Synthetic and Systems Biotechnology 3 (2018) 163–178

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Synthetic and Systems Biotechnology

journal homepage: http://www.keaipublishing.com/synbio

Activation of microbial secondary metabolic pathways: Avenues and T

challenges
Bikash Baral, Amir Akhgari, Mikko Metsä-Ketelä∗
Department of Biochemistry, University of Turku, FIN-20014, Turku, Finland

A R T I C LE I N FO A B S T R A C T

Keywords: Microbial natural products are a tremendous source of new bioactive chemical entities for drug discovery. Next
Biosynthesis generation sequencing has revealed an unprecedented genomic potential for production of secondary metabo-
Chemical diversity lites by diverse micro-organisms found in the environment and in the microbiota. Genome mining has further led
Genome mining to the discovery of numerous uncharacterized ‘cryptic’ metabolic pathways in the classical producers of natural
Metabolic engineering
products such as Actinobacteria and fungi. These biosynthetic gene clusters may code for improved biologically
Synthetic biology
active metabolites, but harnessing the full genetic potential has been hindered by the observation that many of
the pathways are ‘silent’ under laboratory conditions. Here we provide an overview of the various biotechno-
logical methodologies, which can be divided to pleiotropic, biosynthetic gene cluster specific, and targeted
genome-wide approaches that have been developed for the awakening of microbial secondary metabolic path-
ways.

1. Microbes: promising and prolific source of new drug leads therapeutic areas comprise the anticancer agent doxorubicin, the im-
munosuppressants rapamycin and cyclosporine and the anthelmintic
Natural products (also referred to as secondary metabolites or spe- drug avermectin B1 (Fig. 1).
cialized metabolites; SMs) represent a group of low-molecular weight The chemical entities described above (Fig. 1) were discovered
structurally diverse and complex bioactive compounds occupying an several decades ago, most during the “Golden Era of Antibiotics” in the
unusual chemical property space [1]. Microbes in particular are prolific 1950s and 1960s. Starting from the 1980s and 1990s, traditional
antibiotic factories [2], and have proven to be a bountiful source of SMs bioactivity-based screening of microbial culture extracts became se-
that have been successfully developed as crucial drug leads [3,4]. To verely affected by diminishing returns due to high probability of dis-
date, more than 5000 antibiotics have been identified from the genus covering previously known metabolites [17]. Structure elucidation of
Actinobacteria, while 500 natural products have been isolated from hit molecules is time consuming and ultimately the high rediscovery
Myxobacteria [5–7]. Soil-dwelling Streptomyces bacteria are particularly rate of known molecules led to decreased interest of pharmaceutical
proficient producers with 7600 SMs identified until 2005 [8], while companies in natural products [2], which turned their attention to
computational predictions have estimated that these bacteria may have structure-based drug design and combinatorial chemistry instead
the capability to produce 150,000 chemically distinct antimicrobial [18,19]. However, despite very strong investments into this field in the
agents [5–7]. In addition to classical antibiotics, microbial cultures last 15 years, these efforts have not provided a single novel synthetic
have been a source for immune-suppressive agents [9], lantibiotics, antimicrobial agent that has proceeded beyond preliminary clinical
anti-proliferative [10], cytotoxic [11], anti-hypertensive [12], antiviral trials [20]. One key problem has been that while many synthetic
compounds [13,14] and various enzyme inhibitors [15]. Circa 35% of compounds with high efficacies towards their molecular targets have
drugs approved by the FDA/EMA are estimated to be either natural been successfully developed, it has become apparent that these com-
products or their derivatives [16], and > 50% of clinical antibiotics are pounds have difficulties in reaching their target site in vivo and in pe-
of Actinomycetes origin [8]. Widely used antibiotics include ery- netrating bacterial cell membranes [17].
thromycin A, penicillin G and streptomycin, while examples of micro- However, recent technological advancements have created a re-
bial metabolites that have been successfully launched for other naissance of interest in natural products and new chemical entities with

Peer review under responsibility of KeAi Communications Co., Ltd.

∗
Corresponding author.
E-mail address: mianme@utu.fi (M. Metsä-Ketelä).

https://doi.org/10.1016/j.synbio.2018.09.001
Received 13 July 2018; Received in revised form 28 August 2018; Accepted 4 September 2018
2405-805X/ © 2018 Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
B. Baral et al. Synthetic and Systems Biotechnology 3 (2018) 163–178

Fig. 1. Chemical structures of selected microbial natural products.

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novel mode of action [21,22]. The foundation was laid in the 2000s by characterization of pathways thus facilitating future research efforts.
the diligent work of the Academic sector in elucidating the biosynthetic
logic of the main classes of microbial natural products made by poly- 3. Metabolic gene clusters in microbes
ketide synthase (PKS) [23,24], non-ribosomal peptide synthetase
(NRPS) [25] and ribosomally synthesized and post-translationally The genome mapping approaches are facilitated by the fact that all
modified peptide (RiPP) pathways [26]. The classical pre-genomics genes required for the biosynthesis of the metabolite, its regulation,
view was that an individual microbial strain could produce only a resistance and transport are typically located in within the BGCs
limited number of SMs, but the first genome sequences of Streptomyces [47–49]. The size of metabolic gene clusters varies greatly depending
[27] and Aspergillus [28] published in the 2000s and the subsequent on the complexity of the end product of the pathway. As an example,
explosion of genome sequencing data in the 2010s has demonstrated the gene cluster responsible for biosynthesis of the glycosylated an-
that these micro-organisms have the genetic capability to produce a thracycline nogalamycin encodes 32 enzymes [50]. In addition, the
significantly larger number of compounds with great strain-to-strain type of biosynthetic machinery has a significant influence to the size of
variations. These unknown metabolic pathways are likely to encode the BGC with large differences noted between the 30-kb thiostrepton
numerous bioactive molecules, which could be used to solve the current and 128-kb daptomycin gene clusters [51,52], even though both are
problems with drug resistant pathogens and to obtain improved che- assembled from amino acid building blocks and are of similar com-
motherapy agents with reduced side effects. The key question re- plexity. The difference can be explained with the fact that thiostrepton
maining to answer in the field is how to access this hidden potential, is RiPP [51], whereas daptomycin is made through a NRPS pathway.
since it would appear that the most of these cryptic pathways are silent Wide variation may also be found within the same classes of biosyn-
or poorly expressed under laboratory conditions. In our current dis- thetic machineries, since the chemically simple NRP showdomycin is
course, we attempt to provide crucial insights into methodologies that encoded within a 12-kb fragment of DNA, while the pristinamycin su-
have been developed in an attempt to harness this tremendous chemical percluster, which encodes two complex hybrid PK-NRP molecules,
diversity. spans as large as 210-kb [53].
The onset of SMs production typically occurs during the early sta-
2. Genomics-driven natural products discovery tionary phase, and involves complex metabolic changes within the or-
ganism [54]. Pleiotropic (or global) regulators are localized distantly
The paradigm shift to genomics-based discovery of microbial SMs from the biosynthetic cluster, and control the expression of secondary
may be considered to be initiated by the sequencing of the genomes of metabolism by responding to diverse signals [55]. These pleiotropic
S. coelicolor and S. avermitilis, which led to the discovery of 22 [27] and regulators have wider coverage, simultaneously controlling the ex-
25 [29] putative biosynthetic gene clusters (BGC), respectively, that pression of several BGCs. Events of nitrogen or phosphate starvation or
could code for secondary metabolic pathways. The genome of the fi- distinct signaling compounds like N-acetylglucosamine (GlcNAc),
lamentous fungi Aspergillus nidulans harbored an even greater potential stressors like heat, pH and damage to the cell wall provide proper sti-
with 56 putative pathways, as observed by genome mining [30]. muli for triggering the expression of these regulatory genes [56,57].
However, recent next generation sequencing efforts have vastly ex- The production is also guided by regulatory genes present in the bio-
panded this phenomenon in an unparalleled scale to diverse microbial synthetic clusters, which are pathway-specific and may either be acti-
genus that have not typically been associated with natural products vators or repressors [58].
such as Burkholderia, Clostridium and Pseudomonas [31,32]. In addition, The genes responsible for providing resistance and for transporting
probing microbial communities in various ecological niches such as the SMs outside of the cells are also typically clustered within BGCs. In
human microbiota [33] and environmental samples [34] have revealed particular, if the end product of the pathway has biological activity
exceptional metabolic diversity. Many lactic acid bacteria, including against the producing host, then the resistance genes for that particular
species of Enterococcus, Lactobacillus and Streptococcus associated with metabolite may be encoded in the genomic locus. Similarly, genes en-
the gastrointestinal tract have been reported to produce bioactive coding efflux pumps (for instance, ATP-binding cassette (ABC) and
modified peptides [33], in particular those synthesized via RiPP path- Major Facilitator Superfamily proteins) have also been traced along the
ways [35,36]. The marine sponge Theonella swinhoei has been shown to BGCs [59] and are responsible for the reduction of both toxicity [60]
host many uncultivated bacterial symbionts, which harbor pathways for and feedback inhibition effects. For instance, in S. avermitilis, the ABC
production of exotic SMs [37]. It should also be noted that the potential transporter AvtAB responsible for the exporting avermectin is found
of soil microbes for production of SMs remains elusive, since ca. 99% of within the biosynthesis gene cluster [61].
environmental microbes are un-cultured under laboratory settings. In
order to circumvent this barrier, environmental DNA isolated directly 4. Strategies for the activation of unknown metabolic pathways
from soil samples may be used for cloning and expression of BGCs, as in
the case of the malacidins pathway [38]. In another approach, the A growing body of evidence has indicated that the activation of
broad spectrum antibiotic teixobactin was discovered from an un- gene clusters has the potential to greatly facilitate discovery of new
cultured soil bacteria ‘Eleftheria terrae’ by growing the microbe directly natural products of high-therapeutic leads. Several methods have been
in situ in the soil using a bacterial iChip [39]. developed for the activation of silent or poorly expressed cryptic gene
The development of bioinformatics software for analysis of se- clusters, which can be broadly classified to three major categories
quencing data for the presence of BGCs has been instrumental in (Fig. 2). In one group, there are methodologies that aim to modify the
genomics-driven natural products discovery. Programs such as whole metabolome of the target strain and generate pleiotropic effects
antiSMASH [40,41], SMURF [42], BAGEL3 [43] and PRISM [44,45] are to activate randomly any pathway residing in the strain. These methods
able to detect and decipher all of the most common biosynthetic types. are generally technically simple and are therefore suitable for scaling
In some instances, such as type I polyketides and NRPSs and RiPPs, up to high-throughput systems. Unfortunately, these techniques gen-
these programs are able to even provide predictions for the chemical erally forfeit the advantages provided by next-generation sequencing
scaffolds of the metabolites [45], but for other compound classes such and genome mining, since they are not able to target the most inter-
as type II PKSs and glycosylated natural product, the information ac- esting gene clusters for activation. They also suffer from the same
quired through computational studies is limited. Linking genomic data metabolite rediscovery issues as traditional natural products discovery.
to chemistry has also been aided by community standards such as In contrast, the methodologies lying under the second category are able
MIBiG (Minimum Information about a Biosynthetic Gene cluster) [46], to focus on desired pathways, but are technically challenging and suffer
which define standardized annotation and metadata procedures for from low throughput. Finally, the techniques in the third group aim to

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Fig. 2. Methodologies for the activation of silent gene clusters to obtain new natural products. Petals within the circle represents different techniques that have been
developed with three major categories, viz., pleiotropic, BGC-specific and targeted-genome wide approaches highlighted. Abbreviations: OSMAC, One strain many
compounds; P, Promoters; PPtases, Phosphopantetheinyl transferases; RGMS, Reporter-Guided Mutant Selection.

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gain the benefits of both of the approaches mentioned above, usually by clustered at subtelomeric regions and are co-regulated [86], where
combining pleiotropic activation methods to gene cluster specific re- histone acetylation and methylation largely impact transcription [87].
porter systems. The diversity of these methodologies offer many tools For activation of SMs, the state of chromatin packaging has been
for the research community, but to date no single superior method for influenced by artificial expression of histone modification genes and by
activation of biosynthetic pathways has been presented. utilizing small molecule inhibitors against histone deacetylase enzymes.
In A. nidulans, inactivation of the histone deacetylase (HDAC) hdaA
4.1. Genome-wide pleiotropic methods down-regulated secondary metabolic pathways [88], while loss-in-
function of cclA (an ortholog of bre2 involved in histone H3 lysine 4
4.1.1. Ribosome engineering and its applicability methylation) activated the expression of the cryptic secondary meta-
One of the most important adaptations in bacteria to environmental bolic clusters generating monodictyphenone, emodin and its derivatives
stresses, e.g. depletion of amino acids, is the ‘‘stringent response’’ [89]. The use of the HDAC inhibitor suberoylanilide hydroxamic acid
governed by an intracellular transient accumulation of guanosine tet- (SAHA) led to stimulation of production of new cladochromes and
raphosphate (ppGpp) [62]. The bacterial alarmone ppGpp is synthe- calphostin B in Cladosporium cladosporioides [90] and histone deacety-
sized by the ribosomal enzyme ppGpp synthetase (relA) and is initiated lase (HDAC) inhibitors have also caused over-expression of SM genes in
by the binding of an uncharged tRNA molecule to the aminoacyl A. nidulans [88,91].
binding site (A-site) on the ribosome [63]. Subsequent binding of Unlike in fungi and other eukaryotes, prokaryotes are entirely de-
ppGpp to the RNA polymerase (RNAP) modulates transcription by up- prived of histones. However, the bacterial genome, for instance,
regulating and downregulating various promoters [64,65] through Streptomyces sp. comprises their own versions of HDAC [92] and the
mechanisms that can vary between species [66]. Investigations into the structure of the nucleoid may have a role in the global regulation of
RNA polymerase/ppGpp complex in a thermophilic bacterium Thermus metabolism [93]. Access to chromosomal DNA may be restricted by
thermophilus revealed three modes of transcriptional regulation by nucleoid-associated proteins, various RNAs and differential super-
ppGpp depending on the nucleic acid composition of the promoter, coiling in the nucleoid, which may prevent aberrant transcription of
which influenced the conformation of the RNAP [64]. Therefore it is many gene clusters [94]. In Streptomyces, a bifunctional nucleoid-as-
unsurprising that ppGpp has also been linked to secondary metabolism sociated protein (DdbA) has also been characterized that comprises of
and initiation of antibiotic production [67]. In Streptomyces, relA/relC an N-terminal DNA-binding histone H1-like domain and a C-terminal
mutants with significantly decreased ppGpp contents display lowered DksA-like domain, which could modulate the RNA polymerase activity
production of SMs when transferred to a media with nutritional shift- along with ppGpp to induce transcription [95].
down [68–70] (Fig. 3A and B). It has been revealed that mutations
conferring resistance to rifampicin in the rpoB gene, which encodes the 4.1.3. OSMAC approach and environmental cues
RNA polymerase β-subunit, alter the conformation of the RNAP to re- One strain many compounds “OSMAC” is a relatively simple and
semble ppGpp bound status. Therefore, the requirement for ppGpp for versatile approach that allows activation of diverse-metabolic pathways
the expression of silent or poorly expressed gene clusters is bypassed in [96]. The biosynthesis of SMs is stimulated through alterations in cul-
these mutants in various actinomycetes [71–73]. tivation parameters [97,98], e.g. media composition, aeration rate, type
In an analogous manner, the acquisition of mutations in ribosomal of culturing vessel or a combination of these factors. In some cases,
proteins conferring resistance to antibiotics targeting the ribosome has even subtle changes may result in drastic changes, as demonstrated in
also been reported to promote the expression of silent genes. However, fermentations of Paraphaeosphaeria quadriseptata, where switching from
the molecular mechanism of this phenomena remains obscure. The the use of tap water to distilled water lead to the isolation of six new
introduction of streptomycin-resistance mutations in rel mutants of S. SMs [99]. These experiments aim to mimic more precisely the natural
coelicolor and S. griseus reinstated actinorhodin production without the growth conditions found in the environment to trigger the onset of
requirement of ppGpp [74,75]. On the contrary, elevated concentra- secondary metabolism, and has been successfully applied to both bac-
tions of ppGpp (30-fold) and actinorhodin (180-fold) has been detected teria and fungi, as reviewed elsewhere [97,98].
in S. coelicolor, when the strain was exposed to eight antibiotics in a The classical OSMAC approach may be extended to more drastic
successive manner, where all of the antibiotics were targeting the measures by modulating the culture conditions with stress or chemicals.
translational machinery with the exception of rifampicin [76]. In ad- Heat shock has been shown to induce jadomycin production [100] and
dition, relA disruption resulted in the total loss of ppGpp accumulation increase the yields of validamycin [101], whereas limitation of nu-
as well as a significant decrease in actinorhodin production, indicating trients such as alanine and/or an acidic pH shock led to methyleno-
the key role of ppGpp in the activation of biosynthesis. Cluster-acti- mycin production in Streptomyces coelicolor A3(2) [102]. In particular,
vated strains resulting from mutations in the ribosome have been re- numerous SMs have been shown to be regulated negatively by in-
ported in Streptomycetes and other bacteria including Bacillus spp., organic phosphate concentrations [57], while GlcNAc appears to
Mycobacterium spp. and Pseudomonas spp. [74,77,78]. Strikingly, two function as an important sensory molecule for the onset of secondary
single rpoB mutants, an rpoB+rpsL double mutant, and a single gen- metabolism [103]. Both biotic (microbial lysates or soil extracts) and
tamicin-resistant mutant induced the production of piperidamycins in abiotic (antibiotics, synthetic compounds) chemical elicitors have been
S. mauvecolor, whereas the wild-type does not produce these metabo- proved to be effective strategies for the induction of silent gene clusters.
lites at any detectable level in various media [79]. Two small molecule elicitors have been particularly successful; ARC2, a
synthetic compound discovered through large scale screening [104],
4.1.2. Chromatin remodeling and goadsporin [105,106], a natural product that promotes secondary
Chromatin remodeling is a vital mechanism for the modulation of metabolism in Streptomyces. Sub-inhibitory concentrations of other xe-
transcription of eukaryotic genomes [80]. Chromatin is composed of nobiotic antibiotics, such as jadomycin B [107] and monensin [108]
repeating nucleosome units, which consist of histones (2 copies each of have been shown to induce endogenous production of SMs. Interest-
H2A, and H2B, H3 and H4 [80]) wrapped around chromosomal DNA ingly, it has been shown that the addition of rare earth elements, e.g.
and linked together through a linker histone (H1) (Fig. 3C) [81,82]. scandium and lanthanum, at low concentrations resulted in the acti-
Histones cause dynamic variations to packaging and accessibility of vation of the cryptic secondary metabolite biosynthetic gene clusters
DNA in response to physiological and developmental cues [83,84]. [78,109].
These are mediated via post-translational modifications such as dea- Co-cultivation, mimicking the ecological habitats of micro-organ-
cetylation and methylation of histone tails, which ultimately regulate isms, has been emerged as an effective strategy to increase the chemical
gene expression [85]. Gene clusters for secondary metabolism are diversity of secondary metabolites (Fig. 4A). It is believed that the

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Fig. 3. Pleiotropic approaches for activation of silent gene clusters: A) Events that occur in the RNAP when an organism is grown in alternative conditions: (i)
Nutrient rich-medium, where the conditions suppress secondary metabolism and transcription levels from BGCs are low; (ii) Microbe subjected to nutritional down-
shift (depletion of amino-acids) conditions, which leads to the production of ppGpp (Guainosine tetraphosphate), that ultimately binds to RNAP, giving rise to high
transcription levels from the BGC; (iii) Addition of antibiotics like rifampicin induces a mutation in the β-subunit of the polymerase, which in turn increases the
transcription level of the BGC. B) A mutation in the 30S subunit of ribosome stabilizes the 70S ribosomal complex and causes upregulation of protein synthesis in the
stationary phase. C) Packaging of DNA around histones prevents gene transcription and is regulated through post-translational modifications of histone tails. In
chromatin remodeling, enzymes responsible for histone modifications are manipulated to allow histone release, which enables expression of BGCs from the un-
covered stretch of DNA.

social networking in ecological environment is mediated by diffusible antibiotic production in another strain by cross-feeding [114]. Inter-
low-molecular-weight signaling molecules that are secreted from and estingly, dialysis experiments and electron microscopy have revealed
received by cells [110,111]. For example, co-cultivation of the marine- that not only diffusible signals, but also intimate physical interactions
derived fungal isolate A. fumigatus MR2012 with S. leeuwenhoekii strain contribute to the networking and induction of silent clusters during co-
C34 led to induction of metabolite production in both organisms, while cultures of A. nidulans with S. hygroscopicus [115] or A. fumigatus with S.
co-cultivation of the fungus with bacterial strain C58 resulted in the rapamycinicus [116].
detection of pentalenic acid from the bacteria [112]. Another marine-
derived fungus Emericella sp. was induced to produce emericellamides A
4.1.4. Phosphopantheteine transferases
and B upon co-culture with Salinispora arenicola [113], whereas pro-
The biosynthesis of polyketides and nonribosomal peptides only
momycin produced by one Streptomyces strain led to initiation of
occurs if the essential acyl - and peptidyl carrier proteins, respectively,

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Fig. 4. Pleiotropic approaches for the activation of the silent gene clusters: Global regulators, PPTases and the OSMAC approach. A) In the OSMAC approach,
cultivation conditions are manipulated to uncover the environmental signal required for activation of biosynthesis. As an example, co-cultivation of two or more
microbes may induce microbial chemical communication, where one or even both participants produce novel compounds. Abbreviations: A, Activator; BGC,
Biosynthetic gene cluster; P, Promoter. B) PPTases catalyze the post-translational modification of carrier proteins, which is essential for the biosynthesis of poly-
ketides and nonribosomal peptides. Overexpression of PPTases may influence the production of SMs under conditions where the native PPTase may be down-
regulated. Abbreviations: A, Adenylation domain; ACP, Acyl-carrier protein; AT, Acyltransferase; C, Condensation domain; KS, Ketosynthase domain; NRPS, Non-
ribosomal peptide synthetase; PCP, Peptidyl-carrier protein; PKS, Polyketide synthases; TE, Thioesterase domain. C) Pleiotropic regulators control many aspects in
the life cycle of microbes. Manipulation of these regulatory networks may lead to the activation of BGCs.

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are post-translationally modified into active holo-form [117]. This re- transcription factors. Eighty families of transcription factors were re-
action is catalyzed by phosphopantetheinyl transferases (PPtases) vealed through a whole-genome analysis of more than 200 fungal
through covalent attachment of a 4′-phosphopantetheine group to a genomes [134]. For example, the filamentous fungi A. nidulans and N.
highly conserved serine residue (Fig. 4B). Microbial genomes may crassa contain 81 and 58 transcription factors, respectively [135]. These
contain up to three PPtases, which harbor specificity to various bio- aforementioned mechanisms enable alterations in transcription pat-
synthetic pathways [118], but these genes are typically not found terns in response to environmental cues.
within BGCs. Since PPtases regulate primary metabolism that leads to The σ-factors influence the expression of larger groups of genes,
physiological changes and metabolic flux alterations, overexpression of therefore σ-factor-based engineering methods have been utilized to
PPtases may also influence secondary metabolism [119]. Indeed, ex- activate gene expressions associated with secondary metabolite bio-
pression of two PPtase genes sfp and svp resulted in activation of silent synthesis. For example, overexpression of the σ-factor ‘Orf21’ in
pathways with a success rate of 70% in a study using 33 Actinomycetes Streptomyces clavuligerus NRRL3585 resulted in a slight increase of
strains [120]. The sfp and svp gene products, from Bacillus subtilis and clavulanic acid production through the induction of early biosynthetic
Streptomyces verticillus, respectively, exhibit broad substrate specificity genes (ceas2, cas2) and the activator gene ccaR [136]. Moreover, the
to guarantee sufficient phosphopantetheinylation of diverse biosyn- overexpression of σ-factors led to the accumulation of higher level of
thetic enzymes. Recently, PPtase-based activation also led to the iden- FK506, a hybrid polyketide-nonribosomal peptide and carotenoid pro-
tification of three nucleosides including puromycin A and two new ductions in Streptomyces sp. KCCM 11116P [137] and Corynebacterium
congeners in S. alboniger NRRL B-1832 [121]. Interestingly, the bio- glutamicum, respectively [138]. Successful expression of a polyketide
synthetic pathways of nucleosides do not contain carrier proteins, biosynthetic pathway (oxytetracycline) was achieved for the first time
suggesting that the result was achieved indirectly via modulation of in a surrogate host E. coli by overexpressing the host σ54-factor [139].
cellular regulatory networks by the PPtase. The deletion of σ-factor ‘Sig6’ caused over-production of avermectin by
2–2.7 fold in S. avermitilis, while overexpression of an extra copy lead to
4.1.5. Manipulation of global regulatory systems significant decrease in yields [140]. In addition, S. avermitilis hrdB
The engineering of global regulatory genes aims to influence the mutants lacking σhrdB exhibited higher levels of avermectin production
same regulatory networks as the OSMAC approach, but instead of than the parental strain [141].
trying to find the environmental cues, it does it at a more refined mo- The promoter sequences residing in BGCs do not typically promote
lecular level through over-expression or inactivation of the genes con- transcription in all growth conditions and are highly influenced by in-
trolling the cellular response to these phenomena (Fig. 4C). Investiga- tracellular regulation networks. Manipulation of promoter sequences by
tions into pleiotropic regulatory networks in bacteria and fungi have exchanging native silent promoters with functionally active ones di-
revealed that production of SMs is linked to morphological develop- rectly in the producing organism has proven to be a useful strategy to
ment and nutrient availability (carbon and nitrogen sources, phosphate drive expression of the silent genes by circumventing the complexity of
starvation, amino acid and iron availability) [55]. Other examples in- the regulatory genes (Fig. 5B). The methodology requires advanced
clude response to pH stress in fungi mediated by the PacC zinc finger genome editing tools that might not be available for the target organ-
transcription factor [122] and the master regulator DasR in Strepto- isms and is best suited for small BGCs that are composed of very few
myces, which responds to the presence of N-acetylglucosamine that may operons. The activation of a silent polycyclic tetramate macrolactam
be considered as a signal for autolytic degradation of the vegetative (PTM) gene cluster in Streptomyces albus has been achieved by the in-
mycelium [103]. An important distinction to cluster situated regulatory dependent introduction of ermEp* upstream of ftdA and ftdB encoding
genes is that the global regulatory systems also control the transcription putative desaturase and hybrid polyketide synthase-nonribosomal
of many other genes not related to secondary metabolism. In fungi, peptide synthetase, respectively, and led to the identification of 6-epi-
pathway-specific regulatory genes have been discovered only in about alteramides A and B. Furthermore, the biosynthesis of a blue pigment
60% BGCs [123], which indicates that the production of many SMs is ‘indigoidine’, which is not typically produced in standard laboratory
modulated strictly by global regulatory networks. Similarly, the moe- conditions, has been induced in S. albus by expressing the NRPS gene
nomycin biosynthetic gene cluster does not harbor a pathway-specific bpsA under ermEp* promoter [142]. The introduction of strong en-
regulator and is under the control of pleiotropic regulators in Strepto- gineered promoter kasOp* also resulted in successful activation of silent
myces ghanaensis [124]. lycopene biosynthetic cluster in S. avermitilis [143]. Recently, replace-
In Streptomyces, manipulation of regulatory elements that respond ment of the native promoters of the silent actinorhodin, un-
to butyrolactone hormones such as adpA [125] have been particularly decylprodigiosin and PTM clusters with kasOp* produced targeted
successful with cryptic oviedomycin [126] and germicidin [127] gene compounds in S. albus, S. lividans and S. roseosporus, respectively [144].
clusters recently activated. Disruption of whiB-like regulatory genes, In Aspergillus nidulans, replacing the native promoter of the non-
which are associated with control of morphological development [128], ribosomalpeptide synthetase, acvA, with a promoter from the ethanol
have also led to the discovery novel violapyrones [129] and antimycin- dehydrogenase, alcAp, from the same host led to a 30-fold increase in
type depsipeptides [130]. penicillin production [145]. In addition, sequential exchange of pro-
moters for six genes in the silent fellutamide B gene cluster (inp) with
4.2. Biosynthetic gene cluster specific approaches alcAp enabled the activation of the cluster and led to the production of
the target compound in A. nidulans [146].
4.2.1. Transcription factors and promoter exchange experiments
In eukaryotes, each gene is transcribed from its own promoter, 4.2.2. Manipulation of pathway-specific regulatory genes
whereas in prokaryotes (e.g., Streptomyces sp.) several genes are often Pathway-specific transcriptional regulatory genes are typically
clustered together to form operons (Fig. 5A). In all bacterial species, the found within the BGCs and they may either repress or activate the
promoter sequences residing within BGCs are typically associated with biosynthesis of the corresponding SM. These genes represent the lowest
specific sigma factors (σ-factors) that are vital for binding of the RNA organizational level in the cellular regulatory networks and are typi-
polymerase (RNAP) in the formation of the translation initiation com- cally tightly regulated by environmental sensors and pleiotropic reg-
plex [131]. The various σ-factors confer promoter selectivity and are ulators. However, in reality the regulatory circuits are likely to be even
thereby responsible for switching on specific regulons [132]. For in- more complex with cross-talk between distinct pathways [147] and
stance, the filamentous prokaryotes, S. albus J1074, S. coelicolor and S. even between global and BGC specific regulatory genes reported [148].
avermitilis comprise 35, 65 and 60 σ-factors, respectively [27,133]. On The co-localization of pathway-specific regulatory genes offers a pos-
the other hand, promoter recognition in fungi relies on more diverse sibility for targeted activation of the desired BGC either by inactivation

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Fig. 5. Gene cluster specific approaches for activating silent biosynthetic pathways. A) The target gene cluster is silent under laboratory conditions and no tran-
scription or product formation can be observed. B) The gene cluster may be activated by exchanging the promoters from the silent gene cluster to constitutively active
promoters. C) In instances where the gene cluster is controlled by a repressor, inactivation of the regulatory element may lead to expression of the biosynthetic genes.
D) Overexpression of cluster situated activator regulatory genes may also lead to activation of biosynthesis. E) Cloning of the whole biosynthetic gene cluster and
heterologous expression in a surrogate host can be used for artificial activation of the BGC. Abbreviations: AntR, Antibiotic selection marker; BGC, Biosynthetic gene
cluster; Ori, Origin of replication; P, Promoters. F) Synthetic approaches include refactoring of the gene cluster and the use of constitutive or strong promoters to
drive expression of the silent gene cluster.

of the repressor or over-expression of the activator genes (Fig. 5C and domain and a receptor domain that responds to the presence of a small
D). The caveat is that for many families of transcriptional regulators it is molecule ligand [150]. In the absence of the specific signaling mole-
not possible to identify whether the gene is a repressor or an activator cule, TetR dimers bind to DNA and prevent transcription of downstream
based on sequence information alone. The diversity of microbial reg- genes. Binding of the ligand induces a conformational shift that re-
ulatory proteins is expansive [149], and only two systems are described positions the helix-turn-helix motifs of the dimer far apart in such a way
here to demonstrate the functional differences. that the proteins lose their ability to bind DNA [151]. Interestingly,
The TetR family repressors consist of a helix-turn-helix DNA binding some TetR proteins have been proven to respond to γ-butyrolactone

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autoinducers during the onset of secondary metabolism [152], while PPTase genes [176]. Finally, cloning genes in S. albus is facilitated by a
others bind directly to both endogenous and exogenous SMs [107,153]. defective restriction modification system and the naturally minimized
Inactivation of TetR family repressors have led to the activation of ja- genome may help increasing product titres [177]. In filamentous fungi,
domycin [153,154], kinamycin [155], auricin [156] and ceoelimycin Aspergillus species have been widely used as heterologous hosts. An
biosynthesis [157,158]. early example includes the transfer of the entire penicillin gene cluster
Another predominant class of regulators is homologous to LuxR to A. nidulans, while more recently geodin has been heterologously
from Vibrio fischeri, which is a transcription factor involved in quorum produced in this host [178–180]. A. oryzae has gained interest for
sensing [159]. The protein consists of an α/β/α sandwich domain that heterologous expression, as it has traditionally been used in the food
binds the activator ligand and a DNA binding response domain. In industry and has been given the status of GRAS (generally regarded as
contrast to the TerR family, most characterized LuxR proteins are safe) organism. Selected recent examples of SMs produced in this host
transcriptional activators, where ligand binding induces homo- include paxilline [181], alfatrem [182] and pleuromutilin [183]. The
dimerization, which enables binding of the protein to DNA in so-called heterologous expression of A. terreus asperfuranone cluster in the model
tra box sequences and initiate transcription [160]. An important sub- ascomycete, A. nidulans, resulted in awakening of the asperfuranone
family of LuxR regulators in Actinobacteria are LAL–proteins (Large biosynthetic pathway when several non-reducing polyketide synthase
ATP-binding regulators of the LuxR family) [161], originally discovered genes were placed under the control of alcAp [184].
in the regulon for maltose utilization, that contains an additional ATP- An ideal host for heterologous expression would provide sufficient
binding domain at the N-terminus responsible for the interaction with metabolic building blocks for the synthesis of SMs, but would not have
the inducers, maltotriose and ATP [162]. In a recent example, LuxR- native pathways that could interfere with the biosynthesis or generate
type activators were over-expressed and TetR-type repressors were in- background. To this end, several strains have been genome minimized
activated to initiate production of totopotensamides [163]. to function better as expression chasses. Four gene clusters have been
deleted from the genomes of S. coelicolor M1152 and M1154, which
4.2.3. Heterologous expression in surrogate hosts have been further improved for metabolite production by ribosome
Many microbes that have the capability to produce natural pro- engineering [185]. Extensive manipulation, including deletion of a
ducts, such as strains of Cyanobacteria and filamentous fungi, are ge- 1.5 Mb subtelomeric region and 78% of putative transposase genes of
netically intractable [164]. Heterologous expression provides an op- wild type S. avermitilis led to high success rates and product yields in the
portunity to access the genomic potential these organisms harbor in a expression of exogenous biosynthetic pathways [186]. These ongoing
genetically compliant host. The capture of target BGCs was classically efforts are likely to result in a handful of “superhosts” for the hetero-
done through screening of cosmid gene libraries, which was both time- logous expression of diverse metabolic pathways.
consuming and could not accurately clone the desired DNA fragment,
but advances in direct cloning strategies and DNA assembly tools have 4.2.4. Refactoring of gene clusters
provided attractive alternatives. These methodologies are described in The emergence of new metabolic engineering methods and the
more detail in Section 4.2.4., since changing the expression host may continually decreasing costs of DNA synthesis have enabled complete
require manipulation of control elements such as promoter sequences to refactoring of entire BGCs, which typically includes three distinct
ensure sufficient transcription levels (Fig. 5E). stages. The first step involves the architectural design of the synthetic
The choice of expression host is far from trivial and many factors gene cluster from biological part libraries to obtain desired promoter,
need to be considered. The genetically malleable Escherichia coli and gene and terminator sequences, which may be ordered synthetically or
Saccharomyces cerevisiae provide attractive hosts for production of amplified using PCR. In the next step, the pathway is assembled to-
bacterial and fungal SMs, respectively, but since neither is naturally gether with advanced cloning methods such as recombineering or TAR-
equipped for production of complex chemicals, issues with metabolic cloning typically in E. coli or Sacc. cerevisiae. TAR cloning takes ad-
flux, codon usage, self-resistance and activation of biosynthetic acyl/ vantage of the high efficiency natural homologous recombination in
peptidyl carrier proteins need to be resolved. E. coli seems to be well- yeast that allows for both direct capture of BGCs and simultaneous
suited for expression of pathways originating from Cyanobacteria that cloning of multiple DNA fragments [187]. In contrast, recombineering
are challenging to modify genetically, with production of microcystin utilizes recombination systems from phages to enable homologous re-
[165] and lyngbyatoxin reported [166]. Several fungal terpene syn- combination to occur in E. coli [188]. In addition, the combination of
thases have also been expressed in E. coli, but multi-gene clusters are long-amplicon PCR and DNA recombination (DiPaC) has recently been
much more challenging due to the requirement to remove introns and utilized for the direct cloning of the anabaenopeptins and erythromycin
modify promoter regions. E. coli has had less success with BGCs origi- pathways [189]. The ultimate step involves the transfer of the artificial
nating from high-GC Actinobacteria, even though the entire ery- BGC to the expression host selected for the production of SMs (Fig. 5F).
thromycin pathways has been expressed in this host [167–169]. The Reports on fully synthetic refactoring of BGCs have still been scarce
titres of SMs produced in E. coli have been generally low. Other pro- due to the significant costs associated with the method. Even though
mising heterologous hosts for production of prokaryotic SMs include this approach would allow for unprecedented freedom in pathway de-
Myxococcus xanthus, Pseudomonas putida [170] and Bacillus subtilis sign, the complexity of metabolic pathways required for the production
[171]. of even simple SMs makes the task of designing functional BGC in silico
In many instances, it would appear that hosts that are evolutionarily far from trivial. For these reasons, the methodology appears to be best
closely linked to the organism harbouring the target pathway may be suited for improvements in the production titres of known value-added
best suited for heterologous expression. Several well-characterized metabolites and not for the activation of unknown pathways. Examples
Streptomyces sp. are easily amenable and are efficient surrogate hosts of fully synthetic refactored gene clusters include rebeccamycin and
for the heterologous characterization of different genes from pyrrolnitrin [190].
Actinobacteria. In particular, S. albus J1074 [172], S. lividans TK24 A more widely used approach in refactoring has been to modify
[173], S. venezuelae ATCC10712 and S. coelicolor A3(2) have been regulatory elements or promoter regions of natural gene clusters prior
widely used for the expression of cryptic pathways. S. coelicolor is ge- to heterologous expression. Expression of the unmodified taromycin
netically the most studied actinomycete and a large array of tools are gene cluster directly in S. coelicolor M1146 did not lead to the pro-
available for manipulation of the organism [174], whereas the closely duction of the lipopeptide, but activation of the pathway was achieved
related S. lividans TK24 has particularly excelled at expression of pro- by deletion of pathway –specific repressors using in vivo yeast re-
teins [175]. S. venezuelae, is well-suited for the heterologous expression combination-mediated PCR targeting [191]. Conversely, refactoring of
of the PKs and NRPS due to the presence of large number of innate a strong constitutive promoter in front of a cluster situated activator

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Fig. 6. Targeted genome-wide approaches for the acti-

vation of the silent gene clusters. A) In Reporter Guided
Mutant selection, promoters identified from the target
pathway are utilized to report increased transcription
from the BGC. The selected BGC is activated (i) either by
random chemical mutagens or UV irradiation to generate
mutants where the pathway has been upregulated. (ii)
The double-reporter system (xylE-neo) allows for selec-
tion of mutants in the presence of kanamycin and
screening of the mutant library for highest expression
levels of xylE. Abbreviation: RGMS, Reporter-guided
mutant selection. B) In high-throughput elicitor
screening, activation of the gene clusters is achieved by
finding a small-molecular weight compound that triggers
expression. (iv) Elicitors from a chemical library are
added to individual wells for performing the elicitation
response, which is (v) followed by monitoring tran-
scription levels from a promoter probe construct ex-
pressing GFP. (vi) Metabolic profiling can be used to
detect novel SMs from cultivations of wild type and
biosynthesis activated strains, followed by (vii) structural
elucidation of the target metabolite. Abbreviations: GFP,
Green fluorescence protein; HiTES, High-throughput eli-
citor screening; WT, wild-type strain.

enabled the heterologous production of pacidamycin D [190]. In a more of the novobiocin gene cluster, where a cosmid harbouring the BGC was
arduous approach, individual genes or short operons from BGCs may modified using recombineering to facilitate the generation of plug-and-
also be amplified by PCR and placed in front of active promoters. After play expression modules [195]. Through a plug-and-play promoter in-
reassembly of full length pathways, polycyclic tetramate macrolactams sertion strategy, introduction of three novel constitutive promoters of S.
[192,193] and spectinabilin [194] have been produced in heterologous coelicolor, upstream of jadJ in the cryptic jadomycin B gene cluster in S.
hosts. The PCR –amplification step was circumvented in the refactoring venezuelae, led to cluster activation. Importantly, expression of jadJ

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from these three promoters led to higher transcription levels than when 4.3.2. High-throughput elicitor screening
the widely used ermEp* was utilized [196]. The benefit of strong con- Many SMs are produced in response to various chemical signals,
stitutive promoters is that they may lead to increased transcription le- which may either be communication molecules or chemical weapons
vels and product yields, as has been shown in the engineering of made by other organisms cohabiting the same environment. If the en-
polycyclic tetramate macrolactams [193]. vironmental signal can be identified in the laboratory using small mo-
lecule libraries, expression of silent BGCs can be achieved (Fig. 6B). The
chemogenetic HiTES (high-throughput elicitor screening) method does
4.3. Targeted genome-wide methodologies this by coupling the screening to a promoter-probe system, where either
lacZ [205] or eGFP [206] is utilized to report increased transcription
To date, only few approaches have been developed that are able to from the target BGC. The approach has been successfully applied to two
focus on the desired cryptic BGC detected by genome mining, which are gene clusters in the Gram-negative pathogenic model bacterium Bur-
able to utilize pleiotropic methods to widely probe conditions that lead kholderia thailandensis E264, where a silent virulence factor, namely
to activation of the pathway. These targeted genome-wide methodol- malleilactone (mal) and a poorly expressed burkholdac (bhc) cluster
ogies (Fig. 6) typically take advantage of a biosensor to report increased were targeted [205]. The strategy has also been implemented in S. albus
transcription or other activity from the desired BGC, but differ in the J1074, where the nonribosomal peptide surugamide (sur) cluster was
ways of generating alterations to the global transcriptome. awakened [207]. Interestingly, the cytotoxins etoposide and ivermectin
were found to be effective inducers resulting the occurrence of 14 novel
compounds in the culture media.
4.3.1. Reporter guided systems
A pioneering Reporter-Guided Mutant Selection (RGMS) method 5. Concluding thoughts and future perspectives
developed in the group of Prof. Keqian Yang provides a widely ap-
plicable approach for targeted activation of silent gene clusters in the The abundance of silent and cryptic pathways in microbial genomes
native organism [197]. RGMS combines two effective yet simple tech- provides an exciting opportunity for the discovery of new chemical
niques: cloning of key promoter sequences from the gene cluster of entities with high therapeutic potential. The diverse strategies devel-
interest in front of a double xylE-neo reporter system and the use of oped for awakening these pathways offer an excellent starting point,
genome-wide mutagenesis to generate a library of mutants that contain but to date no single superior methodology has been developed. This is
tremendous genomic diversity. The core rationale relies on randomly not unexpected, since the BGCs reside in vastly diverse micro-organisms
modulating the complex regulatory cascades that are controlling sec- and in many cases are strictly regulated to be activated only under
ondary metabolism and converting expression of the target pathway highly specific conditions. The availability of the numerous methods
into a signal that enables selection of a mutant with the desired phe- described above, which all have their strengths and weaknesses, can
notype. The reporter cassette allows initial selection by kanamycin re- therefore be considered as a significant advantage and it will remain the
sistance (neo), followed by colour-based detection of catechol oxidase responsibility of the researcher to choose the most appropriate one for
(xylE) activity, which allows for distinction between spontaneous ka- any given project.
namycin-resistant and genuine target-activated mutants (Fig. 6A).
RGMS was initially developed for industrial strain improvement Acknowledgements
purposes by taking advantage of the correlation between increased
transcription levels from the target BGC and product yields. The The authors would like to dedicate this article to the memory of
method has been used to improve production of lovastatin [198], cla- Prof. Keqian Yang, who made significant contributions to our under-
vulanic acid [199] and natamycin [200] in Aspergillus terreus, S. cla- standing of secondary metabolic pathways in Streptomyces. The authors
vuligerus and S. gilvosporeus, respectively. In addition, the methodology would like to thank the Jane and Aatos Erkko Foundation for their fi-
has also been successfully carried out in combination with ribosome nancial support.
engineering, through exposure to streptomycin, to achieve over-pro-
duction of daptomycin in S. roseosporus NRRL11379 [201]. Recently, References
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