Sylvie Lautru - Academia.edu (original) (raw)

Papers by Sylvie Lautru

Chemistry Biology, 2003

Dé partement d'Ingé nierie et d'Etudes des Proté ines F91191 Gif-sur-Yvette Cedex France mechanis... more Dé partement d'Ingé nierie et d'Etudes des Proté ines F91191 Gif-sur-Yvette Cedex France mechanisms [4-6]. Although the number of newly isolated, naturally oc-2 Institut de Gé né tique et Microbiologie CNRS UMR 8621, Bâ t. 400 curring DKPs has increased during the last few years, the biosynthetic pathways of these molecules remain Université Paris-Sud F91405 Orsay Cedex largely unexplored. In bacteria and fungi, DKP derivatives generally seem to be produced by nonribosomal France pathways, although their biosynthesis has never been shown to be directly catalyzed by NRPS megacomplexes. Studies of ergot alkaloid biosynthesis in the fun-Summary gus Claviceps purpurea have shown that the D-lysergyl peptide synthetase, which is responsible for the forma-Albonoursin [cyclo(⌬Phe-⌬Leu)], an antibacterial peptide produced by Streptomyces noursei, is one of the tion of the D-lysergyl peptide-DKP, an intermediate in the biosynthesis of ergopeptines, is devoid of a C-terminal simplest representatives of the large diketopiperazine (DKP) family. Formation of ␣,␤ unsaturations was pre-thioesterase domain. DKP cyclization is thus thought to occur spontaneously, leading to the release of the viously

Since the discovery of the streptomycin produced by Streptomyces griseus in the middle of the las... more Since the discovery of the streptomycin produced by Streptomyces griseus in the middle of the last century, members of this bacterial genus have been largely exploited for the production of secondary metabolites with wide uses in medicine and in agriculture. They have even been recognized as one of the most prolific producers of natural products among microorganisms. With the onset of the genomic era, it became evident that these microorganisms still represent a major source for the discovery of novel secondary metabolites. This was highlighted with the complete genome sequencing of Streptomyces coelicolor A3(2) which revealed an unexpected potential of this organism to synthesize natural products undetected until then by classical screening methods. Since then, analysis of sequenced genomes from numerous Streptomyces species has shown that a single species can carry more than 30 secondary metabolite gene clusters, reinforcing the idea that the biosynthetic potential of this bacterial genus is far from being fully exploited. This review highlights our knowledge on the potential of Streptomyces ambofaciens ATCC 23877 to synthesize natural products. This industrial strain was known for decades to only produce the drug spiramycin and another antibacterial compound, congocidine. Mining of its genome allowed the identification of 23 clusters potentially involved in the production of other secondary metabolites. Studies of some of these clusters resulted in the characterization of novel compounds and of previously known compounds but never characterized in this Streptomyces species. In addition, genome mining revealed that secondary metabolite gene clusters of phylogenetically closely related Streptomyces are mainly species-specific.

Antimicrob Agents Chemother, 2010

Streptomyces ambofaciens synthesizes spiramycin, a 16-membered macrolide antibiotic used in human... more Streptomyces ambofaciens synthesizes spiramycin, a 16-membered macrolide antibiotic used in human medicine. The spiramycin molecule consists of a polyketide lactone ring (platenolide) synthesized by a type I polyketide synthase, to which three deoxyhexoses (mycaminose, forosamine, and mycarose) are attached successively in this order. These sugars are essential to the antibacterial activity of spiramycin. We previously identified four genes in the spiramycin biosynthetic gene cluster predicted to encode glycosyltransferases. We individually deleted each of these four genes and showed that three of them were required for spiramycin biosynthesis. The role of each of the three glycosyltransferases in spiramycin biosynthesis was determined by identifying the biosynthetic intermediates accumulated by the corresponding mutant strains. This led to the identification of the glycosyltransferase responsible for the attachment of each of the three sugars. Moreover, two genes encoding putative glycosyltransferase auxiliary proteins were also identified in the spiramycin biosynthetic gene cluster. When these two genes were deleted, one of them was found to be dispensable for spiramycin biosynthesis. However, analysis of the biosynthetic intermediates accumulated by mutant strains devoid of each of the auxiliary proteins (or of both of them), together with complementation experiments, revealed the interplay of glycosyltransferases with the auxiliary proteins. One of the auxiliary proteins interacted efficiently with the two glycosyltransferases transferring mycaminose and forosamine while the other auxiliary protein interacted only with the mycaminosyltransferase.

Journal of Biotechnology, 2015

Streptomyces ambofaciens ATCC23877 is a soil bacterium industrially exploited for the production ... more Streptomyces ambofaciens ATCC23877 is a soil bacterium industrially exploited for the production of the macrolide spiramycin which is used in human medicine as an antibacterial and anti-toxoplasmosis chemical. Its genome consists of a 8.3Mbp linear chromosome and a 89kb circular plasmid. The complete genome sequence reported here will enable us to investigate Streptomyces genome evolution and to discover new secondary metabolites with potential applications notably in human medicine.

Albonoursin [cyclo(ΔPhe-ΔLeu)], an antibacterial peptide produced by Streptomyces noursei, is one... more Albonoursin [cyclo(ΔPhe-ΔLeu)], an antibacterial peptide produced by Streptomyces noursei, is one of the simplest representatives of the large diketopiperazine (DKP) family. Formation of α,β unsaturations was previously shown to occur on cyclo(L-Phe-L-Leu), catalyzed by the cyclic dipeptide oxidase (CDO). We used CDO peptide sequence information to isolate a 3.8 kb S. noursei DNA fragment that directs albonoursin biosynthesis in Streptomyces lividans. This fragment encompasses four complete genes: albA and albB, necessary for CDO activity; albC, sufficient for cyclic dipeptide precursor formation, although displaying no similarity to non ribosomal peptide synthetase (NRPS) genes; and albD, encoding a putative membrane protein. This first isolated DKP biosynthetic gene cluster should help to elucidate the mechanism of DKP formation, totally independent of NRPS, and to characterize novel DKP biosynthetic pathways that could be engineered to increase the molecular diversity of DKP deri...

Nature chemical biology, Jan 18, 2015

Microbiology (Reading, England), 2004

Nonribosomal peptide synthetases (NRPSs) are giant multi-domain enzymes that catalyse the biosynt... more Nonribosomal peptide synthetases (NRPSs) are giant multi-domain enzymes that catalyse the biosynthesis of many commercially important peptides produced by bacteria and fungi. Several studies over the last decade have shown that many of the individual domains within NRPSs exhibit significant substrate selectivity, which impacts on our ability to engineer NRPSs to produce new bioactive microbial peptides. Adenylation domains appear to be the primary determinants of substrate selectivity in NRPSs. Much progress has been made towards an empirical understanding of substrate selection by these domains over the last 5 years, but the molecular basis of substrate selectivity in these domains is not yet well understood. Perhaps surprisingly, condensation domains have also been reported to exhibit moderate to high substrate selectivity, although the generality of this observation and its potential impact on engineered biosynthesis experiments has yet to be fully elucidated. The situation is le...

ACS Chemical Biology, 2014

The pyrrolamides constitute a small family of secondary metabolites that are known for their abil... more The pyrrolamides constitute a small family of secondary metabolites that are known for their ability to bind noncovalently to the DNA minor groove with some sequence specificity. To date, only a single pyrrolamide biosynthetic gene cluster has been reported, directing the synthesis of congocidine (netropsin) in Streptomyces ambofaciens. In this study, we improve our understanding of pyrrolamide biosynthesis through the identification and characterization of the gene cluster responsible for the production of distamycin in Streptomyces netropsis DSM40846. We discover that the strain produces two other pyrrolamides, the well-characterized congocidine and a congocidine/distamycin hybrid that we named disgocidine. S. netropsis DSM40846 genome analysis led to the identification of two distinct pyrrolamide-like biosynthetic gene clusters. We show here that these two clusters are reciprocally dependent for the production of the three pyrrolamide molecules. Furthermore, based on detailed functional analysis of these clusters, we propose a biosynthetic route to congocidine and distamycin and an updated model for pyrrolamide assembly. The synthesis of disgocidine, the distamycin/congocidine hybrid, appears to constitute the first example of "natural combinatorial biosynthesis" between two related biosynthetic pathways. Finally, we analyze the genomic context of the two biosynthetic gene clusters and suggest that the presently interdependent clusters result from the coevolution of two ancestral independent pyrrolamide gene clusters.

European Journal of Biochemistry, 2001

Cyclic dipeptide oxidase is a novel enzyme that specifically catalyzes the formation of a,b-dehyd... more Cyclic dipeptide oxidase is a novel enzyme that specifically catalyzes the formation of a,b-dehydro-Phe (DPhe) and a,b-dehydro-Leu (DLeu) residues during the biosynthesis of albonoursin, cyclo(DPhe-DLeu), an antibiotic produced by Streptomyces noursei. It was purified 600-fold with a 30% overall recovery, and consists of the association of a single type of subunit with a relative molecular mass of 21 066 resulting in a large homopolymer of relative molecular mass over 2 000 000. The enzyme exhibits a typical flavoprotein spectrum with maxima at 343.5 and 447.5 nm, the flavin prosthetic group being covalently bound to the protein. The catalytic reaction of the natural substrate cyclo(l-Phe-l-Leu) occurs in a two-step sequential reaction leading first to cyclo(a,b-dehydro-Phe-l-Leu) and finally to albonoursin. Kinetic parameters for the first step were determined (K m 53 mm; k 0.69 s 21 ). The enzyme was shown to catalyze the conversion of a variety of cyclo(dipeptides) and can be reoxidized at the expense of molecular oxygen by producing H 2 O 2 . This reaction mechanism, which differs from those already described for the formation of a,b-dehydro-amino acids, might consist of the transient formation of an intermediate imine followed by its rearrangement into an a,b-dehydro-residue.

Nature Chemical Biology, 2005

Analyses of microbial genome sequences reveal numerous examples of gene clusters encoding protein... more Analyses of microbial genome sequences reveal numerous examples of gene clusters encoding proteins typically involved in complex natural product biosynthesis but not associated with the production of known natural products 1-3 . In Streptomyces coelicolor M145 there are several gene clusters encoding new nonribosomal peptide synthetase (NRPS) systems not associated with known metabolites. Application of structure-based models for substrate recognition by NRPS adenylation domains 4-6 predicts the amino acids incorporated into the putative peptide products of these systems 7,3 , but the accuracy of these predictions is untested. Here we report the isolation and structure determination of the new trishydroxamate tetrapeptide iron chelator coelichelin from S. coelicolor using a genome mining approach guided by substrate predictions for the trimodular NRPS CchH, and we show that this enzyme, which lacks a C-terminal thioesterase domain, together with a homolog of enterobactin esterase (CchJ), are required for coelichelin biosynthesis. These results demonstrate that accurate prediction of adenylation domain substrate selectivity is possible and raise intriguing mechanistic questions regarding the assembly of a tetrapeptide by a trimodular NRPS.

Natural Product Reports, 2012

We review here work on the biosynthesis of diketopiperazines (DKPs), a large class of natural pro... more We review here work on the biosynthesis of diketopiperazines (DKPs), a large class of natural products with noteworthy biological activities, focusing on the biosynthetic pathways involving cyclodipeptide synthases (CDPSs), a newly defined family of enzymes. Distinct from nonribosomal peptide synthetases (NRPSs), the other family of enzymes synthesizing DKPs, CDPSs bridge the primary and secondary metabolic pathways by hijacking aminoacyl-tRNAs to produce DKPs. This review includes a comprehensive description of the state of the art for CDPS-dependent pathways, and highlights the ways in which this knowledge could be used to increase the diversity of natural DKPs by pathway engineering.

Microbiology, 2006

Siderophore-mediated iron acquisition has been well studied in many bacterial pathogens because i... more Siderophore-mediated iron acquisition has been well studied in many bacterial pathogens because it contributes to virulence. In contrast, siderophore-mediated iron acquisition by saprophytic bacteria has received relatively little attention. The independent identification of the des and cch gene clusters that direct production of the tris-hydroxamate ferric iron-chelators desferrioxamine E and coelichelin, respectively, which could potentially act as siderophores in the saprophyte Streptomyces coelicolor A3(2), has recently been reported. Here it is shown that the des cluster also directs production of desferrioxamine B in S. coelicolor and that very similar des and cch clusters direct production of desferrioxamines E and B, and coelichelin, respectively, in Streptomyces ambofaciens ATCC 23877. Sequence analyses of the des and cch clusters suggest that components of ferric-siderophore uptake systems are also encoded within each cluster. The construction and analysis of a series of mutants of S. coelicolor lacking just biosynthetic genes or both the biosynthetic and siderophore uptake genes from the des and cch clusters demonstrated that coelichelin and desferrioxamines E and B all function as siderophores in this organism and that at least one of these metabolites is required for growth under defined conditions even in the presence of significant quantities of ferric iron. These experiments also demonstrated that a third siderophore uptake system must be present in S. coelicolor, in addition to the two encoded within the cch and des clusters, which show selectivity for coelichelin and desferrioxamine E, respectively. The ability of the S. coelicolor mutants to utilize a range of exogenous xenosiderophores for iron acquisition was also examined, showing that the third siderophore-iron transport system has broad specificity for tris-hydroxamate-containing siderophores. Together, these results define a complex system of multiple biosynthetic and uptake pathways for siderophore-mediated iron acquisition in S. coelicolor and S. ambofaciens.

Microbiology, 2007

MbtH-like proteins are a family of small proteins encoded by genes found in many, but not all, no... more MbtH-like proteins are a family of small proteins encoded by genes found in many, but not all, non-ribosomal peptide synthetase-encoding gene clusters that direct the biosynthesis of peptide antibiotics and siderophores. Studies published to date have not elucidated the function of MbtH-like proteins, nor have they clarified whether they are required for metabolite biosynthesis.

Journal of Industrial Microbiology & Biotechnology, 2014

Since the discovery of the streptomycin produced by Streptomyces griseus in the middle of the las... more Since the discovery of the streptomycin produced by Streptomyces griseus in the middle of the last century, members of this bacterial genus have been largely exploited for the production of secondary metabolites with wide uses in medicine and in agriculture. They have even been recognized as one of the most prolific producers of natural products among microorganisms. With the onset of the genomic era, it became evident that these microorganisms still represent a major source for the discovery of novel secondary metabolites. This was highlighted with the complete genome sequencing of Streptomyces coelicolor A3(2) which revealed an unexpected potential of this organism to synthesize natural products undetected until then by classical screening methods. Since then, analysis of sequenced genomes from numerous Streptomyces species has shown that a single species can carry more than 30 secondary metabolite gene clusters, reinforcing the idea that the biosynthetic potential of this bacterial genus is far from being fully exploited. This review highlights our knowledge on the potential of Streptomyces ambofaciens ATCC 23877 to synthesize natural products. This industrial strain was known for decades to only produce the drug spiramycin and another antibacterial compound, congocidine. Mining of its genome allowed the identification of 23 clusters potentially involved in the production of other secondary metabolites. Studies of some of these clusters resulted in the characterization of novel compounds and of previously known compounds but never characterized in this Streptomyces species. In addition, genome mining revealed that secondary metabolite gene clusters of phylogenetically closely related Streptomyces are mainly species-specific.

Nature chemical biology, 2009

Cyclodipeptides and their derivatives belong to the diketopiperazine (DKP) family, which is compr... more Cyclodipeptides and their derivatives belong to the diketopiperazine (DKP) family, which is comprised of a broad array of natural products that exhibit useful biological properties. In the few known DKP biosynthetic pathways, nonribosomal peptide synthetases (NRPSs) are involved in the synthesis of cyclodipeptides that constitute the DKP scaffold, except in the albonoursin (1) pathway. Albonoursin, or cyclo(alpha,beta-dehydroPhe-alpha,beta-dehydroLeu), is an antibacterial DKP produced by Streptomyces noursei. In this pathway, the formation of the cyclo(Phe-Leu) (2) intermediate is catalyzed by AlbC, a small protein unrelated to NRPSs. We demonstrated that AlbC uses aminoacyl-tRNAs as substrates to catalyze the formation of the DKP peptide bonds. Moreover, several other bacterial proteins, presenting moderate similarity to AlbC, also use aminoacyl-tRNAs to synthesize various cyclodipeptides. Therefore, AlbC and these related proteins belong to a newly defined family of enzymes that w...

Chemistry & Biology, 2009

Congocidine (netropsin) is a pyrrole-amide (oligopyrrole, oligopeptide) antibiotic produced by St... more Congocidine (netropsin) is a pyrrole-amide (oligopyrrole, oligopeptide) antibiotic produced by Streptomyces ambofaciens. We have identified, in the right terminal region of the S. ambofaciens chromosome, the gene cluster that directs congocidine biosynthesis. Heterologous expression of the cluster and in-frame deletions of 8 of the 22 genes confirm the involvement of this cluster in congocidine biosynthesis. Nine genes can be assigned specific functions in regulation, resistance, or congocidine assembly. In contrast, the biosynthetic origin of the precursors cannot be easily inferred from in silico analyses. Congocidine is assembled by a nonribosomal peptide synthetase (NRPS) constituted of a free-standing module and several single-domain proteins encoded by four genes. The iterative use of its unique adenylation domain, the utilization of guanidinoacetyl-CoA as a substrate by a condensation domain, and the control of 4-aminopyrrole-2-carboxylate polymerization constitute the most original features of this NRPS.

Chemistry & Biology, 2002

Dé partement d'Ingé nierie et d'Etudes des Proté ines F91191 Gif-sur-Yvette Cedex France mechanis... more Dé partement d'Ingé nierie et d'Etudes des Proté ines F91191 Gif-sur-Yvette Cedex France mechanisms [4-6]. Although the number of newly isolated, naturally oc-2 Institut de Gé né tique et Microbiologie CNRS UMR 8621, Bâ t. 400 curring DKPs has increased during the last few years, the biosynthetic pathways of these molecules remain Université Paris-Sud F91405 Orsay Cedex largely unexplored. In bacteria and fungi, DKP derivatives generally seem to be produced by nonribosomal France pathways, although their biosynthesis has never been shown to be directly catalyzed by NRPS megacomplexes. Studies of ergot alkaloid biosynthesis in the fun-Summary gus Claviceps purpurea have shown that the D-lysergyl peptide synthetase, which is responsible for the forma-Albonoursin [cyclo(⌬Phe-⌬Leu)], an antibacterial peptide produced by Streptomyces noursei, is one of the tion of the D-lysergyl peptide-DKP, an intermediate in the biosynthesis of ergopeptines, is devoid of a C-terminal simplest representatives of the large diketopiperazine (DKP) family. Formation of ␣,␤ unsaturations was pre-thioesterase domain. DKP cyclization is thus thought to occur spontaneously, leading to the release of the viously

Chemistry Biology, 2003

Dé partement d'Ingé nierie et d'Etudes des Proté ines F91191 Gif-sur-Yvette Cedex France mechanis... more Dé partement d'Ingé nierie et d'Etudes des Proté ines F91191 Gif-sur-Yvette Cedex France mechanisms [4-6]. Although the number of newly isolated, naturally oc-2 Institut de Gé né tique et Microbiologie CNRS UMR 8621, Bâ t. 400 curring DKPs has increased during the last few years, the biosynthetic pathways of these molecules remain Université Paris-Sud F91405 Orsay Cedex largely unexplored. In bacteria and fungi, DKP derivatives generally seem to be produced by nonribosomal France pathways, although their biosynthesis has never been shown to be directly catalyzed by NRPS megacomplexes. Studies of ergot alkaloid biosynthesis in the fun-Summary gus Claviceps purpurea have shown that the D-lysergyl peptide synthetase, which is responsible for the forma-Albonoursin [cyclo(⌬Phe-⌬Leu)], an antibacterial peptide produced by Streptomyces noursei, is one of the tion of the D-lysergyl peptide-DKP, an intermediate in the biosynthesis of ergopeptines, is devoid of a C-terminal simplest representatives of the large diketopiperazine (DKP) family. Formation of ␣,␤ unsaturations was pre-thioesterase domain. DKP cyclization is thus thought to occur spontaneously, leading to the release of the viously

Since the discovery of the streptomycin produced by Streptomyces griseus in the middle of the las... more Since the discovery of the streptomycin produced by Streptomyces griseus in the middle of the last century, members of this bacterial genus have been largely exploited for the production of secondary metabolites with wide uses in medicine and in agriculture. They have even been recognized as one of the most prolific producers of natural products among microorganisms. With the onset of the genomic era, it became evident that these microorganisms still represent a major source for the discovery of novel secondary metabolites. This was highlighted with the complete genome sequencing of Streptomyces coelicolor A3(2) which revealed an unexpected potential of this organism to synthesize natural products undetected until then by classical screening methods. Since then, analysis of sequenced genomes from numerous Streptomyces species has shown that a single species can carry more than 30 secondary metabolite gene clusters, reinforcing the idea that the biosynthetic potential of this bacterial genus is far from being fully exploited. This review highlights our knowledge on the potential of Streptomyces ambofaciens ATCC 23877 to synthesize natural products. This industrial strain was known for decades to only produce the drug spiramycin and another antibacterial compound, congocidine. Mining of its genome allowed the identification of 23 clusters potentially involved in the production of other secondary metabolites. Studies of some of these clusters resulted in the characterization of novel compounds and of previously known compounds but never characterized in this Streptomyces species. In addition, genome mining revealed that secondary metabolite gene clusters of phylogenetically closely related Streptomyces are mainly species-specific.

Antimicrob Agents Chemother, 2010

Streptomyces ambofaciens synthesizes spiramycin, a 16-membered macrolide antibiotic used in human... more Streptomyces ambofaciens synthesizes spiramycin, a 16-membered macrolide antibiotic used in human medicine. The spiramycin molecule consists of a polyketide lactone ring (platenolide) synthesized by a type I polyketide synthase, to which three deoxyhexoses (mycaminose, forosamine, and mycarose) are attached successively in this order. These sugars are essential to the antibacterial activity of spiramycin. We previously identified four genes in the spiramycin biosynthetic gene cluster predicted to encode glycosyltransferases. We individually deleted each of these four genes and showed that three of them were required for spiramycin biosynthesis. The role of each of the three glycosyltransferases in spiramycin biosynthesis was determined by identifying the biosynthetic intermediates accumulated by the corresponding mutant strains. This led to the identification of the glycosyltransferase responsible for the attachment of each of the three sugars. Moreover, two genes encoding putative glycosyltransferase auxiliary proteins were also identified in the spiramycin biosynthetic gene cluster. When these two genes were deleted, one of them was found to be dispensable for spiramycin biosynthesis. However, analysis of the biosynthetic intermediates accumulated by mutant strains devoid of each of the auxiliary proteins (or of both of them), together with complementation experiments, revealed the interplay of glycosyltransferases with the auxiliary proteins. One of the auxiliary proteins interacted efficiently with the two glycosyltransferases transferring mycaminose and forosamine while the other auxiliary protein interacted only with the mycaminosyltransferase.

Journal of Biotechnology, 2015

Streptomyces ambofaciens ATCC23877 is a soil bacterium industrially exploited for the production ... more Streptomyces ambofaciens ATCC23877 is a soil bacterium industrially exploited for the production of the macrolide spiramycin which is used in human medicine as an antibacterial and anti-toxoplasmosis chemical. Its genome consists of a 8.3Mbp linear chromosome and a 89kb circular plasmid. The complete genome sequence reported here will enable us to investigate Streptomyces genome evolution and to discover new secondary metabolites with potential applications notably in human medicine.

Albonoursin [cyclo(ΔPhe-ΔLeu)], an antibacterial peptide produced by Streptomyces noursei, is one... more Albonoursin [cyclo(ΔPhe-ΔLeu)], an antibacterial peptide produced by Streptomyces noursei, is one of the simplest representatives of the large diketopiperazine (DKP) family. Formation of α,β unsaturations was previously shown to occur on cyclo(L-Phe-L-Leu), catalyzed by the cyclic dipeptide oxidase (CDO). We used CDO peptide sequence information to isolate a 3.8 kb S. noursei DNA fragment that directs albonoursin biosynthesis in Streptomyces lividans. This fragment encompasses four complete genes: albA and albB, necessary for CDO activity; albC, sufficient for cyclic dipeptide precursor formation, although displaying no similarity to non ribosomal peptide synthetase (NRPS) genes; and albD, encoding a putative membrane protein. This first isolated DKP biosynthetic gene cluster should help to elucidate the mechanism of DKP formation, totally independent of NRPS, and to characterize novel DKP biosynthetic pathways that could be engineered to increase the molecular diversity of DKP deri...

Nature chemical biology, Jan 18, 2015

Microbiology (Reading, England), 2004

Nonribosomal peptide synthetases (NRPSs) are giant multi-domain enzymes that catalyse the biosynt... more Nonribosomal peptide synthetases (NRPSs) are giant multi-domain enzymes that catalyse the biosynthesis of many commercially important peptides produced by bacteria and fungi. Several studies over the last decade have shown that many of the individual domains within NRPSs exhibit significant substrate selectivity, which impacts on our ability to engineer NRPSs to produce new bioactive microbial peptides. Adenylation domains appear to be the primary determinants of substrate selectivity in NRPSs. Much progress has been made towards an empirical understanding of substrate selection by these domains over the last 5 years, but the molecular basis of substrate selectivity in these domains is not yet well understood. Perhaps surprisingly, condensation domains have also been reported to exhibit moderate to high substrate selectivity, although the generality of this observation and its potential impact on engineered biosynthesis experiments has yet to be fully elucidated. The situation is le...

ACS Chemical Biology, 2014

The pyrrolamides constitute a small family of secondary metabolites that are known for their abil... more The pyrrolamides constitute a small family of secondary metabolites that are known for their ability to bind noncovalently to the DNA minor groove with some sequence specificity. To date, only a single pyrrolamide biosynthetic gene cluster has been reported, directing the synthesis of congocidine (netropsin) in Streptomyces ambofaciens. In this study, we improve our understanding of pyrrolamide biosynthesis through the identification and characterization of the gene cluster responsible for the production of distamycin in Streptomyces netropsis DSM40846. We discover that the strain produces two other pyrrolamides, the well-characterized congocidine and a congocidine/distamycin hybrid that we named disgocidine. S. netropsis DSM40846 genome analysis led to the identification of two distinct pyrrolamide-like biosynthetic gene clusters. We show here that these two clusters are reciprocally dependent for the production of the three pyrrolamide molecules. Furthermore, based on detailed functional analysis of these clusters, we propose a biosynthetic route to congocidine and distamycin and an updated model for pyrrolamide assembly. The synthesis of disgocidine, the distamycin/congocidine hybrid, appears to constitute the first example of "natural combinatorial biosynthesis" between two related biosynthetic pathways. Finally, we analyze the genomic context of the two biosynthetic gene clusters and suggest that the presently interdependent clusters result from the coevolution of two ancestral independent pyrrolamide gene clusters.

European Journal of Biochemistry, 2001

Cyclic dipeptide oxidase is a novel enzyme that specifically catalyzes the formation of a,b-dehyd... more Cyclic dipeptide oxidase is a novel enzyme that specifically catalyzes the formation of a,b-dehydro-Phe (DPhe) and a,b-dehydro-Leu (DLeu) residues during the biosynthesis of albonoursin, cyclo(DPhe-DLeu), an antibiotic produced by Streptomyces noursei. It was purified 600-fold with a 30% overall recovery, and consists of the association of a single type of subunit with a relative molecular mass of 21 066 resulting in a large homopolymer of relative molecular mass over 2 000 000. The enzyme exhibits a typical flavoprotein spectrum with maxima at 343.5 and 447.5 nm, the flavin prosthetic group being covalently bound to the protein. The catalytic reaction of the natural substrate cyclo(l-Phe-l-Leu) occurs in a two-step sequential reaction leading first to cyclo(a,b-dehydro-Phe-l-Leu) and finally to albonoursin. Kinetic parameters for the first step were determined (K m 53 mm; k 0.69 s 21 ). The enzyme was shown to catalyze the conversion of a variety of cyclo(dipeptides) and can be reoxidized at the expense of molecular oxygen by producing H 2 O 2 . This reaction mechanism, which differs from those already described for the formation of a,b-dehydro-amino acids, might consist of the transient formation of an intermediate imine followed by its rearrangement into an a,b-dehydro-residue.

Nature Chemical Biology, 2005

Analyses of microbial genome sequences reveal numerous examples of gene clusters encoding protein... more Analyses of microbial genome sequences reveal numerous examples of gene clusters encoding proteins typically involved in complex natural product biosynthesis but not associated with the production of known natural products 1-3 . In Streptomyces coelicolor M145 there are several gene clusters encoding new nonribosomal peptide synthetase (NRPS) systems not associated with known metabolites. Application of structure-based models for substrate recognition by NRPS adenylation domains 4-6 predicts the amino acids incorporated into the putative peptide products of these systems 7,3 , but the accuracy of these predictions is untested. Here we report the isolation and structure determination of the new trishydroxamate tetrapeptide iron chelator coelichelin from S. coelicolor using a genome mining approach guided by substrate predictions for the trimodular NRPS CchH, and we show that this enzyme, which lacks a C-terminal thioesterase domain, together with a homolog of enterobactin esterase (CchJ), are required for coelichelin biosynthesis. These results demonstrate that accurate prediction of adenylation domain substrate selectivity is possible and raise intriguing mechanistic questions regarding the assembly of a tetrapeptide by a trimodular NRPS.

Natural Product Reports, 2012

We review here work on the biosynthesis of diketopiperazines (DKPs), a large class of natural pro... more We review here work on the biosynthesis of diketopiperazines (DKPs), a large class of natural products with noteworthy biological activities, focusing on the biosynthetic pathways involving cyclodipeptide synthases (CDPSs), a newly defined family of enzymes. Distinct from nonribosomal peptide synthetases (NRPSs), the other family of enzymes synthesizing DKPs, CDPSs bridge the primary and secondary metabolic pathways by hijacking aminoacyl-tRNAs to produce DKPs. This review includes a comprehensive description of the state of the art for CDPS-dependent pathways, and highlights the ways in which this knowledge could be used to increase the diversity of natural DKPs by pathway engineering.

Microbiology, 2006

Siderophore-mediated iron acquisition has been well studied in many bacterial pathogens because i... more Siderophore-mediated iron acquisition has been well studied in many bacterial pathogens because it contributes to virulence. In contrast, siderophore-mediated iron acquisition by saprophytic bacteria has received relatively little attention. The independent identification of the des and cch gene clusters that direct production of the tris-hydroxamate ferric iron-chelators desferrioxamine E and coelichelin, respectively, which could potentially act as siderophores in the saprophyte Streptomyces coelicolor A3(2), has recently been reported. Here it is shown that the des cluster also directs production of desferrioxamine B in S. coelicolor and that very similar des and cch clusters direct production of desferrioxamines E and B, and coelichelin, respectively, in Streptomyces ambofaciens ATCC 23877. Sequence analyses of the des and cch clusters suggest that components of ferric-siderophore uptake systems are also encoded within each cluster. The construction and analysis of a series of mutants of S. coelicolor lacking just biosynthetic genes or both the biosynthetic and siderophore uptake genes from the des and cch clusters demonstrated that coelichelin and desferrioxamines E and B all function as siderophores in this organism and that at least one of these metabolites is required for growth under defined conditions even in the presence of significant quantities of ferric iron. These experiments also demonstrated that a third siderophore uptake system must be present in S. coelicolor, in addition to the two encoded within the cch and des clusters, which show selectivity for coelichelin and desferrioxamine E, respectively. The ability of the S. coelicolor mutants to utilize a range of exogenous xenosiderophores for iron acquisition was also examined, showing that the third siderophore-iron transport system has broad specificity for tris-hydroxamate-containing siderophores. Together, these results define a complex system of multiple biosynthetic and uptake pathways for siderophore-mediated iron acquisition in S. coelicolor and S. ambofaciens.

Microbiology, 2007

MbtH-like proteins are a family of small proteins encoded by genes found in many, but not all, no... more MbtH-like proteins are a family of small proteins encoded by genes found in many, but not all, non-ribosomal peptide synthetase-encoding gene clusters that direct the biosynthesis of peptide antibiotics and siderophores. Studies published to date have not elucidated the function of MbtH-like proteins, nor have they clarified whether they are required for metabolite biosynthesis.

Journal of Industrial Microbiology & Biotechnology, 2014

Since the discovery of the streptomycin produced by Streptomyces griseus in the middle of the las... more Since the discovery of the streptomycin produced by Streptomyces griseus in the middle of the last century, members of this bacterial genus have been largely exploited for the production of secondary metabolites with wide uses in medicine and in agriculture. They have even been recognized as one of the most prolific producers of natural products among microorganisms. With the onset of the genomic era, it became evident that these microorganisms still represent a major source for the discovery of novel secondary metabolites. This was highlighted with the complete genome sequencing of Streptomyces coelicolor A3(2) which revealed an unexpected potential of this organism to synthesize natural products undetected until then by classical screening methods. Since then, analysis of sequenced genomes from numerous Streptomyces species has shown that a single species can carry more than 30 secondary metabolite gene clusters, reinforcing the idea that the biosynthetic potential of this bacterial genus is far from being fully exploited. This review highlights our knowledge on the potential of Streptomyces ambofaciens ATCC 23877 to synthesize natural products. This industrial strain was known for decades to only produce the drug spiramycin and another antibacterial compound, congocidine. Mining of its genome allowed the identification of 23 clusters potentially involved in the production of other secondary metabolites. Studies of some of these clusters resulted in the characterization of novel compounds and of previously known compounds but never characterized in this Streptomyces species. In addition, genome mining revealed that secondary metabolite gene clusters of phylogenetically closely related Streptomyces are mainly species-specific.

Nature chemical biology, 2009

Cyclodipeptides and their derivatives belong to the diketopiperazine (DKP) family, which is compr... more Cyclodipeptides and their derivatives belong to the diketopiperazine (DKP) family, which is comprised of a broad array of natural products that exhibit useful biological properties. In the few known DKP biosynthetic pathways, nonribosomal peptide synthetases (NRPSs) are involved in the synthesis of cyclodipeptides that constitute the DKP scaffold, except in the albonoursin (1) pathway. Albonoursin, or cyclo(alpha,beta-dehydroPhe-alpha,beta-dehydroLeu), is an antibacterial DKP produced by Streptomyces noursei. In this pathway, the formation of the cyclo(Phe-Leu) (2) intermediate is catalyzed by AlbC, a small protein unrelated to NRPSs. We demonstrated that AlbC uses aminoacyl-tRNAs as substrates to catalyze the formation of the DKP peptide bonds. Moreover, several other bacterial proteins, presenting moderate similarity to AlbC, also use aminoacyl-tRNAs to synthesize various cyclodipeptides. Therefore, AlbC and these related proteins belong to a newly defined family of enzymes that w...

Chemistry & Biology, 2009

Congocidine (netropsin) is a pyrrole-amide (oligopyrrole, oligopeptide) antibiotic produced by St... more Congocidine (netropsin) is a pyrrole-amide (oligopyrrole, oligopeptide) antibiotic produced by Streptomyces ambofaciens. We have identified, in the right terminal region of the S. ambofaciens chromosome, the gene cluster that directs congocidine biosynthesis. Heterologous expression of the cluster and in-frame deletions of 8 of the 22 genes confirm the involvement of this cluster in congocidine biosynthesis. Nine genes can be assigned specific functions in regulation, resistance, or congocidine assembly. In contrast, the biosynthetic origin of the precursors cannot be easily inferred from in silico analyses. Congocidine is assembled by a nonribosomal peptide synthetase (NRPS) constituted of a free-standing module and several single-domain proteins encoded by four genes. The iterative use of its unique adenylation domain, the utilization of guanidinoacetyl-CoA as a substrate by a condensation domain, and the control of 4-aminopyrrole-2-carboxylate polymerization constitute the most original features of this NRPS.

Chemistry & Biology, 2002

Dé partement d'Ingé nierie et d'Etudes des Proté ines F91191 Gif-sur-Yvette Cedex France mechanis... more Dé partement d'Ingé nierie et d'Etudes des Proté ines F91191 Gif-sur-Yvette Cedex France mechanisms [4-6]. Although the number of newly isolated, naturally oc-2 Institut de Gé né tique et Microbiologie CNRS UMR 8621, Bâ t. 400 curring DKPs has increased during the last few years, the biosynthetic pathways of these molecules remain Université Paris-Sud F91405 Orsay Cedex largely unexplored. In bacteria and fungi, DKP derivatives generally seem to be produced by nonribosomal France pathways, although their biosynthesis has never been shown to be directly catalyzed by NRPS megacomplexes. Studies of ergot alkaloid biosynthesis in the fun-Summary gus Claviceps purpurea have shown that the D-lysergyl peptide synthetase, which is responsible for the forma-Albonoursin [cyclo(⌬Phe-⌬Leu)], an antibacterial peptide produced by Streptomyces noursei, is one of the tion of the D-lysergyl peptide-DKP, an intermediate in the biosynthesis of ergopeptines, is devoid of a C-terminal simplest representatives of the large diketopiperazine (DKP) family. Formation of ␣,␤ unsaturations was pre-thioesterase domain. DKP cyclization is thus thought to occur spontaneously, leading to the release of the viously