Abstract
Actinomycetes are soil-dwelling Gram-positive bacteria, industrially relevant as producers of a wide range of bioactive secondary metabolites, including many antibiotics of clinical and commercial importance.
Keywords
- Antibiotic production
- Actinomycetes
- genetic engineering
- regulation
- heterologous expression
1. Introduction
The discovery by Alexander Fleming of penicillin opened up a completely new era of chemotherapy. Antibiotics have saved a large number of lives and also contributed to the increase in life expectancy. They are mainly produced by the fermentation of fungi (e.g.,
Actinomycetes are soil-dwelling Gram-positive bacteria that have extensive arsenals of secondary metabolites, metabolism products that, differently from primary metabolites such as vitamins, amino acids, nucleotides, etc., are not essential for the bacterial growth, at least in laboratory conditions; indeed, many mutants in antibiotic biosynthesis have been generated revealing that they are still vital and were used as models to understand molecular mechanisms governing antibiotic production.
Secondary metabolites include antitumorals (e.g., doxorubicin and bleomycin), antifungals (e.g., amphotericin B and nystatin), immunosuppressives (e.g., FK-506 and rapamycin), insecticides (e.g., spinosyn A and avermectin B), herbicides (e.g., phosphinotricin) and many antibiotics of clinical and commercial importance.
This chapter reviews common regulatory mechanisms that control antibiotic production in actinomycetes and both genetic and physiological methods to improve antibiotic yields.
2. Antibiotics and their targets
Antibiotics are molecules that selectively inhibit bacterial growth without damaging the eukaryotic organisms. The selectivity of action of these substances is given by the fact that they interfere with processes essential for the bacterial cell and absent or different in the eukaryotic cell.
Antibiotics essentially target bacterial structures or functions, such as cell wall biosynthesis (e.g., vancomycin), translation (e.g., streptomycin), RNA transcription (e.g., rifampicin), DNA replication and synthesis (e.g., novobiocin and metronidazole), membrane (polimyxins), and in general they inhibit bacterial growth (Figure 1).
Among the antibiotics that target the cell wall, glycopeptides are a class of drugs produced by Actinomycetes and are composed of glycosylated cyclic or polycyclic non-ribosomal peptides. Glycopeptides bind to the dipeptide D-alanyl--D-alanine (D-Ala-D-Ala) within the cell wall of Gram-positive bacteria preventing the addition of new units to the peptidoglycan and inhibiting the peptidoglycan synthesis. Significant glycopeptide antibiotics include the anti-infective antibiotics vancomycin, teicoplanin, telavancin, ramoplanin, decaplanin, and the antitumor antibiotic bleomycin. Vancomycin is used as antibiotic of last resort for infections with methicillin-resistant
Cycloserin, produced by
Penicillins and cephalosporins are the most important antibiotics obtained from fungi P
Many different classes of antibiotics block protein synthesis. Tetracycline (produced by
Rifampicin is a semisynthetic antibiotic produced by the fermentation of a strain of
Novobiocin, also known as albamycin or cathomycin, is an aminocoumarin antibiotic that is produced by the actinomycete
Polymyxins are antibiotics produced by nonribosomal peptide synthetase systems in Gram-positive bacteria, such as
A few antibiotics are produced by chemical synthesis (e.g., quinolone and metronidazole). Quinolones are synthetic, bactericidal agents that inhibit the enzyme topoisomerase II, a DNA gyrase necessary for the replication of the microorganism. Topoisomerase II enzyme produces a negative supercoil on DNA, permitting transcription or replication; thus, by inhibiting this enzyme, DNA replication and transcription are blocked.
Metronidazole is a synthetic compound used in the treatment of infections caused by Gram-negative anaerobic bacteria and protozoa. It was shown to induce base-pair substitutions [7] and to be a potent mutagen in bacteria and low eukaryotic systems [8].
3. Genetic organization of antibiotic biosynthesis
Genes involved in the biosynthesis of antibiotics and other secondary metabolites are typically clustered within the respective genome or, rarely, in circular plasmid. A biosynthetic gene cluster contains many genes, often located within a few thousand base pairs of each other that encode for proteins participating in a metabolic pathway that leads to the production of a particular secondary metabolite (Figure 2).
The size of gene clusters can vary significantly, from a few to several hundred genes. Commonly, 10–50 genes are required for the synthesis of an antibiotic. A gene cluster usually contains genes involved in the precursor biosynthesis, tailoring steps, export, resistance, and regulation.
Some peptide antibiotics are formed by amino acidic precursors that are then assembled by non-ribosomal machinery. In the case of non-ribosomal peptide synthesis, non-proteinogenic amino acids, such as 3,5-dihydroxyphenylglycine (DPG) and 4-hydroxyphenylglycine (HPG), can be found. In many cases, the backbone of the antibiotic is modified by the so-called tailoring steps, i.e., chlorination, methylation, glycosylation, N-acylation, and so on.
The polyketides are another class of natural antibiotics synthesized through the decarboxylative condensation of malonyl-CoA-derived extender units in a process similar to the fatty acid synthesis. The polyketide chains produced by a minimal polyketide synthase are often further modified (e.g., glycosylated) into bioactive natural products.
Actinorhodin and undecylprodiginines are two of the antibiotics produced by
Usually, a gene cluster for antibiotic production encodes for regulatory genes, named pathway specific, with positive or negative control on the cluster. Moreover, there could be some pleiotropic regulators that affect antibiotic production, morphological development, and primary metabolism of the bacteria. As examples, actinorhodin biosynthesis is regulated by the transcriptional activator ActII-ORF4 [9–10], while the undecylprodigiosin pathway is regulated via a minicascade of two cluster specific regulators, with RedZ activating the expression of
In the bacteria producers of antibiotics, resistance genes are necessary to avoid the suicide, while transport genes are used to export the antibiotic outside the cell. Resistance to antibiotics can be caused by several general mechanisms (Figure 3): increased efflux or decreased influx of the antibiotic, target site alteration, target amplification, or antibiotic inactivation/modification [12].
The production of β-lactamase is a common mechanism found in many pathogens. This enzyme is capable of hydrolyzing and destroying the β-lactam ring of the antibiotic avoiding its antibacterial activity.
As example of alteration of the target site, the methylation of an adenine of the ribosomal RNA prevents the interaction between macrolides and ribosome.
Resistance to glycopeptides is frequently due to the presence of genes encoding for enzymes involved in the synthesis of alternative forms of peptidoglycan, with low affinity for glycopeptides. For example, the C terminal D-Ala-D-Ala is replaced by D-Ala-D-Lac or D-Ala-D-Ser [13]. Glycopeptide resistance has been explored in three different actinomycetes:
The best known efflux system regards the tetracycline, the gene
4. Morphological and physiological differentiation
Actinomycetes represent an important model of bacterial development; they display an unusual complex life cycle with different cell types (spores, vegetative and reproductive mycelium) and with the morphological changes strictly connected to the physiological differentiation. The understanding of Actinomycetes biology has been based on extensive studies on the model organism
Genetic studies of
Morphological differentiation in Actinomycetes is strictly related to physiological differentiation: indeed the onset of morphological differentiation generally coincides with the production of secondary metabolites.
If on one hand, many factors with pleiotropic activity were identified as key players to control both morphological and physiological differentiation in
5. General approaches to overproduce natural antibiotic
Natural bacterial strains often produce only small amounts of antibiotic (µg/l), while production rates in the range of g/l are needed to set up a cost-effective production process. In order to increase the industrial yield of products, different strategies can be adopted.
Random mutagenesis for the selection of overproducing mutants remains the preferred method when molecular genetic tools have not been developed for the producer microorganism. Although random mutagenesis and screening procedures have been widely used for genetic improvement of antibiotic production, there are certain disadvantages, such as the time necessary to obtain a favorable mutation. The knowledge-driven genetic manipulation can make the optimization of strains and conditions more efficient.
In general, many approaches have been used to improve antibiotic production as schematically represented in Figure 5.
The tuning of media composition and fermentation conditions (carbon source, phosphate and nitrogen concentrations, pH, temperature) and the supply of specific precursors are the first approaches used in order to increase the yield in fermentation. Moreover, genetic manipulation of primary or secondary metabolism can be applied. Regarding primary metabolism, mutations in pathways for amino acids or other molecules that are used as precursors in antibiotic biosynthesis or mutations in the ribosome can improve indirectly the yield of secondary metabolites.
Regarding secondary metabolism, the over-expression of biosynthetic genes, such as the genes that codify for antibiotic specific precursors, the over-expression of pathway-specific positive regulators or the inactivation of pathway-specific negative regulators can result in an increase of antibiotic yield. Increasing self-resistance levels in producing organisms has been also used for improving production yields. Manipulation of pleiotropic regulators, involved in both primary and secondary metabolisms, was also successfully used to improve antibiotic yields.
The production of antibiotics in some
6. Media composition effect on antibiotic production
In bacteria, several sugars can be used as carbon sources. Although glucose is often an excellent carbon and energy source for microbial growth, it is infrequently utilized as the major carbon and energy source in secondary metabolite fermentation. When incubated in media containing glucose and another carbon source, bacteria metabolize first glucose that represses the transcription of genes required for the utilization of the secondary carbon sources. When glucose is exhausted, the metabolism of the second carbon source is activated, and generally this correlates with the onset of antibiotic production. This phenomenon is referred to as carbon catabolite repression and is mediated via components of the phosphoenolpyruvate:carbohydrate phosphotransferase system, which transports and phosphorylates carbohydrates.
Glucose repression in
Glucose and other carbon sources have been found to suppress production of many secondary metabolites, e.g., actinorhodin in
cAMP, ATP, and adenosine were reported to regulate antibiotic production [26]. When glucose is the carbon source, inhibition of the cAMP-producing enzyme, adenylate cyclase, occurs and cAMP levels are low (Figure 7). cAMP is important to activate the transcription factor cAMP receptor protein (CRP). In the absence of cAMP, CRP does not activate the transcription of target genes. When glucose is absent, cAMP is accumulated and it forms a complex with CRP, thereby activating the expression of a large number of genes, including some encoding enzymes that can supply energy independently from glucose and trigger spore germination, aerial mycelium formation, and actinorhodin production [27–30]. Extracellular ATP (exATP) was reported to massively increase actinorhodin and lightly increase undecylprodigiosin yields in
Several microorganisms’ nutrients, such as phosphate and nitrogen compounds, affect the production of antibiotics and other secondary metabolites. The lack of specific nutrients is perceived by microorganisms through complex signaling mechanisms. The study of these pathways is often the key in the understanding of regulatory processes underlying the synthesis of secondary metabolites.
Streptomycetes sense and respond to the stress of phosphate starvation via the two-component PhoR-PhoP signal transduction system (Figure 8). In
The biosynthesis of many antibiotics is very sensitive to phosphate. In
A simple strategy to improve antibiotic production is to alter the PhoP concentration, by disrupting the
High concentration of nitrogen sources (such as ammonium or amino acids) also suppresses the secondary metabolism. Complex fermentation media include proteins as nitrogen sources. For example, production of streptomycin antibiotic in
In
Nitrogen metabolism under phosphate control exerted by the binding of PhoP to the promoter region of
In some bacteria of the phylum Actinobacteria, such as
7. Genetic engineering
Antibiotic production can be improved by metabolic engineering in several ways. A flux increase in the biosynthetic pathway can be improved by directed mutagenesis or by elevated precursor availability. As an example, acetyl-CoA carboxylase was cloned into an expression vector and introduced into
In
A promising method to increase antibiotic production is the ribosome engineering developed by Ochi and colleagues [50]. This method consists of the isolation of spontaneous mutants that are resistant to sub-lethal levels of antibiotic that targets the ribosomal proteins (such as streptomycin, gentamicin, kanamicin, cloramphenicol) or the RNA polymerase. Rifampicin resistance mutation in
Some actinomycetes possess two
The constitutive expression of
The basic knowledge of phosphate and nitrogen metabolic pathway can be used for rational manipulation. For example, amplification of
8. Secondary metabolism to control antibiotic production
Antibiotic production is controlled at two main levels: pleiotropic regulators controlling the production of more than one antibiotic and cluster-situated regulator modulating the antibiotic biosynthetic genes of the cluster in which they are included.
The complex gene cluster for the biosynthesis of each antibiotic usually contains regulatory and resistance genes. Typically, there may be more than one such pathway-specific regulatory gene per cluster. Overexpression of positive regulators or deletion of genes that codify for repressors can be a strategy to improve antibiotic production.
Among the regulatory genes, two-component systems are the most important transduction signal mechanism in bacteria. Typically, the two-component system comprises a membrane-bound histidine kinase and a cognate response regulator. The receptor senses specific environmental stimuli, it auto-phosphorylates and activates by phosphorylation the response regulator that mediates the cellular response, mainly through the transcriptional regulation of target genes in the cluster for antibiotic [56].
The AbrC1 protein is a histidine kinase part of a two-component system in
Other examples of regulators are those of the StrR and LuxR families. StrR was initially identified in
The filamentous actinomycete
The increase of self-resistance levels in producing organisms was used for improving production yields. This strategy was used for
To increase the balhimycin production by
9. γ butyrolactones to control the onset of antibiotic biosynthesis
The production of antibiotics in some
So far, various γ-butyrolactone molecules, synthases, and receptors have been identified [67–68].
γ butyrolactones have been applied for improvement of secondary metabolite production. A factor from
10. Heterologous expression of actinomycetes biosynthetic gene clusters
Many antibiotic producing actinomycetes are recalcitrant to manipulation and suitable protocols for their genetic manipulation are not always available. The transfer of the genetic information for secondary metabolite production from the original producer to a model host represents a successful strategy to manipulate biosynthetic gene clusters. Heterologous expression of large biosynthetic pathways could be also useful in all those cases in which bacteria are not cultivable or to produce cryptic metabolites, revealed by genome sequencing and mining. Actinomycetes are characterized by large genomes that are GC-rich [70] and their genes are not easily expressed in
In some cases, a successful heterologous expression of actinomycetes biosynthetic gene clusters was obtained after changing fermentation conditions, that is, feeding with a biosynthetic precursor, minimizing background endogenous activities, or after cloning strong promoters upstream of production genes weakly transcribed (for a review see [75]). Heterologous expression in amenable hosts can be useful to exploit and to explore the genetic potential of actinomycetes.
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