AIST Tsukuba Central 6, National Institute of Advanced Industrial Science and Technology, Higashi 1-1-1, Tsukuba, Ibaraki, 305-8566, Japan.
Appl Microbiol Biotechnol. 2013 Sep;97(18):8193-203. doi: 10.1007/s00253-013-4987-6. Epub 2013 May 23.
In an effort to produce ricinoleic acid (RA), an important natural raw material with great values as a petrochemical replacement, in Schizosaccharomyces pombe, we introduced Claviceps purpurea oleate Δ12-hydroxylase gene (CpFAH12) to S. pombe, putting it under the control of an inducible nmt1 promoter. However, RA was toxic to S. pombe and the cells expressing CpFAH12 grew poorly at the normal growth temperature 30 °C. To address its toxic mechanism in S. pombe, we screened for a S. pombe cDNA library and identified plg7, which encodes a phospholipase A2, as a suppressor that restored the growth defect without affecting the RA production. A lacZ fusion experiment showed that the expression of plg7 was inducible by RA. Thin layer chromatographic analysis confirmed a reduction in RA moiety in phospholipids and a concomitant increase in free RA in the plg7 overexpressed strain. Since RA is synthesized at the sn-2 position of phosphatidylcholine by Fah12p, and phospholipase A2 hydrolyzes the sn-2 acyl bond of phospholipids, we speculate that plg7 is a stress-responsive gene, and removal of RA moieties from phospholipids, major components of lipid bilayer membrane, by Plg7p would be its suppression mechanism.
为了在裂殖酵母中生产蓖麻酸(RA),这是一种作为石化替代品具有巨大价值的重要天然原料,我们将角堇菜油醇 Δ12-羟化酶基因(CpFAH12)引入裂殖酵母,使其受到诱导型 nmt1 启动子的控制。然而,RA 对裂殖酵母有毒性,表达 CpFAH12 的细胞在正常生长温度 30°C 下生长不良。为了解决其在裂殖酵母中的毒性机制,我们筛选了裂殖酵母 cDNA 文库,并鉴定出 plg7,它编码一种磷脂酶 A2,作为一种抑制剂,可以恢复生长缺陷而不影响 RA 的产生。β-半乳糖苷酶融合实验表明,plg7 的表达可被 RA 诱导。薄层层析分析证实,在过表达 plg7 的菌株中,磷脂中的 RA 部分减少,而游离 RA 增加。由于 RA 是由 Fah12p 在磷脂酰胆碱的 sn-2 位合成的,而磷脂酶 A2 水解磷脂的 sn-2 酰键,我们推测 plg7 是一种应激响应基因,Plg7p 将 RA 部分从磷脂中去除,而磷脂是脂质双层膜的主要成分,这可能是其抑制机制。