Living GenoChemetics by hyphenating synthetic biology and synthetic chemistry in vivo

Abstract

Marrying synthetic biology with synthetic chemistry provides a powerful approach toward natural product diversification, combining the best of both worlds: expediency and synthetic capability of biogenic pathways and chemical diversity enabled by organic synthesis. Biosynthetic pathway engineering can be employed to insert a chemically orthogonal tag into a complex natural scaffold affording the possibility of site-selective modification without employing protecting group strategies. Here we show that, by installing a sufficiently reactive handle (e.g., a C–Br bond) and developing compatible mild aqueous chemistries, synchronous biosynthesis of the tagged metabolite and its subsequent chemical modification in living culture can be achieved. This approach can potentially enable many new applications: for example, assay of directed evolution of enzymes catalyzing halo-metabolite biosynthesis in living cells or generating and following the fate of tagged metabolites and biomolecules in living systems. We report synthetic biological access to new-to-nature bromo-metabolites and the concomitant biorthogonal cross-coupling of halo-metabolites in living cultures.

Targeted Enzyme Engineering Unveiled Unexpected Patterns of Halogenase Stabilization

“…Recently Goss et al . developed an in vivo approach for the generation of a brominated natural product analogue and its subsequent cross‐coupling for diversification of halogenated natural products however, without mentioning turnover or efficiency . Despite substantial efforts to improve in vitro compatibility of halogenases, their application still suffers from severe shortcomings such as low activity, enzyme stability and their limitation to act on relatively electron rich aromatic compounds.…”

Heck Diversification of Indole‐Based Substrates under Aqueous Conditions: From Indoles to Unprotected Halo‐tryptophans and Halo‐tryptophans in Natural Product Derivatives

“…In this study free, unprotected 7‐bromo‐tryptophan was derivatised with 6 different styrenes . Excitingly, as observed with the Suzuki–Miyaura cross‐coupling of tryptophan, these tryptophan‐7‐styrene products were shown to be fluorogenic, thus again opening up the way for fluorescence modulation of halo‐tryptophans. There is considerable potential for the development of Heck methodologies for the functionalisation of free and biomolecule embedded halo‐tryptophans.…”

“…Though considerable research has been carried out on selective modification of halogenated phenylalanines and tyrosines through the application of cross‐coupling chemistry, the functionalisation of tryptophan has, until recently, remained largely unexplored . One potential reason for this is the challenge that such metal mediated cross‐coupling reactions present with tryptophan; indeed, tryptophan has been demonstrated to poison the Suzuki–Miyaura cross‐coupling of halo‐indoles . This may be attributed to the amino acid coordinating to the palladium catalyst .…”

“…This may be attributed to the amino acid coordinating to the palladium catalyst . Notably, esterification of the carboxylate and acylation of the primary amine reduces reaction poisoning, however even this species is not fully innocent and its incorporation in a reaction is still seen to impact upon conversion …”

“…In recent years, series of studies enabling the cross‐coupling of halo‐tryptophans through application of Suzuki–Miyaura and Sonogashira chemistries in aqueous media have been reported . Combination of these chemistries with enzymatic halogenation has been used powerfully for selective C−H activation and diversification of small molecules The application of Heck cross‐coupling would provide a valuable addition to this growing portfolio of reactions for tryptophan functionalisation, enabling the potential for extension of conjugation and tuning of electronic and fluorescence properties as well as the opportunity to potentially modulate conformations of small molecules, peptides and proteins.…”