State Key Laboratory of Protein and Plant Gene Research, School of Advanced Agricultural Sciences and School of Life Sciences, Peking University, Beijing 100871, China.
Department of Molecular Microbiology, John Innes Centre, NR4 7UH Norwich, United Kingdom.
Proc Natl Acad Sci U S A. 2023 Aug 22;120(34):e2305142120. doi: 10.1073/pnas.2305142120. Epub 2023 Aug 16.
Introducing nitrogen fixation ( ) genes into eukaryotic genomes and targeting Nif components to mitochondria or chloroplasts is a promising strategy for engineering nitrogen-fixing plants. A prerequisite for achieving nitrogen fixation in crops is stable and stoichiometric expression of each component in organelles. Previously, we designed a polyprotein-based nitrogenase system depending on Tobacco Etch Virus protease (TEVp) to release functional Nif components from five polyproteins. Although this system satisfies the demand for specific expression ratios of Nif components in , we encountered issues with TEVp cleavage of polyproteins targeted to yeast mitochondria. To overcome this obstacle, a version of the Nif polyprotein system was constructed by replacing TEVp cleavage sites with minimal peptide sequences, identified by knowledge-based engineering, that are susceptible to cleavage by the endogenous mitochondrial-processing peptidase. This replacement not only further reduces the number of genes required, but also prevents potential precleavage of polyproteins outside the target organelle. This version of the polyprotein-based nitrogenase system achieved levels of nitrogenase activity in , comparable to those observed with the TEVp-based polyprotein nitrogenase system. When applied to yeast mitochondria, stable and balanced expression of Nif components was realized. This strategy has potential advantages, not only for transferring nitrogen fixation to eukaryotic cells, but also for the engineering of other metabolic pathways that require mitochondrial compartmentalization.
将固氮（ ）基因导入真核生物基因组，并将 Nif 组件靶向线粒体或叶绿体，是工程固氮植物的一种有前途的策略。在作物中实现固氮的一个前提条件是细胞器中每个组件的稳定和化学计量表达。以前，我们设计了一种基于多蛋白的氮酶系统，依赖烟草蚀纹病毒蛋白酶（TEVp）从五个多蛋白中释放功能性 Nif 组件。虽然该系统满足了细胞器中 Nif 组件特定表达比例的需求，但我们在靶向酵母线粒体的多蛋白中遇到了 TEVp 切割多蛋白的问题。为了克服这一障碍，通过用基于知识的工程鉴定的、对内在线粒体加工肽酶敏感的最小肽序列替换 TEVp 切割位点，构建了 Nif 多蛋白系统的一个版本。这种替换不仅进一步减少了所需基因的数量，而且还防止了多蛋白在靶细胞器外的潜在预切割。这种基于多蛋白的氮酶系统在酵母线粒体中实现了与基于 TEVp 的多蛋白氮酶系统相当的氮酶活性水平。当应用于酵母线粒体时，实现了 Nif 组件的稳定和平衡表达。该策略不仅具有将固氮作用转移到真核细胞的潜在优势，而且还具有工程化需要线粒体区室化的其他代谢途径的潜在优势。
