农用抗生素2-16高产菌株选育及发酵优化组合研究
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摘要
农用抗生素2-16是从不吸水链霉菌黄山变种(Streptomyces ahygroscopicus var．huangshanensis)发酵产物中发现的一种具有广谱抗菌作用的抗生素。农抗2-16对多种作物病原真菌有较强抑制作用，对酵母菌及部分革兰氏阳性细菌和革兰氏阴性细菌也有一定的抑制作用。农抗2-16用于防治油菜菌核病，防效大于65％。在进行本研究之前农抗2-16的发酵单位仅有2000u／ml，相对较低。为提高农抗2-16产生菌的单位效价，本研究通过四种诱变方法选育出一株农抗2-16的高产菌株515~#，并利用SAS软件提供的Plackett-Burman设计和响应面分析法对该菌株的摇瓶发酵培养基进行了优化组合。研究结果如下：
1．由出发菌株0~#(效价2000 u／ml)依次进行原生质体再生处理、紫外线诱变、NTG诱变、低能碳离子注入处理，每步筛选出高产菌株用于下一步诱变处理。原生质体再生处理未获得高产菌株，紫外线诱变获得菌株301~#(效价2317u／ml)，NTG诱变获得菌株442~#(效价4084u／ml)，低能碳离子注入获得菌株515~#(效价6462u／ml)。最终获得的目标菌株515~#效价较出发菌株0~#提高223.10％。四种诱变方法中，低能碳离子注入的诱变贡献率最高，达到53.29％，NTG次之，达到39.60％，本研究中低能碳离子注入和NTG诱变的作用效率显著高于紫外线诱变和原生质体再生处理，是进行农抗2-16产生菌诱变育种的高效方法。
2．利用SAS软件中的Plackett-Burman设计和响应面分析法对515~#菌株的摇瓶发酵培养基进行优化组合，从九个考察因素中选出化合物K和化合物F为重要因素，通过响应面分析法建立模型Y=8914.80—0.2749 x_2—298.65 X_9—170.53x_2~2—19.75 x_2 x_9—299.78 X_9~2，推测出在化合物K浓度为0.018％、化合物F浓度为0.03％时预测的最大响应值为8989 u／ml，经摇瓶发酵实验证实此点的实测值为(8952±94)u／ml，证明模型是有效的，并且存在极大值点。采用优化培养基，515~#菌株效价较原始发酵培养基提高38.53％。
3．高效液相色谱分析发酵液组份表明农用抗生素2-16主要含有A、B两个组份，B组份含量高于A组份。菌株442~#、301~#的A组份含量高于出发菌株0~#，B组份含量低于出发菌株，515~#和515~#(优化发酵培养基)的B组份含量高于出发菌株0~#，A组份含量低于出发菌株。B组份的含量对效价的影响更大，B组份是决定农抗2-16效价高低的关键因素。
Agro-antibiotic 2-16 is an agricultural antibiotic with extensive inhibition effect, which was discovered from the culture filtrate of Streptomyces ahygroscopicus var. huangshanensis. Agro-antibiotic 2-16 exhibits strong effects on many pathogenic fungi of crops, and also some extent of inhibition effects on yeast, some G+ bacteria and G bacteria. When agro-antibiotic 2-16 was used to control Sclerotinia sclerotiorum in the field test, the control effect was more than 65%. Before this study, the production of 2-16 is low, with the titre of only 2000u/ml. In order to improve its production, four mutagenic methods were used in this work. At last, a high-yield strain 515# was obtained. By using Plackett-Burman design and Response Surface Analysis provided by SAS software, the cultivation condition of 515# was optimized. The results were as follows:
1 .The initial strain 0# (with titre of 2000u/ml) was treated by means of protoplast regeneration, ultraviolet radiation, NTG mutagenesis and low energy C+ ions implantation respectively. Every time, a strain with higher titre acqucired from one step was selected as the starting strain of the next step. No high-yield mutant was found after protoplast regeneration, strain 301# (with titre of 2317u/ml) was got by ultraviolet radiation, strain 442# (with titre of 4084u/ml) was got by NTG mutagenesis, strain 515# (with titre of 6462u/ml) was got by low energy C+ ions implantation. The production of 515# was increased by 223.10% in comparision with that of 0#. Low energy C+ ions implantation contributed most to the improvement of the production among the four methods, which was 53.29%, and NTG was the second one, which was 39.60 %. In this study, the effects of NTG mutagenesis and low energy C+ ions implantation were better than those of protoplast regeneration and ultraviolet radiation obviously, and were effect
ive methods for the mutation and breeding of the agro-antibiotic 2-16 producing strain.
2.By using Plackett-Burman design and Response Surface Analysis provided by SAS software, the cultivation condition of strain 515# was optimized. Compound K and compound F were chosen as the major components from nine variables, a regression model equation was obtained after regression analysis performed on the data from RSA experiment, which is: Y=8914.80-0.2749 x2-298.65 x9-170.53x22-19.75 x2 x9-299.78 x92. The optimum concentrations of compound K and compound F were predicted to be 0.018% and 0.03%, respectively, for the maximum antibiotic production, which is 8989 u/ml. The model was validated experimentally by the maximum production of (8952 94)u/ml. The agro-antibiotic 2-16 production was increased by 38.53% when the strain 515 #was cultivated in the optimum medium instead of the initial one.
3.The analysis of the culture filtrates of different strains with High Performance Liquid
Chromatogram indicted that there were two main components--A and B in agro-antibiotic 2-16, and
the content of B was higher than that of A. The contents of A in the culture filtrate of strain 442# and strain 301# were both higher than that of the initial strain 0#, while the contents of B were both lower.
However, when it came to strain 515# and 515# in the optimum medium, the case was the opposite one. The contents of B in the culture filtrates of strain 515# and 515# in the optimum medium were both higher than that of the initial strain 0#, while the contents of A were both lower. The content of B has greater impact on the titre of agro-antibiotic 2-16 than that of A, and was the key factor that dominated the titre of agro-antibiotic 2-16.
1．由出发菌株0~#(效价2000 u／ml)依次进行原生质体再生处理、紫外线诱变、NTG诱变、低能碳离子注入处理，每步筛选出高产菌株用于下一步诱变处理。原生质体再生处理未获得高产菌株，紫外线诱变获得菌株301~#(效价2317u／ml)，NTG诱变获得菌株442~#(效价4084u／ml)，低能碳离子注入获得菌株515~#(效价6462u／ml)。最终获得的目标菌株515~#效价较出发菌株0~#提高223.10％。四种诱变方法中，低能碳离子注入的诱变贡献率最高，达到53.29％，NTG次之，达到39.60％，本研究中低能碳离子注入和NTG诱变的作用效率显著高于紫外线诱变和原生质体再生处理，是进行农抗2-16产生菌诱变育种的高效方法。
2．利用SAS软件中的Plackett-Burman设计和响应面分析法对515~#菌株的摇瓶发酵培养基进行优化组合，从九个考察因素中选出化合物K和化合物F为重要因素，通过响应面分析法建立模型Y=8914.80—0.2749 x_2—298.65 X_9—170.53x_2~2—19.75 x_2 x_9—299.78 X_9~2，推测出在化合物K浓度为0.018％、化合物F浓度为0.03％时预测的最大响应值为8989 u／ml，经摇瓶发酵实验证实此点的实测值为(8952±94)u／ml，证明模型是有效的，并且存在极大值点。采用优化培养基，515~#菌株效价较原始发酵培养基提高38.53％。
3．高效液相色谱分析发酵液组份表明农用抗生素2-16主要含有A、B两个组份，B组份含量高于A组份。菌株442~#、301~#的A组份含量高于出发菌株0~#，B组份含量低于出发菌株，515~#和515~#(优化发酵培养基)的B组份含量高于出发菌株0~#，A组份含量低于出发菌株。B组份的含量对效价的影响更大，B组份是决定农抗2-16效价高低的关键因素。
Agro-antibiotic 2-16 is an agricultural antibiotic with extensive inhibition effect, which was discovered from the culture filtrate of Streptomyces ahygroscopicus var. huangshanensis. Agro-antibiotic 2-16 exhibits strong effects on many pathogenic fungi of crops, and also some extent of inhibition effects on yeast, some G+ bacteria and G bacteria. When agro-antibiotic 2-16 was used to control Sclerotinia sclerotiorum in the field test, the control effect was more than 65%. Before this study, the production of 2-16 is low, with the titre of only 2000u/ml. In order to improve its production, four mutagenic methods were used in this work. At last, a high-yield strain 515# was obtained. By using Plackett-Burman design and Response Surface Analysis provided by SAS software, the cultivation condition of 515# was optimized. The results were as follows:
1 .The initial strain 0# (with titre of 2000u/ml) was treated by means of protoplast regeneration, ultraviolet radiation, NTG mutagenesis and low energy C+ ions implantation respectively. Every time, a strain with higher titre acqucired from one step was selected as the starting strain of the next step. No high-yield mutant was found after protoplast regeneration, strain 301# (with titre of 2317u/ml) was got by ultraviolet radiation, strain 442# (with titre of 4084u/ml) was got by NTG mutagenesis, strain 515# (with titre of 6462u/ml) was got by low energy C+ ions implantation. The production of 515# was increased by 223.10% in comparision with that of 0#. Low energy C+ ions implantation contributed most to the improvement of the production among the four methods, which was 53.29%, and NTG was the second one, which was 39.60 %. In this study, the effects of NTG mutagenesis and low energy C+ ions implantation were better than those of protoplast regeneration and ultraviolet radiation obviously, and were effect
ive methods for the mutation and breeding of the agro-antibiotic 2-16 producing strain.
2.By using Plackett-Burman design and Response Surface Analysis provided by SAS software, the cultivation condition of strain 515# was optimized. Compound K and compound F were chosen as the major components from nine variables, a regression model equation was obtained after regression analysis performed on the data from RSA experiment, which is: Y=8914.80-0.2749 x2-298.65 x9-170.53x22-19.75 x2 x9-299.78 x92. The optimum concentrations of compound K and compound F were predicted to be 0.018% and 0.03%, respectively, for the maximum antibiotic production, which is 8989 u/ml. The model was validated experimentally by the maximum production of (8952 94)u/ml. The agro-antibiotic 2-16 production was increased by 38.53% when the strain 515 #was cultivated in the optimum medium instead of the initial one.
3.The analysis of the culture filtrates of different strains with High Performance Liquid
Chromatogram indicted that there were two main components--A and B in agro-antibiotic 2-16, and
the content of B was higher than that of A. The contents of A in the culture filtrate of strain 442# and strain 301# were both higher than that of the initial strain 0#, while the contents of B were both lower.
However, when it came to strain 515# and 515# in the optimum medium, the case was the opposite one. The contents of B in the culture filtrates of strain 515# and 515# in the optimum medium were both higher than that of the initial strain 0#, while the contents of A were both lower. The content of B has greater impact on the titre of agro-antibiotic 2-16 than that of A, and was the key factor that dominated the titre of agro-antibiotic 2-16.
引文
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59. Banic S, Lunder M. Additive effect of the combination of griseofulvin and ket-oconazole against Microsporum canis in vitro. Mycoses, 1989, 32(9): 47～49
60. Banerjee R, Bhattacharyya. Optimization of mutiple inducers effect on protease biosynthesis by Rhizopus oryzae. Bioprocess Eng, 1992, 7:225～228
61. Bibb M. The regulation of antibiotic production in Streptomyces coelicolor A3(2). Microbiology, 1996, 142:1335～1344
62. Bowyer J, Trumpower B. Rapid reduction of cytochrome c1 in the presence of antimycin and its implication for the mechanism of election transfer in the cytochrome b1 segment of the mitochondrial respiratory chain. Biol Chem, 1981,256(5): 2245～2251
63. Box GEP, Hunter WG & Hunter JS. Statistics for Experimenters. New York: John Wiley and Sons Inc., 1978, 510～539
64. Burg RW, Miller BM, Baker EE, et al. Avermetins, new family of potent anthelmitic agents:producing organism and fermentation. Antimicrob Ag, Chemother, 1979, 15(13): 361～367
65. Cabib, E. Differential inhibition of chitin synthetases 1 and 2 from Saccharomyces cerevisiae by polyoxin D and nikkomycins. Antimicrob Agents Chemother, 1991, 35(1): 170～173
66. Chater KF. Genetics of differentiation in Streptomyces. Annu Rev Microbiol, 1993, 47:685～713
67. Chater KF. The improving prospects for yield increase by genetic engineering in antibiotic-producing Streptomyces. Biotechnology, 1990, 8:115～121
68. Chater KF & Bruton CJ. Resistance regulatory and production genes for the antibiotic methylenomycin are clustered. Embo J, 1985, 4:1893～1897
69. Chen KC, Lee TC & Houng JY. Search method for the optimal medium for production of lactase by Kluyveromyces fragilis. Enzyme Microb Technol, 1992, 14:659～664
70. Cundliffe E. How antibiotic-producing organisms avoid suicide. Annu Rev Microbiol, 1989, 43:207～233
71. Cundliffe E. Resistance to macrolides and lincosamides in Streptomyces livikans and to aminoglycosides in Macromonospora purpurea. Gene, 1992, 115:75～84
72. De Coninck J, Bouquelet S, Dumortier V, et al. Industrial media and fermentation process for improved growth and protease production by Tetrahymena thermophila BⅢ. J Ind Microbiol Biotechnol, 2000, 24:285～290
73．Fisher MH．Avermectin研究的最新进展．农药译丛，1991，13(3)：19～26
74. Gigras PG,Sahai V & Gupta R. Statistical Media Optimization and Production of ITS α-Amylase from Aspergillus oryzae in a Bioreactor. Current Microbiology, 2002, 45:203～208
75. Gomi S, Ikeda D, Nakamura H, et al. Isolation and structure of a new antibiotic, indolizomycin, produced by a strain SK-52 obtianed by interspecies fusion treatment. J Antibiot, 1984, 37(11):513～520
76. Haaland PD. Experimental design in biotechnology. New York: Marcel Dekker Inc., 1989, 1～18
77. Hopwood DA, Bibb MJ, Chater KF, et al. Genetic Manipulation of Streptomyces------a laboratory manual. Norwich: John Innes Institute, 1985, 12～14, 40～41
78. Hopwood DA, Bibb MJ, Chater KF, et al. Regulation of Gene Expression, 25 Years On.Cambridge, UK: Cambidge Univ Press, 1986, 251～276
79. Hori M, Equchi J & Kakik K. Studies on the mode of action of polyoxins Ⅵ.Effect of polyoxin on chitin synthesis in polyoxin-sensitive and resistant strains of Alternaria kikuchiana. J Antibiot, 1974, 27(4): 260～266
80. Ikeda H, Takada Y, Pang CH, et al. Transposon mutagenesis by Tn4560 and applications with avermectin-producing Streptomyces avermitilis. J Bacteriol, 1993, 175:2077～2082
81. Iwasa T, Suetomi K & Kusaka T. Taxonomic study and fermentation of producing organization and antimicrobiol activity of mildiomycin. J Antibiot, 1978, 31 (6): 511～518
82. Kennedy M & Krouse D. Strategies for improving fermentation medium performance. J Ind
Microbiol Biotechnol, 1999,23:456～475
83. Kumada Y, Anzai H, Takano E, et al. The bialaphos resistance gene(bar) plays a role in both self-defense and bialophos biosynthesis in Streptomyces hygroscopicus. J Antibiot,1988, 41(12):1838～1845
84. Kuo MS, Yurek DA, Chirby DG, et al. Mirobial O-carbamylation of novobiocin. J Antibiot, 1991, 44:1096～1100
85. Malmberg LH, Hu WS & Sherman DH. Percursor flux control through targeted chromosomal insertion of the lysine a-aminotransferase (lac) gene in Cephamycin C biosynthesis. J Bacteriol, 1993, 175(21): 6916～6924
86. Martin JF & Liras P. Oraganization and expression of genes involved in the biosynthesis of antibiotics and other secondary metabolites. Annu Rev Microbiol, 1989, 43:173～206
87. Misra AK. Agricultural Uses of Antibiotics. Washington D C, U.S.A.: American Chemical Society, 1986, 49～60
88. Puri S, Khalil BQ, Gupta R. Optipization of alkaline protease production from Bacillus sp. by response surface methodology. Current Microbiology, 2002, 44:286～290
89. Recknagel R, Pitt D, Bormann. Improvement of medium composition by random balance designs. J Basic Microbiol, 1988, 28:659～665
90. Skatrud PL & Queener SW. An electrophoretic molecular karyotype for an industrial strain of Cephalosporium acremonium. Gene, 1989, 78(2): 331～338
91. Takiguchi Y, Mishima H, Okuda M, et al. Milbemycins, a new family of macrolide antibiotics:fermentation, isolation and physico-chemical properties. J Antibiot, 1980, 33: 1120～1127
92. Varela H, Ferrari MD, Belobradjic L, et al. Effect of medium composition on the production by a new bacillus subtilis isolate of protease with promising unhairing activity. World J Microbiol Biotechnol, 1996, 12:643～645
93. Vilches C, Hernandez C, Mendez C, et al. Role of glycosylation and deglycosylation in biosynthesis of and resistance to oleandomycin in the producer organism. J Bacteriol, 1992, 174:161～165
94. Yu ZL, Shao CL. Dose effect of the tyrosine sample implanted by a low energy N~+ ion beam. Radiat Phys Chem, 1994, 43(4): 349～351
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57．邹美云，朱卫民，俞学琴，等．红霉素链霉菌原生质体的形成和再生及其对紫外光的敏感性．中国抗生素杂志，1989，14(1)：1～5
58. Ando K, Oishi H, Hirano S, et al. Tetranactin, a new miticidal antibiotic Ⅰ. Isolation, characterization and properties of Tetranactin. J Antibiot, 1971, 24(6): 347～352
59. Banic S, Lunder M. Additive effect of the combination of griseofulvin and ket-oconazole against Microsporum canis in vitro. Mycoses, 1989, 32(9): 47～49
60. Banerjee R, Bhattacharyya. Optimization of mutiple inducers effect on protease biosynthesis by Rhizopus oryzae. Bioprocess Eng, 1992, 7:225～228
61. Bibb M. The regulation of antibiotic production in Streptomyces coelicolor A3(2). Microbiology, 1996, 142:1335～1344
62. Bowyer J, Trumpower B. Rapid reduction of cytochrome c1 in the presence of antimycin and its implication for the mechanism of election transfer in the cytochrome b1 segment of the mitochondrial respiratory chain. Biol Chem, 1981,256(5): 2245～2251
63. Box GEP, Hunter WG & Hunter JS. Statistics for Experimenters. New York: John Wiley and Sons Inc., 1978, 510～539
64. Burg RW, Miller BM, Baker EE, et al. Avermetins, new family of potent anthelmitic agents:producing organism and fermentation. Antimicrob Ag, Chemother, 1979, 15(13): 361～367
65. Cabib, E. Differential inhibition of chitin synthetases 1 and 2 from Saccharomyces cerevisiae by polyoxin D and nikkomycins. Antimicrob Agents Chemother, 1991, 35(1): 170～173
66. Chater KF. Genetics of differentiation in Streptomyces. Annu Rev Microbiol, 1993, 47:685～713
67. Chater KF. The improving prospects for yield increase by genetic engineering in antibiotic-producing Streptomyces. Biotechnology, 1990, 8:115～121
68. Chater KF & Bruton CJ. Resistance regulatory and production genes for the antibiotic methylenomycin are clustered. Embo J, 1985, 4:1893～1897
69. Chen KC, Lee TC & Houng JY. Search method for the optimal medium for production of lactase by Kluyveromyces fragilis. Enzyme Microb Technol, 1992, 14:659～664
70. Cundliffe E. How antibiotic-producing organisms avoid suicide. Annu Rev Microbiol, 1989, 43:207～233
71. Cundliffe E. Resistance to macrolides and lincosamides in Streptomyces livikans and to aminoglycosides in Macromonospora purpurea. Gene, 1992, 115:75～84
72. De Coninck J, Bouquelet S, Dumortier V, et al. Industrial media and fermentation process for improved growth and protease production by Tetrahymena thermophila BⅢ. J Ind Microbiol Biotechnol, 2000, 24:285～290
73．Fisher MH．Avermectin研究的最新进展．农药译丛，1991，13(3)：19～26
74. Gigras PG,Sahai V & Gupta R. Statistical Media Optimization and Production of ITS α-Amylase from Aspergillus oryzae in a Bioreactor. Current Microbiology, 2002, 45:203～208
75. Gomi S, Ikeda D, Nakamura H, et al. Isolation and structure of a new antibiotic, indolizomycin, produced by a strain SK-52 obtianed by interspecies fusion treatment. J Antibiot, 1984, 37(11):513～520
76. Haaland PD. Experimental design in biotechnology. New York: Marcel Dekker Inc., 1989, 1～18
77. Hopwood DA, Bibb MJ, Chater KF, et al. Genetic Manipulation of Streptomyces------a laboratory manual. Norwich: John Innes Institute, 1985, 12～14, 40～41
78. Hopwood DA, Bibb MJ, Chater KF, et al. Regulation of Gene Expression, 25 Years On.Cambridge, UK: Cambidge Univ Press, 1986, 251～276
79. Hori M, Equchi J & Kakik K. Studies on the mode of action of polyoxins Ⅵ.Effect of polyoxin on chitin synthesis in polyoxin-sensitive and resistant strains of Alternaria kikuchiana. J Antibiot, 1974, 27(4): 260～266
80. Ikeda H, Takada Y, Pang CH, et al. Transposon mutagenesis by Tn4560 and applications with avermectin-producing Streptomyces avermitilis. J Bacteriol, 1993, 175:2077～2082
81. Iwasa T, Suetomi K & Kusaka T. Taxonomic study and fermentation of producing organization and antimicrobiol activity of mildiomycin. J Antibiot, 1978, 31 (6): 511～518
82. Kennedy M & Krouse D. Strategies for improving fermentation medium performance. J Ind
Microbiol Biotechnol, 1999,23:456～475
83. Kumada Y, Anzai H, Takano E, et al. The bialaphos resistance gene(bar) plays a role in both self-defense and bialophos biosynthesis in Streptomyces hygroscopicus. J Antibiot,1988, 41(12):1838～1845
84. Kuo MS, Yurek DA, Chirby DG, et al. Mirobial O-carbamylation of novobiocin. J Antibiot, 1991, 44:1096～1100
85. Malmberg LH, Hu WS & Sherman DH. Percursor flux control through targeted chromosomal insertion of the lysine a-aminotransferase (lac) gene in Cephamycin C biosynthesis. J Bacteriol, 1993, 175(21): 6916～6924
86. Martin JF & Liras P. Oraganization and expression of genes involved in the biosynthesis of antibiotics and other secondary metabolites. Annu Rev Microbiol, 1989, 43:173～206
87. Misra AK. Agricultural Uses of Antibiotics. Washington D C, U.S.A.: American Chemical Society, 1986, 49～60
88. Puri S, Khalil BQ, Gupta R. Optipization of alkaline protease production from Bacillus sp. by response surface methodology. Current Microbiology, 2002, 44:286～290
89. Recknagel R, Pitt D, Bormann. Improvement of medium composition by random balance designs. J Basic Microbiol, 1988, 28:659～665
90. Skatrud PL & Queener SW. An electrophoretic molecular karyotype for an industrial strain of Cephalosporium acremonium. Gene, 1989, 78(2): 331～338
91. Takiguchi Y, Mishima H, Okuda M, et al. Milbemycins, a new family of macrolide antibiotics:fermentation, isolation and physico-chemical properties. J Antibiot, 1980, 33: 1120～1127
92. Varela H, Ferrari MD, Belobradjic L, et al. Effect of medium composition on the production by a new bacillus subtilis isolate of protease with promising unhairing activity. World J Microbiol Biotechnol, 1996, 12:643～645
93. Vilches C, Hernandez C, Mendez C, et al. Role of glycosylation and deglycosylation in biosynthesis of and resistance to oleandomycin in the producer organism. J Bacteriol, 1992, 174:161～165
94. Yu ZL, Shao CL. Dose effect of the tyrosine sample implanted by a low energy N~+ ion beam. Radiat Phys Chem, 1994, 43(4): 349～351