| Project/Area Number |
23810026
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| Research Category |
Grant-in-Aid for Research Activity Start-up
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| Allocation Type | Single-year Grants |
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| Research Field |
Chemical biology
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| Research Institution | University of Shizuoka |
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Principal Investigator |
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| Project Period (FY) |
2011 – 2012
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| Project Status |
Completed (Fiscal Year 2012)
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| Budget Amount *help |
¥3,250,000 (Direct Cost: ¥2,500,000、Indirect Cost: ¥750,000)
Fiscal Year 2012: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
Fiscal Year 2011: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
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| Keywords | シンセティックバイオロジー / 生合成 / 天然物化学 / コチバクチン / 病原微生物 / 創薬 / コリバクチン / ポリケタイド / ペプチド / 大腸ガン |
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| Research Abstract |
The target of our heterologous production system is colibactin. Colibactin is a hybrid molecule of a polyketide-nonribosomal peptideisolated from Escherichia coliIHE3034, that causes DNA double-strand breaks and activation of the DNA damage checkpoint pathway, leading to cell cycle arrest and eventually to cell death. The colibactingene cluster was sequenced. The colibactingene cluster spanned 55-kilobases-long and eighteen open-reading frames were ecoded in the cluster.The biosynthetic gene cluster wastranscribed by seven promoters. Assembly of biosynthesis genes: For the production of colibactinin E. coli, each of the seven operons wasindividually expressed in E. colito confirm expression. The seven operonswere assembled into three separate plasmids with each operoncarrying its own T7 promoter, ribosomal binding site and T7 transcriptional terminator. The multimonocistronic arrangement was an important improvement from previous systems for the heterologous production of 6-dEB, yersiniabactin and rifamycin precursor in E. coli. The multimonocistronic arrangement simplify the assembly process and also minimize the potential premature terminations and mRNA degradation in transcribing excessively long polycistronic gene. In addition as an improvement of previous systems we used orthogonal origins of replication and antibiotic resistance genes to ensure the stable retention of all three plasmids in E. coli.
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