Bottleneck/Challenge: Lack of knowledge about the function and expression timing of many genes, as well as their impact on pathway/organism function, which may impact guild membership.

Potential Solution: Incorporate activity-based chemical probes coupled with proteomics to discover functions in complex communities.

Bottleneck/Challenge: Predicting the downstream perturbations (e.g., metabolic burden, community interactions) following the introduction of a new capability (e.g., heterologous metabolic pathway) into an organism.

Potential Solution: Machine learning approach using a metabolic flux model and trained using empirical data from perturbations.

Bottleneck/Challenge: Genome sequences are available, but we lack much of the necessary information (particularly for accessory genomes) needed to generate functional predictions (e.g., cell morphology, metabolism, proteomics).

Potential Solution: Design technologies that enable testing microbiomes at scale, so protein biochemistry, cellular phenotypes, and gene functions can be examined to increase the amount of data available for annotation prediction programs.

Bottleneck/Challenge: Assays needed to take measurements are not well defined, or existing assays are poorly scaled to take appropriate measurements.

Potential Solution: Identify minimal measurement requirements using inputs from knowledge base of measured trait/taxon associations.

Bottleneck/Challenge: Significant advances (e.g., microscopy, sample preparation, image analysis) will be needed to enable higher-resolution imaging in difficult-to-access environments (e.g., gut microbiome, soil microbiomes), so it can be used to deconvolute complex biological structures such as biofilms.

Potential Solution: Libraries of specifically labeled probes that allow tracking of many diverse metabolisms and activities simultaneously so functionally similar species can be defined at multiple levels of abstraction. The minimal set of these is partly defined by the 2030 milestone. Develop micron-scale magnetic and radioactive imaging in situ.

Bottleneck/Challenge: Relatively few tools can specifically target individual species (e.g., antibiotics target kingdoms of organisms).

Potential Solution: Use a natural or specifically engineered mobile system (e.g., phages, mobile plasmids) to deliver cytotoxic payloads (e.g., phage tail-like bacteriocins, antimicrobial peptides).

Bottleneck/Challenge: Many existing technologies cannot fully penetrate a microbiome, so wild-type cells will grow to repopulate the community.

Potential Solution: Expand the use of bacterial gene drive technology¹²Valderrama, J. A., Kulkarni, S. S., Nizet, V., & Bier, E. (2019). A bacterial gene-drive system efficiently edits and inactivates a high copy number antibiotic resistance locus. Nature Communications, 10(1), 5726. https://doi.org/10.1038/s41467-019-13649-6 or broad host range conjugative plasmids¹³Leonard, S. P., Perutka, J., Powell, J. E., Geng, P., Richhart, D. D., Byrom, M., Kar, S., Davies, B. W., Ellington, A. D., Moran, N. A., & Barrick, J. E. (2018). Genetic Engineering of Bee Gut Microbiome Bacteria with a Toolkit for Modular Assembly of Broad-Host-Range Plasmids. ACS Synthetic Biology, 7(5), 1279–1290. https://doi.org/10.1021/acssynbio.7b00399^,14Sheth, R. U., Cabral, V., Chen, S. P., & Wang, H. H. (2016). Manipulating Bacterial Communities by in situ Microbiome Engineering. Trends in Genetics, 32(4), 189–200. https://doi.org/10.1016/j.tig.2016.01.005 to enable more consistent genetic manipulation in complex environments.

Bottleneck/Challenge: Functional guilds may contain species from multiple kingdoms, and multiple techniques may be needed to manipulate a guild.

Potential Solution: Engineer systems that allow for rapid manipulation and testing cycles, so different methods can be applied in succession to ensure that a function is eliminated from a microbiome.

Bottleneck/Challenge: Functional genomics are relatively new, so it is challenging to target functionally related species, especially across kingdoms, without off-target effects.

Potential Solution: Use high-throughput microbiome model systems and genetic or small molecule inhibitor screens to improve databases of gene functions in complex environments.

Bottleneck/Challenge: Most ecological communities are robust to intrusion, and new organisms will need to outcompete or displace other species in the microbiome.

Potential Solution: Identify mild environmental (e.g., pH change, heat shock), chemical (e.g., low concentration antibiotics, nutrient stress), or biological (e.g., phages, bacteria secreting antimicrobial peptides) perturbations that temporarily disrupt homeostasis and allow another organism to invade.

Bottleneck/Challenge: Existing organism functions will likely have more mechanisms to resist change, especially for mechanisms that contribute to growth or replication.

Potential Solution: Engineer microbiomes to use multiple mechanisms to achieve its function (e.g., stimulate increased glucose uptake while driving higher concentrations of biofuel production).

Bottleneck/Challenge: Requires efficient in situ editing of a community, excellent metabolic knowledge of metabolic capabilities, timing of editing vs. augmenting with target microbes.

Potential Solution: Engineer microbiomes that facilitate succession by using one engineered microbe to disrupt the natural microbiome, creating a niche for the new functional guild.

Bottleneck/Challenge: Many factors limit our ability to identify what guilds exist in a natural community (e.g., unculturable organisms, species may have overlapping functions, very limited models, limits methods to manipulate organisms physically/genetically).

Potential Solution: Improve techniques for manipulating organisms in situ (see Spatiotemporal Control) and ensure that techniques function across kingdoms of life.

Potential Solution: Introduce metabolic pathways for alternative carbon source utilization, to selectively enrich species organisms that can use specific nutrients.¹⁵Shepherd, E. S., DeLoache, W. C., Pruss, K. M., Whitaker, W. R., & Sonnenburg, J. L. (2018). An exclusive metabolic niche enables strain engraftment in the gut microbiota. Nature, 557(7705), 434–438. https://doi.org/10.1038/s41586-018-0092-4

Bottleneck/Challenge: Current DNA delivery capabilities are limited to existing conjugative plasmids or specific “phagemid” systems with very defined and narrow host ranges. The efficacy of delivery is also dependent on many poorly defined factors (e.g., environmental conditions, DNA methylation).

Potential Solution: Isolate natural or engineered tail fiber proteins that interface with existing phagemid systems, harness new phagemid systems, and develop naturally-occurring or engineered conjugative systems to expand DNA delivery tools.

Bottleneck/Challenge: Several environments have microbiomes of interest, but tools for delivering DNA are significantly underdeveloped (e.g., Clostridiales spp. in the human gut).

Potential Solution: Create generalized genome editing tools (e.g., vectors, genetic markers, gene promoters) and orthologous systems that are specifically designed to function under particular environmental conditions (e.g., microaerophilic or anaerobic environments).

Bottleneck/Challenge: Existing delivery vehicles only deliver DNA to microbes, while the ability to introduce other biomolecules could introduce other capabilities and circumvent some host defense systems.

Potential Solution: Further develop existing phages known to deliver RNA or proteins. Combine with prior advances to expand or tailor the host range.

Potential Solution: Use nanotechnology and extracellular vesicles to target cells specifically.

Potential Solution: Combine cell-penetrating peptides with peptide nucleic acids or other smaller molecules to deliver DNA.

Bottleneck/Challenge: Standard approaches require culturing microbes under laboratory conditions, often to high densities.

Potential Solution: Engineer a broad range of donor strains and conjugative proteins that can efficiently deliver genetic cargo to different hosts, even at low cell densities.

Bottleneck/Challenge: Existing DNA delivery technologies are uncontrollable once the delivery vehicle is added to an environment, but the ability to influence timing, location, and actuation of delivery would significantly improve manipulation.

Potential Solution: Use optogenetics or spatially heterogeneous chemical components to direct DNA to specific locations.

Potential Solution: Engineer specific delivery mechanisms, such as conditionally conjugative plasmids or DNA attached to tail fiber proteins, to control which organisms take up the DNA.

Bottleneck/Challenge: Combining components to evaluate how they potentially cross-talk.

Potential Solution: Develop computational methods for considering interactions between delivery vehicles and delivered components.

Bottleneck/Challenge: Models that can capture changes from the level of nucleic acids up to whole cells will require significant computational and mathematical advances.

Potential Solution: Develop conceptual and mathematical frameworks that specifically aim to determine how genetic changes impact cell physiology, with minimal computational processing.