Project Topics
Get Complete Project Material File(s) Now! »
From genes to molecules
Classically the approach used for the discovery of specialized metabolites consisted in the screening of supernatant cultures, or cell extracts for specific bioactivities (such as antibiotic or enzyme inhibition), followed by bio-guided fractionation and purification of the compound of interest, which are then structurally characterized by spectroscopic methods (UV/IR spectroscopy, mass spectrometry, nuclear magnetic resonance (NMR) and X-ray crystallography). Improvements to this approach have been developed such as dereplication (37,38) ,which scans crude extracts for the presence of known compounds in order to focus on the discovery of novel ones.
More recently and stimulated by developments in NGS and the resulting rich dataset of microbial genomes, genome mining strategies have emerged as an alternative to classical bio-guided approaches for the discovery of microbial specialized secondary metabolites (39–41). The genome analysis is carried out by searching homologs of genes and proteins involved in established biosynthetic pathways. The screening is achieved with bioinformatics tools such as Blast, antiSMASH 3.0 or Bagel3 (42,43). This approach has revealed that genes involved in the biosynthesis of specialized metabolites are typically clustered in the genomes of bacteria and fungi. Furthermore, it is also clear that larger microbial genomes, such as those of Actinobacteria, appear to carry more biosynthesis gene clusters when compared to smaller ones, revealing that actinobacteria may produce even more specialized metabolite than expected (12,15,44–48).
Genomic sequences also revealed, many biosynthesis gene clusters, termed cryptic or silent, do not appear to be significantly expressed under standard laboratory conditions of cultivation (1), in which the producing organisms are generally cultivated in conditions lacking environmental cues such as chemicals and other microorganism. Although the factors that control the specialized metabolite production remain elusive, environmental factors have been shown to up- or down-regulate the expression of the biosynthetic genes via complex regulation networks (49). Therefore, one way for discovery of novel microbial natural products via genome mining is the activation of the expression of these untapped reservoirs of bioactive molecules. In that respect, two main strategies have been proposed: the global approach or the pathway-specific approach (1). The former consists of changing the standard conditions of cultivation (media composition, temperature of incubation, cocultures with competitors or host cells), with the idea to mimic environmental condition or to stress the organisms. The latter relies on a specific strategy for each gene cluster, such as inactivation of repressor proteins, overexpression of transcriptional factors and replacement of the promoter by a strong promoter (Figure 5).
Promoter replacement and inactivation of repressor strategies are shown in a and b.
In this targeted approach, the heterologous expression of putative gene clusters can also be achieved (12,50–52). Such strategy lefts a host that can be more readily genetically engineered and also extends genome mining approaches towards the production of metabolites from uncultivable microorganisms for which genomic information exists. The strategies to improve the expression of cryptic gene clusters are be presented in more detail in the introduction of Chapter I. Finally, the detection and identification of the produced metabolites can also be facilitated with a biosynthetic gene knockout that permits a comparison between extracts of the wild type and mutant by liquid chromatography – mass spectrometry (LC-MS) profiling (53,54).
Streptomyces, a prolific metabolite producer
Streptomyces is a genus within Actinobacteria, a phylum of Gram-positive bacteria with a high G/C DNA base ratio (Figure 6) (55,56). Actinobacteria produce more than 70% of all natural product scaffolds used in clinically-relevant antibiotic molecules (48). Streptomyces sp. are non-motile and aerobic bacteria that grow at neutral pH and are mesophilic (optimal growth temperature between 25 °C to 30 °C).
Figure 6 – Intraclass relatedness of the class Actinobacteria showing the presence of five orders based on 16S rRNA gene sequence comparison (57).
The phylogenetic relatedness of the families of the class Actinobacteria is outlined. Taxa newly described are highlighted in bold. Bootstrap values of 50 % or more are indicated at branch points. Bar, 2 substitutions per 100 nucleotide positions.
Streptomyces are distributed both in soil and aquatic habitats, where they typically use complex organic compounds from the environment as a source of energy. Their genomes displays a 60-70% G/C and their 8-9 Mb chromosomes are linear, with a « core » containing essential genes and « arms » carrying conditionally adaptive genes (58). Since the discovery of actinomycin, streptomycin and other antibiotics from actinobacteria by Selman Waksman and co-workers in the 1940s, intensive screening of soil habitat for antibiotic producers has been performed, showing that Streptomyces is a prolific producer of specialized metabolites. The development of genetic methods initiated in the 1960s by David Hopwood (59,60), followed by the genomic area in the 2000s (61,62) has revolutionized the species classification (63) as well as the methods to discover new specialized metabolites and to explore the biotechnological potential of these bacteria. Genome mining of Streptomyces sp. has revealed as many as 30-50 gene clusters per strain that are potentially involved in the biosynthesis of specialized metabolites from all four major chemical classes presented in section I.1 (39,64,65).
The life cycle of Streptomyces
treptomyces show a complex morphology consisting of highly differentiated mycelia that form spores under coordination of physiological processes (Figure 7) (66,67). There are three major steps in the life cycle of Streptomyces: vegetative growth, aerial mycelium erection and sporulation. The life cycle of Streptomyces starts from a dormant spore that meets suitable liquid or solid environment to form a vegetative mycelium (56,68,69). The spore germinates by a swelling process followed by the installation of cell polarity and apical growth. One or more hyphae arise from the spores and grow by tip extension and branching, in contrast to unicellular bacteria such as E. coli that grow by elongation of the lateral wall and fission. The vegetative Streptomyces cells are separated into compartments that are connected by cross-walls, each compartment containing multiple copies of the chromosome. The vegetative mycelium can differentiate into aerial hyphae which show a hair-like surface, generally white, on the top of the colonies. The events that trigger the differentiation from the vegetative mycelium to the aerial hyphae remain unclear, but it typically occurs under unfavourable conditions. This differentiation induces a form of programmed cell death in which the vegetative mycelium is autolysed to release nutrient in order to help the development of aerial hyphae (70).
The erection of aerial hyphae is helped by a hydrophobic sheath that enables the hyphae to break the surface water tension and to grow into the air. In S. coelicolor, this sheath is composed of two proteins, the chaplins and the rodlins and one surfactant peptide SapB (67,69). Eventually, the multigenomic apical cell of aerial hyphae is converted into unigenomic spores by several synchronously coordinated biological processes such as chromosome segregation and sporulation septation (69).
Regulation of specialized metabolite biosynthesis in Streptomyces
In many bacteria, antibiotic production responds to stress situations in coordination with primary metabolic responses (71). One of the main pathways linking a specific type of environmental stimulus to a transcriptional response involves two-component systems (TCSs), which consist of a sensor histidine kinase (HK) and a cognate response regulator (RR) (72,73). Each HK responds to specific types of environmental stimuli via an extracytoplasmic sensor domain. The signal is transferred via a phosphorelay that involves the autophosphorylation of a conserved His residue of the HK, which is then transferred to an Asp residue in the receiver domain of the cognate RR, which then activates transcription of target genes (Figure 8 b). Generally, two-component systems regulate gene expression at the level of transcription, through the DNA-binding domain of the RR. The analysis of prokaryote genomes also revealed the presence of unpaired HKs and RRs. In addition, certain TCSs components, although not encoded by adjacent genes, have been functionally associated (74). Finally, the occurrence of genes encoding single proteins containing sensor and output domains and lacking phosphotransfer domains typical of TCSs has conducted to propose an additional signal transduction pathway involving one-component systems (OCSs) (75) (Figure 8 a).
Figure 8 – One-component and two-component signal transduction systems.
a. A prototypical one-component system consists of one protein with a sensor domain and a regulator domain. b. A typical two-component system includes a sensor histidine kinase (HK) and cognate response regulator (RR), usually encoded by a pair of adjacent genes. ATP or GTP is used for autophosphorylation by the HK, and the phosphoryl group is transferred to the receiver domain of the RR (blue hexagon), which then controls the transcription of target genes through its output domain (in green) (76).
The biosynthesis of specialized metabolites in Streptomyces is subjected to subtle and precise regulatory events, involving a network of complex interconnected pathways of pathway-specific regulatory proteins or pleiotropic regulators or both (71,76–79) (77,79,80). Many studies on the biosynthesis and the regulation of specialized metabolites were performed in S. coelicolor A3(2) (64,73,81), which produces several specialized metabolites with diverse structures (Figure 9): three groups of chromosomally-encoded antibiotics: actinorhodin (ACT), undecylprodigiosin (RED) and calcium dependent antibiotics (CDA) and a group of plasmid-encoded antibiotics: methylenomycins. More recently, a yellow pigment, coelimycin P1, has been isolated upon activation of a cryptic gene cluster (82).
ACT is an aromatic polyketide synthesized by a type II PKS that displays a pH-sensitive colour (red at pH below 8.5, blue at pH above), from which S. coelicolor gets its name (81,82). RED are red hydrophobic tripyrroles made by a fatty acid synthase-like pathway involving a 22-genes (83). CDA are acidic lipopeptides synthesized by the NRPS pathway containing an N-terminal 2,3-epoxyhexanoyl fatty acid side-chain (84–86). Methylenomycins are cyclopentanone antibiotics made by an unusual pathway involving the condensation of a diketide with an pentulose, encoded by the gene cluster Mmy located on the plasmid SCP1 (87,88). Coelimycin P1 is a yellow alkaloid containing a unique functionalized 1,5-oxathiocane heterocycle, recently associated with a type I PKS cluster (cpk) (80).
Here, I use the regulatory mechanism of these molecules among other as example to illustrate the regulation of specialized metabolites by specific-pathway regulators and pleiotropic regulators.
Pathway-specific regulators
The pathway-specific regulators are typically encoded within the gene cluster they controlled and in Streptomyces most of them belong to the Streptomyces antibiotic regulatory protein (SARP) family, which group paralogous proteins involved in antibiotic production only found in actinomycetes (mainly within streptomycetes) (77,83). SARPs bind to sequences in the target promoters via their N-terminal winged Helix-Turn-Helix (HTH) domain, and activate transcription through a C-terminal activation domain. SARPs can be subdivided into three classes in function of their size: small SARPs (< 300 aa), medium SARPs (around 600 aa) which share end-to-end similarity to each other and large SARPs (around 1000 aa) which share an N-terminal SARP domain, an AAA domain (ATPase Associated with diverse cellular Activities), and a C-terminal domain of unknown function (78).
Five pathway-specific regulators have been characterized in S. coelicolor A3(2) (Table 1). The proteins ActII-ORF4, RedD, CdaR and KasO, controlling ACT, RED, CDA and coelimycin P1 production, respectively, are representants of the SARP family. ActII-ORF4 and RedD are small SARPs binding to a repeated TCGA motif spaced by 11 bp at the -35 nt region of their target promoters while CdaR is a medium SARPs (78,83).
An example of large SARP is PimR involved in the biosynthesis of pimaricin, a tetraene produced by Streptomyces natalensis (89). The SARP domain of PimR was shown to bind three heptameric direct repeats of the consensus CGGCAAG spaced by 4 bp and interacts with the – 55 nt region instead of the classical -35 nt region of target promoters. Unlike most of other large SARPs, PimR targets the promoter of another regulator pimM, which in turn activates the expression of the biosynthetic gene of primaricin (90). Interestingly, PimM orthologues are encoded and are functionally conserved in all known biosynthetic gene clusters of antifungal polyketides (91).
In addition to the SARP model described above, the biosynthesis of RED is controlled by two pathway-specific transcriptional activators, RedZ and RedD, with RedZ activating RedD, the direct activator (86). RedZ, is an atypical orphan RR lacking a set of conserved residues for phosphorylation, is an example of one-component system. Note that the mRNA of redZ present a very rare leucine codon, UUA, which can be only read by the specific tRNA BldA that is actively expressed when culture enters the stationary phase and initiates programmed cell death (86,92).
Pleiotropic regulators
A pleiotropic regulator function over a larger set of genes in the genome and in prokaryotes many are related to the metabolism of carbon, nitrogen and/or phosphate or correlated with morphological differentiation. Indeed, nutrient limitation is one of the principal signal to activate specialized metabolism inducing the developmental differentiation (56,77,80). An important representant of pleiotropic regulators in S. coelicolor is DasR, which is primarily involved in the metabolism of N-acetylglucosamine (GlcNAc) (Figure 10) (93– 95). GlcNAc is the building block of the abundant natural polymer chitin and a major constituent of peptidoglycan constituting a primary source of carbon and nitrogen for streptomycetes. GlcNAc released during programmed cell death (see section I.3.a), activates a cascade response leading to expression of specific-pathway-regulatory genes such as actII-ORF4 or redZ (78,93,96).
(a) A complete signalling cascade from extracellular nutrients to the activation of ACT and RED production, from (79,93). N-acetylglucosamine (GlcNAc) enters the cell and is subsequently phosphorylated via the GlcNAc-specific phosphoenolpyruvate-dependent phosphotransferase system (PTS). N-acetylglucosamine-6-phosphate (GlcNAc-6P) is then deacetylated by GlcN-6P deacetylase NagA to glucosamine-6-phosphate (GlcN-6P), the effector molecule that inhibits DasR DNA-binding. This results in the derepression of actII-ORF4 and redZ/redD, the pathway-specific transcriptional activator genes for the ACT and RED biosynthetic gene clusters, respectively. (b) Summary of the DasR-controlled processes, from (95). Red lines indicate direct binding and transcriptional repression of the target genes by DasR. Derepression of control by DasR in response to the accumulation of GlcN-6P results in activation (green lines) of morphological development and antibiotic production, among other processes..
Ribosomally synthesized and post-translationally modified peptides
Diversity
As discussed previously in section I.1, RiPPs share a common biosynthetic strategy that starts from a linear precursor synthesized by classical transcription/translation processes, which then undergoes different levels of PTM, that results in a great diversity of molecules (5,97) (Table 2). Lanthipeptides and thiopeptides will be described the next sub-section given that they are a great source of information to understand the biosynthesis and the regulatory mechanism of RiPPs and followed by the description of lasso peptides as they are the subject of this thesis.
Lanthipeptides
Structure
Lanthipeptides (also called lantibiotics for those with antibacterial activities) are a major class of RiPPs containing meso-lanthionine (Lan) and 3-methyllanthionine (MeLan) (106). The first reported lanthipeptide was nisin in 1928 but their ribosomal origin was only confirmed in 1988 with the sequencing of the epidermin gene cluster (116,117). Lan and MeLan are residues formed by intramolecular connection of a cysteine and the post-translational modified amino acid 2,3-didehydroalanine (Dha) or (Z)-2,3-dehydrobutyrine (Dhb) formed by dehydration of a Ser and Thr residue, respectively (Figure 11) (118). Nisin is a lanthipeptide produced by Lactococcus lactis and is considered as a model to illustrate the biosynthesis of lanthipeptides, their regulation and their biological activities.
Biosynthesis
The gene cluster for the production of nisin consists of 11 genes: nisABTCIPRKFEG encoding the 57-amino acids long precursor NisA, the maturation enzymes NisB, -C and –P, two ABC transporter family NisT and NisFEG, an immunity lipoprotein involved in the self-protection NisI and a two-component system that comprises a sensor kinase (NisK) and a response regulator (NisR) (118–122). The nisin gene cluster is under the control of three promoters PnisA, PnisR and PnisF that promote the transcription of three operons nisABTCIP, nisRK and nisFEG, respectively. A terminator is probably present after nisA that allows restricted read-through of the downstream genes nisBTCIP (Figure 12).
The structural lantibiotic genes (filled black), the genes encoding proteins involved in precursor maturation and transport (vertical hatching), processing (backward tilted hatching, producer immunity (horizontal hatching), and regulation (forward tilted hatching) are indicated. The positions of the promoters that have been identified within the nisin gene clusters are indicated (123).
The precursor NisA is composed of a 23 amino acid long leader peptide and the 34 amino acid long structural peptide. The leader peptide has been shown to be requisite for secretion and complete maturation (124–126). NisA undergoes a series of dehydrations by the lanthipeptide dehydratase NisB, which utilizes glutamyl-tRNAGlu as the glutamate donor to activate Ser/Thr. Then, glutamate elimination results in DhA/DhB residues (Figure 13) (127,128).
Cyclization is performed by NisC through thioether crosslinks, by addition of Cys thiols to the resulting DhA or DhB to form Lan or MeLan, respectively. The modified NisA is then transported across the cytoplasm membrane by NisT and the leader is cleaved by the extracellular peptidase NisP (129,130).
Classification
Lanthipeptides are subdivided into 4 classes depending on the post-translational modification enzymes which install (Me)Lan motif. For class I lanthipeptides, (Me)Lan motif is installed by the action of LanB and LanC enzyme, a lanthipeptide dehydratase and a lanthipeptide cyclase, respectively. For class II, III and IV lanthipeptides, dehydration and cyclization are carried out by bifunctional lanthionine synthetase LanM, LanKC and LanL, respectively. LanM is a large protein with an N-terminal dehydration domain that does not show homology to LanB proteins and a C-terminal cyclase domain which displays weak homology to LanC proteins. LanKC and LanL carry out the dehydration by successive action of a central kinase domain and an N-terminal phosphoSer/phosphoThr lyase domain, but differs in the cyclase domain (5,118). Although LanKC show LanC-like cyclase domain, it lacks the three zinc ligands binding domains conserved in the other cyclase domains.
Biological activities
Nisin displays antibacterial activity against Gram-positive bacteria and has been used for more that 50 years as food preservative. Its mechanism of action is dual: nisin binds to the peptidoglycan intermediate building block lipid II, thus causing inhibition of cell wall biosynthesis and can insert into the membranes, leading to pore formation (131). Cytolysin is produced by Enterococcus faecalis and belongs to the group of two-component lanthipeptides. By name, cytolysin, consists of two modified peptides, which together display strong antibiotic activity but have weak or no antimicrobial activity when separated (118,132). Cytolysin displays antibacterial activity against Gram-positive bacteria and also functions as a virulence factor, lysing erythrocytes and polymorphonuclear leukocytes (133). Interestingly, the matured precursors of lantibiotics do not show antimicrobial activity. Thus, for those that are processed extracellularly, the leader peptide may prevent the activity of the mature peptide until it is outside the cell (120,134).
Thiopeptides
The first thiopeptide isolated was micrococcin in 1948, but thiostrepton isolated from Streptomyces azureus in 1954 is referred as the archetype compound (5,99). Although thiopeptide are mainly isolated from soil bacteria, novel representative has been isolated from marine environment (135).
Structure
Thiopeptides, also termed thiazolyl peptide, contain a nitrogenous macrocycle which present different oxidative states, fully saturated piperidine, didehydropiperidine, imidazopiperidine, pyridine and pyridine with hydroxyl at position 5 (Figure 16). Thiopeptides can harbour additional modifications such as dehydroamino acids similar to those of lanthipeptides (5,99).
Biosynthesis
The thiostrepton biosynthetic gene cluster identified from whole-genome scanning of Streptomyces laurentii is one example demonstrating that thiopeptides are ribosomally-synthesised then posttranslationally modified (135,136). Like the other RiPPs, thiopeptide biosynthetic gene clusters contain one gene encoding a precursor and other genes encoding the enzymatic machinery. For example, the gene cluster of thiomuracins contains one gene encoding the precursor TpdA, and 6 other genes encoding TpdB-G proteins (Figure 14).
In the precursor peptide sequence, the structural peptide is numbered with positive figures and the leading peptide with negative ones. Residues that appear in the mature thiopeptide are underlined.
In this biosynthesis, oxazoles, thiazoles, thiazoline rings and DhA and Dhb are first installed. Then, the cycloaddition between distant Dha residues is performed and finally further side-chain modifications are installed (Figure 15) (137). Dehydroamino acids residues are probably installed by TpdB and TpdC which show weak similarities with LanB dehydratase from class I lanthipeptides (5,138,139).
Classification
Thiopeptides have been regrouped into five series according to the oxidative state of the nitrogenous macrocycle, fully saturated piperidine, didehydropiperidine, imidazopiperidine, pyridine and pyridine with hydroxyl at position 5, respectively series a, b, c, d, and e (Figure 16).
Biological activities
The main biological activity of thiopeptide is the antibacterial. Thiopeptides efficiently inhibit protein synthesis in Gram-positive bacteria by either direct interaction with the 50S ribosomal subunit or by inhibiting elongation factor Tu (EF-Tu), whereas they display almost no activity against Gram-negative bacteria (5,99,135). Interestingly, some have been shown to possess other pharmacological activities such as anticancer (142) or immunosuppressive (143).
Interestingly, a thiopeptide containing a Dha in the tail close to six-membered central scaffold, such as thiostrepton, appears to induce the tipA gene promotion by binding to the regulatory protein TipAL (135,145,146). TipAL is a transcriptional regulator which contains a N-terminal helix-turn-helix DNA-binding domain and a C-terminal drug recognition domain. The binding of thiostrepton-like molecule to TipAL induce the recruitment of RNA polymerase (RNAP) to the promoter tipAp, thus resulting in autogenous transcriptional activation of the tipA gene. The feature was widely used in Streptomyces as an inducible promoter.
Lasso peptides
Diversity
Lasso peptides represent an emerging class of RiPPs. They possess a unique topology reminiscent to a lasso and were isolated from Proteobacteria, Actinobacteria and more recently from Firmicutes (100,147–149). The topology of a lasso peptide consists of an N-terminal macrolactam ring threaded by the C-terminal chain. The macrolactam ring is formed by the condensation of the N-terminal amino group of the core peptide with the side chain carboxylate of an Asp or Glu at position 7, 8 or 9. The N-terminal amino acid involved in the isopeptide bond is mostly a Gly and less frequently a Cys, Ser or Ala. The C-terminal tail is locked inside the ring by large amino acid(s) and/or disulphide bond(s) stabilizing the lasso topology (150– 153). The size of isolated lasso peptides range from 15 to 24 residues with varied amino acid composition (147). The minimal gene cluster for lasso peptide biosynthesis consists of three genes encoding the precursor A and two processing enzymes B and C (154–156). Lasso peptides show diverse biological activities: antimicrobial, antiviral, receptor antagonist or enzyme inhibitor. Lasso peptides are subdivided into 3 classes according to the number of disulphide bonds (Figure 17 and Table 3) (5,100,147,148). Class I lasso peptides present two disulphide bonds while class II and III contain none or one, respectively. Class I lasso peptide are represented by five members, sviceucin, aborycin (RP71955), siamycins and humidimycin which share highly similar amino acid compositions and are only produced by Streptomyces species (7,157–162). Moreover, the structural elucidation of aborycin, siamycins and sviceucin showed that disulphide bonds Cys1-Cys13 and Cys7-Cys19 connecting the ring to the C-terminal tail are strictly conserved. The structure for humidimycin is not available but the primary sequence showed exact same position of the cysteine residues suggesting the same secondary structure. Class II contains over 20 structurally diverse lasso peptides from different bacteria ranging from Proteobacteria to Actinobacteria and Firmicutes (163–165). A typical class II lasso peptide is represented by microcin J25 (MccJ25). Its structure consists of an 8-residue long N-terminal macrolactam ring, closed by an isopeptide bond formed between N-terminal amine of Gly1 and the γ-carboxyl group of Glu8, which is threaded by the 13 amino acids C-terminal tail (150,151,166). The structure is stabilized by bulky amino acid(s) located on each side of the ring, such as in the case of Mccj25 where C-terminal tail is trapped by residues Phe19 and Tyr20. Finally, BI-32169 is the sole member of the class III lasso peptide containing a single disulphide bond.
Class I, II and III assemble lasso peptides with two, none or one disulfide bond(s). Macrolactam ring is shown in green, C-terminal tail in blue, disulfide bonds in black. The bulky amino acids acting as plugs of the lasso topology are shown in pink.
The length of the N-terminal macrolactam ring of the known lasso peptides varies from 7 to 9 amino acids and the C-terminal tails length varies from 8 to 15 amino acids. The size of the tail above the ring, termed loop, can vary greatly between lasso peptides. For example, the loop of MccJ25 consists of 11 amino acids whereas the loop of capistruin consists of 4 amino acids.
The lasso topology is a very constrained structure and is reported to be remarkably stable against degradation by protease, heat shock and denaturating agents (166–169). While, thermal stability has been re-evaluated with the identification of new lasso peptides with different physicochemical properties. Indeed, several lasso peptides, such as caulosegnins, astexins, caulonodins, rhodanodin and rubrivinodin, can unthread to a branched-cyclic topology upon heating (163,169–171).
Table of contents :
Literature review
I. Microbial natural product discovery in the genomics era
I.1. Diversity of microbial specialized metabolites
I.2. From genes to molecules
I.3. Streptomyces, a prolific metabolite producer
I.3.a. The life cycle of Streptomyces
I.3.b. Regulation of specialized metabolite biosynthesis in Streptomyces
I.3.b.i Pathway-specific regulators
I.3.b.ii Pleiotropic regulators
II. Ribosomally synthesized and post-translationally modified peptides
II.1. Diversity
II.2. Lanthipeptides
II.2.a. Structure
II.2.b. Biosynthesis
II.2.c. Classification
II.2.d. Biological activities
II.3. Thiopeptides
II.3.a. Structure
II.3.b. Biosynthesis
II.3.c. Classification
II.3.d. Biological activities
II.4. Lasso peptides
II.4.a. Diversity
II.4.b. Discovery
II.4.c. Biosynthesis
II.4.c.i Gene cluster organization
II.4.c.ii Maturation process
II.4.c.iii Additional modifications
II.4.d. Biological activities
II.4.e. Bioengineering using lasso peptides
III. Ecological role of RiPPs
IV. Objectives of the thesis
Materials and methods
I. Chemicals and biological materials
II. General DNA and microbiology methods
II.1. Purification of plasmids and cosmids
II.2. Isolation of genomic DNA
II.3. General Polymerase Chain Reaction methods (PCR)
II.4. Agarose gel electrophoresis
II.5. General cloning procedure
II.6. Preparation and transformation of competent E. coli cells
II.6.a. Electrocompetent cells
II.6.b. Chemical competent cells
II.7. Site-directed mutagenesis
II.8. Modification of cosmids using PCR targeting
II.9. Conjugation methods for Streptomyces strains
III. RNA methods
III.1. RNA extraction
III.2. Reverse transcription PCR (RT-PCR)
III.3. Real-time quantitative PCR (qPCR)
IV. Heterologous expression of lasso peptide gene clusters
IV.1. Generation of lasso peptide expression system
IV.1.a. Cosmid libraries
IV.1.b. Plasmid vectors
IV.2. Lasso peptide production in heterologous host
IV.3. Lasso peptide production and extraction
V. Liquid chromatography mass spectrometry (LC-MS)
VI. Generation of deletion mutants in S. sviceus-
VI.1. Inactivation using pKGLP2-based suicide plasmids
VI.2. Inactivation using modified cosmids
VI.2.a. Generation of P4H8, a P4H7 based suicide cosmid
VI.2.b. Modification of P4H8 by PCR Targeting
VI.2.c. Inactivation using P4H8-based cosmids
VI.2.d. Gene complementation in the deletion mutants of S. sviceus
VII. Gene reporter assays
VIII. Oxidative sensibility assay
IX. Morphology characterization
IX.1. Optic microscopy
IX.2. Scanning Electronic microscopy (SEM)
X. Autoinduction of sviceucin
X.1. Gene reporter assay
X.2. qPCR
Chapter I: Heterologous expression of lasso peptides from Actinobacteria
I. Introduction
I.1. Common strategies to trigger and improve the expression of cryptic or silent BGCs
I.1.a. Optimization of cultivation conditions
I.1.b. Heterologous expression
I.1.c. Metabolic engineering
I.1.d. Synthetic biology
I.2. Heterologous expression of lasso peptides
II. Results and discussion
II.1. Genome mining
II.1.a. Stackebrandtia nassauensis cluster
II.1.b. Nocardiopsis alba cluster
II.1.c. Actinoalloteichus sp. cluster
II.1.d. Streptomyces noursei cluster
II.1.e. Streptomyces venezuelae cluster
II.2. Heterologous production based on native gene clusters
II.2.a. Cosmid-based method
II.2.b. Co-expression with the pathway-specific regulator
II.3. Heterologous expression using genetically-engineered clusters
II.3.a. Promoter exchange strategy
II.3.b. Orthogonal two-plasmid expression system
III. Conclusion and perspectives
Chapter II: Regulation mechanism and ecological role of sviceucin in Streptomyces sviceus
I. Introduction
I.1. Sviceucin as a good model to study LP regulation and ecological roles
I.2. Overview of growth and development in Streptomyces
II. Results and discussion
II.1. Determining the operon structure in the sviceucin gene cluster
II.2. Growth dependence of sviceucin production
II.3. Probing the regulation mechanism of sviceucin biosynthesis
II.3.a. Is sviceucin biosynthesis controlled by SviR1 and SviR2 ?
II.3.b. Attempts to determine the SviR1 and SviR2 regulon in the svi cluster
II.3.c. Effects of deleting other genes on sviceucin production
II.3.d. Is sviceucin an autoinducing peptide?
II.4. Morphological characterization of S. sviceus mutants
II.5. Sensitivity to oxidative stress of S. sviceus mutants
III. Conclusions and perspectives
Conclusion and perspectives
I. Heterologous expression of lasso peptides from Actinobacteria
II. Regulation mechanism and physiological role of sviceucin
