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

ME：酿酒酵母和大肠杆菌中花青素生产的代谢工程

• 批准号: 0331404
• 负责人: Mattheos Koffas
• 金额: $ 10万
• 依托单位: SUNY at Buffalo
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-10-01 至 2006-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0331404&HistoricalAwards=false
• 关键词: ME; Metabolic; Engineering; Anthocyanin; Production

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.

本探索性项目将集中于两个目标：1.阐明参与花青素生物合成的蛋白质的酶学性质；2.构建并实现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(黄素离子)的花青素生物合成途径的异源表达。第一个目标的主要目标是二氢黄酮醇4-还原酶(DFR)，它能将二氢黄酮醇转化为亮氨酸花青素。该酶可能是整个花青素途径中最重要和最有趣的原因是：(I)它不在大肠杆菌中功能性表达(但在酵母中表达)，(Ii)它可以利用其他各种底物，如黄烷酮甚至花青素，(Iii)它是原核生物基因组中为数不多的类黄酮类生物合成酶之一。首席调查员(PI)现在有实验证据表明，植物和原核生物的DFR基因成功地翻译成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物和通透性细胞对重组蛋白进行酶学表征，并正在努力阐明该酶在酵母中可能正在经历的翻译后修饰(在那里它似乎具有功能)，如糖基化和磷酸化。第二个目标的目标是在酵母中构建具有功能的花青素生物合成途径。PI现在已经在他的实验室中获得了一套完整的四种基因，这些基因来自各种植物，可以将黄烷酮(柚皮苷元)转化为花青素，并将把这四种基因放在酵母中，研究柚皮脂素(发酵介质中提供的)转化为花青素3-葡萄糖苷的过程。此外，PI将获得在拟南芥中将苯丙氨酸转化为柚皮素的5个完整基因集(由日本RIKEN和Kazusa研究所提供)。该途径的构建将通过同源重组和染色体整合来实现。