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.

Expanding the repertoire of aromatic chemicals by microbial production

“…In their study, the vioABCDE cluster with Ptrc promoter and strong RBS motif was overexpressed in L-Trp hyperproducer strain (C. glutamicum ATCC 21850) and a titer of 5436 mg L −1 violacein was achieved in a 3 L bioreactor. Rodrigues and co-workers 60 reported the production of deoxyviolacein in an engineered E. coli strain by expression of the deoxyviolacein cluster vioABCE with ParaBAD promoter. In a glycerol-based fed-batch process, the mutant strain dVio-8 produced 1.6 g L −1 of deoxyviolacein after 200 h. The microbial production of other L-Trp derivatives is usually derived from L-Trp via whole-cell bioconversion ( Table 2).…”

Optoregulated Drug Release from an Engineered Living Material: Self‐Replenishing Drug Depots for Long‐Term, Light‐Regulated Delivery

“…E. coli has been engineered for metabolic production of drugs such as artemisinin, [20] cinnamaldehyde, [21] carbapenem, [22] violacein, [23,24] deoxyviolacein (dVio), [23,24] etc., at industrial scales. [25] Among these, the l-tryptophan-derived bisindoles dVio and violacein are attractive due to their antibacterial, [26] antifungal, [27] and antitumoral properties [26,28] and the possibility to produce them at gram scales.…”

“…[25] Among these, the l-tryptophan-derived bisindoles dVio and violacein are attractive due to their antibacterial, [26] antifungal, [27] and antitumoral properties [26,28] and the possibility to produce them at gram scales. [23,24] dVio is synthesized from endogenously produced l-tryptophan by expressing four enzymes, vioA, vioB, vioC, and vioE, in E. coli, genetically encoded as a vioABCE operon. [23] For our study, the vio-ABCE operon was incorporated within the blue-light (470 nm) responsive optogenetic plasmid, pDawn, [16] to develop a genetic circuit within an endotoxin-free E. coli strain capable of lightactivated production of dVio.…”

“…E. coli has been engineered for metabolic production of drugs such as artemisinin, cinnamaldehyde, carbapenem, violacein, deoxyviolacein (dVio), etc., at industrial scales . Among these, the l ‐tryptophan‐derived bisindoles dVio and violacein are attractive due to their antibacterial, antifungal, and antitumoral properties and the possibility to produce them at gram scales . dVio is synthesized from endogenously produced l ‐tryptophan by expressing four enzymes, vioA, vioB, vioC, and vioE, in E. coli , genetically encoded as a vioABCE operon .…”

Influence of Environmental Factors on the Production of Violacein Synthesized By Janthinobacterium lividum

“…Violacein is a purple pigment product of the secondary metabolism of bacteria, whose biological activities have been widely reported, including antimicrobial activity (antibacterial, antiviral, antiprotozoal and antifungal) [3,4,[6][7][8][9][10][11][12][13][14] anticancer activity [15][16][17][18][19] antioxidant activity [20] and recent studies also show insecticidal activity [5]. Due to its biotechnological potential, numerous studies have been done indicating that Chromobacterium violaceum and Janthinobacterium lividum are primarily responsible for its synthesis [3][4][5]9,21,22].…”