Regulating Tacrolimus Production in Streptomyces Species

Streptomyces species are bacteria growing in the soil

These bacteria are among the most abundant organisms in soil. They are heterotrophic, i.e., they decompose the organic matter of other living beings (like plants). Their morphology and way of living resemble that of fungi: filamentous cells that grow by the tips and penetrates the substrates, secreting hydrolytic enzymes to decompose big cell structures or organic molecules.

As occurs with fungi, the Streptomyces filaments differentiate into spores when the environmental conditions are no longer supporting growth. Spores are a specialized cellular form to survive until favorable conditions for growth appear.

Soil is a very crowded place to live and showing drastic variations in the growth conditions

The initiation of sporulation is usually linked to the production of antibiotics. These compounds seem to serve to sporulating bacteria as chemical weapons to protect the colony against bacterial or fungal competitors during this delicate stage. Antibiotics might act also as signaling molecules for cell-to-cell communication. In any case, about two-thirds of all known antibiotics are produced by species of Streptomyces genus, so the medical importance of this bacteria. Moreover, some of the antibiotic-related compounds show other useful bioactivities, such as antitumor, insecticidal or immunosuppressant activities.

Regulation of antibiotic production is important from both the bacterial point of view and the human point of view

Antibiotics are expensive for the cell because they are complex structures which biosynthesis required energy and nutrients. So, antibiotics are produced by Streptomyces only when needed, such as when nutrients are scarce and sporulation begins.

It is not strange that when the environmental conditions favor growth, e.g., an abundance of carbon and energy sources such as sugars, sporulation and antibiotic production do not initiate. In terms of laboratory cultures, a growth medium with a richness of carbon sources is good for bacterial growth but bad for antibiotic production. Nutrients that favor growth are bad for antibiotic production. The paradigmatic case is glucose. Glucose can be readily used by the metabolism of most organisms cells and support a good growth, but usually represses antibiotic production. This is one aspect of the regulatory phenomenon called carbon catabolite repression.

Streptomyces tsukubensis (or tsukubaensis) is an important industrial species

Due to the interest of the antibiotic production by species, the genus has been the focus of many works on regulatory subjects. Most of these studies have been carried out in the model species Streptomyces coelicolor. This species lacks any industrial importance, however, it has been used as the model since decades ago because it produces two pigmented antibiotics, actinorhodin and undecylprodigionsin, that are easily identified visually: colonies on plates and also liquid cultures become reddish (undecylprodigionsin) or blueish (actinorhodin; “coelicolor” means sky color).

For the current work, we focused our efforts on the producer of tacrolimus (or FK506). This compound shows certain antifungal activity, but its medical utility relies on its immunosuppressant activity. Tacrolimus is widely used in the prevention of graft rejection; also in the treatment of skin diseases and shows potential properties as a neuroprotective, neuroregenerative, and anticancer agent.

Glucose and glycerol caused the repression of tacrolimus production

Our primary interest is the study of the regulation of tacrolimus production. For this work, we were interested in carbon catabolite repression. What we made first was to establish which carbon sources and at what concentration caused repression of tacrolimus. In the medium and conditions used for liquid cultures, tacrolimus is produced after growth is limited by the depletion of phosphate (about 89 h of culture; phosphate is the only source of essential nutrient phosphorous).

We tested the effect on tacrolimus production of the addition of different carbon sources. We tried the addition at 70 h of culture, close to the end of a rapid growth phase, and before the onset of tacrolimus production. We found that addition of glucose completely blocked production (during all the time course of the cultures, 9 and a half days); glycerol addition also resulted in a strong repression, although not complete. Meanwhile, maltose, a glucose disaccharide, did not affect production.

Thus, the experimental setup was established: we conducted a transcriptomic analysis using time series cultures with added glucose, glycerol or maltose, the latter as the control (no repression condition). Transcriptomics allow us to study the transcription of genes, that is to say, an intermediate step from genes to proteins. DNA is transcribed to RNA, most of which is translated into proteins in a further step. Thus, we can determine which genes are more or less transcribed in a particular experimental condition.

For transcriptomic studies, we used microarray technology. Microarrays consist of thousands of short DNA sequences, named as probes, fixed on a solid surface. Probe sequences represent different regions of the genome. Complementary DNA sequences obtained from cells of each experimental conditions are fixed to probes by hybridization. The amount of DNA hybridized is measured by means of fluorescence.

Genome-wide transcription profiles reveal hints on the complex regulatory mechanisms

Samples of the cultures were taken at certain time points. Total RNA was purified from the cells and the RNA preparations were labeled and hybridized against microarrays. The resulting profiles of the genes that encode the biosynthetic enzymes reflected the production features of the cultures. Thus, in maltose-added cultures the expression of the biosynthetic genes coincide with the timing of the tacrolimus appearance in the culture; in contrast, in glucose and glycerol cultures there was not expression of the biosynthetic genes.

Since the lack of tacrolimus production in repressing conditions was caused by the lack of gene expression, regulatory genes that 1) were only active in maltose condition, and 2) its expression was activated in advance to that of biosynthetic genes, could be positive regulators of tacrolimus biosynthesis genes. That is the case of fkbN, a gene located in the same chromosomal region than that of the biosynthesis genes, the biosynthesis cluster. This gene is well known as encoding the required transcriptional activator protein which activates the transcription of the biosynthesis genes. By means of measuring the Pearson correlation among transcriptional values, we identified other genes showing similar transcription profiles to that of fkbN. Up to 80 genes which profiles were correlated to those of fkbN were found, and they might be useful candidates for genetic engineering of the strain to increase production.

A multitude of genes and functions were affected by the carbon source addition. In order to be very brief, it can be said that the not only genes for tacrolimus production but also genes responsible for the morphological differentiation were strongly and permanently downregulated, as expected.

Streptomyces is a diverse genus with around 600 species characterized. Interestingly, some observed gene responses to repressing carbon sources were different between S. tsukubensis and those previously observed in the model species S. coelicolor. These discrepancies reinforce the importance of using distinct and industrially important Streptomyces species for regulatory studies, which are useful not only for the discovery of new antibiotics but also for the improvement of production yields of already-known bioactive compounds.

These findings are described in the article entitled Streptomyces tsukubaensis as a new model for carbon repression: transcriptomic response to tacrolimus repressing carbon sources, recently published in the journal Applied Microbiology and Biotechnology. This work was conducted by M. Ordóñez-Roblez, F. Santos-Beneit, S. M. Albillos, P. Liras, J. F. Martín, and A. Rodríguez-García from the Instituto de Biotecnología de León, INBIOTEC.