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Streptomyces as a Source of Antimicrobials: Novel Approaches to Activate Cryptic Secondary Metabolite Pathways

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Ángel Manteca and Paula Yagüe

Submitted: September 4th, 2018Reviewed: October 2nd, 2018Published: March 1st, 2019

DOI: 10.5772/intechopen.81812

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Streptomyces is the most important bacterial genus for bioactive compound production. These soil bacteria are characterized by a complex differentiation cycle. Streptomyces is extremely important in biotechnology, producing approximately two thirds of all antibiotics, as well as many compounds of medical and agricultural interest. Drug discovery from streptomycetes became challenging once the most common compounds were discovered, and the system was basically abandoned by industry. Simultaneously, antibiotic resistance is increasing dramatically, and new antibiotics are required. Screening from nature is being resumed (exploring new environments, looking for elicitors, metagenome, etc.). Secondary metabolism is conditioned by differentiation; although the relationship between both has long remained elusive, differentiation as a trigger for antibiotic production remains basically unexplored. Most cultures used in screening campaigns for new bioactive molecules have been performed empirically, and workflow was extremely productive during the so-called golden age of antibiotics; however, currently there is a bottleneck. Streptomyces is still the most important natural source of antibiotics, and it also harbors many cryptic secondary metabolite pathways not expressed under laboratory conditions. In this chapter, we review strategies based on differentiation, one of the keys improving secondary metabolite production and activating cryptic pathways to face the challenges of drug discovery.


  • Streptomyces
  • screening
  • antibiotics
  • secondary metabolism
  • differentiation
  • elicitors
  • morphology
  • liquid cultures

1. Introduction

1.1 Streptomycesdevelopment

1.1.1 Solid cultures

The complex Streptomyceslife cycle on solid-grown cultures is made up of different phases.

Microorganisms on soils are exposed to different stress factors: chemical, physical and/or biological. Of these variable conditions, sometimes nutrients can be limited, preventing growth [1]. A good survival strategy of some bacteria and fungi is the latency stage of spores. Under this stage, cells stop growing and their metabolism is mainly inactive [2]. Spores are very resistant to stress conditions. In Streptomycetes, they are formed by thick hydrophobic covers that preserve genetic information during unfavorable conditions. Spores are dispersed by wind, water or insects and remain in latency until germination in favorable situations [3]. Besides the thickness of the cell wall, their resistance to extreme temperatures and other physicochemical factors is because of their low water content [4]. In this dehydrated environment, molecules have inactive conformations (denaturalized proteins, etc.) that are stabilized and protected by the sugar trehalose [2]. From this null or limited metabolism, spores are able to germinate, as the macromolecules needed have already been synthesized before the latency stage [2]. For germination, optimal hydration conditions are required. However, it can be faster and more successful in the presence of nutrients and specific stimuli. Spores also need enough levels of intracellular trehalose [5], and polyphosphates [6] that allow metabolic activity to commence before cells are able to detect and assimilate an external source of energy [2].

Despite the fact that germination is a very important stage in the Streptomyceslife cycle, it has remained largely unstudied, because it is not an industrially important stage. When environmental conditions are prosperous, spores on soils/laboratory conditions (petri plates with solid culture medium) germinate. Germination of Streptomycesspores is a sequential process, divided into at least four stages: darkening, swelling, emission of the germination tube [7] and reduced swelling [8].

Darkening is the stage in which spores allow the entry of water into the cell losing their impermeability and heat resistance. After a few minutes, spores change their optical characteristics and the cells are able to reactive their metabolism. Darkwelling requires bivalent cations such as Ca+2, Mg+2, Mn+2, Zn+2 and Fe+2 as well as energy reserves of the spore [7, 9, 10]. Degradation of the cover leads to the reactivation of the cell wall hydrolases. These enzymes, like Rpf (Resuscitation-promoting factors), are involved in cell wall degradation, facilitating peptidoglycan remodeling by hydrolyzing glycosylic bonds B-1,4 between cell wall components, N-acetylglucosamine and N-acetylmuramic acid, which in turn, allow the access to external nutrients [2].

Swelling is the stage in which spores increase its volume due to the entrance of water. This increases the levels of glucose, as a result of trehalose hydrolysis [11]. This seems to be an essential step in germination since only when trehalose concentrations decrease and proteins able to recover their functional conformations [12, 13]. Ribosomes are inactive inside the spores [14, 15]. However, only few minutes after germination starts (30–60 min), they are fully functional and new proteins can be translated [6]. During this stage, spores are metabolically active and are able to use trehalose as main energy source [11].

Emission of the germination tube: This stage begins at the same time as the first DNA replication, when spores are able to detect external sources of nutrients and adjust their metabolic pathways as a result [2]. The accurate point from which the germination tube is emitted is indicated by the accumulation of the protein, SsgA [3]. SsgA is not essential for germination; nevertheless, its absence significantly reduces the number of germ tubes in each spore, and its overexpression has the opposite effect [3].

Reduced swelling: Once the germ tube emerges, the spore starts to gradually reduce its swelling. This stage depends on the quantity of peptidoglycan cross-link that involves a carboxypeptidase (which corresponds to the SCO4439 gene in the Streptomyces coelicolorgenome A deletion of this carboxypeptidase causes spores to continue swelling until 5 μm diameter (2.5 times more than the wild-type strain), after the emission of the germ tube [8]. The existence of this stage demonstrates that the regulation of water entering into the spores is complex and highly regulated.

During germination, several important morphological changes occur, including the degradation of the spore cover, which is mainly formed of peptidoglycan, and causing a huge increase of metabolic activity [2]. On the other hand, shortly after the emission of the germ tube, the expression of the protein DivIVA starts [6]. This protein plays a critical function in the vegetative growth and is located at the apex of the hyphae [16, 17, 18]. After germination, genes involved in cell division (like ftsZ) and proteins involved in cellular growth (like FilP) are expressed [2]. The expression of these genes is considered as the final of the germination phase [2, 6].

Curiously, during germination, three different secondary metabolites are produced: albaflavenone (a terpenoid) with an antibacterial effect against Bacillus subtilis, giving an important advantage in competitive environments, and two polyketides (germicidin A and chancone) with a germination inhibitory effect. These compounds are de novogenerated during germination and are good examples of compounds produced during vegetative growth [19].

After germination, the Streptomycesmycelium starts to grow apically (regulated by divIVA gene) as a very transitory, multinucleated stage called first mycelium (MI), which is multinucleated [20]. This is a very transitory stage in which dead and living segments alternate in the same hyphae [21]. There are two kinds of septa separating segments during the MI stage: one of them is composed of a cell membrane and cell wall (with peptidoglycan) and the other, recently discovered, is a 1-μm spacing cross-membrane-based septa, without peptidoglycan [20]. FtsZ participates in this septation, but it is not essential, as cross-membranes were observed in the FtsZ null mutant. Later, the dead segments are completely disintegrated and living segments continue apical growth. In this phase, the growth is especially branched [18].

After this early stage of branching growth, there is a morphological differentiation into a second mycelium (MII), a sort of multinucleated mycelium with sporadic and apparently randomly positioned septa (formed of cell membrane and cell wall), which is the secondary metabolite producer [22]. This mycelium has two different populations: one of which remains as a branching mycelium growing on surfaces and is called substrate mycelium and the other develops hydrophobic covers and starts to grow into the air without branching (aerial mycelium) [18]. This is known as the “sky-pathway,” which regulates the expression of genes encoding proteins forming hydrophobic proteins (rodlins, chaplins and ramS) that cover the aerial hyphae. Some of the so-called “bald” (bld) mutants among others (defective in aerial growth) regulate the activation of this pathway [23]. At the end of the cycle, aerial hyphae undergo extensive synchronous septation (cell division) to create up to 100 uninucleoid compartments, which finally differentiate to create chains of spores [24]. At the end of the cycle, the “white” (whi) genes are activated (whose mutants are defective in the formation of mature gray spores on the fluffy aerial mycelium) and the hyphae septate and sporulate [25].

1.1.2 Liquid cultures

Streptomycesdifferentiation in liquid cultures has hardly been studied, mainly due to the fact that most strains do not sporulate under these conditions. However, the fact that Streptomyces venezuelaeis capable of sporulation in liquid cultures has made it a new study model in recent years [26].

The applications of novel methodologies (confocal microscopy, proteomics, transcriptomics) to the study of Streptomycesbiology have extended the knowledge regarding the development during the phases preceding sporulation in liquid cultures [27, 28, 29, 30, 31].

In these conditions, in most Streptomycesstrains, aerial mycelium formation and sporulation are blocked [28]; so, there is no hydrophobic cover formation, or sporulation, but there is a first exponential growth phase corresponding to the compartmentalized MI morphology, and then there is a growth arrest phase corresponding to programmed cell death (PCD). After that, the hyphae, which remain alive, continue growing in a second exponential growth phase in which the morphology of the hyphae becomes MII multinucleated mycelium, which corresponds to the antibiotic production [27, 30].

1.2 Correlation between Streptomyceslife cycle and antibiotic production

Most industrial processes for secondary metabolite production are performed in liquid (flasks or bioreactors). This fact makes the knowledge of the Streptomycesbehavior essential in these conditions. The optimization of Streptomycesliquid cultures has been empiric for decades; however, the end of the so-called “golden age of antibiotics,” which meant fewer and fewer compound discoveries or the advances in genome sequences revealing the existence of cryptic biosynthetic pathways that are not expressed under laboratory conditions, calls the attention of the scientific community.

Work on Streptomycesdifferentiation in liquid cultures has largely focused on the analysis of mycelial macroscopic morphology (pellets/clumps formation), media composition and bioreactor design [31]. The differentiation of the hyphae during the life cycle is essential for secondary metabolism [30]; however, there is still some controversy over which is the best morphology for production. Some authors affirm that pellets are better for production, while others report about clumps or even disaggregated growth. Hence, these conditions seem to be strain dependent. For instance, in Streptomyces olidensis(retamycin) [32], Streptomyces tendae(nikkomycins) [33], Streptomyces lividans(hybrid antibiotics) [34] and Streptomyces coelicolor(Undecylprodigiosin, Actinorhodin) [30], pellet formation is essential for good production. However, in Streptomyces noursei(nystatin) [35] and Streptomyces fradiae(tylosin) [36], formation of pellets leads to worse antibiotic production than disaggregated growth. The lack of a reliable developmental model in streptomycetes liquid cultures has hindered the precise identification of reliable phenotypes for use in the analysis and optimization of industrial fermentations. It has been demonstrated that antibiotics are produced by the substrate mycelium at the end of the proliferation phase, i.e., second mycelium morphology (after PCD process) [27, 30, 37]. However, despite the fact that there is a distinctive mycelium producing antibiotics (MII) [27, 30] (hence there is not secondary metabolite production until the differentiation of MII), antibiotic production has additional regulations, and each Streptomycesstrain does not display its entire potential secondary metabolism at a specific developmental condition.

1.3 Screening for new bioactive compounds and drug discovery approaches

Most antibiotics and other bioactive compounds (antitumorals, immunosuppressors, etc.) were discovered from actinomycetes, but this source of drugs became challenging once the most common compounds were discovered. New antibiotic scaffolds are required, and resuming screening from nature is mandatory.

During the past 30 years, only two new classes of antibiotics have been brought to the clinic for treatment systemic infections [38]. At the same time, microbial resistance to existing antibiotics has increased dramatically, rendering some microbial infections that are extremely hard to treat. The current scenario looks gloomy, and there is a concrete risk of humanity returning to the pre-antibiotic era, with a high mortality from routine surgical or chemotherapeutic procedures because of infections by antibiotic-resistant pathogens. There is a general consensus that new antibiotics are urgently required and are the best chance in the fight against resistance.

Only a fraction of secondary metabolite pathways is active in actinomycete laboratory cultures, and there are a large number of strains whose potential to produce bioactive compounds remains unexplored [39]. When whole genome sequencing became available at the start of the twenty-first century, the existence of many silent or cryptic biosynthetic gene clusters in actinomycetes was revealed, which may encode antibiotic-like substances but are not, or only poorly, expressed under laboratory growth conditions. Each Streptomycesstrain harbors 20–50 biosynthetic pathways for natural products, for which only some products have been identified [39]. The big challenge is to find ways to activate these pathways so as to allow screening for new secondary metabolites from nature to resume.


2. Strategies in drug discovery

Once the most common antibiotics and bioactive compounds were discovered, drug discovery has become more challenging, and industry had mostly discontinued the search for new drugs, with Streptomycesas the source. Nevertheless, the urgent necessity of new antibiotics in the clinic has caused screening from nature to be resumed [39]. New environments are being explored and some new actinomycete strains have been discovered [40]; the potential of the marine environment has been highly explored during last decade, as well as some, a priorihostile soils, such as high mountains, deserts or icy places. The scientific community is also paying more attention to symbiotic relationships.

Thinking of natural conditions of growth is another important point of view. Laboratory/industrial conditions in which microorganisms are typically grown are very different conditions from nature, which imply the dormancy of most of the genetic pathways involved in antibiotic production, since these substances are related to stress conditions or defense against niche competitors. Furthermore, it is necessary to take into account that the majority of environmental bacteria are not easily culturable or are even nonculturable in laboratory conditions. This broad view is now possible, thanks to the access to the metagenome in different environments [41].

Another current strategy is to look for elicitors. Elicitors are small molecules, which are able to induce Streptomycesdifferentiation as well as trigger cryptic antibiotic pathways.

Additionally, secondary metabolism is conditioned by differentiation, so it is another aspect that requires special attention. As mentioned above, the life cycle stages and the mycelium morphology are keys for antibiotic production.

At present, different complementary strategies are being developed to improve secondary metabolite production and active cryptic biosynthetic gene clusters. One way to classify them is by unselective and selective methods [42], considering unselective methods are used in the improvement of screening of new activities and selective methods are used in the improvement of production of known molecules.

2.1 Methods for searching for new bacteria/compounds

2.1.1 Exploring new environments

The actinomycetes are widely distributed in the environment; they were discovered at the end of the nineteenth century as a group of soil-living microorganisms. However, their importance started in 1943 with the discovery of streptomycin, the first effective treatment against tuberculosis by the most important actinomycetes genus, Streptomyces. Researchers have isolated different Streptomycesas soil inhabitants during decades as the most important source of antibiotics and bioactive compounds in nature. Clavulanic acid (Streptomyces clavuligerus[43]), neomycin (S. marinensis[44]), chloramphenicol (Streptomyces venezuelae[45]), the insecticide avermectin (Streptomyces avermitilis[46]), the immunosuppressant tacrolimus (Streptomyces tsukubaensis[47]), kanamycin (Streptomyces kanamyceticus[48]) and potent antitumoral platenolides (Streptomyces platensis[49]) are only few examples of the 12,400 bioactive compounds produced by the genus Streptomycesthat society is routinely using in the clinic or agriculture. The rest of the phylum produces 3600 bioactive compounds (being 3400 antibiotics) [50]. Until the 1980s, new bioactive compounds were discovered relatively easily; however, since 1985, only three new compounds have been discovered, with the last one, platensimycin (S. platensis[40]), in 2006.

Once the most common compounds are discovered and producer strains are isolated, one of the strategies for founding new activities is the exploration of new environments, under-exploited habitats, generally difficult to access. Marine environment are an important niche of new species of Streptomyces[51]. Besides marine actinomycetes, they have been isolated from Himalaya Mountains [52], several islands [53], Atacama Desert [54], Antarctica [55] and several extremophile habitats [56, 57, 58].

Promising results and new species are being discovered from these unexplored areas, in combination with next-generation genome sequencing, metagenomics and new ways of bacterial culture [59], being one of the most important approaches in the new age of bioactive compound research.

2.1.2 Symbiosis relationships

As mentioned above, Streptomycesare ubiquitous in soils. However, it has been discovered that they are not only free-living soil bacteria but many have also evolved to live in symbiosis with plants, fungi and animals [60]. Their secondary metabolism is a consequence of these complex interactions [60].

There are many and very diverse symbiotic relationships, which involve Streptomyces. One of the first discovered is its relationship with plant roots providing a natural defense against fungal infections [61]; it is very common to find bacteria of the Streptomycesgenus in the rhizosphere. This fact raises the possibility, currently discussed, about its use as biofertilizers in crops [62, 63]. Furthermore, genome mining is now allowing us to know on one hand which genes are involved in this interaction and on the other hand the discovery of potential novel molecules produced only in these conditions [64].

Other important interactions of Streptomycesgenera take place between bacteria and insects. For instance, in the case of Beewolf digger wasps, Streptomycesprovides protection against pathogens to the larval cocoon producing a mixture of nine antibiotic compounds [65, 66].

In Streptomycesisolates from colonies of the South African termite Macrotermes natalensis, a number of interesting novel compounds including natalamycin A [67] and the potent antitumor geldanamycin [67] have been discovered. Another Streptomycesisolate from the same termite produces a novel cyclic analogue of dentigerumycin [68], the antifungal from the fungus-growing ant system. Finally, a series of glycosylated isoflavonoid compounds, including termisoflavone A [69], were recovered from a third Streptomycesisolate.

In 2012, 30 Streptomycesstrains were isolated from a different South African fungus-growing termite, Microtermessp., and two novel polyketides, microtermolides A and B [69], were identified.

Two Streptomycesstrains, isolated from Dendroctonus frontalisbeetles, inhibit the antagonistic Ophiostomafungus, but not the mutualistic fungus Entomocorticium, suggesting a defensive role that supports the beetle-fungus mutualism. The molecules responsible for this selective antifungal activity are the polyketide mycangimycin [70] and the polycyclic tetramate macrolactams, frontalamides A and B [7172].

Other important symbiotic relationship takes place between ants and Streptomyces. In recent years, new strains and compounds have been isolated from ant head, legs and nests [73, 74, 75, 76].

2.1.3 Nonculturable microorganisms

Since the beginning of microbiology, it was known that many microorganisms do not grow under laboratory conditions and we cannot cultivate them. The new techniques of massive sequencing have revealed the existence of a huge amount of nonculturable microorganisms. Nonculturable microorganisms are, in fact, a majority, and they represent an important challenge in the screening for new secondary metabolites. Next generation sequencing (NGS) has revealed the big pharmacological potential of uncultured bacteria; hence, approaches to improve cultivation-based methods, such as isolation chip (iChip), which is a method of culturing bacteria within its soil environment, or co-cultures, culturing actinomycetes with other bacteria (generally species with which they naturally coexist), are being used successfully [70, 71, 72]. The combination of iChip and unexplored ecosystems or symbiotic relationships, mentioned above, is promising as well.

2.2 Methods for screening improvement in a known microorganism (unselective)

Unselective methods include classical strategies for improving the production of a certain compound by modifying the growth of the Streptomycesstrain. This strategy used to be empiric.

2.2.1 Changing media components

Streptomycesgrow well in rich culture mediums; nevertheless, antibiotic production often needs some specific nutrients. Sometimes defined medium can be better for determined compounds. Optimization of nutritional requirements has been one of the most useful changes for secondary metabolite activities [73, 74, 75, 76].

2.2.2 Inducing stress response

Stressful situations are one of the reasons why microorganisms start to produce and export bioactive compounds. For this reason, the induction of cultures with several treatments of stress is one of the improvement strategies for synthesis of secondary metabolite compounds in bacteria. Heat and ethanol shock treatments, high salt conditions, increased hydrostatic pressure, acidic pH shock or feed limitations are some examples of this classical strategy (reviewed in [77]).

2.2.3 Random mutagenesis

There are different random mutagenesis methods used in Streptomyces: chemical mutagenesis, in which are used (e.g., by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), or nitrous acid (NA)) and physical mutagenesis (e.g., by ultraviolet (UV) light or X-rays)) that remain being robust methods to generate very high-producing strains [78, 79, 80].

On the other hand, there is transposon mutagenesis, a powerful tool for random mutagenesis of bacterial genomes and insertion of foreign DNA. A plasmid containing the enzyme transposase and the transposon (small fragment of DNA with transposase target regions) is extracted and inserted randomly into the host chromosome. Transposition creates single mutations that can be identified and mapped by plasmid rescue and DNA sequencing, generating a direct link between the higher yield (phenotype) and the transposon insertion (genotype) [81, 82].

2.2.4 Ribosomal engineering

The fact that some antibiotic resistances trigger some cryptic pathways of secondary metabolites has been recently used as another strategy in the search for new compounds.

It has been demonstrated that this occurs in strains affected by antibiotics, which target a ribosomal protein (for instance, streptomycin, paromomycin and gentamicin [42]), so the effects of the mutations at this level have had very good results, for example, in Streptomyces coelicolor, for improving secondary metabolite production [83]. The mechanism of this strategy is still unknown, but it has been proved that there is activation of the positive regulators like actII-orf4 gene at least in S. coelicolor[42]. Through this approach, the antibacterial piperidamycins family compounds have been discovered [84].

2.2.5 Elicitors

Elicitors are small diffusible molecules, which are being used as signals for improving secondary metabolite production or even activating cryptic antibiotic pathways [85].

Elicitors are also present in Streptomycesnatural environments; hence one of the strategies for stimulating bioactive compound production is the co-culture of different bacteria [85, 86]. Co-cultures usually include species that have symbiotic relationships with Streptomycesin nature [87, 88] or pathogenic partners to activate the production of antimicrobial compounds against them [71, 89, 90].

Other kinds of elicitors are also useful, for instance, fungal elicitors (a complex mix of fungal cell walls and filtered fungi cultures) can positively affect the production of natamycin [91]; small molecules, such as GlcNac or phosphate, can activate differentiation and antibiotic production in S. coelicolorthrough the activation of actII-ORF4/redZ genes in some conditions [92].

At the end of the 1980s, the discovery of new compounds reached a bottleneck, and screening processes often led to the rediscovery of compounds already known. To avoid rediscovery, Pimentel-Elardo et al. [93] developed a workflow based on the use of chemical elicitors combined with activity-independent screening [93]. Activity-independent screening is based on the low-abundant compounds found in biological samples. The use of elicitors increases the production of these low-abundant compounds, distinguishing them from the most abundant ones. The elicitor “CI-ARC” has been identified as being responsible for triggering several cryptic biosynthetic genes [93].

2.2.6 Differentiation strategies based on morphology Streptomyces behavior in liquid cultures

Large-scale antibiotic production is mostly performed in liquid cultures. Macroscopic morphology of the mycelium (pellets and clumps) correlates with the production of secondary metabolites [30]. The genes controlling mycelium aggregation have been characterized in the S. coelicolor matgene cluster [94], and the cslA, glxAand dtpAgenes [95, 96, 97] are responsible for pellet formation. This knowledge can be used for controlling and improving morphology in industrial fermentations.

Since spores are inoculated in liquid medium, there is an aggregation trend [98]. Germlings determine the macroscopic morphology (pellets and clumps) of the culture, which involves the highly conserved genes matA, matB[94, 98] and the cslA/glxAoperon [98].

In liquid cultures, there are some strains able to sporulate [99], but even those which aren’t able to sporulate in flasks can sporulate in bioreactors, beacuse of the stess conditions inside the fermenter, such as lack of oxygen, strong agitation, etc. [100]. When Streptomycesbacteria sporulate, all the metabolic efforts are focused on that, which means that the secondary metabolism stops by. Therefore, one of the main things for taking into account during a screening for new secondary metabolites is to avoid sporulation in the industrial fermentations. Hence, it is very important to look out the cultures for keeping secondary metabolism as much as possible [100].

Morphology in liquid cultures can be monitored by several tools. Laser diffraction has been used to measure pellet size, an improvement over image analysis, which is trickier and requires more time [101], and a technique based on flow cytometry has been used to establish the pellet size distribution of a culture population [102, 103]. Recently, a useful algorithm has been developed as a plugin for the open-source software ImageJ to characterize the morphology of submerged growing cells [104]. On the other hand, mathematical models have been performed to predict the behavior of Streptomycesliquid cultures based on pellet/clump morphology [105, 106].

Furthermore, it has been reported that the oxygen transfer rate (OTR) and oxygen uptake rate (OUR) are crucial for the development and production in liquid cultures [100]. Controlling these rates, which directly affects morphology, was described as being crucial for scaling up production to industrial conditions [107]. Biophysical parameters, including pH, viscosity, agitation, dissolved oxygen levels and surface tension, directly affect mycelium morphology. The optimization of these factors is another factor to consider [31]. Programmed cell death and MII differentiation

As mentioned in the introduction of this chapter, programmed cell death (PCD) is a key event, triggering the differentiation of antibiotic producer mycelium (MII) in liquid and solid cultures [30]. However, the specific signals derived from cell death are not yet known. The N-acetylglucosamine resulting from peptidoglycan dismantling accelerates the development and antibiotic production [108, 109] and might be one of the signals released during PCD. A simple methodology based on fluorimetric measures was designed to quantify PCD in liquid cultures [37], allowing prediction of the efficiency of antibiotic production based on the level of PCD.

PCD and MII differentiation can be modulated by modifying culture conditions and morphology. Modifying developmental conditions to enhance PCD and MII differentiation leads to the improvement of secondary metabolite production. This approach was recently applied in a study combining the heterologous expression of plant flavonoids in Streptomyces albuswith a strategy to enhance PCD and MII differentiation [110]. The same strategy was successful at improving mTGase production from Streptomyces mobaraensis[111]. Therefore, PCD and MII differentiation were demonstrated to be crucial for the streamlining of secondary metabolite production in bioreactors [100].

2.3 Methods for production improvement in a known compound (selective)

Selective methods with a specific target include regulatory engineering, heterologous expression, genome mining and combinatorial biosynthesis.

2.3.1 Regulatory engineering

Each active compound, for example, antibiotic or antitumor, etc., has several molecules synthesized first in the pathway, and sometimes these molecules are a limited source for the compound of interest. In this sense, one strategy widely used is the addition of these precursors, previously synthetized from an external source. In the same way, it is possible to enhance the production of a specific compound through the overexpression of its positive regulators. Inversely, knocking out the suppressors (negative regulation) of the gene of interest has also had good results increasing the final compound production [112, 113].

2.3.2 Heterologous expression

In microbiology, this molecular genetic technique is based on the expression of a gene or a group of genes (gene cluster) in a host microorganism that does not have this particular gene or gene cluster in its own genome. This procedure, performed by recombinant DNA technology, is very useful when hosts are bacteria with simple developmental cycles, such as E. colior Bacillus[114]. However, in the case of Streptomyces,due to its complex pathways to produce antibiotics, heterologous expression usually works only among Streptomycesstrains [115, 116], not solving the problem of its tricky growth.

Nevertheless, good results have been achieved in activating cryptic metabolites, for example, by using the widely conserved Streptomyces coelicolorpleiotropic regulator AfsQ, which activates the production of new compounds in several streptomycetes [117]. Despite the fact that heterologous expression in Streptomycesis a challenge, this technique continues to be improved [118].

2.3.3 Genome mining

Genome mining is defined as the obtaining of information about an organism, based on genome analysis. In the postgenomic era, the combination of easier genome sequencing and bioinformatics analyses allows researchers to uncover the biosynthetic potential of the microorganisms, i.e., the gene clusters for producing secondary metabolites into actinomycetes strains that are in the genome, but silent. Genome mining is nowadays one of the more powerful tools in the screening for new antibiotics [119, 120, 121, 122].

2.3.4 Combinatorial biosynthesis

Combinatorial biosynthesis can be defined as the application to modify biosynthetic pathways to natural products to produce new and altered structures using nature’s biosynthetic machinery [123]. The chemical modification of existing chemical scaffolds is challenging and sometimes provides only a temporary solution against resistant organisms [124]. Combinatorial biosynthesis has been largely developed over the last 20 years, and it has been enhanced, thanks to genome mining and synthetic biology [125, 126, 127].


3. Conclusions

Humanity faces the great challenge of fighting antibiotic resistance, which is growing at a faster rate than our capacity to find new antimicrobials and new strategies to solve this problem.

The Streptomycesgenus is still a huge source of natural bioactive compounds, but we need to elaborate new multidisciplinary strategies to avoid rediscovering the same compounds. Therefore, we need to invest efforts into developing alternative strategies to resume screening from natural actinomycetes.

There is not a “magic” methodology to activate cryptic pathways and improve the discovery of new bioactive compounds, but multidisciplinary approaches combining the methodologies discussed in this chapter will play a key role in the improvement of screening for new bioactive compounds from streptomycetes.



We thank the Spanish “Ministerio de Economía y Competitividad” (MINECO; BIO2015-65709-R) and the “Marie Curie cofund Clarin” Grant for financial support and for proofreading the final manuscript.


Conflict of interest

There is no conflict of interest in this work.


  1. 1.Challis GL, Hopwood DA. Synergy and contingency as driving forces for the evolution of multiple secondary metabolite production byStreptomycesspecies. Proceedings of the National Academy of Sciences of the United States of America. 2003;100(Suppl 2):14555-14561
  2. 2.Bobek J, Smidova K, Cihak M. A waking review: Old and novel insights into the spore germination inStreptomyces. Frontiers in Microbiology. 2017;8:2205
  3. 3.Noens EE, Mersinias V, Willemse J, Traag BA, Laing E, Chater KF, et al. Loss of the controlled localization of growth stage-specific cell-wall synthesis pleiotropically affects developmental gene expression in an ssgA mutant ofStreptomyces coelicolor. Molecular Microbiology. 2007;64(5):1244-1259
  4. 4.Kalakoutskii LV, Agre NS. Comparative aspects of development and differentiation in actinomycetes. Bacteriological Reviews. 1976;40(2):469-524
  5. 5.Ranade N, Vining LC. Accumulation of intracellular carbon reserves in relation to chloramphenicol biosynthesis byStreptomyces venezuelae. Canadian Journal of Microbiology. 1993;39(4):377-383
  6. 6.Strakova E, Bobek J, Zikova A, Vohradsky J. Global features of gene expression on the proteome and transcriptome levels inS. coelicolorduring germination. PLoS One. 2013;8(9):e72842
  7. 7.Hardisson C, Manzanal MB, Salas JA, Suarez JE. Fine structure, physiology and biochemistry of arthrospore germination inStreptomyces antibioticus. Journal of General Microbiology. 1978;105(2):203-214
  8. 8.Rioseras B, Yague P, Lopez-Garcia MT, Gonzalez-Quinonez N, Binda E, Marinelli F, et al. Characterization of SCO4439, a D-alanyl-D-alanine carboxypeptidase involved in spore cell wall maturation, resistance, and germination inStreptomyces coelicolor. Scientific Reports. 2016;6:21659
  9. 9.Grund AD, Ensign JC. Properties of the germination inhibitor ofStreptomyces viridochromogenesspores. Journal of General Microbiology. 1985;131(4):833-847
  10. 10.Salas JA, Guijarro JA, Hardisson C. High calcium content inStreptomycesspores and its release as an early event during spore germination. Journal of Bacteriology. 1983;155(3):1316-1323
  11. 11.McBride MJ, Ensign JC. Effects of intracellular trehalose content onStreptomyces griseusspores. Journal of Bacteriology. 1987;169(11):4995-5001
  12. 12.Nwaka S, Holzer H. Molecular biology of trehalose and the trehalases in the yeastSaccharomyces cerevisiae. Progress in Nucleic Acid Research and Molecular Biology. 1998;58:197-237
  13. 13.Singer MA, Lindquist S. Multiple effects of trehalose on protein folding in vitro and in vivo. Molecular Cell. 1998;1(5):639-648
  14. 14.Mikulik K, Janda I, Weiser J, Stastna J, Jiranova A. RNA and ribosomal protein patterns during aerial spore germination inStreptomycesgranaticolor. European Journal of Biochemistry. 1984;145(2):381-388
  15. 15.Bobek J, Halada P, Angelis J, Vohradsky J, Mikulik K. Activation and expression of proteins during synchronous germination of aerial spores ofStreptomycesgranaticolor. Proteomics. 2004;4(12):3864-3880
  16. 16.Flardh K. Essential role of DivIVA in polar growth and morphogenesis inStreptomyces coelicolorA3(2). Molecular Microbiology. 2003;49(6):1523-1536
  17. 17.Flardh K. Growth polarity and cell division inStreptomyces. Current Opinion in Microbiology. 2003;6(6):564-571
  18. 18.Flardh K, Richards DM, Hempel AM, Howard M, Buttner MJ. Regulation of apical growth and hyphal branching inStreptomyces. Current Opinion in Microbiology. 2012;15(6):737-743
  19. 19.Cihak M, Kamenik Z, Smidova K, Bergman N, Benada O, Kofronova O, et al. Secondary Metabolites Produced during the Germination ofStreptomyces coelicolor. Frontiers in Microbiology. 2017;8:2495
  20. 20.Yague P, Willemse J, Koning RI, Rioseras B, Lopez-Garcia MT, Gonzalez-Quinonez N, et al. Subcompartmentalization by cross-membranes during early growth ofStreptomyces hyphae. Nature Communications. 2016;7:12467
  21. 21.Manteca A, Fernandez M, Sanchez J. A death round affecting a young compartmentalized mycelium precedes aerial mycelium dismantling in confluent surface cultures ofStreptomyces antibioticus. Microbiology. 2005;151(Pt 11):3689-3697
  22. 22.Yague P, Rodriguez-Garcia A, Lopez-Garcia MT, Martin JF, Rioseras B, Sanchez J, et al. Transcriptomic analysis ofStreptomyces coelicolordifferentiation in solid sporulating cultures: First compartmentalized and second multinucleated mycelia have different and distinctive transcriptomes. PLoS One. 2013;8(3):e60665
  23. 23.Claessen D, de Jong W, Dijkhuizen L, Wosten HA. Regulation ofStreptomycesdevelopment: Reach for the sky! Trends in Microbiology. 2006;14(7):313-319
  24. 24.Flardh K, Buttner MJ.Streptomyces morphogenetics: Dissecting differentiation in a filamentous bacterium. Nature Reviews. Microbiology. 2009;7(1):36-49
  25. 25.Kaiser BK, Stoddard BL. DNA recognition and transcriptional regulation by the WhiA sporulation factor. Scientific Reports. 2011;1:156
  26. 26.Schlimpert S, Flardh K, Buttner J. Fluorescence time-lapse imaging of the completeS. venezuelaelife cycle using a microfluidic device. Journal of Visualized Experiments. 2016;108:53863
  27. 27.Yague P, Rodriguez-Garcia A, Lopez-Garcia MT, Rioseras B, Martin JF, Sanchez J, et al. Transcriptomic analysis of liquid non-sporulatingStreptomyces coelicolorcultures demonstrates the existence of a complex differentiation comparable to that occurring in solid sporulating cultures. PLoS One. 2014;9(1):e86296
  28. 28.Manteca A, Jung HR, Schwammle V, Jensen ON, Sanchez J. Quantitative proteome analysis ofStreptomyces coelicolornonsporulating liquid cultures demonstrates a complex differentiation process comparable to that occurring in sporulating solid cultures. Journal of Proteome Research. 2010;9(9):4801-4811
  29. 29.Rioseras B, Shliaha PV, Gorshkov V, Yague P, Lopez-Garcia MT, Gonzalez-Quinonez N, et al. Quantitative proteome and phosphoproteome analyses ofStreptomyces coelicolorreveal proteins and phosphoproteins modulating differentiation and secondary metabolism. Molecular & Cellular Proteomics. 2018;17(8):1591-1611
  30. 30.Manteca A, Alvarez R, Salazar N, Yague P, Sanchez J. Mycelium differentiation and antibiotic production in submerged cultures ofStreptomyces coelicolor. Applied and Environmental Microbiology. 2008;74(12):3877-3886
  31. 31.van Dissel D, Claessen D, van Wezel GP. Morphogenesis ofStreptomycesin submerged cultures. Advances in Applied Microbiology. 2014;89:1-45
  32. 32.Giudici R, Pamboukian CR, Facciotti MC. Morphologically structured model for antitumoral retamycin production during batch and fed-batch cultivations ofStreptomyces olindensis. Biotechnology and Bioengineering. 2004;86(4):414-424
  33. 33.Vecht-Lifshitz SE, Sasson Y, Braun S. Nikkomycin production in pellets ofStreptomyces tendae. The Journal of Applied Bacteriology. 1992;72(3):195-200
  34. 34.Sarra M, Casas C, Godia F. Continuous production of a hybrid antibiotic byStreptomyces lividansTK21 pellets in a three-phase fluidized-bed bioreactor. Biotechnology and Bioengineering. 1997;53(6):601-610
  35. 35.Jonsbu E, McIntyre M, Nielsen J. The influence of carbon sources and morphology on nystatin production byStreptomyces noursei. Journal of Biotechnology. 2002;95(2):133-144
  36. 36.Stratigopoulos G, Gandecha AR, Cundliffe E. Regulation of tylosin production and morphological differentiation inStreptomyces fradiaeby TylP, a deduced gamma-butyrolactone receptor. Molecular Microbiology. 2002;45(3):735-744
  37. 37.Yague P, Manteca A, Simon A, Diaz-Garcia ME, Sanchez J. New method for monitoring programmed cell death and differentiation in submergedStreptomycescultures. Applied and Environmental Microbiology. 2010;76(10):3401-3404
  38. 38.Procopio RE, Silva IR, Martins MK, Azevedo JL, Araujo JM. Antibiotics produced byStreptomyces. The Brazilian Journal of Infectious Diseases. 2012;16(5):466-471
  39. 39.Genilloud O. The re-emerging role of microbial natural products in antibiotic discovery. Antonie Van Leeuwenhoek. 2014;106(1):173-188
  40. 40.Takahashi Y, Omura S. Isolation of new actinomycete strains for the screening of new bioactive compounds. The Journal of General and Applied Microbiology. 2003;49(3):141-154
  41. 41.McMahon MD, Guan C, Handelsman J, Thomas MG. Metagenomic analysis ofStreptomyces lividansreveals host-dependent functional expression. Applied and Environmental Microbiology. 2012;78(10):3622-3629
  42. 42.Craney A, Ahmed S, Nodwell J. Towards a new science of secondary metabolism. Journal of Antibiotics (Tokyo). 2013;66(7):387-400
  43. 43.Reading C, Cole M. Clavulanic acid: A beta-lactamase-inhiting beta-lactam fromStreptomyces clavuligerus. Antimicrobial Agents and Chemotherapy. 1977;11(5):852-857
  44. 44.Waksman SA, Lechevalier HA. Neomycin, a new antibiotic active against streptomycin-resistant bacteria, including tuberculosis organisms. Science. 1949;109(2830):305-307
  45. 45.Shapiro S, Vining LC. Nitrogen metabolism and chloramphenicol production inStreptomyces venezuelae. Canadian Journal of Microbiology. 1983;29(12):1706-1714
  46. 46.Ikeda H, Nonomiya T, Usami M, Ohta T, Omura S. Organization of the biosynthetic gene cluster for the polyketide anthelmintic macrolide avermectin inStreptomyces avermitilis. Proceedings of the National Academy of Sciences of the United States of America. 1999;96(17):9509-9514
  47. 47.Karlsson H, Truedsson L, Nassberger L. The immunosuppressive agent FK506 inhibits in vitro expression of membrane-bound and soluble interleukin-2 receptors on resting but not on activated human lymphocytes. Immunology Letters. 1991;30(1):129-132
  48. 48.Kharel MK, Subba B, Basnet DB, Woo JS, Lee HC, Liou K, et al. A gene cluster for biosynthesis of kanamycin fromStreptomyces kanamyceticus: Comparison with gentamicin biosynthetic gene cluster. Archives of Biochemistry and Biophysics. 2004;429(2):204-214
  49. 49.Sakai T, Sameshima T, Matsufuji M, Kawamura N, Dobashi K, Mizui Y. Pladienolides, new substances from culture ofStreptomyces platensisMer-11107. I. Taxonomy, fermentation, isolation and screening. Journal of Antibiotics (Tokyo). 2004;57(3):173-179
  50. 50.Hamedi J, Poorinmohammad N, Wink J. The role of actinobacteria in biotechnology. In: Wink J, Mohammadipanah F, Hamedi J, editors. Biology and Biotechnology of Actinobacteria. Cham: Springer; 2017. pp. 269-328. DOI: 10.1007/9783319603391
  51. 51.Lam KS. Discovery of novel metabolites from marine actinomycetes. Current Opinion in Microbiology. 2006;9(3):245-251
  52. 52.Bhattacharjee K, Banerjee S, Joshi SR. Diversity ofStreptomycesspp. in eastern Himalayan region–computational RNomics approach to phylogeny. Bioinformation. 2012;8(12):548-554
  53. 53.Charousova I, Medo J, Halenarova E, Javorekova S. Antimicrobial and enzymatic activity of actinomycetes isolated from soils of coastal islands. Journal of Advanced Pharmaceutical Technology & Research. 2017;8(2):46-51
  54. 54.Idris H, Labeda DP, Nouioui I, Castro JF, Del Carmen Montero-Calasanz M, Bull AT, et al.Streptomyces aridussp. nov., isolated from a high altitude Atacama Desert soil and emended description ofStreptomyces noboritoensisIsono et al. 1957. Antonie Van Leeuwenhoek. 2017;110(5):705-717
  55. 55.Sanyal A, Antony R, Samui G, Thamban M. Microbial communities and their potential for degradation of dissolved organic carbon in cryoconite hole environments of Himalaya and Antarctica. Microbiological Research. 2018;208:32-42
  56. 56.Bonilla JO, Kurth DG, Cid FD, Ulacco JH, Gil RA, Villegas LB. Prokaryotic and eukaryotic community structure affected by the presence of an acid mine drainage from an abandoned gold mine. Extremophiles. 2018;22(5):699-711
  57. 57.Ben Abdallah M, Karray F, Kallel N, Armougom F, Mhiri N, Quemeneur M, et al. Abundance and diversity of prokaryotes in ephemeral hypersaline lake Chott El Jerid using Illumina Miseq sequencing, DGGE and qPCR assays. Extremophiles. 2018;22(5):811-823
  58. 58.Bull AT, Idris H, Sanderson R, Asenjo J, Andrews B, Goodfellow M. High altitude, hyper-arid soils of the Central-Andes harbor mega-diverse communities of actinobacteria. Extremophiles. 2018;22(1):47-57
  59. 59.Stewart EJ. Growing unculturable bacteria. Journal of Bacteriology. 2012;194(16):4151-4160
  60. 60.Seipke RF, Kaltenpoth M, Hutchings MI.Streptomycesas symbionts: An emerging and widespread theme? FEMS Microbiology Reviews. 2012;36(4):862-876
  61. 61.Schrey SD, Tarkka MT. Friends and foes: Streptomycetes as modulators of plant disease and symbiosis. Antonie Van Leeuwenhoek. 2008;94(1):11-19
  62. 62.Rey T, Dumas B. Plenty is no plague:Streptomycessymbiosis with crops. Trends in Plant Science. 2017;22(1):30-37
  63. 63.Vurukonda S, Giovanardi D, Stefani E. Plant growth promoting and biocontrol activity ofStreptomycesspp. as endophytes. International Journal of Molecular Sciences. 2018;19(4)
  64. 64.Ceapa CD, Vazquez-Hernandez M, Rodriguez-Luna SD, Cruz Vazquez AP, Jimenez Suarez V, Rodriguez-Sanoja R, et al. Genome mining ofStreptomyces scabrisporusNF3 reveals symbiotic features including genes related to plant interactions. PLoS One. 2018;13(2):e0192618
  65. 65.Kroiss J, Kaltenpoth M, Schneider B, Schwinger MG, Hertweck C, Maddula RK, et al. Symbiotic Streptomycetes provide antibiotic combination prophylaxis for wasp offspring. Nature Chemical Biology. 2010;6(4):261-263
  66. 66.Engl T, Kroiss J, Kai M, Nechitaylo TY, Svatos A, Kaltenpoth M. Evolutionary stability of antibiotic protection in a defensive symbiosis. Proceedings of the National Academy of Sciences of the United States of America. 2018;115(9):E2020-E2029
  67. 67.Kim KH, Ramadhar TR, Beemelmanns C, Cao S, Poulsen M, Currie CR, et al. Natalamycin A, an ansamycin from a termite-associatedStreptomycessp. Chemical Science. 2014;5(11):4333-4338
  68. 68.Wyche TP, Ruzzini AC, Beemelmanns C, Kim KH, Klassen JL, Cao S, et al. Linear peptides are the major products of a biosynthetic pathway that encodes for cyclic depsipeptides. Organic Letters. 2017;19(7):1772-1775
  69. 69.Kang HR, Lee D, Benndorf R, Jung WH, Beemelmanns C, Kang KS, et al. Termisoflavones A-C, isoflavonoid glycosides from termite-associatedStreptomycessp. RB1. Journal of Natural Products. 2016;79(12):3072-3078
  70. 70.Kealey C, Creaven CA, Murphy CD, Brady CB. New approaches to antibiotic discovery. Biotechnology Letters. 2017;39(6):805-817
  71. 71.Sung AA, Gromek SM, Balunas MJ. Upregulation and identification of antibiotic activity of a marine-derivedStreptomycessp. via co-cultures with human pathogens. Marine Drugs. 2017;15(8)
  72. 72.Onaka H, Mori Y, Igarashi Y, Furumai T. Mycolic acid-containing bacteria induce natural-product biosynthesis inStreptomycesspecies. Applied and Environmental Microbiology. 2011;77(2):400-406
  73. 73.Vastrad BM, Neelagund SE. Optimization of medium composition for the production of neomycin byStreptomycesfradiae NCIM 2418 in solid state fermentation. Biotechnology Research International. 2014;2014:674286
  74. 74.Alduina R, Sosio M, Donadio S. Complex regulatory networks governing production of the glycopeptide A40926. Antibiotics (Basel). 2018;7(2)
  75. 75.Jakeman DL, Graham CL, Young W, Vining LC. Culture conditions improving the production of jadomycin B. Journal of Industrial Microbiology & Biotechnology. 2006;33(9):767-772
  76. 76.Saudagar PS, Singhal RS. Optimization of nutritional requirements and feeding strategies for clavulanic acid production byStreptomyces clavuligerus. Bioresource Technology. 2007;98(10):2010-2017
  77. 77.Yoon V, Nodwell JR. Activating secondary metabolism with stress and chemicals. Journal of Industrial Microbiology & Biotechnology. 2014;41(2):415-424
  78. 78.Baltz RH. Genetic manipulation of secondary metabolite biosynthesis for improved production inStreptomycesand other actinomycetes. Journal of Industrial Microbiology & Biotechnology. 2016;43(2-3):343-370
  79. 79.Khaliq S, Akhtar K, Afzal Ghauri M, Iqbal R, Mukhtar Khalid A, Muddassar M. Change in colony morphology and kinetics of tylosin production after UV and gamma irradiation mutagenesis ofStreptomyces fradiaeNRRL-2702. Microbiological Research. 2009;164(4):469-477
  80. 80.Korbekandi H, Darkhal P, Hojati Z, Abedi D, Hamedi J, Pourhosein M. Overproduction of clavulanic acid by UV mutagenesis ofStreptomyces clavuligerus. Iranian Journal of Pharmaceutical Research. 2010;9(2):177-181
  81. 81.Fedashchin A, Cernota WH, Gonzalez MC, Leach BI, Kwan N, Wesley RK, et al. Random transposon mutagenesis of theSaccharopolyspora erythraeagenome reveals additional genes influencing erythromycin biosynthesis. FEMS Microbiology Letters. 2015;362(22)
  82. 82.Xu Z, Wang Y, Chater KF, Ou HY, Xu HH, Deng Z, et al. Large-scale transposition mutagenesis ofStreptomyces coelicoloridentifies hundreds of genes influencing antibiotic biosynthesis. Applied and Environmental Microbiology. 2017;83(6)
  83. 83.Wang G, Hosaka T, Ochi K. Dramatic activation of antibiotic production inStreptomyces coelicolorby cumulative drug resistance mutations. Applied and Environmental Microbiology. 2008;74(9):2834-2840
  84. 84.Hosaka T, Ohnishi-Kameyama M, Muramatsu H, Murakami K, Tsurumi Y, Kodani S, et al. Antibacterial discovery in actinomycetes strains with mutations in RNA polymerase or ribosomal protein S12. Nature Biotechnology. 2009;27(5):462-464
  85. 85.Onaka H. Novel antibiotic screening methods to awaken silent or cryptic secondary metabolic pathways in actinomycetes. Journal of Antibiotics (Tokyo). 2017;70(8):865-870
  86. 86.Marmann A, Aly AH, Lin W, Wang B, Proksch P. Co-cultivation--a powerful emerging tool for enhancing the chemical diversity of microorganisms. Marine Drugs. 2014;12(2):1043-1065
  87. 87.Piel J. Metabolites from symbiotic bacteria. Natural Product Reports. 2004;21(4):519-538
  88. 88.Yu J, Liu Q, Chen C, Qi X. Antifungal activity change ofStreptomyces rimosusMY02 mediated by confront culture with other microorganism. Journal of Basic Microbiology. 2017;57(3):276-282
  89. 89.Perez J, Munoz-Dorado J, Brana AF, Shimkets LJ, Sevillano L, Santamaria RI.Myxococcus xanthusinduces actinorhodin overproduction and aerial mycelium formation byStreptomyces coelicolor. Microbial Biotechnology. 2011;4(2):175-183
  90. 90.Wang Y, Wang L, Zhuang Y, Kong F, Zhang C, Zhu W. Phenolic polyketides from the co-cultivation of marine-derivedPenicilliumsp. WC-29-5 andStreptomyces fradiae007. Marine Drugs. 2014;12(4):2079-2088
  91. 91.Wang D, Yuan J, Gu S, Shi Q. Influence of fungal elicitors on biosynthesis of natamycin byStreptomyces natalensisHW-2. Applied Microbiology and Biotechnology. 2013;97(12):5527-5534
  92. 92.Liu G, Chater KF, Chandra G, Niu G, Tan H. Molecular regulation of antibiotic biosynthesis inStreptomyces. Microbiology and Molecular Biology Reviews. 2013;77(1):112-143
  93. 93.Pimentel-Elardo SM, Sorensen D, Ho L, Ziko M, Bueler SA, Lu S, et al. Activity-independent discovery of secondary metabolites using chemical elicitation and cheminformatic inference. ACS Chemical Biology. 2015;10(11):2616-2623
  94. 94.van Dissel D, Claessen D, Roth M, van Wezel GP. A novel locus for mycelial aggregation forms a gateway to improvedStreptomycescell factories. Microbial Cell Factories. 2015;14:44
  95. 95.Chaplin AK, Petrus ML, Mangiameli G, Hough MA, Svistunenko DA, Nicholls P, et al. GlxA is a new structural member of the radical copper oxidase family and is required for glycan deposition at hyphal tips and morphogenesis ofStreptomyces lividans. The Biochemical Journal. 2015;469(3):433-444
  96. 96.Petrus ML, Vijgenboom E, Chaplin AK, Worrall JA, van Wezel GP, Claessen D. The DyP-type peroxidase DtpA is a Tat-substrate required for GlxA maturation and morphogenesis inStreptomyces. Open Biology. 2016;6(1):150149
  97. 97.Petrus ML, Claessen D. Pivotal roles forStreptomycescell surface polymers in morphological differentiation, attachment and mycelial architecture. Antonie Van Leeuwenhoek. 2014;106(1):127-139
  98. 98.Zacchetti B, Willemse J, Recter B, van Dissel D, van Wezel GP, Wosten HA, et al. Aggregation of germlings is a major contributing factor towards mycelial heterogeneity ofStreptomyces. Scientific Reports. 2016;6:27045
  99. 99.Geneviève Girard BAT, Sangal V, Mascini N, Hoskisson PA, Goodfellow M, van Wezel GP. A novel taxonomic marker that discriminates between morphologically complex actinomycetes. Open Biology. 2013;3(10):130073
  100. 100.Rioseras B, Lopez-Garcia MT, Yague P, Sanchez J, Manteca A. Mycelium differentiation and development ofStreptomyces coelicolorin lab-scale bioreactors: Programmed cell death, differentiation, and lysis are closely linked to undecylprodigiosin and actinorhodin production. Bioresource Technology. 2014;151:191-198
  101. 101.Ronnest NP, Stocks SM, Lantz AE, Gernaey KV. Comparison of laser diffraction and image analysis for measurement ofStreptomyces coelicolorcell clumps and pellets. Biotechnology Letters. 2012;34(8):1465-1473
  102. 102.van Veluw GJ, Petrus ML, Gubbens J, de Graaf R, de Jong IP, van Wezel GP, et al. Analysis of two distinct mycelial populations in liquid-grownStreptomycescultures using a flow cytometry-based proteomics approach. Applied Microbiology and Biotechnology. 2012;96(5):1301-1312
  103. 103.Petrus ML, van Veluw GJ, Wosten HA, Claessen D. Sorting ofStreptomycescell pellets using a complex object parametric analyzer and sorter. Journal of Visualized Experiments. 2014;84:e51178
  104. 104.Willemse J, Buke F, van Dissel D, Grevink S, Claessen D, van Wezel GP. SParticle, an algorithm for the analysis of filamentous microorganisms in submerged cultures. Antonie Van Leeuwenhoek. 2017;111(2):171-182
  105. 105.Celler PC. K, van Loosdrecht MC, van Wezel GP., Structured morphological modeling as a framework for rational strain design ofStreptomycesspecies. Antonie Van Leeuwenhoek. 2012;102(3):409-423
  106. 106.Nieminen L, Webb S, Smith MC, Hoskisson PA. A flexible mathematical model platform for studying branching networks: Experimentally validated using the model actinomycete,Streptomyces coelicolor. PLoS One. 2013;8(2):e54316
  107. 107.Ha S, Lee KJ, Lee SI, Gwak HJ, Lee JH, Kim TW, et al. Optimization of Herbicidin A Production in Submerged Culture ofStreptomycesscopuliridis M40. Journal of Microbiology and Biotechnology. 2017;27(5):947-955
  108. 108.Rigali S, Nothaft H, Noens EE, Schlicht M, Colson S, Müller M, et al. The sugar phosphotransferase system ofStreptomyces coelicoloris regulated by the GntR-family regulator DasR and links N-acetylglucosamine metabolism to the control of development. Molecular Microbiology. 2006;61(5):1237-1251
  109. 109.Rigali S, Titgemeyer F, Barends S, Mulder S, Thomae AW, Hopwood DA, et al. Feast or famine: The global regulator DasR links nutrient stress to antibiotic production byStreptomyces. EMBO Reports. 2008;9(7):670-675
  110. 110.Marin L, Gutierrez-Del-Rio I, Yague P, Manteca A, Villar CJ, Lombo F. De novo biosynthesis of apigenin, luteolin, and eriodictyol in the actinomyceteStreptomyces albusand production improvement by feeding and spore conditioning. Frontiers in Microbiology. 2017;8:921
  111. 111.LuciaTreppiccione AO, Luongo D, Maurano F, Manteca Á, FelipeLombó MR. Development of gluten with immunomodulatory properties using mTG-active food grade supernatants fromStreptomyces mobaraensiscultures. Journal of Functional Foods. 2017;34
  112. 112.Yi JS, Kim M, Kim EJ, Kim BG. Production of pikromycin using branched chain amino acid catabolism inStreptomyces venezuelaeATCC 15439. Journal of Industrial Microbiology & Biotechnology. 2018;45(5):293-303
  113. 113.Razmilic V, Castro JF, Andrews B, Asenjo JA. Analysis of metabolic networks ofStreptomyces leeuwenhoekiiC34 by means of a genome scale model: Prediction of modifications that enhance the production of specialized metabolites. Biotechnology and Bioengineering. 2018;115(7):1815-1828
  114. 114.Ece S, Lambertz C, Fischer R, Commandeur U. Heterologous expression of aStreptomyces cyaneuslaccase for biomass modification applications. AMB Express. 2017;7(1):86
  115. 115.Novakova R, Nunez LE, Homerova D, Knirschova R, Feckova L, Rezuchova B, et al. Increased heterologous production of the antitumoral polyketide mithramycin A by engineeredStreptomyces lividansTK24 strains. Applied Microbiology and Biotechnology. 2018;102(2):857-869
  116. 116.Kawahara T, Izumikawa M, Kozone I, Hashimoto J, Kagaya N, Koiwai H, et al. Neothioviridamide, a polythioamide compound produced by heterologous expression of aStreptomycessp. cryptic RiPP biosynthetic gene cluster. Journal of Natural Products. 2018
  117. 117.Daniel-Ivad M, Hameed N, Tan S, Dhanjal R, Socko D, Pak P, et al. An engineered allele of afsQ1 facilitates the discovery and investigation of cryptic natural products. ACS Chemical Biology. 2017;12(3):628-634
  118. 118.Mevaere J, Goulard C, Schneider O, Sekurova ON, Ma H, Zirah S, et al. An orthogonal system for heterologous expression of actinobacterial lasso peptides inStreptomyceshosts. Scientific Reports. 2018;8(1):8232
  119. 119.Yu Y, Tang B, Dai R, Zhang B, Chen L, Yang H, et al. Identification of the streptothricin and tunicamycin biosynthetic gene clusters by genome mining inStreptomycessp. strain fd1-xmd. Applied Microbiology and Biotechnology. 2018;102(6):2621-2633
  120. 120.Liu W, Sun F, Hu Y. Genome mining-mediated discovery of a new avermipeptin analogue inStreptomyces actuosusATCC 25421. ChemistryOpen. 2018;7(7):558-561
  121. 121.Gran-Scheuch A, Trajkovic M, Parra L, Fraaije MW. Mining the genome ofStreptomyces leeuwenhoekii: Two new type I Baeyer-Villiger Monooxygenases from Atacama Desert. Frontiers in Microbiology. 2018;9:1609
  122. 122.Ye S, Molloy B, Brana AF, Zabala D, Olano C, Cortes J, et al. Identification by genome mining of a type I polyketide gene cluster fromStreptomyces argillaceusinvolved in the biosynthesis of pyridine and piperidine alkaloids argimycins P. Frontiers in Microbiology. 2017;8:194
  123. 123.Floss HG. Combinatorial biosynthesis--potential and problems. Journal of Biotechnology. 2006;124(1):242-257
  124. 124.Genilloud O. Actinomycetes: Still a source of novel antibiotics. Natural Product Reports. 2017;34(10):1203-1232
  125. 125.Olano C, Mendez C, Salas JA. Post-PKS tailoring steps in natural product-producing actinomycetes from the perspective of combinatorial biosynthesis. Natural Product Reports. 2010;27(4):571-616
  126. 126.González A, Rodríguez M, Braña AF, Méndez C, Salas JA, Olano C. New insights into paulomycin biosynthesis pathway inStreptomycesalbus J1074 and generation of novel derivatives by combinatorial biosynthesis. Microbial Cell Factories. 2016:15
  127. 127.Baltz RH. Synthetic biology, genome mining, and combinatorial biosynthesis of NRPS-derived antibiotics: A perspective. Journal of Industrial Microbiology & Biotechnology. 2017;45(7):635-649

Written By

Ángel Manteca and Paula Yagüe

Submitted: September 4th, 2018Reviewed: October 2nd, 2018Published: March 1st, 2019