Streptomyces bingchenggensis produced milbemycins, including a 1 (A 3 ), a 3 (A 4 ), b 13 , b 14 , a 28 , a 29 , a 30 and ST 906, four secomilbemycins A, B, C and D, and two cyclic pentapeptides. [1][2][3][4][5] Milbemycins belong to a 16-membered macrolide antibiotic with an outstanding activity against various kinds of mites. 6 During a screening program for high production of A 3 and A 4 , a mutant S. bingchenggensis X-4 was obtained by UV treated, N-methyl-N¢-nitroso-N-nitroso-guanidine mutation and genetic manipulative techniques. Significant differences of phenotype, such as the morphology of aerial mycelia, and the metabolite HPLC profiles were observed between the wild-type S. bingchenggensis and its mutant strain X-4. In the course of investigating the metabolites of this mutant strain, three new interesting compounds milbemycin b 15 (1), seco-milbemycins E (2) and F (3) were isolated from the fermentation broth of S. bingchenggensis X-4. The structure of compound 1 was similar with milbemycin D, which is a highly selective and potent nematocide and insecticide. 7 So the bioactivity of compound 1 should be further investigated. Compared with the seco-milbemycins isolated previously, 2,3 the hydroxy groups at C-5 were absent in compounds 2 and 3. Furthermore, all the milbemycins 3,8 and avermectins 9,10 obtained from microorganisms contain the hydroxyl at C-5, and the 5-dehydroxyl derivatives of milbemycins and avermectins can only be obtained using the synthetic methods. 11 So seco-milbemycins E and F may have an important role in understanding and perfecting the proposed biosynthesis pathways of milbemycins.The producing strain S. bingchenggensis X-4 was maintained on an 1/2YM slant agar consisting of sucrose 0.4%, skim milk 0.1%, yeast extract (OXOID Basingstoke, Hampshire, UK) 0.2%, malt extract (BD Biosciences, San Jose, CA, USA) 0.5%, agar (BD Biosciences) 2.0% at 28 1C for 12 days. 12 A seed 15-l fermentor (FUS-15 L (A), Shanghai Guoqiang Bioengineering Equipment, Shanghai, China) containing 10 l of seed medium (sucrose 1.0%, polypepton 0.2%, K 2 HPO 4 0.05%, skim milk 0.05%) 12 was inoculated with 0.5 l of broth cultured in flask with seed medium. The flask with seed medium (2-6Â10 7 spores per ml) was cultured for 30 h at 28 1C on a rotary shake at 250 r.p.m.After incubation for 32 h, the seed broth (3 l) in the 15-l fermentor was transferred into the production 50-l fermentor (FUS-50 L (A), Shanghai Guoqiang Bioengineering Equipment) containing 30-l production medium (16.0% sucrose, 2.0% soybean powder, 0.5% yeast extract, 0.5% meat extract, 0.05% K 2 HPO 4 , 0.05% MgSO 4 7H 2 O, 0.005% FeSO 4 7H 2 O and 0.3% CaCO 3 ). 12 The culture temperature was 28 1C and the initial pH was 7.40 sterilized by sparging with steam at 121 1C for 30 min. The dissolved oxygen was maintained above 35% by adjusting the agitation speed. The initial aeration and agitation rate in the 15-l reactor was 1 vvm and 180 r.p.m., whereas those in the 50 l were 0.8 vvm and 150 r.p.m., respectively. In the process of fermenta...