SLAS Technology 29 (2024) 100120

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SLAS Technology
journal homepage: www.elsevier.com/locate/slast

Employing synthetic biology to expand antibiotic discovery

Greta D. Cook , Nikolas M. Stasulli *
Department of Biology and Environmental Science, University of New Haven, 300 Boston Post Rd, Dodds Hall 316, West Haven 06516 USA

A R T I C L E I N F O A B S T R A C T

Keywords: Antimicrobial-resistant (AMR) bacterial pathogens are a continually growing threat as our methods for
Synthetic biology combating these infections continue to be overcome by the evolution of resistance mechanisms. Recent thera­
Biosynthetic gene cluster (BGC) peutic methods have not staved off the concern of AMR infections, so continued research focuses on new ways of
Cryptic BGCs
identifying small molecules to treat AMR pathogens. While chemical modification of existing antibiotics is
Natural products (NPs)
Mutagenesis
possible, there has been rapid development of resistance by pathogens that were initially susceptible to these
Endogenous gene expression compounds. Synthetic biology is becoming a key strategy in trying to predict and induce novel, natural anti­
Heterologous gene expression biotics. Advances in cloning and mutagenesis techniques applied through a synthetic biology lens can help
characterize the native regulation of antibiotic biosynthetic gene clusters (BGCs) to identify potential modifi­
cations leading to more potent antibiotic activity. Additionally, many cryptic antibiotic BGCs are derived from
non-ribosomal peptide synthase (NRPS) and polyketide synthase (PKS) biosynthetic pathways; complex, clus­
tered genetic sequences that give rise to amino acid-derived natural products. Synthetic biology can be applied to
modify and metabolically engineer these enzyme-based systems to promote rapid and sustainable production of
natural products and their variants. This review will focus on recent advances related to synthetic biology as
applied to genetic pathway characterization and identification of antibiotics from naturally occurring BGCs.
Specifically, we will summarize recent efforts to characterize BGCs via general genomic mutagenesis, endoge­
nous gene expression, and heterologous gene expression.

1. Introduction antibiotic resistant genes, bacteria possessing multiple ARGs become

The current antibiotic resistance crisis can largely be attributed to
the overuse, misuse, and lack of new discoveries of antibiotic treat­
ments. The identification of ‘modern’, biologically produced antibiotics
began with Sir Alexander Fleming’s discovery of penicillin in 1928
[1–3]. After the first sulfonamide antibiotic was introduced for use in
humans in 1937, it didn’t take more than a hand full of years before
resistance was reported [4]. Alongside the use of antibiotics to treat
infection, antimicrobial-resistant (AMR) pathogens have evolved to
overcome many of the antibiotics that are now commonly used [1,5,6].
Antibiotic resistance can occur through genetic mutations within the
genome or through horizontal gene transfer (HGT), acquiring foreign
DNA containing antibiotic resistance genes (ARGs) via transformation,
transduction, or conjugation [6,7]. With the increase in use of antibi­
otics across human populations, animal populations, and within the
environment (agriculturally) the likelihood of bacteria interacting via
HGT to evolve, acquire, and accumulate new ARGs, making them
resistant to multiple antibiotics, increases. Through the accumulation of
labeled multidrug-resistant (MDR) or extremely drug resistant (XDR) [1,
6]. One problem with current broad-spectrum antibiotics is they lack
chemical diversity [8,9]; over 70 % of antibiotics developed between
1981 and 2005 being modifications of four basic chemical scaffolds
(cephalosporins, macrolides, penicillins, and quinolones) [10]. More
distinct chemical scaffolds such as glycopeptides, lipopeptides, amino­
glycosides, and tetracyclines are also produced in some more specific
antibiotics, but they are applied narrowly in certain circumstances for
selected bacterial infections [8].
Many antibiotics are naturally occurring, secreted compounds from
bacteria and fungi and are therefore considered natural products (NPs).
They are produced by microbes in response to interactions within their
communities and have vast chemical diversity and complexity, giving
them more diverse bioactivity than we can currently generate on an
industrial level [8]. Antibiotic NPs are considered secondary, or
specialized, metabolites and are synthesized from metabolic in­
termediates. They are considered secondary metabolites because they
are not required for the growth or survival of the bacteria, but rather

* Corresponding author.
E-mail address: nstasulli@newhaven.edu (N.M. Stasulli).

https://doi.org/10.1016/j.slast.2024.100120
Received 16 June 2023; Received in revised form 4 January 2024; Accepted 7 February 2024
Available online 8 February 2024
2472-6303/© 2024 The Authors. Published by Elsevier Inc. on behalf of Society for Laboratory Automation and Screening. This is an open access article under the
CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
G.D. Cook and N.M. Stasulli
play important roles in protection, molecular signaling, and environ­
mental interactions [11]. Natively produced antibiotics are small mol­
ecules containing complex chemical structures that act as a type of
microbial warfare in the fight for resources. These small molecule NPs
have evolved to interact with biological macromolecules, making novel
NPs excellent candidates for new antibiotics [8]. As noted previously,
the NPs we currently know of produce chemical diversity around basic
antibiotic molecule scaffolds: peptides, polyketides, carbohydrates, al­
kaloids, or terpenes backbones [8]. These structural cores of secondary
metabolite antibiotics are produced by biosynthetic enzyme families:
nonribosomal peptide synthetases (NRPS), polyketide synthases (PKS),
terpene synthases (TPS), and dimethylallyl-tryptophan synthases
(DMATS) [12–14]. This core is further modified by tailoring enzymes
and regulatory proteins to create chemical diversity. The genes encoding
the biosynthetic enzymes, tailoring enzymes, regulatory proteins, and
transportation genes are all clustered around the same location in the
chromosome [12]. These clusters are collectively known as biosynthetic
gene clusters (BGCs).
Biosynthetic gene clusters encode many small molecule NPs, or
secondary metabolites, in bacteria [15]. These BGCs are being targeted
for their efficient production of complex natural products that would be
much harder to generate under laboratory settings. One issue that arises
when studying BGC is that many are transcriptionally and translation­
ally silent under typical laboratory growth conditions, making any po­
tential secondary metabolite, itself, inaccessible and difficult to study
[15,16]. Secondary metabolites are usually regulated by an external
stimulus; if the stimulus is absent the metabolite will not be synthesized
[12]. BGCs that produce secondary metabolites are detectable in mi­
crobial genome sequences and can be identified using genomic
sequencing, and automated genome mining tools [16]. Programs such as
Secondary Metabolite Unique Regions Finder (SMURF) [17] and Anti­
biotics and Secondary Metabolite Analysis Shell (antiSMASH) [18,19]
use advanced computer bioinformatics algorithms to predict and iden­
tify BGCs within genomic data [12]. BGCs that are identified, but not
associated with known secondary metabolites, are termed “cryptic”
because they have been previously undetectable or transcriptionally
silent. Once these cryptic BGCs are located in the genome bioengi­
neering techniques can be applied to manipulate the genome, stimu­
lating the expression of the encoded NP or potentially modifying it to
generate more chemical diversity.
Bioengineering and molecular cloning principles and techniques can
be applied to biological systems that produce protein or small molecule
products that have defined and controllable properties. These tech­
niques generally aim to transform genes, gene circuits, and metabolic
pathways to modify, reinvent, or build novel biological systems [20,21].
Synthetic biology can take advantage of these techniques to contribute
to the fight against AMR bacterial pathogens by expanding the chemical
diversity of antibiotics through activating and manipulating cryptic
BGCs encoding potentially novel antibiotic secondary metabolites.
Techniques focused on genomic mutagenesis, endogenous gene
expression, and heterologous gene expression can be utilized to elicit
and study potentially novel antibiotics from understudied and envi­
ronmental microbes [22,23]. Outside of antibiotic discovery and pro­
duction, synthetic biology also has potential applications in biofuel
production, industrial chemical/natural product substitution synthesis,
biomedical and bacterial infection applications, disease diagnosis, and
treatment through the same methods that can be used to address the
AMR crisis [20].
In this review, synthetic biology’s role in studying cryptic antibiotic
BGCs through genomic mutagenesis, endogenous gene expression, and
heterologous gene expression will be explored. Within mutagenesis
strategies, genetically modifying bacteria, we review recombination-
mediated genetic engineering (recombineering), Multiplex Automated
Genome Engineering (MAGE), Targetrons, Global transcription ma­
chinery engineering (gTME), and clustered regularly interspaced short
palindromic repeats (CRISPR-Cas9) systems. Endogenous gene
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SLAS Technology 29 (2024) 100120
expression focuses on the manipulation of antibiotic BGC transcription
through cultural condition modifications, classical genetic techniques,
and chemical genetic techniques [12,24]. Heterologous gene expression
focuses on the reconstruction of BGCs into model microbial strains and
involves cloning and reconstructing biosynthetic pathways [25,26].
Being able to activate, characterize, and manipulate antibiotic BGCs
from microbes is one of our best strategies for identifying novel, or more
potent, antibiotics that can help address the growing concern of AMR
pathogens. Synthetic biology can offer new options to fight the rapid
emergence of AMR by genetically engineering bacteria to identify new
scaffolds, enzymatically modify existing scaffolds, and purify com­
pounds to generate chemical libraries for antibiotic screening. With the
help of these technologies, the efforts around BGC manipulation and
characterization are developing rapidly and can be the driver for the
next generation of antibiotics. Recent reviews on activating cryptic BGCs
[27] and genomic engineering through synthetic biology [28] have been
thorough, however, in this review we focus specifically on recent
application of synthetic biology for unlocking BGCs to identify and
characterize novel antibiotics.
2. Flexible genomic mutagenesis strategies
Mutations in an organism’s DNA, via various mutagenesis mecha­
nisms, are responsible for generating both genotypic and phenotypic
variation in a population [29]. Through molecular editing techniques
bacterial genomes can be artificially edited by purposefully inducing
mutations. Synthetic biologists can take advantage of mutagenesis
mechanisms in the lab setting to search for novel antibiotic variants after
inducing mutations into genomes [30]. While this is the optimal
outcome in a lab setting, naturally occurring mutations can be either
advantageous or deleterious to the bacteria’s survival depending on its
environment. Mutagenesis strategies employed in synthetic biology,
whether random or site-directed, aim to create and identify advanta­
geous mutations that have affected the BGCs involved in antibiotic
production. Techniques used for inducing directed mutations to genet­
ically modify bacteria include CRISPR systems [31], recombineering
[32], MAGE [33], and Targetron vectors [34]. While some of the tech­
niques discussed in this section have direct applications to endogenous
and heterologous gene expression systems, we aim to give an overview
of common mutagenesis strategies synthetic biologists could take
advantage of when activating and studying cryptic BGCs (Table 1).
Recombineering, or recombination-mediated genetic engineering,
describes the incorporation of mutations into a genome by surrounding
the desired modifications with homologous regions of DNA. [32,35].
These homologous nucleotide sequences are identical to sequences
present in the genome where the mutation or genetic element being
introduced will be located. Homologous recombination-based strategies
introduce specific genetic elements (full genes, small mutations, new or
modified promoters, etc.) into the genome and require various, some­
times bacteria specific, recombination proteins along with the known
homologous DNA sequences [35,36]. One common example of this type
of system is the λ-Red recombinase system [37] that can be used for
introducing insertions, deletions, or point mutations. This mechanism
utilizes the introduction of the desired mutation amongst homologous
genomic regions of DNA for recombination [35]. The λ-Red recombinase
system uses the lambda bacteriophage and Exo, Beta, and Gam proteins
to introduce genomic mutations [36,37]. The Gam protein protects
linear DNA from endogenous RecBCD and SbcCD nucleases [38]. The
Exo protein is a double stranded DNA-dependent exonuclease possessing
5′ to 3′ activity to degrade dsDNA to create ssDNA or partially degraded
dsDNA with 3′ overhangs [38,39]. The Beta protein protects the ssDNA
created by Exo protein and aids in annealing within the target cell [39].
By introducing mutations, the λ-Red recombinase system aims to modify
genes within the biosynthetic gene cluster to exchange modules of BGCs
to produce second-generation derivatives of existing antibiotics. As an
example, the λ-Red recombinase method was used to inactivate clo-hal
G.D. Cook and N.M. Stasulli SLAS Technology 29 (2024) 100120

Table 1 protein-based transcriptional interference system that can control pro­

Summary of flexible mutagenesis strategies. tein expression levels. This CRISPRi technology silences transcription
Category Strategy Examples of use using designed guide-RNA and a mutant Cas9 protein without replacing
or modifying the gene promoter region [30,50]. While this system is
Recombination-mediated Methods of DNA mutagenesis [32,35–42]
genetic engineering relying on homologous more in line with endogenous expression control, it will be covered here
(recombineering) sequences surrounding the for content consistency. CRISPRi systems have the potential to offer
region of interest specific gene knockouts with little to no off-target binding [51]. The
CRISPR/Cas9 systems Methods of DNA mutagenesis • Basic CRISPR/ mutant dCas9 protein lacks nuclease activity but retains the ability to
relying on specific targeting of Cas [43–46]
the exact DNA sequence that is to • with EvolvR
form a complex with the guide-RNA. When binding to protein-coding
be modified. Relies on designed [47,48] genes, the CRISPRi technique can block transcription, controlling gene
RNA molecules for targeting • CREATE [31, expression without altering the genome sequence. The CRISPRi tech­
DNA and a Cas endonuclease for 49] nique has been successful in knocking out gene expression within yeast
cleavage of DNA at the site of • CRISPRi
[52] and E. coli [50]. CRISPRi methods have the potential to be utilized
mutagenesis. [50–52]
Multiplex automated High-throughput methods, using • Basic MAGE in studying BGCs by selectively knocking down the expression-specific
genome engineering specifically designed pools of [55] genes within a gene cluster to study regulation or modifications of
(MAGE) synthetic oligonucleotides, that • Cos-MAGE antibiotic compounds. CRISPR/Cas9 systems have become a tool in
induce mutations by taking [33] synthetic biology for their precise editing, multigene editing, and precise
advantage of natural cellular • with CRISPR
processes. [56,58]
regulating as recently reviewed thoroughly by Li et al. [53]. They are
• Port-MAGE effective tools for generating targeted mutations with potentially
[57] high-throughput capacity [54].
• dReaMGE A more high-throughput technique (a feature that is always desirable
[60]
when bio-prospecting for new or modified molecules) the Multiplex
Targetrons Methods of using retrohoming • Basic
with a modified type-II intronic targetron [34, Automated Genome Engineering (MAGE) method, was developed at the
RNA, with ribozyme and reverse 61–63,67] Wyss Institute, Harvard Medical School, to mimic the natural principles
transcriptase activity, to target • ClosTron [64] of evolution in genome editing [55]. MAGE uses synthetic oligonucle­
mutation incorporation into the • with CRISPR otide pools and the intrinsic bacterial cell cycle, along with nucleotide
genome. [63,65,66]
introduction (cell induction, incorporation of ssDNA, and trans­
Global transcription Using error-prone polymerase [58,68–71]
machinery engineering chain reaction to generate a formation via electroporation) and cell recovery (outgrowth) methods,
(gTME) mutant library, usually targeting to genetically modify cells and grow them over generations. The MAGE
a transcription factor that may system, for the first time, allowed multiple, small mutational insertions
regulate BGCs.
to be introduced at different locations simultaneously [30,55]. Multiple
constructed, site-specific DNA segments are introduced to a bacterial
and cloz genes within the biosynthetic gene cluster responsible for population and the cells are manipulated, via transformation tech­
producing the antibiotic clorobiocin. The BGC was then engineered to niques, to take up the DNA, ultimately leading to homologous recom­
add a methyl group to create a clorobiocin analog. Unfortunately, in this bination and introduction of genomic diversity into the population.
case, this modification reduced potency compared to the original com­ After each recovery period, post mutation incorporation, more DNA can
pound [40]. This method has also been used for the biosynthesis of the be transformed to promote the accumulation of insertions. All of the
antibiotic daptomycin (cubicin), which has been approved in the US to oligonucleotides are specifically created, not randomly generated se­
treat skin infections caused by Staphylococcus aureus [41]. quences, so this method can be used by synthetic biologists to target
Second-generation derivatives of daptomycin have been made using the various parts of a BGC at once. The MAGE method has been modified
lambda-red recombinase system by modifying multiple modules of its and refined to tailor to new hypotheses and desired outcomes of ex­
NRPS gene cluster [42]. periments. The Co-selection MAGE (CoS-MAGE) [33],
The clustered regularly interspaced short palindromic repeats CRISPR-optimized MAGE-recombineering [56], and pORTMAGE [57]
(CRISPR)/Cas9 system is becoming more and more popular and is used systems are examples of refined MAGE techniques. CoS-MAGE selects
as a genome editing tool. CRISPR gene editing will be mentioned several subpopulations of cells that already have acquired mutations and are
times as part of the improvement of other techniques, but is also useful known to be more-highly competent. The selecting of subpopulations
on its own, allowing for site-specific gene editing within bacterial cells already containing a mutation increases the likelihood of adding more
[43]. The CRISPR system is relatively easy to use and constantly being mutated sites through further rounds of MAGE; the selected clones being
refined and modified to manipulate the genome [44,45]. This system more electrocompetent or more amenable to olgio-based editing. This
targets specific DNA sites through complementary base pairing of selection of known, recombination-competent cells allows for a higher
designed guide-RNA, inducing a double-strand DNA break, via the Cas9 recombination efficiency of larger insertions (<2 % efficiency for >20
nuclease, at a specific protospacer adjacent motif (PAM) sequence. bp insertion in the original MAGE protocol) during subsequent rounds of
Either non-homologous end-joining or homology-directed repair MAGE DNA incorporation. This increased efficiency of incorporation of
mechanisms are used to complete the editing procedure after the large base pair insertions can allow for directed insertion of entire
introduction of the desired mutation [43,46]. The CRISPR/Cas system promoter sequences at more than one site in the genome [30,33].
can be applied with the EvolvR system [47] for the introduction of However, the CoS-MAGE system was not sufficient to improve the
semi-random mutations at locations targetable by CRISPR/Cas9. A overall recombineering efficiency of the first round of MAGE. An addi­
mutant nicking Cas9 protein (nCas9) is coupled to an error-prone DNA tional adaptation using CRISPR/Cas system was applied to further
polymerase I to introduce mutations [48]. This EvolvR system could be optimize recombineering [56,58]. The CRISPR-optimized MAGE-r­
used to target and induce mutations to activate and/or modify BGCs. ecombineering system allows for the introduction of mutations within
CRISPR has also been used to generate and identify genetic variations multiple genomic locations at a much higher efficiency [58]. The MAGE
using the CRISPR-enabled trackable genome engineering (CREATE) system also developed into the portable MAGE (pORTMAGE) system
system [30,49]. The CREATE system can be used to produce genetic [57] to introduce multiple mutations to many bacterial genomes
variant libraries for evaluation [31]. A variation of the CRISPR system, simultaneously using a single plasmid for transformation with little to no
CRISPR interference (CRISPRi), uses a nuclease-deficient Cas9 (dCas9) off-target mutagenesis [57]. Antithetical to the goals of this review, the
pORTMAGE system has been used to introduce 10 antibiotic-resistance

3
G.D. Cook and N.M. Stasulli
mutations into the genomes of Salmonella enterica and Escherichia coli to
study the conservation of antibiotic-resistant molecular mechanisms
within these bacteria [57]. Overall, the MAGE system edits the bacterial
genome by inducing mutations at multiple target sites to generate
functional changes in the cell and can be applied in synthetic biology
research to modify promoters, ribosomal-binding sites (RBSs), or protein
coding genes to optimize biosynthetic pathways for NP production [59].
Most recently, the double-stranded DNA Recombineering-assisted
Multiplex Genome Engineering (dReaMGE) system allows for rapid
mutagenesis through insertions and deletions of kilobases at multiple
sites without double-strand breaks or mismatch repair [60]. The
dReaMGE system has been applied to manipulate BGCs for inactivation,
promoter replacement, and combinatorial modification of transcrip­
tional regulators for the improved production of NPs and the discovery
of additional derivatives [60]. Specifically, In Schlegelella brevitalea DSM
7029 dReaMGE was used to simultaneously replace native promoters of
the glbC, glbF, and glbB genes within the glb BGC to enhance the pro­
duction of the antibiotic glidobactin A [60].
Site-specific mutagenesis can also be achieved through Targetrons,
modified mobile type-II introns that target specific DNA sites by RNA-
DNA base pairing. Type-II introns are capable of inserting themselves
into DNA sequences after they are spliced out of RNA during post-
transcriptional processing. The RNA intron utilizes its ribozyme activ­
ity, breaking and forming bonds with nucleic acids, to reverse splice and
insert directly into the target DNA site [34,61]. This ability of ribozymes
to insert into specific DNA target sequences is termed retrohoming and is
the basis of this technique [62]. Targetrons are redesigned type-II
intronic RNA, used for site-specific mutagenesis, consisting of an auto­
catalytic intronic RNA (ribozyme) and an intron-encoded reverse tran­
scriptase [28,63]. Utilizing RNA-based introns is advantageous because
they are independent of the host’s recombination machinery and
contain heterologous sequences that can be easily re-targeted for future
gene delivery and insertions [63]. ClosTron technology is one
Targetron-based system used to successfully edit bacterial genomes [28,
64]. The ClosTron system incorporates a specialized
retrotransposition-activated selectable marker (RAM) site within the
type-II intron. The RAM is an inactivated marker that becomes activated
by the relocation and insertion of the type-II intron [64]. The ClosTron
system was initially based on the ermB gene encoding for erythromycin
resistance [64]. The ermB gene is inactivated by the insertion of a DNA
fragment including a type-I intron in a way that blocks splicing of the
mRNA transcript, resulting in no production of the ErmB protein
(leaving the bacteria now susceptible to erythromycin) [64]. RAM ele­
ments specific to other antibiotic resistance genes should theoretically
be able to be created using specific exon sequences required for splicing
within the targeted structural gene relating to antibiotic resistance [64].
Targetron systems can also be paired with CRISPR to overcome an in­
tron’s limitation of splicing and insertion when containing foreign DNA
[63,65,66]. The system has also recently been modified to include
temperature induction of targetron mutagenesis (Thermotargetron)
[67]. Synthetic biologists can take advantage of the ability of targetrons
to knock in and knock out sequences, or full genes, and may be able to
manipulate gene function or regulation to optimize the expression of
BGCs.
A final mutagenesis strategy useful to synthetic biologists may be the
Global transcription machinery engineering (gTME) system, a muta­
genesis strategy using error-prone polymerase chain reaction (PCR) to
produce a random mutant library with genetic variation in a desired
transcription factor(s) [30,58]. These mutated transcription factors will
ideally generate transcriptomic expression changes during growth to
unlock specific phenotypes [68,69]. The gTME strategy was first used in
yeast to improve its ethanol tolerance and the production efficiency of
glucose to ethanol for applications in biofuel production [70]. It has also
been successfully used in bacteria to engineer organic-solvent tolerant
E. coli strains for industrial applications [68,71]. While not yet used in
the search for naturally created antibiotic variations, the gTME method
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SLAS Technology 29 (2024) 100120
has the potential to modulate BGC expression by employing random
mutagenesis to activate BGC transcription and produce novel antibiotic
diversity. Synthetic biology has been applied in and along with muta­
genesis strategies to try and induce mutations at specific locations,
ideally, not randomly, to activate cryptic BGCs. Mutagenesis strategies
optimally stimulate changes within the genome to alter gene expression
and are being applied to cryptic BGCs to activate them directly or
indirectly by knocking out or in genomic content in hopes of inducing
the novel NP production.
3. Endogenous gene expression
Endogenous, or homologous, gene expression specifically focuses
around altering the levels of a gene being transcribed within an organ­
ism [72], and generally refers to overexpression or repression of genes
within the organism it originates. The advantage of studying a gene
endogenously is that it is tested in the context in which it resides and
physiological relevance is more likely to be observed. However,
endogenous studies are not always possible. This may be most relevant
when trying to identify novel antibiotics since some environmentally
identified NP producers are not culturable under lab conditions; pre­
venting the use of endogenous expression studies [27,73–78]. Within
synthetic biology BGC studies, procedures around endogenous expres­
sion focus mainly on the manipulation of promoters, the overexpression
of transcriptional activators, and the elimination of transcriptional re­
pressors to control transcription [12]. In the context of searching for
novel antibiotic BGCs, these techniques are most effectively utilized in
cases where a culturable and genetically manipulatable organism has
been found to contain cryptic gene clusters (Table 2).
As noted previously, cultivation conditions of an organism can
impact gene expression and the expression of BGCs. More times than
not, what is ‘optimal’ growth as defined in a lab setting is not optimal for
the expression of BGCs. Alterations in temperature and nutrient avail­
ability have been shown to contribute to the activation of BGCs and their
natural product synthesis [74,79,80]. For example, a 2 ◦ C increase in the
culture temperature of Streptomyces hygroscopicus increased the tran­
scription of the validamycin BGC [79]. Typically, culture modification is
the first step in trying to activate cryptic BGCs by replicating environ­
mental stressors. One method of altering culturing conditions to stim­
ulate NP production is called one strain-many compounds (OSMAC)
[81]. OSMAC has enhanced the production of terrosamycin, through test
growth Streptomyces sp. RKND004 in 14 different media, which has
antibacterial activity similar to other polyether ionophores and also
demonstrated anticancer activates [82]. OSMAC methods can be effec­
tive at mimicking the environmental stress conditions to activate cryptic
BGCs but they lack the presence of other microbes and their interactions;
another contributing factor to the modulation of gene expression. Mi­
crobial cocultures, which contain two or more bacterium, take advan­
tage of inter-bacterial interactions to try and activate cryptic metabolites
[83–88]. The paradigms for interaction are most often nutrient
changes/starvation, signaling molecules, physical contact, and antibi­
osis via cytotoxic compounds [27,89]. For example, the biosynthesis of
NPs is altered when colonies of Streptomyces coelicolor and Myxococcus
xanthus are grown next to each other in low-iron conditions. In this
environment, M. xanthus upregulates a BGC expressing an iron-chelating
siderophore, myxochelin, while S. coelicolor upregulates a BGC
expressing an antibiotic, actinorhodin, that inhibits M. xanthus growth
[90,91]. Environmental stress and the presence of different microbes
can obviously influence the NP synthesis, potentially unlocking cryptic
antibiotics. However, when altering culture conditions fails to activate
BGCs and their natural product expression we can turn to synthetic
biology for other endogenous expression methods via both classical and
chemical genetics [27].
The alteration of bacterial genomes through classical genetics, to
manipulate endogenous gene expression, includes techniques such as
promoter manipulation, transcription factor manipulation, and
G.D. Cook and N.M. Stasulli
Table 2
Summary of endogenous gene expression strategies.
Category Strategy Implementation
Cultural Condition Growth condition • Culture the same bacterial
Modifications alterations: one strain strain in media with
many compounds differing growth conditions
(OSMAC) to elicit or modify the
production of metabolites
[74,79–82]
○ Temperature
○ Carbon source and other
nutrients
○ Inducing stress
Coculture • Involves multiple organisms
to model bacterial
interactions [83–88]
• May allow for both physical
and/or chemical
communication [27,89–91]
Classical Genetics Direct DNA • Reporter-guided mutant
using cloning modifications selection of BGCs [92–95]
strategies • Regulatory gene deletion,
(mutagenesis and duplication or modification
reporters) [96–104,108]
• Promoter manipulation,
introduction, or
replacement [105,106,108]
Transcription factor • Creation of transcription
manipulation factor decoys to manipulate
repressor binding [12,103]
• Manipulation of
transcription factor levels
[105,107]
• Usage of synthetic
transcription factor [105,
107]
RNAP engineering • Mutations in ribosomal or
RNA polymerase genes that
lead to transcriptional
changes [109–113]
Chemical Genetics ‘Microbial hormone’ • The use of bacterial
treatment signaling molecules to
manipulate gene expression
[114–118]
High throughput • Use reporter gene under
elicitor screening native promoter to detect
(HiTES) transcription during
challenge with a screen of
natural product molecules
[119–122]
• Detection of transcriptional
changes from natural
products without
mutagenesis: Bioactivity
HiTES [123,124],
HiTES-imagine mass spec­
trometry [125]
repressor deletion [27]. Strategies such as genome-wide and
transposon-based mutagenesis have been combined with reporter-based
assays and mass spectrometry to activate and discover new novel
products possessing antimicrobial activity [73,92–95]. In one example,
Guo et al. successfully applied reporter-guided mutant selection (RGMS)
to activate the pga silent BGC, encoding an unknown product, in Strep­
tomyces sp. PGA64. This technique employs random mutagenesis and a
promoter-reporter system to monitor transcription of targeted gene
clusters. This led to the discovery of two new anthraquinone amino­
glycoside antibiotics: gaudimycin D and E [94]. Silent BGC regulators
can be induced to elevate natural product production through the
introduction of synthetic promoters or the insertion of multiple copies of
a regulatory gene [96–101]. For example, large ATP binding regulators
from the LuxR family (LAL) of proteins were constitutively overex­
pressed to encourage binding to promoter regions upstream/within a
BGC in Streptomyces ambofaciens to identify glycosylated macrolides,
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SLAS Technology 29 (2024) 100120
stambomycins [101], and in Streptomyces venezuelae to discover a new
biaryl polyketide, venemycin [102]. The inactivation of repressors
through gene deletion or transcription factor decoys can also increase
the expression of BGCs and their natural product synthesis [103].
Recently, hybrid regulatory proteins like LuxR- and LysR-type regulators
with native DNA binding domains and ligand-binding domains have
been used to control the expression of BGCs [104]. For example, a LuxR
response regulator, which regulates a cryptic BGC in Pseudomonas sp.
SZ57, was replaced with a mutated LuxR containing its DNA-binding
domain and ligand-binding domains from a LasR protein and a Lux
protein capable of binding to 3-oxo-decanoyl-homoserine lactone (HSL)
[104]. In the presence of the HSL ligand, the LuxR protein upregulated
the cryptic BGC in Pseudomonas sp. SZ57 to discover the NP pseudo­
momol [104]. In addition to upregulated hybrid proteins, synthetic and
artificial transcription factors can also upregulate the expression of BGCs
[12]. Furthermore, genomic sequences can be specifically modified. For
instance, upstream activation sequences (UASs) have been integrated
into native promoters to reprogram the transcriptional regulation sys­
tem of targeted genes to interact with specific transcription factors. This
method had been successful in Aspergilli [105], Ustilago maydis [106],
and Trichoderma reesei [107]. However, the replacement of native pro­
moters with inducible or constitutively active promoters, rather than
their modification, can be the most direct way to control BGC expression
[108]. The native promoter responsible for the production of 6‑epi-­
alteramides was replaced with a constitutive promoter, through
conjugation-mediated homologous recombination, using plasmid
pOJ313, to enhance its endogenous NP production [108]. An advantage
to inserting new promoters is that the cryptic NP BGC being stimulated
can be more directly controlled. However, because of the number of
genes present in some BGCs, not all genes are under control of a single
promoter. Having multiple separate genes or operons can limit some
classical genetic methods since several promoter modifications may be
needed to address the different gene regions within a cluster; that is if all
of the promoter regions can even be identified within the full BGC [27].
Along with promoter manipulation, other alterations to transcriptional
and translational proteins are being explored for the activation and
production of NPs. Ribosome engineering or RNA polymerase (RNAP)
engineering target and manipulate core biology dogma enzymes to
create mutated transcription or translation machinery complexes that
can be more stable under stressful environmental conditions, like amino
acid deprivation, to activate BGCs and encourage NP synthesis
[109–111]. This method is based on the observation that development of
resistance to streptomycin, through an accrued mutation in ribosomal
protein S12 (RpsL), not only made Streptomyces lividans
streptomycin-resistant but upregulated the production of an unrelated
antibiotic, actinorhodin [110]. This mutation, mapped to the Lysine-88
residue in RpsL, provides more structural stability and leads to increased
levels of off-target protein synthesis [110]. RNAP is responsible for
discovering the antibiotic NPs, piperidamycins, through the increased
promoter affinity of mutated RNAP for secondary metabolite BGCs
biosynthesis [112,113].
Alternative methods of modifying endogenous gene expression are
through chemical genetics where small compounds are employed to
activate silent BGCs. The aim is to test these small molecules in hopes
that they elicit a modification in transcriptional and/or translational
host machinery to activate silent BGCs. Mass spectrometry (MS), or
some similar technique, can then be used for the detection of natural
products after the chemical challenge of the desired microbe [27].
Similarly to culture modifications, the addition of chemical signals can
affect BGC activation. For example, ‘microbial hormone’ treatment has
been used to inactivate transcriptional regulators, inhibiting the tran­
scription of BGCs to express natural products. In one example, the TetR
family of transcriptional regulators has been shown to regulate the
expression of NP BGCs and keep them silent under laboratory growth
conditions [114,115]. ArpA, a TetR family transcriptional repressor
protein that regulates the production of streptomycin in Streptomyces
G.D. Cook and N.M. Stasulli
griseus [116], can be inactivated by ‘microbial hormone’ treatment using
the signaling molecule A-factor [117]. The A-factor molecule binds to
ArpA causing it to release from the promoter of adpA, which encodes
global activators of secondary metabolism. With ArpA inactivated, the
production of streptomycin increases [118]. Being able to artificially
encourage the overproduction of potentially novel antibiotics would
make it easier to study their structure, chemical properties, and the
factors contributing to their production. Chemical genetics can also
incorporate synthetic biology approaches and utilize more high
throughput methods that rely on mutagenesis. One example is the high
throughput elicitor screening (HiTES) method that specifically utilizes
small, synthetic elicitor molecules to induce BGC expression [119].
Through this technique a reporter gene is cloned inside the targeted BGC
where it could be transcribed under a native promoter. This reporter
strain, for the specific BGC, is then challenged with a predetermined set
of elicitor molecules and the bacteria cultures are monitored for
Fig. 1. A visual depiction of a heterologous gene expression scheme indicating strategies that could be utilized in synthetic biology to target BGCs for expression
and study.
6
SLAS Technology 29 (2024) 100120
production of the reporter molecule. Any output from the reporter gene
is then initially assumed to be indicative of an activation of the targeted
BGC [119]. The HiTES method was applied to Burkholderia thailandensis,
using a library of NPs and clinical agents as elicitors, to express the
cryptic malleilactone (mal) BGC [119]. Using a malL-lacZ reporter strain
of B. thailandensis, which created a translational fusion of the LacZ re­
porter to the MalL protein that is essential for malleilactone synthesis,
low-dose antibiotics trimethoprim and piperacillin were discovered to
be the best elicitors of the mal BGC. [119–121]. While the BGC in this
example encodes a virulence factor involved in nematode infections
[119] this technique is adaptable to study antibiotic BGCs as well [122].
In fact, this technique has been adapted to even more high-throughput
approaches that don’t require mutagenesis of the organism in ques­
tion: Bioactivity-HiTES [123,124] and HiTES-IMS [125]. Overall,
endogenous expression techniques aim to activate silent BGCs natively
to produce novel antibiotics within the original bacterial strain.
G.D. Cook and N.M. Stasulli
Synthetic biology is applied to endogenous expression methods by
building promoters, repressing transcriptional regulators, modifying
receptor proteins, and employing small chemical molecules to elicit a
transcriptional or translational change, generally in tandem with culti­
vation conditions and coculturing, to activate cryptic BGCs.
4. Heterologous gene expression
In opposition to endogenous expression, heterologous gene expres­
sion relates to incorporating and altering the levels of a non-native gene,
or genes, that originate(s) from a distinct species or cell type. With the
development of synthetic biology, heterologous gene expression has
been able to better characterize NPs produced by BGCs, especially those
from unculturable organisms [25,71,126–133]. Heterologous gene
expression has been the main focus of synthetic biology to discover
novel antibiotics. These heterologous expression systems generally use
cloning to express the BGCs from unculturable microbes in a heterolo­
gous host [16,134]. In short, this process involves first identifying and
mobilizing target BGCs onto a cloning vector for genetic exchange with a
more easily manipulated and cultured host. The regulatory elements of
the BGC must then be modified on the cloning vector to match the new
host bacteria before genetic exchange can occur. After horizontal gene
transfer of the final modified cloning vector into the heterologous host,
the BGC can be expressed so the produced NP can be studied further [16,
24–26] (Fig. 1). For the best outcomes of heterologous expression, the
BGC’s boundaries need to be accurately defined to include all genes
required for NP biosynthesis. The heterologous host also needs to have
all necessary machinery for expression of the BGC products. Bio­
informatic programs previously mentioned, antiSMASH [18,19], and
SMURF [17] can be used to identify the BGCs and their surrounding
genomic content. Typically, heterologous gene expression has been used
to link the natural product chemotype with its genotype [25].
The first step in heterologous gene expression is identifying target
BGCs and cloning them into DNA vectors, which can be done through
both targeted and untargeted methods. Targeted methods for identifying
BGCs include genomic sequencing followed by DNA synthesis or direct
cloning [135–138]. Untargeted methods for identifying BGCs include
constructing random genomic libraries via mutagenesis followed by
select clone recovery [138,139]. CONKAT-seq, a targeted sequence
workflow using library-wide co-occurrence analysis, has been used to
identify, locate, and assess multiple BGCs concurrently [138]. This
software uses amplicon barcodes to identify the domain sequence (i.e.,
NRPS, PKS, TPS, and DMATS) and uses this information to locate whole
BGCs within the original genome library [138].
After the identification of BGCs, and their boundaries, they need to
be mobilized to prepare them for integration into the host. Techniques
involved in mobilization of BGCs commonly include Gibson assembly
[140] or insertion of BGCs into a cloning vector (cosmid [62,141], fos­
mid [16,75,142], bacterial artificial chromosome (BAC) [143,144],
P1-derived artificial chromosome (PAC) libraries [145,146], yeast arti­
ficial chromosome (YAC) [147,148]), or CRISPR/cas9 associated tar­
geting of chromosome segments (CATCH) [149]). Gibson assembly joins
PCR-amplified genes directly from genomic DNA [150]. Cosmid vectors
contain specific Lambda phage cos regions for packaging DNA into
phages for DNA transduction [141]. Fosmid vectors are similar to cos­
mid vectors, but instead contain the F-factor genes necessary for
conjugation as a DNA transfer method instead of transduction [142].
CRISPR/Cas9-assisted targeting of chromosome segments (CATCH) can
excise ~150 kb sized BGCs and ligate them into cloning vectors [25].
Mobilizing pieces of DNA that contain full BGCs can require even larger
cloning vectors to be constructed and may have to rely on BACs or YACs
depending on the size or application [148,151]. One method of large
DNA segment mobilization, transformation-associated recombination
(TAR), utilizes homologous recombination in yeast to combine small
TAR vectors with target BGCs [152]. The TAR approach is useful in
mobilizing silent BGCs in unculturable bacteria [16,153]. The ends of
7
SLAS Technology 29 (2024) 100120
linearized TAR cloning vectors contain homologous sequences of target
BGCs to be used as recognition sequences for homologous recombina­
tion [16,152]. These sequences are also called “homology arms” and
sequentially match either end of the targeted BGC. The TAR vector is
linearized and homologously combined with the target BGC of isolated
host DNA to form a BAC or YAC to be transformed into a heterologous
host for NP production research [152].
After BGCs have been mobilized, they are genetically modified to fit
the needs of the heterologous bacterial host. Generally, native regula­
tory regions, like promotors, are replaced with inducible or
constitutively-active promoters. Typically, Red/ET recombination and λ
phage proteins (Redα and Redβ) or Rec phage proteins (RecE and RecT)
are utilized to replace or inactivate sections of the target BGC [16].
These promoters are reprogrammed with the goal of optimizing the
expression of BGCs within the heterologous host. The availability of
synthetic parts, such as promoters and ribosome binding sites (RBSs),
has allowed for the manipulation of BGCs for specific gene expression,
can increase and improve NP production, or can activate silent BGCs
[25]. A suitable heterologous host is necessary for successful, high-level
NP production [16,154].
After editing the vectors, they need to be delivered into the heter­
ologous host for cloning and induction of potential NP expression.
Transformation via electroporation and conjugation are the typical
horizontal gene transfer methods used to insert the BGC-containing
plasmid into the host [27]. The first case of successful heterologous
expression of a natural product in Streptomyces salinispora used the
TAR-mediate pCAP01 vector to capture the whole enterocin BGC and
conjugate the plasmid into S. lividans TK23 and S. coelicolor M1146[16,
132]. Synthetic biology is used in heterologous gene expression methods
to help activate and better characterize the production of NPs through
BGC activation. These heterologous methods, while not always
high-throughput, may represent the best possibility for discovering
novel antibiotic BGCs because of the ability to study gene clusters from
organisms without the need to culture them. Most of the bacterial world
is still unculturable to us and may be hiding many genomic secrets in
plain sight.
5. Final thoughts
Synthetic biology can be applied to existing genome editing tools by
using created and engineered components for controlling and manipu­
lating gene expression. Synthetic biology can also be used to manipulate
gene circuits or construct metabolic pathways in non-native organisms.
There are abundant techniques that can be modified and tailored for
cryptic BGC activation. These methods are being developed and applied
within the ground-breaking, rapidly expanding field of synthetic
biology. Genomic mutagenesis strategies are being applied in synthetic
biology to stimulate genetic variations within a bacterium’s genome to
activate BGCs and produce their encoded natural product. Endogenous
and heterologous expression systems are being used in synthetic biology
to activate cryptic BGCs using cloning and genetic engineering tech­
niques. While each technique has its advantages and disadvantages for
activating cryptic BGCs, the drawbacks of mutagenesis strategies,
endogenous expression, and heterologous expression systems may prove
to be challenging for synthetic biologists to overcome. Mutagenesis
strategies may be successful in producing novel products if the BGCs
have been located and defined. The biggest obstacle involves identifying
the BGC and being certain it’s responsible for producing a novel product
[138]. Endogenous gene expression systems may prove challenging for
synthetic biologists to produce enough natural products to be studied
efficiently [119]. With Heterologous expression techniques, synthetic
biologists may find producing novel products time-consuming and
difficult due to a large amount of manipulation in different locations of
the BGC to ensure the mobilization vector is complementary to the
heterologous host [27]. Efforts should be made to improve the ability to
locate and identify BGCs and make techniques more high throughput.
G.D. Cook and N.M. Stasulli
The goal of activating these cryptic BGCs is to discover, characterize,
and use novel natural products as antibiotics. If these techniques can be
effectively adapted and harnessed to focus on antibiotic BGC discovery
and characterization, we may be able to enter a new generation of
antibiotic production that can turn back the clock and prevent the
looming danger of the ever-expanding list of MDR and XDR bacterial
pathogens.
Funding
GDC is supported by a Provost Office Research Assistantship within
the Department of Biology & Environmental Science at the University of
New Haven, West Haven, CT, USA. This funding source had no input in
this manuscript.
CRediT authorship contribution statement
Greta D. Cook: Writing – review & editing. Nikolas M. Stasulli:
Writing – review & editing.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
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