抗生素产生菌AP19-1的鉴定及UV诱变育种的研究
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摘要
本文以一株抗生素产生菌AP19-1为出发菌株，对其进行了系统分类学以及UV诱变育种的研究。
形态学特征、生理生化性状和16S rDNA核苷酸序列分析表明，菌株AP19-1属于链霉菌属(Streptomyces sp.)。
用紫外线对AP19-1的孢子及原生质体分别进行诱变育种，获得了产抗生素的高产菌株。经纸片法和最小抑菌浓度(MIC)测定，其中一株由原生质体诱变得到的菌株，其发酵液中抗生素的相对效价是原始菌株的4倍，且有较高的遗传稳定性。实验结果表明，原生质体的紫外诱变比孢子的紫外诱变更易得到抗生素相对效价提高较多的突变株。
我们同时还研究了不同的培养温度对孢子的紫外诱变正向突变率的影响。将UV诱变后的AP19-1孢子，分别置于适宜的生长温度27℃与接近抑制生长的胁迫温度33℃下培养，结果表明：在33℃下培养获得的子代菌株中，产抗生素水平超过其出发菌株的正向突变体所占的比例，明显高于在27℃下培养的结果。27℃下培养，正向突变体占总子代菌株数的百分率为25.8％，而在33℃下培养则为58.1％。用17种10bp的随机引物对出发菌株与UV诱变后的子代菌株进行总DNA的RAPD测验证明，在接近抑制生长的胁迫温度33℃下培养获得的子代，发生在其DNA水平上的变异程度要比在27℃培养下的子代高得多，与对照菌株RAPD条带的相似性从27℃的66.4％下降到了33℃的57.4％。这一方法较大幅度地提高了链霉菌AP19-1紫外诱变育种的工作效率，同时也为链霉菌紫外诱变后突变形成机制的进一步研究提供了新途径。
Studies on UV mutagenic breeding and identification of an antibiotic-producing strain AP19-1 were carried out.
Based on the results of phenotypic characteristics and 16S rDNA sequence analysis, the isolate was assigned to the genus Streptomyces.
In order to improve the antibiotic-producing trait of strain AP19-1, we exposed its protoplasts and spores to UV and isolated a mutant strain Y207. The antibiotic's relative liter of the mutant's fermented broth was 4 times as high as the parent strain through MIC experiment. And its high capacity for producing antibiotic remained stable after 4 times of subcultures. It was also found that the effect of protoplasts' UV mutagenic breeding was better than spores'.
UV irradiated spores of strain AP19-1 were incubated at 27℃ and 33℃ close to inhibiting growth temperature. Results showed that among the offspring from UV irradiated spores grown at 33℃ , the amount of forward mutants whose antibiotics-producing ability were over the original strain's were many more than that at 27℃. The percentage of the forward mutants was 25.8% at 27℃ and 58.1% at 33 ℃ respectively. Total DNA of the progeny strain and the original strain was tested by RAPD with 17 primers. It was demonstrated that the variations occurred in the chromosomal DNA of the progeny strains grown at 33℃ were more than that at 27 ℃. This method improved the efficiency of UV mutagenic breeding and put forward a new way in researching into the mechanisms of mutation formation in Streptomyces sp. cells irradiated by UV.
形态学特征、生理生化性状和16S rDNA核苷酸序列分析表明，菌株AP19-1属于链霉菌属(Streptomyces sp.)。
用紫外线对AP19-1的孢子及原生质体分别进行诱变育种，获得了产抗生素的高产菌株。经纸片法和最小抑菌浓度(MIC)测定，其中一株由原生质体诱变得到的菌株，其发酵液中抗生素的相对效价是原始菌株的4倍，且有较高的遗传稳定性。实验结果表明，原生质体的紫外诱变比孢子的紫外诱变更易得到抗生素相对效价提高较多的突变株。
我们同时还研究了不同的培养温度对孢子的紫外诱变正向突变率的影响。将UV诱变后的AP19-1孢子，分别置于适宜的生长温度27℃与接近抑制生长的胁迫温度33℃下培养，结果表明：在33℃下培养获得的子代菌株中，产抗生素水平超过其出发菌株的正向突变体所占的比例，明显高于在27℃下培养的结果。27℃下培养，正向突变体占总子代菌株数的百分率为25.8％，而在33℃下培养则为58.1％。用17种10bp的随机引物对出发菌株与UV诱变后的子代菌株进行总DNA的RAPD测验证明，在接近抑制生长的胁迫温度33℃下培养获得的子代，发生在其DNA水平上的变异程度要比在27℃培养下的子代高得多，与对照菌株RAPD条带的相似性从27℃的66.4％下降到了33℃的57.4％。这一方法较大幅度地提高了链霉菌AP19-1紫外诱变育种的工作效率，同时也为链霉菌紫外诱变后突变形成机制的进一步研究提供了新途径。
Studies on UV mutagenic breeding and identification of an antibiotic-producing strain AP19-1 were carried out.
Based on the results of phenotypic characteristics and 16S rDNA sequence analysis, the isolate was assigned to the genus Streptomyces.
In order to improve the antibiotic-producing trait of strain AP19-1, we exposed its protoplasts and spores to UV and isolated a mutant strain Y207. The antibiotic's relative liter of the mutant's fermented broth was 4 times as high as the parent strain through MIC experiment. And its high capacity for producing antibiotic remained stable after 4 times of subcultures. It was also found that the effect of protoplasts' UV mutagenic breeding was better than spores'.
UV irradiated spores of strain AP19-1 were incubated at 27℃ and 33℃ close to inhibiting growth temperature. Results showed that among the offspring from UV irradiated spores grown at 33℃ , the amount of forward mutants whose antibiotics-producing ability were over the original strain's were many more than that at 27℃. The percentage of the forward mutants was 25.8% at 27℃ and 58.1% at 33 ℃ respectively. Total DNA of the progeny strain and the original strain was tested by RAPD with 17 primers. It was demonstrated that the variations occurred in the chromosomal DNA of the progeny strains grown at 33℃ were more than that at 27 ℃. This method improved the efficiency of UV mutagenic breeding and put forward a new way in researching into the mechanisms of mutation formation in Streptomyces sp. cells irradiated by UV.
引文
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2 Stackebrandt E, Woese CR. Current Microbiology, 1981, 5:197-202.
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4 Labeda DP. DNA relatedness among the Streptomyces fulvissimus and Streptomyces griseoviridis phenotypic cluster groups[J]. International journal of systematic bacteriology, 1998, 48: 829-832.
5 Oscar H, Martinez-Costa, Magdalena Z, et al. The promoter of a cold-shock-like gene has pleiotropic effects on Streptomyces antibiotic biosynthesis [J]. FEMS microbiology letters, 2003, 220:215-221.
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7 施巧琴，吴松刚．工业微生物育种学[M]，北京：科学出版社，2003，125-126．
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10 崔大鹏，石莲英．原生质体再生和诱变在硫霉素产生菌选育中的应用[J]．中国抗生素杂志，1999，24(4)：265-268．
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32 van Mellaert L, Anné J. Gram-positive bacteria as host cells for heterologous production of biopharmacuticals [M]. In: van Broekhoven A, Shapiro F, AnnéJ(Eds.), Novel Frontiers in the Production of Compounds for Biomedical Use. Focus on Biotechnology, Kluwer Academic Publishing, Dordrecht, The Netherlands, 2001, vol.Ⅰ: 277-300.
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37 Lee SH, Rho YT. Improvement of tylosin fermentation by mutation and medium optimization [J]. Letters in applied microbiology, 1999, 28(2): 142-144.
38 Aharonowitz Y, Cohen G. The microbial production of pharmaceuticals [J], Scientific American, 1981, 245(3): 141-152.
39 Demain AL, Production of new antibiotics by directed biosynthesis and by the use of mutants [M]. London: Academic Press, 1981.
40 Omura S, Ikeda H, Malpartida F, et al. Production of new hybrid antibiotics, mederrhodins A and B, by a genetically engineered strain [J]. Antimicrobial agents and chemotherapy, 1986, 29(1): 13-19.
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2 Stackebrandt E, Woese CR. Current Microbiology, 1981, 5:197-202.
3 Vandamme P, Pot B, Gillis M, .et al. Polyphasic taxonomy, a consensus approach to bacterial systematics [J]. Microbiol Review, 1996, 60(2): 407-438.
4 Labeda DP. DNA relatedness among the Streptomyces fulvissimus and Streptomyces griseoviridis phenotypic cluster groups[J]. International journal of systematic bacteriology, 1998, 48: 829-832.
5 Oscar H, Martinez-Costa, Magdalena Z, et al. The promoter of a cold-shock-like gene has pleiotropic effects on Streptomyces antibiotic biosynthesis [J]. FEMS microbiology letters, 2003, 220:215-221.
6 Zohreh H, Claire M, Barbara H, et al. Structure, biosynthetic origin, and engineered biosynthesis of calcium-dependent antibiotics from Streptomyces coelicolor [J]. Chemistry & Biology, 2002, 9: 1175-1187.
7 施巧琴，吴松刚．工业微生物育种学[M]，北京：科学出版社，2003，125-126．
8 周德庆．微生物学实验手册[M]．上海：科学技术出版社，1983．
9 粱蓉芳．龟裂链霉菌原生质体诱变育种的研究[J]．微生物学研究与应用，1991，1：28-32．
10 崔大鹏，石莲英．原生质体再生和诱变在硫霉素产生菌选育中的应用[J]．中国抗生素杂志，1999，24(4)：265-268．
11 Hopwood D A(邓子新，唐纪良译)．Genetic manipulation of Streptomyces：A laboratory manual[M]．湖南：科学技术出版社，1988．
12 Frederick M，Ausubel，et al．(严子颖，王海林 译)．Short protocols in molecular biology[M]．北京：科学出版社，1998，39．
13 Altschul, Stephen F, Thomas L, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs [J]. Nucleic Acids Research, 1997, 5: 3389-3402.
14 Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting,
position-specific gap penalties and weight matrix choice [J]. Nucleic Acid Research, 1994, 22: 4673-4688.
15 Felsenstein J. PHYLIP: Phylogeny Inference Package, version 3.5c. Seattle: Department of Genetics, University of Washington.
16 Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees [J]. Molecular biology and evolution, 1987, 4(4): 406-425.
17 Page, RD. TREEVIEW: An application to display phylogenetic trees on personal computers [J]. Computer Applications in the Biosciences, 1996, 12:357-358.
18 马绪荣，苏德模．药品微生物学检验手册，北京：科学出版社，2000，201-203；198-199．
19 Carl WD，Gabriela SD(黄培堂等 译)．PCR primer: A laboratory manual[M]. 北京：科学出版社，1998，138-145．
20 http://www.ncbi.nlm.nih.gov/Taxonomy/Browser.
21 Dalevi D, Hugenholtz P, Blackall LL. A multiple-outgroup approach to resolving division-level phylogenetic relationships using 16S rDNA data [J], International journal of systematic and evolutionary microbiology, 2001, 51: 385-391.
22 Buchanan RE, Gibbons NE．(中国科学院微生物研究所《伯杰细菌鉴定手册》翻译组 译)Bergey's Manual of Determinative Bacteriology, (Eight Edition)[M]，北京科学出版社，1984，1038-1161．
23 陈志高，沈建新．紫外线和氯化锂对核糖甙链霉菌原生质体诱变作用的探讨[J]．中国医药工业杂志，2001，32(4)：150-152．
24 Peter Strike, Microbiology today, 2003, 30: 8-10.
25 Vierling S, Weber T, Wohlleben W, et al. Evidence that an additional mutation is required to tolerate insertional inactivation of the Streptomyces lividans recA gene [J]. Journal of Bacteriology, 2001, 183(14): 4374-4381.
26 Pelaez AI, Ribas-Aparicio RM, Gomez A, et al. Structural and functional characterization of the recR gene of Streptomyces [J]. Mollecular Genetic Genomics, 2001, 265(4): 663-672.
27 A．N．格拉泽，二介堂弘著，陈守文，喻子牛等译．微生物生物技术[M]，上海：科学出版社，2002，7．
28 Hopwood DA, Charier KH. Genetic and Breeding of Industrial Microorganisms [M]. Florida.C.R.C.Press. 1984.
29 Kieser T, Bibb MJ, Buttner MJ, et al. Practical Streptomyces Genetics[M]. The John Innes Foundation, Norwich, 2000, 613.
30 Gelbert M, Morosoli R, Shareck F, et al. Production and secretion of proteins by Streptomycetes [J]. Critical reviews in biotechnology. 1995, 15: 13-39.
31 Hopwood DA. Forty years of genetics with Streptomyces: from in vivo through in vitro to in silico [J]. Microbiology, 1999, 145:2183-2202.
32 van Mellaert L, Anné J. Gram-positive bacteria as host cells for heterologous production of biopharmacuticals [M]. In: van Broekhoven A, Shapiro F, AnnéJ(Eds.), Novel Frontiers in the Production of Compounds for Biomedical Use. Focus on Biotechnology, Kluwer Academic Publishing, Dordrecht, The Netherlands, 2001, vol.Ⅰ: 277-300.
33 Kieser T, Moss MT, Dale JW, et al. Cloning and expression of Mycobacterium bovis BCG DNA in "Streptomyces lividans" [J]. Journal of Bacteriology, 1986, 168(1): 72-80.
34 Gray G, Seizer G, Buell G, et al. Synthesis of bovine growth hormone by Streptomyces lividans [J], Gene, 1984, 32: 21-30.
35 Bulock JD, et al. Bioactive microbial products, search and discovery [M]. London and New York Academic press, 1982.
36 Zvenigorodskii VI, Tyaglov BV, Voeikova TA. Isolation of components of the peptide antibiotic virginiamycin and breeding of their producer, Streptomyces virginiae [J]. Applied biochemistry and microbiology, 2001, 37(3): 260-266.
37 Lee SH, Rho YT. Improvement of tylosin fermentation by mutation and medium optimization [J]. Letters in applied microbiology, 1999, 28(2): 142-144.
38 Aharonowitz Y, Cohen G. The microbial production of pharmaceuticals [J], Scientific American, 1981, 245(3): 141-152.
39 Demain AL, Production of new antibiotics by directed biosynthesis and by the use of mutants [M]. London: Academic Press, 1981.
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