Living GenoChemetics by hyphenating synthetic biology and synthetic chemistry in vivo.

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.

Revealing the first uridyl peptide antibiotic biosynthetic gene cluster and probing pacidamycin biosynthesis.

There is an urgent need for new antibiotics with resistance continuing to emerge toward existing classes. The pacidamycin antibiotics possess a novel scaffold and exhibit unexploited bioactivity rendering them attractive research targets. We recently reported the first identification of a biosynthetic cluster encoding uridyl peptide antibiotic assembly and the engineering of pacidamycin biosynthesis into a heterologous host. We report here our methods toward identifying the biosynthetic cluster. Our initial experiments employed conventional methods of probing a cosmid library using PCR and Southern blotting, however it became necessary to adopt a state-of-the-art genome scanning  and in silico hybridization approach  to pin point the cluster. Here we describe our "real" and "virtual" probing methods and contrast the benefits and pitfalls of each approach. 

Pacidamycin biosynthesis: identification and heterologous expression of the first uridyl peptide antibiotic gene cluster.

The pacidamycins are antimicrobial nucleoside antibiotics produced by Streptomyces coeruleorubidus that inhibit translocase I, an essential bacterial enzyme yet to be clinically targeted. The novel pacidamycin scaffold is composed of a pseudopeptide backbone linked by a unique exocyclic enamide to an atypical 3'-deoxyuridine nucleoside. In addition, the peptidyl chain undergoes a double inversion caused by the incorporation of a diamino acid residue and a rare internal ureido moiety. The pacidamycin gene cluster was identified and sequenced, thereby providing the first example of a biosynthetic cluster for a member of the uridyl peptide family of antibiotics. Analysis of the 22 ORFs provided an insight into the biosynthesis of the unique structural features of the pacidamycins. Heterologous expression in Streptomyces lividans resulted in the production of pacidamycin D and the newly identified pacidamycin S, thus confirming the identity of the pacidamycin biosynthetic gene cluster. Identification of this cluster will enable the generation of new uridyl peptide antibiotics through combinatorial biosynthesis. The concise cluster will provide a useful model system through which to gain a fundamental understanding of the way in which nonribosomal peptide synthetases interact.

New pacidamycins biosynthetically: probing N- and C-terminal substrate specificity.

Feeding phenylalanine analogues to Streptomyces coeruleorubidus reveals the remarkable steric and electronic flexibility of this biosynthetic pathway and leads to the generation of a series of new halopacidamycins.

New pacidamycin antibiotics through precursor-directed biosynthesis.

Pacidamycins, mureidomycins and napsamycins are structurally related uridyl peptide antibiotics that inhibit translocase I, an as yet clinically unexploited target. This potentially important bioactivity coupled to the biosynthetically intriguing structure of pacidamycin make this natural product a fascinating subject for study. A precursor-directed biosynthesis approach was employed in order to access new pacidamycin derivatives. Strikingly, the biosynthetic machinery exhibited highly relaxed substrate specificity with the majority of the tryptophan analogues that were administered; this resulted in the production of new pacidamycin derivatives. Remarkably, 2-methyl-, 7-methyl-, 7-chloro- and 7-bromotryptophans produced their corresponding pacidamycin analogues in larger amounts than the natural pacidamycin. Low levels or no incorporation was observed for tryptophans substituted at positions 4, 5 and 6. The ability to generate bromo- and chloropacidamycins opens up the possibility of further functionalising these compounds through chemical cross-coupling in order to access a much larger family of derivatives.