Manipulating Bacterial Communities by in situ Microbiome Engineering

Abstract

Microbial communities inhabit our entire planet and have a crucial role in biogeochemical processes, agriculture, biotechnology, and human health. Here, we argue that 'in situ microbiome engineering' represents a new paradigm of community-scale genetic and microbial engineering. We discuss contemporary applications of this approach to directly add, remove, or modify specific sets of functions and alter community-level properties in terrestrial, aquatic, and host-associated microbial communities. Specifically, we highlight emerging in situ genome engineering approaches as tractable techniques to manipulate microbial communities with high specificity and efficacy. Finally, we describe opportunities for technological innovation and ways to bridge existing knowledge gaps to accelerate the development of in situ approaches for microbiome manipulations.

Keywords: genome engineering; microbial communities; microbiome engineering; phage; prebiotics; probiotics; synthetic biology; xenobiotics.

Figure 1. In situ microbiome perturbations vary in magnitude, specificity and degree of rational design required

A variety of approaches, based on chemical (green), cellular (blue) and DNA (orange) methods can be applied to manipulate microbial communities in their native context. Each method can vary in its magnitude of perturbation to the native microbiome, shown increasing on the horizontal axis; its specificity of targeting to particular community members, shown increasing on the vertical axis; and its degree of required rational design, shown with increasing shading density. Particular combinations of magnitudes and specificities may be desirable for given target applications. Chemical-based approaches such as xenobiotics, prebiotics, and nutritional variation yield relatively broad spectrum changes, with varying magnitudes. Antibiotics, a class of xenobiotics, can yield larger magnitude changes with higher specificity. Cellular-based techniques such as probiotics and engineered probiotics can yield low magnitude, specific perturbations, while large-scale microbiota transplants or synthetic communities can yield to larger, but less specific changes. Finally, DNA-based methods such as phages can yield highly specific, albeit low magnitude perturbations, while engineered mobile DNA can yield perturbations over a large range of magnitudes and specificity. This flexible control of magnitude and specificity implies that engineered mobile DNA may be a desirable and tractable method for manipulating microbial communities in comparison to other methods.

Figure 2. The in situ genome engineering toolbox

The genomic content of native microbial communities can be directly engineered via in situ genome engineering. As we depict in the top panel, mobile genetic elements can be delivered and transferred to an endogenous microbiome, where they elicit desired function via a combination of regulation and actuation strategies; these elements can then maintain themselves over time to achieve long term desired function. A toolbox of existing and novel genetic tools will enable engineering of mobile genetic elements for in situ genome engineering methods. Transfer methods such as phages, plasmids and transposons can be used to deliver and circulate engineered DNA sequences to microbial communities, via processes such as transduction, transformation and conjugation. Regulatory parts, including transcription and translation parts and sensors of endogenous and exogenous chemical ligands will enable the construction of more complex genetic devices to tune host-range and endow higher order functions such as logic and memory. Actuation of genomes, including addition and removal of genes, modulation of expression, targeted mutations, and episomal modifications will allow for changes to underlying community metabolic function, or introduction of wholly new functions such as reporting on the state of an environment. These functional manipulations can further alter communities at the ecological level by altering the abundances of specific strains or introducing competitive or cooperative interactions. Maintenance of these mobile elements allows for dynamic and long-term control of engineered genetic content; with replication, integration and optimized immune evasion, vectors can be stably propagated over time. Alternatively, lysis can quickly disseminate phages across a community, or engineered circuits such as kill-switches could be used to eliminate circuits as a safe-guard.

Figure 3. Key Figure. Principles and knowledge gaps in in situ genome engineering

Key principles for in situ microbiome engineering include community ecology (blue), systems engineering (red), and quantitative modeling (orange). On the left, we illustrate the direct interplay between each of the three principles; these directional interactions and knowledge that inform one-another are denoted via color coded arrows and text. On the right, we detail specific biological and ecological and engineering and modeling knowledge gaps.

Figure 4. Applications of in situ genome engineering

In situ genome engineering approaches could be utilized to address fundamental basic science questions and key applications in medicine, health, food, farming, water and energy. Engineered vectors introduced into native environments could allow for manipulation of metagenomic content, and would propagate within the community over time. These engineered vectors could then endow these communities with desirable alterations to community-level function, stability, and dynamics, detailed in the upper right panel.