Institute of Molecular Biosciences, Goethe University Frankfurt, 60438 Frankfurt am Main, Germany.
Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstraße 3, 06466 Gatersleben, Germany.
FEMS Yeast Res. 2018 Mar 1;18(2). doi: 10.1093/femsyr/foy017.
A wide range of commercially relevant aromatic chemicals can be synthesized via the shikimic acid pathway. Thus, this pathway has been the target of diverse metabolic engineering strategies. In the present work, an optimized yeast strain for production of the shikimic acid pathway intermediate 3-dehydroshikimate (3-DHS) was generated, which is a precursor for the production of the valuable compounds cis, cis-muconic acid (CCM) and gallic acid (GA). Production of CCM requires the overexpression of the heterologous enzymes 3-DHS dehydratase AroZ, protocatechuic acid (PCA) decarboxylase AroY and catechol dioxygenase CatA. The activity of AroY limits the yield of the pathway. This repertoire of enzymes was expanded by a novel fungal decarboxylase. Introducing this enzyme into the pathway in the optimized strain, a titer of 1244 mg L-1 CCM could be achieved, yielding 31 mg g-1 glucose. This represents the highest yield of this compound reported in Saccharomyces cerevisiae to date. To demonstrate the applicability of the optimized strain for production of other compounds from 3-DHS, we overexpressed AroZ together with a mutant of a para-hydroxybenzoic acid hydroxylase with improved substrate specificity for PCA, PobAY385F. Thereby, we could demonstrate the production of GA for the first time in S. cerevisiae.
通过莽草酸途径可以合成广泛的具有商业应用价值的芳香族化学品。因此，该途径一直是多种代谢工程策略的目标。在本工作中，生成了一种用于合成莽草酸途径中间体 3-脱氢莽草酸（3-DHS）的优化酵母菌株，3-DHS 是生产有价值的化合物顺式，顺式-粘康酸（CCM）和没食子酸（GA）的前体。生产 CCM 需要过表达异源酶 3-DHS 脱水酶 AroZ、原儿茶酸（PCA）脱羧酶 AroY 和儿茶酚 1,2-双加氧酶 CatA。AroY 的活性限制了该途径的产率。通过一种新型真菌脱羧酶扩展了该酶的作用范围。将该酶引入到优化的菌株中的途径中，可达到 1244mg/L CCM 的浓度，葡萄糖得率为 31mg/g。这是迄今为止在酿酒酵母中报道的该化合物的最高产量。为了证明优化菌株用于 3-DHS 生产其他化合物的适用性，我们过表达了 AroZ 以及对 PCA 具有改善的底物特异性的对羟基苯甲酸羟化酶的突变体 PobAY385F。由此，我们首次在酿酒酵母中证明了 GA 的生产。
