Daptomycin biosynthesis in Streptomyces roseosporus: cloning and analysis of the gene cluster and revision of peptide stereochemistry

Abstract

Daptomycin is a 13 amino acid, cyclic lipopeptide produced by a non-ribosomal peptide synthetase (NRPS) mechanism in Streptomyces roseosporus. A 128 kb region of S. roseosporus DNA was cloned and verified by heterologous expression in Streptomyces lividans to contain the daptomycin biosynthetic gene cluster (dpt). The cloned region was completely sequenced and three genes (dptA, dptBC, dptD) encoding the three subunits of an NRPS were identified. The catalytic domains in the subunits, predicted to couple five, six or two amino acids, respectively, included a novel activation domain and amino-acid-binding pocket for incorporating the unusual amino acid L-kynurenine (Kyn), three types of condensation domains and an extra epimerase domain (E-domain) in the second module. Novel genes (dptE, dptF ) whose products likely work in conjunction with a unique condensation domain to acylate the first amino acid, as well as other genes (dptI, dptJ) probably involved in supply of the non-proteinogenic amino acids L-3-methylglutamic acid and Kyn, were located next to the NRPS genes. The unexpected E-domain suggested that daptomycin would have D-Asn, rather than L-Asn, as originally assigned, and this was confirmed by comparing stereospecific synthetic peptides and the natural product both chemically and microbiologically.

The action mechanism of daptomycin

“…The C-terminal ten amino acids form a macrocyclic core, which contains a ring-closing depsi (ester) bond between the side chain of Thr (4) and the α-COOH of the C-terminal Kyn (13) . 23,24 The N-terminal tripeptide protrudes from the ring and carries the variable fatty acyl residue, which is attached to Trp (1) .…”

“…50 At the time of this report, it was not known that daptomycin contains D-Asn rather than L-Asn at position 2; this was discovered only a short time afterwards. 24 Moreover, in all of their analogues, mGlu was replaced with Glu. Consequently, most of their analogues were triple mutants containing Glu in place of mGlu (12) , the unintended L-Asn (2) substitution, as well as an additional substitution of interest at a third position within the peptide.…”

Structural characterization of a lipopeptide antibiotic A54145E(Asn3Asp9) produced by a genetically engineered strain of Streptomyces fradiae

“…12,14 In addition, bioinformatic analysis predicted that A54145 factors would contain D-amino acids at position 2, 8 and 11 (D-Glu 2 , D-Lys 8 and D-Asn 11 ), based on the presence of epimerase domains in the respective NRPS modules. 9 The predicted locations of the D-amino acids coincide with the locations of the three D-amino acids in daptomycin, 16 suggesting that A54145 and daptomycin may have similar three-dimensional structures. The prominent natural variants have either n-decanoic (nC 10 ), iso-decanoic (iC 10 ) or anteiso-undecanoic (aC 11 ) acids attached to the terminal amino group of Trp 1 .…”

Molecular beacons to identify gifted microbes for genome mining

“…The NRPS mega-enzyme involved in daptomycin biosynthesis in S. roseosporus has three subunits, DptA, DptBC and DptD, 47 which have been exploited in combinatorial biosynthesis. 48 The three subunit arrangement has also been observed in the highly related cryptic daptomycin-like pathway in the finished genome of Saccharopolyspora viridis 49 and the finished biosynthetic gene cluster for taromycin A in the marine Saccharopolyspora sp.…”