Cloning and Characterization of Genes Involved in Nostoxanthin Biosynthesis of <em>Sphingomonas elodea</em> ATCC 31461

Most Sphingomonas species synthesize the yellow carotenoid nostoxanthin. However, the carotenoid biosynthetic pathway of these species remains unclear. In this study, we cloned and characterized a carotenoid biosynthesis gene cluster containing four carotenogenic genes (crtG, crtY, crtI and crtB) and a β-carotene hydroxylase gene (crtZ) located outside the cluster, from the gellan-gum producing bacterium Sphingomonas elodea ATCC 31461. Each of these genes was inactivated, and the biochemical function of each gene was confirmed based on chromatographic and spectroscopic analysis of the intermediates accumulated in the knockout mutants. Moreover, the crtG gene encoding the 2,2′-β-hydroxylase and the crtZ gene encoding the β-carotene hydroxylase, both responsible for hydroxylation of β-carotene, were confirmed by complementation studies using Escherichia coli producing different carotenoids. Expression of crtG in zeaxanthin and β-carotene accumulating E. coli cells resulted in the formation of nostoxanthin and 2,2′-dihydroxy-β-carotene, respectively. Based on these results, a biochemical pathway for synthesis of nostoxanthin in S. elodea ATCC 31461 is proposed.大多数鞘氨醇单胞菌属（Sphingomonas）菌种可合成黄色类胡萝卜素诺斯特黄素（nostoxanthin），但该类群的类胡萝卜素生物合成途径仍未明确。本研究从产结冷胶（gellan-gum）的鞘氨醇单胞菌（Sphingomonas elodea）ATCC 31461中，克隆并鉴定了一个包含4个类胡萝卜素合成基因（crtG、crtY、crtI与crtB）的类胡萝卜素生物合成基因簇，以及位于该簇外的β-胡萝卜素羟化酶基因crtZ。我们对上述各基因分别进行了失活处理，并通过对敲除突变株中积累的中间产物进行色谱与光谱分析，验证了各基因的生化功能。此外，针对两种均参与β-胡萝卜素羟化反应的酶——编码2,2'-β-羟化酶的crtG基因与编码β-胡萝卜素羟化酶的crtZ基因，我们通过利用产不同类胡萝卜素的大肠杆菌（Escherichia coli）进行互补实验，验证了其功能。在分别积累玉米黄质（zeaxanthin）与β-胡萝卜素的大肠杆菌菌体中表达crtG基因，分别可生成诺斯特黄素与2,2'-二羟基-β-胡萝卜素。基于上述实验结果，我们提出了鞘氨醇单胞菌ATCC 31461中诺斯特黄素的生物合成途径。