Despite the fact that most industrial processes for secondary metabolite production are performed with submerged cultures, a reliable developmental model for Streptomyces under these culture conditions is lacking. With the exception of a few species which sporulate under these conditions, it is assumed that no morphological differentiation processes take place. In this work, we describe new developmental features of Streptomyces coelicolor A3(2) grown in liquid cultures and integrate them into a developmental model analogous to the one previously described for surface cultures. Spores germinate as a compartmentalized mycelium (first mycelium). These young compartmentalized hyphae start to form pellets which grow in a radial pattern. Death processes take place in the center of the pellets, followed by growth arrest. A new multinucleated mycelium with sporadic septa (second mycelium) develops inside the pellets and along the periphery, giving rise to a second growth phase. Undecylprodigiosin and actinorhodin antibiotics are produced by this second mycelium but not by the first one. Cell density dictates how the culture will behave in terms of differentiation processes and antibiotic production. When diluted inocula are used, the growth arrest phase, emergence of a second mycelium, and antibiotic production are delayed. Moreover, pellets are less abundant and have larger diameters than in dense cultures. This work is the first to report on the relationship between differentiation processes and secondary metabolite production in submerged Streptomyces cultures.Streptomyces is a soil bacterium that produces numerous clinically useful antibiotics (1, 54, 66), as well as many molecules that affect eukaryotic systems, such as inducers of eukaryotic cellular differentiation, inducers and inhibitors of apoptosis (59), and protein C kinase inhibitors with antitumor activity (such as staurosporine and others) (48, 57). Moreover, its remarkably complex developmental cycle makes this microorganism an interesting subject for study. The traditional developmental cycle of this bacterium describes two differentiated mycelial structures, a substrate (vegetative) mycelium and an aerial (reproductive) mycelium (10,25,33). In the substrate mycelium, septa are thought to be widely spaced and to define compartments containing several nucleoids (10, 63). After a short growth arrest phase characterized by reduced macromolecular synthesis (25), aerial hyphae develop from simple branching from substrate mycelium (29). Finally, the tips of the aerial mycelium differentiate into hydrophobic spore chains (8). Recently, we have been able to extend what is known about the developmental cycle in surface confluent cultures a great deal. Our main contribution has been to reveal the existence of a very young compartmentalized mycelium that dies following an orderly pattern, leaving alternating live and dead segments in the same hypha (37). Subsequently, the remaining live mycelium grows in successive waves that vary according to the density of the ...

Streptomycetes are mycelium-forming bacteria that produce two thirds of clinically relevant secondary metabolites. Secondary metabolite production is activated at specific developmental stages of Streptomyces life cycle. Despite this, Streptomyces differentiation in industrial bioreactors tends to be underestimated and the most important parameters managed are only indirectly related to differentiation: modifications to the culture media, optimization of productive strains by random or directed mutagenesis, analysis of biophysical parameters, etc. In this work the relationship between differentiation and antibiotic production in lab-scale bioreactors was defined. Streptomyces coelicolor was used as a model strain. Morphological differentiation was comparable to that occurring during pre-sporulation stages in solid cultures: an initial compartmentalized mycelium suffers a programmed cell death, and remaining viable segments then differentiate to a second multinucleated antibiotic-producing mycelium. Differentiation was demonstrated to be one of the keys to interpreting biophysical fermentation parameters and to rationalizing the optimization of secondary metabolite production in bioreactors.

Streptomycetes comprise very important industrial bacteria, producing two-thirds of all clinically relevant secondary metabolites. They are mycelial microorganisms with complex developmental cycles that include programmed cell death (PCD) and sporulation. Industrial fermentations are usually performed in liquid cultures (large bioreactors), conditions in which Streptomyces strains generally do not sporulate, and it was traditionally assumed that there was no differentiation. In this work, we review the current knowledge on Streptomyces pre-sporulation stages of Streptomyces differentiation.

Nutraceutical compounds as plant flavonoids play an important role in prevention and modulation of diverse heath conditions, as they exert interesting antifungal, antibacterial, antioxidant, and antitumor effects. They also possess anti-inflammatory activities in arthritis, cardiovascular disease or neurological diseases, as well as modulatory effects on the CYP450 activity on diverse drugs. Most flavonoids are bioactive molecules of plant origin, but their industrial production is sometimes hindered due to reasons as low concentration in the plant tissues, presence in only some species or as a complex mixture or inactive glycosides in plant vacuolae. In this work, we describe the de novo biosynthesis of two important flavones, apigenin and luteolin, and one known flavanone, eriodictyol. Their plant biosynthetic pathways have been reconstructed for heterologous expression in Streptomyces albus, an actinomycete bacterium manageable at industrial production level. Also, production levels for apigenin have been improved by feeding with naringenin precursor, and timing for settlement of secondary metabolism has been advanced by spore conditioning. In the cases of eriodictyol and luteolin, their production in this important type of biotechnology-prone bacteria, the actinomycetes, had not been described in the literature yet.

Streptomycetes are very important industrial bacteria, which produce two thirds of all clinically relevant secondary metabolites. They have a complex developmental-cycle in which an early compartmentalized mycelium (MI) differentiates to a multinucleated mycelium (MII) that grows inside the culture medium (substrate mycelium) until it starts to growth into the air (aerial mycelium) and ends up forming spores. Streptomyces developmental studies have focused mainly on the later stages of MII differentiation (aerial mycelium and sporulation), with regulation of pre-sporulation stages (MI/MII transition) essentially unknown. This work represents the first study of the Streptomyces MI transcriptome, analyzing how it differs from the MII transcriptome. We have used a very conservative experimental approach to fractionate MI from MII and quantify gene expressions. The expression of well characterized key developmental/metabolic genes involved in bioactive compound production (actinorhodin, undecylprodigiosin, calcium-dependent antibiotic, cpk, geosmin) or hydrophobic cover formation-sporulation (bld, whi, wbl, rdl, chp, ram) was correlated with MII differentiation. Additionally, 122 genes conserved in the Streptomyces genus, whose biological function had not been previously characterized, were found to be differentially expressed (more than 4-fold) in MI or MII. These genes encoded for putative regulatory proteins (transcriptional regulators, kinases), as well as hypothetical proteins. Knowledge about differences between the MI (vegetative) and MII (reproductive) transcriptomes represents a huge advance in Streptomyces biology that will make future experiments possible aimed at characterizing the biochemical pathways controlling pre-sporulation developmental stages and activation of secondary metabolism in Streptomyces.

Streptomyces is a diverse group of gram-positive microorganisms characterised by a complex developmental cycle. Streptomycetes produce a number of antibiotics and other bioactive compounds used in the clinic. Most screening campaigns looking for new bioactive molecules from actinomycetes have been performed empirically, e.g., without considering whether the bacteria are growing under the best developmental conditions for secondary metabolite production. These screening campaigns were extremely productive and discovered a number of new bioactive compounds during the so-called “golden age of antibiotics” (until the 1980s). However, at present, there is a worrying bottleneck in drug discovery, and new experimental approaches are needed to improve the screening of natural actinomycetes. Streptomycetes are still the most important natural source of antibiotics and other bioactive compounds. They harbour many cryptic secondary metabolite pathways not expressed under classical laboratory cultures. Here, we review the new strategies that are being explored to overcome current challenges in drug discovery. In particular, we focus on those aimed at improving the differentiation of the antibiotic-producing mycelium stage in the laboratory.

Streptomycetes are multicellular bacteria with complex developmental cycles. They are of biotechnological importance as they produce most bioactive compounds used in biomedicine, antibiotic, antitumoral and immunosupressor compounds. genomes encode many Ser/Thr/Tyr kinases, making this genus an outstanding model for the study of bacterial protein phosphorylation events. We used mass spectrometry based quantitative proteomics and phosphoproteomics to characterize bacterial differentiation and activation of secondary metabolism of We identified and quantified 3461 proteins corresponding to 44.3% of the proteome across three developmental stages: vegetative hypha (first mycelium); secondary metabolite producing hyphae (second mycelium); and sporulating hyphae. A total of 1350 proteins exhibited more than 2-fold expression changes during the bacterial differentiation process. These proteins include 136 regulators (transcriptional regulators, transducers, Ser/Thr/Tyr kinases, signaling proteins), as well as 542 putative proteins with no clear homology to known proteins which are likely to play a role in differentiation and secondary metabolism. Phosphoproteomics revealed 85 unique protein phosphorylation sites, 58 of them differentially phosphorylated during differentiation. Computational analysis suggested that these regulated protein phosphorylation events are implicated in important cellular processes, including cell division, differentiation, regulation of secondary metabolism, transcription, protein synthesis, protein folding and stress responses. We discovered a novel regulated phosphorylation site in the key bacterial cell division protein FtsZ (pSer319) that modulates sporulation and regulates actinorhodin antibiotic production. We conclude that manipulation of distinct protein phosphorylation events may improve secondary metabolite production in industrial streptomycetes, including the activation of cryptic pathways during the screening for new secondary metabolites from streptomycetes.

Streptomyces species produce many clinically relevant secondary metabolites and exhibit a complex development that includes hyphal differentiation and sporulation in solid cultures. Industrial fermentations are usually performed in liquid cultures, conditions in which Streptomyces strains generally do not sporulate, and it was traditionally assumed that no differentiation took place. The aim of this work was to compare the transcriptomes of S. coelicolor growing in liquid and solid cultures, deepening the knowledge of Streptomyces differentiation. Microarrays demonstrated that gene expression in liquid and solid cultures were comparable and data indicated that physiological differentiation was similar for both conditions. Eighty-six percent of all transcripts showed similar abundances in liquid and solid cultures, such as those involved in the biosynthesis of actinorhodin (actVA, actII-4) and undecylprodigiosin (redF); activation of secondary metabolism (absR1, ndsA); genes regulating hydrophobic cover formation (aerial mycelium) (bldB, bldC, bldM, bldN, sapA, chpC, chpD, chpE, chpH, ramA, ramC, ramS); and even some genes regulating early stages of sporulation (wblA, whiG, whiH, whiJ). The two most important differences between transcriptomes from liquid and solid cultures were: first, genes related to secondary metabolite biosynthesis (CDA, CPK, coelichelin, desferrioxamine clusters) were highly up-regulated in liquid but not in solid cultures; and second, genes involved in the final stages of hydrophobic cover/spore maturation (chpF, rdlA, whiE, sfr) were up-regulated in solid but not in liquid cultures. New information was also provided for several non-characterized genes differentially expressed in liquid and solid cultures which might be regulating, at least in part, the metabolic and developmental differences observed between liquid and solid cultures.