Biosynthetic Gene Cluster of the Herbicide Phosphinothricin Tripeptide from Streptomyces viridochromogenes Tü494

Actinoplanes friuliensis produces the lipopeptide antibiotic friulimicin, which is a cyclic peptide with one exocyclic amino acid linked to a branched-chain fatty acid acyl residue. The structural relationship to daptomycin and the excellent antibacterial performance of friulimicin make the antibiotic an attractive drug candidate. The complete friulimicin biosynthetic gene cluster of 24 open reading frames from A. friuliensis was sequenced and analyzed. In addition to genes for regulation, self-resistance, and transport, the cluster contains genes encoding peptide synthetases, proteins involved in the synthesis and linkage of the fatty acid component of the antibiotic, and proteins involved in the synthesis of the nonproteinogenic amino acids pipecolinic acid, methylaspartic acid, and 2,3-diaminobutyric acid. By using heterologous gene expression in Escherichia coli, we provide biochemical evidence for the stereoselective synthesis of L-pipecolinic acid by the deduced protein of the lysine cyclodeaminase gene pip. Furthermore, we show the involvement of the dabA and dabB genes in the biosynthesis of 2,3-diaminobutyric acid by gene inactivation and subsequent feeding experiments.In recent years, the sequences of numerous gene clusters involved in the synthesis of many secondary metabolites have become available for comparison and exploitation. The programmed manipulation of genes encoding enzymes in the biosynthetic pathways offers promise for redesigning existing antibiotic structures to create antibiotics with new activities and the ability to overcome bacterial resistance (50). Therefore, each newly analyzed gene cluster represents a new tool for combinatorial biosynthesis and can provide information about the synthesis of unusual building blocks, such as nonproteinogenic amino acids, acyl residues, and sugar moieties. An interesting group of secondary metabolites for such experiments seems to be bioactive lipopeptides isolated from streptomycetes. So far, only a few biosynthetic gene clusters corresponding to these lipopeptides have been isolated and characterized, such as the clusters for calcium-dependent antibiotic (CDA) from Streptomyces coelicolor (17), daptomycin from Streptomyces roseosporus (26), and A54145 from Streptomyces fradiae (24). By targeted modification and gene exchange, it is possible to generate new lipopeptide structures (12,25).Another member of this group of secondary metabolites is the antibiotic friulimicin that is produced by the actinomycete Actinoplanes friuliensis. This compound is highly active against multidrug-resistant gram-positive bacteria, such as methicillinresistant Staphylococcus aureus strains (4). The biosynthesis of this lipopeptide is catalyzed by nonribosomal peptide synthetases (NRPS) (15).The eight bioactive lipopeptides isolated from A. friuliensis (4) consist of 10 amino acids that form a ring structure with one exocyclic amino acid linked to an acyl residue of various chain lengths. The acyl residue is a branched-chain fatty acid with a ⌬cis3 double bond. The ...

Section: Vol 51 2007 Friulimicin Biosynthesis In Actinoplanes Friulmentioning

confidence: 99%

Sequencing and Analysis of the Biosynthetic Gene Cluster of the Lipopeptide Antibiotic Friulimicin in Actinoplanes friuliensis

Müller

Nolden

Gebhardt

et al. 2007

“…Instead, one of the ORFs (psf2) encodes a protein that has homology with the citrate synthase-like proteins FrbC and Pms (see Fig. S1 in the supplemental material) that are encoded in the biosynthetic gene clusters of the phosphonate natural products FR-900098 and phosphinothricin, respectively (5,14,24,48). These enzymes have been shown to add an acetyl group from Ac-CoA to the carbonyl group of phosphonopyruvate and phosphinopyruvate, respectively (14,24).…”

Section: Resultsmentioning

confidence: 99%

Different Biosynthetic Pathways to Fosfomycin in Pseudomonas syringae and Streptomyces Species

Kim

Metcalf

et al. 2012

cFosfomycin is a wide-spectrum antibiotic that is used clinically to treat acute cystitis in the United States. The compound is produced by several strains of streptomycetes and pseudomonads. We sequenced the biosynthetic gene cluster responsible for fosfomycin production in Pseudomonas syringae PB-5123. Surprisingly, the biosynthetic pathway in this organism is very different from that in Streptomyces fradiae and Streptomyces wedmorensis. The pathways share the first and last steps, involving conversion of phosphoenolpyruvate to phosphonopyruvate (PnPy) and 2-hydroxypropylphosphonate (2-HPP) to fosfomycin, respectively, but the enzymes converting PnPy to 2-HPP are different. The genome of P. syringae PB-5123 lacks a gene encoding the PnPy decarboxylase found in the Streptomyces strains. Instead, it contains a gene coding for a citrate synthase-like enzyme, Psf2, homologous to the proteins that add an acetyl group to PnPy in the biosynthesis of FR-900098 and phosphinothricin. Heterologous expression and purification of Psf2 followed by activity assays confirmed the proposed activity of Psf2. Furthermore, heterologous production of fosfomycin in Pseudomonas aeruginosa from a fosmid encoding the fosfomycin biosynthetic cluster from P. syringae PB-5123 confirmed that the gene cluster is functional. Therefore, two different pathways have evolved to produce this highly potent antimicrobial agent.

“…Interestingly, a role of OrfM in PTT synthesis was demonstrated by disruption of orfM, which led to a block in PTT production in S. viridochromogenes (34). As a matter of fact, the biochemical data shown here clearly point to the fact that the functional interactions of the PPT synthases are governed by the structural intactness of the cellular organization because neither purified enzymes nor crude extracts of dialyzed cell homogenates displayed synthetic activity in terms of formation of free or enzyme-bound reaction products.…”

Section: Discussionmentioning

confidence: 99%

“…They lie about 20 kb from phsA in the PTT biosynthesis gene cluster (34) and in addition are separated by a gene, orfM, encoding a membrane protein of unknown function (Fig. 2).…”

Section: Discussionmentioning

confidence: 99%

Phosphinothricin Tripeptide Synthetases in Streptomyces viridochromogenes Tü494

Schwartz

Grammel

Heinzelmann

et al. 2005

Self Cite

The tripeptide backbone of phosphinothricin (PT) tripeptide (PTT), a compound with herbicidal activity from Streptomyces viridochromogenes, is assembled by three stand-alone peptide synthetase modules. The enzyme PhsA (66 kDa) recruits the PT-precursor N-acetyl-demethylphosphinothricin (N-Ac-DMPT), whereas the two alanine residues of PTT are assembled by the enzymes PhsB and PhsC (129 and 119 kDa, respectively). During or after assembly, the N-Ac-DMPT residue in the peptide is converted to PT by methylation and deacetylation. Both phsB and phsC appear to be cotranscribed together with two other genes from a single promoter and they are located at a distance of 20 kb from the gene phsA, encoding PhsA, in the PTT biosynthesis gene cluster of S. viridochromogenes. PhsB and PhsC represent single nonribosomal peptide synthetase elongation modules lacking a thioesterase domain. Gene inactivations, genetic complementations, determinations of substrate specificity of the heterologously produced proteins, and comparison of PhsC sequence with the amino terminus of the alanine-activating nonribosomal peptide synthetase PTTSII from S. viridochromogenes confirmed the role of the two genes in the bialanylation of Ac-DMPT. The lack of an integral thioesterase domain in the PTT assembly system points to product release possibly involving two type II thioesterase genes (the1 and the2) located in the PTT gene cluster alone or in conjunction with an as yet unknown mechanism of product release.The peptide antibiotic phosphinothricin tripeptide (PTT; bialaphos) consists of the unusual amino acid phosphinothricin (PT) and two alanine residues (Fig. 1). It is produced by Streptomyces viridochromogenes and Streptomyces hygroscopicus (3,16). PTT penetrates bacterial cells as a prodrug via peptide uptake systems with subsequent release of PT, which, due to its structural similarity to glutamate, inhibits glutamine synthetase, a key enzyme of nitrogen metabolism (10). By contrast, plant cells can take up both PTT and PT directly. The inhibition of glutamine synthetase in their case results in the accumulation of ammonium ions, which are toxic to plant cells (42). Based on this mechanism, both PTT and PT have found commercial applications as broad-spectrum herbicides (known as Herbiace and Basta, respectively).Previous work on the biosynthesis of PTT indicated that it is derived from a precursor peptide, N-acetyl-demethylphosphinothricin tripeptide (N-Ac-DMPTT), which is assembled in a nonribosomal mechanism (1,11,32,43). In contrast to PTT, NAc-DMPTT contains N-acetyldemethylphosphinothricin (NAc-DMPT) instead of PT. N-Ac-DMPTT was never isolated, however, the occurrence of DMPT or DMPTT in fermentation broth of PTT-producing S. hygroscopicus and accumulation of N-AcDMPT in a PTT-nonproducing mutant of S. viridichromogenes (NTG1) obtained by mutational cloning point indirectly to its existence (1). Genetic complementation of the latter mutant in trans revealed the gene phsA (1, 43). The deduced gene product of phsA represented a 66-kDa no...