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

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.

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Phosphinothricin Tripeptide Synthetases in Streptomyces viridochromogenes Tü494

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.11.4598-4607.2005 ◽

2005 ◽

Vol 49 (11) ◽

pp. 4598-4607 ◽

Cited By ~ 15

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.

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Comparison of Cyanopeptolin Genes in Planktothrix, Microcystis, and Anabaena Strains: Evidence for Independent Evolution within Each Genus

Applied and Environmental Microbiology ◽

10.1128/aem.01475-07 ◽

2007 ◽

Vol 73 (22) ◽

pp. 7322-7330 ◽

Cited By ~ 38

Author(s):

Trine B. Rounge ◽

Thomas Rohrlack ◽

Ave Tooming-Klunderud ◽

Tom Kristensen ◽

Kjetill S. Jakobsen

Keyword(s):

Gene Cluster ◽

Cyclic Peptide ◽

Phylogenetic Analyses ◽

Gene Clusters ◽

Binding Pocket ◽

Nonribosomal Peptide Synthetase ◽

Glyceric Acid ◽

Nonribosomal Peptide ◽

Independent Evolution ◽

Peptide Synthetase

ABSTRACT The major cyclic peptide cyanopeptolin 1138, produced by Planktothrix strain NIVA CYA 116, was characterized and shown to be structurally very close to the earlier-characterized oscillapeptin E. A cyanopeptolin gene cluster likely to encode the corresponding peptide synthetase was sequenced from the same strain. The 30-kb oci gene cluster contains two novel domains previously not detected in nonribosomal peptide synthetase gene clusters (a putative glyceric acid-activating domain and a sulfotransferase domain), in addition to seven nonribosomal peptide synthetase modules. Unlike in two previously described cyanopeptolin gene clusters from Anabaena and Microcystis, a halogenase gene is not present. The three cyanopeptolin gene clusters show similar gene and domain arrangements, while the binding pocket signatures deduced from the adenylation domain sequences and the additional tailoring domains vary. This suggests loss and gain of tailoring domains within each genus, after the diversification of the three clades, as major events leading to the present diversity. The ABC transporter genes associated with the cyanopeptolin gene clusters form a monophyletic clade and accordingly are likely to have evolved as part of the functional unit. Phylogenetic analyses of adenylation and condensation domains, including domains from cyanopeptolins and microcystins, show a closer similarity between the Planktothrix and Microcystis cyanopeptolin domains than between these and the Anabaena domain. No clear evidence of recombination between cyanopeptolins and microcystins could be detected. There were no strong indications of horizontal gene transfer of cyanopeptolin gene sequences across the three genera, supporting independent evolution within each genus.

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Nonribosomal peptide synthetase biosynthetic clusters of ESKAPE pathogens

Natural Product Reports ◽

10.1039/c7np00029d ◽

2017 ◽

Vol 34 (8) ◽

pp. 981-1009 ◽

Cited By ~ 23

Author(s):

Andrew M. Gulick

Keyword(s):

Natural Products ◽

Gene Clusters ◽

Nonribosomal Peptide Synthetase ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Nonribosomal Peptide ◽

Peptide Synthetase ◽

Eskape Pathogens

This review describes the peptide natural products produced by NRPS biosynthetic gene clusters from the ESKAPE pathogens.

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Biosynthesis of isonitrile lipopeptides by conserved nonribosomal peptide synthetase gene clusters in Actinobacteria

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1705016114 ◽

2017 ◽

Vol 114 (27) ◽

pp. 7025-7030 ◽

Cited By ~ 31

Author(s):

Nicholas C. Harris ◽

Michio Sato ◽

Nicolaus A. Herman ◽

Frederick Twigg ◽

Wenlong Cai ◽

...

Keyword(s):

Gene Cluster ◽

Acyl Chain ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Nonribosomal Peptide Synthetase ◽

Biosynthetic Gene ◽

Nonribosomal Peptide ◽

Peptide Synthetase

A putative lipopeptide biosynthetic gene cluster is conserved in many species of Actinobacteria, including Mycobacterium tuberculosis and M. marinum, but the specific function of the encoding proteins has been elusive. Using both in vivo heterologous reconstitution and in vitro biochemical analyses, we have revealed that the five encoding biosynthetic enzymes are capable of synthesizing a family of isonitrile lipopeptides (INLPs) through a thio-template mechanism. The biosynthesis features the generation of isonitrile from a single precursor Gly promoted by a thioesterase and a nonheme iron(II)-dependent oxidase homolog and the acylation of both amino groups of Lys by the same isonitrile acyl chain facilitated by a single condensation domain of a nonribosomal peptide synthetase. In addition, the deletion of INLP biosynthetic genes in M. marinum has decreased the intracellular metal concentration, suggesting the role of this biosynthetic gene cluster in metal transport.

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Antimicrobials Inspired by Nonribosomal Peptide Synthetase Gene Clusters

Journal of the American Chemical Society ◽

10.1021/jacs.6b11861 ◽

2017 ◽

Vol 139 (4) ◽

pp. 1404-1407 ◽

Cited By ~ 18

Author(s):

Xavier Vila-Farres ◽

John Chu ◽

Daigo Inoyama ◽

Melinda A. Ternei ◽

Christophe Lemetre ◽

...

Keyword(s):

Gene Clusters ◽

Nonribosomal Peptide Synthetase ◽

Nonribosomal Peptide ◽

Peptide Synthetase

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Distribution and Evolution of Nonribosomal Peptide Synthetase Gene Clusters in the Ceratocystidaceae

Genes ◽

10.3390/genes10050328 ◽

2019 ◽

Vol 10 (5) ◽

pp. 328 ◽

Cited By ~ 1

Author(s):

Mohammad Sayari ◽

Magriet A. van der Nest ◽

Emma T. Steenkamp ◽

Nicole C. Soal ◽

P. Markus Wilken ◽

...

Keyword(s):

Gene Clusters ◽

Nonribosomal Peptide Synthetase ◽

Main Element ◽

Biosynthetic Pathways ◽

Biosynthetic Gene Clusters ◽

Nrps Gene ◽

Nonribosomal Peptide ◽

Peptide Synthetase ◽

The Family ◽

Multifunctional Enzymes

In filamentous fungi, genes in secondary metabolite biosynthetic pathways are generally clustered. In the case of those pathways involved in nonribosomal peptide production, a nonribosomal peptide synthetase (NRPS) gene is commonly found as a main element of the cluster. Large multifunctional enzymes are encoded by members of this gene family that produce a broad spectrum of bioactive compounds. In this research, we applied genome-based identification of nonribosomal peptide biosynthetic gene clusters in the family Ceratocystidaceae. For this purpose, we used the whole genome sequences of species from the genera Ceratocystis, Davidsoniella, Thielaviopsis, Endoconidiophora, Bretziella, Huntiella, and Ambrosiella. To identify and characterize the clusters, different bioinformatics and phylogenetic approaches, as well as PCR-based methods were used. In all genomes studied, two highly conserved NRPS genes (one monomodular and one multimodular) were identified and their potential products were predicted to be siderophores. Expression analysis of two Huntiella species (H. moniliformis and H. omanensis) confirmed the accuracy of the annotations and proved that the genes in both clusters are expressed. Furthermore, a phylogenetic analysis showed that both NRPS genes of the Ceratocystidaceae formed distinct and well supported clades in their respective phylograms, where they grouped with other known NRPSs involved in siderophore production. Overall, these findings improve our understanding of the diversity and evolution of NRPS biosynthetic pathways in the family Ceratocystidaceae.

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A genome-wide analysis of nonribosomal peptide synthetase gene clusters and their peptides in a Planktothrix rubescens strain

BMC Genomics ◽

10.1186/1471-2164-10-396 ◽

2009 ◽

Vol 10 (1) ◽

pp. 396 ◽

Cited By ~ 63

Author(s):

Trine B Rounge ◽

Thomas Rohrlack ◽

Alexander J Nederbragt ◽

Tom Kristensen ◽

Kjetill S Jakobsen

Keyword(s):

Gene Clusters ◽

Nonribosomal Peptide Synthetase ◽

Nonribosomal Peptide ◽

Genome Wide Analysis ◽

Planktothrix Rubescens ◽

Genome Wide ◽

A Genome ◽

Peptide Synthetase

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Polyketide Synthase and Nonribosomal Peptide Synthetase Gene Clusters in Type Strains of the Genus Phytohabitans

Life ◽

10.3390/life10110257 ◽

2020 ◽

Vol 10 (11) ◽

pp. 257

Author(s):

Hisayuki Komaki ◽

Tomohiko Tamura

Keyword(s):

Secondary Metabolites ◽

Polyketide Synthase ◽

Gene Clusters ◽

Nonribosomal Peptide Synthetase ◽

Nrps Gene ◽

Nonribosomal Peptide ◽

Rare Actinomycetes ◽

Whole Genomes ◽

Peptide Synthetase ◽

Established Genus

(1) Background: Phytohabitans is a recently established genus belonging to rare actinomycetes. It has been unclear if its members have the capacity to synthesize diverse secondary metabolites. Polyketide and nonribosomal peptide compounds are major secondary metabolites in actinomycetes and expected as a potential source for novel pharmaceuticals. (2) Methods: Whole genomes of Phytohabitans flavus NBRC 107702T, Phytohabitans rumicis NBRC 108638T, Phytohabitans houttuyneae NBRC 108639T, and Phytohabitans suffuscus NBRC 105367T were sequenced by PacBio. Polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) gene clusters were bioinformatically analyzed in the genome sequences. (3) Results: These four strains harbored 10, 14, 18 and 14 PKS and NRPS gene clusters, respectively. Most of the gene clusters were annotated to synthesis unknown chemistries. (4) Conclusions: Members of the genus Phytohabitans are a possible source for novel and diverse polyketides and nonribosomal peptides.

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