Examples of realized projects

Daunomycin is a fermentation-derived anthracycline antibiotic that is clinically useful in the treatment of human leukaemias. Daunomycin itself is found rarely in microbial fermentations but is present normally in the form of glycoside derivatives that yield the free drug on simple acid hydrolysis.

At present the research mainly improves the method for daunomycin yield mainly by strain improvement, gene mutation strain screening, optimization of the formula of fermentation medium or use of recombinant strains etc. Although obtaining certain achievement effects are low, fermentation level is generally 1000- 2 500 μg/mL, and fermentation level is unstable due to a toxic effect of metabolite on producing organism.

We have explored the affinity of daunomycin toward the iron and developed the green process based on a spontaneous formation of oil/iron/daunomycin/phytic acid nanoparticles. The process is robust, cheap, and easy to run, characterized by a stable production of about 5 g daunomycin per litre. In comparison with the current process, daunomycin remains inside the sediment and can be removed from the medium by conventional methods which simplifies separation and results in very significant reduction of used organic solvents during the extraction process.

Due to complexity of processes behind daunomycin production the method of adaptive laboratory evolution has been applied to develop and optimize both the strain and process.

The wild strain was obtained from river sediment (sandy-clayey sediment) in the foothills of the Šumava mountain range (Teplá Vltava valley, Czech Republic) after the spring floods in 2018. The isolation was carried out in the same year. R2A was used as isolation medium and PDA and M2 as growth media. Strain identification was performed using 16S RNA (100% identity with Streptomyces coeruleorubidus). The strain is deposited in Czech Culture Collection according to Budapest Treaty requirements.

Wild train was highly unstable characterized by spontaneous formation of both white and bald mutants producing either sterile mycelium or conidiophores with degenerated or un-maturated conidia (Figure 1) with highly variable production of daunomycin.

The strain was screened and selected for presence of daunomycin regulatory genes dnrN, dnrO, and dnrI, daunomycin resistance genes drrA, drrB, drrC and for absence of dauW gene in combination with sporulation behaviour and fast and homogenous growth, daunomycin resistance and production of daunomycin at least 2 000 mg/L in flasks.

The strain was isolated and stabilized by optimization of cultivation medium which forms spontaneously a complex with daunomycin, by neutralizing its toxic effect towards the cells, and allowing the strain to grow and produce daunomycin in rather high concentration.

Producing strain shows a stable phenotype characterized by formation of chains of typical conidia (Figure 2). When cultivated on optimized medium production of daunomycin is up to 5 g/l. At the end of fermentation process majority (95 %) of daunomycin is present in the debris and can be concentrated by any common concentration method. In fact, the majority is deposited in structures spontaneously form during the fermentation process and separated from the cultivation environment by a kind of particle surface incrustation. Presence of daunomycin can be experimentally proven after treatment of the particles with peroxide when the coloured droplets containing daunomycin are formed and visible under the light microscope (Figure 3).

Figure 1 Morphology of the wild strain with visible unmatured spore chains (SEM)

Figure 2 Morphology of S. coeruleorubidus producing strain with typical chain of pores (SEM)

Figure 3 Visualization of daunomycin containing droplet in H₂O₂ treated particle

Figure 4 HPLC profile of purified daunomycin