安普霉素产生菌的研究
|
摘要
本文以尼拉霉素单一组分—安普霉素产生菌S.tenebrarius A04(发酵单位为2847u／ml)为出发菌株，通过对单孢子悬液和超声波破碎菌体的诱变处理并复合筛选模型，获得了遗传特性稳定的单组分高产菌株：A2-23、A2-30、ASM6、A1-16，摇瓶发酵单位达4800-5200u／ml。证明了UV处理复合耐受自身产物、UV处理复合耐受链霉素能获得较好的筛选结果。
以菌株A1-16为实验菌株，运用均匀设计方法，对发酵培养基、通气量、接种量等进行了考察，得到发酵培养基和种子培养基的优化配方。对三级发酵工艺、补料工艺等发酵工艺条件进行了优化，进一步确定了中试发酵工艺条件，4m~3发酵罐上平均发酵单位达4478u／ml。
通过氨基酸添加实验研究了安普霉素生物合成途径中的氮的来源及调节机制。Glu、Gln及α—酮戊二酸添加实验结果表明Glu、Gln对菌体的生长无明显的影响，但能强烈促进安普霉素的生物合成，且相同浓度条件下Gln的促进作用明显高于Glu，而α—酮戊二酸则对安普霉素的生物合成有抑制作用。因而我们认为Gln可能是安普霉素生物合成氮元素的供体。Arg添加实验结果表明，Arg可能通过两种途径影响黑暗链霉菌体内的氮代谢：(1)Arg可能影响胞外蛋白酶的活性，进而促进含氮大分子物质的分解代谢，补充发酵过程中的氮素来源。(2)Arg可能在体内转化为Glu，同时增加了游离氨的含量，进而促进安普霉素的生物合成。
Streptomyces tenebrarius A04, the producer (with a titer of 2874u/ml) of single component of nebramycin ?apramycin was studied in this paper. After treatment of spore suspension and mycelia shivered by supersonic with mutagen ,combined with the application of screening models, some stable high yield apramycin-producing strains (with a fermentation titer of 4800-5200u/ml by shaking flask) such as A2-23, A2-30, ASM6 and Al-16 were obtained.
This result proved that UV treatment combined with tolerating apramycin or with tolerating streptomycin could be effective way to obtain high yield apramycin-producing strains.
With strain Al-16, the fermentation medium, aeration and inoculation volume etc. were investigated and the optimized vegetative medium and fermentation medium were obtained by uniform design.
The tertiary fermentation techniques and the FBC (fed-batch culture) and other fermentation techniques were also optimized. A scale-up fermentation technique was set up and the apramycin productivity reached 4478u/ml in a 4m3 fermentator.
Amino acid feeding experiments were also performed in this study in order to investigate the pathway, by which nitrogen incoperated into apramycin molecule, and the regulatory mechanism of nitrogen metabolism.
The feeding of glutamic acid, glutamine and a -keto- glutaric acid respectively showed that glutamic acid and glutamine had no obvious effects
on cell growth , but stimulated apramycin production greatly.With the same concentration of amino acids supplemented ,glutamine showed a stronger stimulation effect than glutamic acid, while a -keto-glutaric acid showed a repression effect on apramycin production. It could be deduced from above results that glutamine possibly is the donor of nitrogen element for the biosynthesis of apramycin. Arginine feeding experiment showed that nitrogen metabolism in the S.tenebraius was obviously affected by arginine through two possible ways:(l)pronase activity in vitro could be influnced by arginine, as a result, the catabolism of nitrogen-containing macro-molecule was promoted and the nitrogen element in the broth was increased.(2)arginine could be transformed into glutamic acid, so that the biosynthesis of apramycin was promoted.
以菌株A1-16为实验菌株，运用均匀设计方法，对发酵培养基、通气量、接种量等进行了考察，得到发酵培养基和种子培养基的优化配方。对三级发酵工艺、补料工艺等发酵工艺条件进行了优化，进一步确定了中试发酵工艺条件，4m~3发酵罐上平均发酵单位达4478u／ml。
通过氨基酸添加实验研究了安普霉素生物合成途径中的氮的来源及调节机制。Glu、Gln及α—酮戊二酸添加实验结果表明Glu、Gln对菌体的生长无明显的影响，但能强烈促进安普霉素的生物合成，且相同浓度条件下Gln的促进作用明显高于Glu，而α—酮戊二酸则对安普霉素的生物合成有抑制作用。因而我们认为Gln可能是安普霉素生物合成氮元素的供体。Arg添加实验结果表明，Arg可能通过两种途径影响黑暗链霉菌体内的氮代谢：(1)Arg可能影响胞外蛋白酶的活性，进而促进含氮大分子物质的分解代谢，补充发酵过程中的氮素来源。(2)Arg可能在体内转化为Glu，同时增加了游离氨的含量，进而促进安普霉素的生物合成。
Streptomyces tenebrarius A04, the producer (with a titer of 2874u/ml) of single component of nebramycin ?apramycin was studied in this paper. After treatment of spore suspension and mycelia shivered by supersonic with mutagen ,combined with the application of screening models, some stable high yield apramycin-producing strains (with a fermentation titer of 4800-5200u/ml by shaking flask) such as A2-23, A2-30, ASM6 and Al-16 were obtained.
This result proved that UV treatment combined with tolerating apramycin or with tolerating streptomycin could be effective way to obtain high yield apramycin-producing strains.
With strain Al-16, the fermentation medium, aeration and inoculation volume etc. were investigated and the optimized vegetative medium and fermentation medium were obtained by uniform design.
The tertiary fermentation techniques and the FBC (fed-batch culture) and other fermentation techniques were also optimized. A scale-up fermentation technique was set up and the apramycin productivity reached 4478u/ml in a 4m3 fermentator.
Amino acid feeding experiments were also performed in this study in order to investigate the pathway, by which nitrogen incoperated into apramycin molecule, and the regulatory mechanism of nitrogen metabolism.
The feeding of glutamic acid, glutamine and a -keto- glutaric acid respectively showed that glutamic acid and glutamine had no obvious effects
on cell growth , but stimulated apramycin production greatly.With the same concentration of amino acids supplemented ,glutamine showed a stronger stimulation effect than glutamic acid, while a -keto-glutaric acid showed a repression effect on apramycin production. It could be deduced from above results that glutamine possibly is the donor of nitrogen element for the biosynthesis of apramycin. Arginine feeding experiment showed that nitrogen metabolism in the S.tenebraius was obviously affected by arginine through two possible ways:(l)pronase activity in vitro could be influnced by arginine, as a result, the catabolism of nitrogen-containing macro-molecule was promoted and the nitrogen element in the broth was increased.(2)arginine could be transformed into glutamic acid, so that the biosynthesis of apramycin was promoted.
引文
1、刘诗通：托普霉素(综述)，国外药学抗生素分册，1987，8(5)：346～354
2、Stark W.M., Knox N.G. et al: Strains of Streptomyces tenebrarius and Biosynthesis. Folia Mierobiol, 1971,6(3):205～217
3、K. F. Koch, K. E. Merkel, S. C. O'Connor et al: Structure of Some of the Minor Aminoglycoside Factors of the Nebramycin Fermentation, J.Org. Chem. Vol.43,No.7,1978
4、Sean O'conner, L.K.T. Lam et al: Apramycin, a Unique Aminocyclitol Antibiotic, J. Org. Chem. 1976,Vol 41 No.12:2087～2092
5、Pashov D., Bor A. et al :J Vet Phamaeol Ther :1985,36(4):153～164
6、Sazonova E.M.Kovalev V.F. et al: Vestinik Nauki Mokva, 1991,(4): 82～86
7、章慧德，吴兴，刘肃：一株托普霉素高产菌株特性的研究，微生物学报，1987，27(4)：357～361
8、韩漪云、母连军、李俊英等：抗生素83-1050B的研究Ⅰ 抗生素83-1050B产生菌的鉴定与体外抗菌活性研究，抗生素，1986，11(3)：183～189
9、魏同：托普霉素新菌株U-135技术鉴定会，微生物学通报，1991，18(2)：119
10、USP,3962247
11、武慧渊，安普霉素产生菌的研究，[学位论文]，沈阳药科大学，1998，6
12、许铭轩，安普霉素生物合成途径的研究，[学位论文]，沈阳药科大学，2000，6
13、李相丰，安普霉素生物合成途径的研究，[学位论文]，沈阳药科大学，2001，6
14、张德安等，生物大分子实验手册，吉林大学出版社，1991，pp429-430
15、Haifeng Hu and Kozo Ochi,Novel approach for improving the productivity of antibiotic-producing strains by inducing combined mutations,Applied And
Environmental Microbiology,Apr.2001,pp1885-1892
16、邬行彦，熊宗贵，抗生素生产工艺学，化学工业出版社，1982，pp31-47
17、沈同，生物化学，高等教育出版社，1992，pp217-254
18、A.怀特等著，张澄波等译，生物化学原理，，中册pp439-508
19、吴章秀、王蓓蓓，抗生素83-1050B的研究Ⅱ抗生素83-1050B的提取分离及其组份鉴别，抗生素，1986，11(3)，190～195
20、陈代杰主编，微生物药物学，华东理工大学出版社，pp
21、David H.,Shermen等：国外医药抗生素分册，1990，11(5)：321-327
22、Jun Shima,Rew Hesketh,Susumu Okamoto et al,Induction of actionrhddin production by rpsL (encoding ribosomal protein S12) mutations that confer streptomycin resistance in streptomyces lividans and streptomyces coelicolor A3(2),Journal Of Bacteriology, Dce. 1996.pp7276-7284
23、Adamidis,T.P. Riggle,and W. Champness,Mutation in a new Streptomyces coelicolor locus which globally block antibiotic biosynthesis but not sporulation.J.Bacteriol. 1990,172:2962-2969
24、Brlan,P.J.Riggle,R.A. Santos,and W.C.Champness,Global negative regulation of Streptomyces coelicolor antibiotic synthesis mediated by an absA-encoded putative signal transduction system. J.Bacteriol, 1996,178:3221-3231
25、Bystrykh,L.V .,M .A .Femandez-Moreno,et al,Production of actionrhodin-related "blue-pigment" by Streptomyces coelicolor A3(2).J.Baceriol. 1996,178: 2238—2244
26、Beauclerk,A.A.D.,E.Cundliffe,Sites of action of two ribosomal RNA methylases responsible for resistance to aminoglycosides,1987,J.Mol. Biol. 193:661-671.
27、Buckel,P.A.Buchberger,H.G.Wittmann,Alterafion of ribosomal protein L6 in mutants of Escherichia coli resistant to gentamicin,1977,Mol.Gen. Genet.158:47-54
28、Champness,W.P. Riggle,T. Adamidis,Loci involved in regulation of antibiotic synthesis,1990,J.Cell. Biochem.14A:88
29、Chater, K.E,The improving prospects for yield increasee by genetic engineering in antibiotic-producing streptomycetes, 1990,Bio/Technology 8:115-121
30、Davies,J.,L. Gorini,B.D.Davis,Misreading of RNA codewords induced by aminoglycoside antibiofics,1965,Mol. Pharmacol.1:93-106
2、Stark W.M., Knox N.G. et al: Strains of Streptomyces tenebrarius and Biosynthesis. Folia Mierobiol, 1971,6(3):205～217
3、K. F. Koch, K. E. Merkel, S. C. O'Connor et al: Structure of Some of the Minor Aminoglycoside Factors of the Nebramycin Fermentation, J.Org. Chem. Vol.43,No.7,1978
4、Sean O'conner, L.K.T. Lam et al: Apramycin, a Unique Aminocyclitol Antibiotic, J. Org. Chem. 1976,Vol 41 No.12:2087～2092
5、Pashov D., Bor A. et al :J Vet Phamaeol Ther :1985,36(4):153～164
6、Sazonova E.M.Kovalev V.F. et al: Vestinik Nauki Mokva, 1991,(4): 82～86
7、章慧德，吴兴，刘肃：一株托普霉素高产菌株特性的研究，微生物学报，1987，27(4)：357～361
8、韩漪云、母连军、李俊英等：抗生素83-1050B的研究Ⅰ 抗生素83-1050B产生菌的鉴定与体外抗菌活性研究，抗生素，1986，11(3)：183～189
9、魏同：托普霉素新菌株U-135技术鉴定会，微生物学通报，1991，18(2)：119
10、USP,3962247
11、武慧渊，安普霉素产生菌的研究，[学位论文]，沈阳药科大学，1998，6
12、许铭轩，安普霉素生物合成途径的研究，[学位论文]，沈阳药科大学，2000，6
13、李相丰，安普霉素生物合成途径的研究，[学位论文]，沈阳药科大学，2001，6
14、张德安等，生物大分子实验手册，吉林大学出版社，1991，pp429-430
15、Haifeng Hu and Kozo Ochi,Novel approach for improving the productivity of antibiotic-producing strains by inducing combined mutations,Applied And
Environmental Microbiology,Apr.2001,pp1885-1892
16、邬行彦，熊宗贵，抗生素生产工艺学，化学工业出版社，1982，pp31-47
17、沈同，生物化学，高等教育出版社，1992，pp217-254
18、A.怀特等著，张澄波等译，生物化学原理，，中册pp439-508
19、吴章秀、王蓓蓓，抗生素83-1050B的研究Ⅱ抗生素83-1050B的提取分离及其组份鉴别，抗生素，1986，11(3)，190～195
20、陈代杰主编，微生物药物学，华东理工大学出版社，pp
21、David H.,Shermen等：国外医药抗生素分册，1990，11(5)：321-327
22、Jun Shima,Rew Hesketh,Susumu Okamoto et al,Induction of actionrhddin production by rpsL (encoding ribosomal protein S12) mutations that confer streptomycin resistance in streptomyces lividans and streptomyces coelicolor A3(2),Journal Of Bacteriology, Dce. 1996.pp7276-7284
23、Adamidis,T.P. Riggle,and W. Champness,Mutation in a new Streptomyces coelicolor locus which globally block antibiotic biosynthesis but not sporulation.J.Bacteriol. 1990,172:2962-2969
24、Brlan,P.J.Riggle,R.A. Santos,and W.C.Champness,Global negative regulation of Streptomyces coelicolor antibiotic synthesis mediated by an absA-encoded putative signal transduction system. J.Bacteriol, 1996,178:3221-3231
25、Bystrykh,L.V .,M .A .Femandez-Moreno,et al,Production of actionrhodin-related "blue-pigment" by Streptomyces coelicolor A3(2).J.Baceriol. 1996,178: 2238—2244
26、Beauclerk,A.A.D.,E.Cundliffe,Sites of action of two ribosomal RNA methylases responsible for resistance to aminoglycosides,1987,J.Mol. Biol. 193:661-671.
27、Buckel,P.A.Buchberger,H.G.Wittmann,Alterafion of ribosomal protein L6 in mutants of Escherichia coli resistant to gentamicin,1977,Mol.Gen. Genet.158:47-54
28、Champness,W.P. Riggle,T. Adamidis,Loci involved in regulation of antibiotic synthesis,1990,J.Cell. Biochem.14A:88
29、Chater, K.E,The improving prospects for yield increasee by genetic engineering in antibiotic-producing streptomycetes, 1990,Bio/Technology 8:115-121
30、Davies,J.,L. Gorini,B.D.Davis,Misreading of RNA codewords induced by aminoglycoside antibiofics,1965,Mol. Pharmacol.1:93-106