The herbicide phosphinothricin-tripeptide - a model for the analysis of the evolution of secondary metabolism in bacteria

WOLFGANG WOHLLEBEN
KLAUS SCHAD
EVA SCHINKO
Lehrstuhl für Mikrobiologie/Biotechnologie
Universität Tübingen
Auf der Morgenstelle 28
D-72076 Tübingen
wowo@biotech.uni-tuebingen.de
http://www.mikrobio.uni-tuebingen.de/ag_wowo/index.php

References
D. Schwartz, S. Berger, E. Heinzelmann, K. Muschko, K. Welzel und W. Wohlleben (2004). The biosynthetic gene cluster of the herbicide phosphinothricin tripeptide from Streptomyces viridochromogenes Tü494. Appl. Environ. Microbiol. 70, 7093-102.
The antibiotic phosphinothricin tripeptide (PTT) consists of two molecules of L-alanine and one molecule of the unusual amino acid phosphinothricin (PT) which are nonribosomally combined. The bioactive compound PT has bactericidal, fungicidal, and herbicidal properties and possesses a C-P-C bond, which is very rare in natural compounds. Previously uncharacterized flanking and middle regions of the PTT biosynthetic gene cluster from Streptomyces viridochromogenes Tü494 were isolated and sequenced. The boundaries of the gene cluster were identified by gene inactivation studies. Sequence analysis and homology searches led to the completion of the gene cluster, which consists of 24 genes. Four of these were identified as undescribed genes coding for proteins that are probably involved in uncharacterized early steps of antibiotic biosynthesis or in providing precursors of PTT biosynthesis (phosphoenolpyruvate, acetyl-coenzyme A, or L-alanine). The involvement of the genes orfM and trs and of the regulatory gene prpA in PTT biosynthesis was analyzed by gene inactivation and overexpression, respectively. Insight into the regulation of PTT was gained by determining the transcriptional start sites of the pmi and prpA genes. A previously undescribed regulatory gene involved in morphological differentiation in streptomycetes was identified outside of the left boundary of the PTT biosynthetic gene cluster.

D. Schwartz, N. Grammel, E. Heinzelmann, U. Keller and W. Wohlleben (2005). Phosphinothricin tripeptide synthetases in Streptomyces viridochromogenes Tü494.
Antimicrob. Agents Chemother. 49, 4598-607.
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.

S. Eys, D. Schwartz, W. Wohlleben and E. Schinko. Three Thioesterases are involved in the Biosynthesis of Phosphinothricin Tripeptide in Streptomyces viridochromogenes Tü494, submitted.
Phosphinothricin tripeptide (PTT) is a peptide antibiotic, produced by Streptomyces viridochromogenes Tü 494, which is synthesized by non-ribosomal peptide synthetases. The PTT biosynthetic gene cluster contains three peptide synthetase genes phsA, phsB and phsC. Each of these peptide synthetases comprises only one module. Neither in PhsB nor in PhsC is a typical C-terminal thioesterase domain present. In contrast, a single thioesterase motif GXSXG has been identified in the N-terminus of the first peptide synthetase PhsA. In addition, two external thioesterase genes, the1 and the2, are located within the PTT biosynthetic gene cluster. To analyze the thioesterase function as well as the assembly of the peptide synthetases within the PTT biosynthesis, several mutants were generated and analysed. A phsA deletion mutant MphsA was complemented with two in the thioesterase motif mutated phsA constructs. In one construct, the thioesterase motif comprising 45 amino acids of phsA was deleted. In a second construct, the conserved serine residue of the GXSXG motif was replaced by an alanine. In both cases, complementation of MphsA did not restore PTT biosynthesis, revealing that the thioesterase motif in the N-terminus of PhsA is required for PTT production, probably for the release of the antibiotic from the peptide synthetase complex. In contrast, The1 and The2 might have an editing function as an interruption of the the1 and the2 genes led to reduced PTT production, whereas an overexpression of both genes in the wild type enhanced PTT yield.

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