Characterization of bacterial β-carotene 3,3′-hydroxylases, CrtZ, and P450 in astaxanthin biosynthetic pathway and adonirubin production by gene combination in Escherichia coli
Characterization of bacterial β-carotene 3,3′-hydroxylases, CrtZ, and P450 in astaxanthin biosynthetic pathway and adonirubin production by gene combination in Escherichia coli
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DOI：
10.1007/s00253-006-0426-2
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发表时间：
2006-10-01
影响因子：
5
通讯作者：
Misawa, Norihiko
中科院分区：
文献类型：
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作者：
Choi, Seon-Kang;Matsuda, Satoru;Misawa, Norihiko
β-胡萝卜素羟化酶（β-Carotene hydroxylase，CrtZ）是虾青素合成的限速酶之一。使用大肠杆菌转化体进行互补分析以比较来自Brevundimonas sp.SD212、Paracoccus sp.PC1（以前称为产碱菌属PC-1）、副球菌属N81106（Agrobacterium aurantiacum）、Pantoea ananatis（Erwinia uredovora 20 D3）、海洋细菌P99-3和来自嗜热栖热菌HB 27的P450单加氧酶（CYP 175 A1）。crtZ和CYP 175 A1基因在大肠杆菌中均有表达。由于质粒pAC-Cantha和pACCAR 16 Δ crtX的分别存在，大肠杆菌转化体合成了黄质和β-胡萝卜素。重组E.通过色谱和光谱分析检查大肠杆菌细胞。E.在检测的转化子中，携带Brevundimonas sp.SD212 crtZ的大肠杆菌显示出最高的虾青素生产效率，而在从β-胡萝卜素转化为玉米黄素的催化效率上没有显著差异。重组大肠表达CYP 175 A1基因的大肠杆菌，除了合成黄质的基因外，令人惊讶地积累了作为主要产物的侧金盏花红色素（phoenicoxanthin），尽管其它重组E.大肠杆菌中没有积累任何阿多胆红素。本研究结果表明，Brevundimonas sp.SD212 crtZ和T.嗜热菌HB 27的CYP 175 A1基因可分别用于在异源宿主中高效生产虾青素和adonirubin。
beta-Carotene hydroxylase (CrtZ) is one of rate-limiting enzymes for astaxanthin production. A complementation analysis was conducted using Escherichia coli transformants to compare the catalytic efficiency of bacterial CrtZ from Brevundimonas sp. SD212, Paracoccus sp. PC1 (formerly known as Alcaligenes sp. PC-1), Paracoccus sp. N81106 (Agrobacterium aurantiacum), Pantoea ananatis (Erwinia uredovora 20D3), marine bacterium P99-3, and P450 monooxygenase (CYP175A1) from Thermus thermophilus HB27. Each crtZ or CYP175A1 gene was expressed in E. coli transformants synthesizing canthaxanthin and beta-carotene due to the respective presence of plasmids pAC-Cantha and pACCAR16 Delta crtX. The carotenoids that accumulated in the resulting recombinant E. coli cells were examined by chromatographic and spectroscopic analyses. E. coli carrying Brevundimonas sp. SD212 crtZ showed the highest astaxanthin production efficiency among the transformants examined, while there was no significant difference in the catalytic efficiency for conversion from beta-carotene to zeaxanthin. Recombinant E. coli expressing the CYP175A1 gene, in addition to the genes for canthaxanthin synthesis, surprisingly accumulated adonirubin (phoenicoxanthin) as the main product, although the other recombinant E. coli did not accumulate any adonirubin. The present results suggest that the Brevundimonas sp. SD212 crtZ and T. thermophilus HB27 CYP175A1 genes could, respectively, be used for the efficient production of astaxanthin and adonirubin in heterologous hosts.