WEN Shuping,CHEN Hui,WANG Yang,et al.Research of glycosyltransferase gene tobM2 in tobramycin[J].Journal of Yanbian University,2016,42(01):39-44.
妥布霉素糖基转移酶基因tobM2的研究
- Title:
- Research of glycosyltransferase gene tobM2 in tobramycin
- Keywords:
- Streptomyces tenebrarius; tobramycin; tobM2; gene dirsrupted
- 分类号:
- Q93
- 文献标志码:
- A
- 摘要:
- 为探索妥布霉素生物合成过程中糖基转移的作用,对糖基转移酶基因tobM2进行了阻断研究.以妥布霉素生物合成基因簇为模板,构建了同源重组质粒pMB4,通过接合转移将质粒pMB4导入黑暗链霉菌Tt-49,然后利用红霉素抗性筛选并经PCR验证,获得了一株基因tobM2被阻断的工程菌TW402.发酵及代谢产物分析结果表明:工程菌总效价略有下降,约为出发菌株的85%.TLC检测与质谱分析显示,工程菌TW402主产安普霉素,并积累中间产物尼泊拉胺.TobM2转化尼泊拉胺生成妥布霉素,tobM2基因的缺失阻断了妥布霉素的生物合成,获得一株主产安普霉素工程菌.
- Abstract:
- The glycosyltransferase gene tobM2 was disrupted to study the reaction of glycosylation in the tobramycin biosynthesis. A recombinant plasmid pMB4 was constructed with tobramycin biosynthesis gene cluster as the template. Then, the plasmid pMB4 was transformed into Streptomyces tenebrarius Tt-49 by conjugation. A mutant strain TW402 was obtained by the gene tobM2 was disrupted, screened and confirmed by erythromycin resistance and PCR amplification. Ferment and metabolite analysis showed that the total titer of mutant strain TW402 decreased slightly, which was about 85% of that of the wild-type strain. TLC detection and MS analyses indicated that the mutant strain TW402 mainly produced apramycin, and nebramine was accumulated. TobM2 converted nebramine into tobramycin, and apramycin-producing engineering bacterium was obtained by disruption of tobM2 gene, which blocked the tobramycin biosynthesis.
参考文献/References:
[1] Kharel M K, Subba B, Lee H C, et al. An approach for cloning biosynthetic genes of 2-deoxystreptamine-containing aminocyclitol antibiotics: isolation of a biosynthetic gene cluster of tobramycin from Streptomyces tenebrarius[J]. Biotechnology Letters, 2003,25(24):2041-2047.
[2] Kharel M K, Basnet D B, Lee H C, et al. Isolation and characterization of the tobramycin biosynthetic gene cluster from Streptomyces tenebrarius[J]. FEMS Microbiology Letters, 2004,230(2):185-190.
[3] Flatt P M, Mahmud T. Biosynthesis of aminocyclitol-aminoglycoside antibiotics and related compounds[J]. Natural Product Reports, 2007,24(2):358-392.
[4] Kudo F, Eguchi T. Biosynthetic genes for aminoglycoside antibiotics[J]. The Journal of Antibiotics, 2009,62(9):471-481.
[5] 倪现朴.黑暗链霉菌aprD3和aprD4基因的研究及卡那霉素B高产菌株的构建[D].沈阳:沈阳药科大学,2011.
[6] 林玉双,洪文荣.黑暗链霉菌tobS1-C基因缺失突变株的构建[J].中国药科大学学报,2012,43(1):92-96.
[7] 李辉.黑暗链霉菌次级代谢产物生物合成基因的研究[D].福州:福州大学,2013.
[8] Flett F, Mersinias V, Smith C P. High efficiency intergeneric conjugal transfer of plasmid DNA from Escherichia coli to methyl DNA-restricting streptomycetes[J]. FEMS Microbiology Letters, 1997,155(2):223-229.
[9] Paranthaman S, Dharmalingam K. Intergeneric conjugation in Streptomyces peucetius and Streptomyces sp. strain C5: chromosomal integration and expression of recombinant plasmids carrying the chiC gene [J]. Applied and Environmental Microbiology, 2003,69(1):84-91.
[10] 林玉双,洪文荣,林烨,等.氨甲酰妥布霉素高产菌株选育及其发酵特性研究[J].中国抗生素杂志,2008,33(7):442-445.
[11] Kieser T, Bibb M J, Mark J, et al. Practical Streptomyces Genetics[M]. Norwich, England: The John Innes Foundation, 2000:249-250.
[12] Sambrook J F, Russell D W. Molecular Cloning: A Laboratory Manual[M]. Third Edition. Cold Spring Harbor Laboratory Press, 2001:27-99.
[13] Hong W R, Yan S D. Engineering Streptomyces tenebrarius to synthesize single component of carbamoyltobramycin[J]. Lett Appl Microbiol, 2012,55(1):33-39.
[14] Ni X P, Li D, Yang L H, et al. Construction of kanamycin B overproducing strain by genetic engineering of Streptomyces tenebrarius[J]. Applied Microbiology and Biotechnology, 2011,89:723-731.
[15] 国家药典委员会.中华人民共和国药典[M].2部.北京:中国医药科技出版社,2010.
备注/Memo
收稿日期: 2015-09-25 *通信作者: 洪文荣(1956—),男,博士,教授,研究方向为微生物制药. 基金项目: 国家自然科学基金资助项目(31070093); 国家“重大新药创制”科技重大专项项目(2012ZX09201101-008)