Available online at www.sciencedirect.com ScienceDirect Principles for designing synthetic microbial communities 1,2 1,2 1 Nathan I Johns , Tomasz Blazejewski , Antonio LC Gomes 1,3 and Harris H Wang Advances in synthetic biology to build microbes with defined applications that involve complex substrates may require and controllable properties are enabling new approaches to the use of multiple pathways and processes, which may be design and program multispecies communities. This emerging difficult or impossible to execute efficiently using single field of synthetic ecology will be important for many areas of strains. These and other complex applications may be biotechnology, bioenergy and bioremediation. This endeavor best tackled by cohorts of different microbes, each pro- draws upon knowledge from synthetic biology, systems grammed with specialized sub-functions that synergize biology, microbial ecology and evolution. Fully realizing the towards an overall population-level function. This fact is potential of this discipline requires the development of new evident in natural systems where single species do not strategies to control the intercellular interactions, occupy all niches in an environment, but rather multiple spatiotemporal coordination, robustness, stability and species coexist and perform complementary roles, creat- biocontainment of synthetic microbial communities. Here, we ing intricate ecological networks [4]. review recent experimental, analytical and computational advances to study and build multi-species microbial With a greater understanding of natural microbial inter- communities with defined functions and behavior for various actions, dynamics, and ecology, we are poised to expand applications. We also highlight outstanding challenges and microbial engineering to mixed consortia in order to future directions to advance this field. perform more complex and challenging functions in both Addresses closed and defined bioreactors as well as open and natural 1 Department of Systems Biology, Columbia University Medical Center, environments. This emerging field of synthetic ecology New York, USA 2 builds upon gene circuit design strategies [5] and further Integrated Program in Cellular, Molecular and Biomedical Studies, integrates ecological and evolutionary principles [6]. Columbia University Medical Center, New York, USA 3 Department of Pathology and Cell Biology, Columbia University These population-scale considerations involve microbial Medical Center, New York, USA interactions with complex dynamics and stability proper- ties manifesting over different time and length scales. Corresponding author: Wang, Harris H ([email protected]) In this perspective, we explore how these properties can be applied to design and construct microbial commu- Current Opinion in Microbiology 2016, 31:146–153 nities relevant to emerging biotechnology applications (Figure 1). We specifically discuss four key considerations This review comes from a themed issue on Environmental microbiology for building synthetic microbial communities: engineer- ing various interspecies and intraspecies interactions, Edited by Monica Vasquez and Steven Hallam constructing spatiotemporal dynamics, modeling and maintaining community-wide functional robustness, and developing population control and biocontainment http://dx.doi.org/10.1016/j.mib.2016.03.010 measures. We discuss recent examples of experimental 1369-5274/# 2016 Elsevier Ltd. All rights reserved. and quantitative modeling advances that have enhanced our foundational capabilities to understand, develop and exploit synthetic microbial consortia in different settings. Engineering intercellular interactions Introduction Organisms in nature interact with one another through a Genetically modified microbial organisms are used in variety of modes ranging from competitive or predatory many applications in industrial and environmental bio- behaviors to commensal and mutualistic exchanges that technology, from synthesis of materials, chemicals, med- have been extensively explored in ecological studies [7]. icines, and fuels, to remediation of waste products and Microbes living within communities are involved in many toxins. Recent advances in synthetic biology have sub- interactions simultaneously — competing for some stantially improved our ability to program these microbes resources while exchanging others. Over time, these quickly and cheaply on a large scale with greater control tradeoffs create interspecies dependencies manifested [1,2]. While many successes are documented for single- by differing specialized phenotypes across various step microbial bioconversion reactions [3], potential microbes. A key challenge for engineering consortia with Current Opinion in Microbiology 2016, 31:146–153 www.sciencedirect.com Design principles for synthetic microbial communities Johns et al. 147 Figure 1 Engineering New Synthetic Microbial Augmenting Open Functions Communities Microbiome Systems Synthesis Catalysis Remediation Sensing Recording Reporting Features of Synthetic Microbial Communities Diversification of function & tasks Engineerable interdependencies complex substrate products Compartmentalization of function Spatiotemporal control surface Modular systems optimization Reduced individual burden divide & conquer Current Opinion in Microbiology A summary of the design and utility of synthetic microbial communities. In addition to principles used in single-strain engineering, community engineering allows for diversification of biochemical roles in breaking down complex substrates, and optimized compartmentalization of pathways between individuals for simultaneous execution of multiple functions with reduced individual burden. Synthetic communities can be further engineered with increased robustness through interdependencies and spatiotemporal control. www.sciencedirect.com Current Opinion in Microbiology 2016, 31:146–153 148 Environmental microbiology stable interactions has been to understand how functions and respond to specific signaling molecules in a density- can be partitioned across a microbial population in pro- dependent fashion, offers a means to program cell–cell ductive compartments to achieve desirable population- communication and coordinate population-level behav- level behaviors (Figure 2a). ior. These simple bacterial communication systems pos- sess modular and engineerable features that have been Several studies have recently explored strategies to divide exploited extensively for rational design [16]. In a recent metabolic roles across different individuals in a consor- example, Saeidi et al. engineered an E. coli strain that was tium toward generation of a desired biochemical product. able to sense naturally produced quorum sensing mole- Minty et al. showed that a two-member microbial consor- cules from Pseudomonas aeruginosa and respond by turning tium containing a cellulase-secreting fungi, Trichoderma on a self-lysis kill-switch to release a pyocin compound reesei, and an engineered Escherichia coli strain that pro- that inhibited the growth of the target pathogen [17]. duced isobutanol could be used for the direct conversion Many opportunities exist to extend these approaches for of plant biomass into biofuels [8]. Zhou et al. engineered cooperative intercellular interactions. an E. coli and Saccharomyces cerevisiae consortium that can more effectively produce natural products than the indi- To model these microbial interactions, computational and vidual strains alone [9 ]. In this study, the biosynthetic genome-scale approaches may be used to inform the pathway for oxygenated taxanes, a medically valuable rational engineering of simple and complex consortia. diterpene chemotherapeutic, was partitioned into two Constraint-based methods such as flux balance analysis separate pathways in E. coli and S. cerevisiae. A taxadiene (FBA) use in silico metabolic reconstructions of cellular intermediate was produced and secreted by E. coli and metabolism based on a set of known stoichiometrically then taken up by the yeast to complete the necessary balanced reactions to assess steady-state metabolite fluxes oxidation steps using more optimal eukaryotic cyto- within the cell during growth. When extending these chromes. Studies like these demonstrate that consortia approaches to model synthetic or natural communities, containing members with specialized tasks can succeed in each cell can be compartmentalized and fluxes between applications where single-strains would struggle. the compartments can be evaluated across the population to predict community-wide behaviors [18]. Currently, for A number of studies have explored syntrophic interac- most microbial species, incomplete or missing information tions using model bacterial and yeast systems that about the metabolic network components and their exchange essential metabolites [6]. Each strain is engi- biochemical functions make FBA-based methods chal- neered to produce some but not all essential metabolites lenging. Furthermore, drawing realistic inferences from (e.g. amino acids). When these different auxotrophic these models requires constraints for maximizing commu- strains are grown together, those with complementary nity-wide objective functions (e.g. growth), which may be metabolic functions are able to support the growth of one difficult to define or can change on an individual level another as a syntrophic co-culture. These systems have across a dynamic community. Nevertheless, these been characterized in synthetic communities of two