Tao Luan, Wilczek Jolanta, Odom J Martin, Cheng Qiong
Biological and Chemical Sciences and Engineering, Central Research and Development, E. I. DuPont de Nemours Inc., Wilmington, DE 19880-0328, USA.
Metab Eng. 2006 Nov;8(6):523-31. doi: 10.1016/j.ymben.2006.06.001. Epub 2006 Jun 27.
A new beta-carotene ketolase gene (crtW) was cloned from an environmental isolate Sphingomonas sp. DC18. A robust and reliable color screen was developed for protein engineering to improve its activity on hydroxylated carotenoids for astaxanthin production. Localized random mutagenesis was performed on the crtW gene including the upstream ribosomal binding site (RBS). Six mutations (H96L, R203W, A205V, A208V, F213L and A215T) in the crtW gene were isolated multiple times that showed improved astaxanthin production. These mutations were localized near the conserved histidine motifs, which were proposed for binding iron required for enzymatic activity. Combination of two of the mutations (R203W/F213L) further improved astaxanthin production. One mutation at the RBS (a438t) was shown to have additional effect on improving astaxanthin production. Most of the mutants still retained high activity on beta-carotene, however, the F213L single mutant and the R203W/F213L double mutant that yielded the highest improvement for astaxanthin production showed decreased activity for canthaxanthin production.
从环境分离株鞘氨醇单胞菌属DC18中克隆出一个新的β-胡萝卜素酮醇酶基因（crtW）。开发了一种强大且可靠的颜色筛选方法用于蛋白质工程，以提高其对羟基化类胡萝卜素的活性，用于虾青素生产。对包括上游核糖体结合位点（RBS）在内的crtW基因进行了定位随机诱变。crtW基因中的六个突变（H96L、R203W、A205V、A208V、F213L和A215T）被多次分离出来，这些突变显示出虾青素产量有所提高。这些突变位于保守组氨酸基序附近，推测该基序用于结合酶活性所需的铁。两个突变（R203W/F213L）的组合进一步提高了虾青素产量。RBS处的一个突变（a438t）显示出对提高虾青素产量有额外作用。大多数突变体对β-胡萝卜素仍保持高活性，然而，虾青素产量提高最大的F213L单突变体和R203W/F213L双突变体对角黄素生产的活性降低。
