Di Sansebastiano Gian Pietro, Rizzello Francesca, Durante Miriana, Caretto Sofia, Nisi Rossella, De Paolis Angelo, Faraco Marianna, Montefusco Anna, Piro Gabriella, Mita Giovanni
DiSTeBA (Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali), University of Salento, Campus ECOTEKNE, 73100 Lecce, Italy.
CNR - ISPA (Istituto di Scienze delle Produzioni Alimentari), Via Monteroni, 73100 Lecce, Italy.
J Biotechnol. 2015 May 20;202:146-52. doi: 10.1016/j.jbiotec.2014.11.016. Epub 2014 Nov 29.
Plants are ideal bioreactors for the production of macromolecules but transport mechanisms are not fully understood and cannot be easily manipulated. Several attempts to overproduce recombinant proteins or secondary metabolites failed. Because of an independent regulation of the storage compartment, the product may be rapidly degraded or cause self-intoxication. The case of the anti-malarial compound artemisinin produced by Artemisia annua plants is emblematic. The accumulation of artemisinin naturally occurs in the apoplast of glandular trichomes probably involving autophagy and unconventional secretion thus its production by undifferentiated tissues such as cell suspension cultures can be challenging. Here we characterize the subcellular compartmentalization of several known fluorescent markers in protoplasts derived from Artemisia suspension cultures and explore the possibility to modify compartmentalization using a modified SNARE protein as molecular tool to be used in future biotechnological applications. We focused on the observation of the vacuolar organization in vivo and the truncated form of AtSYP51, 51H3, was used to induce a compartment generated by the contribution of membrane from endocytosis and from endoplasmic reticulum to vacuole trafficking. The artificial compartment crossing exocytosis and endocytosis may trap artemisinin stabilizing it until extraction; indeed, it is able to increase total enzymatic activity of a vacuolar marker (RGUSChi), probably increasing its stability. Exploring the 51H3-induced compartment we gained new insights on the function of the SNARE SYP51, recently shown to be an interfering-SNARE, and new hints to engineer eukaryote endomembranes for future biotechnological applications.
植物是生产大分子的理想生物反应器，但运输机制尚未完全了解且不易操控。多次尝试过量生产重组蛋白或次生代谢产物均告失败。由于储存区室的独立调节，产物可能会迅速降解或导致自我中毒。黄花蒿植物产生的抗疟化合物青蒿素就是一个典型例子。青蒿素的积累自然发生在腺毛的质外体中，可能涉及自噬和非常规分泌，因此通过未分化组织如细胞悬浮培养物来生产青蒿素可能具有挑战性。在此，我们表征了黄花蒿悬浮培养物原生质体中几种已知荧光标记物的亚细胞区室化，并探索使用修饰的SNARE蛋白作为分子工具来改变区室化的可能性，以便用于未来的生物技术应用。我们着重观察了体内液泡的组织情况，并使用AtSYP51的截短形式51H3来诱导一个由内吞作用的膜以及从内质网到液泡运输的膜共同作用产生的区室。这个跨越胞吐作用和内吞作用的人工区室可能会捕获青蒿素并使其稳定直至提取；实际上，它能够提高液泡标记物（RGUSChi）的总酶活性，可能是提高了其稳定性。通过探索51H3诱导的区室，我们对SNARE蛋白SYP51的功能有了新的认识，最近研究表明它是一种干扰性SNARE，同时也为未来生物技术应用中改造真核生物内膜提供了新的线索。
