Ye Rick W, Stead Kristen J, Yao Henry, He Hongxian
DuPont Experimental Station, Route 141 and Henry Clay Road, Wilmington, DE 19880, USA.
Appl Environ Microbiol. 2006 Sep;72(9):5829-37. doi: 10.1128/AEM.00918-06.
Biosynthesis of the commercial carotenoids canthaxanthin and astaxanthin requires beta-carotene ketolase. The functional importance of the conserved amino acid residues of this enzyme from Paracoccus sp. strain N81106 (formerly classified as Agrobacterium aurantiacum) was analyzed by alanine-scanning mutagenesis. Mutations in the three highly conserved histidine motifs involved in iron coordination abolished its ability to catalyze the formation of ketocarotenoids. This supports the hypothesis that the CrtW ketolase belongs to the family of iron-dependent integral membrane proteins. Most of the mutations generated at other highly conserved residues resulted in partial activity. All partially active mutants showed a higher amount of adonixanthin accumulation than did the wild type when expressed in Escherichia coli cells harboring the zeaxanthin biosynthetic gene cluster. Some of the partially active mutants also produced a significant amount of echinenone when expressed in cells producing beta-carotene. In fact, expression of a mutant carrying D117A resulted in the accumulation of echinenone as the predominant carotenoid. These observations indicate that partial inactivation of the CrtW ketolase can often lead to the production of monoketolated intermediates. In order to improve the conversion rate of astaxanthin catalyzed by the CrtW ketolase, a color screening system was developed. Three randomly generated mutants, carrying L175M, M99V, and M99I, were identified to have improved activity. These mutants are potentially useful in pathway engineering for the production of astaxanthin.
商业类胡萝卜素角黄素和虾青素的生物合成需要β-胡萝卜素酮酶。通过丙氨酸扫描诱变分析了来自副球菌属菌株N81106（以前归类为橙色土壤杆菌）的这种酶的保守氨基酸残基的功能重要性。参与铁配位的三个高度保守的组氨酸基序中的突变消除了其催化酮类胡萝卜素形成的能力。这支持了CrtW酮酶属于铁依赖性整合膜蛋白家族的假设。在其他高度保守残基处产生的大多数突变导致部分活性。当在含有玉米黄质生物合成基因簇的大肠杆菌细胞中表达时，所有部分活性突变体积累的adonixanthin量都比野生型高。一些部分活性突变体在产生β-胡萝卜素的细胞中表达时也产生了大量的海胆酮。事实上，携带D117A的突变体的表达导致海胆酮作为主要类胡萝卜素的积累。这些观察结果表明，CrtW酮酶的部分失活通常会导致单酮化中间体的产生。为了提高CrtW酮酶催化虾青素的转化率，开发了一种颜色筛选系统。鉴定出携带L175M、M99V和M99I的三个随机产生的突变体具有提高的活性。这些突变体在虾青素生产的途径工程中可能有用。
