Efthimia Stegmann

The Biosynthesis of Vancomycin-Type Glycopeptide Antibiotics—A Model for Oxidative Side-Chain Cross-Linking by Oxygenases Coupled to the Action of Peptide Synthetases

Vancomycin (Scheme 1) and teicoplanin are “last resort” antibiotics for the treatment of severe infections with enterococci and methicillin-resistant Staphylococcus aureus (MRSA) strains. However, over the past 15 years, vancomycin-resistant enterococci (VRE) and intermediate resistant staphylococci (VISA) have emerged. One approach to counter such resistance is the generation of novel glycopeptides with altered antibiotic activity by combinatorial biosynthesis, that is, the reprogramming of glycopeptide biosynthesis, a basic requirement for which is the understanding of the process. The recent sequencing of glycopeptide biosynthesis gene clusters has provided deeper insights into glycopeptide antibiotic biosynthesis. Subsequent biosynthesis investigations have been performed by heterologous expression and characterization of enzymes, as well as gene inactivation combined with the characterization of accumulated peptide intermediates. The latter approach has mainly been performed with balhimycin (Scheme 1) produced by Amycolatopsis balhimycina, formerly referred to as A. mediterranei. Glycopeptides are assembled from amino acid precursors by the action of nonribosomal peptide synthetases (NRPS), and modified by the action of so-called “tailoring enzymes”. The tailoring enzymes include three P450-dependent oxygenases responsible for the cross linking of the aromatic side chains, glycosyl transferases for the attachment of carbohydrate residues and an N-methyl transferase that introduces a methyl group at the amino group of leucine. The three oxidative sidechain cyclizations were assigned to three oxygenase genes (oxyA/B/C). A sequence for the assembly of the glycopeptide aglycon from linear peptide precursors was deduced as: 1) CDring (OxyB), 2) DE-ring (OxyA) and 3) AB-ring (OxyC) coupling. Whether oxidative formation of AB, CD and DE rings occurs before or after cleavage of the linear peptide from the NRPS complex has not been determined. Our previous observation of considerable amounts of various linear and cyclized hexaand heptapeptides isolated from oxygenase mutants (oxyA/B/ C) cast doubt on whether side-chain-cyclized hexapeptides were degradation products or were rather related to true biosynthesis intemediates. 7] Here we report on the characterization of metabolites accumulated from balhimycin biosynthesis mutants inactivated in the central step of heptapeptide formation. These studies lead to the important conclusion that peptide assembly on the NRPS appears to be intimately coupled to the action of the oxygenases (OxyA/B/C). Two A. balhimycina in-frame deletion mutants, described in earlier work and both inactivated in different stages of heptaScheme 1. Structural formulae of glycopeptide antibiotics balhimycin and vancomycin.

Biosynthesis of Chloro-β-Hydroxytyrosine, a Nonproteinogenic Amino Acid of the Peptidic Backbone of Glycopeptide Antibiotics

The role of the putative P450 monooxygenase OxyD and the chlorination time point in the biosynthesis of the glycopeptide antibiotic balhimycin produced by Amycolatopsis balhimycina were analyzed. The oxyD gene is located directly downstream of the bhp (perhydrolase) and bpsD (nonribosomal peptide synthetase D) genes, which are involved in the synthesis of the balhimycin building block beta-hydroxytyrosine (beta-HT). Reverse transcriptase experiments revealed that bhp, bpsD, and oxyD form an operon. oxyD was inactivated by an in-frame deletion, and the resulting mutant was unable to produce an active compound. Balhimycin production could be restored (i) by complementation with an oxyD gene, (ii) in cross-feeding studies using A. balhimycina JR1 (a null mutant with a block in the biosynthesis pathway of the building blocks hydroxy- and dihydroxyphenylglycine) as an excretor of the missing precursor, and (iii) by supplementation of beta-HT in the growth medium. These data demonstrated an essential role of OxyD in the formation pathway of this amino acid. Liquid chromatography-electrospray ionization-mass spectrometry analysis indicated the biosynthesis of completely chlorinated balhimycin by the oxyD mutant when culture filtrates were supplemented with nonchlorinated beta-HT. In contrast, supplementation with 3-chloro-beta-HT did not restore balhimycin production. These results indicated that the chlorination time point was later than the stage of free beta-HT, most likely during heptapeptide synthesis.

Development of three different gene cloning systems for genetic investigation of the new species Amycolatopsis japonicum MG417-CF17, the ethylenediaminedisuccinic acid producer

For the first time gene cloning systems have been developed for Amycolatopsis japonicum. Direct transformation, polyethyleneglycol (PEG) induced protoplast transformation and conjugal transfer was established for A. japonicum MG417-CF17, the ethylenediaminedisuccinic acid (EDDS) producer. The direct transformation procedure was modified to introduce DNA. The most important parameter for an efficient DNA uptake was the age of the culture. Using of mycelium from 36-h old cultures resulted in the highest transformation frequencies. Further, conditions for transformation of A. japonicum protoplasts were established. The efficiency of transformation depended mainly on the source of PEG and the components of the regeneration agar. The replicative plasmid pULVK2A carrying pA-rep and the apramycin resistance gene was transferred into the EDDS producer with a frequency of 0.38 colonies microg(-1) DNA by using the direct transformation procedure and with a frequency of 0.56 colonies microg(-1) DNA by using the PEG induced protoplast transformation. The plasmid was genetically stable, and could easily be reisolated from A. japonicum. We also demonstrated that conjugal transfer of the plasmid pSET152 from Escherichia coli ET12567 (pUB307) to Amycolatopsis spores is possible. The plasmid pSET152 integrated in the A. japonicum chromosome. A titre of 2.4 x 10(-4) exconjugants per recipient was obtained.

The border sequence of the balhimycin biosynthesis gene cluster from Amycolatopsis balhimycina contains bbr, encoding a StrR-like pathway-specific regulator

Balhimycin, produced by the actinomycete Amycolatopsis balhimycina DSM5908, is a glycopeptide antibiotic highly similar to vancomycin, the antibiotic of ‘last resort’ used for the treatment of resistant Gram-positive pathogenic bacteria. Partial sequence of the balhimycin biosynthesis gene cluster was previously reported. In this work, cosmids which overlap the region of the characterized gene cluster were isolated and sequenced. At the ‘left’ end of the cluster, genes were identified which are involved in balhimycin biosynthesis, transport, resistance and regulation. The ‘right’ end border is defined by a putative 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (dahp) gene. The proximate gene is similar to a type I polyketide synthase gene of the rifamycin producer Amycolatopsis mediterranei indicating that another biosynthesis gene cluster might be located directly next to the balhimycin gene cluster. The newly identified StrR-like pathway-specific regulator, Bbr, was characterized to be a DNA-binding protein and may have a role in balhimycin biosynthesis. Purified N-terminally His-tagged Bbr shows specific DNA-binding to five promoter regions within the gene cluster. By in silico analysis and by comparison of the DNA sequences binding Bbr, conserved inverted repeat sequences for the Bbr-binding site are proposed. The putative Bbr consensus sequence differs from that published for StrR.

Differential proteomic analysis reveals novel links between primary metabolism and antibiotic production in Amycolatopsis balhimycina

A differential proteomic analysis, based on 2‐DE and MS procedures, was performed on Amycolatopsis balhimycina DSM5908, the actinomycete producing the vancomycin‐like antibiotic balhimycin. A comparison of proteomic profiles before and during balhimycin production characterized differentially and constitutively expressed protein isoforms, which were associated with 203 ORFs in the A. balhimycina genome. These data, providing insights on the major metabolic pathways/molecular processes operating in this organism, were used to compile 2‐DE reference maps covering 3–10, 4–7 and 4.5–5.5 pH gradients available over the World Wide Web as interactive web pages (http://www.unipa.it/ampuglia/Abal‐proteome‐maps). Functional clustering analysis revealed that differentially expressed proteins belong to functional groups involved in central carbon metabolism, amino acid metabolism and protein biosynthesis, energetic and redox balance, sugar/amino sugar metabolism, balhimycin biosynthesis and transcriptional regulation or with hypothetical and/or unknown function. Interestingly, proteins involved in the biosynthesis of balhimycin precursors, such as amino acids, amino sugars and central carbon metabolism intermediates, were upregulated during antibiotic production. qRT‐PCR analysis revealed that 8 out of 14 upregulated genes showed a positive correlation between changes at translational and transcriptional expression level. Furthermore, proteomic analysis of two nonproducing mutants, restricted to a sub‐set of differentially expressed proteins, showed that most proteins required for the biosynthesis of balhimycin precursors are downregulated in both mutants. These findings suggest that primary metabolic pathways support anabolic routes leading to balhimycin biosynthesis and the differentially expressed genes are interesting targets for the construction of high‐yielding producer strains by rational genetic engineering.