Streptomycetes are mycelium-forming sporulating 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 liquid nonsporulating cultures (flasks and 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 chapter, we review the relationship between differentiation and antibiotic production in liquid cultures. Morphological differentiation in liquid cultures is 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 is one of the keys to interpreting biophysical fermentation parameters and to rationalizing the optimization of secondary metabolite production in liquid cultures.
- programmed cell death
Streptomycetes are gram-positive, environmental soil bacteria that play important roles in the mineralization of organic matter.
Streptomycetes have a complex developmental cycle that makes this bacterium a multicellular prokaryotic model. The classical
The main objective of this work is to review the state of the art of
Streptomycesdevelopment in liquid cultures
During the last decade, new knowledge regarding
Microbial multinucleated structures such as the substrate and aerial mycelia are fragile, uncommon, and usually related to transitory reproductive stages . Contrary to what was postulated during the last 50 years,
Streptomycesdifferentiation and industrial fermentations
The absence of an understanding of
Only recently has basic knowledge about differentiation in liquid cultures been generated. As introduced above, MII was demonstrated to be the antibiotic-producing mycelium. In a recent work, our group demonstrated that differentiation is one of the keys to interpreting typical fermentation parameters (growth, antibiotic production, dissolved oxygen tension, agitation, and oxygen uptake rates)  in bioreactors. Pellet and clump formation greatly influences PCD, usually occurring in the center of the mycelium pellet  and MII differentiation from MI living cells at the pellet periphery . We proposed a general consensus to improve secondary metabolite production in
3.1 Differentiation of
There are important differences between liquid cultures in laboratory flasks and bioreactors. Despite the obvious hydrodynamic differences between the two systems, there are also differences in the culture media. In this sense, one of the most important differences between flasks and bioreactors is the use of antifoams. Antifoams are often used in bioreactors to prevent foam formation and its interference with the bioreactor probes . Apparently, antifoams do not affect development and they are usually added automatically in small amounts when foam is detected by a specific probe, and in some cases, they are added directly to the culture
3.1.1 Differentiation of
S. coelicolorin antifoam-free media
Mycelium differentiation in bioreactors is comparable to differentiation in laboratory flasks  (outlined in Figure 2): at early time points, hyphae presented the regular discontinuities and gaps previously described for MI hyphae ; MI differentiates into a second multinucleated mycelium (MII) after a programmed cell death (reviewed in Yagüe et al. ). However, there are important differences between development in flask and bioreactor.
One of the most important differences observed in the bioreactor with respect to laboratory flasks for
MII differentiation and antibiotic production is accelerated in the
Biophysical fermentation parameters, such as dissolved oxygen tension (DOT), agitation, and oxygen uptake rates (OURs), correlated well with differentiation : DOT falls from saturation at time zero to the fixed level (50% saturation), due to hyphal growth and respiration; there is a concomitant increase in agitation to maintain oxygen levels at the fixed level; once pellet disintegration starts, biological oxygen consumption and agitation decrease gradually, and dissolved oxygen levels increase suddenly to saturation. OUR values fall during the MI PCD and are not recovered until the MII differentiation. This description of mycelium differentiation and OUR in bioreactors constitutes an elegant example of the importance of understanding
3.1.2 Differentiation of
S. coelicolorin bioreactors supplemented with antifoam
Rioseras et al.  modified growing conditions in bioreactors to prevent the early massive lysis described above. The most obvious difference between bioreactors and laboratory flasks is the impellers used for agitation in the case of the bioreactor, so the first experimental approach to trying to prevent lysis was to reduce agitation to minimum levels (50 rpm); however, the same extension of pellet disintegration was observed . Similar results were observed at different agitation rates (50, 100, 200, or 300 rpm) or by replacing Rushton impellers by a gentle impeller (pitched blade impellers) . The only modification that worked to avoid the massive mycelial lysis observed in bioreactors was the modification of the culture medium’s rheology reducing surface tension by means of an antifoam agent (Biospumex 153 K, BASF). This effect of preventing early fragmentation/lysis was only observed at relatively high antifoam concentrations (1%) (outlined in Figure 2).
The reason why antifoam prevents pellet disintegration is as yet unknown. However, the antifoam tended to coat the mycelial pellets, and the hydrophobic forces generated may have prevented this phenomenon. Antifoams are often used with
Biophysical fermentation parameters also correlated well with differentiation in cultures with antifoam: the absence of pellet disintegration prolonged the oxygen consumption phase, generating two peaks of OUR (MI and MII stages) separated by a stage of low oxygen consumption . These two maxima in OUR are very unusual in industrial fermentation, and are another nice illustration of the necessity of understanding
3.2 Sporulation of
Another important difference between bioreactor- and laboratory flask-S
Streptomycesdifferentiation and screening for new bioactive compounds
Streptomycetes are important biotechnological bacteria from which two thirds of the bioactive secondary metabolites used clinically (mainly antibiotics, but also antitumourals, immunosuppressors, etc.) were discovered . Drug discovery became challenging once the most common antibiotics were discovered. In fact, during the past 30 years, only three new classes of antibiotics have been brought to the clinic (mutilins, lipopeptides, and oxazolidinones) [38, 39]. At the same time, microbial resistance to existing antibiotics has increased dramatically, rendering some microbial infections extremely hard to treat.
New antibiotics are urgently needed in the clinic. No valid alternatives to screening natural strains have emerged to find new scaffolds and families of antibiotics . New workflows are needed to access the natural secondary metabolites that remain inaccessible in the laboratory . Nonnatural synthetic antibiotics obtained by chemical/combinatorial biosynthesis exist, but most of them are variations of natural molecules . The best way to find structurally novel bioactive compounds is to resume screening from natural streptomycetes. The most obvious approach to look for new bioactive compounds from natural streptomycetes is to study streptomycetes isolated from relatively lowly explored niches such as marine ecosystems , symbiotic streptomycetes , etc., an approach followed by several research groups and biotechnology companies. On the other hand, genomic analyses revealed that Streptomyces genomes encode an average of 30 secondary metabolite pathways , but only a fraction of these pathways (around four per strain ) is active in laboratory cultures. Consequently, there is a huge amount of potentially bioactive compounds produced by streptomycetes that are never observed in the lab (cryptic pathways) and remain unexplored. There is a consensus in the scientific community about the necessity to activate the expression of these cryptic pathways in order to overcome the present bottleneck in drug discovery.
Several research groups and biotechnology companies face the challenge of activating cryptic pathways to try to mimic the ecological niche of the bacteria by making co-cultures of different microbes , looking for elicitor activating pathways (nutrients such as glucose, xylose, and small molecules such as GlcNac and phosphate)  or making heterologous expression . As stated above,
Different streptomycetes show different behaviors in liquid cultures: some species form large pellets, such as
We thank the Spanish “Ministerio de Economía y Competitividad” (MINECO; BIO2015-65709-R) and the “Marie Curie cofund Clarin” Grant for financial support and Proof-Reading-Service.com for proofreading the final manuscript.
Conflict of interest
There is no conflict of interest in this work.