Institute of Life Sciences, Scuola Superiore Sant'Anna, Pisa 56127, Italy.
Chemistry Department, Università di Pisa, Pisa 56124, Italy.
Plant Physiol. 2019 Mar;179(3):986-1000. doi: 10.1104/pp.18.01003. Epub 2018 Nov 20.
Due to the involvement of oxygen in many essential metabolic reactions, all living organisms have developed molecular systems that allow adaptive physiological and metabolic transitions depending on oxygen availability. In mammals, the expression of hypoxia-response genes is controlled by the heterodimeric Hypoxia-Inducible Factor. The activity of this transcriptional regulator is linked mainly to the oxygen-dependent hydroxylation of conserved proline residues in its α-subunit, carried out by prolyl-hydroxylases, and subsequent ubiquitination via the E3 ligase von Hippel-Lindau tumor suppressor, which targets Hypoxia-Inducible Factor-α to the proteasome. By exploiting bioengineered versions of this mammalian oxygen sensor, we designed and optimized a synthetic device that drives gene expression in an oxygen-dependent fashion in plants. Transient assays in Arabidopsis () mesophyll protoplasts indicated that a combination of the yeast Gal4/upstream activating sequence system and the mammalian oxygen sensor machinery can be used effectively to engineer a modular, oxygen-inducible transcriptional regulator. This synthetic device also was shown to be selectively controlled by oxygen in whole plants when its components were expressed stably in Arabidopsis seedlings. We envision the exploitation of our genetically encoded controllers to generate plants able to switch gene expression selectively depending on oxygen availability, thereby providing a proof of concept for the potential of synthetic biology to assist agricultural practices in environments with variable oxygen provision.
由于氧气参与了许多重要的代谢反应，所有的生物体都发展出了分子系统，使它们能够根据氧气的可用性进行适应性的生理和代谢转变。在哺乳动物中，缺氧反应基因的表达受缺氧诱导因子的异二聚体控制。这种转录调节剂的活性主要与它的α亚基中保守脯氨酸残基的氧依赖性羟化有关，由脯氨酰羟化酶完成，随后通过 E3 连接酶 von Hippel-Lindau 肿瘤抑制因子进行泛素化，将缺氧诱导因子-α靶向蛋白酶体。通过利用这种哺乳动物氧传感器的生物工程版本，我们设计并优化了一种合成装置，使其能够在植物中以氧气依赖的方式驱动基因表达。在拟南芥（）叶肉原生质体中的瞬时测定表明，酵母 Gal4/上游激活序列系统和哺乳动物氧传感器机制的组合可以有效地用于构建模块化的、氧诱导的转录调节剂。当该合成装置的组件在拟南芥幼苗中稳定表达时，它也被证明可以在整个植物中被氧气选择性地控制。我们设想利用我们的遗传编码控制器来生成能够根据氧气可用性选择性地切换基因表达的植物，从而为合成生物学在氧气供应变化的环境中辅助农业实践的潜力提供了一个概念验证。
