Biosynthetic Pathway for γ-Cyclic Sarcinaxanthin in Micrococcus luteus: Heterologous Expression and Evidence for Diverse and Multiple Catalytic Functions of C50 Carotenoid Cyclases
Biosynthetic Pathway for γ-Cyclic Sarcinaxanthin in Micrococcus luteus: Heterologous Expression and Evidence for Diverse and Multiple Catalytic Functions of C50 Carotenoid Cyclases
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DOI：
10.1128/jb.00724-10
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发表时间：
2010-11-01
影响因子：
3.2
通讯作者：
Brautaset, Trygve
中科院分区：
文献类型：
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作者：
Netzer, Roman;Stafsnes, Marit H.;Brautaset, Trygve
我们报告了藤黄微球菌 NCTC2665 中γ-环 C-50 类胡萝卜素八叠球菌黄素生物合成基因簇（crtE、crtB、crtI、crtE2、crtYg、crtYh 和 crtX）的克隆和表征。完整和部分基因簇在大肠杆菌宿主中的表达表明，前体分子法尼基焦磷酸 (FPP) 的八叠球菌黄素生物合成是通过 C-40 番茄红素、C-45 九黄黄素、C-50 黄黄素和 C-50 八叠球菌黄素进行的。八叠球菌黄素的糖基化是通过 crtX 基因产物完成的。这是第一份描述 γ-环 C-50 类胡萝卜素生物合成途径的报告。来自海洋藤黄杆菌分离株 Otnes7 的相应基因在产生番茄红素的大肠杆菌宿主中表达，导致摇瓶中产生高达 2.5 mg/g 细胞干重的八叠球菌黄素。为了通过实验了解八叠球菌黄素和结构相关的ε-环十癸烯黄素生物合成途径之间的具体差异，我们构建了一个杂合基因簇，其中来自M. uteus的γ-环C-50类胡萝卜素环化酶基因crtYg和crtYh被替换为来自天然植物的类似ε-环C-50类胡萝卜素环化酶基因crtYe和crtYf。十癸烯黄素生产者谷氨酸棒杆菌。令人惊讶的是，该杂合基因簇在大肠杆菌宿主中的表达不仅导致十癸烯黄素的积累，而且还导致八叠球菌黄素和不对称ε-和γ-环C-50类胡萝卜素八叠球菌黄素的积累，这是本研究中首次描述的。总之，这些数据有助于人们对细菌 C-50 类胡萝卜素环化酶作为合成结构不同类胡萝卜素的关键催化剂的多样性和多功能性有了新的认识。
We report the cloning and characterization of the biosynthetic gene cluster (crtE, crtB, crtI, crtE2, crtYg, crtYh, and crtX) of the gamma-cyclic C-50 carotenoid sarcinaxanthin in Micrococcus luteus NCTC2665. Expression of the complete and partial gene cluster in Escherichia coli hosts revealed that sarcinaxanthin biosynthesis from the precursor molecule farnesyl pyrophosphate (FPP) proceeds via C-40 lycopene, C-45 nonaflavuxanthin, C-50 flavuxanthin, and C-50 sarcinaxanthin. Glucosylation of sarcinaxanthin was accomplished by the crtX gene product. This is the first report describing the biosynthetic pathway of a gamma-cyclic C-50 carotenoid. Expression of the corresponding genes from the marine M. luteus isolate Otnes7 in a lycopene-producing E. coli host resulted in the production of up to 2.5 mg/g cell dry weight sarcinaxanthin in shake flasks. In an attempt to experimentally understand the specific difference between the biosynthetic pathways of sarcinaxanthin and the structurally related epsilon-cyclic decaprenoxanthin, we constructed a hybrid gene cluster with the gamma-cyclic C-50 carotenoid cyclase genes crtYg and crtYh from M. luteus replaced with the analogous epsilon-cyclic C-50 carotenoid cyclase genes crtYe and crtYf from the natural decaprenoxanthin producer Corynebacterium glutamicum. Surprisingly, expression of this hybrid gene cluster in an E. coli host resulted in accumulation of not only decaprenoxanthin, but also sarcinaxanthin and the asymmetric epsilon- and gamma-cyclic C-50 carotenoid sarprenoxanthin, described for the first time in this work. Together, these data contributed to new insight into the diverse and multiple functions of bacterial C-50 carotenoid cyclases as key catalysts for the synthesis of structurally different carotenoids.