expertise of the academic collaborators is focused on Aspergillus techniques. This is a logical and wise start, but as many genera may require other technologies, it is recommended that multiple ARL team members acquire additional

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⁵¹ B.J. Caliando and C.A. Voigt, 2015, “Targeted DNA Degradation Using a CRISPR Device Stably Carried in the Host Genome,” Nature Communications 6:6989.

expertise in the molecular biology and physiology of filamentous fungi. This expertise could be grown through visits to the laboratories of experts in the field, who are broadly trained in work with diverse species. It is suggested that such visits be at least 2–3 months long in order to achieve immersion in the wide array of techniques and growth conditions necessary to work with a large group of diverse fungi. Conversely, ARL may consider hosting such experts, if it is feasible; however, this is a less attractive option because visits to external laboratories will provide access to more fungal researchers and more opportunities to view demonstrations of fungal techniques.

In addition to visits, ARL team members could consider attending either the biannual Gordon Research Conference or the Fungal Genetics Conference in order to keep abreast of current technological approaches for filamentous fungi. Synthetic biology conferences tend to focus on bacteria and yeast-like fungi, and the approaches will be different when working with filamentous organisms, thus it will be important for ARL team members to attend these two conferences. Several references are provided in Box 3-1, to provide ARL with research references and recommendations of researchers that may help inform fungal research at ARL.

Opportunities Identified for Individual Projects

• Organism Onboarding for Synthetic Biology. In considering the fungal aspects of this intramural overview presentation, standardizing production of genetic constructs is a strong feature of the work and will speed progress in the long run. However, there are conceptual issues with the slide presenting transformation methods for filamentous fungi; for example, the premise that the protoplasting approach takes longer than an Agrobacterium tumefaciens-mediated transformation (ATMT) methodology. Protoplasting is faster than ATMT, as the coculture step is omitted. In the end, for slow-growing species, both of these methods are dependent on the time it takes for the organism to form colonies. It therefore may be useful to develop both lines of strategies concurrently.
• Advances in Filamentous Fungal Engineering Through a Modular Cloning Toolkit and Agrobacterium Mediated Fungal Transformation. There were three stated goals for this project: (1) development of transformation protocols that can be applied to a diverse set of organisms; (2) development of a fungal toolkit that can be deployed in a diverse set of organisms; and (3) development of precision editing and gene expression control systems in filamentous fungal organisms.⁵² Goals 2 and 3 share significant overlap, due to the need to test the different parts (toolkit) of the transformation vector for activity in the various species. As mentioned, the inclusion of filamentous fungi as target organisms for synthetic biology is a ripe and timely research direction. The team has made significant progress on this relatively new project and is poised to make important contributions to the field, as more than fast followers. While the names of the external collaborators were not included in the presentation, it was learned that both external researchers have experience with Aspergillus nidulans. However, the extent of their involvement in the research was not made clear. Ideas on ways to expand the knowledge base to additional organismal groups are presented below.

  The rationale for the choice of test organisms was not explicitly stated. It was also not clear that the team had mined available fungal genomes and the literature for information on gene groups of interest for downstream applications. It is suggested that the downstream goal needs to drive the selection of organisms. Additionally, a few well studied and tractable organisms (e.g., A. nidulans or other Aspergillus, possibly certain Trichoderma, Fusarium, or Magnaporthe) could be included as controls for the various genetic manipulations. As a

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⁵² J. Baumbach, 2024, “Advances in Filamentous Fungal Engineering Through a Modular Cloning Toolkit and Agrobacterium Mediated Fungal Transformation,” DEVCOM ARL presentation to the committee, July 9.

• caution, it should be noted that approaches that have worked for baker’s yeast are not always transferable to filamentous fungi. ARL could also look at the Neurospora deletion collection (in this collection every gene predicted in the genome has been deleted in both mating types as well as essential genes, which are maintained as heterokaryons).⁵³ It is publicly available from the Fungal Genetics Stock Center. The knock-out collection provides an incredibly useful resource to test phenotype of conserved genes.

  ARL operates in Biosafety Level 2 (BSL-2) facilities and BSL-2 fungi can be used. Most fungi are BSL-1, but some opportunistic fungi are BSL-2, including A. fumigatus, which contains the best characterized melanin gene cluster. Through experience, A. fumigatus, has proven to be safe and simple fungus to work with, and one needs only to use a biosafety hood when transferring spores. Commentary on each project goal is included below.

  • Goal 1: Development of Transformation Protocols. While the team presented this goal as being largely achieved with the use of ATMT, some suggestions are presented for consideration. The team stated that they tried four protocols but only provided results for ATMT transformation. The hygromycin B resistance/hygromycin B phosphotransferase (hph) gene was communicated to be the marker, but the promoter was not provided. It was not clear that the team tried multiple promoters to drive expression of hph—this might impact whether any transformants are obtained or increase the number of transformants and the speed of their growth on selective medium. If the promoter is not driving good expression of hph, a method (such as protoplasting or electroporation) will appear not to work. Also, not all fungi are sensitive to hygromycin B, which may necessitate use of other antibiotic markers (such as the pleomycin/ble R gene, or many others). It is also useful to work with auxotrophic strains and complement the auxotroph via deletion of the gene of choice. The easiest auxotroph to make is a pyrG/pyr4 mutant. In addition, there are recent publications (in press) reporting greatly increased ATMT efficiencies that will greatly expedite this work as it would eliminate the need to test all of the reagents for a particular species, which is incredibly time consuming.⁵⁴
  • Goal 2: Development of a Fungal Toolkit. This objective is still under development, but with significant progress being made. The apical membrane antigen 1 (AMA1) episomal replication plasmid that is included in the ATMT vector is active in Aspergillus and Penicillium spp., and more recently in Fusarium spp., which makes use of this sequence attractive.^55,56 However, there are many fungi in which AMA1 is not active and/or with no known episomal DNA sequences, presumably including some of the species under study by the team. Unless there is a reason to believe that the AMA1 DNA will be active as an episomal origin of replication in most of the organisms, the team needs to consider eliminating it from the primary

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⁵³ H.V. Colot, G. Park, G.E. Turner, C. Ringelberg, C.M. Crew, L. Litvinkova, R.L. Weiss, J.A. Borkovich, and J.C. Dunlap, 2006, “A High-Throughput Gene Knockout Procedure for Neurospora Reveals Functions for Multiple Transcription Factors,” Proceedings of the National Academy of Sciences 103:10352–10357.

⁵⁴ M.J. Szarzanowicz, L.M. Waldburger, M. Busche, G.M. Geiselman, L.D. Kirkpatrick, A.J. Kehl, C. Tahmin, et al., 2024, “Binary Vector Copy Number Engineering Improves Agrobacterium-Mediated Transformation,” Nature Biotechnology, https://doi.org/10.1038/s41587-024-02462-2.

⁵⁵ S. Tong, K. An, W. Chen, W. Zhou, Y. Sun, Q. Wang, and D. Li, 2022, “Evasion of Cas9 Toxicity to Develop an Efficient Genome Editing System and Its Application to Increase Ethanol Yield in Fusarium venenatum TB01,” Applied Microbiology and Biotechnology 106:6583–6593.

⁵⁶ C. Hao, J. Yin, M. Sun, Q. Wang, J. Liang, Z. Bian, H. Liu, and J.-R. Xu, 2019, “The Meiosis-Specific APC Activator FgAMA1 Is Dispensable for Meiosis but Important for Ascosporogenesis in Fusarium graminearum,” Molecular Microbiology 111:1245–1262.

• • ATMT vector, as it increases the size and complexity of the plasmid and may negatively impact the overall success of the initial transformations.

    In the work presented on the slide “Characterizing Promoters in Diverse Organisms,” control of green fluorescent protein (GFP) expression was assessed by fluorescent microscopy. A total of six promoters were tested, but the species origin of each was not included. It appears that most, if not all, are from Aspergillus nidulans. This may influence whether the promoter is active in more distant species. The species used for the thiamine repressible promoter work was not indicated. Additionally, it is important to keep abreast of high-throughput technologies that enable the manipulation of parts and landing pads in the genome (promoters, terminators, etc.) to obtain a graded level of production, form, and tunable level of gene expression and protein and product/production (e.g., melanin). This will help with the assessment of optimal strains for production purposes.

    In the work presented in the poster “Metabolic Pathway Engineering for Non-fluorescent Reporters,” it appears that Hormoconis resinae has been successfully transformed with a beta-glucuronidase gene (GUS) construct that is expressed. However, no quantitation of expression was provided.

  • Goal 3: Development of Precision Editing and Gene Expression Control Systems. This goal is not yet realized, but the work is progressing. In the presented subproject poster “Toward a Fungal CRISPR Toolkit,” the constructs included the Cas9 gene and the guide RNA. The stated goal was to target melanin biosynthetic genes. Although not explicitly indicated, it was assumed that the test transformation of H. resinae was performed using ATMT. GFP was expressed under the control of the GAPDH (glyceraldehyde 3-phosphate dehydrogenase) promoter, and the plan for implementing CRISPR inference (CRISPRi) was presented. In nonfungal work, successful CRISPR-based gene integration was demonstrated in an algal species. Another slide of a subproject presented the plans to implement PRIME and Programmable Addition via Site-specific Targeting Elements (PASTE) editing in filamentous fungi. Prime editing uses a prime editor protein and a modified CRISPR guide ribonucleic acid (pegRNA) to make edits in a target sequence. PASTE combines prime editing with a pair of enzymes to integrate large DNA segments without creating double-stranded DNA breaks.

    A critical consideration with CRISPR approaches using ATMT is the number of copies of the Cas9 construct and/or the guide ribonucleic acid (RNA) that were integrated in the genome. It is not clear that the team performed Southern analysis using the transforming DNA to check for ectopic integration events that might influence the growth or physiology of the transformants. Another issue with ATMT approaches where the Cas9 construct is integrated in the genome is off-target effects due to the nuclease. This can be mitigated by performing the transformation in a genetic background deficient in non-homologous end joining DNA repair, either through mutation of a Ku gene or treatment of the recipient cells with a Ku inhibitor. An alternative to CRISPR mutation via ATMT transformation is protoplast transformation with ribonucleoproteins (RNPs) containing Cas9 and the guide RNA.⁵⁷

• Engineering of Escherichia coli BL21 for Melanin Production on Glucose Media Using CRISPR/Cas9 (Poster). This poster is relevant to Goal 3 in the presentation, “Advances in

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⁵⁷ V. Maini Rekdal, C.R.B. van der Luijt, Y. Chen, R. Kakumanu, E.E.K. Baidoo, C.J. Petzold, P. Cruz-Morales, and J.D. Keasling, 2024, “Edible Mycelium Bioengineered for Enhanced Nutritional Value and Sensory Appeal Using a Modular Synthetic Biology Toolkit,” Nature Communications 15(1):2099.

• Filamentous Fungal Engineering Through a Modular Cloning Toolkit and Agrobacterium Mediated Fungal Transformation.” The work was well-performed, mature, and used state-of-the-art approaches. The team has made great progress in engineering E. coli using CRISPR to funnel intracellular tyrosine to DOPA melanin production during growth on several media.
• B. subtilis Synbio Tools: Applications in Melanin Synthesis, Secretion Systems, and Engineered Biofilms (Poster). This poster created a Golden Gate–based toolkit for genetic engineering of B. subtilis, including 5–6 promoters. This project was also well-performed, mature, and used state-of-the-art approaches. The team used a supervised learning approach to identify the peak conditions for B. subtilis growth and the optimal sites in the genome for construct integration. A Rhizobium tyrosinase gene was introduced into B. subtilis to allow synthesis of DOPA melanin and production of bacilli coated with melanin. The team was able to conjugate the B. subtilis transformant with an undomesticated soil isolate. The team also reported testing surface properties of several B. subtilis strains lacking genes implicated in regulation of biofilm formation.

COMPETENCY PORTFOLIO OF SCIENTIFIC EXPERTISE

Biology in Military Environments Core Competency

Within the biology in military environments core competency, the strongest areas of expertise are among the extramural scientists working in molecular genetics, those working with complex microbial systems studies, and those performing exquisite analytical instrumentation for field measurements. The strongest areas of expertise within the intramural teams were in molecular biology and synthetic biology. There is a need to hire or leverage dedicated expertise in the areas of fungal biology, complex microbial community research, bioinformatics (computational applications in particular), and AI/ML, as well as fungal geneticists, and molecular biologists for identifying and characterizing novel enzymes. The computational infrastructure overall within the ARL portfolio could serve as a remarkable resource if the competency could access this expertise and capitalize on it.

Biosynthesis and Biomaterials Core Competency

The biosynthesis and biomaterials core competency has solid scientific expertise in the areas of well-established fields of biological chemistry, materials science, and computational chemistry. The work on demonstrating the role of biology in templating materials, developing novel bio-orthogonal chemistry, and deploying pipelines is connecting to well established tools that enable rapid response. To move into emerging scientific opportunities, it will be important to diversify the expertise by bringing on leaders in the fields of supramolecular chemistry and supramolecular polymers, protein design, and modern data science.

Synthetic Biology Tools Core Competency

The qualifications of the teams supporting the synthetic biology tools core competency to meet existing goals is generally adequate, however, the core competencies bioinformatic support appears to be overextended. Data science personnel will be needed to build the requisite infrastructure to support modern AI and foundational models, and additional bioinformatics support (personnel) is required to effectively use these tools. At the time of the assessment, there was only one bioinformatics expert who was trained in data analysis. While this individual was competent in those aspects (e.g., genome annotation, transcriptomics), they were increasingly being tasked with raw data processing to facilitate the exponential growth of data acquisition (e.g., genome sequencing). The required skills such as genome assembly were new to this individual, whose training was in standard tools. For some of the

environmental microbes (e.g., fungi), those tools are insufficient, and this individual was too over committed to sufficiently expand their skillset. There is a need for data “processors” that can convert raw instrument data (e.g., sequence reads) into data that can then be analyzed and mined (which is where this individual’s skillset is). Thus, the current growing needs of the competency will require more than a single generalist.

There is also a need for a general data scientist who can create a data architecture for the core competency. At current rates of data generation, the data will become underutilized because its growth will outpace human capacity to analyze it. The competency will need to utilize ML, which requires the creation of a specific framework for it to be machine readable. This will require a trained data scientist, as opposed a scientist who looks exclusively at bio data sets. While ARL mentioned that other competencies within ARL could/would handle this, a coherent plan, as opposed to organic collaborations, will be needed here. There also appears to be a need for both data scientists and data architects to help build an intramural biofoundry, which was described in the “Research Portfolio Opportunities” section above, and ARL may want to consider hiring staff in these areas to increase research productivity.

ARL (both intramural and extramural) is off to a good start with this new focus on using fungi for synthetic biology to address big picture thrusts. Fungal technology is a hot topic across the microbiology and technology world and holds incredible promise for advancing the goals of all technology-oriented fields. The Kingdom Fungi is vast and covers organisms that are equally, if not more, diverse than species in the Kingdom Animalia. Thus, as mentioned, it is critical for the core competency to align with experienced mycologists with extensive years with molecular biology and physiology of filamentous fungi of different genera. Some potential mentors are mentioned in Box 3-1.

In general, filamentous fungal experts group into model systems (Aspergillus nidulans, Neurospora crassa), medical fungi (Cryptococcus, Candida, Aspergillus fumigatus, Blastomyces, Histoplasma, Coccidiodes), industrial/food (Aspergillus niger, Aspergillus oryzae, some Penicillium, some Fusarium, N. intermedia), or plant pathology (all genera, largest variety). There are other subgroupings, but these may be the easiest to consider when starting out to develop fungal expertise. Some experts span these groupings. The team needs to determine which expertise would be most useful for collaborations and mentorship, particularly if the goal is to expand to environmental fungi or food biotechnology.

It was also previously suggested that ARL team members could attend either the biannual GRC or the Fungal Genetics Conference in order to keep abreast of current technological approaches for filamentous fungi. Synthetic biology conferences tend to focus on bacteria and yeast-like fungi, and the approaches will be different when working with filamentous organisms.

FACILITIES AND RESOURCES

Biology in Military Environments Core Competency

The research facilities at ARL’s Adelphi Laboratory Center that were toured during the review are more than adequate to carry out the proposed research and hold potential for additional research endeavors. The equipment and resources are new and on-par or better than with current technology used in university research facilities. It is understood that there are also excellent ARL facilities in Austin, Texas, but the review took place in Maryland, so those facilities were not toured for this assessment.

Biosynthesis and Biomaterials Core Competency

The facilities and resources are adequate to support current endeavors in the biosynthesis and biomaterials core competency. As in-house data science capabilities are being developed, it will be important to bring in modern high-performance computing capabilities (i.e., graphics processing unit-based clusters).

Synthetic Biology Tools Core Competency

Relevant to the synthetic biology tools core competency, it is understood that ARL is building a biofoundry for non-model chassis microbes or a (distributed) facility that combines biology, automation, AI, synthetic biology, materials science, and chemistry to rapidly prototype and test engineered biological systems. The creation of such a facility is highly encouraged. In the “Research Portfolio Opportunities” subsection of the “Synthetic Biology Tools Core Competency” section above, there is a larger discussion on how ARL can help to ensure the success of this facility through its focused efforts.

As mentioned, ARL operates in BSL-2 facilities, and BSL-2 fungi can be used, but this capability is currently underutilized. The facilities observed during a tour during the review appeared more than adequate to perform the proposed work. In particular, the close proximity of autoclaves, DNA and RNA sequencing instruments, fluorescent microscopes, etc., is ideal for rapid progress on multiple fronts.

THEMES THAT CUT ACROSS CORE COMPETENCIES

This section discusses important themes that were observed in more than one core competency within the biological and biotechnology sciences competency.

Enhanced conference attendance could greatly benefit the biological and biotechnology sciences competency. There is potential for acquisition of new extramural collaborations afforded by greater knowledge of and connections to leading-edge researchers; expansion of ARL’s intramural fungal genetics research veins by staying abreast of cutting-edge research and interfacing with fungal researchers at the leading edge, and addition of supramolecular polymers into the research portfolio by developing more of an understanding of this material’s potential from leading scientists at conferences specific to this field.

Across the core competencies, ARL needs to bolster its efforts in data science, bioinformatics, and AI/ML approaches (e.g., there is a need to integrate both large language models [LLMs] and AI-based optimization in various disciplines. LLM can help with organizing the content, and more efficient search, as well as fine tuning for specific applications). For the biology in military environments core competency, it was found that modern computational biology, bioinformatics, and AI approaches are not yet being harnessed to their potential, and incorporating these approaches is an imperative. Within the biosynthesis and biomaterials core competency, it was found that all efforts would benefit from a clear data strategy, which could be informed by connecting with leaders in the data science field. It was also noted that current projects within the core competency either did not utilize AI or they considered legacy ML approaches as an afterthought. To keep at pace with the field, it was suggested that dedicated internal staff within the biotechnology and biosciences competency and external efforts to build programming with AI in the center will benefit not only this competency but ARL more broadly. This chapter also discussed scientific areas (e.g., protein capture agents) where there are opportunities to expand beyond traditional methods and adopt modern AI-driven approaches.

The synthetic biology tools core competency also noted that expertise in bioinformatics is currently overextended in the core competency and leveraging additional expertise will be needed. In addition, a general data scientist is needed to create a data architecture that can help ARL manage future volumes of data generation, because its growth will outpace human capacity to analyze it, and a data scientist can help the core competency lean into ML by creating a specific framework for data to be machine readable. The core competency could also leverage data science experts to inform the development of its plans to build a biofoundry.

It was also found that all of the projects in the biosynthesis and biomaterials core competency would benefit from closer integration with the synthetic biology tools core competency. Closer collaborations will increase throughput and standardization of data generation to improve interoperability and robustness. For projects such as Biomanufactured Cellulose as a Versatile Feedstock for Military Applications, the integration

with the synthetic biology capability is likely to be essential for success given the inherent limitations with the natural organism evolutionary constraints.

Finally, ARL could consider whether extramural laboratories working in the areas addressed by ARL have similar meetings aimed at learning the latest advances. ARL biologists could perhaps approach these extramural laboratories to form a standing committee with monthly meetings aimed at communicating advances that are relevant to ARL’s scientific goals. Doing so may forge a path to future collaborations focused on solutions to ARL’s scientific goals with those inside and outside of this standing committee.