Processing

Bottleneck/Challenge: Secretion systems in model organisms are limited in yield and sequence requirements.

Potential Solution: Develop better tools for protein secretion and for polymer formation in non-model organisms (e.g., Bacillus subtilis).

Bottleneck/Challenge: Limited number of chassis used for non-standard amino acid incorporation.

Potential Solution: Expand orthogonal translational systems to other organisms such as Bacillus subtilis, an efficient protein secretor.

Bottleneck/Challenge: Limited secretion products have resulted in under-exploration of in situ separation methods to improve production.

Potential Solution: Test techniques such as organic overlay, used for in situ small molecule extraction.

Bottleneck/Challenge: Few conjugation, functionalization, and/or assembly strategies well-suited to occur in or around cells.

Potential Solution: Identify or design new coupling chemistries that function in fermentative environments.

Bottleneck/Challenge: Incomplete understanding of, and design tools, for complex regulation that controls spatiotemporal patterning in biological systems.

Potential Solution: Identify a series of model materials with design features that require spatiotemporal patterning and compartmentalization, to quantify progress.

Bottleneck/Challenge: Lack of cooperativity in growth and material production and patterning in organisms that do not come by it naturally; inconsistent growth medium requirements and replication rates that confound such coordinated consortia.

Potential Solution: Engineer mutualistic dependencies between disparate organisms that link growth and development, providing the framework on which to build spatiotemporal coordination biomaterial synthesis and deposition.

Potential Solution: Template inorganic structures with unique stoichiometry and potential to access novel ceramics or catalysts.

Bottleneck/Challenge: Design rules of self-assembly are not fully elucidated.

Potential Solution: Development of experimental and computational frameworks to support materials discovery for biomolecule-based and/or embedded materials (e.g., machine learning, computational biology, and protein-folding algorithms).

Bottleneck/Challenge: Fundamental gaps in knowledge about how specific protein sequences and tissue inhibitors affect cellular properties and cell-matrix interactions.

Potential Solution: Controlled engineering of three-dimensional networks that resemble the ECM.

Potential Solution: Standardized characterization of cellular properties in natural and engineered networks with site-specific cellular/protein interactions.

Bottleneck/Challenge: Gaps in knowledge remain about how specific physical properties of ECM (stiffness, porosity, viscoelasticity) affect cell functions from different cell types.

Potential Solution: Enable more thorough characterization of ECM physical properties.

Bottleneck/Challenge: Limited understanding of the complex multiscale interactions that drive self-assembly.

Potential Solution: Molecular-level investigations of the self-assembly of stimuli-responsive biopolymers or amphiphiles with conformational adaptability.

Bottleneck/Challenge: Understanding of the interactions of biocomponents with the substrate, including covalent, ionic, and van der Waals interactions.

Potential Solution: Characterize the bonding in categories of the type of interaction, with the aim that similar biocomponents will bond similarly to the same substrate.

Bottleneck/Challenge: Tunable protein-based stimulus-responsive nanostructures.

Potential Solution: Characterize and engineer existing proteins that are responsive to light, pH, mechanical stimuli, etc.

Bottleneck/Challenge: Lack adhesive biological and/or synthetic moieties that allow for specific interactions.

Potential Solution: Engineer a series of orthogonal membrane proteins on living cells to create autonomous patterns on surfaces.

Bottleneck/Challenge: Limited characterization methods for patterned/deposited surfaces.

Potential Solution: Establish and expand dimension, thickness, periodicity, and spectroscopic methods.

Bottleneck/Challenge: Understanding of amino acid and traditional chemical reactivity to build controlled structures.

Potential Solution: Full characterization of the suites of materials produced to date and analyze with artificial intelligence and machine learning techniques.

Bottleneck/Challenge: Biological materials do not withstand the processing conditions that semiconductors do.

Potential Solution: Stepwise generation of hybrid systems to account for the fragility of biocomponents.

Potential Solution: Depending on application/desired outcome, inorganic semiconductor may not be needed, and a more biocompatible one, such as an organic semiconductor, may be used with processing conditions closer to that of biomaterials.

Bottleneck/Challenge: Molecular platforms to transmit electrical signals between cells and electronic materials are not universal and maybe difficult to integrate with signal transduction pathways.

Potential Solution: Develop interchangeable tools to allow for integration of common electrical signals (e.g., redox, ion conduction).

Bottleneck/Challenge: The utilization of what we already know in templating materials on biological systems to produce the material size and shapes desired.

Potential Solution: Utilize techniques such as DNA origami or very selective chemistry to combine biological materials into non-natural shapes and sizes (e.g., combining linear phage into the vertices of a pyramid).

Potential Solution: Manipulate an organism to autonomously form a desired size or shape.

Potential Solution: Use bilayers, micelles, and vesicles to constrain shape.

Potential Solution: Use a patterned and sacrificial substrate to constrain systems to certain shapes.

Potential Solution: Use asymmetric polymeric materials (e.g., tapered bottlebrushes similar to scaffolding proteins) as molecular units for templating and scaffolding.

Bottleneck/Challenge: Multiple biological processes need to work in tandem, molecular precursors need to be present and accessible, and the kinetics of many processes need to be improved.

Potential Solution: Improve engineered communication between singular intracellular functions.

Potential Solution: Perform processes on an intelligent substrate, such as one that can monitor activity of components or precursors and update components’ information to each other.

Bottleneck/Challenge: Controlled interactions between individual materials would be required in order to stack materials in a desired array without outside stimuli.

Potential Solution: Innovation in additive manufacturing (e.g., in situ printing of a defined layer of materials directly onto a living organism).

Bottleneck/Challenge: Maintaining the structure and function of cells in the template.

Potential Solution: ‘Ink-jet printing’ of bacterial inoculum using acoustic liquid-handling.

Potential Solution: ‘Photo-masking’ enabled selection of spatially-resolved surviving organisms from a mixed population using light.

Potential Solution: Imprint lithography to generate a scaffold with spatially differentiated layers or regions through the step of coating these regions with an appropriate chemical.

Bottleneck/Challenge: Cellular behavior in printing conditions (in shear field, solidification, or polymerization) remains to be explored.

Potential Solution: Develop an analysis flow for systematic studies on the relevant variables (e.g., printing condition and viability, scaffold molecular composition and viability, viability and motility relationships with metabolic activity).

Potential Solution: Develop methodology to assess cellular viability during or shortly following printing.

Bottleneck/Challenge: Engineering microstructures within the printed biological composite materials.

Potential Solution: Enable block-copolymer, shear-induced pre-alignment.

Potential Solution: Introduce acoustic, magnetic, or electric fields into synthesis protocols.

Bottleneck/Challenge: ’Externally’ defined 3D patterns (i.e., those determined by printing) are not currently reinforced by intracellular genetic circuits that preserve patterning in different environments.

Potential Solution: Introduce asymmetric control of cell growth and reproduction, such that one class of microbes constrains physiology of other community members in a consortium.

Bottleneck/Challenge: Engineering persistence of designed and structured 3D cellular communities over time.

Potential Solution: Control individual strains in a mixed-culture pattern with feedback circuits from environmental sensors to preserve deposited patterns in diverse environmental conditions.

Bottleneck/Challenge: Need to enable a wider variety of cell types that can be printed.

Potential Solution: Further characterization of multi-species and multicellular communities and structures.

Bottleneck/Challenge: Many of these enzymes/couplings function only in conditions incompatible with protein and cell stability.

Potential Solution: Investigate associated enzyme family members under a broader range of conditions.¹Jia Liu, J., Kim, Y.S., Richardson, C.E., Tom, A., Ramakrishnan, C., Birey, F., Katsumata, T., Chen, S., Wang, C., Wang, X., Joubert, L., Jiang, Y,, Wang, H., Fenno, L.E., Tok, J.B.H., Pașca, S.P., Shen, K., Bao, Z., & Deisseroth, K. (2020). Genetically targeted chemical assembly of functional materials in living cells, tissues, and animals. Science, 367(6484), 1372-1376. View Publication

Bottleneck/Challenge: Expanding types and host chassis for creating minimal cell systems is required.

Potential Solution: Explore types and host chassis for creating minimal cell systems and test their resilience, robustness, and performance in/with materials.

Bottleneck/Challenge: Engineering of non canonical organisms for isolation of novel molecules.

Potential Solution: Determine synthesis chassis for extremophile organisms that allows production of unique molecules.

Bottleneck/Challenge: Building of models with experimental data from a wide-range of organisms.

Potential Solution: Import of useful functionalities from less conventional organisms into other chassis.

Bottleneck/Challenge: Biological structure-function-property relationships are driven by solvophobic/hydrogen bonding interactions; very difficult to translate to other solvent systems.

Potential Solution: Explore polar, non-volatile solvent systems for ability to maintain enzymatic function, structure in non-aqueous environments; initial substrates may include dimethyl sulfoxide, ethylene glycol, and low-molecular-weight polyethylene glycols.

Potential Solution: Develop predictive capability for folding correlated to solvent dielectric, protein analogs.

Bottleneck/Challenge: Standardization of cell lysates collection and optimization of reaction conditions.

Potential Solution: Systems biology approaches (e.g., proteomics, metabolomics, transcriptomics) to analyze cell-free expression systems to assess and identify limiting factors.

Bottleneck/Challenge: Large-scale culture may require industry scale equipment not readily available in academic labs.

Potential Solution: Develop continuous bioprocessing strategies for routine, large-scale production of cell-free systems.

Bottleneck/Challenge: Post-translational modifications are difficult to precisely control.

Potential Solution: Engineer systems in which cell-free synthesized proteins go through a series of desired modifications by passing through microfluidic channels with enzymes at specific locations to modify the proteins passing through, akin to a conveyer belt in a manufacturing process.

Potential Solution: Develop batch processes with multiple distinct enzymes together that enables synthesis of homogeneous glycoproteins.

Bottleneck/Challenge: Compartmentalization is typically lacking in cell-free systems.

Potential Solution: Engineer encapsulation into cell-free systems or minimal cells to permit material separation from enzymes or cofactors for optimized utilization.

Bottleneck/Challenge: Humidity and reaction stability limit use.

Potential Solution: Develop abiotic materials that can maintain biological conditions.

Bottleneck/Challenge: High costs limit adoption.

Potential Solution: Develop strategies based on energy substrates and strain engineering to reduce costs an order of magnitude from present day.

Bottleneck/Challenge: Reaction stability constrains utility in resource limited settings.

Potential Solution: Develop materials that stabilize cell-free systems for extended periods of time while freeze-dried (>1 year).

Bottleneck/Challenge: Most bioprocesses still use reduced-carbon compounds like glucose.

Potential Solution: Capture carbon from CO₂ fixation pathways.

Potential Solution: Combine electrolyzers with cell-free systems to fuel cost-effective, manufacturing strategies at large-scale.

Bottleneck/Challenge: Co-factor regeneration and stability is a concern.

Potential Solution: Develop new stable cofactors.

Bottleneck/Challenge: Enzyme stability is limiting.

Potential Solution: Develop continuous bioprocesses to replenish catalysts.

Potential Solution: Develop materials to stabilize catalysts for one month continuous operation.

Bottleneck/Challenge: Studies of enzymatic degradation of polymeric scaffolds exist, yet their performance and substrate scope remains to be improved and engineered for various contexts; current approaches involve trial and error and oftentimes substrates are not selective but instead responsive to a variety of enzymes.

Potential Solution: Develop predictive models that help develop structure-property relationships in enzymatically degradable systems that allow for development of materials with increased selectivity.

Bottleneck/Challenge: Complete degradation of polymers (e.g., ester and carbonyl bonds) may require multi-enzyme systems or chimeric enzymes.

Potential Solution: Enable engineering of expanded classes of enzymes, including incorporation of non-standard and/or non-natural amino acids.

Bottleneck/Challenge: Spore-forming microbes and associated tools have been underdeveloped for synthetic biology applications.

Potential Solution: Engineer spore-display and genetic circuit tools for responsive functionalization of spores to abiotic interfaces or within abiotic materials.

Bottleneck/Challenge: Click chemistries used for bioconjugation through the use of non-standard amino acids are infrequent/nonexistent in nature, enzymes that can specifically degrade/reverse a click reaction have not been identified or developed.

Potential Solution: Scour sequence databases for DNA sequences that could encode enzymes with needed putative activities.

Potential Solution: Pursue a protein engineering effort to generate an enzyme capable of degrading or reversing a click reaction.

Bottleneck/Challenge: Requires development of feedback loops for new materials.

Potential Solution: Studies to explore dynamic equilibria exhibited by actin filaments, application to motility and force generation; develop principles for governing dynamics, required kinetics, expand from baseline understanding.

Bottleneck/Challenge: Control of sense-response activity for extracellular matrices.

Potential Solution: Identification of signaling nodes that receive input from force sensing (i.e., via a mechanosensitive channel) and ECM binding that can be coupled to secretion of matrix metalloproteinases.

Bottleneck/Challenge: Degradative enzymes are often intolerant of solvents required to solubilize synthetic polymers.

Potential Solution: Design strategies to functionalize enzyme surfaces for increased solvent tolerance (e.g., via non-standard amino acids such as fluoro-phenylalanine, post-translational modifications, or synthetic modification).²Deepankumar, K., Shon, M., Nadarajan, S.P., Shin, G., Mathew, S., Ayyadurai, N., Kim, B., Choi, S., Lee, S., & Yun, H. (2014). Enhancing thermostability and organic solvent tolerance of ω‐transaminase through global incorporation of fluorotyrosine. Advanced Synthesis & Catalysis, 356(5), 993-998. View Publication

Potential Solution: Enable a co-polymer approach to enzyme stabilization.³Panganiban, B., Qiao, B., Jiang, T., DelRe, C., Obadia, M.M., Nguyen, T.D., Smith, A.A.A., Hall, A., Sit, I., Crosby, M.G., Dennis, P.B., Drockenmuller, E., Olvera de la Cruz, M., & Xu, Ting. (2018). Random heteropolymers preserve protein function in foreign environments. Science, 359(6381), 1239-1243. View Publication

Bottleneck/Challenge: Efficient product isolation and purification requires biocatalysts compatible with advanced materials and systems engineering strategies spanning multiple scales.

Potential Solution: Bioreactors with cells suspended in extruded pluronic hydrogels with tunable rates of substrate and product diffusion.

Potential Solution: 3D-printed biocompatible nanostructures encapsulating engineered cell- or cell-free biocatalysts.

Bottleneck/Challenge: Currently there is not a large economic incentive for industry to iterate at this scale.

Potential Solution: Government and academic investments to produce such facilities.

Bottleneck/Challenge: Characterization of performance metrics for materials within context of application.

Potential Solution: Spectroscopy methods that enable high-throughput analysis of materials characterization.

Bottleneck/Challenge: Currently there are very few facilities in the US that provide fermentation and downstream processing capabilities.

Potential Solution: Regional facilities that enable the utilization of various feedstocks and increase accessibility.

Bottleneck/Challenge: Pilot scale facilities need to be self sustaining.

Potential Solution: A robust number of academic, industrial, and government led projects continuing for the next decade will allow for such facilities to gain traction.

Bottleneck/Challenge: Coordinated technologies and platforms for bioprocessing.

Potential Solution: Optimization and molecular design of hydrogel materials, bioreactor platforms and integration of separations.

Potential Solution: Incorporate process monitoring and control schemes.

Bottleneck/Challenge: Insufficient demand for such infrastructure due to the way supply chains are handled and the fact that the bioeconomy is still a small section of the overall industrial base.

Potential Solution: Development of less expensive and more modular equipment will increase accessibility of fermentation and downstream processing equipment to be stood up at the point-of-need.

Bottleneck/Challenge: The investment to stand up a biomanufacturing facility with downstream and feedstock processing is timely and costly.

Potential Solution: Development of chemical engineering processes that are more flexible and modular for downstream processing.

Potential Solution: Development of fermentation equipment that is disposable or recyclable.

Potential Solution: High titer yields would require a much smaller scale of production.