title: Systems metabolic engineering of Escherichia coli for production of the antitumor drugs violacein and deoxyviolacein.
abstract: Violacein and deoxyviolacein are interesting therapeutics against pathogenic bacteria and viruses as well as tumor cells. In the present work, systems-wide metabolic engineering was applied to target Escherichia coli, a widely accepted recombinant host in pharmaceutical biotechnology, for production of these high-value products. The basic producer, E. coli dVio-1, that expressed the vioABCE cluster from Chromobacterium violaceum under control of the inducible araC system, accumulated 180 mg L(-1) of deoxyviolacein. Targeted intracellular metabolite analysis then identified bottlenecks in tryptophan supporting pathways, the major product building block. This was used for comprehensive engineering of serine, chorismate and tryptophan biosynthesis and the non-oxidative pentose-phosphate pathway. The final strain, E. coli dVio-6, accumulated 320 mg L(-1) deoxyviolacein in shake flask cultures. The created chassis of a high-flux tryptophan pathway was complemented by genomic integration of the vioD gene of Janthinobacterium lividum, which enabled exclusive production of violacein. In a fed-batch process, the resulting producer E. coli Vio-4 accumulated 710 mg L(-1) of the desired product. With straightforward broth extraction and subsequent crystallization, violacein could be obtained with 99.8% purity. This demonstrates the potential of E. coli as a platform for production of tryptophan based therapeutics.
authors.affiliation: Institute of Biochemical Engineering, Technische Universität Braunschweig, Germany.
authors.forename: André L
authors.initials: AL
authors.lastname: Rodrigues
authors.forename: Nathalie
authors.initials: N
authors.lastname: Trachtmann
authors.forename: Judith
authors.initials: J
authors.lastname: Becker
authors.forename: Ananta F
authors.initials: AF
authors.lastname: Lohanatha
authors.forename: Jana
authors.initials: J
authors.lastname: Blotenberg
authors.forename: Christoph J
authors.initials: CJ
authors.lastname: Bolten
authors.forename: Claudia
authors.initials: C
authors.lastname: Korneli
authors.forename: André O
authors.initials: AO
authors.lastname: de Souza Lima
authors.forename: Luismar M
authors.initials: LM
authors.lastname: Porto
authors.forename: Georg A
authors.initials: GA
authors.lastname: Sprenger
authors.forename: Christoph
authors.initials: C
authors.lastname: Wittmann
mesh_terms: D000970:Antineoplastic Agents / Q000378:metabolism*; D002861:Chromobacterium / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D007211:Indoles / Q000378:metabolism*; D060847:Metabolic Engineering; D005810:Multigene Family
chemical_list: D000970:Antineoplastic Agents; D007211:Indoles; C063155:violacein
keywords: Chorismate; Chromobacterium violaceum; Fed-batch; Janthinobacterium lividum; Pathway deregulation; Tryptophan