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Engineering probiotics for therapeutic applications:
recent examples and translational outlook
Nikhil Aggarwal1,2, Amy M Ehrenworth Breedon3,4,
Christina M Davis3,4, In Young Hwang1,2 and
Matthew Wook Chang1,2
Engineered probiotics are the next generation of live
biotherapeutics that have been genetically modified to target
specific diseases. With the advancements in synthetic biology,
the engineering of probiotics has become increasingly
sophisticated which has led to the development of therapies for
treating cancer, infection, metabolic disorders and
inflammation, as well as for diagnosing and preventing them.
Herein, we review some of the recent examples of probiotics
which have been engineered to target such diseases. Although
there are numerous examples of engineered probiotics
showing efficacy in animal models, there are no approved
products on the market with very few in clinical trials. Therefore,
we also discuss a set of features that may be incorporated into
engineered probiotics to aid in clinical translation and
ultimately, realizing the potential of these biotherapeutics.
Addresses
1
Department of Biochemistry, Yong Loo Lin School of Medicine,
National University of Singapore, 8 Medical Drive, Singapore, 117596,
Singapore
2
NUS Synthetic Biology for Clinical and Technological Innovation
(SynCTI), Centre for Life Sciences, National University of Singapore,
28 Medical Drive, Singapore, 117456, Singapore
3
UES, Inc., Dayton, OH, 45432, United States
4 promising approach as it enables functionalization of the
Air Force Research Laboratory, 711th Human Performance Wing,
Wright-Patterson Air Force Base, OH, 45433, United States
Corresponding author: Chang, Matthew Wook (bchcmw@nus.edu.sg)
Current Opinion in Biotechnology 2020, 65:171–179
This review comes from a themed issue on Chemical Biotechnology
Edited by Christoph Wittmann and Sang Yup Lee
For a complete overview see the Issue and the Editorial
Available online 15th April 2020
https://doi.org/10.1016/j.copbio.2020.02.016
0958-1669/ã 2020 Elsevier Ltd. All rights reserved.
Introduction
Probiotics are living microorganisms that confer a health
benefit to the host when administered in adequate amounts
[1]. Some of the commonly used probiotics are Escherichia
coli Nissle 1917, Saccharomyces boulardii, and species
belonging to the Lactobacillus and Bifidobacterium genera,
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such as Lactobacillus reuteri, Lactobacillus casei, Lactobacillus
rhamnosus, Bifidobacterium infantis and Bifidobacterium
breve. In recent years, these wild-type probiotics, either
individually or in combination, have been assessed in
numerous clinical trials against a plethora of diseases rang-
ing from infections and allergies to metabolic diseases and
inflammation. Meta-analyses of these clinical trials have
shown that probiotics are effective in preventing antibiotic-
associated and traveller’s diarrhoea, alleviating symptoms
of irritable bowel syndrome, treating paediatric acute
diarrhoea, eradication of Helicobacter pylori infection and
protection against atopic dermatitis [2,3]. Although it is
seen as a promising strategy to treat different diseases, other
meta-analyses have shown that probiotics had no significant
effect in managing dental caries [4], prevention of allergies
[5] or treatment of depression [6] and anxiety [7].
The efficacy of the probiotics is both strain and disease
dependent [8], and often, the mechanism of action of the
probiotic is unknown, hindering further development of
the therapeutic product. Genetically engineering the
probiotic strains to target a specific disease may be a
probiotic with multiple beneficial attributes, depending
upon the disease to be targeted. This may result in higher
efficacy compared to the wild-type strains. Therapies
based on engineered probiotics can offer several advan-
tages over existing therapies, such as cheaper formula-
tions with fewer associated negative side effects. In case
of diseases without existing treatment options, engi-
neered probiotics may be a novel method to treat the
disease. In this review, we highlight some of the recent
examples of engineered probiotics developed to target
various diseases. We also discuss a set of characteristics of
engineered probiotics, such as safety and regulated
expression of the genetic modifications, that may be
required for the successful approval of the live biother-
apeutics by regulatory bodies.
Engineered probiotics for treating diseases
Genetic engineering of probiotics is becoming easier with
the advancements made in the field of synthetic biology
[9]. It is now feasible to reprogram probiotics with increas-
ingly complex functionalities. As such, engineered pro-
biotics have moved from simple design of constitutive
expression of therapeutic molecules or enzymes to more
complex design that enables the combined use of sense
Current Opinion in Biotechnology 2020, 65:171–179
172 Chemical biotechnology
and respond features [10]. Recent examples of such
engineered probiotics for various therapeutic interven-
tions are discussed below (summarized in Table 1).
Cancer
The concept of treating cancer with bacteria has been
around for decades (for reviews, see Refs. [11,12,13,14]).
This is especially due to the tendency of obligate and
facultative anaerobic bacteria, such as Clostridium, Salmo-
nella, Bifidobacterium and Escherichia, to colonize in the
solid tumours. With an increased versatility in genetic
engineering, there has been an increase in efforts to
engineer these bacteria for targeted cancer treatments.
The modified bacteria can be used to either deliver anti-
cancer compounds, elicit an immune response against
cancer or complement with existing therapies for superior
efficacy [14].
In many cases, the tumour-targeting capabilities of E.
coli Nissle 1917 and attenuated Salmonella typhimurium
have been exploited to deliver anti-tumour payloads in
tumour-bearing mice. Li et al. engineered E. coli Nissle
1917 to produce cytotoxic compounds whereas He et al.
engineered the bacteria to secrete tumour suppressor
protein p53 and anti-angiogenic protein Tum-5 within
the tumours [15,16]. S. typhimurium has also been used to
secrete Vibrio vulnificus flagellin B (FlaB) in tumour
tissues, which induces the innate immune response
leading to suppressed tumour growth and metastasis
in animal models, with no observed toxicity [17]. A more
complex genetic engineering was performed by
Chowdhury et al. who engineered an E. coli strain to
lyse specifically within the tumours and release a nano-
body antagonist of CD47, resulting in anti-cancer effects
in syngenic mouse tumour models [18]. The authors
placed a bacteriophage lysis protein, fX174E, under a
promoter which is activated by the quorum-sensing
molecule, N-acyl homoserine lactone (AHL), produced
by the bacteria. As the E. coli strain colonizes the
tumours, the levels of AHL will increase and after
crossing a particular threshold, lysis of the bacterial cells
and subsequent release of the nanobody will be
triggered.
To enhance the binding of the probiotics to the
tumours, Ho et al. engineered E. coli Nissle 1917 to
express histone-like protein A (HlpA) from Streptococcus
gallolyticus which enables the probiotic to bind to the
heparan sulphate proteoglycan (HSPG) on the colorec-
tal cancer (CRC) cell surface. In addition, the authors
engineered E. coli Nissle to secrete myrosinase to
convert the inactive glucosinolate, found in cruciferous
plants, into the anti-cancer agent sulforaphane [19].
The engineered probiotic induced cancer cell death in
vitro and reduced tumour size and occurrence in CRC
mice model.
Current Opinion in Biotechnology 2020, 65:171–179
Infections
With the mounting concern of antibiotic-resistant bacte-
ria and lack of novel antibiotics, there has been an
increase in the efforts to develop novel therapeutics to
target pathogens. Engineered probiotics provide a prom-
ising approach as therapies can be developed with high
specificity without concomitant damage to the microbiota
of the host [20]. One such example is E. coli Nissle
1917 engineered to kill Enterococcus faecium and Entero-
coccus faecalis, the two prominent species responsible for
vancomycin-resistant Enterococcus (VRE) infections [21].
The probiotic was engineered to secrete three antimicro-
bial peptides which specifically kill Enterococcus. Peng
et al. overexpressed the myosin cross-reactive antigen
gene (mcra) in L. casei to enhance the production of
conjugated linoleic acids which have antimicrobial
activity against enteric pathogens, Campylobacter jejuni,
S. typhimurium and enterohaemorrhagic E. coli [22,23].
Apart from the production of anti-microbial molecules,
probiotics have also been engineered to sense and respond
to pathogens, such as Pseudomonas aeruginosa, Vibrio cholerae
and Candida albicans. Hwang et al. improved their sense and
respond circuit comprising an anti-P. aeruginosa toxin,
pyocin S5, released in response to the AHL produced by
P. aeruginosa by additionally incorporating an anti-biofilm
enzyme, dispersin B in E. coli Nissle 1917 [24]. The
engineered probiotic showed therapeutic as well as pro-
phylactic effects in both Caenorhabditis elegans and mouse
infection models. A similar strategy was adopted by Jayara-
man et al. who engineered an E. coli strain to sense CAI-1,
the quorum-sensing molecule produced by V. cholerae, and
induce self-lysis to secrete an endolysin with antibacterial
activity against the pathogen [25]. In the first reported use
of an engineered microbe targeting fungal infections, an
E. coli strain was engineered to detect hydroxyphenylacetic
acid, a novel molecule secreted by C. albicans. This sensory
system was coupled to the production of a hypha inhibitor,
cis-2-dodecenoic acid, which inhibits filamentation and
virulence gene expression in C. albicans. In an in-vitro
model, the engineered microbe was shown to protect
epithelial cells from fungal-induced damage [26].
Inflammation
Probiotics have been shown to ameliorate inflammation as
they can correct the resultant dysbiosis and influence the
host immune system. As such, probiotics have found
application in treating allergies and inflammatory bowel
diseases (IBD), including Crohn’s disease and ulcerative
colitis [27,28].
Studies have been undertaken to engineer bacteria for
diagnosis of inflammation and IBD. E. coli NGF-1 was
engineered to detect tetrathionate, produced during
inflammation, by incorporating the TtrR-S two-component
system from S. typhimurium and retain the memory of
exposure which is determined by the expression of
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Table 1
Summary of probiotics engineered for diagnosing, preventing or treating diseases
Disease Engineered microbe
Cancer E. coli Nissle 1917
E. coli Nissle 1917
Attenuated S. typhimurium
E. coli Pir1+
E. coli Nissle 1917
Infection E. coli Nissle 1917
L. casei
E. coli Nissle 1917
E. coli strains
E. coli NGF-1.
E. coli Nissle 1917
Inflammation E. coli Nissle 1917
E. coli NGF-1
E. coli Nissle 1917
E. coli strains
E. coli W3110
L. lactis NZ9000/IL1403
B. longum HB25
Metabolic disorders L. plantarum CM_PUJ411
L. reuteri 100 23C
E. coli Nissle 1917
E. coli Nissle 1917
Diagnosis L. reuteri DSM20016
L. lactis subsp. cremoris
MG1363
Vaccination S. cerevisiae strains
L. lactis NZ3900
L. casei ATCC 393
B. thetaiotaomicron VPI-5482
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Engineering probiotics for therapeutic application Aggarwal et al. 173
Result Ref.
Probiotic engineered to secrete cytotoxic compounds. Anti-tumour effect [15]
was observed in-vitro and in tumour-bearing mice.
Engineered bacteria secreting therapeutic proteins showed antitumor effects [16]
in tumour-bearing mice.
Engineered bacteria expressing flagellin B suppressed tumour growth and [17]
metastasis in mice models.
Probiotic engineered to produce a nanobody antagonist of CD47 which [18]
elicits an immune response resulting in tumour regression in a syngenic
tumour mice model.
Probiotic engineered to bind to cancer cells and produce cytotoxic [19]
sulphoraphane from cruciferous vegetables. The probiotic induced cancer
cell death in vitro and reduced tumour size and occurrence in colorectal
cancer mice models.
Engineered microbe secreting antimicrobial peptides against vancomycin- [21]
resistant Enterococcus was effective in a mouse model.
Probiotic producing linoleic acid to target enteric pathogens was effective [22,23]
in-vitro.
Probiotic engineered to sense and kill P. aeruginosa showed therapeutic and [24]
prophylactic effects in C. elegans and mouse infection models.
Engineered microbe was able to sense and kill V. cholerae in-vitro. [25]
Engineered probiotic could detect C. albincans and produce a hypha [26]
inhibitor to prevent damage to Caco-2 epithelial cells.
a
Engineered probiotic showed the ability to inhibit growth of Salmonella in- [58]
vitro in response to the inflammation caused by the pathogen.
a
Intranasal administration of an engineered microbe expressing a birch-grass [59]
pollen chimera reduced inflammation and poly sensitization in mice.
Engineered probiotic detected tetrathionate and retained memory of [29]
exposure in mice models (analysed by faecal testing).
The bacteria engineered to detect thiosulfate or tetrathionate, with the former [30]
being a likely biomarker for inflammation in DSS-treated mice.
The bacteria engineered to have motility and secrete a biotherapeutic protein [31,32]
in response to nitric oxide, an intestinal biomarker for Crohn’s disease.
Engineered microbe showed H2O2 induced directional motility in transwell [33]
and microfluidic models.
The microbe was engineered for cell surface expression or secretion of anti- [34–36]
inflammatory molecules and demonstrated activity in in-vitro or in-vivo
models.
Production of a human superoxide dismutase by the probiotic penetrated the [37]
cells reduced DSS-induced ulcerative colitis in mice.
Secreted enzyme from the engineered bacteria transported through a Caco- [40]
2 cell monolayer and showed in vitro reduction of Phe for the treatment of
PKU.
Engineered bacteria expressing phenylalanine lyase reduced blood Phe [41]
levels in PKU model mice.
Engineered probiotic expressing enzymes for breakdown of Phe functioned [42]
in both PKU mouse models and healthy monkeys.
Engineered probiotic decreased blood ammonia levels in [43]
hyperammonaemia mice models and was well tolerated in healthy monkeys
and human volunteers.
Engineered microbe was able to sense the quorum sensing molecule AIP-I [44]
produced by Staphylococcus aureus in the range of 10 9-10 7 M.
Engineered bacteria detected the quorum sensing molecule CAI-1 from V. [45]
cholerae with the output enzymatic reporter observed in the mice faeces.
Surface expression of the HIV-1 Gag protein on the probiotic matured [48]
dendritic cells with antigen presentation in-vitro.
Probiotic expressing H. pylori adhesin A increased IgG levels in mice. [49]
Probiotic expressing the toxoid of C. perfringens a-toxin induced an immune [50]
response in mice which provides protection against the natural toxin of the
pathogen.
The microbe was engineered to express antigens from the plague causing [51]
pathogen Y. pestis. Intranasal delivery of the probiotic elicited antibody
production which killed the pathogen in the non-human primate host.
Current Opinion in Biotechnology 2020, 65:171–179
174 Chemical biotechnology

Table 1 (Continued )
Disease Engineered microbe Result Ref.
a
Other diseases E. coli Nissle 1917 An engineered heme-sensing probiotic produced a fluorescence signal [60]
which was detected by an ingestible bio-electronic micro device to wirelessly
transmit the signal outside the body. The device communicated signal in
response to gastrointestinal blood in pigs and also demonstrated extended
applications when combined with thiosulfate-sensing and AHL-sensing
probiotics for sensing inflammation and infection.
a
L. lactis subsp. cremoris Engineered probiotic expressing glucagon-like peptide-1 demonstrated [61,62]
therapeutic efficacy in mice model of neuro-inflammation and Parkinson’s
disease.
a
L. reuteri Production of IL-22 by the bacteria led to reduced liver damage and [63]
inflammation in mice fed an ethanol diet.
a
L. paracasei ATCC 27092 The microbe was engineered to secrete and transport angiotensin converting [64]
enzyme 2 and showed therapeutic effects in diabetic retinopathy mice
models, including reduced inflammatory cytokine expression and acellular
capillaries.
a
Denotes references not discussed in the text.

b-galactosidase analysed in the faecal samples from mice Metabolic disorders

[29]. E. coli Nissle 1917 was also engineered to sense both
thiosulfate and tetrathionate by identifying novel sensors
from marine Shewanella species and incorporating them into
the E. coli probiotic strain. The study showed that thiosul-
fate but not tetrathionate can be a novel biomarker for the
widely used dextran sodium sulfate (DSS) mouse model of
colitis [30]. A cell-based sensor for Crohn’s disease was
developed by McKay et al. by using a nitric oxide (NO)-
responsive promoter to drive the expression of a reporter
gene. Levels of NO are elevated in the intestinal fluids of
patients with IBD and thus, a probiotic-based sensor of NO
can be used to diagnose the disease [31]. The group further
engineered the NO sensor to control pseudotaxis, targeting
the probiotic to sites of elevated NO, and to secrete a
therapeutic protein, granulocyte macrophage-colony stim-
ulating factor, in response to the signalling molecule [32]. In
another example of programming motility and directional-
ity via pseudotaxis, Virgile et al. engineered E. coli to target
the microbe to hydrogen peroxide (H2O2), a reactive oxy-
gen species associated with IBD and other diseases and
showed induced directional motility in trans-well and
microfluidic models [33].
Apart from diagnostics, probiotics can also be engineered to
treat inflammation. For example, a Lactococcus lactis strain
was engineered to express blockers of IL-23 receptor on its
surface to suppress a pro-inflammatory cascade and prevent
the development of inflammatory diseases [34]. L. lactis was
also used to deliver pentadecapeptide, BPC-157 to treat
gastrointestinal (GI) inflammation and IL-35 to ameliorate
arthritis [35,36]. Bifidobacterium longum was used as a deliv-
ery vehicle for human manganese superoxide dismutase
(MnSOD) to reduce the levels of reactive oxygen species,
which are associated with ulcerative colitis. A cell-
penetrating peptide was fused to MnSOD for transport
into the host cells. The probiotic was found to successfully
express the therapeutic protein and reduce the DSS-
induced ulcerative colitis in mice [37].
Metabolic disorders are caused by abnormalities in the
enzymes involved in the host metabolism which results in
a build-up or deficiency of specific metabolites [38].
These disorders can have debilitating and potentially
lethal effects and can be difficult to treat, often requiring
severe lifestyle restrictions and expensive treatments
[39]. Engineered probiotics can provide a promising
alternative treatment for such disorders.
Phenylketonuria (PKU) is an inherited genetic disorder
caused by the loss of function of phenylalanine hydroxylase
(PAH). This results in an accumulation of L-phenylalanine
(Phe) in the blood, which can cause neurotoxicity at high
concentrations. Reduction of Phe to treat PKU has been a
common target of engineered probiotics in recent years. For
example, Lactobacillus plantarum was engineered to secrete
human PAH in the GI tract [40]. The secreted enzyme was
found to be active and able to cross the monolayer of colon
cancer cells in-vitro. L. reuteri was also genetically modified
to produce phenylalanine lyase (PAL), an enzyme which
breaks down Phe, and was able to reduce blood Phe levels
in a PKU mice model [41]. In a more advanced study,
Isabella et al. developed SYNB1618 by engineering E. coli
Nissle 1917 to degrade Phe by having the probiotic to
highly express PAL and L-amino acid deaminase (LAAD)
under anoxic conditions [42]. SYNB1618 successfully
reduced serum Phe levels in a PKU mouse model and
healthy Cynomolgus monkeys challenged with dietary
Phe. The authors also encoded a biocontainment module
in the probiotic by rendering the bacteria auxotrophic for
diaminopimelate, which is required for cell wall biosynthe-
sis and growth but not found in the intestine of mice and
soil. SYNB1618 is currently being evaluated for efficacy and
safety for human use in clinical trials (ClinicalTrials.gov
identifier NCT03516487).
Hyperammonaemia, or high ammonia levels in the blood,
is associated with diseases such as urea cycle disorders

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 Engineering probiotics for therapeutic application Aggarwal et al. 175

(UCDs) and hepatic encephalopathy (HE). Kurtz and co- [48]. Other examples of oral vaccine development include
workers engineered E. coli Nissle 1917 to treat hyper- engineering L. lactis and L. casei as vaccines for H. pylori
ammonaemia by converting ammonia into L-arginine and Clostridium perfringens, respectively [49,50]. In both
[43]. In their engineered probiotic SYNB1020, the cases, an antigen from the pathogen (H. pylori adhesin A
authors upregulated arginine biosynthesis and introduced protein and toxoid of C. perfringens a-toxin, respectively)
thymidine auxotrophy for biocontainment. SYNB1020 was expressed by the probiotic which elicited an immune
decreased blood ammonia levels in both HE and UCD response in mice models. Carvalho et al. used a gut
mice models and was well tolerated in healthy mice, commensal bacterium, Bacteroides thetaiotaomicron, to
monkeys, and human volunteers. deliver plague protective antigens in the respiratory tract
of non-human primates and observed the generation of
serum antibodies that are able to kill the plague bacte-
Diagnosis and prevention of infections
rium Yersinia pestis [51].
Rapid detection of infection is an essential step in its
treatment regime as it will enable early administration of
therapy potentially saving lives and preventing further
Translation of engineered probiotics
Although there have been numerous examples of
spread of infection. Engineered probiotics can be used to
engineered probiotics targeting different diseases and
detect both Gram-negative and Gram-positive pathogens
showing high efficacy in in-vivo models, there are no
by incorporating mechanisms to sense novel metabolites
commercially available products that have been approved
or quorum-sensing molecules produced by the pathogen.
by the regulatory bodies, with very few currently in
In the study by Lubkowicz et al., the authors engineered
clinical trials (Table 2). The majority of engineered
L. reuteri to sense autoinducer peptide -1 (AIP-1), the
biotherapeutics have not been evaluated in clinical trials
quorum-sensing molecule produced by Staphylococcus
due to various reasons, including time and financial
aureus [44]. L. reuteri was engineered to detect AIP-1
considerations and design of the biotherapeutic. Depend-
by adapting the agr quorum sensing (agrQS) system from
ing upon the disease and the proposed use of the engi-
S. aureus into the probiotic, functionalizing it to sense
neered probiotics, the design of the clinical trials to
AIP-1 in nanomolar to micromolar range. Mao et al.
evaluate the therapeutic outcome will vary. However,
reported a diagnostic for V. cholerae by engineering
certain features may be incorporated into every
L. lactis to detect CAI-1 from the pathogen [45]. The
engineered probiotic for successful drug development
sensing system was developed by constructing a two-
(Figure 1). These features are in accordance with the
component system comprising the sensor kinase from
guidelines set out by the US Food and Drug Administra-
V. cholera and the response regulator from L. lactis. The
tion (Guidance on Live Biotherapeutic Organisms,
engineered probiotic was able to detect the pathogen in
docket number FDA-2010-D-0500) as well as the Euro-
mice resulting in the secretion of the b-lactamase reporter
pean Pharmacopoeia Commission (EPC) [52] and are
in the faecal pellets, the activity of which can be visual-
discussed below.
ized by a colorimetric assay.

Probiotics have also been previously engineered as oral
vaccine vectors for infections from numerous bacterial
pathogens and viruses [46,47]. Recently, probiotic Sac-
charomyces cerevisiae was genetically engineered to express
the HIV Gag protein on its surface and was found to be
able to induce specific T cell immune response ex-vivo
Absence of plasmids and antibiotic resistance genes
Both the US-FDA and EPC require the absence of
antibiotic resistance genes in the engineered probiotic
as they can potentially be transferred to the natural
microbial flora of the humans leading to the emergence
of antimicrobial-resistant organisms. Antibiotic resistance

Table 2

Examples of engineered probiotics in clinical trials

Engineered probiotic Disease Company/university Stage ClinicalTrials.gov

identifier
B. longum to deliver the plasmid Solid tumours IQVIA Phase 1 NCT04025307
for IL-12 expression
E. coli Nissle 1917 to metabolize Phenylketonuria Synlogic Phase 1/2a NCT03516487
phenylalanine
E. coli Nissle 1917 to metabolize Cirrhosis and hepatic Synlogic Phase 1/2 NCT03447730
ammonia insufficiency
L. lactis to secrete human Trefoil Oral mucositis in patients Actobio therapeutics/Oragenics Phase 2 NCT03234465
Factor 1 with head and neck cancer
L. lactis to deliver human proinsulin Type 1 diabetes Actobio therapeutics Phase 1b/2a NCT03751007
and hIL-10

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176 Chemical biotechnology

Figure 1

(a) (b)

R
GOI Ab

Input 1 Input 2 Input 1 Input 2 Input 1 Input 2 Input 1 Input 2

Cell death Cell death Cell death

Cell death

Regulated Biocontainment
expression
Current Opinion in Biotechnology

(a) Engineered probiotics should be devoid of plasmids with antibiotic resistance genes (AbR). Instead, the gene of interest (GOI) should be
integrated into the genome with its expression regulated by an inducer/repressor system along with a biocontainment module. (b) Pathway of
engineered probiotics after in vivo testing. During the growth in a bioreactor both the expression of GOI and the biocontainment module should be
switched OFF. In the human gut, only the expression of GOI should be switched ON to exert the therapeutic effect. When the probiotic is released
into the environment the biocontainment module should be switched ON to prevent the growth of the probiotic strain.

genes are usually present on plasmids and other mobile
genetic elements that can be transferred from one
microbial cell to another [53]. Due to a close physical
proximity and increased cell-to-cell contact in the GI
tract, the gene transfer can occur at high frequency
among the gut microbes [54]. To prevent this transfer of
genes, the engineered probiotic should be devoid of
mobile genetic elements, and the genetic modifica-
tions, either overexpression of existing genes or intro-
duction of new genes, should be integrated into the
genome of the probiotic. This may lead to a reduction
in the expression levels of the gene, but the levels can
be further improved by introducing multiple copies of
the gene or by using stronger promoters and ribosome
binding sites.
Genetic stability
Before being prepared for administration to humans,
engineered probiotics may be grown in fed-batch bior-
eactors wherein the genetic modifications introduced may
be mutated as the probiotic is grown to high densities,
resulting in loss of therapeutic efficacy. This is highly
probable particularly in the cases where the genetic
modifications exert a high metabolic burden on the cell
and hinder its growth. Therefore, to provide stability to
the genetic modifications, these genes can be dynamically
regulated by using inducible or repressor systems. In both
cases, the expression of the genes can be switched off
when the probiotic is grown in the bioreactor, but the
expression may be switched on when administered to
humans. In the absence of the expression during the
growth in the bioreactor, the genes will not exert a
metabolic burden on the cell and thus, are less likely
to be mutated. Previously, the anaerobic-inducible pro-
moter has been used to regulate the expression of the
enzymes in E. coli Nissle [42].
Human safety
Probiotics are usually non-pathogenic and safe for
human consumption. However, there are studies that
engineer attenuated pathogens to develop therapies for
different diseases [17,55]. In these cases, the safety
profile of the micro-organism should be evaluated in
clinical trials to ensure that the attenuated pathogen is
safe for use in humans. In addition, the colonization
time of the probiotic strains in humans should also be
assessed. Although it is known that the commonly used
probiotic strains transiently colonize the GI tract [56],
novel probiotics or attenuated pathogens maybe long-
term persistors, and methods to eliminate them from
the GI tract, such as by using specific antibiotics, should
be known to prevent overdosing and other harmful
effects.

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 Engineering probiotics for therapeutic application Aggarwal et al. 177
Environmental safety
After being eliminated from humans, the engineered
probiotics will be released into the environment through
the faecal matter. These probiotics should be devoid of
the proliferative activity in the environment to ensure
that no harm is caused to the natural flora and fauna. To
enable this, biocontainment strategies can be adopted
which include genetically modifying the probiotic to
replicate under permissive conditions only. This can be
achieved by introducing synthetic auxotrophy into the
probiotic, wherein the probiotic requires an essential
nutrient for growth which is not found in the natural
environment. Another method of biocontainment is to
use synthetic kill switches in which a self-killing product
is produced by the probiotic in response to external
stimuli from the environment [57].
Conclusion and future perspective
As seen from the examples discussed above, probiotics
have been engineered to target a wide range of diseases
with some demonstrating high efficacy in animal models.
The engineering ranges from simple constitutive expres-
sion of proteins to complex genetic circuits combining
sense and respond features. In the future, more complex
systems, such as those involving the use of multi-input/
output circuits and microbial consortia, are highly likely.
On the basis of the encouraging results observed in in-
vivo studies, engineered probiotics are well-suited to fulfil
an unmet need of novel therapeutics for diseases, such as
cancer, infections and metabolic disorders. However,
before these probiotics can proceed to clinical translation
safety concerns will have to be addressed along with
ensuring compliance with the guidelines set out by vari-
ous health regulatory agencies.
Moving forward, it is anticipated that probiotics will be
engineered to develop safer, cheaper and more effective
therapeutics for broader applications, including other
diseases and perhaps even conditions related to human
performance.
Conflict of interest statement
Nothing declared.
Acknowledgements
We’d like to thank Dr Michael Goodson (AFRL) for guidance and
feedback. This work was supported by the Synthetic Biology Initiative of
the National University of Singapore (DPRT/943/09/14), Summit Research
Program of the National University Health System (NUHSRO/2016/053/
SRP/05) the Ministry of Defence of Singapore (MINDEF, RE2016-074),
the National Research Foundation Investigatorship of the National
Research Foundation of Singapore (NRF-NRFI05-2019-0004), the US Air
Force Office of Scientific Research - Asian Office of Aerospace Research
and Development (FA2386-18-1-4058), and the US Office of the Secretary
of Defense’s Applied Research for the Advancement of Science and
Technology Priorities program.
www.sciencedirect.com
References and recommended reading
Papers of particular interest, published within the period of review,
have been highlighted as:
 of special interest
 of outstanding interest
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