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1 微生物学通报 DEC 20, 2009, 36(12): 1859~1864 Microbiology 2009 by Institute of Microbiology, CAS 研究报告基因工程 FR-008/ 杀念菌素脱羧衍生物 CS103 的分离提取及毒性和抗白色念珠菌活性研究毛相朝 1, 2 陶欣艺 1 杨亮 1 沈亚领 1* 魏东芝 1, 2* 邓子新 ( ) ( ) ( ) 3 摘要 : 本文在建立了基因工程 FR-008/ 杀念菌素脱羧衍生物 CS103 分离提取工艺的基础上, 经进一步纯化, 获得一定供试样品通过对比脱羧 FR-008/ 杀念菌素 CS103 FR-008/ 杀念菌素和两性霉素 B 三种化合物对人胚肾细胞毒性对人血红细胞的溶血活性和对白色念珠菌的抗菌效果, 发现脱羧 FR-008/ 杀念菌素 CS103 的毒性较 FR-008/ 杀念菌素和两性霉素 B 大大降低, 且保持了较高的抗真菌 (Candida albicans) 活性关键词 : FR-008/ 杀念菌素, 脱羧衍生物 CS103, 两性霉素 B, 毒性, 抗真菌活性 Research on the Isolation, Cell Toxicity and Antifungal Activity Against Candida albicans of Novel Decarboxylated FR-008/Candicidin Derivative CS103 MAO Xiang-Zhao 1,2 TAO Xin-Yi 1 YANG Liang 1 SHEN Ya-Ling 1* WEI Dong-Zhi 1,2* DENG Zi-Xin 3 (1. State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai , China) (2. Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong , China) (3. Laboratory of Microbial Metabolism and School of Life Science & Biotechnology, Shanghai Jiao Tong University, Shanghai , China) Abstract: Based on founding the methods for isolation and purification of the novel decarboxylated FR-008/Candicidin derivative CS103, we obtained enough samples for testing from the culture mycelia of the mutant of Streptomyces FR-008. Through comparing the cell toxicities on Human Embryonic Kidney Cells 293, haemolytic activities on human erythrocytes and antifungal activities on C. albicans, we found that the toxicity of decarboxylated FR-008/Candicidin derivative CS103 had been lower than FR-008/Candicidin and Amphoteicin B, while it still had high antifungal activity on C. albicans. 基金项目 : (No. 2007DZ ) * 通讯作者 :Tel: , Fax: ; : dzhwei@ecust.edu.cn; Tel: , Fax: ; : ylshen@ecust.edu.cn 收稿日期 : ; 接受日期 :

2 1860 微生物学通报 2009, Vol.36, No.12 Keywords: FR-008/Candicidin, Novel decarboxylated derivative, Amphoteicin B, Toxicities, Antifungal activities,, [1 3],, [4] FR [5], FR-008 FR-008 IMRU3570(Streptomyces. griseus IMRU3570), 38 [6,7], B, 1 FR-008/,, ; ; ( ) [8,9],, FR-008/ 3 : C-21 C-9 ( C-10 ) C-18, FR-008/, P450 FscP C-18 [6] fscp CS103 FR-008/, FR-008/ CS103 FR-008/, FR-008/ CS103 FR-008/ [6,10] FR-008/ CS103,, FR-008/ CS103 FR-008/ B () [6,10] 图 1 FR-008/ 杀念菌素及其脱羧衍生物 CS103 的结构 Fig. 1 Chemical structures of FR-008/candicidin and the novel decarboxylated derivatives CS103 [6,10] : FR-008/ : R=COOH; CS103: R=CH 3. Note: R=COOH is FR-008/candicidin; R=CH3 is its novel decarboxylated derivatives CS103., 1 材料和方法 1.1 菌种和细胞 fscp FR-008 CS103 ; (C. albicans), ; (Human embryonic kidney cells)293:, 5% CO 2, 37 C, DMEM, 10% 1.2 抗生素发酵生产方法 300 μl 50 ml ( 1%, 0.3%, 0.3%, 0.5%, NaOH ph 7.2~7.5) 250 ml, 28 C, 200 r/min 30 h 10% 2% 1% 1.39% 0.3% 0.5% 0.1% NaCl 1% FeSO 4 7H 2 O 0.05% CuSO %, 450 r/min 200 L/h, 28 C [11 14], [15] 1.3 分析测定方法抗生素待测溶液配置 : FR-008/ CS103 FR-008/ B DMSO () 5000 μg/ml,, 细胞毒性实验 : 96,

3 : FR-008/ CS %, 10% DMEM /ml, 96, 0.1 ml FR-008/ CS103 FR-008/ B,, 5~6 ; ( DMSO), 96, 200 μl, 6, 37 C 5% CO 2, 18 h 36 h, 5 g/l MTT 10 μl, 4 h,, DMSO 150 μl, 10 min, MTT, BioRad 550, 550 nm, 655 nm, HEK293 (IC 50 ) = OD / OD 100%, = 1, (IC 50 ) = 溶血活性实验 : ( 20 ml),, 0.9% 10,, 1500 r/min~2000 r/min 5 min,, 0.9% 2~3, 0.9% 2%,, [16, 17] ( ABS ABS0 ) : 溶血率 (%) = ( ABS100 ABS0) 100, ABS, ABS 100, ABS 抗白色念珠菌活性实验 : DMSO, ( 4% 1% %) 0.4 μg/ml, 96 10, 10 4 CFU/mL~ 10 5 CFU/mL (C. albicans), 35 C 24 h, 90% MBC ( ) 2 结果和讨论 2.1 抗生素分离工艺的确定发酵上清液和菌丝体中目标产物的分布状态 :, FR-008/ CS103,,, ( ) 2 h, 1,, FR-008/ CS103,, 3.55% 表 1 发酵上清液和菌丝体中目标产物的分布比例 Table 1 Distributing of the antibiotic in the broth and mycelium FR-008/ CS103 Decarboxylated FR-008/Candicidin Run Derivative CS103 (μg/ml) The ratio of supernant Supernant Mycelium Total (%) Average ,, 菌丝体内抗生素分离提取工艺的确定 :,, ( 1), ( ),,,, (80%) 6 80%, 0.25 h 0.5 h 1 h 2 h 4 h 8 h 2,,, 2 h,,

4 1862 微生物学通报 2009, Vol.36, No.12 图 2 甲醇 (80%) 萃取参数的确定 Fig. 2 The extraction parameters of antibiotic with methanol (80%), 80% 2 h 80%, 100 g 300 ml 80% 2 h, 5 ( 2), 3,,, 3, [18],, 3 : ;,,,,,,,,, ;, 2.2 细胞毒性实验 1.3.2, ( 4),, FR- 008/ CS103 FR-008/, B, HEK h,, FR-008/ CS103 FR-008/ B 3 (IC 50 ) μg/ml, 0.26 μg/ml 5.94 μg/ml, FR-008/, FR-008/ CS 溶血活性实验 (Hemolysis), Fig. 3 图 3 产物分离纯化流程 The process of separation and purification of antibiotic CS103

5 : FR-008/ CS Fig. 4 图 4 脱羧 FR-008/ 杀念菌素 CS103 FR-008/ 杀念菌素和两性霉素 B 对细胞存活率的影响 The effect of Decarboxylated FR-008/Candicidin derivative CS103, FR-008/Candicidin and Amphotericin B on cell viability, 1.3.3, FR-008/ CS103 FR-008/ B 200 μg/ml 150 μg/ml 100 μg/ml 50 μg/ml 10 μg/ml 5 μg/ml 0.5 μg/ml 0.1 μg/ml 0.05 μg/ml, ( 5) 5, FR-008/ CS103 FR-008/ B B 1 μg/ml~ 5 μg/ml 100%, FR-008/ CS μg/ml 100% FR-008/ CS103 FR-008/ B 3 50% : μg/ml 0.69 μg/ml 0.40 μg/ml [10], FR-008/, FR-008/ CS 抗白色念珠菌活性实验 FR-008/ CS103 FR-008/ B (C. albicans) 2, FR-008/,, , FR-008/ CS103 FR-008/ B, FR-008/ CS103, B,, FR-008/ CS103 B, 图 5 脱羧 FR-008/ 杀念菌素 CS103 FR-008/ 杀念菌素和两性霉素 B 的溶血活性 Fig. 5 Hemolysis of Decarboxylated FR-008/Candicidin derivative CS103, FR-008/Candicidin and Amphotericin B 表 2 脱羧 FR-008/ 杀念菌素 CS103 FR-008/ 杀念菌素和两性霉素 B 抗白色念珠菌活性比较 Table 2 Antifungal activities against C. albicans of Decarboxylated FR-008/Candicidin derivative CS103, FR-008/Candicidin and Amphotericin B on C. albicans C. albicans MBC (μg/ml) Decarboxylated FR-008/Candicidin derivative CS ~0.05 FR-008/Candicidin ~ Amphotericin B 0.05

6 1864 微生物学通报 2009, Vol.36, No.12 50, FR-008/ CS103 FR-008/ B 3 结论和展望 FR-008/ CS103,,,,,,, FR-008/ CS103 FR-008/ B HEK293 (IC 50 ) μg/ml 0.26 μg/ml 5.94 μg/ml, 3 50% : μg/ml 0.69 μg/ml 0.40 μg/ml; (C. albicans) FR-008/ CS103 B, Candicidin,, FR-008/ CS103,, 参考文献 [1] Hopwood D. Genetic contributions to understanding polyketide synthases. Chem Rev, 1997, 97(7): [2] Bailey J, Birnbaum S, Galazzo J, et al. Strategies and challenges in metabolic engineering. Ann N Y Acad Sci, 1990, 589: [3],.., 2006, 31(2): [4] Singhal A, Mosbach A, Schaffner C. Effect of candicidin on cholesterol and bile acid metabolism in the rat. Lipids, 1981, 16(6): [5], , 1987, 3: [6] Chen S, Huang X, Zhou X, et al. Organizational and mutational analysis of a complete FR-008/Candicidin gene cluster encoding a structurally related polyene complex. Chem Biol, 2003, 10(11): [7],,. FR-008., 1999, 24(5): [8] Fox J. Candicidin, a new antifungal antibiotic: first clinical report. Antibiotic Med Clin Ther, 1955, 1(6): [9] Gordon H, Schaffner C. The effect of polyene macrolides on the prostate gland and canine prostatic hyperplasia. Proc Natl Acad Sci USA, 1968, 60(4): [10] Chen S, Mao X, Shen Y, et al. Tailoring the P450 monooxygenase gene for FR-008/Candicidin biosynthesis. Appl Environ Microbiol, 2009, 75(6): [11] Mao X, Shen Y, Yang L, et al. Optimizing the medium compositions for accumulation of the novel FR-008/Candicidin derivatives CS101 by a mutant of Streptomyces sp. using statistical experimental methods. Proc Biochem, 2007, 42(5): [12] Mao X, Wang F, Zhang J, et al. The ph shift and precursor feeding strategy in a low-toxicity FR-008/Candicidin derivative CS103 fermentation bioprocess by a mutant of Streptomyces sp. FR-008. Appl Biochem Biotechnol, (Online) [13],,,. FR-008/ CS103., 2008, 33(6): [14],,,. FR-008/ CS103., 2009, 39(2): 1 6. [15] Mao X, Shen Y, Wei D, et al. Determination of Candicidin/FR-008 and their ramification in fermentation broth by RP-HPLC. J Chin Pharm Sci, 2005, 42(2): [16] 2005 : 118. [17] Larabi M, Yardley V, Loiseau P, et al. Toxicity and antileishmanial activity of a new stable lipid suspension of amphotericin B. Antimicrob Agents Chemother, 2003, 47(12): [18] Wagman G, Testa R, Patel M, et al. New polyene antifungal antibiotic produced by a species of Actinoplanes. Antimicrob Agents Chemother, 1975, 7(4):

1860 微生物学通报 2009, Vol.36, No.12 Keywords: FR-008/Candicidin, Novel decarboxylated derivative, Amphoteicin B, Toxicities, Antifungal activities 随

1860 微生物学通报 2009, Vol.36, No.12 Keywords: FR-008/Candicidin, Novel decarboxylated derivative, Amphoteicin B, Toxicities, Antifungal activities 随微生物学通报 DEC 20, 2009, 36(12): 1859~1864 Microbiology 2009 by Institute of Microbiology, CAS tongbao@im.ac.cn 研究报告基因工程 FR-008/ 杀念菌素脱羧衍生物 CS103 的分离提取及毒性和抗白色念珠菌活性研究毛