Genome Mining and Metabolomics Uncover a Rare d-Capreomycidine Containing Natural Product and Its Biosynthetic Gene Cluster

Abstract

We report the metabolomics-driven genome mining of a new cyclic-guanidino incorporating non-ribosomal peptide synthetase (NRPS) gene cluster and full structure elucidation of its associated hexapeptide product, faulknamycin. Structural studies unveiled that this natural product contained the previously unknown (R,S)-stereoisomer of capreomycidine, D-capreomycidine. Furthermore, heterologous expression of the identified gene cluster successfully reproduces faulknamycin production without an observed homologue of VioD, the pyridoxal phosphate (PLP)dependent enzyme found in all previous L-capreomycidine biosynthesis. An alternative NRPS-dependent pathway for D-capreomycidine biosynthesis is proposed.

Cyclofaulknamycin with the Rare Amino Acid D-capreomycidine Isolated from a Well-Characterized Streptomyces albus Strain

“…Until recently, the products of both gene orthologs (VioC and VioD) were considered to be required for capreomycidine biosynthesis [ 26 ]. During the preparation of this manuscript, an article describing the new hypothetical pathway for capreomycidine biosynthesis was published [ 20 ]. From the identified clusters, two flk BGCs that had a very similar structure and NRPS domain organization attracted our attention; however, they were identified in different microorganisms, S. albus J1074 and Streptomyces koyangensis VK-A60T ( Figure 1 ).…”

“…Furthermore, the recently published faulknamycin gene cluster (fau BGC) [ 20 ] appeared to be very similar to the flk BGCs. Based on adenylation domain analysis, the two clusters were predicted to encode hexapeptides composed of the same amino acids ( Figure 1 ).…”

“…A search for compounds with such masses in the dictionary of natural products did not reveal any hits; therefore, the products were considered to be new. However, during the preparation of this manuscript, a linear compound with one of these masses (750.4 Da), referred to as faulknamycin, was published [ 20 ].…”

“…The amino acid mixture was compared with commercially acquired standards derivatized with L-FDLA ( Figure S11 ). The configuration of capreomycidine was determined as described previously by using standards derived from the hydrolysis of capreomycidine and chymostatin and derivatization by L-FDLA and D-FDLA [ 20 ] ( Figure S13 ). The amino acid β-phenylserine has been identified by Tryon et al as the D- threo- isomer; therefore, DL- threo -β-phenylserine was used as a standard to confirm the reported stereochemistry ( Figures S14 and S15 ).…”

“…To identify the product of this cluster, the metabolomes of two previously developed S. albus mutant strains [ 19 ] with and without the abovementioned cluster were compared. The analysis and comparison of the secondary metabolite profiles of the two strains allowed us to identify compounds with molecular masses of 731.4 and 749.4 Da, which correspond to the molecular weights of the predicted but never-identified cyclic faulknamycin compound and the recently described linear faulknamycin, respectively [ 20 ]. This is the first identified cyclic peptide compound with the rare D-capreomycidine amino acid.…”