Activation of a silent cluster and isolation of a novel bioactive macrolide of Streptomyces ambofaciens ATCC2387771

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Interconnection between primary and secondary metabolism

Secondary metabolite biosynthesis is closely related to the so called “primary” metabolism, which is responsible for the production of the essential biological components, such as amino acids, nucleotides, vitamins and small organic acids. These molecules are produced by all living cells and are intermediates or end products of the metabolic pathways, or are the building blocks for essential macromolecules (Demain and Adrio, 2007).
It clearly appears that primary and secondary metabolisms are deeply interconnected, both in terms of precursor supply and through nutrient regulation (chapter 4 of introduction). For instance, antibiotics are synthesized from 12 precursor metabolites which are all used for the synthesis of the key cellular constituents (Rokem et al., 2007). These precursors come in turn from different carbon sources used by the cell, as well described by the bow-tie structure of Figure 2. Another link between primary and secondary metabolisms is represented by the common use of cofactors: ATP for the energy input to drive secondary metabolite biosynthesis, NADH and NADPH as electron acceptors (see also Fig. 2). In particular, ATP was shown to be a key cofactor of polyphosphate kinase Ppk, which is involved in the repression of antibiotic production in Streptomyces lividans (Ghorbel et al., 2006).

Mechanisms of bacterial resistance

Microorganisms that produce bioactive compounds show no sensitivity to their own product; therefore they must contain a self-protection system, which probably co-evolved together with the biosynthetic genes. Indeed, genes encoding enzymes for bacterial resistance are often identified inside secondary metabolite clusters. In the same time, non-producing microbes, to be able to compete and survive in their environment, need to have a bacterial resistance system (Hopwood, 2007). Three general strategies have been described and characterized, which might sometimes overlap and be used for self-protection and resistance at one time.

Active-efflux pumps

Levy and co-workers (McMurry et al., 1980) were the first to demonstrate that an active efflux pump, mediated by a transmembrane protein, named TET, was responsible for tetracycline resistance in E. coli. In gram-positive bacteria, these transmembrane proteins are located in the cytoplasmic membranes, while in gram-negative bacteria they are also found in the outer membranes (Walsh, 2003). The pumps can be driven by proton motive force or by ATP hydrolysis, in order to export antibiotics or other bioactive compounds, thus decreasing their concentration inside the cell. The pumps can be specific for one chemical class, like the TET pump for tetracyclines, but more often they have a broader range of substrate specificity, typically found in multi-drug pathogens (Van Bambeke et al., 2000).
Bacterial efflux pumps can be divided in five protein families, according to the amino acid sequence identity and mode of energy coupling: the major facilitator subfamily (MFS), the small multidrug regulator family (SMR), the resistance-nodulation-cell division family (RND) and the multidrug and toxic compound extrusion family (MATE) use an electrochemical gradient of cations (H+ or Na+) for drug transport; whereas the ATP-binding cassette family (ABC) hydrolyzes ATP (Mascaretti, 2003). Some ABC transporters, whose genes were found in secondary metabolite clusters, have been reported to have a resistance-independent role. OleB is an ABC transporter from S. antibioticus responsible for the secretion of the glycosylated inactive form of the antibiotic oleandomycin (Hernandez et al., 1993; Quiros et al., 1998). Recently, Menges and co-workers (2007) have characterized the ABC transporter Tba from Amycolatopsis balhimycin, which exports the glycopeptide balhimycin, without taking part in any resistance mechanism to the antibiotic. Intracellular accumulation of balhimycin was observed in the tba mutant strain.

Enzymatic inactivation of the antibiotic

Another strategy that bacteria can employ is to inactivate the secondary metabolite changing its chemical structure. Synthetic antibacterials, as fluoroquinolones or oxazolidinones, are the only class not affected by this resistance mechanism (Walsh, 2003). β-lactamases are well characterized bacterial enzymes that hydrolyze the amide bond of the four-membered β-lactam ring of penicillins, cephalosporins and carbapenems, which are no longer able to bind and inactivate their cellular target, the transpeptidases (Wilke et al., 2005). With an analogous mechanism the reactive epoxide ring of fosfomycin is opened by a glutathione S-transferase (Arca et al., 1990). Three types of enzymatic modification are known to inactivate aminoglycoside antibiotics which specifically interact with the 16S rRNA, inhibiting protein biosynthesis (Kotra et al., 2000). The -OH groups can undergo phosphorylation or adenylation, the -NH2 group an acetylation; all these reactions are irreversible.

Modification of the target site

Bacteria are also able to modify the drug target, without affecting any cellular function. These modifications often occur to the cell wall components or the ribosomal subunits (Walsh, 2003). Methicillin-resistant Staphylococcus aureus (MRSA) strains have acquired, through horizontal transfer, a 30-40 kb mobile DNA element containing the mecA gene which encodes a novel type of transpeptidase, insensitive to all β-lactam antibiotics (Hiramatsu et al., 2001). Macrolides interact with the 23S rRNA in the 50S ribosomal subunit: mono or di-methylation of the amino group of the adenine residue A2058 decrease the affinity of the antibiotic for the RNA. This mechanism was identified and characterized in erythromycin and tylosin producer strains (Zalacain and Cundliffe, 1989).

Gene inactivation and comparative metabolic profiling approach

This alternative method does not necessarily require structural predictions of the expected metabolite and directly indicates the link between the metabolite and the orphan biosynthetic cluster, identified by genome mining. A gene of interest in the cluster is disrupted and the metabolic profile of the mutant strain is compared with that one of the wild type strain, employing analytical techniques such as LC-MS or DAD-HPLC to identify the potential natural product. The limits can derive from the level of expression, which is the case for silent clusters, and from the construction of the mutant strain, since for many organisms genetic tools are still unavailable. This method was applied successfully for the discovery of the cathecolic siderophore bacillibactin, from Bacillus subtilis (May et al., 2001) and for the discovery of three new metabolites of S. coelicolor, isogermicidin A, B and germicidin C (Song et al., 2006) (See Fig. 7).


Screening of rare genera of actinomycetes and other untapped sources

In order to enlarge and increase the chance to discover novel secondary metabolites, in the last few years, industrial and academic attention was focused on improving the traditional screening approach aiming to exploit rare and untapped sources of microbial diversity.
Actinomycetes are responsible for the production of more than half of the discovered bioactive molecules (Berdy, 2005), especially those isolated from terrestrial environments. More than 10% of these secondary metabolites are isolated from the so called “rare” actinomycetes, which belong to the families of Micromonosporaceae (e.g. Micromonospora and Actinoplanes), Pseudonocardiaceae (e.g. Amycolaptopsis and Saccharopolyspora), Thermomonosporaceae (e.g. Actinomadura), Nocardiaceae (e.g. Nocardia) and Streptosporangiaceae (e.g. Streptosporangium) (Fig. 10). The term “rare” does not refer to their abundance in the environment, but mostly to their isolation frequency using conventional methods, which is much lower compared to the genus of Streptomyces. The importance of these strains is demonstrated by the discovery of many successful antibacterial agents derived from them, such as erythromycin from Saccharopolyspora erythraea or vancomycin from Amycolatopsis orientalis (Lazzarini et al., 2001).

Table of contents :

1. General context
2. Secondary metabolism and microbial natural products
2.1 Interconnection between primary and secondary metabolism
2.2 The antibiotics
2.3 Mechanisms of bacterial resistance
2.3.1 Active-efflux pumps
2.3.2 Enzymatic inactivation of the antibiotic
2.3.3 Modification of the target site
3. Strategies to discover novel natural bioactive molecules
3.1 Genome mining
3.1.1 OSMAC approach
3.1.2 Genomisotopic approach
3.1.3 Gene inactivation and comparative metabolic profiling approach
3.1.4 Heterologous expression
3.1.5 Modification of the regulatory network
3.2 Metagenomics
3.3 Combinatorial biosynthesis
3.4 Screening of rare genera of actinomycetes and other untapped sources
4. Insights in the regulation of secondary metabolism
4.1 Nutritional regulation
4.2 Global regulation
4.3Extracellular signals
4.3.1 γ-butyrolactones
4.3.2 Furans
4.3.3 PI factor
4.4 Pathway-specific regulators
4.4.1 SARP regulators
4.4.2 LAL regulators
5. Polyketide synthases versus non-ribosomal peptide synthases
5.1 The core domains
5.2 The auxiliary domains
5.3 The initiation and termination domains
5.3.1 Type II thioesterase
6. Polyketide synthases
6.1 Typology of polyketide synthases
6.1.1 Modular type I PKS
6.1.2 Iterative type I PKS
6.1.3 Type II PKS
6.1.4 Type III PKS
6.2 Polyketide-tailoring genes
6.2.1 Glycosyltransferases
6.2.2 Methyltransferases and oxygenases
7. Streptomyces, a prolific producing genus
7.1 General characteristics
7.2 Streptomyces ambofaciens
8. Objectives of the thesis
Chapter 1 In silico characterization of a large type I PKS cluster
1.1 Sequence analysis of the enzymatic domains
1.1.1 Ketosynthase domains
1.1.2 Acyl transferase domains
1.1.3 Acyl carrier protein domains
1.1.4 Ketoreductase domains
1.1.5 Dehydratase domains
1.1.6 Enoyl reductase domains
1.1.7 Thioesterase domain
1.2 Prediction of the linear polyketide structure
1.3 Analysis of the other genes putatively involved in the biosynthesis
1.3.1 Glycosyltransferase and sugar genes
1.3.2 Cytochrome P450
1.3.3 Thioesterase type II
1.3.4 Additional genes
1.4 The resistance genes
1.5 The regulatory genes
1.5.1 A two component system
1.5.2 A LAL regulator
Chapter 2 Activation of a silent cluster and isolation of a novel bioactive macrolide of Streptomyces ambofaciens ATCC2387771
2.1 Activation of a silent cluster
2.2 Detection and isolation of a novel metabolite
2.3 The detected metabolites are synthetised by the type I PKS cluster
2.4 Structural elucidation of the novel compounds
2.5 Biological properties of the macrolide sambomycin
Article: Discovery of a novel macrolide in Streptomyces ambofaciens by awaking a sleeping giant
Chapter 3 Characterization of sambomycin biosynthesis
3.1 Limits of the sambomycin cluster
3.2 The phosphopantetheinyl transferase
3.3 The cytochromes P450
3.4 The acyl-CoA synthetase and the acyl-CoA carboxylase
3.5 Secretion of sambomycin
3.6 The resistance genes
Chapter 4 Regulation of the sambomycin cluster
4.1 The LAL regulator is an activator of the sambomycin gene cluster
4.2 Searching for the targets of the LAL regulator
4.3 In vitro characterization of the LAL regulator
4.4 Sambomycin production in R2 medium
4.5 Effect of SAMR0484 on spiramycin production
4.6 The two component system
1. Sambomycin, a peculiar 50-membered macrolide
1.1 A new cyclization mechanism for polyketides
1.2 Biosynthesis of an unusual extender unit
1.3 Glycosylation of the sambomycin aglycones
2. Macrolides: biological properties and resistance mechanisms
2.1 Biological properties
2.2 Resistance mechanisms
3. The LAL regulator is a positive pathway-specific regulator of the sambomycin cluster
4. Combinatorial biosynthesis of the sambomycin gene cluster
5. The role of sambomycin in nature
6. Streptomyces ambofaciens and its fascinating secondary metabolites


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