56. Pichia pastorisによるブラシノステロイドを中心とした植物ホルモン生合成P450酵素類の機能解析(口頭発表)

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Brassinosteroids (BRs) are a plant hormone that is essential for normal plant growth and development. We suppose a new BRs pathway involving lactonization reaction at an earlier step. It has been shown that mAmbidopsis thahana, CYP85A2 converts castasterone to brassinolide. In this study, we demonstrated that CYP85A2 also catalyzes the lactonization of teasterone (TE) and typhasterol (TY) to 7-oxateasterone (7-OXTE) and 7-oxatyphasterol (7-OXTY), respectively. To reveal the new biosynthesis pathway, we used Pichia pastoris, a methylotrophic yeast for analysis of the functions of the P450s. Plasmids were constructed using pPICZ vector (Zeocin resistance) for CYP85A2 and pPIC6 vector (Blasticidin resistance) for ATR1. Both constructs were introduced, first ATR1, then CYP85A2, into P. pastoris by electroporation. A suitable transformant that showed tolerant to both zeocin and blasticidin was isolated and insertion of CYP85A2 andATRl genes in the genome was confirmed by colony PCR. The transformant was incubated with substrates under induction culture condition for 4 days. The products were extracted from the supernatant and analyzed by GC-MS, resulting that CYP85A2 converts TE and TY to 7-OXTE and 7-OXTY, respectively. A gibberellin biosynthesis enzyme, enMcaurene oxidase and ent-kcaurenoic acid oxidase, was also analyzed by P. pastoris.
植物化学調節学会の論文
2007-10-05