. 202. 2012 3 37 3 1001-8689(2012)03-0202-05 * ( 361005) (Streptomyces roseosporus ATCC 11379) D1000-S3-2 59mg/L 63.8% Q939 A Screening of high daptomycin-producing strains by combined antibiotics resistance screening method Zhang Zhi-xiang, Duan Ran, Jing Ke-ju, Wu Yi-xun and Lu Ying-hua (Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005) Abstract Objective Combined antibiotics resistance screening method was applied to screen daptomycin high-producing strain. Methods Based on the biosynthetic pathway and metabolization mechanism of daptoomycin, combined antibiotics resistance screening method was applied to screen daptomycin high-producing strain from original strain of Streptomyces roseosporus ATCC 11379. Results A mutant strain D1000-S3-2 was obtained. The yield of daptomycin was 59 mg/l in flask by the mutant strain, which increased 63.8%, compared to that of the parental strain. Conclusion It was demonstrated that the method provides a fast and effective way of screening S. roseosporus. Key words Daptomycin; Streptomyces roseosporus; Combined antibiotics resistance screening ( Streptomyces roseosporus) 13 β N- [1] [2-3] (VRE) (MRSA) (GISA) (CNS) (PRSP) [4-5] 2011-04-21 (No. 31071488) 1985E-mail: net7@xmu.edu.cn * E-mail: jkj@xmu.edu.cn
. 203. 1997 Cubist [6] 2003 (FDA) (daptomycin)( cubicin) 1 VRE [7] 1989 Huber [8] 10 A21978C 1940mg/L Cubist [9] 20mW (D-38)20min 81.2mg/L 39% [10] N + N + N3-36 26% Ochi [11-13] rel rel (ppgpp) ppgpp ppgpp (1) (2) Ochi S. coelicolor A3(2) (1.7~2.5 ) [14] 1 1.1 1.1.1 (Streptomyces roseosporus)atcc 11379 1.1.2 DA1(g/L) 4 10 4 CaCO 3 2 15 ph7.0 (g/l) 5 30 ph7.0 (g/l) 11 Fe(NH 4 ) 2 (SO 4 ) 2 6H 2 O 1.5 10.7 30 3.0 ph7.0 LB(g/L) 5 10 NaCl 10 8 ph6.8 1.2 1.2.1 30 30 50mL/250mL 200r/min 48~56 h 30 50mL/250mL 5% 200r/min 8~10d 37 16~24 h 1.2.2 50mL DA1 20% -20 1.2.3 (MIC) 10~100
. 204. 2012 3 37 3 7~9d 30 1.2.4 LB 50~60 0.1mL ( ) 4 1h 37 1~2d 1.2.5 1mL 10000r/min 5min 2~3 0.22μm HPLC 50μL 2 2.1 2.2.1 (MIC) 0 1 400mg/L 500mg/L (MIC) 400mg/L 2.2.2 (MIC) 0 2 1.0mg/L 1.2mg/L (MIC) 1.0mg/L 2.2 2.2.1 MIC MIC 800 1000 1200 1400 1600mg/L 1400 1600mg/L 800 1000 1200mg/L 38 2.0 2.5 3.0 3.5 4.0 4.5 5.0mg/L 5.0mg/L 2.0 2.5 3.0 3.5 4.0 4.5mg/L 74 2.2.2 - - 3 31 - - 1-4 2.3 2.3.1 1.2.4 15 5 2.3.2 15 1 Tab. 1 Effect of daptomycin on the cultivation of Streptomyces roseosporus /(mg/l) 0 100 200 300 400 500 600 700 +++ +++ +++ ++ + - - - +++ ++ + - 2 Tab. 2 Effect of streptomycin on the cultivation of Streptomyces roseosporus /(mg/l) 0 02 0.4 0.6 0.8 1.0 1.2 1.4 +++ +++ +++ +++ ++ + - - +++ ++ + -
. 205. 3 Tab. 3 Result of Streptomyces roseosporus screening on daptomycinstreptomycin combined resistant plates /(mg/l) 800 800 800 1000 1000 1000 1200 1200 /(mg/l) 2.0 3.0 4.0 2.0 3.0 4.0 2.0 3.0 12 5 1 7 5 1 0 0 1 - (A) (B) Fig. 1 Morphological character comparison between mutant strain(a) and the original strain(b) 4 Tab. 4 Morphological character comparison between mutant strain and the original strain " " ( 1.2.1) HPLC ( 1.2.5) 2 HPLC 6 15 HPLC 11 D1000-S3-2 30g/L 59mg/L 63.8% 2.4 4 5 3 7 D1000-S3-2 5 5 Tab. 5 Resistant-selection results /mm 18 D1000-7 20 D1200-4 21 D1200-5 21 S2.5-6 20 S3-1 20 S4-1 19 S4-5 20 D800-S2-3 19 D800-S2-5 20 D800-S2-10 23 D800-S3-4 21 D800-S4-1 23 D1000-S2-5 21 D1000-S3-2 23 D1000-S3-5 21 D S 2 (A) (B)HPLC Fig. 2 HPLC results of standard daptomycin(a) and sample(b) 57.5~59.0mg/L 58.6mg/L 7
. 206. 2012 3 37 3 6 Tab. 6 Daptomycin production and biomass of resistant mutants and the original strain HPLC /(mau s) /(mg/l) /(g/l) /(mg/g DCW) 275.6 36.1 26 1.4 D1000-7 268.6 35.2 25 1.4 D1200-4 231.8 30.5 30 1.0 D1200-5 363.1 47.3 25 1.9 S2.5-6 276.4 36.2 27 1.3 S3-1 309.2 40.4 28 1.4 S4-1 297.5 38.9 29 1.3 S4-5 427.9 55.6 25 2.2 D800-S2-3 386.5 50.3 30 1.7 D800-S2-5 323.2 42.2 26 1.6 D800-S2-10 239.6 31.5 27 1.2 D800-S3-4 194.3 25.7 28 0.9 D800-S4-1 324.1 42.3 28 1.5 D1000-S2-5 295.2 38.6 25 1.5 D1000-S3-2 454.5 59.0 30 2.0 D1000-S3-5 307.6 40.2 28 1.4 3 CaCO 3 D1000-S3-2 30g/L 59mg/L 63.8% [1] Micklefield J. Daptomycin structure and mechanism of 7 Tab. 7 Genetical stability experiment /(mg/l) D1000-S3-2 S4-5 D800-S2-3 D1200-5 1 59.0 55.6 50.3 47.3 2 59.2 55.5 50.7 47.1 3 58.7 54.5 48.0 47.3 4 58.8 54.0 47.2 46.2 5 57.5 54.0 47.2 46.0 action revealed[j]. Chem Biol, 2004, 11: 949-957. [2] Raja A, LaBonte J, Lebbos J, et al. Fresh from the pipeline: Daptomycin[J]. Nat Rev Drug Discov, 2003, 2: 943-944. [3] Cunha B A., Hamid N, Kessler H, et al. Daptomycin cure after cefazolin treatment failure of methicillin-sensitive Staphylococcus aureus (MSSA) tricuspid valve acute bacterial endocarditis from a peripherally inserted central catheter (PICC) line[j]. Heart Lung, 2005, 34(6): 442-447. [4] Tally F P, Zeckel M, Wasilewski M M, et al. Daptomycin: a novel agent for Gram-positive infections[j]. Exp Opin Invest Drugs, 1999, 8: 1223-1238. [5] Jeu L A, Fung H B. Daptomycin: a cyclic lipopeptide antimicrobial agent[j]. Clin Ther, 2004, 26(11): 1728-1757. [6] Nichols R L. Optimal treatment of complicated skin and skin structure infections[j]. Antimicrob Chemother, 1999, ( 219 )
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