title: Engineering of methionine chain elongation part of glucoraphanin pathway in E. coli.
abstract: 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.
authors.affiliation: DynaMo Center, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.
authors.forename: Nadia
authors.initials: N
authors.lastname: Mirza
authors.affiliation: DynaMo Center, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.
authors.forename: Christoph
authors.initials: C
authors.lastname: Crocoll
authors.affiliation: Section for Plant Biochemistry, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.
authors.forename: Carl
authors.initials: C
authors.lastname: Erik Olsen
authors.affiliation: DynaMo Center, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark. Electronic address: bah@plen.ku.dk.
authors.forename: Barbara
authors.initials: B
authors.lastname: Ann Halkier
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005961:Glucosinolates / Q000235:genetics / Q000378:metabolism*; D007096:Imidoesters / Q000378:metabolism*; D060847:Metabolic Engineering; D008715:Methionine / Q000096:biosynthesis / Q000235:genetics; D010091:Oximes; D013454:Sulfoxides
chemical_list: D005961:Glucosinolates; D007096:Imidoesters; D010091:Oximes; D013454:Sulfoxides; D008715:Methionine; C119494:glucoraphanin
keywords: Aliphatic glucosinolates; Chain elongation pathway; Dihomo-methionine; Glucoraphanin