title: Systems metabolic engineering of Escherichia coli for gram scale production of the antitumor drug deoxyviolacein from glycerol.
abstract: Deoxyviolacein is a microbial drug with biological activity against tumors, gram-positive bacteria, and fungal plant pathogens. Here, we describe an Escherichia coli strain for heterologous production of this high-value drug from glycerol. Plasmid-based expression of the deoxyviolacein cluster vioABCE was controlled by the araBAD promoter and induction by L-arabinose. Through elimination of L-arabinose catabolism in E. coli, the pentose sugar could be fully directed to induction of deoxyviolacein biosynthesis and was no longer metabolized, as verified by (13) C isotope experiments. Deletion of the araBAD genes beneficially complemented with previously described (i) engineering of the pentose phosphate pathway, (ii) chorismate biosynthesis, (iii) tryptophan biosynthesis, (iv) improved supply of L-serine, (v) elimination of tryptophan repression, and (vi) of tryptophan catabolism. Subsequent screening of the created next-generation producer E. coli dVio-8 identified glycerol as optimum carbon source and a level of 100 mg L(-1) of L-arabinose as optimum for induction. Transferred to a glycerol-based fed-batch process, E. coli dVio-8 surpassed the gram scale and produced 1.6 g L(-1) deoxyviolacein. With straightforward extraction from culture broth and purification by flash chromatography, deoxyviolacein was obtained at >99.5% purity. Biotechnol. Bioeng. 2014;111: 2280-2289. © 2014 Wiley Periodicals, Inc.
authors.affiliation: Institute of Systems Biotechnology, Saarland University, Saarbrücken, Germany; Institute of Biochemical Engineering, Technische Universität Braunschweig, Braunschweig, Germany.
authors.forename: André Luis
authors.initials: AL
authors.lastname: Rodrigues
authors.forename: Judith
authors.initials: J
authors.lastname: Becker
authors.forename: André Oliveira
authors.initials: AO
authors.lastname: de Souza Lima
authors.forename: Luismar Marques
authors.initials: LM
authors.lastname: Porto
authors.forename: Christoph
authors.initials: C
authors.lastname: Wittmann
mesh_terms: D000970:Antineoplastic Agents / Q000378:metabolism*; D001089:Arabinose / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D005990:Glycerol / Q000378:metabolism*; D007211:Indoles / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D005810:Multigene Family; D010957:Plasmids; D011401:Promoter Regions, Genetic
chemical_list: D000970:Antineoplastic Agents; D007211:Indoles; D001089:Arabinose; D005990:Glycerol; C063155:violacein
keywords: 13C flux; L-arabinose; deoxyviolacein; glycerol; natural product; tryptophan