Microbial communities drive diverse processes that impact nearly everything on this planet, from global biogeochemical cycles to human health. Harnessing the power of these microorganisms could provide solutions to many of the challenges that face society. However, naturally occurring microbial communities are not optimized for anthropogenic use. An emerging area of research is focusing on engineering synthetic microbial communities to carry out predefined functions. Microbial community engineers are applying design principles like top-down and bottom-up approaches to create synthetic microbial communities having a myriad of real-life applications in health care, disease prevention, and environmental remediation. Multiple genetic engineering tools and delivery approaches can be used to ‘knock-in' new gene functions into microbial communities. A systematic study of the microbial interactions, community assembling principles, and engineering tools are necessary for us to understand the microbial community and to better utilize them. Continued analysis and effort are required to further the current and potential applications of synthetic microbial communities.
Microbes are omnipresent. They are present not only on the surface of Earth but also underwater, in the soil, in the atmosphere, as well as in multicellular organisms [1–7]. In nature, microbes can benefit from living in a community due to the collective behavior and action of the community. Diverse microbial communities composed of multiple species are advantageous over monocultures due to gene bank diversity and for allowing individual microbes to metabolically specialize, making survival in harsh and variable environments easier. Microbes in communities can interact with each other through the exchange of metabolites, genetic materials, or even information like in the case of quorum sensing. However, we still lack a clear understanding of the molecular basis of microbe interactions in the community, community-level stability, and functions despite the fact that natural microbe communities affect every aspect of ecology and human life. Natural microbial communities can contain tens to more than thousands of species [8–10] and making them difficult to characterize and understand the interactions between community members. Recently there has been a keen interest in understanding more about these communities and developing synthetic microbial communities that can be used to solve problems for real-world applications. Synthetic microbial communities are microbial communities that are artificially assembled using natural or engineered microbes. They can perform complex tasks requiring multiple steps, can accomplish tasks that are not feasible for individual strains through a division of labors, and are robust to environmental fluctuations if well designed [2–5,9,11]. By engineering microbial communities, synthetic biologists are gaining a better understanding of the rules that govern microbial interactions, the design principles needed to engineer these communities, and how to expand the functionality of these diverse and dynamic communities. Here we present recent progress on understanding synthetic microbial communities, the tools needed to engineer them, and emerging applications of these engineered communities [11,12].
Designing microbial communities
Many factors affect microbe-microbe interactions as well as the overall behavior of microbial communities. Changes in the physical environment and resource availability alter interactions between members of the microbial community and affect the overall performance of the microbial community [13–15]. Surplus resources result in cooperative behaviors among the members of the community whereas, scarcity of resources results in competition [7,16–19]. Moreover, members of the community can have positive, negative, or neutral effects on other members of the community. To study these interactions or to assemble a community with specific functions in mind, we can assemble microbe communities with top-down or bottom-up approaches.
Function driven approach
The priority for function driven synthetic community assembly is to design the community driven by the overall function, stability, and performance needs. Function Drive Approach is also described as Top-Down Approach. Top-Down takes the community as a whole although fewer details about the individual strain interaction are provided (Figure 1a). (1) One commercial example of this approach is to assemble a synthetic community for anaerobic biowaste digestion and biogas synthesis. By assembling a synthetic community with natural and/or engineered strains, the community can collectively degrade biowaste/biomass to produce valuable products [20–23]. In 2013, Minty et al.  designed a synthetic consortia consisting of fungus Trichoderma reesei and engineered Escherichia coli wherein the former secreted cellulase enzymes to hydrolyze lignocellulosic biomass into soluble saccharides, and the latter metabolized soluble saccharides into isobutanol. Other functional community has proved to convert human waste into Biohydrogen  and to produce hydrogen and butanol from wheat straw . (2) Other than assembling functional microbes into the community, researchers achieved targeted knockdown of undesired species from a community to modulate the community function in vivo and in vitro. In 2019, Hsu et al.  used bacteriophage to modulate gut microbiome components in mouse model gut and thus modulate metabolites production including amino acids and bile acids, which are known to affect the host. While Hsu et al.  used natural bacteriophage, Citorik et al.  utilized engineered phage to deliver CRISRP-cas system into synthetic bacteria communities to selectively knockdown targeted strains based on genetic signatures, providing higher selectivity toward the species. (3) Other than strain level modulation on the community, researchers can ‘knock-in' new functions into the community without changing the strain components. Ronda et al.  applied a universal bacterial conjugation platform to modify mouse gut microbiome in situ. They were able to modify the mouse gut microbiome with green fluorescence protein-antibiotic resistance payloads, demonstrating the capability to modulate the community function without altering strain components of the system. Advantages of the top-down approach for engineering synthetic microbial communities include: (1) the ability to engineer functionality into a microbial community despite having limited knowledge of that community; (2) optimize the performance of the community by carefully pick the strains/proteins with desired functions de novo; (3) often increased robustness of the community due to genetic and metabolic diversity of member organisms . Limitations of the top-down approach that may arise include: (1) Coexistence of natural and engineered strains in the synthetic community could lead to unforeseen and undesired interactions; (2) temporal changes of the components in the microbial community could adversely affect the community performance and functionality.
Schematic representation of the top-down and bottom-up approach.
Interaction driven approach
Interaction Driven Approach, which is also described as Bottom-Up approach, focuses on identifying microbial interaction patterns and utilize this information to understand microbial communities [9,30]. Species interactions in microbial communities can happen through small molecules, small peptides or genetic information. species interactions have a strong influence on community behaviors. When two strains are involved, there are six possible interactions including commensalism, cooperation, competition, predation, amensalism, and neutralism (Figure 1b). Commensalism is observed when one of the interacting species excretes or produces metabolites that benefit the other species. Amensalism is contrary to commensalism. In amensalism the first species secretes toxins that negatively affect the growth of the second species [6,12]. In neutralism, the presence of either species neither benefits nor harms the other species. In competition, both strains compete for the same substrate which leads to reduced growth in either strain [31,32]. For predator–prey interaction, one of the strains produces a metabolite that is beneficial for the other strain whereas the other produces toxin that is harmful to the first strain. Cooperation is a type of cell–cell interaction in which cells help each other grow better when grown together [19,33,34]. The possible interaction states involving more strains increase exponentially with the number of strains with four strains system being over half a million . The interaction could either be unidirectional or bidirectional. In unidirectional interaction, the presence of the second species does not affect the growth of the first strain while for bidirectional interaction the presence of either species affects the other. Commensalism, amensalism, and neutralism are categorized as unidirectional interactions, whereas, cooperation, competition and predator prey model are categorized as bidirectional interaction (Figure 1b).
Engineering these cell–cell communications can greatly enhance our understanding of the interaction mechanism and help construct synthetic microbial communities. Kong et al.  engineered gene circuits in bacteria to study all the possible six two-strain interactions including commensalism, amensalism, neutralism, cooperation, competition, and predation. It was achieved through bacterial produced molecules including lactococcin A (lcnA), which is an antimicrobial peptide, and nisin, which is an antimicrobial and quorum-sensing molecule. In order to produce these molecules, the synthetic gene circuits were constructed by assembling individual DNA parts and transformed into the species, so that the quorum-sensing and/or antimicrobial molecules could be produced to form these six interactions. They later upgraded to study the population dynamics of three- and four-strain ecosystems, validating the translatability of engineered community from laboratory conditions to realistic settings with more species. Other than that, bacterial communication has been engineered to study oscillation , biofilm-based community , pattern formation , and even cancer therapies . The bottom-up approach is to understand component interaction within a synthetic microbial community, providing knowledge as step stones to build a synthetic community. Advantages to the bottom-up approach include: (1) detailed understanding of individual interaction between strains serves as designing rules for constructing synthetic community; (2) the ability to protect key strains of the synthetic community by looking at the effects of other strains on these strains; (3) the dynamics study predicts the stability and robustness of the community after long term evolution with environment and resource change. Limitations of the bottom-up approach that may arise include: (1) the inability to accurately predict the community performance when complexity goes up; (2) lack of overview on functions of the community as a whole.
Engineering microbial communities through gene editing
Although there are multiple ways of assembling microbial communities including bottom-up and top-down approaches, these may still not result in the desired functional outcome. In this case, engineered microbes may be needed and we will need to ‘knock-in' new functional genes into the community. To achieve this, we need genetic engineering tools to modify individual microbes and delivery platforms to deliver these genetic engineering tools to the target communities.
Genetic editing tools
Recombinases are enzymes that bind to DNA and catalyze directionally sensitive and highly specific splicing reactions, typically delivered via a plasmid or viral vectors (Figure 2a). It can be used to insert DNA sequences into target areas and thus has been successfully used to genetically edit microbial communities . Santos et al.  utilized recombinase to develop a microbial platform to convert brown microalgae to ethanol, while in a different study, Brenner et al.  used recombinase to construct knockout strains of Escherichia coli for biofilm construction. In particular, Santos et al.  used Cre recombinase to implement recombinase-assisted genome engineering (RAGE), with the goal of facilitating the precise integration of large genetic fragments into the bacterial genome. Using the RAGE method, Santos et al.  successfully integrated a 34 kb heterologous alginate degradation and ethanol production heterologous pathway into E. coli strains. The resulting strain achieves a ∼40% higher titre than its plasmid-based counterpart and enables substantial improvements in titre (∼330%) and productivity (∼1200%) after 50 generations, demonstrating the robustness of the system. Recombinase's reliance on a specific recognition site for successful gene editing represents a significant drawback for this tool [42,43] and to address this, directed evolution has been utilized to allow the recombinase to recognize alternative sites to make recombinase more applicable [43,44].
Gene editing tools.
Zinc finger nucleases and transcription activator-like (TAL) effector nucleases (TALEN) are DNA-binding proteins with enabled DNA cleavage or modification proteins (Figure 2b) [39,45]. Gene editing occurs when the zinc finger or TAL DNA-binding domain couples with the FokI restriction enzyme's non-specific cleavage domain [39,46,47]. This method has been successfully used in a variety of microbes and organisms ranging from nematodes like Caenorhabditis elegans  up to humans [47,49–52]. These methods provide the possibility of DNA modification on specific sites but lack the flexibility of easily changing cleavage sites. Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) utilizes RNA guide molecules to recognize and target sequences after a short specific DNA sequence known as a protospacer adjacent motif (PAM) sequence (Figure 2c) [39,53,54]. Next, the Cas9 or other cleavage proteins binds to the target site and cuts the DNA [55,56]. From there, researchers can delete, correct, or insert genes into the break. Deletion occurs with non-homologous end-joining (NHEJ), where the strands reattach at the break. Correction and insertion occur through the process of homology-directed repair (HDR), in which cases donor DNA template will be used as a template for inserting the correction or added sequence into the break. By modifying the RNA guide sequence, the Cas9 can be programmed to ‘search' out specific locations even on very complex genomes and thus provides a higher level of flexibility compared with recombinase, ZFN, or TALENs. This flexibility provides great potential for microbial community genetic engineering applications [57,58]. CRISPR-Cas9 has already proven useful when editing microbial communities, allowing researchers to perform a variety of functions such as implementing an adaptive immunity system for microbes [49,59,60] or genome modification of microbes for industrial purposes [59,61–63]. All of these genetic engineering tools serve as platforms for efficient engineering of individual microbes. Delivery of these tools to a microbial community to genetically engineer them in situ can be a powerful approach to add new functions to the community.
Many genetic engineering tools can be encoded into DNA for transmission to the microbe of interest. Horizontal gene transfer (HGT), the process in which genetic material is passed ‘horizontally' from a donor organism to a recipient, is one of the primary delivery methods for engineering microbial communities . In nature, conjugation is a key pathway by which microbes exchange genetic material through the cell to cell contact. This allows for the transfer of larger genetic payloads, even the entire CRISPR-Cas9 system . For example, this method was applied in situ to deliver GFP and an antibiotic-resistant gene through F+ conjugation within a mouse gut .
Phages are viruses that infect and replicate within microbes, transferring genetic material via the process of HGT transduction. Phages function by infecting a host cell and hijack it to create more phages until the infected cell eventually bursts, releasing phages that can then infect other cells . Phages can also remove a portion of the infected cell's DNA and transfer it to another host cell through either generalized or specific transduction. Phages have key advantages serving as genetic engineering tools, including their high specificity and delivery capacity [67–69]. A good example of this was mentioned in an earlier section, where Citorik et al.  utilized phages to deliver the CRISPR-Cas system into a synthetic bacteria community to modulate community components by selective knockdown of targeted strains based on genetic signatures. Bikard et al.  explored delivering type II CRISPR systems to microbiota, using a special phage called a phagemid. Using this method, they successfully delivered their target payloads and showed that it could successfully target and kill microbiota with virulence genes, but not the avirulent strains. Phage delivery allows the targeting of specific members of the community for the purpose of engineering the community functions .
Engineering microbial communities requires careful considerations of the tools and delivery methods that are the most useful and effective when modifying the community. To successfully engineer and study our desired community, we must carefully consider and select the proper tools to most effectively engineer our experiments and communities.
Applications of engineered microbial communities
Microbial communities have been used for the production of various chemicals including biofuels and pharmaceuticals [24,71–75]. One major advantage of using microbial communities for chemical production over single strains is the capability of labor divisions. This results in less metabolic burdens on the microbes and improved efficiency in the intermediates and final product yield. Several attempts have been made to produce butanol, a potential biofuel source with lower vapor pressure and high energy content, in a single engineered strain of bacteria. Butanol produced from renewable lignocellulosic feedstock can help reduce the society's dependence on fossil fuels at a relatively low cost, however, bacteria that are excellent at producing butanol often cannot degrade cellulose. When the jobs of both cellulose degradation and butanol production are assigned into a single bacteria strain, the bacteria suffer from carbon and electron flux imbalance, causing low butanol production [74,76–79]. Synthetic microbial communities become an apparent alternative to address this problem. Using the co-culture of cellulose-degrading Clostridium thermocellum and butanol producing Clostridium saccharoperbutylacetonicum, Nakayama et al. [80,81] and Kiyoshi et al.  were able to produce butanol from crystalline cellulose and rice straw with higher yield and less demanding temperature requirement (Figure 3A). Other than biofuels, synthetic bacterial communities have also been used to make pharmaceuticals , bioplastics , and more.
Application examples of microbial communities.
In addition to making products, bacterial communities can also be used to treat waste such as heavy metals, municipal waste, and oil spill contaminants [85–87]. Hydrocarbon contamination from oil exploitation, petroleum products transportation, waste emission, and oil spill accidents has led to detrimental effects on the ecosystem such as the permanent marsh area loss caused by the BP oil spill in 2010 [88,89]. Traditional methods to remove contaminants using physical skimming or chemical dispersants are often labor-intensive and expensive . Fortunately, many of the bacteria strains, found in the contaminated area, are able to degrade hydrocarbon contaminants [91–96]. Due to the complex nature of contamination that usually consists of more than one component including alkanes, cycloalkanes, and polycyclic aromatic hydrocarbons, an engineered community of microbes with diverse functions becomes an ideal choice for this type of situations . Another factor that limits the biodegradation efficiency of petroleum hydrocarbons is the low solubility, and thus low bioavailability, of the contaminants. Biosurfactants that have both hydrophobic and hydrophilic groups are able to increase the solubility of hydrophobic contaminants, resulting in a higher biodegradation efficiency . Compared with chemically synthesized surfactants, biosurfactants have low toxicity, high biocompatibility, and biodegradability, which suit the purpose of biodegradation of petroleum hydrocarbons . By combining biosurfactant producer Rhodococcus erythropolis OSDS1 with petroleum hydrocarbon degraders Serratia proteamaculans S1BD1, Alcaligenes sp. OPKDS2, Rhizobium sp. PNS1, and Pseudomonas sp. BSS9BS1, Xia et al.  developed a consortium that had higher efficiency and a broader spectrum in degrading petroleum hydrocarbons (Figure 3b). The consortium achieved a higher oil depletion efficiency than any of the strain alone did.
Breaking down pollutants is a useful function of microbial communities, but these communities can do even better by producing useful products from the waste. One such application of a microbial community is to convert human waste to biohydrogen (BioH2) . Until now, human waste generated during space missions is often discarded into the Earth's atmosphere for destruction, but for future farther space travels, the onsite resource recycling also becomes crucial. BioH2 produced from crew's waste can be used to generate electricity, and provide the crew members with portable water . Wang et al.  developed a synthetic microbial community that was mainly comprised of Thermoanaerobacterium, Caloribacterium, and Caldanaerobius, which together showed the highest activity of endoglucanase to hydrolyze the cellulose that accounts for a major portion of human waste. Although Thermoanaerobacterium alone could also be used for BioH2 production, the consortium that included Caloribacterium and Caldanaerobius was able to continue producing H2 in a wider range of pH .
Human health is significantly impacted by microbial communities residing in or on the human body. Humans have ten times more bacteria than it does its own body cells . These bacteria form a community themselves and affect the body's function and health through metabolism, immunity, and gut-brain axis [104–106]. Bacteria have been engineered to perform diagnostic and therapeutic functions, but a microbial community will have special advantages with designed components and metabolites . Fecal microbial transplant (FMT), a procedure that transfers the microbes from a healthy donor's fecal sample to the colon of patients, has shown positive effects in helping gastrointestinal infection and inflammatory bowel disease . Caballero et al.  found that a consortium of Blautia producta, Clostridium bolteae, Bacteroides sartorii, and Parabacteroides disasonis restored colonization resistance against Enterococcus in a mice model through inhibition of the Enterococcus growth (Figure 3c). In this case, a bacterial community is especially useful because B. producta can inhibit Enterococcus growth potentially through direct bacterial interactions including nutrient competition or production of inhibitory factors, while B. producta needs C. bolteae’s assistance in colonizing the gut from a cooperative interaction, which although works well, still requires additional investigation . However, both B. producta and C. bolteae are antibiotic sensitive strains, and they cannot survive in ampicillin treated mice without B. sartorii and P. disasonis that produced β-lactamase.
In addition to protection against infection, Besseling-van der Vaart et al.  developed a consortium including Bifidobacterium lactis W51, Lactobacillus acidophilus W22, Lactobacillus plantarum W21, and Lactococcus lactis W19. This consortium has been shown to strengthen the gut barrier function and provide health benefits for patients with food intolerance.
Developing microbial communities has been shown to be a powerful approach to carry out complex tasks such as producing chemicals efficiently, degrading chemical wastes that have multiple components, and protect our health through close interaction with the body. Although top-down approach is often used when the microbial community is engineered to perform a specific task, the bottom-up approach helps the researchers understand the mechanism of how each member within the community interacts with the other strains, and optimize the overall performance.
Synthetic microbial communities can perform complex tasks that are more feasible for individual strains and are more robust in harsh environments. Although with many advantages, challenges associated with synthetic communities include the lack of understanding of bacteria interaction patterns and mechanisms, designing rules for efficient and functional communities, and applications of the community in different areas. By putting natural and engineered strains together, researchers can look into details of microbial interactions, providing a better understanding of the rules governing microbial interactions. The function driven assembly of synthetic community looks at the community as a whole piece to achieve the desired functions of the community. There are also progress on genetic engineering tools and delivery methods to engineer the community both in vitro and in vivo. All the gained knowledge and designing rules have been used to expand the functionality of these diverse and dynamic communities. Looking into the future, the challenges will be to study bacterial interactions with more strains and complicated environments, to have a robust and functional community with long-period standing, and to improve the in vivo engineering efficiency. Solving these challenges will level up the potential applications of the synthetic community in manufacturing, environmental and biomedical areas.
Synthetic microbial communities can help understanding microbial community assembling principles and can perform complex tasks that microbes cannot achieve individually.
By engineering microbial communities, researchers are studying communities from function and interaction perspectives, developing genetic engineering platforms they can use to engineer the community in vitro and in vivo, and applying synthetic communities to solve challenge problems.
Looking into the future, by better understanding the community at a higher complex level and in more complicated environments, researchers can build robust and functional communities with long-period standing towards biomedical, environmental and manufacturing applications.
The authors declare that there are no competing interests associated with the manuscript.
Clustered Regularly Interspaced Short Palindromic Repeat
fecal microbial transplant
horizontal gene transfer
protospacer adjacent motif
recombinase-assisted genome engineering
zinc finger nuclease
These authors contributed equally to this work.