Micro

nature biotechnology
Review article https://doi.org/10.1038/s41587-023-01932-3
Soil microbiome engineering for

sustainability in a changing environment
Received: 22 February 2023 Janet K. Jansson , Ryan McClure & Robert G. Egbert
Accepted: 1 August 2023
Published online: 30 October 2023 Recent advances in microbial ecology and synthetic biology have the
potential to mitigate damage caused by anthropogenic activities that
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are deleteriously impacting Earth’s soil ecosystems. Here, we discuss
challenges and opportunities for harnessing natural and synthetic soil
microbial communities, focusing on plant growth promotion under
different scenarios. We explore current needs for microbial solutions in
soil ecosystems, how these solutions are being developed and applied, and
the potential for new biotechnology breakthroughs to tailor and target
microbial products for specific applications. We highlight several scientific
and technological advances in soil microbiome engineering, including
characterization of microbes that impact soil ecosystems, directing how
microbes assemble to interact in soil environments, and the developing
suite of gene-engineering approaches. This Review underscores the
need for an interdisciplinary approach to understand the composition,
dynamics and deployment of beneficial soil microbiomes to drive efforts
to mitigate or reverse environmental damage by restoring and protecting
healthy soil ecosystems.
To feed Earth’s growing human population that is estimated to reach and alkaline soils, changes in precipitation patterns that result in
8 and 9.8 billion by 2025 and 2050, respectively1, new solutions pro- more intense periods of flooding and drought, and increased wildfire
vided by microorganisms are required. One promising approach to frequency and intensity6. Increased global temperatures cause perma-
sustainably improve crop production and soil health is to harness frost thaw at the poles, resulting in changes in landscapes, together
the beneficial properties of soil microorganisms (Fig. 1). Certain soil with associated shifts in aboveground vegetation and belowground
microorganisms can be harnessed to promote plant growth by acting microbial communities7. Thawing permafrost also releases addi-
as fertilizers or pesticides, thereby reducing reliance on synthetic tional carbon dioxide and methane to the atmosphere as microbial
chemicals. These microorganisms may also be used to improve plant activity increases, leading to feedback cycling of increased warming
productivity in marginal soils, such as saline or alkaline soils, and/or as atmospheric carbon drives a greenhouse effect8. Other negative
during periods of drought2. In addition, soil microorganisms have anthropogenic impacts that are compounding those directly caused
been used for remediation of organic and heavy metal pollutants in by climate change include deforestation, intensive agricultural prac-
soil3,4. Because soil microorganisms are integral to carbon cycling tices that result in soil erosion and loss of carbon9,10, and exposure
and sequestration, they also hold potential for capturing and storing of soil ecosystems to pollutant chemicals3,11. The increased stresses
atmospheric carbon belowground5. placed on natural and managed soil ecosystems from anthropogenic
Our planet is currently experiencing unprecedented impacts have spurred research into alternative approaches to reduce
anthropogenic-induced changes, such as climate change and intro- these negative environmental impacts.
duction of pollutants, that are having dramatic impacts on soil eco- Here, we will explore current needs for microbial solutions in
systems and the beneficial services that they provide. Examples soil ecosystems, report how these solutions are being developed
of climatic changes that impact crop-production systems include and applied and elaborate on the potential for new biotechnology
rising seawater levels in coastal areas, increases in areas of saline breakthroughs to tailor and target microbial products for specific
Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory, Richland, WA, USA. e-mail: [email protected]
Nature Biotechnology | Volume 41 | December 2023 | 1716–1728 1716

Nutrient runoff Salinity Drought
Heavy metal Pesticide Overfertilization

contamination contamination
Nutrient Osmotolerance Drought
Pesticide turnover tolerance
degradation
+
Low organic C C sequestration
Heavy metal
binding C
C
C
Pathogen N fixation
resistance
Nutrient limitation
(P, N micronutrients) P acquisition
Biopesticides Pests/pathogens
Micronutrient
Induced systemic acquisition
resistance
Fig. 1 | Soil microorganisms carry out key ecosystem services and have microorganisms that are indicated by the pink and yellow symbols. Top left,
the potential to help mitigate a variety of deleterious anthropogenic heavy metals (gray hexagons) that can be bound by heavy metal-binding bacteria
impacts on soil ecosystems. White-boxed text indicates different deleterious (red rods) and pesticides (light green dots) that can be degraded by rhizosphere
anthropogenic impacts. Yellow-boxed text indicates different biological microorganisms. Bottom left, potential pests that include viruses (dark green
mitigation strategies. The rhizosphere is highlighted in the circle in the center, sphere with knobs), bacteria (blue rod) and nematodes (green ribbon). C, carbon;
with associated mycorrhizal fungi (green threads), nitrogen-fixing nodule- N, nitrogen; P, phosphorus.
forming bacteria (brown nodules on the root) and a variety of root-associated
applications. We also highlight scientific and technological advances atmospheric nitrogen and provide it in a form that can be used by plants
in soil microbiome engineering, which we classify into three pillars and other microorganisms1. Other species carry out specific steps in
(Fig. 2): (1) the discovery and characterization of microbes that impact cycling the resulting nitrogenous compounds15 and other essential
soil ecosystems, (2) understanding and directing how microbes assem- nutrients. Some soil microorganisms decompose carbon compounds
ble in structured communities to coexist and interact in soil environ- that comprise soil organic matter. However, most functions carried
ments, and (3) the developing suite of tools to genetically manipulate out by specific microorganisms in soil remain largely uncharacter-
soil microbiomes to control composition, function and environmental ized. This knowledge gap is primarily due to the difficulties in studying
persistence. An interdisciplinary approach to understand the compo- soil microorganisms due to (1) the physical heterogeneity of the soil
sition, dynamics and deployment of beneficial soil microbiomes will matrix, (2) the high taxonomic and chemical diversity, with thousands
drive efforts to mitigate or reverse severe impacts of anthropogenic of species and metabolites and millions of potential interactions,
activity by restoring and protecting healthy soil ecosystems. (3) the difficulty in studying microorganisms at the microscale at which
they function in situ, and (4) the fact that most soil microorganisms
The potential and challenges of harnessing soil have not yet been cultivated in a laboratory setting, and their functions
microorganisms therefore remain uncharacterized. Because uncultured microorgan-
Soil microorganisms are highly abundant and taxonomically diverse, isms may have beneficial functions that are not yet characterized, they
with estimates of billions of microbial cells and thousands of species represent a potential opportunity that could be further explored16.
occurring in a single gram of soil12. However, the majority of soil micro- Despite these challenges, several methods have been developed
organisms have not yet been cultivated, which makes them difficult to and applied for the study of microorganisms in soil. Cultivation on
study. In addition, microbial abundance and community composition solid or liquid medium has traditionally been used to obtain specific
vary widely across different soil types and geographic regions13. The microbial isolates. The advantage of cultivation is that it facilitates
soil microbial community, or soil microbiome, is also influenced by biochemical and physiological studies of the isolated strains. Advances
environmental variables. Increases or decreases in types and func- in culture-based approaches have helped to increase the number of
tional activities of soil microorganisms can occur depending on how known soil microbial isolates. For example, dilution-to-extinction
the environment is altered by changes in temperature, soil moisture culturing was successfully used to isolate hundreds of heterotrophic
and other variables14. bacterial taxa from soil17. Microcultivation is an approach that relies
Soil microorganisms include bacteria, archaea, fungi, protists and on in situ systems that mimic the natural environment to favor growth
their respective viruses. Together, soil microorganisms interact to per- of difficult-to-cultivate microorganisms. This was used to isolate
form roles that are essential for normal ecosystem function. These roles new strains that degrade oil from contaminated soil18. In addition, a
include cycling of carbon and other nutrients, support of plant growth, high-throughput cultivation approach, ‘culturomics’, was recently
degradation of pollutants and others. Some microbial populations developed that uses machine learning and robotics to rapidly obtain
carry out specific keystone functions for agriculture production and/ isolates as colonies on microtiter plates19. A culturomic approach may
or ecosystem sustainability. For example, some species of rhizobia fix also be used to identify soil taxa.

function (reviewed in ref. 24). Depth of coverage is especially critical for

metatranscriptomics, as individual genes are expressed at levels span-
ning orders of magnitude, thereby making identification of expressed
genes that are transcriptionally regulated and/or from low-abundance
species even more challenging. A promising technique that overcomes
several of these issues is to incorporate stable isotopes (for example,
13
C or 3H) into DNA or RNA during incubation under specific conditions.
Subsequent extraction and sequencing of the heavy-labeled DNA or
Ecosystem
services RNA provides information on specific microbial populations that are
growing or incorporating specific substrates25.
Study of community proteins (metaproteomics) provides infor-
mation about specific microbial functions in soil. For a protein to be
Soil microbiome expressed, it must have already passed regulatory bottlenecks during
engineering transcription and translation. Therefore, detection of a protein pro-
vides an additional level of confidence that a function conferred by a
particular protein actually occurred in the system of study. However,
coverage issues and extraction challenges can also affect recovery
and analysis of proteins expressed by soil microbiome constituents
Isolate Assembled
discovery communities used for metaproteomic analyses26. Another option for detection of
specific proteins produced by microorganisms is to use activity-based
probes27. These are molecules that mimic enzyme targets and bind
to them, allowing for their subsequent isolation and analysis28. If the
enzymes of interest are intracellular, the producing microorganisms
can also be isolated, thereby providing information on which taxa are
expressing a function of interest27. Even when overcoming these chal-
Genetic
manipulation lenges, obtaining high-quality omics data is only the first step toward
gaining an understanding of microbial communities and functions in
Fig. 2 | The three pillars of soil microbiome engineering are isolate discovery,
assembled communities and genetic manipulation. The interplay among soil. Computational annotation and bioinformatic interpretation of
these pillars will lead to advances in providing soil ecosystem services, including omics data are also substantial challenges. This is largely due to the
(1) mapping microbial functions to organisms and genes, (2) improved ecosystem lack of fundamental knowledge regarding soil microbial processes.
function through the establishment of naturally evolved and synthetic microbial Many of these processes are carried out by species that are either
communities enhanced by engineered isolates, and (3) promoting biosafety and completely unknown or, if they have been identified, our understand-
biosecurity goals by understanding colonization and containment dynamics at ing of their fundamental biology is only partial, thus leading to gaps
the ecosystem scale. in our interpretation of data and the role of certain species in driving
observed outcomes.
Several bioinformatic tools have been developed for interrogation
DNA sequencing is an approach widely used to determine the (DRAM)29 and integration (MEMPIS, XCMS) of omics datasets30,31. The
composition of soil microbiomes20. Specifically, amplicon sequenc- increasing availability of omics data provides an opportunity to mine
ing of 16S rRNA genes for bacteria and archaea and 18S rRNA genes or the soil microbiome for specific species, genes, enzymes and/or func-
intervening sequences for Eukarya is routinely undertaken20. Sequenc- tions that can be exploited for engineering applications. For example,
ing bypasses some of the limitations of cultivation because it captures metagenomes from saline soil were mined for genes that encode toler-
the entire community, including cells that are inactive and/or dormant. ance to salt stress32, from which a new cellulase was cloned from a sug-
However, a drawback is that dead cells may also be represented in arcane soil metagenome33, and a new esterase was isolated from a plant
the resulting sequence data. Another disadvantage of this approach rhizosphere soil metagenome34. Several studies have also used ampli-
is that it is only able to identify taxonomic signatures. This does not con sequencing to target specific microbial populations for isolation,
directly provide information on their functional potential. By contrast, such as previously uncultivated Verrucomicrobia spp. from the potato
metagenomic sequencing of DNA has the advantage of ascertaining rhizosphere35. Ideally, microorganisms with desired functions can be
the taxonomic and functional–genomic structure of the soil micro- used as microbial inoculants for environmental-engineering appli-
biome. However, metagenomic approaches are still challenging for cations in soil. Here, we will discuss different microbial-inoculation
certain soil types and for identifying low-abundance but potentially strategies that have been developed, tested and, in some cases, applied.
important species21. Higher-throughput sequencing can help with the
depth-of-coverage issue, but this can lead to other problems related to Characterization of specific isolates for improved
high computational demands for large datasets22. In addition, quantita- plant performance
tive analysis is challenging in the absence of absolute-abundance data. Traditionally, microbial inoculants for soil applications have relied on
Most sequencing-based studies primarily rely on relative-abundance single organisms with beneficial functions that are isolated in culture,
data. This can make it difficult to distinguish the actual abundance grown in large volumes and applied to the field. A classic example is the
changes of a specific taxon from changes due to one species reducing use of rhizobial inoculants to provide fixed nitrogen to legume crops in
in abundance, leading to greater fractional abundance for other com- place of synthetic fertilizers1. Arbuscular mycorrhizal inoculants have
munity members23. also been widely applied36 to supplement provision of nutrients to plants,
Many of the same issues that hinder DNA sequencing also apply especially phosphorus in P-limited soils. Other fungal inoculants include
to microbiome RNA sequencing (metatranscriptomics). Additional biocontrol strains of Trichoderma that attack plant-pathogenic fungi37.
challenges include the fact that RNA is generally more unstable Several methods have been developed for cultivation of ben-
than DNA, is more difficult to extract from soil and is composed eficial microorganisms for environmental applications. For example,
primarily of ribosomal RNA, which can make sequencing more dif- members of the barley root microbiome were isolated using a trap–
ficult for more-informative messenger RNA transcripts that indicate bait approach38. This approach used clay chips to capture most of the

microorganisms present in the rhizosphere. Potentially beneficial and (2) deriving naturally enriched natural communities with reduced
strains can also be isolated by selective enrichment. For example, complexity (‘NatComs’) (Fig. 3). SynComs are typically built from a
beneficial rhizosphere microorganisms were isolated following enrich- relatively simple (2–5-member) pre-existing collection of isolates to
ment on plant root exudates39. facilitate their formulation and environmental application and to aid in
Microbial inoculants for applications that benefit plants are often studies of interspecific interactions56–59. Technological developments
targeted to the root zone (rhizosphere) and are thus referred to as plant for building SynComs include high-throughput screening of potential
growth-promoting rhizobacteria (PGPR). Successful examples include combinations of species60 and selective culturing. Once the optimal
the use of rhizobial inoculants to enhance soybean yields in Kenya40 species are identified, they can be combined into specific formulations
and Brazil41. Additional applications of PGPR include alleviation of to carry out a given environmental function. SynComs built from char-
abiotic stress in plants, promotion of plant growth by increasing nutri- acterized isolates offer several benefits over less-characterized systems.
ent availability and production of plant hormones and siderophores1. As they are built from individual isolates, the community composition
For example, auxin biosynthesized by microorganisms is thought to is already known. In addition, the individual isolates can be character-
be involved in beneficial plant–microbe interactions either indirectly ized separately for their performance and genomic data. The role of
via signaling pathways or directly by changing the root architecture each member can be defined using leave-out experiments or genetic
and growth of the host42. Auxin from microorganisms (indole-3-acetic manipulation61. An example of a SynCom for soil is a collection of three
acid) also has several positive effects on plant growth, including pro- isolates from the soybean rhizosphere that was named the hitchhik-
moting formation of root hairs and lateral roots43. Other PGPR have ers of the rhizosphere (THOR)62. The individual constituents of THOR
been applied to mitigate plant salinity stress in a variety of studies were selected based on their potential influence in the rhizosphere and
(reviewed in ref. 44). For example, a strain of Bacillus licheniformis their root-distribution patterns. Only isolates that were genetically
could improve the water-use efficiency of maize plants when compared tractable were selected. THOR has since proven to be a valuable model
to uninoculated plants45. community for understanding microbial community interactions in a
Although several microbial species have been commercialized genetically tractable system. Another example of a SynCom is a com-
for delivery as PGPR to crop fields, their effects on plant performance munity of 16 species built from soil surrounding switchgrass plants63.
are often inconsistent1. In some cases, the applied strains perform While defined SynComs do have some benefits, their main drawback
poorly in the field environment in which they must compete with is that sets of isolates must first be collected, purified and studied in
locally adapted microorganisms46, and therefore the desired increases isolation. Only species that grow in pure culture in a laboratory setting
in crop yields are not realized47. For example, tracking the abundance are practical for SynComs. Furthermore, the species that are combined
of inoculants added to promote growth of maize plants showed that may not represent species that naturally interact, which may reduce
levels of inoculants peaked at 50–100 d after inoculation but began to their ability to persist together in the soil environment.
fall thereafter48. Alternatively, some single-strain inoculants have even An alternative approach for development of mixed inoculants is
been shown to survive for years. This ability is not well understood to enrich microorganisms from the soil environment in which com-
and depends on the environment and type of inoculant, among other munity members have naturally evolved to interact with each other
factors. For example, a single fungal strain added to soil was detected (NatComs) (Fig. 3). A selective enrichment process combined with a
up to 15 years after the initial inoculation49. A similar result was found dilution approach can be used to reduce community richness and to
with a Bacillus strain that was inoculated into soil and detected up to select for communities with a specific desired trait or combination of
3 years after application50. traits64. For example, Model Soil Consortium 1 (MSC-1) was enriched
One of the major complications with application of PGPR is that from soil using chitin as a substrate65. The resulting community con-
individual formulations with a specific mode(s) of action may therefore tained approximately 30 members that demonstrated several positive
limit their application to specific locations and/or soil and plant types1. interactions during chitin decomposition.
Introduction of non-native inoculants can also have unintended con- NatComs have some key advantages over SynComs; the major one
sequences on the local soil microbiome and the ecosystem functions being that, as they are allowed to develop naturally, they are more likely
they carry out51. This was found to be the case for some single inoculants to represent native interactions. The user or developer has little input
of Rhizobium that led to long-lasting changes in both the bacterial and on which species are retained or lost in the NatCom as this is driven
fungal communities of the soil52,53. In addition, certain widely used primarily by microbial ecology. In some cases, communities can be
fungal inoculants, such as arbuscular mycorrhizal fungi, have not developed that have advantages of both NatComs and SynComs. This
been adequately assessed for their potential ecological impacts54. is the case for Model Soil Consortium 2, which is a combination of
The introduced fungi may behave as invasive species if applied in high eight isolates derived from the MSC-1 NatCom66. As this consortium
enough concentrations. Because the diversity of soil fungi is known is a combination of individual isolates, it has elements of a SynCom.
to positively correlate with ecosystem services, an imbalance could However, because the isolates were selected from a naturally evolved
have negative repercussions55. Therefore, selection of field inoculants community (MSC-1), this implies that the interactions between com-
should not only be based on their beneficial properties but also on their munity members are more likely to reflect those driven by microbial
potentially negative impacts on the soil ecosystem. ecology in nature rather than an arbitrary assembly of soil isolates.
Examination of Model Soil Consortium 2 has shown that only a subset
Assembly of natural and artificial microbial of species contributes to the metabolism of abundant carbon and
communities nitrogen sources, likely sharing these breakdown products with other
Although most soil inoculants to date comprise single isolates, there is members of the consortium66. These results suggest that the ability to
increasing interest in application of communities of microorganisms metabolize a major nutrient source is not the only factor determining
(‘consortia’), to provide greater resilience to stress and to improve their whether a species will be successful in a community context. Rather,
ability to survive competition from resident microorganisms to enable this study suggested that the fundamental niche size of a species (how
them to persist and perform their intended functions. Multi-strain many carbon sources it can metabolize) is the main determinant of
inoculations also allow for a greater breadth of ecological functions success, at least as measured by abundance.
to be added to the soil microbiome and a larger palette for genetic One of the main advantages of reducing the soil microbiome com-
manipulation of functions that can be introduced to soil. Development plexity is the ability to better delineate possible interactions between
of microbial consortia for soil applications proceeds along one of two species that give rise to the functions of the greater soil microbiome.
routes: (1) combining isolates into synthetic communities (‘SynComs’) Sequencing data based on the 16S rRNA gene are normally used to

NatCom SynCom
Reduced-complexity community
Complete soil microbiome

Community A
Pathogen resistance
Community B Community C
Microbial interaction network
Bioremediation Drought resistance
Fig. 3 | Design of tractable communities of soil microbes. The design of a community. A SynCom leads to much greater control of the community’s
tractable communities usually proceeds along one of two routes. In the naturally constituents as well as more inherent knowledge of the potential processes,
evolved route (NatCom), naturally interacting members of the soil microbiome but the individual SynCom members may not naturally interact and may be less
are enriched from soil. In a SynCom, individual isolates are combined to form stable in the environment than a NatCom.
infer a network in which taxa are linked based on their co-abundances members in the environment. One approach that can circumvent this
across a range of samples and/or experimental conditions. Networks is to design SynComs based on knowledge of the core microbiome,
can be built using Pearson or Spearman correlations, but other which consists of a specific combination of microorganisms that are
network-inference tools have also been used (reviewed in refs. 67,68). recruited by a given plant species (reviewed in ref. 63). For example,
Once inferred, networks have been used to predict species interactions the core microbiome associated with 12 cultivated varieties belonging
(that is, those that are strongly correlated in abundance), the nature to the Citrus genus has been defined as Pseudomonas, Agrobacterium,
of the interaction (a positive correlation may indicate cooperation, Cupriavidus, Bradyrhizobium, Rhizobium, Meorhizobium, Burkholderia,
and a negative correlation may indicate competition), and keystone Cellvibrio, Sphingomonas, Variovorax and Paraburkholderia71. By con-
members in a community (by identifying central nodes in the network) trast, that of potato has been defined as Bradyrhizobium, Sphingobium,
(Fig. 4). For example, network analysis was used to identify potential Microvirga, Blastococcus and SMB53 (ref. 72). The hypothesis behind
keystone species in MSC-1, such as members of the Streptomyces and this strategy is that the core microorganisms are adapted to a given
Rhodococcus genera65. plant species and can serve to benefit the growth of that plant across
Network inference can be a powerful tool to view potential inter- a variety of habitats. By focusing on species that comprise the core
actions between species, but care must be taken when interpreting microbiome, it is possible to circumvent the assembly challenges with a
coexpression networks. Species co-abundance networks are normally greater diversity of microorganisms. While these approaches have been
derived from macroscale amounts of soil (0.1–1 g) and thus include spe- met with success, there are drawbacks related to the fact that there can
cies that may not normally co-occur on a microbiological scale. In addi- be minor differences between strains and species of the same genus
tion, mechanical processing of soil samples (sieving, homogenizing, that can lead to major differences in function, particularly in special-
bead beating, etc.) destroys the native structure of the soil. Therefore, ized metabolism. This means that building a SynCom from available
co-occurrence networks are not in themselves definitive proof of spe- isolates defined at the genus (or even species) level can be misleading.
cies interactions. Indeed, new studies have suggested that interaction One alternative is to use isolates cultivated directly from the soil site of
networks, in some cases, may be driven more by the richness of the interest for construction of native SynComs to increase the potential
samples rather than by individual interactions69. The main advantage for inclusion of site-adapted strains with desired functions.
with network analyses is that they provide an integrated and high-level There are several examples of SynComs that have been built for
view of the system that can point researchers in the right direction to specific soil environmental applications. A SynCom was developed
pursue more targeted experiments to test specific hypotheses. to provide tomato plants protection against bacterial wilt disease
caused by Ralstonia solanacearum73. The SynCom consisted of four
Environmental applications of soil inoculants Gram-positive bacteria and helped to confer disease resistance to
Soil inoculants have been developed for a variety of applications, tomato plants. Another example was the use of a SynCom to help
including bioremediation of pollutants, improvement of soil fertility alleviate water-deficit stress in maize74. This SynCom consisted of
and support of plant growth (Fig. 1). For example, the toxic pollutant 17 isolates from the sugarcane core microbiome75. Application of this
2,4-dinitrotoluene was degraded in contaminated soil by an engineered SynCom may have helped to stimulate production of plant osmolytes,
strain of Pseudomonas putida in association with plants70. However, the as suggested by the enrichment of ABC-type transporters for different
majority of recent studies have instead focused on promotion of plant osmolyte molecules in the genomes of the community members76. This
growth. We therefore mainly highlight that application in this Review. is an example of cross-species protection because the same SynCom
There is increasing interest in designing SynComs and NatComs was beneficial to both sugarcane and maize (Poaceae)75,76.
(Fig. 3) to promote plant growth and resilience in the face of climate While applications of SynComs to soil have led to some benefits,
change and other stress conditions. Design of SynComs for soil appli- there are also important concerns about applying non-indigenous
cations can, however, be impeded by lack of understanding of the microbial consortia to a soil ecosystem. In some cases, the applied
complexity of natural interactions that can occur between community SynComs can alter the existing microbiome, shifting the abundances

do so83. Because multi-species communities have been used for far

Positive
co-abundance less time than single-strain inoculants, a direct comparison of the two
(interaction) approaches does not provide equal weight to the scientific history of
each approach. Another approach to consider is inoculation with fully
High-degree
species complex communities. This approach relied on soil translocation to
Negative successfully to restore degraded soils84. Ultimately, the use of single
co-abundance
(interaction) species or communities comes down to the specifics of the plant being
targeted, the environmental conditions and the desired output.
High-
betweenness Strategies for genetic manipulation of
species microorganisms
New synthetic biology tools are continually being developed for
genetic transformation, strain optimization and biocontainment of
soil microorganisms85 that are designed to address current environ-
Fig. 4 | Schematic of a co-abundance network for microbial taxa. Microbial
taxa are represented with distinct shapes and colors. Potential interactions mental and sustainability challenges (Fig. 5). Synthetic biology is a field
between species (for example, exchange of metabolites, predation, cross- of science that incorporates aspects of both molecular biology and
feeding) are indicated by lines connecting taxa. These lines are drawn based on genetic engineering86. For example, advances in synthetic biology and
co-abundances of these species across a range of conditions or samples. Gray genetic engineering tools are being used to discover and enhance native
lines indicate positive co-abundances and may point to positive interactions, microbial functions87,88, introduce new traits89, develop biological sen-
while yellow lines indicate negative co-abundances and putative negative sors90, eliminate bottlenecks that limit critical pathways91 and combine
interactions. By using this approach at a community level, taxa that are critical multiple beneficial traits into a single organism92. Additionally, the
to the community can be identified. These may be species of ‘high betweenness’ environmental persistence of engineered functions can be controlled
(acting as a bridge point connecting two separate clusters of taxa) or ‘high through containment measures at the species and community levels.
degree’ (having a large number of connections to other taxa). To meet growing needs for synthetic biology studies on environmen-
tal isolates93, considerable advances have been made in the follow-
ing areas: (1) developing genetic tools for new soil microbial isolates,
of certain rhizosphere members based on interactions and recruiting (2) high-throughput genome-scale gene editing, (3) in situ microbial
new members from the existing rhizosphere community. This was community editing using conjugation and CRISPR–Cas technologies
found to be the case with the maize SynComs described above74,75. and (4) platforms to reproducibly interrogate plant–microbe interac-
Here, the added SynCom competed with the existing microbiome tions via microscopy and multiomics. An important ethical consid-
and displaced it, in the process promoting maize growth. However, eration for engineered microbes in soil environments is the potential
these experiments were carried out in a laboratory setting and over a ecological and biosecurity impacts of their environmental prolifera-
relatively short timeframe (4 weeks). In another case, SynComs directly tion. Despite substantial advances in the field, challenges remain in
added to field sites had a positive effect on plant growth77. To what predicting soil microbiome functions and interactions from available
extent the members of the added SynCom were still present was not data and algorithms and in optimizing engineered microbiome func-
evaluated in this case, but the effect of the SynCom addition was clear tions in the field.
even when applied in the context of an existing community77. Another Most synthetic biology to date has been performed on model labo-
study found a similarly positive effect of SynComs in promoting the ratory isolates that are used as an experimental chassis for DNA circuit
growth of wheat. The persistence of the SynCom was monitored in the and metabolic pathway design. For soil applications, the bacterium
native soil, finding that it dropped dramatically after 2 weeks78. These P. putida has been used extensively as a model soil microbial chassis94.
conclusions and other work79 suggest that some SynComs may be short Among other relevant applications for soil, P. putida has been used as
lived in native soil, and their use may therefore be limited by the need a model strain for bioremediation of environmental pollutants3. The
for repeated applications. explosion of interest and momentum in studying soil isolates and
Although a single-strain inoculum has the advantage of ease communities are likely to add hundreds of new microbial hosts com-
of growth and formulation compared to a multi-species inoculum, patible with synthetic biology practices. To date, a maize-associated,
consortia potentially allow for a wider range of desirable traits to be nitrogen-fixing strain of Klebsiella variicola was engineered to increase
added to soil and take advantage of the inherent benefits of micro- fixation of atmospheric nitrogen in the field by engineering the genome
bial interactions driving emergent functions. However, it must be to relieve gene-regulatory barriers91. Furthermore, root-associated
acknowledged that use of single inoculants or consortia depends Ralstonia and Pseudomonas strains have been engineered to increase
on the specific environmental application. A meta-analysis of over bioavailable phosphate for plant growth by breaking down phytate89.
400 experiments showed that single-species inoculations increased
crop growth up to 41% more compared80 to inoculations with con- Genetic transformation of new hosts
sortia of microorganisms. In addition, a combined treatment with Synthetic biology approaches have advanced considerably in the last
three fungal strains was less efficient at promoting strawberry plant decade to enable targeted optimization and design of microorganisms,
growth than a treatment consisting of a single strain81. Another study including soil isolates, for specific sensing or metabolic functions. Gene
with tomato plants found no synergistic effect of adding two species editing is a fundamental tool of synthetic biology that has undergone
(Bacillus subtilis and Azospirillum brasilense) compared to each spe- major recent advances for non-model hosts. Directed gene edits are
cies individually82. It should be noted that many of the studies in these primarily carried out through native recombination of homologous
analyses were focused on plants in controlled environments over short DNA, enhanced by approaches that increase recombination efficiency92
timeframes. In natural field soils, combined inoculations may perform or allow the selection of gene edits without antibiotic markers95. Preci-
better. For example, three strains of bacteria (belonging to Microbac- sion gene editing requires whole-genome sequence data and methods
terium, Stenotrophomonas and Xanthomonas) that were specifically for genetic transfer into the cell.
recruited in the rhizosphere of Arabidopsis thaliana plants provided Genetic transformation efficiency can be highly variable across
control of the downy mildew plant pathogen when they were inoculated microbial taxa, even at the genus level, and remains a technical
together in soil, but the individual-strain inoculants were not able to challenge for environmental isolates. Optimization of intracellular

Isolate engineering High-throughput genetics
Fabricated
ecosystems
NH3
Community gene editing Ecological containment
Rhizosphere Bulk soil
Fig. 5 | Platforms to edit and evaluate genome modifications of soil can indicate microbial function in the context of microbial communities and
microbiome isolates. Platforms and approaches in the image, starting accelerate the development of engineered functions. Community contexts
clockwise from the top left: (1) isolate engineering: establishing genetic tools provide insight into strategies to control the environmental persistence of
for soil microbiome isolates must overcome transformation barriers, such as engineered functions. (4) Ecological containment: specific strains can be
host-specific methylation patterns (colored dots on DNA strands), and use engineered to carry out a desired function at the target site, for example, the
genetic parts for gene editing and gene expression that are compatible with the rhizosphere. In this example, either engineering metabolic dependency on the
target host. (2) Fabricated ecosystems: the design, build, test and learn cycle plant or introduction of genes for which the products kill the cells when they
for soil microbiome engineering will be substantially accelerated by field- leave the rhizosphere can be effective biocontainment options. (5) Community
simulant fabricated ecosystems that reveal plant–microbe interactions and can gene engineering: this approach relies on plasmids to introduce functional
be reproducibly deployed across research laboratories. (3) High-throughput genes to other members of the soil community. Plants and bacteria are not
genetics: when employed at the genome scale, high-throughput genetic tools drawn to scale.
DNA-delivery efficiency has been realized through advances in elec- Multiple genome-editing approaches have been applied to engi-
troporation methods96,97. Low-volume microfluidic electroporation neer genetically stable bacterial strains that could be used for soil
enabled the transformation of combinatorial libraries97, whereas microbiome engineering. Chromosomally integrated DNA is more
large-volume, continuous-flow microfluidic electroporation enabled genetically stable and does not require antibiotics that are generally
the transformation of large mutant libraries at scale for strains with necessary to maintain plasmid DNA and impractical for deployment in
lower transformation efficiencies96. A major barrier to DNA stability soil environments. Recombineering is a short-homology recombina-
after entry in the cell is cellular defense mechanisms, such as the deg- tion method designed to integrate single-stranded oligonucleotides
radation of introduced nucleic acids via restriction endonucleases. or double-stranded DNA into microbial genomes and can be accom-
Restriction endonucleases degrade intracellular DNA that does not plished efficiently using homologous sequences as short as 35 bp106.
contain the host’s native methylation patterns. To overcome this bar- This approach was adapted to create CRISPR–Cas9 gene-edited mutant
rier, heterologous expression of methyltransferases from a target libraries that are genetically barcoded to map functions to individ-
host in a surrogate strain, such as Escherichia coli, enabled efficient ual genes in a high-throughput, genome-wide manner107. Another
transformation of plasmid DNA by mimicking the methylation patterns successful approach to genetic transformation is the construction
of the target host98. Native methylation patterns have been decoded of an engineered or minimal integrative and conjugative element
through single-molecule pore sequencing and custom workflows99, (mini-ICE) in B. subtilis to specifically engineer the genomes of target
which are likely to extend genetics to new soil isolates by creating hosts108. The mini-ICE system removes fragments that enable jumping
surrogate methylation strains that generate host-compatible DNA. from organism to organism, resulting in ‘one-hop’ modifications of
microbial genomes, with efficacy in Gram-positive bacilli, including in
High-throughput strain optimization soil environments.
Certain technologies allow for high-throughput and tailored opti- Phage recombinase-based methods have emerged as power-
mization of microbial strain phenotypes (Fig. 6). High-throughput ful techniques for engineering large gene clusters and creating
approaches for genome integration and transformation enable opti- high-throughput genome variant libraries. Recombinase-assisted
mization of strain function and genetic stability in genetically tracta- approaches are more universally effective than recombineering
ble hosts. These approaches include the creation and evaluation of strategies, which have primarily found success in model hosts such
gene expression libraries across taxonomically diverse microbes100,101, as E. coli92. By contrast, chassis-independent recombinase-assisted
engineered protein libraries102 and combinatorial pathway-expression genome engineering (CRAGE)109 demonstrated the integration of
libraries103. These approaches overcome uncertainties regarding large biosynthetic gene clusters in dozens of proteobacteria hosts
sequence–function relationships by creating and screening mas- using Cre–lox recombination and was used to show that engineered
sive collections of functional variants. The advent of deep-learning bacteria can release phosphate to plants89. Using a collection of up
approaches applied to synthetic biology104,105 could rapidly accelerate to ten independent integration sites and their cognate recombi-
the realization of designer functions in soil microorganisms through nases, serine recombinase-assisted genome engineering (SAGE)101
genome engineering. has been demonstrated to integrate heterologous DNA constructs in

a Site-specific recombination b Homologous recombination
SAGE CRAGE Cargo
Target
Cargo
CRISPR–Cas
attP
Target Cargo
attB
ssCRISPR Genome-wide
CRISPR–Cas
attL Cargo attR
c Conjugative transfer
MAGIC
d CRISPR–Cas transposase
INTEGRATE
DART
PAM
Mini-ICE
PAM
Fig. 6 | Genome-editing approaches for soil microbiome engineering. a, Site- to selectively promote or kill strains in microbial communities (ssCRISPR) or to
specific recombination uses serine recombinases (SAGE, shown in graphic) or create genome-wide pooled mutant libraries. c, Direct community editing has
tyrosine recombinases (CRAGE) to integrate engineered DNA into specific sites been demonstrated using conjugative transfer, using an E. coli host (MAGIC)
on bacterial chromosomes, including those of soil isolates, with high efficiency. to transfer a replicative or integrative plasmid to the mouse microbiome
b, Engineered homologous recombination, or recombineering, relies on native or a Bacillus host expressing a mini-ICE to Gram-positive bacteria in soil
or engineered proteins to create deletions, insertions or directed mutations in communities. d, Engineered CRISPR–Cas transposase complexes can create
DNA using single-stranded or double-stranded substrates with homology for the directed gene edits without homologous recombination (INTEGRATE) and have
target site. Recombineering methods enhanced by CRISPR–Cas counterselection been demonstrated to engineer members of microbial consortia (DART). PAM,
ease introduction of mutation without selection markers and can be employed protospacer-adjacent motif.
five taxonomically diverse hosts as efficiently as plasmid transforma- that, under some circumstances, persisted in a mouse host for at least
tion, which conventionally transforms orders of magnitude more 11 days. Although MAGIC was demonstrated in the mouse gut, similar
efficiently. SAGE enables construction of combinatorial strain librar- approaches could be extended to transform a soil microbiome with
ies through iterative integration of DNA constructs and was used in an engineered function in a host-agnostic manner.
multiple plant-derived soil isolates to evaluate a panel of hundreds Direct genome edits within complex communities have been
of promoters from taxonomically diverse species as tools for genetic enabled by engineering advances in high-efficiency CRISPR–Cas sys-
circuits or metabolic pathways. tems. The insertion of transposable elements by guide RNA-assisted
targeting (INTEGRATE), an engineered version of recently discov-
Engineering microbial communities in situ ered CRISPR–transposon systems, enabled high-efficiency genome
Recent developments show promise for in situ control of microbial integration of large genetic constructs without selection markers95.
community composition, host-agnostic community function and DNA-editing all-in-one RNA-guided CRISPR–Cas transposase (DART),
host-specific genome edits, including those that originate from soil a similar CRISPR–transposase system, was exploited to establish
(Fig. 6). Strain-specific CRISPR (ssCRISPR) was developed to edit the host-specific genome editing within a nine-member, synthetic soil
membership of a microbiome community and selectively purified or microbial community and extended to infant gut microbiome sam-
eliminated members of a synthetic collection of model hosts110. The ples112. Efficient DART targeting was enabled by environmental trans-
approach uses CRISPR–Cas guide RNA species unique to a target host formation sequencing of the microbial community, a method to
to elicit strain-specific DNA cleavage and cell death or to introduce an identify genetically transformable members of a mixed community
antibiotic-resistance marker gene to selectively enrich a target strain. by mapping genome-integration events of a transposable element
Metagenomic alteration of the gut microbiome by in situ conjugation by high-throughput sequencing. Genome editing in microbial com-
(MAGIC) was developed to transfer a function to the community via munities enabled by environmental transformation sequencing could
conjugation without specific knowledge of the metagenome or the take advantage of accessibility of metagenome-assembled genomes
genetic tractability of community members111. In MAGIC, a replicative (MAGs) from soil metagenomes, although rare species might not be
or integrative plasmid was transferred by an E. coli donor to a complex represented. While these advances represent unprecedented access
community to confer a fluorescence or antibiotic-resistance phenotype to genome editing within a microbial community, substantial hurdles

remain to assess the efficacy of these in situ editing approaches in other specialized chamber systems have been developed to study
non-laboratory environments. plant–microbe interactions123. Testing systems are also available to
determine the fate and efficacy of engineered and native soil micro-
Containment of engineered microbes organisms for environmental applications. For example, fabricated
When deploying microbial systems to promote soil and plant health, ecosystems (ecoFABs) provide standardized platforms for plant–
biosecurity is often a critical consideration. In this context, ‘biosecurity’ microbiome studies across laboratories that promote reproducible
refers to preserving the resilience of native ecosystems and restrict- findings (Fig. 5)124. Another platform is the ‘rhizochip’, which uses
ing the proliferation of engineered functions beyond the expected structured polymer molds to mimic irregular soil particles and leads to
spatiotemporal operating bounds. Developing effective containment root growth and exudation profiles that mimic soil growth125. Rhizochip
approaches for engineered microbial functions could help minimize growth assays are compatible with light microscopy and spatial mass
regulatory hurdles and public hesitation regarding engineered micro- spectrometry. To enable spatially resolved investigations of plant–
bial systems being used in the environment. Risk assessment involves microbe interactions, three-dimensional-printed ‘rhizogrid’ scaffolds
several steps that need to be evaluated before field application. These allow integration of X-ray computed tomography, high-throughput
include extensive identification and evaluation of potential adverse taxonomic profiling and metabolomics data from segmented soil and
consequences113. Therefore, despite the original promise and excite- rhizosphere samples at multiple depths126. Currently, an opportunity
ment about the potential applications of genetically modified micro- exists to increase the field relevance of these platforms by integrating
organisms, there have been few recent examples of field releases. The synthetic, reproducible soil systems to emulate the geochemistry of
majority have focused on non-food applications, such as bioremedia- regional soils using laboratory reagents and resources127.
tion of heavy metals and xenobiotic compounds and/or biostimulation
of the rate of microbial degradation of pollutants113–115. Currently, it Engineering the rhizosphere of the plant host
is difficult to gauge how readily future applications that target crop Another approach for engineering the soil microbiome is to engineer
productivity and climate-change mitigation will be approved. the plant to produce specific compounds in the rhizosphere that select
Traditionally, containment measures for soil microorganisms have for beneficial members of the community. This approach relies on the
relied on introduction of amino acid auxotrophies, environmentally ability of plant root exudates to shape the composition of soil micro-
triggered toxins or combined use of these approaches116. For example, organisms associated with their roots. For example, root exudation
a strain of P. putida was engineered to be metabolically dependent on of sucrose promoted colonization of the beneficial soil microorgan-
phosphite, a rare compound in nature117. The strain was engineered both ism B. subtilis on the roots of A. thaliana plants128. Differences in root
to assimilate phosphite and disable its ability to transport and grow on exudation of specific flavonoid compounds also influenced the ability
phosphate. Synthetic biology has introduced other new approaches, of arbuscular mycorrhiza to colonize the invasive plant Triadica sebif-
including the integration of multiple signals, and inherent redundancy era128. Production of benzoxazinoids that are defensive metabolites
to suppress escape via individual mutations. These approaches include produced in roots resulted in alteration of the rhizosphere microbiomes
introduction of non-canonical amino acids118 and environmentally of maize plants129. Barley engineered to produce rhizopine130 coupled
sensitive kill switches that incorporate logic gates119. More recently, with a synthetic nitrogen-fixation phenotype regulated by rhizopine
increased genetic stability of kill switches has been demonstrated by in the bacterium Azorhizobium caulinodans enabled activation of
introducing redundancy120. Despite early interest in biocontainment of nitrogen fixation by the microbe only in the presence of the modified
genetically engineered microorganisms designed for soil applications, barley131,132. These studies suggest that plants may be engineered to
there are only a few recent examples85. One example is the control of the produce specific root-exudate compounds for targeted recruitment
persistence of Bacillus thuringiensis spores by introducing a genetic of beneficial microbes, natural or engineered. A related approach is
circuit that prevented sporulation121. An important consideration for to stimulate growth of beneficial microbes by conditioning soil with
biocontainment is to avoid the use of antibiotic-resistance genes that specific root exudates that are known to recruit beneficial microbes.
could potentially spread and contribute to the growing health concern For example, combinations of fatty acids and amino acids enriched
of antibiotic-resistant pathogens122. While these technical advances different rhizosphere microbes when compared to combinations of
provide new tools to control the function of engineered microbial organic acids and sugars. Several bacterial isolates were isolated from
systems in the environment, efficacy in field environments remains a the soil enrichments, and some of them protected the host from a foliar
frontier for biosecurity research related to the release of engineered pathogen infection91. Identification of plant quantitative trait loci that
microbial functions. are responsive to specific beneficial microorganisms and/or environ-
mental conditions can also be used for plant selection132.
Soil microbiome-testing platforms
A challenge is scaling results from the laboratory and the greenhouse Remaining gaps and opportunities for soil
to the field. We acknowledge that laboratory studies are invaluable microbiome engineering
for gaining detailed knowledge regarding species interactions, how Although this Review has summarized the current state of the sci-
they can be harnessed and modified, what the keystone species are ence, several hurdles remain before wide-scale implementation of
and the development of tools to change these species to gain more soil microorganisms can be applied for tackling pressing issues in the
from the soil. However, laboratory studies are not ‘the field’, and care face of climate change, an increasing global human population and a
must be taken that the information gained is physiologically and eco- decline in wildlife habitat and biodiversity. Soil microorganisms have
logically relevant to field processes. Although direct analysis of soil the potential to alleviate issues associated with these challenges, but we
microorganisms in the field, for example, using multiomic analyses, are still largely working in context with several unknowns with respect
is progressing, it remains a daunting challenge due to the complexity to understanding the functional potential of soil microorganisms, how
of the natural environment. they work together as a community and how they can best be collected
The development of new testing platforms allows for a detailed and formulated to provide the desired beneficial outcome. Further-
study of complex, field-relevant systems in a laboratory setting. As more, with respect to working with genetically engineered strains,
these tools become more common, they can be used to generate spe- regulatory hurdles remain that limit their widespread application.
cific hypotheses that can be tested in a native field system, bridging the The largely untapped potential of soil microorganisms presents
gap between laboratory and field for a more detailed but translational a tremendous opportunity for mitigating many of the challenges fac-
understanding of soil microbial systems. For example, rhizotrons and ing the environment. These opportunities include using natural or

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128. Tian, B., Pei, Y., Huang, W., Ding, J. & Siemann, E. Increasing Additional information
flavonoid concentrations in root exudates enhance associations Correspondence and requests for materials should be addressed
between arbuscular mycorrhizal fungi and an invasive plant. to Janet K. Jansson.
ISME J. 15, 1919–1930 (2021).
129. Hu, L. et al. Root exudate metabolites drive plant–soil feedbacks Peer review information Nature Biotechnology thanks Giles Oldroyd,
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detection of quantitative trait loci for growth at low phosphorus Springer Nature or its licensor (e.g. a society or other partner) holds
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three to tango: the importance of microbes, host plant, terms of such publishing agreement and applicable law.
and soil management to elucidate manipulation strategies
for the plant microbiome. Can. J. Microbiol. 66, 413–433 © Battelle Memorial Institute, under exclusive licence to Springer
(2020). Nature America, Inc. 2023

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nature biotechnology

Review article https://doi.org/10.1038/s41587-023-01932-3

Soil microbiome engineering for

Accepted: 1 August 2023

Nature Biotechnology | Volume 41 | December 2023 | 1716–1728 1716

Nutrient runoff Salinity Drought

Heavy metal Pesticide Overfertilization

Nature Biotechnology | Volume 41 | December 2023 | 1716–1728 1717

function (reviewed in ref. 24). Depth of coverage is especially critical for

Nature Biotechnology | Volume 41 | December 2023 | 1716–1728 1718

Nature Biotechnology | Volume 41 | December 2023 | 1716–1728 1719

Complete soil microbiome

Nature Biotechnology | Volume 41 | December 2023 | 1716–1728 1720

do so83. Because multi-species communities have been used for far

Nature Biotechnology | Volume 41 | December 2023 | 1716–1728 1721

Isolate engineering High-throughput genetics

Community gene editing Ecological containment

Rhizosphere Bulk soil

Nature Biotechnology | Volume 41 | December 2023 | 1716–1728 1722

a Site-specific recombination b Homologous recombination

SAGE CRAGE Cargo

attL Cargo attR

Nature Biotechnology | Volume 41 | December 2023 | 1716–1728 1723

Nature Biotechnology | Volume 41 | December 2023 | 1716–1728 1724

Nature Biotechnology | Volume 41 | December 2023 | 1716–1728 1725

Nature Biotechnology | Volume 41 | December 2023 | 1716–1728 1726

Nature Biotechnology | Volume 41 | December 2023 | 1716–1728 1727

123. Yee, M. O. et al. Specialized plant growth chamber designs to Acknowledgements

Nature Biotechnology | Volume 41 | December 2023 | 1716–1728 1728

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