Improvement of Sinefungin-Producing Strain of Streptomyces incarnatus by Conferring Rifampicin-Resistance through Ultraviolet Light Irradiation and Protoplast Regeneration

Secondary metabolite production by gram-positive bacteria is strictly regulated at the transcription of the biosynthetic genes to mRNA in response to certain stringent conditions. Therefore, some mutational disruption of regulatory domains of the bacterial RNA polymerase might increase the production of the antibiotics. In this study, we have attempted to improve the sinefungin-producing strain of Streptomyces incarnatus NRRL 8057 by irradiating ultraviolet light on the protoplast, and selecting mutants that acquired the resistance to rifampicin, the antibiotic which specifically binds to the β-subunit of bacterial RNA polymerase. After three rounds of mutation, 10 strains were obtained with varied resistance to rifampicin. A mutant which showed the highest resistance was found to have the highest sinefungin production, which was 2.4 times higher(0.45±0.11μg/ml)than the wild type strain (0.19±0.07μg/ml). The breeding approach by rifampicin-resistance may be advantageous over the classical random screening since it requires much smaller number of candidates to be examined.

グラム陽性菌の二次代謝は，貧栄養条件などの緊縮応答として，その生合成遺伝子の転写段階が厳密な制御を受けている．そこで，これらのバクテリアのRNAポリメラーゼの制御部位を突然変異導入により破壊すれば，二次代謝産物の生産性向上が期待できる．本研究ではシネフンギンを生産するStreptomycesincarnatusNRRL8057プロトプラストに紫外線を照射して，RNAポリメラーゼ阻害剤であるリファンピシンに耐性を獲得した変異株を取得した．突然変異操作を三回行なった結果，異なるリファンピシン耐性を持つ突然変異株10株を得た．この中で最も高い耐性を示した突然変異株が最も高いシネフンギン生産能を示し，その生産性（0.45±0.11μg/ml）は野生株（0.19±0.07μg/ml）の約2.4倍あった．リァンピシン耐性を指標とする菌株の改良は，ランダムなスクリーニングを行なうよりも効率よく選抜を行うことができるという点で有効であるといえる