Expression and membrane-targeting of an active plant cytochrome P450 in the chloroplast of the green alga Chlamydomonas reinhardtii
Expression and membrane-targeting of an active plant cytochrome P450 in the chloroplast of the green alga Chlamydomonas reinhardtii
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DOI：
10.1016/j.phytochem.2014.12.006
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发表时间：
2015-02-01
期刊：
影响因子：
3.8
通讯作者：
Robinson, Colin
中科院分区：
文献类型：
--
作者：
Gangl, Doris;Zedler, Julie A. Z.;Robinson, Colin
单细胞绿色衣藻莱茵衣藻具有作为生产重组蛋白和其他化合物的细胞工厂的潜力，但由于缺乏遗传工具和需要鉴定可以以具有成本效益的方式产生的产品，主流采用受到阻碍。一种有前途的策略是使用藻类叶绿体作为合成高价值生物活性化合物如二萜类化合物的位点，因为这些化合物来源于细胞器内天然存在的代谢构件。然而，这些复杂的植物代谢产物的合成需要引入膜相关酶，包括细胞色素P450酶（P450）。在这里，我们表明，编码模型P450的基因（CYP79A1）可以引入到C reinhardtii叶绿体基因组中使用一个简单的转化系统。该基因稳定表达，P450通过其内源性N-末端锚结构域有效地靶向叶绿体膜，在那里它是有活性的，占总细胞蛋白的0.4%。这些结果为将二萜类化合物的合成途径引入到叶绿体中提供了理论依据。莱因哈德氏菌(C)2014爱思唯尔有限公司版权所有。
The unicellular green alga Chlamydomonas reinhardtii has potential as a cell factory for the production of recombinant proteins and other compounds, but mainstream adoption has been hindered by a scarcity of genetic tools and a need to identify products that can be generated in a cost-effective manner. A promising strategy is to use algal chloroplasts as a site for synthesis of high value bioactive compounds such as diterpenoids since these are derived from metabolic building blocks that occur naturally within the organelle. However, synthesis of these complex plant metabolites requires the introduction of membrane-associated enzymes including cytochrome P450 enzymes (P450s). Here, we show that a gene (CYP79A1) encoding a model P450 can be introduced into the C reinhardtii chloroplast genome using a simple transformation system. The gene is stably expressed and the P450 is efficiently targeted into chloroplast membranes by means of its endogenous N-terminal anchor domain, where it is active and accounts for 0.4% of total cell protein. These results provide proof of concept for the introduction of diterpenoid synthesis pathways into the chloroplast of C. reinhardtii. (C) 2014 Elsevier Ltd. All rights reserved.