Department of Biological Sciences, University of Calgary, 2500 University Dr. NW, Calgary, AB, T2N 1N4, Canada.
Department of Chemical and Petroleum Engineering, University of Calgary, 2500 University Dr. NW, Calgary, AB, T2N 1N4, Canada.
Metab Eng. 2024 Mar;82:286-296. doi: 10.1016/j.ymben.2024.02.011. Epub 2024 Feb 20.
Curcumin is a polyphenolic natural product from the roots of turmeric (Curcuma longa). It has been a popular coloring and flavoring agent in food industries with known health benefits. The conventional phenylpropanoid pathway is known to proceed from phenylalanine via p-coumaroyl-CoA intermediate. Although hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyl transferase (HCT) plays a key catalysis in the biosynthesis of phenylpropanoid products at the downstream of p-coumaric acid, a recent discovery of caffeoyl-shikimate esterase (CSE) showed that an alternative pathway exists. Here, the biosynthetic efficiency of the conventional and the alternative pathway in producing feruloyl-CoA was examined using curcumin production in yeast. A novel modular multiplex genome-edit (MMG)-CRISPR platform was developed to facilitate rapid integrations of up to eight genes into the yeast genome in two steps. Using this MMG-CRISPR platform and metabolic engineering strategies, the alternative CSE phenylpropanoid pathway consistently showed higher titers (2-19 folds) of curcumin production than the conventional pathway in engineered yeast strains. In shake flask cultures using a synthetic minimal medium without phenylalanine, the curcumin production titer reached up to 1.5 mg/L, which is three orders of magnitude (∼4800-fold) improvement over non-engineered base strain. This is the first demonstration of de novo curcumin biosynthesis in yeast. Our work shows the critical role of CSE in improving the metabolic flux in yeast towards the phenylpropanoid biosynthetic pathway. In addition, we showcased the convenience and reliability of modular multiplex CRISPR/Cas9 genome editing in constructing complex synthetic pathways in yeast.
姜黄素是来自姜黄（Curcuma longa）根部的多酚天然产物。它一直是食品工业中常用的着色剂和调味剂，具有已知的健康益处。传统的苯丙素途径被认为是从苯丙氨酸经对香豆酰辅酶 A 中间体进行的。尽管羟基肉桂酰辅酶 A：莽草酸羟基肉桂酰转移酶（HCT）在对香豆酸下游的苯丙素产物生物合成中发挥关键催化作用，但最近发现咖啡酰莽草酸酯酶（CSE）表明存在替代途径。在这里，使用酵母中的姜黄素生产来检查常规和替代途径在产生阿魏酰辅酶 A 方面的生物合成效率。开发了一种新颖的模块化多重基因组编辑（MMG）-CRISPR 平台，以方便在两步中将多达 8 个基因快速整合到酵母基因组中。使用这种 MMG-CRISPR 平台和代谢工程策略，替代的 CSE 苯丙素途径在工程酵母菌株中始终显示出比常规途径更高的姜黄素产量（2-19 倍）。在使用不含苯丙氨酸的合成最小培养基的摇瓶培养中，姜黄素的产量达到了 1.5 mg/L，比非工程基础菌株提高了三个数量级（约 4800 倍）。这是首次在酵母中证明从头合成姜黄素。我们的工作表明 CSE 在提高酵母中苯丙素生物合成途径的代谢通量方面起着关键作用。此外，我们展示了模块化多重 CRISPR/Cas9 基因组编辑在构建酵母中复杂合成途径的便利性和可靠性。
