Statistical optimization of production and isolation of cytotoxic compound from Nocardia ignorata
Résumé
Lichens are symbiotic organisms consisting of fungi and algae (Chlorophyta or Cyanobacteria), and are well-known as rich sources of novel compounds [1]. However, recent studies revealed the presence of bacterial microbiota associated with these organisms [2]. These microorganisms may represent an underexplored reservoir of novel species of potential interest in the discovery of novel lead compounds. However, the biosynthesis of original microbial compounds is influenced by cultivation conditions such as nutrients, incubation periods, pH, temperature, etc. Changing these parameters according to the OSMAC approach [3] (One Strain MAny Compounds) can result in different secondary metabolite profiles. Actinobacteria are known for their ability to produce compounds of clinical and pharmaceutical importance [4]. Thus, we applied the OSMAC approach to Nocardia ignorata, which was isolated from Collema auriforme [5]. The extracts obtained from various culture media (LB, TY, MB, ISP2) showed interesting cytotoxicity against two different cell lines. A statistical optimization of the production of active compound was established using Tagushi L9 orthogonal array design. The four selected parameters were temperature, pH, volume and oxygenation of the medium with three different levels for each parameter. After several days of growth using a 5L bioreactor BioFlo® 115, the culture medium TY was collected and extracted with XAD-7 resin yielding two extracts (supernatant and resin extracts). A bioguided isolation process showed that the cytotoxicity was focused in the supernatant (HaCaT (Human keratinocyte cells): IC50= 8.0 ± 1.5 µg/mL; B16 (Murine melanoma cells): IC50 = 4.6 ± 1.8 µg/mL). This extract was then purified using flash chromatography, semi-preparative reversed-phase HPLC using a water/CH3CN gradient. This process led to the isolation of an active compound which is a novel bromide diketopiperazine identified by NMR and mass spectrometry data.