Polymyxin b: controlled biosynthesis.

The polymyxins form a group of closely related strongly basic cyclicpeptide antibiotics with a rather specific activity against gram-negative bacteria. The most well-known and commercially produced members are poly-myxin B and E (colistin). These two consists of polymyxin B1, B2, B3 and E1, E2, E " the single components of them differing only in the fatty acid moiety of the side chain attached to the cyclic peptides. B1 contains 6-methyloctanoic acid (MOA), B2 isooctanoic acid (IOA), B3, octanoic acid. Corresponding differences exist between the components E1, E2 and E3. The commercial polymyxin B consists of a mixture with varying contents of B1, B2 and B3, the major constituent being B1 (above 70%). Little is known about the activity and toxicity of the single components. The biosynthesis of MOA and IOA is described by ITO et al.11: L-Isoleucine or L-valine is deaminated, decarboxylatcd and condensed with CoASH. Further condensation with 2 malonyl CoA leads to MOA, and IOA respectively. The following experiments describe attempts to change the proportion of B1, B2 and B3 when isoleucinc and valine are added to the culture medium of a polymyxin-producing strain of Bacillus palymyxa. Materials and Methods Culture Bacillus polymyxa (Wellcome Research Foundation Culture no. 1419) was maintained on Casitonc, Soytone, dextrose agar. Seed cultures were prepared in a complex medium containing whey powder 40 g, soybean flower 20 g, CaCO3 20 g, (NH4)2HPO4 7 g, yeast extract 3 g, (NH4)2SO4 1 g, KCl 1 g, NaCl1 g per liter tap water; 500-m1 flasks containing 100 ml of medium (pH was adjusted to 7.0) were inoculated and incubated on a rotary shaker (250 rpm) for 24 hours at 27°C; 100 ml of such a culture was used as inoculum for 4 liters of the same medium in a 5-liter aerated and stirred laboratory fermentor (vol/vol/min and 250 rpm). pH of the medium was adjusted to 7.0 before inoculation and the fermentation temperature was 27°C. Isolation of polymyxin After 48 hours of propagation 9 g of oxalic acid were added to the fermentation broth, the pH was adjusted to 2.0 with conc.H3SO4 and heated for ten minutes at 60°C. After centrifugation the supernatant was neutralized using NaOH, and stirred with the ion-exchanger IRC-50 (75 g wet, sodium form). The IRC-50 was collected by decantation, washed with water and eluated with methanol-water (40:60) acidified with HCI to pH 1.8. After neutralization of the cluate the methanol was …