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.Coupling synthetic biology and chemical reactions in cells is a challenging task. The authors engineer bacteria capable of generating bromo-metabolites, develop a mild Suzuki-Miyaura cross-coupling reaction compatible with cell growth and carry out the cross-coupling chemistry in live cell cultures.

Fig. 1

Biosynthetic halogenation enabling Suzuki–Miyaura cross-coupling. a in vivo generation of Cl-pacidamycin, by the natural producer, transformed with a halogenase, followed by in vitro cross-coupling of the compound as a component of cell free extract, at 80 °C (in vivo: in vitro), b in vitro generation of reactive bromo-aromatics by RebH variant enzymes, followed by in vitro cross-coupling of the compound as a component of the crude extract (in vitro: in vitro), c in vitro generation of reactive bromo-aromatics by stabilized crosslinked enzyme aggregates ((CLEAs) of a series of flavin-dependent halogenases, membrane partitioned from anaerobic, palladium-mediated cross-coupling conditions (in vitro: in vitro), d in vivo incorporation of reactive, synthetic iodophenylalanine into peptides, followed by protein purification and cross-coupling of the iodinated protein (in vivo: in vitro), e in vivo Suzuki–Miyaura modification of synthetically generated, reactive triflate fluoran (synthetic: in vitro), f in vivo generation of reactive 7-Br-tryptophan 2 by engineered E. coli RG-1500 synchronous with the in-culture Suzuki–Miyaura cross-coupling of this reactive metabolite (in vivo: in vivo); and g generation of reactive Br-pacidamycin D 3 by engineered Streptomyces coelicolor RG-1104 concomitant with the in-culture Suzuki–Miyaura cross-coupling of this reactive metabolite (in vivo: in vivo)

Fig. 2

Selected water-soluble catalysts explored in this study. Catalyst stock solutions were prepared in deionized water, stored at ambient temperature and used within two weeks. L1-Pd solution were freshly prepared each day.

Fig. 3

Tryptophan inhibits L1-Pd catalyzed cross-coupling. Inhibition of cross-coupling of 5-I-indole 4 doped with increasing concentrations of unprotected tryptophan 1 (0–1 equiv.) or selectively protected tryptophans 9–11 (1 equiv.). Conversion was determined by ¹H NMR of the crude reaction

Fig. 4

Mild cross-coupling of Br-tryptophans and tripeptides. a Mild cross-coupling of unprotected halo-tryptophans at 45 °C using L2-Pd, ^aconversion is determined by ¹H NMR of the crude reaction, ^bisolated yield after purification by reversed-phase chromatography. b synthetic tripeptides incorporating 7-Br-tryptophan 2 used for cross-coupling studies, c Suzuki–Miyaura cross-coupling products derived from the corresponding tripeptides containing 7-Br-tryptophan. A mixture of Br-tryptophan or bromo-tripeptide, L2-Pd (5 mol%) catalyst, p-Tol-B(OH)₂ (3 equiv.) and K₂CO₃ (6 equiv.) in water-EtOH (4:1) or water was stirred at 45 °C for 48 h. Isolated yields are reported after purification by reversed-phase chromatography

Fig. 5

Synchronous production and cross-coupling of 7-Br-trytpophan. a E. coli PHL644 engineered to produce tryptophan 7-halogenase (prnA) (E. coli RG-1500). b Cross-coupling of the 7-Br-tryptophan 2 produced in living culture. An approximate conversion of 61% can be calculated based on the levels of the cross-coupled product 2a observed and based on the concentration of 7-Br-tryptophan 2 at the start of cross-coupling; however, as 7-Br-tryptophan 2 can be constantly produced and metabolized by living cultures, this can only be an approximation. c UPLC analysis, fluorescence chromatograms of the cross-coupling reaction (top trace), a control culture without Pd-catalyst or boronic acid (middle) and a purified standard of product 2a (bottom trace)

Fig. 6

In culture biosynthesis and cross-coupling of Br-pacidamycin-D. a S. coelicolor M1154 engineered to express the tryptophan 7-halogenase gene (prnA) and the entire pacidamycin gene cluster (pac1–pac22), which encodes the non-ribosomal peptide template/thiol tethered biosynthesis of pacidamycin (S. coelicolor RG-1104). b Cross-coupling of the Br-pacidamycin D 3 produced by the living culture. c Extracted Ion Chromatograms of Br-pacidamycin D 3 and the cross-coupled product p-tolyl-pacidamycin D 3a. This system exemplifies that this chemistry is not only compatible with less robust living cells but also that it is compatible with the very high dilutions and low titers that many natural products are present at. The engineering of bromo-metabolite production enables this exciting advance. Furthermore, the use of this model system exemplifies that the chemistry is compatible with both peptides and nucleosides