Screening of high daptomycin-producing strains by combined antibiotics resistance screening method

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1 (2012) * ( ) (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 ) 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 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] (No ) net7@xmu.edu.cn * jkj@xmu.edu.cn

2 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 + N % Ochi [11-13] rel rel (ppgpp) ppgpp ppgpp (1) (2) Ochi S. coelicolor A3(2) (1.7~2.5 ) [14] (Streptomyces roseosporus)atcc DA1(g/L) CaCO ph7.0 (g/l) 5 30 ph7.0 (g/l) 11 Fe(NH 4 ) 2 (SO 4 ) 2 6H 2 O ph7.0 LB(g/L) 5 10 NaCl 10 8 ph mL/250mL 200r/min 48~56 h 30 50mL/250mL 5% 200r/min 8~10d 37 16~24 h mL DA1 20% (MIC) 10~100

3 ~9d LB 50~60 0.1mL ( ) 4 1h 37 1~2d mL 10000r/min 5min 2~3 0.22μm HPLC 50μL (MIC) mg/L 500mg/L (MIC) 400mg/L (MIC) mg/L 1.2mg/L (MIC) 1.0mg/L MIC MIC mg/L mg/L mg/L mg/L 5.0mg/L mg/L Tab. 1 Effect of daptomycin on the cultivation of Streptomyces roseosporus /(mg/l) Tab. 2 Effect of streptomycin on the cultivation of Streptomyces roseosporus /(mg/l)

4 Tab. 3 Result of Streptomyces roseosporus screening on daptomycinstreptomycin combined resistant plates /(mg/l) /(mg/l) (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% D1000-S Tab. 5 Resistant-selection results /mm 18 D D D S S S S D800-S D800-S D800-S D800-S D800-S D1000-S D1000-S D1000-S 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

5 Tab. 6 Daptomycin production and biomass of resistant mutants and the original strain HPLC /(mau s) /(mg/l) /(g/l) /(mg/g DCW) D D D S S S S D800-S D800-S D800-S D800-S D800-S D1000-S D1000-S D1000-S 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 D action revealed[j]. Chem Biol, 2004, 11: [2] Raja A, LaBonte J, Lebbos J, et al. Fresh from the pipeline: Daptomycin[J]. Nat Rev Drug Discov, 2003, 2: [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): [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: [5] Jeu L A, Fung H B. Daptomycin: a cyclic lipopeptide antimicrobial agent[j]. Clin Ther, 2004, 26(11): [6] Nichols R L. Optimal treatment of complicated skin and skin structure infections[j]. Antimicrob Chemother, 1999, ( 219 )

6 Echinocandin B % 95% 4 HZ830 Echinocandin B Echinocandin B 6 Echinocandin B Fig. 6 The photo of Echinocandin B crystal 3 Tab. 3 The experiment of process validation Echinocandin B Echinocandin B Echinocandin B /(μg/ ml) /L /g HPLC /% /g HPLC /% /% % 95% [1]. [J]. :, 2007, 28(6) : [2],. [J]., 2005, 14(11): [3],,. : CN X [P] [4] PROTIA, LLC, Reno, NV(US). Deuterium-enriched Anidulafungin: US 2009/ A1[P] [5] Eli Lilly and Company Indianapolis Ind. Method of producing the A antibiotics. US [P] [6]. B : CN [P] ( 206 ) 44(Suppl A): [7] Safdar N, Andes D, Craig W A. In vivo pharmacodynamic activity of daptomycin[j]. Antimicrob Agents Chemother, 2004, 48: [8] Floyd M Huber, Richard L Pieper, Anthony J Tietz. Process for producing A-21978C derivatives: US, 4,885,243[P] [9],,,. [J]., 2006, 33(3): [10],,,. [J]., 2008, 26(5): [11] Ocm K, Okamoto S, Tozawa Y, et al. Ribosome engineering and secondary metabolite production[m]. Advances in Applied Microbiology, Academic Press, 2003: [12] Hu H F, Ochi K. Novel approach for improving the productivity of antibiotic-producing strains by inducing combined resistant mutations[j]. Appl Environ Microbiol, 2001, 67(4): [13] Hesketh A, Ochi K. A novel method for improving Streptomyces coelicolor A3(2) for production of actinorhodin by introduction of rpsl mutation conferring resistance to streptomycin[j]. J Antibiot, 1997, 50(6): [14] Chater K F, Bruton C J. Resistance, regulatory and production genes for the antibiotic methylenomycin are clustered[j]. EMBO J, 1985, 4: