The production of jadomycin by Streptomyces venezuelae was carried out by applying various environmental stress conditions on the culture in nutrient deprived, amino acid rich MSM production media. The effects of culture transfer time (18, 21 and 24 h) from the growth media to the production media, heat shock at different temperatures (35, 40, 45 and 50°C) and exposure times (0.5 and 1 h), alcohol shock using ethanol and methanol at various concentrations (3.0, 4.5, 6.0 and 7.5 % v/v), multiple shocking by 3 % v/v ethanol at various times [once (0 h), twice (0, 3 h) and three times (0, 3 and 6 h)] and ethanol shock with various concentrations (3.0, 4.5 and 6.0 % v/v) and varying nitrogen concentrations (45, 60 and 75 mM) in the media on the growth and activity of S.venezuelae and jadomycin production were investigated. The growth of S.venezuelae was determined by measuring cell number and TF yield and the Jadomycin B was determined by measuring the optical density (AU¬526nm). Shocking the culture that was transferred after 18 h of growth in the growth medium produced the highest jadomycin, cell growth and activity compared to those that were transfered after 21 and 24 h. The culture shocked at a temperature of 40°C for 1 h produced the highest jadomycin. Higher temperature and/or longer exposure time resulted in poor cell growth and activity. The ethanol concentration of 4.5 % v/v produced the highest jadomycin. The culture shocked with ethanol once (0 h) produced higher jadomycin, cell growth and activity compared to the cultures shocked twice [(0, 3 h) and three times (0, 3 and 6 h)]. A nitrogen concentration of 75 mM with 4.5 % v/v in the production media increased cell growth and jadomycin production. The results showed that shocking the culture once with ethanol (3 % v/v) produced 81.5 % more jadomycin than shocking with heat at a temperature of 45°C for 1 h. However, increasing the ethanol concentration to 4.5 % v/v produced 10.8 % more jadomycin than 3 % v/v ethanol. By increasing the nitrogen concentration to 75 mM in the production medium and shocking with the 4.5 % v/v ethanol further increased the jadomycin production by 13.9 % above that without nitrogen addition. Therefore, the treatment with ethanol (4.5 % v/v) and nitrogen (75 mM) is recommended for jadomycin production.

Jadomycins are secondary metabolites produced by Streptomyces venezuelae ISP5230 VS1099 in response to conditions of stress such as heat or ethanol shock. They have been shown to exhibit antibiotic and anticancer activity. Unique structural features of the jadomycins include a rare 2,6-dideoxysugar, L-digitoxose, and an oxazolone ring with an amino acid component. Previous studies have revealed that jadomycin derivatives can be produced by altering the amino acid in S. venezuelae ISP5230 VS1099 culture media which becomes incorporated into the oxazolone ring. One jadomycin from a proteogenic amino acid and three new jadomycins from non-proteogenic amino acids have been successfully produced on a large scale (4 mg/L to 12 mg/L, 2 L) and characterized using mass spectrometry, infrared spectroscopy, UV-visible spectroscopy, and nuclear magnetic resonance spectroscopy. One of these contains a terminal alkyne functionality and has been used in cycloaddition reactions with various azides to produce a library of triazole-containing jadomycins.

Multidrug resistance refers to the simultaneous resistance to structurally and mechanistically unrelated cytotoxic drugs. Chronic administration of cytotoxic drugs to patients with metastatic breast cancer results in the development of multidrug resistance, thus rendering chemotherapy unsuccessful. One mechanism by which multidrug resistance is conferred is the decreased intracellular drug accumulation due to the upregulation of the ATP-binding cassette (ABC) transporters. Jadomycins are polyketide-derived natural products produced by the soil actinomycetes Streptomyces venezuelae, ISP 5230. Jadomycins exhibit anticancer, antibacterial and antifungal activities. Pilot work in our laboratory demonstrated that jadomycin B exhibited similar cytotoxic effects in drug-sensitive and drug-resistant cancer cells. We hypothesize that jadomycins are poor substrates of ABCB1, ABCC1 and ABCG2 efflux transporters, and consequently will exhibit higher intracellular accumulation, which results in improved cytotoxic efficacy over existing chemotherapeutics that are rapidly effluxed by ABC transporters. Using methyltetrazolium (MTT) cell viability assays, the cytotoxic efficacy of nine jadomycin analogues (DNV, L, B, SPhG, F, W, S, T and N) in drug-sensitive and drug-resistant MCF7 breast cancer cells was evaluated. Jadomycin B, L, S and T were found to be equally toxic to drug-sensitive and drug-resistant ABCB1, ABCC1 or ABCG2-overexpressing MCF7 breast cancer cells. The inhibition of ABCB1, ABCC1 or ABCG2 efflux transporters with verapamil, MK-571 or ko143, respectively, did not significantly augment the cytotoxic effects of jadomycin DNV, L, B and S in drug-resistant MCF7 cells, suggesting that these jadomycins are poor substrates of the targeted transporter. Furthermore, all nine jadomycin analogues did not increase the intracellular accumulation of ABCB1, ABCC1 or ABCG2 probe fluorescent substrates in HEK-293 cells, indicating that these jadomycins do not inhibit the efflux function of the transporters. We conclude that jadomycins B, L and S are effective agents in the eradication of resistant breast cancer cells grown in culture, and that the ability of specific jadomycins to retain cytotoxic efficacy in resistant cells stems from their limited interactions with ABCB1, ABCC1 or ABCG2 efflux transporters.

The bacteria Streptomyces venezuelae produce the novel antibiotic jadomycin. The study of growth characteristics and metabolic behavior of the bacteria are necessary to scale up antibiotic production and facilitate further research. In this study, a method for producing consistent inoculum was developed that showed good repeatability when used in growth trials. The rod shaped spores of Streptomyces venezuelae were determined to be approximately 0.8 x 0.2 ?m with a smooth surface type. The effects of temperature and pH on bacterial growth and substrate consumption were examined in a 7 L bioreactor. Of the range of parameters tested (28, 32, 36 °C, and media pH of 5, 7 and 9), 32 °C with a media pH of 7 yielded the highest rate of growth (µmax of 1.43 hours-1 with a lag time of 7.7 hours). The results of all trials showed that free glucose was consumed before the maltose, which was the major sugar substrate in the media. The initiation of exponential bacterial growth occurred after rapid consumption of free glucose. A heat balance analysis was also performed over the bioreactor to identify the heat generated through agitation, losses over the vessel and the heat of metabolism from Streptomyces venezuelae. Under normal operating parameters 33 - 24 % of the heat generated through mixing was lost with the exhaust gas, while 56 - 64 % was lost through the bioreactor wall. The heat of mixing was calculated to be 1.62 J•s-1 while the maximum amount of heat generated by Streptomyces venezuelae metabolism and activity during a growth trial was 2.28 J•s-1 for 60 x 109 CFU?mL-1.