Steroids from the Myxobacterium Nannocystis exedens

Squalene and three sterols were isolated from Nannocystis exedens (Myxobacterales). The sterol content was about 0.4% of the dry weight; squalene content was in the same range. The main component was identified as cholest-8(9)-en-3~-01. ( 14C)Mevalonate was readily incorporated into the sterols. The bacterium was not inhibited by nystatin or amphotericin B. METHODS Struins und culture conditions. Nunnocystis r.redens strain Na el( = N. r.wdrns HR 1 = German Collection of Microorganisms DSM 71 ; type strain : Reichenbach, 1970) was used for the chemical investigation. The organism was cultivated in 1 litre Erlenmeyer flasks containing 300 ml PL liquid medium (consisting of Probion LS 600 (single cell protein; Hoechst), 1 %; MgSOJ. 7H10, 0.1 %; and CaCl?. 2H10, 0.1 7;; pH 7.0, autoclaved). The flasks were shaken at 30 "C at 160 r.p.m. From 8.7 1 of culture, 235 g of wet cells were obtained, corresponding to about 60g dry wt. Alternatively the organism was grown in MDI medium (consisting of Casitone (Difco), 0.30;; CaCI?. 2H,O, 0.050,{; MgSO,. 7HZ0, 0.2%; cyanocobalamine, 0.5 mg I-' ; and trace elements solution; pH 7.2, autoclaved). Cell yields were much lower in this medium. The incorporation experiments were performed with N. osetlens strain Na el58 (= N. e.wdens HR 2), isolated in 1980 from field soil collected near Cairo, Egypt. Isolation of'steroids. The wet cell mass (235 g) was extracted three times with 250 ml acetone, and the aqueous acetone solution was extracted three times with 200 ml toluene. After removal of the toluene under vacuum, there remained 1.1 g of crude material. GLC of an aliquot, with cholesterol as a standard, showed that this material contained about 220 mg of steroids. The crude material was dissolved in CH,Cl,/methanol (1 :I, v/v) and chromatographed on a column (850 x 35 mm) of Sephadex LH-20 with CH,Cl,/methanol as the eluant. There were 90 fractions of 15 ml each, and steroids could be demonstrated by TLC in fractions 48 to 65 (total residue 300 mg). This material was chromatographed on a silica gel column (Lobar column size B, self-packed with LiChrosorb Si60 silica gel, 10 pm particles; Merck) with hexane/acetone (6 :4, v/v) as the eluant. GLC of the 170 fractions (1 ml each) showed the steroids to be in fractions 51 to 86 (total residue 210 mg). Atwfj.ricu/ chrcmufogruphy. For TLC, silica gel on aluminium sheets (Merck) was used, with acetone/hexane (35 : 65, v/v) as the solvent, the steroids having an Rf of 0.6.The acetylated steroids were chromatographed on silica gel (Kieselgel 60 HFZSs; Merck) impregnated with 10% (w/w) AgNO, and activated by heating at 110 "C for 30 min; using toluene/ethyl acetate (9 : I, viv), the R, values of the sterol acetates were 0.44, 0-52 and 0.58. For detection the plates were sprayed with a solution of 2% (w/w) SbCl, in CHCI, and heated to I10 "C. GLC was performed on a Hewlett-Packard 5730 chromatograph equipped with a glass column (2 m x 4 mm) containing 3% (w/w) OV-17 on Chromosorb W HP, 80 to 100 mesh. The carrier gas was helium (20 ml min-I ), the temperature was either programmed from 250 to 300 "C (8 "C min-' ) or held at 300 "C, and detection was by