Production improvement of antifungal, antitrypanosomal nucleoside sinefungin by rpoB mutation and optimization of resting cell system of Streptomyces incarnatus NRRL 8089

Koji Fukuda, Takashi Tamura, Hideyuki Ito, Sayaka Yamamoto, Kozo Ochi, Kenji Inagaki

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Sinefungin, a nucleoside antibiotic with potent antifungal, antiviral, and anti-trypanosome activities, has been a target for production enhancement in the past decades through medium optimization and strain improvement. For the purpose of introducing a more rational approach, we induced rpoB mutation in the producer strain, Streptomyces incarnatus NRRL 8089, by optimized UV-irradiation, and a resulting rifampicin-resistant strain rif-400 increased the sinefungin production by 7-fold. The growth and melanin production were obviously accelerated in the rifampicin-resistant high-producer mutant, while the morphological differentiation such as aerial mycelia and spiked-spore formation was retained. Molecular cloning and DNA sequencing identified a single mutation A1340G in the rpoB gene, which encodes the β-subunit of RNA polymerase, and the resulting amino acid substitution Asp447Gly corresponded to one of mutations that reportedly allowed the transcriptional up-regulation of actinorhodin production in S. coelicolor A3(2). Sinefungin production was further enhanced by resting cell system using the rpoB mutant strain in the presence of 10 mM l-Arg. d-Arg or l-ornithine did not enhance the sinefungin production, and > 50 mM urea strongly suppressed the nucleoside antibiotic production, supporting the proposed biosynthetic mechanism by which urea is liberated from the guanidino-group-bearing intermediate that is produced by enzymatic condensation of l-Arg and ATP.

Original languageEnglish
Pages (from-to)459-465
Number of pages7
JournalJournal of Bioscience and Bioengineering
Volume109
Issue number5
DOIs
Publication statusPublished - May 2010

Keywords

  • Rifampicin-resistance
  • Secondary metabolism
  • Sinefungin
  • Streptomyces incarnatus
  • rpoB gene

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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