Mirza Nadia, Crocoll Christoph, Erik Olsen Carl, Ann Halkier Barbara
DynaMo Center, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.
Section for Plant Biochemistry, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.
Metab Eng. 2016 May;35:31-37. doi: 10.1016/j.ymben.2015.09.012. Epub 2015 Sep 26.
The methionine-derived glucosinolate glucoraphanin is associated with the health-promoting properties of broccoli. This has developed a strong interest in producing this compound in high amounts from a microbial source. Glucoraphanin synthesis starts with a five-gene chain elongation pathway that converts methionine to dihomo-methionine, which is subsequently converted to glucoraphanin by the seven-gene glucosinolate core structure pathway. As dihomo-methionine is the precursor amino acid for glucoraphanin production, a first challenge is to establish an expression system for production of dihomo-methionine. In planta, the methionine chain elongation enzymes are physically separated within the cell with the first enzyme in the cytosol while the rest are located in the chloroplast. A de-compartmentalization approach was applied to produce dihomo-methionine by expression of the respective plant genes in Escherichia coli cytosol. Introduction of two plasmids encoding the methionine chain elongation pathway into E. coli resulted in production of 25mgL(-1) of dihomo-methionine. In addition to chain-elongated methionine products, side-products from chain elongation of leucine were produced. Methionine supplementation enhanced dihomo-methionine production to 57mgL(-1), while keeping a steady level of the chain-elongated leucine products. Engineering of the de-compartmentalized pathway of dihomo-methionine in E. coli cytosol provides an important first step for microbial production of the health-promoting glucoraphanin.
源自蛋氨酸的硫代葡萄糖苷萝卜硫素与西兰花的健康促进特性相关。这引发了人们对从微生物来源大量生产这种化合物的浓厚兴趣。萝卜硫素的合成始于一个由五个基因组成的链延伸途径，该途径将蛋氨酸转化为二高蛋氨酸，随后二高蛋氨酸通过由七个基因组成的硫代葡萄糖苷核心结构途径转化为萝卜硫素。由于二高蛋氨酸是萝卜硫素生产的前体氨基酸，第一个挑战是建立一个生产二高蛋氨酸的表达系统。在植物中，蛋氨酸链延伸酶在细胞内物理分隔，第一种酶位于细胞质中，其余的位于叶绿体中。采用去分隔化方法，通过在大肠杆菌细胞质中表达相应的植物基因来生产二高蛋氨酸。将两个编码蛋氨酸链延伸途径的质粒导入大肠杆菌，可产生25mgL(-1)的二高蛋氨酸。除了链延伸的蛋氨酸产物外，还产生了亮氨酸链延伸的副产物。补充蛋氨酸可将二高蛋氨酸产量提高到57mgL(-1)，同时保持链延伸的亮氨酸产物水平稳定。在大肠杆菌细胞质中对二高蛋氨酸的去分隔化途径进行工程改造，为微生物生产具有健康促进作用的萝卜硫素提供了重要的第一步。
