Mijts Benjamin N, Lee Pyung Cheon, Schmidt-Dannert Claudia
Department of Biochemistry, University of Minnesota, St. Paul, Minnesota 55108, USA.
Chem Biol. 2005 Apr;12(4):453-60. doi: 10.1016/j.chembiol.2005.02.010.
A carotenoid desaturase homolog from Staphylococcus aureus (CrtOx) was identified. When expressed in engineered E. coli cells synthesizing linear C(30) carotenoids, polar carotenoid products were generated, identified as aldehyde and carboxylic acid C(30) carotenoid derivatives. The major product in this engineered pathway is the fully desaturated C(30) dialdehyde carotenoid 4,4'-diapolycopen-4,4'-dial. Very low carotenoid yields were observed when CrtOx was complemented with the C(40) carotenoid lycopene pathway. But extension of an in vitro evolved pathway of the fully desaturated 2,4,2',4'-tetradehydrolycopene produced the structurally novel fully desaturated C(40) dialdehyde carotenoid 2,4,2',4'-tetradehydrolycopendial. Directed evolution of CrtOx by error-prone PCR resulted in a number of variants with higher activity on C(40) carotenoid substrates and improved product profiles. These findings may provide new biosynthetic routes to highly polar carotenoids with unique spectral properties desirable for a number of industrial and pharmaceutical applications.
鉴定出一种来自金黄色葡萄球菌的类胡萝卜素去饱和酶同源物（CrtOx）。当在合成线性C(30)类胡萝卜素的工程化大肠杆菌细胞中表达时，会产生极性类胡萝卜素产物，鉴定为醛和羧酸C(30)类胡萝卜素衍生物。该工程途径中的主要产物是完全去饱和的C(30)二醛类胡萝卜素4,4'-二脱辅基多聚番茄红素-4,4'-二醛。当CrtOx与C(40)类胡萝卜素番茄红素途径互补时，观察到类胡萝卜素产量非常低。但是，通过体外进化的完全去饱和的2,4,2',4'-四脱氢番茄红素途径的延伸产生了结构新颖的完全去饱和的C(40)二醛类胡萝卜素2,4,2',4'-四脱氢番茄红素二醛。通过易错PCR对CrtOx进行定向进化，产生了许多对C(40)类胡萝卜素底物具有更高活性和改善产物谱的变体。这些发现可能为具有独特光谱特性的高极性类胡萝卜素提供新的生物合成途径，这些特性对于许多工业和制药应用来说是理想的。
