Rajabi Fatemeh, Heene Ernst, Maisch Jan, Nick Peter
Molecular Cell Biology, Botanical Institute, Karlsruhe Institute of Technology, Germany.
PLoS One. 2017 Jan 12;12(1):e0169778. doi: 10.1371/journal.pone.0169778. eCollection 2017.
The great potential of pharmacologically active secondary plant metabolites is often limited by low yield and availability of the producing plant. Chemical synthesis of these complex compounds is often too expensive. Plant cell fermentation offers an alternative strategy to overcome these limitations. However, production in batch cell cultures remains often inefficient. One reason might be the fact that different cell types have to interact for metabolite maturation, which is poorly mimicked in suspension cell lines. Using alkaloid metabolism of tobacco, we explore an alternative strategy, where the metabolic interactions of different cell types in a plant tissue are technically mimicked based on different plant-cell based metabolic modules. In this study, we simulate the interaction found between the nicotine secreting cells of the root and the nicotine-converting cells of the senescent leaf, generating the target compound nornicotine in the model cell line tobacco BY-2. When the nicotine demethylase NtomCYP82E4 was overexpressed in tobacco BY-2 cells, nornicotine synthesis was triggered, but only to a minor extent. However, we show here that we can improve the production of nornicotine in this cell line by feeding the precursor, nicotine. Engineering of another cell line overexpressing the key enzyme NtabMPO1 allows to stimulate accumulation and secretion of this precursor. We show that the nornicotine production of NtomCYP82E4 cells can be significantly stimulated by feeding conditioned medium from NtabMPO1 overexpressors without any negative effect on the physiology of the cells. Co-cultivation of NtomCYP82E4 with NtabMPO1 stimulated nornicotine accumulation even further, demonstrating that the physical presence of cells was superior to just feeding the conditioned medium collected from the same cells. These results provide a proof of concept that combination of different metabolic modules can improve the productivity for target compounds in plant cell fermentation.
具有药理活性的次生植物代谢产物的巨大潜力常常受到其产生植物的低产量和可获得性的限制。这些复杂化合物的化学合成往往成本过高。植物细胞发酵提供了一种克服这些限制的替代策略。然而，分批细胞培养的生产效率通常仍然很低。一个原因可能是不同细胞类型必须相互作用才能使代谢产物成熟，而悬浮细胞系很难模拟这种情况。利用烟草的生物碱代谢，我们探索了一种替代策略，即基于不同的植物细胞代谢模块，从技术上模拟植物组织中不同细胞类型之间的代谢相互作用。在本研究中，我们模拟了根中分泌尼古丁的细胞与衰老叶片中转化尼古丁的细胞之间的相互作用，在模式细胞系烟草BY-2中产生目标化合物去甲烟碱。当尼古丁脱甲基酶NtomCYP82E4在烟草BY-2细胞中过表达时，会触发去甲烟碱的合成，但程度较小。然而，我们在此表明，通过添加前体尼古丁，可以提高该细胞系中去甲烟碱的产量。对另一个过表达关键酶NtabMPO1的细胞系进行工程改造，可以刺激这种前体的积累和分泌。我们表明，添加来自NtabMPO1过表达细胞的条件培养基可以显著刺激NtomCYP82E4细胞的去甲烟碱产量，且对细胞生理没有任何负面影响。将NtomCYP82E4与NtabMPO1共培养进一步刺激了去甲烟碱的积累，表明细胞的物理存在优于仅添加从相同细胞收集的条件培养基。这些结果提供了一个概念验证，即不同代谢模块的组合可以提高植物细胞发酵中目标化合物的生产率。
