Download PDF Winter Bridge on The Grainger Foundation Frontiers of Engineering December 13, 2024 Volume 54 Issue 4 This issue features articles by The Grainger Foundation US Frontiers of Engineering 2024 symposium participants. The articles examine cutting-edge developments in microbiology and health, artificial intelligence, the gut-brain connection, and digital twins. Precision Microbiome Engineering Thursday, December 12, 2024 Author: Mark Mimee Microbiome engineering strategies are well poised to enable future efforts in causal microbiome research and to lead the translational charge once clear indications and targets are known. The human body is inhabited by trillions of microorganisms from thousands of unique species of all domains of life. During childbirth, we collect microbes from our mothers and our environment, and these bacteria, fungi, protists, archaea, and viruses colonize our skin, respiratory tract, gut, and urogenital tract. The composition of our microbiome changes throughout development, influenced by diet, lifestyle, age, and disease. While microbial-host associations have been noted since Antonie van Leeuwenhoek observed bacteria in tooth scrapings with a prototypical microscope, technological advances in next-generation sequencing catalyzed a new era in microbiome research. Short-read sequencing enabled culture-independent profiling of microbial communities (Eckburg et al. 2005). Scientists and clinicians were quick to catalog the composition of the human microbiome during homeostasis and disease (Human Microbiome Project Consortium 2012; The Integrative HMP (iHMP) Research Network Consortium 2019). These large-scale microbial’omics efforts revealed that many diseases, including inflammatory bowel disease, cancer, metabolic syndrome, autism spectrum disorder, and allergy (amongst many more), profoundly alter the diversity, composition, and activity of the gut microbiome. A rough, multidimensional, and highly variable representation of healthy and unhealthy (dysbiotic) gut microbiomes emerged from these profiling efforts. Alongside the advent of next-generation sequencing technology transforming the field of microbial ecology, crude, early successes in manipulating dysbiotic microbiomes to combat recurrent Clostridioides difficile infections using fecal microbiota transplants (Kassam et al. 2013) garnered more attention for the translational potential of microbiome research. In recent years, the focus of microbiome research has shifted from observational studies to mechanistic ones that can attribute causality to microbiome-disease associations. Phrased more succinctly, does the microbiome affect a given disease? If yes, then gut microbiome-targeted interventions could potentially represent a new therapeutic modality. However, we are currently limited in our ability to make predictable, targeted perturbations to complex microbiomes. These techniques are necessary to not only perform the mechanistic gain- and loss-of-function studies that can attribute causality between microbes and disease outcomes, but also for the translation of microbiome-targeted therapies. A microbiome engineering community has emerged to fill this technology gap and enable the precise manipulation of the world’s microbiome, not just those associated with humans, but also other animals, plants, soil, and marine systems, for applications in human health, agriculture, and sustainability. Microbiome Engineering Microbiome engineering strategies can be described as additive, subtractive, and modulatory (Arnold et al. 2023; Mimee at al. 2016). Additive approaches, such as probiotics, live biotherapeutic products (LBPs), microbial consortia, engineered LBPs, and fecal microbiota, add new strains or functions to microbial communities. Subtractive strategies, such as broad- to narrow-spectrum antimicrobials, remove deleterious strains or functions from the microbiome. Finally, modulatory approaches, such as diet or prebiotics, enrich or deplete the abundance or activity of specific microbes within the community. These approaches span a spectrum of precision, with many of the current commercial modalities (probiotics, fecal microbiota transplants, broad-spectrum antibiotics) crudely altering the gut microbiome, whereas many technologies in development (designer consortia, natural or engineered LBPs, and narrow-spectrum antibiotics) prioritize precise, reproducible changes. Additive Approaches: Natural and Engineered Live Biotherapeutics Live biotherapeutics (LPBs) are next-generation probiotics whose clinical use is supported by mechanistic research, often linking one or several specific microbial products to therapeutic efficacy. However, even a single strain of bacteria codes for thousands of genes, most of unknown function. Administration of the strain therefore not only adds the desired functions, but many others that may positively or negatively affect the host. An approach to increasing specificity is to leverage synthetic biology strategies to add defined, novel functions to a strain of interest. Design criteria for engineered live biotherapeutics include: Choice of Microbial Chassis: The source, cultivability, genetic tractability, anatomical distribution, and intrinsic functions are key factors considered during the choice of the appropriate microbial strain or chassis. While classic Escherichia coli or lactic acid bacteria probiotics were the initial microbial chassis of choice, advances in genetic engineering have enabled probiotic yeast (Scott et al. 2021) and resident gut bacteria (Dong et al. 2022; Jin et al. 2022; Mimee et al. 2015; Russell et al. 2022) as potential strains. Additionally, some have proposed strategies for in situ microbiome engineering wherein horizontal gene transfer via conjugation (Ronda et al. 2019; Rubin et al. 2022) or bacteriophage (Hsu et al. 2020) is leveraged to directly deliver payloads to resident microbes. Availability of Genetic Parts: For each microbial chassis, a genetic toolkit must be available to reliably and reproducibly engineer the organism. These parts include regulatory elements (promoters, ribosome binding sites, operators, transcriptional regulators) to precisely control gene expression as well as genome engineering tools (plasmids, integrases, CRISPR-Cas systems) to stably maintain genetic payloads within the cell. Engineered microbes can additionally be outfitted with sensor systems to program conditional gene expression based on environmental conditions and generate a closed-loop system. For example, both E. coli (Zou et al. 2022) and probiotic yeast (Scott et al. 2021) have been engineered to secrete anti-inflammatory proteins in response to gut inflammation. Payloads to Engineer Host Physiology: Genetic payloads are the key functions programmed into the engineered microbe. These engineered functions act in concert with the intrinsic properties of the microbial chassis. Examples include: a) Metabolic transformations: Microbes can be engineered to convert dietary components into therapeutic agents, to degrade potential toxic compounds (Isabella et al. 2018), or to synthesize metabolites that affect host physiology (Russell et al. 2022); b) Protein delivery: Therapeutic proteins can be secreted from engineered microbes for local activity (Lynch et al. 2023; Steidler et al. 2003; Zou et al. 2022); and c) Colonization resistance: Microbes can be programmed to secrete metabolites or proteins to combat potential pathogens. Engraftment: The stable engraftment of the engineered microbe into the microbiome is a key design consideration. For some applications, if the payload can be delivered in transit, the engineered microbe can be explicitly designed to pass through the host without engraftment. For other applications, a strain is engineered to act as a sentinel that engrafts stably in the community, senses perturbations in homeostasis, and expresses payloads in response. Controlled engraftment is a non-trivial challenge as most microbiomes display colonization resistance that excludes new microbial members from entering the community, either through metabolic competition (Fischbach 2011) or intermicrobial antagonism (García-Bayona 2018). However, controlled engraftment can be achieved by equipping microbes with gene clusters to metabolize rare and orthogonal substrates that establish a new metabolic niche for colonization (Shepherd et al. 2018). Biocontainment: As therapeutic use of genetically modified organisms can lead to their escape into the environment or unintended colonization of new hosts, several strategies have been implicated to control their growth. Auxotrophy, or the engineered dependence on specific metabolic substrates, is a main approach for biocontainment to ensure the engineered organisms can only grow in intended environments with the necessary nutrients (Steidler et al. 2003). Additionally, strains have been outfitted with genetic kill-switches to trigger self-destruction with specific chemical cues (Chan et al. 2016; Rottinghaus et al. 2022; Stirling et al. 2017). Other strategies focus on recoding (Rovner et al. 2015) or eliminating genetic material (Hayashi et al. 2024) to prevent its environmental release. A microbiome engineering community has emerged to fill this technology gap and enable the precise manipulation of the world’s microbiome. Subtractive Approaches: Narrow-Spectrum Antimicrobials While the microbiome contains many beneficial microbes, it can also contain those that cause or exacerbate disease. Removal of these bad actors is a key pillar of microbiome engineering. However, current antibiotics used to treat bacterial infections are broad-spectrum in nature and indiscriminately destroy symbionts in the microbiome alongside the offending pathogen. These off-target effects can contribute to the increased incidence in antibiotic resistance in bacterial populations and lead to difficult-to-treat antibiotic-recalcitrant infections, such as Clostrioides difficile, vancomycin-resistant Enterococci, and carbapenem-resistant Enterobacteriaceae. Narrow-spectrum antimicrobials that spare the microbiome are in development to surgically remove deleterious strains, without harming innocuous or beneficial symbionts. Efforts in programmable-spectrum antimicrobial peptides derived from natural or synthetic sources may offer more specific means to edit microbial communities (King et al. 2023; Ma et al. 2022; Torres et al. 2024). Bacteriophages (phages), viruses that infect and kill bacteria, have seen a revival as an alternative to traditional antibiotics. Despite their discovery over a century ago and continued use to treat bacterial infections, clinical application of phage waned in the Western world, partly due to the ease-of-use of small-molecule antibiotics and the narrow spectrum of phage. However, recent years have seen a resurgence in applications of phage to treat antibiotic-resistant chronic infections (Green et al. 2023; Onallah et al. 2023; Pirnay et al. 2024) and to specifically edit microbiomes (Frederici et al. 2022; Rotman et al. 2024). Additionally, similar to cellular chassis, bacteriophage can be engineered to express payloads to improve their efficacy, including the expression of sequence-specific endonucleases (Bikard et al. 2014; Citorik et al. 2014; Gencay et al. 2014), biofilm-degradation enzymes (Lu and Collins 2007), or altered host range (Ando et al. 2015; Huss et al. 2021; Yehl et al. 2019). Despite their promise, there are many unknowns about the behavior of bacteriophage during the course of therapy. Specifically, phenotypic (Lourenço et al. 2022) and spatial (Lourenço 2020) heterogeneity, differential mucus binding (Green et al. 2020; Wu et al. 2024), and interactions with the host immune system (Berkson et al. 2024; Gogokhia 2019; Roach et al. 2017) can modulate the success or failure of phage therapy in vivo. Conclusions Despite its early clinical use and rapid attempts at translation, most microbiome engineering efforts are still in their infancy, with many precision microbiome therapeutics in preclinical or early clinical stages. Basic, mechanistic research is pivotal to understand the design and operating rules of the microbiome and, ultimately, what parameters should be tuned to affect host physiology. Microbiome engineering strategies are well-poised to enable future efforts in causal microbiome research and to lead the translational charge once clear indications and targets are known. References Ando H, Lemire S, Pires DP, Lu TK. 2015. Engineering Modular Viral Scaffolds for Targeted Bacterial Population Editing. Cell Syst. 1:187–96. Arnold J, Glazier J, Mimee M. 2023. Genetic engineering of resident bacteria in the gut microbiome. J. Bacteriol. 205:e00127–23. Berkson JD, Wate CE, Allen GB, Schubert AM, Dunbar KE, Coryell MP, Sava RL, Gao Y, Hastie JL, Smith EM, and 3 others. 2024. Phage-specific immunity impairs efficacy of bacteriophage targeting Vancomycin Resistant Enterococcus in a murine model. Nat. Commun. 15:2993. Bikard D, Euler CW, Jiang W, Nussenzweig PM, Goldberg GW, Duportet X, Fischetti VA, Marraffini LA. 2014. Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials. Nat Biotechnol 32:1146–51. Chan CTY, Lee JW, Cameron DE, Bashor CJ, Collins JJ. 2016. “Deadman” and “Passcode” microbial kill switches for bacterial containment. Nat. Chem. Biol. 12:82–86. Citorik RJ, Mimee M, Lu TK. 2014. Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases. Nat Biotechnol 32:1141–45. Dong X, Guthrie BGH, Alexander M, Noecker C, Ramirez L, Glasser NR, Turnbaugh PJ, Balskus EP. 2022. Genetic manipulation of the human gut bacterium Eggerthella lenta reveals a widespread family of transcriptional regulators. Nat. Commun. 13:7624. About the Author:Mark Mimee is assistant professor in the Department of Microbiology and the Pritzker School of Molecular Engineering at the University of Chicago.