ME: Metabolic Engineering of Anthocyanin Production in Saccharomyces cerevisiae and Escherichia coli

本探索性项目将重点关注两个目标：1.；阐明参与花青素生物合成的蛋白质的酶性质；在酵母中构建并实现了从黄酮和苯丙氨酸到第一个稳定的有色花青素——花青素3-葡萄糖苷（黄离子）的花青素生物合成途径的异源表达。第一个靶点的主要目标是酶二氢黄酮醇4还原酶（DFR），它将二氢黄酮醇转化为白花青素。该酶可能是整个花青素途径中最重要和最有趣的原因是：(1)它不在大肠杆菌中表达（但在酵母中表达），(2)它可以利用各种其他底物，如黄酮甚至花青素，(3)它是少数出现在原核物种基因组中的类黄酮生物合成酶之一。首席研究员（PI）现在有实验证据表明，植物和原核生物的dfr基因成功地转化为出现在大肠杆菌细胞质中的蛋白质。这消除了最近其他研究人员提出的密码子使用问题和包涵体形成的可能性。PI目前正在使用大肠杆菌和酵母粗提取物和渗透细胞对重组蛋白进行酶学表征，并正在阐明这种酶在酵母中可能发生的翻译后修饰（它似乎是功能性的），例如糖基化和磷酸化。第二个目标的目标是在酵母中构建一个功能性的花青素生物合成途径。PI现在在他的实验室里有一套完整的四种基因，这些基因来自不同的植物，可以将黄酮（柚皮素）转化为花青素，他将把这四种基因放在一起，在酵母中研究柚皮素（在发酵培养基中提供）转化为花青素3-葡萄糖苷。此外，PI将获得拟南芥中将苯丙氨酸转化为柚皮素的完整的五个基因（由日本理研和Kazusa研究所提供）。该途径的构建将通过质粒和染色体整合的同源重组来实现。

This exploratory project will focus on two targets: 1. Elucidate the enzymatic properties of proteins involved in anthocyanin biosynthesis, and 2. Construct and achieve the heterologous expression of the anthocyanin biosynthesis pathway that leads from flavanones and phenylalanine to the first stable colored anthocyanin, anthocyanidin 3-glucoside (flavylium ion) in yeast. The primary goal in the first target is the enzyme dihydroflavonol 4-reductase (DFR) that converts dihydroflavonols to leucoanthocyanidins. The reasons why this enzyme is perhaps the most important and intriguing in the whole anthocyanin pathway are: (i) it does not functionally express in Escherichia coli (but expresses in yeast), (ii) it can utilize various other substrates, such as flavanones and even anthocyanidins and (iii) it is one of the few flavonoid biosynthesis enzymes that appears in the genome of prokaryotic species. The Principal Investigator (PI) now has experimental evidence showing that plant and prokaryotic dfr genes successfully translate into protein that appears in the cytoplasm of Escherichia coli. That eliminates the possibility of codon usage problems and inclusion body formation suggested by other researchers in the very recent past. The PI is currently working on the enzymatic characterization of the recombinant proteins using E. coli and yeast crude extracts and permeabilized cells, and is also working on elucidating possible post-translational modifications this enzyme might be undergoing in yeast (where it appears to be functional), such as glycosylation and phosphorylation. The goal on the second target is the construction of a functional anthocyanin biosynthesis pathway in yeast. The PI now has available in his lab a complete set of four genes from various plants that convert flavanones (naringenin) to anthocyanin, and will be putting these four genes together in yeast and investigate the conversion of naringenin (provided in the fermentation medium) to anthocyanidin 3-glucoside. In addition the PI will be acquiring the complete set of five genes that convert phenylalanine to naringenin in Arabidopsis thaliana (provided by the RIKEN and Kazusa Research Institutes in Japan). The pathway construction will be achieved by homologous recombination using plasmid and chromosome integration.