GenoChemetic Strategy for Derivatization of the Violacein Natural Product Scaffold

Abstract

Natural products and their analogues are often challenging to synthesize due to their complex scaffolds and embedded functional groups. Solely relying on engineering the biosynthesis of natural products may lead to limited compound diversity. Integrating synthetic biology with synthetic chemistry allows rapid access to much more diverse portfolios of xenobiotic compounds, which may accelerate the discovery of new therapeutics. As a proof-of-concept, by supplementing an Escherichia coli strain expressing the violacein biosynthesis pathway with 5-bromo-tryptophan in vitro or tryptophan 7-halogenase RebH in vivo , six halogenated analogues of violacein or deoxyviolacein were generated, demonstrating the promiscuity of the violacein biosynthesis pathway. Furthermore, 20 new derivatives were generated from 5-brominated violacein analogues via the Suzuki–Miyaura cross-coupling reaction directly using the crude extract without prior purification. Herein we demonstrate a flexible and rapid approach to access a diverse chemical space that can be applied to a wide range of natural product scaffolds.

Biotransformation Of l‐Tryptophan To Produce Arcyriaflavin A With Pseudomonas putida KT2440

“…[42,43] In the future, it may be interesting to generate hybrid pathways with homologous enzymes (e. g., VioA, VioB) that also produce CPA (3) but have a higher preference for l-tryptophan, which could lead to an increased production of arcyriaflavin A. [20] In addition, it could be relevant to investigate the production of arcyriaflavin derivatives by conversion of unnatural substrates (e. g., 5-fluoro-, 5-bromo-or 1-methyl-l-tryptophan [42,44] ) in such hybrid pathways. Overall, almost six-fold enhanced productivity (from 5.7 μg DCW À 1 h À 1 to 32.2 μg DCW À 1 h À 1 ) and an eight-fold increase in product titer (from 0.49 mg L À 1 to 3.71 mg L À 1 ) were achieved in the present study (Figure 3B).…”

Expanding the Chemical Diversity of Fasamycin Via Genome Mining and Biocatalysis

“…Since halogens are very useful for expanding the structural diversity via transition-metal-catalyzed cross-coupling reactions, for example, the Suzuki-Miyaura cross-coupling reaction, − we were interested in the generation of new halogenated fasamycins using other halogenases. Our previous study revealed that halogenase Abx (−) H participating in (−)-anthrabenzoxocinone ((−)-ABX) biosynthesis can recognize the DHDA motif to catalyze the C-14 and C-16 halogenation of both (−)-ABX and (+)-ABX, which bears an oxygen-bridged scaffold in different chirality (Figure ).…”

Exploration of the In Vitro Violacein Synthetic Pathway with Substrate Analogues

“…For the remaining tryptophan derivatives, 1-methyl-L-tryptophan showed comparable activity to L-tryptophan while 7-azatryptophan showed a significant decrease in both k cat (0.17 s −1 ) and K M (6.0 × 10 −4 μM −1 x s −1 ) of VioA relative to Ltryptophan (Figure 3C). Overall, the k cat value for the different analogues varied between 0.6 and 2.4-fold to that of tryptophan, while K M varied between 0.2−1.7-fold, and k cat / 34,35 and of 1methyl-L-tryptophan and 7-aza-tryptophan with data reported by Fuller et al 48 Together, the data compiled by ourselves and these other groups allows for an emerging picture of what is and is not tolerated by VioA, see Table S2. Note that many of these tryptophan analogues show some toxicity to strains of E. coli, a common biosynthesis organism.…”