The generation of nitrogen fixing crops is considered a challenge that could lead to a new agricultural ‘green’ revolution. Here, we report the use of synthetic biology tools to achieve and optimize the production of active nitrogenase Fe protein (NifH) in the chloroplasts of tobacco plants. Azotobacter vinelandii nitrogen fixation genes, nifH , M , U and S, were re‐designed for protein accumulation in tobacco cells. Targeting to the chloroplast was optimized by screening and identifying minimal length transit peptides performing properly for each specific Nif protein. Putative peptidyl‐prolyl cis‐trans isomerase NifM proved necessary for NifH solubility in the stroma. Purified NifU, a protein involved in the biogenesis of NifH [4Fe‐4S] cluster, was found functional in NifH reconstitution assays. Importantly, NifH purified from tobacco chloroplasts was active in the reduction of acetylene to ethylene, with the requirement of n ifU and n ifS co‐expression. These results support the suitability of chloroplasts to host functional nitrogenase proteins, paving the way for future studies in the engineering of nitrogen fixation in higher plant plastids and describing an optimization pipeline that could also be used in other organisms and in the engineering of new metabolic pathways in plastids.

中文翻译：

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利用合成生物学工具优化烟草叶片细胞叶绿体中活性固氮酶Fe蛋白的产生。

固氮作物的产生被认为是一项挑战，可能会引发新的农业“绿色”革命。在这里，我们报告了使用合成生物学工具来实现和优化烟草植物叶绿体中活性固氮酶Fe蛋白（NifH）的生产。葡萄固氮菌固氮基因nifH，M，U和S重新设计用于烟草细胞中蛋白质的积累。通过筛选和鉴定对每种特定的Nif蛋白都能正常运行的最小长度的转运肽，可以优化针对叶绿体的靶向性。事实证明，假定的肽基脯氨酰顺反异构酶NifM对于NifH在基质中的溶解度是必需的。纯化的NifU是一种与NifH [4Fe-4S]簇生物发生有关的蛋白质，在NifH重组分析中被发现具有功能。重要的是，从NifH烟草叶绿体中的乙炔还原成乙烯纯化的活性，具有的要求Ñ IFU和Ñ IFS共表达。这些结果支持叶绿体适合宿主功能性固氮酶蛋白，为将来在高等植物质体固氮工程中的研究铺平了道路，并描述了也可用于其他生物体和新代谢途径工程的优化流程。在质体中。