Orikasa Y, Yamada A, Yu R, Ito Y, Nishida T, Yumoto I, Watanabe K, Okuyama H
Graduate School of Environmental Earth Science, Hokkaido University, Sapporo 060-0810, Japan.
Cell Mol Biol (Noisy-le-grand). 2004 Jul;50(5):625-30.
The 38 kb eicosapentaenoic acid (EPA) biosynthesis gene cluster of Shewanella sp. strain SCRC-2738 was cloned into the cosmid vector (pEPA). A 27 kb nucleotide sequence of the XhoI to SpeI region of pEPA showed EPA production (6.3%) in E. coli JM109. Among the nine open reading frames (ORFs) in this sequence, only five (ORFs 2 and 5-8) were essential for EPA production. High levels of production (16%-22%) were found in E. coli JM109 transformed with a multicopy pNEB vector carrying only the five essential ORFs and in that transformed with a pNEB vector that integrated ORFs 3, 5, 6, 7 and 8, and vector pSTV28 that integrated the ORF2 encoding phosphopantetheinyl transferase (PPTase). Thus, production of EPA appears to be regulated by the presence of all the biosynthesis gene products and by the ratio of PPTase to the other gene products. The temperature -EPA production relationship in E. coli strain DH5alpha varied between constructs, suggesting that it is controlled not only by EPA biosynthesis enzymes but also by other factors in vivo. There was a strict upper temperature limit for EPA biosynthesis: no EPA was synthesized at 30 degrees C in E. coli transformants carrying any gene construct for EPA biosynthesis.
将希瓦氏菌属菌株SCRC - 2738的38 kb二十碳五烯酸（EPA）生物合成基因簇克隆到黏粒载体（pEPA）中。pEPA的XhoI至SpeI区域的27 kb核苷酸序列在大肠杆菌JM109中显示出EPA产量（6.3%）。在该序列的9个开放阅读框（ORF）中，只有5个（ORF 2和5 - 8）对EPA的产生至关重要。在用仅携带5个必需ORF的多拷贝pNEB载体转化的大肠杆菌JM109中，以及在用整合了ORF 3、5、6、7和8的pNEB载体以及整合了编码磷酸泛酰巯基乙胺基转移酶（PPTase）的ORF2的载体pSTV28转化的大肠杆菌JM109中，发现了高水平的产量（16% - 22%）。因此，EPA的产生似乎受所有生物合成基因产物的存在以及PPTase与其他基因产物的比例调控。大肠杆菌DH5α菌株中温度与EPA产量的关系在不同构建体之间有所不同，这表明它不仅受EPA生物合成酶的控制，还受体内其他因素的控制。EPA生物合成存在严格的温度上限：在携带任何EPA生物合成基因构建体的大肠杆菌转化体中，30℃时不合成EPA。
