Eliciting the silent lucensomycin biosynthetic pathway in Streptomyces cyanogenus S136 via manipulation of the global regulatory gene adpA

AbstractActinobacteria are among the most prolific sources of medically and agriculturally important compounds, derived from their biosynthetic gene clusters (BGCs) for specialized (secondary) pathways of metabolism. Genomics witnesses that the majority of actinobacterial BGCs are silent, most likely due to their low or zero transcription. Much effort is put into the search for approaches towards activation of silent BGCs, as this is believed to revitalize the discovery of novel natural products. We hypothesized that the global transcriptional factor AdpA, due to its highly degenerate operator sequence, could be used to upregulate the expression of silent BGCs. Using Streptomyces cyanogenus S136 as a test case, we showed that plasmids expressing either full-length adpA or its DNA-binding domain led to significant changes in the metabolome. These were evident as changes in the accumulation of colored compounds, bioactivity, as well as the emergence of a new pattern of secondary metabolites as revealed by HPLC-ESI-mass spectrometry. We further focused on the most abundant secondary metabolite and identified it as the polyene antibiotic lucensomycin. Finally, we uncovered the entire gene cluster for lucensomycin biosynthesis (lcm), that remained elusive for five decades until now, and outlined an evidence-based scenario for its adpA-mediated activation.

Download Full-text

Expanding the Natural Products Heterologous Expression Repertoire in the Model Cyanobacterium Anabaena sp. Strain PCC 7120: Production of Pendolmycin and Teleocidin B-4

10.26434/chemrxiv.11316098.v1 ◽

2019 ◽

Cited By ~ 1

Author(s):

Patrick Videau ◽

Kaitlyn Wells ◽

Arun Singh ◽

Jessie Eiting ◽

Philip Proteau ◽

...

Keyword(s):

Natural Products ◽

Genome Mining ◽

Gene Clusters ◽

Combinatorial Biosynthesis ◽

Test Case ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Cyanobacterium Anabaena ◽

Anabaena Sp ◽

Pcc 7120

Cyanobacteria are prolific producers of natural products and genome mining has shown that many orphan biosynthetic gene clusters can be found in sequenced cyanobacterial genomes. New tools and methodologies are required to investigate these biosynthetic gene clusters and here we present the use of <i>Anabaena </i>sp. strain PCC 7120 as a host for combinatorial biosynthesis of natural products using the indolactam natural products (lyngbyatoxin A, pendolmycin, and teleocidin B-4) as a test case. We were able to successfully produce all three compounds using codon optimized genes from Actinobacteria. We also introduce a new plasmid backbone based on the native <i>Anabaena</i>7120 plasmid pCC7120ζ and show that production of teleocidin B-4 can be accomplished using a two-plasmid system, which can be introduced by co-conjugation.

Download Full-text

Understanding Thioamitide Biosynthesis Using Pathway Engineering and Untargeted Metabolomics

10.1101/2020.12.17.423248 ◽

2020 ◽

Author(s):

Tom H. Eyles ◽

Natalia M. Vior ◽

Rodney Lacret ◽

Andrew W. Truman

Keyword(s):

Biosynthetic Pathway ◽

Gene Clusters ◽

Untargeted Metabolomics ◽

Pathway Engineering ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Heterologous Host ◽

Detailed Understanding ◽

Precursor Peptide ◽

Modified Peptides

ABSTRACTThiostreptamide S4 is a thioamitide, a family of promising antitumour ribosomally synthesised and post-translationally modified peptides (RiPPs). The thioamitides are one of the most structurally complex RiPP families, yet very few thioamitide biosynthetic steps have been elucidated, even though the gene clusters of multiple thioamitides have been identified. We hypothesised that engineering the thiostreptamide S4 gene cluster in a heterologous host could provide insights into its biosynthesis when coupled with untargeted metabolomics and targeted mutations of the precursor peptide. Modified gene clusters were constructed, and in-depth metabolomics enabled a detailed understanding of the biosynthetic pathway, including the identification of an effector-like protein critical for amino acid dehydration. We use this biosynthetic understanding to bioinformatically identify new widespread families of RiPP biosynthetic gene clusters, paving the way for future RiPP discovery and engineering.

Download Full-text

Activation and Identification of a Griseusin Cluster in Streptomyces sp. CA-256286 by Employing Transcriptional Regulators and Multi-Omics Methods

Molecules ◽

10.3390/molecules26216580 ◽

2021 ◽

Vol 26 (21) ◽

pp. 6580

Author(s):

Charlotte Beck ◽

Tetiana Gren ◽

Francisco Javier Ortiz-López ◽

Tue Sparholt Jørgensen ◽

Daniel Carretero-Molina ◽

...

Keyword(s):

Biosynthetic Pathway ◽

Genome Mining ◽

Gene Clusters ◽

Transcriptional Regulators ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Heterologous Host ◽

Streptomyces Sp ◽

Identification And Characterization

Streptomyces are well-known producers of a range of different secondary metabolites, including antibiotics and other bioactive compounds. Recently, it has been demonstrated that “silent” biosynthetic gene clusters (BGCs) can be activated by heterologously expressing transcriptional regulators from other BGCs. Here, we have activated a silent BGC in Streptomyces sp. CA-256286 by overexpression of a set of SARP family transcriptional regulators. The structure of the produced compound was elucidated by NMR and found to be an N-acetyl cysteine adduct of the pyranonaphtoquinone polyketide 3′-O-α-d-forosaminyl-(+)-griseusin A. Employing a combination of multi-omics and metabolic engineering techniques, we identified the responsible BGC. These methods include genome mining, proteomics and transcriptomics analyses, in combination with CRISPR induced gene inactivations and expression of the BGC in a heterologous host strain. This work demonstrates an easy-to-implement workflow of how silent BGCs can be activated, followed by the identification and characterization of the produced compound, the responsible BGC, and hints of its biosynthetic pathway.

Download Full-text

Bagremycin Antibiotics and Ferroverdin iron-chelators are synthetized by the Same Gene Cluster

10.1101/631242 ◽

2019 ◽

Author(s):

Loïc Martinet ◽

Aymeric Naômé ◽

Benoit Deflandre ◽

Marta Maciejewska ◽

Déborah Tellatin ◽

...

Keyword(s):

Environmental Conditions ◽

Biosynthetic Pathway ◽

Gene Clusters ◽

Iron Chelators ◽

Hydroxybenzoic Acid ◽

Iron Availability ◽

Biosynthetic Gene ◽

Single Family ◽

Biosynthetic Gene Clusters ◽

Specialized Metabolites

AbstractBiosynthetic gene clusters (BGCs) are organized groups of genes involved in the production of specialized metabolites. Typically, one BGC is responsible for the production of one or several similar compounds with bioactivities that usually only vary in terms of strength and/or specificity. Here we show that the previously described ferroverdins and bagremycins, which are families of metabolites with different bioactivities, are produced from the same BGC, whereby the fate of the biosynthetic pathway depends on iron availability. Under conditions of iron depletion, the monomeric bagremycins are formed, which are amino-aromatic antibiotics resulting from the condensation of 3-amino-4-hydroxybenzoic acid with p-vinylphenol. Conversely, when iron is abundantly available, the biosynthetic pathway additionally produces a molecule based on p-vinylphenyl-3-nitroso-4-hydroxybenzoate, which complexes iron to form the trimeric ferroverdins that have anticholesterol activity. Thus our work challenges the concept that BGCs should produce a single family of molecules with one type of bioactivity, the occurrence of the different metabolites being triggered by the environmental conditions.

Download Full-text

A Hierarchical Network of Four Regulatory Genes Controlling Production of the Polyene Antibiotic Candicidin in Streptomyces sp. Strain FR-008

Applied and Environmental Microbiology ◽

10.1128/aem.00055-20 ◽

2020 ◽

Vol 86 (9) ◽

Author(s):

Yanping Zhu ◽

Wenhao Xu ◽

Jing Zhang ◽

Peipei Zhang ◽

Zhilong Zhao ◽

...

Keyword(s):

Gene Cluster ◽

Regulatory Network ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Regulatory Genes ◽

Transcriptional Analysis ◽

Antibiotic Biosynthesis ◽

Biosynthetic Gene ◽

Polyene Antibiotic ◽

Biosynthetic Gene Clusters

ABSTRACT The four regulatory genes fscR1 to fscR4 in Streptomyces sp. strain FR-008 form a genetic arrangement that is widely distributed in macrolide-producing bacteria. Our previous work has demonstrated that fscR1 and fscR4 are critical for production of the polyene antibiotic candicidin. In this study, we further characterized the roles of the other two regulatory genes, fscR2 and fscR3, focusing on the relationship between these four regulatory genes. Disruption of a single or multiple regulatory genes did not affect bacterial growth, but transcription of genes in the candicidin biosynthetic gene cluster decreased, and candicidin production was abolished, indicating a critical role for each of the four regulatory genes, including fscR2 and fscR3, in candicidin biosynthesis. We found that fscR1 to fscR4, although differentially expressed throughout the growth phase, displayed similar temporal expression patterns, with an abrupt increase in the early exponential phase, coincident with initial detection of antibiotic production in the same phase. Our data suggest that the four regulatory genes fscR1 to fscR4 have various degrees of control over structural genes in the biosynthetic cluster under the conditions examined. Extensive transcriptional analysis indicated that complex regulation exists between these four regulatory genes, forming a regulatory network, with fscR1 and fscR4 functioning at a lower level. Comprehensive cross-complementation analysis indicates that functional complementation is restricted among the four regulators and unidirectional, with fscR1 complementing the loss of fscR3 or -4 and fscR4 complementing loss of fscR2. Our study provides more insights into the roles of, and the regulatory network formed by, these four regulatory genes controlling production of an important pharmaceutical compound. IMPORTANCE The regulation of antibiotic biosynthesis by Streptomyces species is complex, especially for biosynthetic gene clusters with multiple regulatory genes. The biosynthetic gene cluster for the polyene antibiotic candicidin contains four consecutive regulatory genes, which encode regulatory proteins from different families and which form a subcluster within the larger biosynthetic gene cluster in Streptomyces sp. FR-008. Syntenic arrangements of these regulatory genes are widely distributed in polyene gene clusters, such as the amphotericin and nystatin gene clusters, suggesting a conserved regulatory mechanism controlling production of these clinically important medicines. However, the relationships between these multiple regulatory genes are unknown. In this study, we determined that each of these four regulatory genes is critical for candicidin production. Additionally, using transcriptional analyses, bioassays, high-performance liquid chromatography (HPLC) analysis, and genetic cross-complementation, we showed that FscR1 to FscR4 comprise a hierarchical regulatory network that controls candicidin production and is likely representative of how expression of other polyene biosynthetic gene clusters is controlled.

Download Full-text

A Single Biosynthetic Gene Cluster Is Responsible for the Production of Bagremycin Antibiotics and Ferroverdin Iron Chelators

mBio ◽

10.1128/mbio.01230-19 ◽

2019 ◽

Vol 10 (4) ◽

Cited By ~ 6

Author(s):

Loïc Martinet ◽

Aymeric Naômé ◽

Benoit Deflandre ◽

Marta Maciejewska ◽

Déborah Tellatin ◽

...

Keyword(s):

Natural Product ◽

Gene Cluster ◽

Biosynthetic Pathway ◽

Genome Mining ◽

Gene Clusters ◽

Bioactive Molecules ◽

Bioactive Metabolites ◽

Biosynthetic Gene ◽

Single Family ◽

Biosynthetic Gene Clusters

ABSTRACT Biosynthetic gene clusters (BGCs) are organized groups of genes involved in the production of specialized metabolites. Typically, one BGC is responsible for the production of one or several similar compounds with bioactivities that usually only vary in terms of strength and/or specificity. Here we show that the previously described ferroverdins and bagremycins, which are families of metabolites with different bioactivities, are produced from the same BGC, whereby the fate of the biosynthetic pathway depends on iron availability. Under conditions of iron depletion, the monomeric bagremycins are formed, representing amino-aromatic antibiotics resulting from the condensation of 3-amino-4-hydroxybenzoic acid with p-vinylphenol. Conversely, when iron is abundantly available, the biosynthetic pathway additionally produces a molecule based on p-vinylphenyl-3-nitroso-4-hydroxybenzoate, which complexes iron to form the trimeric ferroverdins that have anticholesterol activity. Thus, our work shows a unique exception to the concept that BGCs should only produce a single family of molecules with one type of bioactivity and that in fact different bioactive molecules may be produced depending on the environmental conditions. IMPORTANCE Access to whole-genome sequences has exposed the general incidence of the so-called cryptic biosynthetic gene clusters (BGCs), thereby renewing their interest for natural product discovery. As a consequence, genome mining is the often first approach implemented to assess the potential of a microorganism for producing novel bioactive metabolites. By revealing a new level of complexity of natural product biosynthesis, we further illustrate the difficulty of estimation of the panel of molecules associated with a BGC based on genomic information alone. Indeed, we found that the same gene cluster is responsible for the production of compounds which differ in terms of structure and bioactivity. The production of these different compounds responds to different environmental triggers, which suggests that multiplication of culture conditions is essential for revealing the entire panel of molecules made by a single BGC.

Download Full-text

Gene Modules Co-regulated with Biosynthetic Gene Clusters for Allelopathy between Rice and Barnyardgrass

International Journal of Molecular Sciences ◽

10.3390/ijms20163846 ◽

2019 ◽

Vol 20 (16) ◽

pp. 3846 ◽

Cited By ~ 1

Author(s):

Most. Humaira Sultana ◽

Fangjie Liu ◽

Md. Alamin ◽

Lingfeng Mao ◽

Lei Jia ◽

...

Keyword(s):

Gene Networks ◽

Regulatory Gene ◽

Crop Protection ◽

Gene Clusters ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Central Process ◽

Coordinated Expression ◽

Gene Modules ◽

Allelopathic Interactions

Allelopathy is a central process in crop–weed interactions and is mediated by the release of allelochemicals that result in adverse growth effects on one or the other plant in the interaction. The genomic mechanism for the biosynthesis of many critical allelochemicals is unknown but may involve the clustering of non-homologous biosynthetic genes involved in their formation and regulatory gene modules involved in controlling the coordinated expression within these gene clusters. In this study, we used the transcriptomes from mono- or co-cultured rice and barnyardgrass to investigate the nature of the gene clusters and their regulatory gene modules involved in the allelopathic interactions of these two plants. In addition to the already known biosynthetic gene clusters in barnyardgrass we identified three potential new clusters including one for quercetin biosynthesis and potentially involved in allelopathic interaction with rice. Based on the construction of gene networks, we identified one gene regulatory module containing hub transcription factors, significantly positively co-regulated with both the momilactone A and phytocassane clusters in rice. In barnyardgrass, gene modules and hub genes co-expressed with the gene clusters responsible for 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) biosynthesis were also identified. In addition, we found three genes in barnyardgrass encoding indole-3-glycerolphosphate synthase that regulate the expression of the DIMBOA cluster. Our findings offer new insights into the regulatory mechanisms of biosynthetic gene clusters involved in allelopathic interactions between rice and barnyardgrass, and have potential implications in controlling weeds for crop protection.

Download Full-text

Fungal Dihydroxynaphthalene-Melanin: Diversity-Oriented Biosynthesis through Enzymatic and Non-enzymatic Transformations

Synlett ◽

10.1055/s-0036-1588526 ◽

2017 ◽

Vol 28 (18) ◽

pp. 2360-2372 ◽

Cited By ~ 3

Author(s):

Michael Müller ◽

Syed Husain

Keyword(s):

Biosynthetic Pathway ◽

Molecular Diversity ◽

Magnaporthe Grisea ◽

Gene Clusters ◽

Extended Model ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Melanin Biosynthesis ◽

Aromatic Polyketides

Tetrahydroxynaphthalene reductase (T4HNR) from Magnaporthe grisea catalyzes the reduction of polyhydroxynaphthalenes, hydroxynaphthoquinones, and 1,4-diketones, with extensive ramifications for the biosynthesis of (shunt) metabolites related to 1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis. Hence, an extended model for DHN-melanin biosynthesis has been developed which is based on a screening hypothesis involving non-enzymatic transformations such as oxidations and tautomerism. This has led to the broadening of the functions of several short-chain dehydrogenases/reductases (SDRs) capable of reducing polyhydroxyanthracenes, polyhydroxynaphthalenes, and polyhydroxybenzenes. Our work, broadening the scope of enzymatic dearomatization reactions, provides access to the biocatalytic synthesis of a variety of natural and natural-like products. Furthermore, the results described in this account provide the basis for the identification of other SDRs amenable to reducing aromatic compounds, and thus enable the identification of biosynthetic gene clusters most likely involved in the biosynthesis of aromatic polyketides.1 Introduction2 Biosynthesis of 1,8-Dihydroxynaphthalene (DHN)3 Biosynthesis of Shunt Metabolites and the Origin of Molecular Diversity3.1 Role of Spontaneous Non-enzymatic Oxidations3.2 Role of T4HNR and T3HNR3.3 Role of Tautomerism in the Biosynthesis of (Shunt) Metabolites4 Extended Melanin Biosynthesis: A Screening Hypothesis5 Useful Outcomes of the Newly Identified Melanin Biosynthetic Pathway5.1 NADP+ Regeneration Using Lawsone as Mediator5.2 Anthrahydroquinone as an Intermediate in the Biosynthesis of Chrysophanol and Other Anthraquinone-Derived Products5.3 Combination of T3HNR and GDH To Access trans-Ketodiols5.4 Phloroglucinol Reductases (PGRs) To Dearomatize Monomeric Phenols6 Conclusion

Download Full-text

Genetic dereplication driven discovery of a tricinoloniol acid biosynthetic pathway in Trichoderma hypoxylon

Organic & Biomolecular Chemistry ◽

10.1039/d0ob01202e ◽

2020 ◽

Vol 18 (28) ◽

pp. 5344-5348 ◽

Cited By ~ 1

Author(s):

Huan Liu ◽

Yu-Han Pu ◽

Jin-Wei Ren ◽

Er-Wei Li ◽

Li-Xia Guo ◽

...

Keyword(s):

Gene Expression ◽

Biosynthetic Pathway ◽

Gene Clusters ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Targeted Deletion ◽

Terpene Cyclase

Three new sesquiterpene tricinoloniol acids were found by a genetic dereplication approach in combination with coordinated gene expression of biosynthetic gene clusters of tri and tra. The biosynthetic pathway was identified by targeted deletion of terpene cyclase traA.

Download Full-text