Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, MN, USA.
Appl Microbiol Biotechnol. 2018 Sep;102(17):7377-7388. doi: 10.1007/s00253-018-9135-x. Epub 2018 Jun 21.
Mesaconate, a branched unsaturated dicarboxylic acid, has drawn great interest because of its versatile applications. In this work, we optimized the fermentation efficiency of Escherichia coli to produce mesaconate from glucose. We first drove the carbon flux to 2-ketoglutarate by overexpressing genes involved in TCA precursor pathway and anaplerotic pathways. Then, to increase the pool of phosphoenolpyruvate (PEP), an upstream precursor for 2-ketoglutarate, the phosphotransferase system (PTS) of E. coli was inactivated by deleting glucose PTS permease and the import of glucose was altered by overexpressing galactose/H symporter GalP. Further, production optimization was achieved by deleting a class I fumarase (FumA) to block the hydration of mesaconate. Finally, we overexpressed PEP synthase (PpsA) to increase the availability of phosphoenolpyruvate and accelerate the production of mesaconate. These genetic modifications led to mesaconate production with a titer of 23.1 g L and a yield of 0.46 g g glucose (64% of the theoretical maximum). This work demonstrates the possibility of engineering a highly efficient bacteria strain that converts glucose into mesaconate with promising titer, rate, and yield.
戊二烯酸，一种支链不饱和二羧酸，因其广泛的应用而受到极大关注。在这项工作中，我们通过过表达三羧酸循环前体途径和补料途径中的基因来优化大肠杆菌生产戊二烯酸的发酵效率。然后，为了增加 2-酮戊二酸的前体磷酸烯醇丙酮酸（PEP）的池，通过敲除葡萄糖磷酸转移酶系统（PTS）中的葡萄糖 PTS 通透酶和过表达半乳糖/H 同向转运蛋白 GalP 来改变葡萄糖的摄取，从而抑制了大肠杆菌的 PTS。此外，通过敲除一种 class I 富马酸酶（FumA）来阻断戊二烯酸的水合作用，进一步实现了生产优化。最后，我们过表达 PEP 合成酶（PpsA）来增加磷酸烯醇丙酮酸的可用性并加速戊二烯酸的生产。这些遗传修饰使戊二烯酸的产量达到 23.1 g/L，葡萄糖得率为 0.46 g/g（理论最大值的 64%）。这项工作证明了工程改造高效细菌菌株将葡萄糖转化为戊二烯酸的可能性，具有很高的浓度、速率和产率。
