A54145, a new lipopeptide antibiotic complex. Discovery, taxonomy, fermentation and HPLC.

Abstract

A54145is a complex of new lipopeptide antibiotics that inhibits Gram-positive bacteria and acts as a growth promotant for broiler chicks. Eight factors; A, B, C, D, E, F, At and B^have been isolated and characterized. They contain four similar peptide nuclei, each of which is acylated with either an 2-decanoyl, rc-decanoyl, or undecanoyl side chain. Taxonomicstudies ascertained that the producing microorganism was a strain of Streptomyces fradiae. Fermentation studies determined that superior antibiotic yields were obtained in stirred bioreactors in a soybean flour-molasses medium employing a continuous glucose feed. These findings, interwoven with the selection of hyper-productive mutants, increased fermentation yields from < 50 /xg/ml to more than 1 mg/ml. An analytical HPLCsystem was developed for the identification and subsequent quantitation of each factor of the A54145 complex.While screening actinomycetes for novel antimicrobial substances, a new culture was isolated from a soil sample collected in Mexico. This isolate produced a complex of lipopeptide antibiotics from which eight factors; A, B, C, D, E, F, Ax and Bx; were isolated and characterized1*.These factors contained four different cyclic peptide nuclei which varied only in valine/isoleucine and/or glutamate/3-CH3-glutamate substitutions. The TV-terminus of each nucleus was acylated with either an 2-decanoyl (z"C10), rc-decanoyl («C10), or undecanoyl (flCn) lipid side chain (Fig. I)1'2*. Major factors were A and Bl9 both of which contained the zC10 fatty acyl units. Although four additional factors could presumably be produced by this combination of nuclei and side chains, these additional factors were not isolated. A54145B,Bl9 C and E exhibited in vitro MICvalues of 0.5~2/zg/ml vs. various strains of Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus. Factors A and B were also shown to be active vs. these organisms in mice3). The factors additionally increased weight gain and feed efficiency in swine and fowl, being especially effective in chicks and turkeys (R. Wellenreiter, personal communication). A natural variant of the original isolated, NRRL18158, was characterized as a strain of Streptomycesfradiae. This paper describes taxonomy and fermentation studies on the A54145-producing cultures and the analytical HPLCsystem developed to monitor the A54145 factors. Materials and Methods Cell Wall AnalysesDiaminopimelic acid (DAP) isomers were determined by the method of Becker et al.4). Cell wall sugars were identified by the procedure of Lechevalier5). Taxonomic MethodsThe methods and media recommended by the International Streptomyces Project (ISP)6) for characterization of Streptomyces species were followed. Color names were assigned to reverse pigments

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

“…In addition, the identification of the acyl group was further confirmed by observing a homogeneous peak at t R 7.01 min after spiking the reaction resultant of 1 with the library mixture ( Figure 3c). As a result, the anteiso-undecanoyl (aC 11 ) group was identified in 1, as expected from fermentation of S. fradiae DA1489 in DSF medium supplemented with L-Ile. 14, 18 Structural characterization of 1 by NMR studies was somewhat challenging because of the observation of two sets of 1 H-NMR signals in DMSO-d 6 .…”

“…9,10 A54145 is a family of cyclic tridecapeptide antibiotics related to daptomycin and produced by Streptomyces fradiae. [11][12][13][14][15] The A54145 lipopeptides contain several nonproteinogenic amino acids, including hydroxyasparagine (hAsn 3 ), sarcosine (Sar 5 ), methoxyaspartic acid (moAsp 9 ) and 3-methylglutamic acid (3mGlu 12 ). 12 Four different cyclic peptide nuclei vary in the ring amino acid residues Glu/3mGlu at position 12 and Ile/Val at position 13.…”

“…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 . 12,14 By acyl tail modification, 13 a few A54145 analogs were semisynthesized for evaluation of in vitro antibacterial activity.…”

“…In this study, to confirm the presence of the three D-stereochemistry amino acids, Glu, Lys and Asp, were specifically selected for determination of their optical purity using an enantiomer-labeling method. 23 In this method, the measured optical purity of Asp was derived from both Asp and Asn residues, as Asn was converted to Asp during the DCl/D 2 Naturally occurring A54145 factors have acyl groups, which consist of n-decanoyl (nC 10 ), iso-decanoyl (iC 10 ) and anteiso-undecanoyl (aC 11 ). 12 The acyl substituent of 1 was determined by acidic hydrolysis of the lipopeptide followed by coupling the resultant FA with Trp to produce a corresponding FA-Trp amide.…”

Production of novel lipopeptide antibiotics related to A54145 by Streptomyces fradiae mutants blocked in biosynthesis of modified amino acids and assignment of lptJ, lptK and lptL gene functions

“…Typically, A54145 factors containing Glu 12 accumulate early in fermentation and those containing 3mGlu 12 accumulate later, with final yields of about 60% of factors containing Glu 12 . 1 The study by Boeck et al 5 demonstrated that the distribution of A54145 factors can be manipulated by adding certain amino acids or lipids to the fermentation. For instance, feeding L-Ile, enriched for factors containing Ile 13 from 89 to 98%, whereas feeding L-Val (which inhibited overall lipopeptide production by about 70%) enriched for compounds containing Val 13 from 11 to 56%, almost exclusively in…”