成果報告書詳細
管理番号20120000001146
タイトル*平成23年度中間年報 「ゲノム創薬加速化支援バイオ産業基盤技術開発/有用天然化合物の安定的な生産技術開発」
公開日2012/12/19
報告書年度2011-2011
委託先名次世代天然物化学技術研究組合
プロジェクト番号P11002
部署名バイオテクノロジー・医療技術部
和文要約和文要約等以下本編抜粋:
1. 1. 研究開発の内容及び成果等
(1) 生合成遺伝子クラスターイブリの構築生合成遺伝子クラスターイブリの構築
1. ギガシークエンサを用いた放線菌ゲノム解析
放線菌ゲノムはGC含有率が高く、通常のシークエンス解析困難であることが知られている。
英文要約Title: Development of stable heterologous production method for industrially useful natural compounds. (FY2011-2012) FY2011 Annual Report
1) Construction of biosynthetic gene cluster library
 The analysis of the genome sequence of actinomycetes including Streptomyces is known to be difficult because of their high G-C content. The high technique developed by OIST (Okinawa Institute of Science Technology) using next generation sequencers, we succeeded in the simultaneous genomic sequencing of multiple strains of actinomycetes. This method enabled us to sequence 16 actinomycetes' genome sequence efficiently and inexpensively.
 We developed an annotation software specific for actinomycetes genome anasis based on the homologous sequence with known gene clusters,. Furthermore, by modifying this software, we are now able to forecast the sites of PKS genes clusters precisely.
 We cloned 23 biosynthetic gene clusters by using cosmid vector method up to 40 kbp of gene clusters and 10 biosynthetic gene clusters by using BAC vector method for longer gene clusters. Because the technique for BAC vector cloning was premature, especially in Japan, we tried to develop the method for BAC vector cloning and finally succeeded in practical BAC vector cloning methodology around 100 kbp length of biosynthetic gene clusters in actinomycetes genome. 
2) Development of stable heterologous expression technique.
 We used SUKA strains as the host of heterologous expression system, which was derived from a strain of Streptomyces avermitilis utilized for industrial production of avermectin by deleting several endogenous biosynthetic genes. By using SUKA17, we confirmed the production of 6 compounds originated from the cosmid vectors out of 10 genes tried, and 2 compounds for BAC vectors out of 4 genes tried. As for 1 gene cluster obtained by BAC vector method, we obtained new compounds instead of the expected compound.
 We compared the transformation efficacy of SUKA17 and Streptomyces albus G153 strain, which is the commonly used in the world as the host of heterologous expression, by using empty comsid vector. As expected, the transformation efficiency of G153 strain was 1/100-1/1000 of that of SUKA17 strain, thereby confirming the superiority of SUKA17 strain. We also confirmed that the transformation efficiency of BAC vector was much lower than that of cosmid vector. We also tried to use Streptomyces reveromyceticus, the producer strain of reveromycin, as a heterologous production host strain, but found that this strain was not suitable for heterologous expression on account of low transformation efficiency.
 We also carried out the synthesis of the starter substance of a biosynthetic pathway and fed it in the heterologous expression host to clarify the novel biosynthetic pathway. Using this technique, we will be able to get new compound by changing the starter substance.
 In conclusion, we developed the world’s best technology for BAC vector cloning. We also confirmed the superiority of SUKA strains as a heterologous expression host.
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