scholarly journals Phosphinothricin Tripeptide Synthetases in Streptomyces viridochromogenes Tü494

2005 ◽  
Vol 49 (11) ◽  
pp. 4598-4607 ◽  
Author(s):  
Dirk Schwartz ◽  
Nicolas Grammel ◽  
Eva Heinzelmann ◽  
Ullrich Keller ◽  
Wolfgang Wohlleben
Keyword(s):  
Gene Cluster ◽  
Nonribosomal Peptide Synthetase ◽  
Herbicidal Activity ◽  
Type Ii ◽  
Biosynthesis Gene Cluster ◽  
Nonribosomal Peptide ◽  
Product Release ◽  
Peptide Synthetase ◽  
Biosynthesis Gene

ABSTRACT The tripeptide backbone of phosphinothricin (PT) tripeptide (PTT), a compound with herbicidal activity from Streptomyces viridochromogenes, is assembled by three stand-alone peptide synthetase modules. The enzyme PhsA (66 kDa) recruits the PT-precursor N-acetyl-demethylphosphinothricin (N-Ac-DMPT), whereas the two alanine residues of PTT are assembled by the enzymes PhsB and PhsC (129 and 119 kDa, respectively). During or after assembly, the N-Ac-DMPT residue in the peptide is converted to PT by methylation and deacetylation. Both phsB and phsC appear to be cotranscribed together with two other genes from a single promoter and they are located at a distance of 20 kb from the gene phsA, encoding PhsA, in the PTT biosynthesis gene cluster of S. viridochromogenes. PhsB and PhsC represent single nonribosomal peptide synthetase elongation modules lacking a thioesterase domain. Gene inactivations, genetic complementations, determinations of substrate specificity of the heterologously produced proteins, and comparison of PhsC sequence with the amino terminus of the alanine-activating nonribosomal peptide synthetase PTTSII from S. viridochromogenes confirmed the role of the two genes in the bialanylation of Ac-DMPT. The lack of an integral thioesterase domain in the PTT assembly system points to product release possibly involving two type II thioesterase genes (the1 and the2) located in the PTT gene cluster alone or in conjunction with an as yet unknown mechanism of product release.

scholarly journals Activation of a Silent Fungal Polyketide Biosynthesis Pathway through Regulatory Cross Talk with a Cryptic Nonribosomal Peptide Synthetase Gene Cluster

2010 ◽  
Vol 76 (24) ◽  
pp. 8143-8149 ◽  
Author(s):  
Sebastian Bergmann ◽  
Alexander N. Funk ◽  
Kirstin Scherlach ◽  
Volker Schroeckh ◽  
Ekaterina Shelest ◽  
...  
Keyword(s):  
Natural Products ◽  
Gene Cluster ◽  
Secondary Metabolite ◽  
Cross Talk ◽  
Regulatory Gene ◽  
Gene Clusters ◽  
Nonribosomal Peptide Synthetase ◽  
Nonribosomal Peptide ◽  
Peptide Synthetase ◽  
Biosynthesis Gene

ABSTRACT Filamentous fungi produce numerous natural products that constitute a consistent source of potential drug leads, yet it seems that the majority of natural products are overlooked since most biosynthesis gene clusters are silent under standard cultivation conditions. Screening secondary metabolite genes of the model fungus Aspergillus nidulans, we noted a silent gene cluster on chromosome II comprising two nonribosomal peptide synthetase (NRPS) genes, inpA and inpB, flanked by a regulatory gene that we named scpR for secondary metabolism cross-pathway regulator. The induced expression of the scpR gene using the promoter of the alcohol dehydrogenase AlcA led to the transcriptional activation of both the endogenous scpR gene and the NRPS genes. Surprisingly, metabolic profiling of the supernatant of mycelia overexpressing scpR revealed the production of the polyketide asperfuranone. Through transcriptome analysis we found that another silent secondary metabolite gene cluster located on chromosome VIII coding for asperfuranone biosynthesis was specifically induced. Quantitative reverse transcription-PCR proved the transcription not only of the corresponding polyketide synthase (PKS) biosynthesis genes, afoE and afoG, but also of their activator, afoA, under alcAp-scpR-inducing conditions. To exclude the possibility that the product of the inp cluster induced the asperfuranone gene cluster, a strain carrying a deletion of the NRPS gene inpB and, in addition, the alcAp-scpR overexpression cassette was generated. In this strain, under inducing conditions, transcripts of the biosynthesis genes of both the NRPS-containing gene cluster inp and the asperfuranone gene cluster except gene inpB were detected. Moreover, the existence of the polyketide product asperfuranone indicates that the transcription factor ScpR controls the expression of the asperfuranone biosynthesis gene cluster. This expression as well as the biosynthesis of asperfuranone was abolished after the deletion of the asperfuranone activator gene afoA, indicating that ScpR binds to the afoA promoter. To the best of our knowledge, this is the first report of regulatory cross talk between two biosynthesis gene clusters located on different chromosomes.

scholarly journals Disruption of a Nonribosomal Peptide Synthetase in Aspergillus fumigatus Eliminates Gliotoxin Production

2006 ◽  
Vol 5 (6) ◽  
pp. 972-980 ◽  
Author(s):  
Robert A. Cramer ◽  
Michael P. Gamcsik ◽  
Rhea M. Brooking ◽  
Laura K. Najvar ◽  
William R. Kirkpatrick ◽  
...  
Keyword(s):  
Invasive Aspergillosis ◽  
Aspergillus Fumigatus ◽  
Regulation Of Gene Expression ◽  
Nonribosomal Peptide Synthetase ◽  
Biosynthesis Gene Cluster ◽  
Nonribosomal Peptide ◽  
Peptide Synthetase ◽  
Impact On Survival ◽  
Fungal Secondary Metabolite

ABSTRACT The fungal secondary metabolite gliotoxin produced by Aspergillus fumigatus has been hypothesized to be important in the development of invasive aspergillosis. In this study, we addressed this hypothesis by disrupting a nonribosomal peptide synthetase (NRPS) (encoded by gliP) predicted to be involved in gliotoxin production. Mutants with a disrupted gliP locus failed to produce gliotoxin, which confirmed the role of the NRPS encoded by gliP in gliotoxin biosynthesis. We found no morphological, developmental, or physiological defects in ΔgliP mutant strains. In addition, disruption of gliP resulted in down regulation of gene expression in the gliotoxin biosynthesis gene cluster, which was restored with addition of exogenous gliotoxin. This interesting result suggests a role for gliotoxin in regulating its own production. Culture filtrates from the ΔgliP mutant were unable to inhibit ionomycin-dependent degranulation of mast cells, suggesting a role for gliotoxin in suppressing mast cell degranulation and possibly in disease development. However, the ΔgliP mutant did not have an impact on survival or tissue burden in a murine inhalational model of invasive aspergillosis. This result suggests that gliotoxin is not required for virulence in an immunosuppressed host with an invasive pulmonary infection.

scholarly journals Gene Cluster Involved in the Biosynthesis of Griseobactin, a Catechol-Peptide Siderophore of Streptomyces sp. ATCC 700974

2009 ◽  
Vol 192 (2) ◽  
pp. 426-435 ◽  
Author(s):  
Silke I. Patzer ◽  
Volkmar Braun
Keyword(s):  
Gene Cluster ◽  
Linear Form ◽  
Streptomyces Griseus ◽  
Biosynthetic Gene Cluster ◽  
Peptide Structure ◽  
Biosynthetic Gene ◽  
Biosynthesis Gene Cluster ◽  
Streptomyces Sp ◽  
Peptide Synthetase ◽  
Biosynthesis Gene

ABSTRACT The main siderophores produced by streptomycetes are desferrioxamines. Here we show that Streptomyces sp. ATCC 700974 and several Streptomyces griseus strains, in addition, synthesize a hitherto unknown siderophore with a catechol-peptide structure, named griseobactin. The production is repressed by iron. We sequenced a 26-kb DNA region comprising a siderophore biosynthetic gene cluster encoding proteins similar to DhbABCEFG, which are involved in the biosynthesis of 2,3-dihydroxybenzoate (DHBA) and in the incorporation of DHBA into siderophores via a nonribosomal peptide synthetase. Adjacent to the biosynthesis genes are genes that encode proteins for the secretion, uptake, and degradation of siderophores. To correlate the gene cluster with griseobactin synthesis, the dhb genes in ATCC 700974 were disrupted. The resulting mutants no longer synthesized DHBA and griseobactin; production of both was restored by complementation with the dhb genes. Heterologous expression of the dhb genes or of the entire griseobactin biosynthesis gene cluster in the catechol-negative strain Streptomyces lividans TK23 resulted in the synthesis and secretion of DHBA or griseobactin, respectively, suggesting that these genes are sufficient for DHBA and griseobactin biosynthesis. Griseobactin was purified and characterized; its structure is consistent with a cyclic and, to a lesser extent, linear form of the trimeric ester of 2,3-dihydroxybenzoyl-arginyl-threonine complexed with aluminum under iron-limiting conditions. This is the first report identifying the gene cluster for the biosynthesis of DHBA and a catechol siderophore in Streptomyces.

scholarly journals The Role of a Nonribosomal Peptide Synthetase inl-Lysine Lactamization During Capuramycin Biosynthesis

ChemBioChem ◽  
2016 ◽  
Vol 17 (9) ◽  
pp. 804-810 ◽  
Author(s):  
Xiaodong Liu ◽  
Yuanyuan Jin ◽  
Zheng Cui ◽  
Koichi Nonaka ◽  
Satoshi Baba ◽  
...  
Keyword(s):  
Nonribosomal Peptide Synthetase ◽  
Nonribosomal Peptide ◽  
Peptide Synthetase

scholarly journals Manipulating Protein–Protein Interactions in Nonribosomal Peptide Synthetase Type II Peptidyl Carrier Proteins

Biochemistry ◽  
2017 ◽  
Vol 56 (40) ◽  
pp. 5269-5273 ◽  
Author(s):  
Matt J. Jaremko ◽  
D. John Lee ◽  
Ashay Patel ◽  
Victoria Winslow ◽  
Stanley J. Opella ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Nonribosomal Peptide Synthetase ◽  
Type Ii ◽  
Carrier Proteins ◽  
Protein Protein Interactions ◽  
Nonribosomal Peptide ◽  
Peptide Synthetase

scholarly journals Nostophycin Biosynthesis Is Directed by a Hybrid Polyketide Synthase-Nonribosomal Peptide Synthetase in the Toxic Cyanobacterium Nostoc sp. Strain 152

2011 ◽  
Vol 77 (22) ◽  
pp. 8034-8040 ◽  
Author(s):  
David P. Fewer ◽  
Julia Österholm ◽  
Leo Rouhiainen ◽  
Jouni Jokela ◽  
Matti Wahlsten ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Polyketide Synthase ◽  
Catalytic Domain ◽  
Phylogenetic Analyses ◽  
Biosynthetic Gene Cluster ◽  
Nonribosomal Peptide Synthetase ◽  
Open Reading Frames ◽  
Nonribosomal Peptide ◽  
Peptide Synthetase ◽  
Biochemical Analyses

ABSTRACTCyanobacteria are a rich source of natural products with interesting pharmaceutical properties. Here, we report the identification, sequencing, annotation, and biochemical analysis of the nostophycin (npn) biosynthetic gene cluster. Thenpngene cluster spans 45.1 kb and consists of three open reading frames encoding a polyketide synthase, a mixed polyketide nonribosomal peptide synthetase, and a nonribosomal peptide synthetase. The genetic architecture and catalytic domain organization of the proteins are colinear in arrangement, with the putative order of the biosynthetic assembly of the cyclic heptapeptide. NpnB contains an embedded monooxygenase domain linking nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) catalytic domains and predicted here to hydroxylate the nostophycin during assembly. Expression of the adenylation domains and subsequent substrate specificity assays support the involvement of this cluster in nostophycin biosynthesis. Biochemical analyses suggest that the loading substrate of NpnA is likely to be a phenylpropanoic acid necessitating deletion of a carbon atom to explain the biosynthesis of nostophycin. Biosyntheses of nostophycin and microcystin resemble each other, but the phylogenetic analyses suggest that they are distantly related to one another.

scholarly journals Biosynthesis Gene Cluster and Oxazole Ring Formation Enzyme for Inthomycins in Streptomyces sp. Strain SYP-A7193

2020 ◽  
Vol 86 (20) ◽  
Author(s):  
Shao-Yang Hou ◽  
Meng-Yue Zhang ◽  
Hong-Da Wang ◽  
Yi-Xuan Zhang
Keyword(s):  
Gene Cluster ◽  
Genome Sequencing ◽  
Gene Disruption ◽  
Polyketide Synthase ◽  
Nonribosomal Peptide Synthetase ◽  
Ring Formation ◽  
Type I ◽  
Oxazole Ring ◽  
Peptide Synthetase ◽  
Biosynthesis Gene

ABSTRACT Inthomycins belong to a growing family of oxazole-containing polyketides and exhibit a broad spectrum of anti-oomycete and herbicidal activities. In this study, we purified inthomycins A and B from the metabolites of Streptomyces sp. strain SYP-A7193 and determined their chemical structures. Genome sequencing, comparative genomic analysis, and gene disruption of Streptomyces sp. SYP-A7193 showed that the inthomycin biosynthetic gene cluster (itm) belonged to the hybrid polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) system. Functional domain comparison and disruption/complementation experiments of itm12 resulted in the complete loss of inthomycins A and B and the subsequent restoration of their production, confirming that itm12 encodes a discrete acyltransferase (AT), and hence, itm was considered to belong to the trans-AT type I PKS system. Moreover, the disruption/complementation experiments of itm15 also resulted in the loss and restoration of inthomycin A and B formation. Further gene cloning, expression, purification, and activity verification of itm15 revealed that Itm15 is a cyclodehydratase that catalyzes a straight-chain dehydration reaction to form an oxazole ring for the biosynthesis of inthomycins A and B. Thus, we discovered a novel enzyme that catalyzes oxazole ring formation and elucidated the complete biosynthetic pathway of inthomycins. IMPORTANCE Streptomyces species produce numerous secondary metabolites with diverse structures and pharmacological activities that are beneficial for human health and have several applications in agriculture. In this study, hybrid nonribosomal peptide synthetase/polyketide synthase metabolites inthomycins A and B were isolated from after fermenting Streptomyces sp. SYP-A7193. Genome sequencing, gene disruption, gene complementation, heterologous expression, and activity assay revealed that the biosynthesis gene assembly line of inthomycins A and B was a 95.3-kb trans-AT type I PKS system in the strain SYP-A7193. More importantly, Itm15, a cyclodehydratase, was identified to be an oxazole ring formation enzyme required for the biosynthesis of inthomycins A and B; it is significant to discover this catalyzation reaction in the PKS/NRPS system in the field of microbiology. Our findings could provide further insights into the diversity of trans-AT type I PKS systems and the mechanism of oxazole cyclization involved in the biosynthesis of natural products.

scholarly journals Characterization of the Saframycin A Gene Cluster from Streptomyces lavendulae NRRL 11002 Revealing a Nonribosomal Peptide Synthetase System for Assembling the Unusual Tetrapeptidyl Skeleton in an Iterative Manner

2007 ◽  
Vol 190 (1) ◽  
pp. 251-263 ◽  
Author(s):  
Lei Li ◽  
Wei Deng ◽  
Jie Song ◽  
Wei Ding ◽  
Qun-Fei Zhao ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Biosynthetic Gene Cluster ◽  
Nonribosomal Peptide Synthetase ◽  
Combinatorial Biosynthesis ◽  
Common Mechanism ◽  
Nonribosomal Peptide ◽  
Streptomyces Lavendulae ◽  
Peptide Synthetase

ABSTRACT Saframycin A (SFM-A), produced by Streptomyces lavendulae NRRL 11002, belongs to the tetrahydroisoquinoline family of antibiotics, and its core is structurally similar to the core of ecteinascidin 743, which is a highly potent antitumor drug isolated from a marine tunicate. In this study, the biosynthetic gene cluster for SFM-A was cloned and localized to a 62-kb contiguous DNA region. Sequence analysis revealed 30 genes that constitute the SFM-A gene cluster, encoding an unusual nonribosomal peptide synthetase (NRPS) system and tailoring enzymes and regulatory and resistance proteins. The results of substrate prediction and in vitro characterization of the adenylation specificities of this NRPS system support the hypothesis that the last module acts in an iterative manner to form a tetrapeptidyl intermediate and that the colinearity rule does not apply. Although this mechanism is different from those proposed for the SFM-A analogs SFM-Mx1 and safracin B (SAC-B), based on the high similarity of these systems, it is likely they share a common mechanism of biosynthesis as we describe here. Construction of the biosynthetic pathway of SFM-Y3, an aminated SFM-A, was achieved in the SAC-B producer (Pseudomonas fluorescens). These findings not only shed new insight on tetrahydroisoquinoline biosynthesis but also demonstrate the feasibility of engineering microorganisms to generate structurally more complex and biologically more active analogs by combinatorial biosynthesis.

scholarly journals Characterization of a Gene Cluster Responsible for the Biosynthesis of Anticancer Agent FK228 in Chromobacterium violaceum No. 968

2007 ◽  
Vol 73 (11) ◽  
pp. 3460-3469 ◽  
Author(s):  
Yi-Qiang Cheng ◽  
Min Yang ◽  
Andrea M. Matter
Keyword(s):  
Gene Cluster ◽  
Genomic Dna ◽  
Polyketide Synthase ◽  
Anticancer Agent ◽  
Biosynthetic Gene Cluster ◽  
Nonribosomal Peptide Synthetase ◽  
Biosynthetic Gene ◽  
Biosynthetic Genes ◽  
Nonribosomal Peptide ◽  
Peptide Synthetase

ABSTRACT A gene cluster responsible for the biosynthesis of anticancer agent FK228 has been identified, cloned, and partially characterized in Chromobacterium violaceum no. 968. First, a genome-scanning approach was applied to identify three distinctive C. violaceum no. 968 genomic DNA clones that code for portions of nonribosomal peptide synthetase and polyketide synthase. Next, a gene replacement system developed originally for Pseudomonas aeruginosa was adapted to inactivate the genomic DNA-associated candidate natural product biosynthetic genes in vivo with high efficiency. Inactivation of a nonribosomal peptide synthetase-encoding gene completely abolished FK228 production in mutant strains. Subsequently, the entire FK228 biosynthetic gene cluster was cloned and sequenced. This gene cluster is predicted to encompass a 36.4-kb DNA region that includes 14 genes. The products of nine biosynthetic genes are proposed to constitute an unusual hybrid nonribosomal peptide synthetase-polyketide synthase-nonribosomal peptide synthetase assembly line including accessory activities for the biosynthesis of FK228. In particular, a putative flavin adenine dinucleotide-dependent pyridine nucleotide-disulfide oxidoreductase is proposed to catalyze disulfide bond formation between two sulfhydryl groups of cysteine residues as the final step in FK228 biosynthesis. Acquisition of the FK228 biosynthetic gene cluster and acclimation of an efficient genetic system should enable genetic engineering of the FK228 biosynthetic pathway in C. violaceum no. 968 for the generation of structural analogs as anticancer drug candidates.

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