Department of Chemical Engineering, The University of Texas at Austin, 200 E Dean Keeton St. Stop C0400, Austin, TX 78712, USA.
Metab Eng. 2013 Jan;15:55-66. doi: 10.1016/j.ymben.2012.10.003. Epub 2012 Nov 17.
The dicarboxylic acid muconic acid has garnered significant interest due to its potential use as a platform chemical for the production of several valuable consumer bio-plastics including nylon-6,6 and polyurethane (via an adipic acid intermediate) and polyethylene terephthalate (PET) (via a terephthalic acid intermediate). Many process advantages (including lower pH levels) support the production of this molecule in yeast. Here, we present the first heterologous production of muconic acid in the yeast Saccharomyces cerevisiae. A three-step synthetic, composite pathway comprised of the enzymes dehydroshikimate dehydratase from Podospora anserina, protocatechuic acid decarboxylase from Enterobacter cloacae, and catechol 1,2-dioxygenase from Candida albicans was imported into yeast. Further genetic modifications guided by metabolic modeling and feedback inhibition mitigation were introduced to increase precursor availability. Specifically, the knockout of ARO3 and overexpression of a feedback-resistant mutant of aro4 reduced feedback inhibition in the shikimate pathway, and the zwf1 deletion and over-expression of TKL1 increased flux of necessary precursors into the pathway. Further balancing of the heterologous enzyme levels led to a final titer of nearly 141mg/L muconic acid in a shake-flask culture, a value nearly 24-fold higher than the initial strain. Moreover, this strain has the highest titer and second highest yield of any reported shikimate and aromatic amino acid-based molecule in yeast in a simple batch condition. This work collectively demonstrates that yeast has the potential to be a platform for the bioproduction of muconic acid and suggests an area that is ripe for future metabolic engineering efforts.
二酸黏康酸因其作为生产几种有价值的消费生物塑料（包括尼龙-6,6 和聚亚安酯（通过己二酸中间产物）和聚对苯二甲酸乙二醇酯（通过对苯二甲酸中间产物）的平台化学品的潜力而受到极大关注。许多工艺优势（包括较低的 pH 值）支持该分子在酵母中的生产。在这里，我们首次在酵母酿酒酵母中异源生产黏康酸。该合成途径由三步组成，包括来自平革菌的脱氢莽草酸脱水酶、阴沟肠杆菌的原儿茶酸脱羧酶和白色念珠菌的儿茶酚 1,2-双加氧酶，该途径由一个合成的复合途径组成。进一步的遗传修饰通过代谢建模和反馈抑制缓解进行了介绍，以增加前体的可用性。具体来说，敲除 ARO3 和过表达反馈抗性突变体 aro4 减少了莽草酸途径中的反馈抑制，zwf1 缺失和 tkl1 的过表达增加了必要前体进入途径的通量。进一步平衡异源酶水平，最终在摇瓶培养中得到近 141mg/L 黏康酸的终浓度，比初始菌株高近 24 倍。此外，在简单的分批条件下，该菌株的黏康酸产量是酵母中报道的任何基于莽草酸和芳香族氨基酸的分子中最高的终浓度和第二高的产率。这项工作共同表明，酵母具有成为黏康酸生物生产平台的潜力，并表明该领域非常适合未来的代谢工程努力。
