在格尔德霉素产生菌吸水链霉菌17997(Streptomyces hygroscopicus 17997)中存在两种3-氨基-5-羟基苯甲酸(3-amino-5-hydroxybenzoic acid, AHBA)的生物合成基因簇, 根据同源性可分为苯醌类和萘醌类。已证明其中苯醌类的AHBA生物合成基因簇负责格尔德霉素(geldanamycin, Gdm)起始
20 ISSN1000-3061 CN11-1998/Q Chin J Biotech January 25, 2008 Vol.24 No.1
流相关。本研究采用基因重组技术, 通过破坏AHBA-N基因, 获得的基因阻断变株ΔSOP, 其苯醌型安莎类抗生素Gdm的发酵产量提高了185%, 表明AHBA的合成可能在吸水链霉菌中是限制因素, 抑制该菌中AHBA-N的供应, 可以促进AHBA-B的合成和供应, 有利于Gdm的合成。
一般链霉菌的生活周期多以孢子起始和告终。孢子萌发后形成菌丝, 在固体培养基上开始长为基内菌丝, 菌丝体发育至一定阶段形成气生菌丝, 部分气生菌丝发展为孢子丝和孢子。链霉菌固体培养物的生长周期对菌种的发酵潜能影响很大。使用以孢子量多和成熟的菌种为佳, 过于年轻的孢子产生抗生素的能力容易波动, 过于成熟的孢子已接近衰老会导致生产能力的降低, 但不同菌种随着培养基的不同其生长规律有变化。尤其是吸水链霉菌在固体培养基上的孢子易自溶而形成湿斑。本研究表明吸水链霉菌17997原株孢子形成较缓慢, 至10 d孢子数达到高峰, 而ΔSOP变株在固体培养基上孢子形成可分为两个周期, 第一代孢子形成期为7 d, 但这时孢子表面颜色较浅, 显示培养物处于比较年轻的状态, 第7天以后孢子数明显减少, 表明第一代孢子又形成气生菌丝, 以后又长成孢子, 至第10天孢子表面颜色变深, 显示第二代孢子比较成熟, 以孢子计数为指标的菌种生长周期与发酵潜力的研究表明, 在本实验条件下17997原株在孢子形成高峰期发酵效价较高(HPLC图谱从略)。ΔSOP在第二代孢子成熟期的发酵能力比第一代孢子高140%。其确切机制尚需进一步深入研究。
[5] Goetz MP, Toft D, Reid J, et al. Phase I trial of 17-allylamino-
17-demethoxygeldanamycin in patients with advanced cancer. Jounal of Clinical Oncology. 2005, 23(6): 1078 1087.
[6] Glaze ER, Lambert AL, Smith AC, et al. Preclinical toxic-ity of a geldanamycin analog, 17-(dimethylaminoethylamino)- 17-demeth oxygeldanamycin (17-DMAG), in rats and dogs: potential clinical relevance. Cancer Chemotherapy Pharmacology, 2005, 56 (6): 637 647.
[7] Gao QJ, Shang GD, Wang YG, et al. Cloning and analysis
of geldanamycin biosynthetic genes from S. hygroscopicus 17997. Chinese Journal Antibiotics, 2002, 27: 13 17. 高群杰, 尚广东, 王以光, 等. Geldanamycin产生菌生物
合成相关基因的克隆与分析. 中国抗生素杂志, 2002, 27: 13 17.
[8] Ghisalba O, Nüesch J. Genetic approach to the biosynthe-sis of the rifamycin-chromophore in Nocardia mediterra-nei. IV, identification of 3-amino-5-hydroxybenzoic acid as a direct precursor of the seven-carbon amino starter- unit. Jounal of Antibiotics, (Tokyo), 1981, 34: 64 71. [9] Becker AM, Herlt AJ, Hilton GL, et al. 3-Amino-5- hy-droxybenzoic acid in antibiotic biosynthesis. VI. Directed biosynthesis studies with ansamycin antibiotics. Jounal of Antibiotics (Tokyo), 1983, 36: 1323 1328.
[10] He WQ, Wu LZ, Wang YG, et al. Identification of AHBA
biosynthetic genes related to geldanamycin biosynthesis in Streptomyces hygroscopicus 17997. Current Microbiology, 2006, 52 (3): 197 203.
[11] Li-Hong Malmberg, Hu WS, David H Sherman. Precursur
flux control through targeted chromosomal insertion of the lysine-ε-aminotransferase (Lat) gene in cephamycin C biosynthesis. Journal of Bacteriology, 19993, 175 (21): 6916 6924.
[12] Mo HB, Bai LQ, Yang KQ, et al. Construction of efficient
conjugal plasmids between Escherichia coli and strepto-mycetes. Chinese Journal of Biotechnology, 2004, 20: 662 666.
莫宏波, 白林泉, 杨克迁, 等. 大肠杆菌-链霉菌高效接
合载体的构建及其应用. 生物工程学报, 2004, 20:
REFERENCES
[1] Sasaki K, Yasuda H, Onodera K. Growth inhibition of vi-rus transformed cells in vitro and antitumor activity in vivo of geldanamycin and its derivatives. Journal of Antibiotics (Tokyo), 1979, 32(8): 849 951.
[2] Tao PZ, Lou ZX, Yao TJ, et al. Antiviral study on the
broad spectrum antiviral antibiotic 17997 in vitro and in vivo. Chinese Journal of Antibiotics, 1997, 22: 368 372. 陶佩珍, 娄志贤, 姚天爵, 等. 广谱抗病毒抗生素17997
体内外抗病毒活性研究. 中国抗生素杂志, 1997, 22:
368 372.
[3] Prodromou C, Roe SM, O’Brien R, et al. Identification and
structural characterization of the ATP/ADP-binding site in the Hsp90 molecular chaperone. Cell, 1997, 90(1): 65 75.
[4] Stebbins CE, Russo AA, Schneider C, et al. Crystal struc-ture of an Hsp90-geldanamycin complex: targeting of a protein chaperone by an antitumor agent. Cell, 1997, 89 (2): 239 250.
662 666.
[13] Bierman M, Logan R, O’Brien K, et al. Plasmid cloning
vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene, 1992, 116: 43 49.
[14] Sambrook J, Russell DW, Sambrook J. Molecular Cloning:
A Laboratory Manual. 2nd ed. Cold Spring Harbor Labo-ratory, 1989.
[15] Tobias Kieser, Mervyn J Bibb, Mark J Buttner, Keith F
Chater, David A Hopwood. Practical Streptomyces Genet-ics. the John Innes Foundation Norwich, 2000.
[16] He WQ, Li JY, Sun GZ, et al. Detection of fermentation
prodcuts from Streptomyces hygroscopicus17997 by HPLC analysis. Chinese Journal of Antibiotic, 2006, 31(3): 168 171.
赫卫清, 李京艳, 孙桂芝, 等. 反相高效液相谱型分析检
测吸水链霉菌17997变株发酵新产物. 中国抗生素杂志,
2006, 31(3): 168 171.
[17] Shuo Chen, Daniel von Bamberg, Valeric Hale, et al.
Biosynthesis of ansatrienin (mycotrienin) and naphthomy-cin Identification and analysis of two separate biosynthetic gene clusters in Streptomyces collinus Tu 1892. European Journal of Biochemistry, 1999, 261: 98 107.