Institute of Molecular Biosciences, Faculty of Biological Sciences, Goethe University Frankfurt, Max-von-Laue-Straße 9, 60438 Frankfurt am Main, Germany.
ACS Synth Biol. 2020 Nov 20;9(11):2909-2916. doi: 10.1021/acssynbio.0c00241. Epub 2020 Oct 19.
Compartmentalization in membrane-surrounded organelles has the potential to overcome obstacles associated with the engineering of metabolic pathways, such as unwanted side reactions, accumulation of toxic intermediates, drain of intermediates out of the cell, and long diffusion distances. Strategies utilizing natural organelles suffer from the presence of endogenous pathways. In our approach, we make use of endoplasmic reticulum-derived vesicles loaded with enzymes of a metabolic pathway ("metabolic vesicles"). They are generated by fusion of synthetic peptides containing the N-terminal proline-rich and self-assembling region of the maize storage protein gamma-Zein ("Zera") to the pathway enzymes. We have applied a strategy to integrate three enzymes of a ,-muconic acid production pathway into those vesicles in yeast. Using fluorescence microscopy and cell fractionation techniques, we have proven the formation of metabolic vesicles and the incorporation of enzymes. Activities of the enzymes and functionality of the compartmentalized pathway were demonstrated in fermentation experiments.
膜包围的细胞器的区室化有可能克服与代谢途径工程相关的障碍，如不需要的副反应、有毒中间产物的积累、中间产物从细胞中排出以及扩散距离长。利用天然细胞器的策略存在内源性途径。在我们的方法中，我们利用内质网衍生的含有代谢途径的酶的小泡（“代谢小泡”）。它们是通过融合含有玉米贮藏蛋白γ-Zein 的 N 端脯氨酸丰富区和自组装区的合成肽与途径酶来生成的。我们已经应用了一种将 - 粘康酸生产途径的三种酶整合到酵母中这些小泡的策略。通过荧光显微镜和细胞分级分离技术，我们已经证明了代谢小泡的形成和酶的掺入。在发酵实验中证明了酶的活性和分隔途径的功能。
