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

该探索性项目将侧重于两个目标：1.阐明参与花青素苷生物合成的蛋白质的酶学性质; 2.构建并实现了从黄烷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（黄酸离子）的花青素生物合成途径在酵母中的异源表达。 第一个靶点的主要目标是将二氢黄酮醇转化为无色花青素的二氢黄酮醇4-还原酶（DFR）。这种酶可能是整个花青素途径中最重要和最有趣的原因是：（i）它在大肠杆菌中不功能性表达（但在酵母中表达），（ii）它可以利用各种其他底物，如黄烷酮甚至花青素，（iii）它是出现在原核物种基因组中的少数类黄酮生物合成酶之一。主要研究者（PI）现在有实验证据表明，植物和原核生物的dfr基因成功地翻译成蛋白质，出现在大肠杆菌的细胞质中。 这消除了密码子使用问题和包涵体形成的可能性，这是最近其他研究人员提出的。PI目前正在使用E.该研究所正在对大肠杆菌和酵母粗提物以及透性化细胞进行研究，并致力于阐明这种酶可能在酵母中经历的翻译后修饰（在酵母中它似乎是功能性的），如糖基化和磷酸化。 第二个目标是在酵母中构建功能性花青素合成途径。PI现在在他的实验室中有一套完整的四个基因，来自各种植物，将黄烷酮（柚皮素）转化为花青素，并将这四个基因放在酵母中，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五个基因的完整集合（由日本的RIKEN和Kazusa研究所提供）。将通过使用质粒和染色体整合的同源重组来实现途径构建。

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.