Characterization and engineering of a carotenoid biosynthesis operon from Bacillus megaterium
Characterization and engineering of a carotenoid biosynthesis operon from Bacillus megaterium
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DOI：
10.1016/j.ymben.2018.07.017
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发表时间：
2018-09-01
影响因子：
8.4
通讯作者：
Hannemann, Frank
中科院分区：
文献类型：
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作者：
Hartz, Philip;Milhim, Mohammed;Hannemann, Frank
巨大芽孢杆菌属于一类产生胡萝卜素的色素杆菌，它能确保自身免受紫外线辐射诱导和伴随的氧化损伤。菌株MS941的代谢产物分析表明，存在C30类胡萝卜素4，4‘-二茂铁和4，4’-双鞘孢子酸。基因功能分析表明，存在相应的C30类胡萝卜素生物合成途径，具有重要的药学意义。我们鉴定了一个基因簇，该基因簇包括一个法尼基二磷酸合成酶(ISPA)、一个双菲洛烯合酶(CRTM)和三个不同的双菲罗烯脱饱和酶(CrtN1-3)。有趣的是，CRTM与两个已识别的crtN基因一起被组织在一个操纵子中。通过异源表达和产物分析，确定了编码酶在非胡萝卜素产生模式生物大肠杆菌中的个体活性。我们的实验数据表明，巨大芽孢杆菌C30类胡萝卜素生物合成的第一步催化步骤与金黄色葡萄球菌相应的生物合成途径有很大的相似之处。鉴定的ISPA和CRTM分别催化法尼基二磷酸酯的生物合成和随后缩合生成4，4‘-双菲罗烯。然而，随后的脱饱和反应生成4，4‘-穿膜孢子烯，需要多个脱饱和酶的活性。设计了一种生物合成操纵子，并在大肠杆菌全细胞系统中成功表达，为C30类胡萝卜素4，4‘-鞘孢子烯的高产生产创造了细胞工厂，该化合物在治疗各种炎症性疾病方面具有潜在的应用前景。
Bacillus megaterium belongs to the group of pigmented bacilli producing carotenoids that ensure self-protection from UV radiation-induced and collateral oxidative damage. Metabolite profiling of strain MS941 revealed the presence of the C30 carotenoids 4,4'-diapophytofluene and 4,4'-diaponeurosporenic acid. A gene function analysis demonstrated the presence of a corresponding C30 carotenoid biosynthetic pathway with pharmaceutical importance. We identified a gene cluster comprising putative genes for a farnesyl diphosphate synthase (IspA), a diapophytoene synthase (CrtM) and three distinct diapophytoene desaturases (CrtN1-3). Intriguingly, crtM was organized in an operon together with two of the identified crtN genes. The individual activities of the encoded enzymes were determined by heterologous expression and product analysis in the non-carotenogenic model organism Escherichia coli. Our experimental data show that the first catalytic steps of C30 carotenoid biosynthesis in B. megaterium share significant similarity to the corresponding biosynthetic pathway of Staphylococcus aureus. The biosynthesis of farnesyl diphosphates and their subsequent condensation to form 4,4'-diapophytoene are catalyzed by the identified IspA and CrtM, respectively. The following desaturation reactions to form 4,4'-diaponeurosporene, however, require the activities of multiple diapophytoene desaturases. A biosynthetic operon was engineered and successfully expressed in an E. coli whole-cell system creating a cell factory for a high-yield production of the C30 carotenoid 4,4'-diaponeurosporene which has promising potential in the treatment of various inflammatory diseases.