Seo Min-Ju, Shin Kyung-Chul, Oh Deok-Kun
Department of Bioscience and Biotechnology, Konkuk University, Seoul, 143-701, South Korea.
Appl Microbiol Biotechnol. 2014 Sep;98(17):7447-56. doi: 10.1007/s00253-014-5709-4. Epub 2014 Apr 3.
Diol synthase from Aspergillus nidulans was cloned and expressed in Escherichia coli. Recombinant E. coli cells expressing diol synthase from A. nidulans converted linoleic acid to a product that was identified as 5,8-dihydroxy-9,12(Z,Z)-octadecadienoic acid by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). The recombinant cells and the purified enzyme showed the highest activity for linoleic acid among the fatty acids tested. The optimal reaction conditions for the production of 5,8-dihydroxy-9,12(Z,Z)-octadecadienoic acid from linoleic acid using whole recombinant E. coli cells expressing diol synthase were pH 7.5, 35°C, 250 rpm, 5 g l(-1) linoleic acid, 23 g l(-1) cells, and 20% (v/v) dimethyl sulfoxide in a 250-ml baffled flask. Under these optimized conditions, whole recombinant cells expressing diol synthase produced 4.98 g l(-1) 5,8-dihydroxy-9,12(Z,Z)-octadecadienoic acid for 150 min without detectable byproducts, with a conversion yield of 99% (w/w) and a productivity of 2.5 g l(-1) h(-1). This is the first report on the biotechnological production of dihydroxy fatty acid using whole recombinant cells expressing diol synthase.
构巢曲霉二醇合酶在大肠杆菌中克隆并表达。表达构巢曲霉二醇合酶的重组大肠杆菌细胞将亚油酸转化为一种产物，通过液相色谱-质谱/质谱（LC-MS/MS）鉴定为5,8-二羟基-9,12（Z,Z）-十八碳二烯酸。在测试的脂肪酸中，重组细胞和纯化的酶对亚油酸表现出最高活性。使用表达二醇合酶的重组大肠杆菌全细胞从亚油酸生产5,8-二羟基-9,12（Z,Z）-十八碳二烯酸的最佳反应条件为：在250 ml带挡板的烧瓶中，pH 7.5、35°C、250 rpm、5 g l(-1)亚油酸、23 g l(-1)细胞和20%（v/v）二甲基亚砜。在这些优化条件下，表达二醇合酶的重组全细胞在150分钟内产生4.98 g l(-1) 5,8-二羟基-9,12（Z,Z）-十八碳二烯酸，无检测到的副产物，转化率为99%（w/w），生产力为2.5 g l(-1) h(-1)。这是关于使用表达二醇合酶的重组全细胞生物技术生产二羟基脂肪酸的首次报道。
