Publications

• Cloning and nucleotide sequence of the gene responsible for chlorination of tetracycline.

  Dairi T., Nakano T., Aisaka K., Katsumata R., Hasegawa M.

  Two cosmid clones containing distinct types of self-defense gene of a 6-demethylchlortetracycline producer, Streptomyces aureofaciens NRRL3203, were isolated. The gene responsible for chlorination of tetracycline (chl gene) was subcloned from one of the cosmid clones by complementation of a chlori ... >> More

  Two cosmid clones containing distinct types of self-defense gene of a 6-demethylchlortetracycline producer, Streptomyces aureofaciens NRRL3203, were isolated. The gene responsible for chlorination of tetracycline (chl gene) was subcloned from one of the cosmid clones by complementation of a chlorination-deficient mutant, using a gene cloning system for strain NRRL3203 developed in this study. The nucleotide sequence analysis of a 4.4-kb SacI-BamHI fragment containing the chl gene showed that the predicted product of the chl gene is a polypeptide of 452 amino acids, and that the chl gene was preceded by two open reading frames, which could endode polypeptides of 50 kDa and 32 kDa, respectively. A search for sequences homologous to these ORFs found that the former product strongly resembles that of the 6-hydroxylation enzyme for oxytetracycline biosynthesis, and that the latter product has a weak but significant similarity to the hydroxyindole O-methyltransferase of bovine pineal gland. By Northern blot analysis, these three genes were suggested to be polycistronically transcribed. << Less

  Biosci. Biotechnol. Biochem. 59:1099-1106(1995) [PubMed] [EuropePMC]

• Deciphering and engineering of the final step halogenase for improved chlortetracycline biosynthesis in industrial Streptomyces aureofaciens.

  Zhu T., Cheng X., Liu Y., Deng Z., You D.

  Chlortetracycline (CTC) is an important member from antibiotics tetracycline (TC) family, which inhibits protein synthesis in bacteria and is widely involved in clinical therapy, animal feeds and aquaculture. Previous works have reported intricately the biosynthesis of CTC from the intermediates i ... >> More

  Chlortetracycline (CTC) is an important member from antibiotics tetracycline (TC) family, which inhibits protein synthesis in bacteria and is widely involved in clinical therapy, animal feeds and aquaculture. Previous works have reported intricately the biosynthesis of CTC from the intermediates in random mutants of Streptomyces aureofaciens and the crucial chlorination remained unclear. We have developed the genetic manipulation in an industrial producer, in which about 15.0g/l CTC predominated along with 1.2g/l TC, and discovered that chlorination by ctcP (an FADH2-dependent halogenase gene) is the last inefficient step during CTC biosynthesis. Firstly, the ΔctcP strain accumulated about 18.9g/l "clean" TC without KBr addition and abolished the production of CTC. Subsequently, CtcP was identified to exhibit a substrate stereo-specificity to absolute TC (4S) rather than TC (4R), with low kcat of 0.51±0.01min(-1), while it could halogenate several TC analogs. Accordingly, we devised a strategy for overexpression of ctcP in S. aureofaciens and improved CTC production to a final titer of 25.9g/l. We anticipate that our work will provide a biotechnological potential of enzymatic evolution and strain engineering towards new TC derivatives in microorganisms. << Less

  Metab. Eng. 19:69-78(2013) [PubMed] [EuropePMC]