Distribution and diversity of dimetal-carboxylate halogenases in cyanobacteria

AbstractHalogenation is a recurring feature in natural products, especially those from marine organisms. The selectivity with which halogenating enzymes act on their substrates renders halogenases interesting targets for biocatalyst development. Recently, CylC – the first predicted dimetal-carboxylate halogenase to be characterized – was shown to regio- and stereoselectively install a chlorine atom onto an unactivated carbon center during cylindrocyclophane biosynthesis. Homologs of CylC are also found in other characterized cyanobacterial secondary metabolite biosynthetic gene clusters. Due to its novelty in biological catalysis, selectivity and ability to perform C-H activation, this halogenase class is of considerable fundamental and applied interest. However, little is known regarding the diversity and distribution of these enzymes in bacteria. In this study, we used both genome mining and PCR-based screening to explore the genetic diversity and distribution of CylC homologs. While we found non-cyanobacterial homologs of these enzymes to be rare, we identified a large number of genes encoding CylC-like enzymes in publicly available cyanobacterial genomes and in our in-house culture collection of cyanobacteria. Genes encoding CylC homologs are widely distributed throughout the cyanobacterial tree of life, within biosynthetic gene clusters of distinct architectures. Their genomic contexts feature a variety of biosynthetic partners, including fatty-acid activation enzymes, type I or type III polyketide synthases, dialkylresorcinol-generating enzymes, monooxygenases or Rieske proteins. Our study also reveals that dimetal-carboxylate halogenases are among the most abundant types of halogenating enzymes in the phylum Cyanobacteria. This work will help to guide the search for new halogenating biocatalysts and natural product scaffolds.Data statementAll supporting data and methods have been provided within the article or through a Supplementary Material file, which includes 14 supplementary figures and 4 supplementary tables.

Download Full-text

Distribution and diversity of dimetal-carboxylate halogenases in cyanobacteria

BMC Genomics ◽

10.1186/s12864-021-07939-x ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Nadia Eusebio ◽

Adriana Rego ◽

Nathaniel R. Glasser ◽

Raquel Castelo-Branco ◽

Emily P. Balskus ◽

...

Keyword(s):

Genome Mining ◽

Gene Clusters ◽

Culture Collection ◽

Type I ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Rieske Proteins ◽

Number Of Genes ◽

Genes Encoding ◽

Biological Catalysis

Abstract Background Halogenation is a recurring feature in natural products, especially those from marine organisms. The selectivity with which halogenating enzymes act on their substrates renders halogenases interesting targets for biocatalyst development. Recently, CylC – the first predicted dimetal-carboxylate halogenase to be characterized – was shown to regio- and stereoselectively install a chlorine atom onto an unactivated carbon center during cylindrocyclophane biosynthesis. Homologs of CylC are also found in other characterized cyanobacterial secondary metabolite biosynthetic gene clusters. Due to its novelty in biological catalysis, selectivity and ability to perform C-H activation, this halogenase class is of considerable fundamental and applied interest. The study of CylC-like enzymes will provide insights into substrate scope, mechanism and catalytic partners, and will also enable engineering these biocatalysts for similar or additional C-H activating functions. Still, little is known regarding the diversity and distribution of these enzymes. Results In this study, we used both genome mining and PCR-based screening to explore the genetic diversity of CylC homologs and their distribution in bacteria. While we found non-cyanobacterial homologs of these enzymes to be rare, we identified a large number of genes encoding CylC-like enzymes in publicly available cyanobacterial genomes and in our in-house culture collection of cyanobacteria. Genes encoding CylC homologs are widely distributed throughout the cyanobacterial tree of life, within biosynthetic gene clusters of distinct architectures (combination of unique gene groups). These enzymes are found in a variety of biosynthetic contexts, which include fatty-acid activating enzymes, type I or type III polyketide synthases, dialkylresorcinol-generating enzymes, monooxygenases or Rieske proteins. Our study also reveals that dimetal-carboxylate halogenases are among the most abundant types of halogenating enzymes in the phylum Cyanobacteria. Conclusions Our data show that dimetal-carboxylate halogenases are widely distributed throughout the Cyanobacteria phylum and that BGCs encoding CylC homologs are diverse and mostly uncharacterized. This work will help guide the search for new halogenating biocatalysts and natural product scaffolds.

Download Full-text

Genome Sequencing of Streptomyces olivaceus SCSIO T05 and Activated Production of Lobophorin CR4 via Metabolic Engineering and Genome Mining

Marine Drugs ◽

10.3390/md17100593 ◽

2019 ◽

Vol 17 (10) ◽

pp. 593 ◽

Cited By ~ 3

Author(s):

Chunyan Zhang ◽

Wenjuan Ding ◽

Xiangjing Qin ◽

Jianhua Ju

Keyword(s):

Metabolic Engineering ◽

Deep Sea ◽

Genome Mining ◽

Gene Clusters ◽

Type I ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Genus Streptomyces ◽

Disruption Method ◽

Streptomyces Olivaceus

Marine-sourced actinomycete genus Streptomyces continues to be an important source of new natural products. Here we report the complete genome sequence of deep-sea-derived Streptomyces olivaceus SCSIO T05, harboring 37 putative biosynthetic gene clusters (BGCs). A cryptic BGC for type I polyketides was activated by metabolic engineering methods, enabling the discovery of a known compound, lobophorin CR4 (1). Genome mining yielded a putative lobophorin BGC (lbp) that missed the functional FAD-dependent oxidoreductase to generate the d-kijanose, leading to the production of lobophorin CR4 without the attachment of d-kijanose to C17-OH. Using the gene-disruption method, we confirmed that the lbp BGC accounts for lobophorin biosynthesis. We conclude that metabolic engineering and genome mining provide an effective approach to activate cryptic BGCs.

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

Discovery of Unusual Cyanobacterial Tryptophan-containing Anabaenopeptins by MS/MS Based Molecular Networking

10.20944/preprints202007.0562.v1 ◽

2020 ◽

Author(s):

Subhasish Saha ◽

Germana Esposito ◽

Petra Urajova ◽

Jan Mareš ◽

Daniela Ewe ◽

...

Keyword(s):

Multidisciplinary Approach ◽

Spectroscopic Analysis ◽

Chemical Space ◽

Genome Mining ◽

Gc Content ◽

Gene Clusters ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Hela Cell Lines ◽

Bioactive Secondary Metabolites

Heterocytous cyanobacteria are among the most prolific source of bioactive secondary metabolites, including anabaenopeptins (APTs). A terrestrial filamentous Brasilonema sp. CT11 collected in Costa Rica bamboo forest, as black mat was studied using a multidisciplinary approach: genome mining and HPLC-HRMS/MS coupled with bionformatic analyses. Herein, we report the nearly complete genome consisting 8.79 Mbp with a GC content of 42.4%. Moreover, we report on three novel tryptophane-containing APTs; anabaenopeptin 788 (1), anabaenopeptin 802 (2) and anabaenopeptin 816 (3). Further, the structure of two homologues, i.e., anabaenopeptin 802 (2a) and anabaenopeptin 802 (2b) was determined by spectroscopic analysis (NMR and MS). Both compounds were shown to exert weak to moderate antiproliferative activity against HeLa cell lines. This study also provides the unique and diverse potential of biosynthetic gene clusters and an assessment of the predicted chemical space yet to be discovered from this genus.

Download Full-text

Global analysis of adenylate-forming enzymes reveals β-lactone biosynthesis pathway in pathogenic Nocardia

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013528 ◽

2020 ◽

Vol 295 (44) ◽

pp. 14826-14839

Author(s):

Serina L. Robinson ◽

Barbara R. Terlouw ◽

Megan D. Smith ◽

Sacha J. Pidot ◽

Timothy P. Stinear ◽

...

Keyword(s):

Machine Learning ◽

Natural Product ◽

Global Analysis ◽

Gene Clusters ◽

Divergent Evolution ◽

Acid Activation ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Predictive Tool ◽

Wide Range

Enzymes that cleave ATP to activate carboxylic acids play essential roles in primary and secondary metabolism in all domains of life. Class I adenylate-forming enzymes share a conserved structural fold but act on a wide range of substrates to catalyze reactions involved in bioluminescence, nonribosomal peptide biosynthesis, fatty acid activation, and β-lactone formation. Despite their metabolic importance, the substrates and functions of the vast majority of adenylate-forming enzymes are unknown without tools available to accurately predict them. Given the crucial roles of adenylate-forming enzymes in biosynthesis, this also severely limits our ability to predict natural product structures from biosynthetic gene clusters. Here we used machine learning to predict adenylate-forming enzyme function and substrate specificity from protein sequences. We built a web-based predictive tool and used it to comprehensively map the biochemical diversity of adenylate-forming enzymes across >50,000 candidate biosynthetic gene clusters in bacterial, fungal, and plant genomes. Ancestral phylogenetic reconstruction and sequence similarity networking of enzymes from these clusters suggested divergent evolution of the adenylate-forming superfamily from a core enzyme scaffold most related to contemporary CoA ligases toward more specialized functions including β-lactone synthetases. Our classifier predicted β-lactone synthetases in uncharacterized biosynthetic gene clusters conserved in >90 different strains of Nocardia. To test our prediction, we purified a candidate β-lactone synthetase from Nocardia brasiliensis and reconstituted the biosynthetic pathway in vitro to link the gene cluster to the β-lactone natural product, nocardiolactone. We anticipate that our machine learning approach will aid in functional classification of enzymes and advance natural product discovery.

Download Full-text

An Update on Molecular Tools for Genetic Engineering of Actinomycetes—The Source of Important Antibiotics and Other Valuable Compounds

Antibiotics ◽

10.3390/antibiotics9080494 ◽

2020 ◽

Vol 9 (8) ◽

pp. 494

Author(s):

Lena Mitousis ◽

Yvonne Thoma ◽

Ewa M. Musiol-Kroll

Keyword(s):

Genetic Engineering ◽

Genome Mining ◽

Gene Clusters ◽

Biosynthetic Gene ◽

Molecular Tools ◽

Biosynthetic Gene Clusters ◽

Streptomyces Antibioticus ◽

The Past ◽

Bioactive Agents ◽

Valuable Compounds

The first antibiotic-producing actinomycete (Streptomyces antibioticus) was described by Waksman and Woodruff in 1940. This discovery initiated the “actinomycetes era”, in which several species were identified and demonstrated to be a great source of bioactive compounds. However, the remarkable group of microorganisms and their potential for the production of bioactive agents were only partially exploited. This is caused by the fact that the growth of many actinomycetes cannot be reproduced on artificial media at laboratory conditions. In addition, sequencing, genome mining and bioactivity screening disclosed that numerous biosynthetic gene clusters (BGCs), encoded in actinomycetes genomes are not expressed and thus, the respective potential products remain uncharacterized. Therefore, a lot of effort was put into the development of technologies that facilitate the access to actinomycetes genomes and activation of their biosynthetic pathways. In this review, we mainly focus on molecular tools and methods for genetic engineering of actinomycetes that have emerged in the field in the past five years (2015–2020). In addition, we highlight examples of successful application of the recently developed technologies in genetic engineering of actinomycetes for activation and/or improvement of the biosynthesis of secondary metabolites.

Download Full-text

Genomic Assemblies of Members of Burkholderia and Related Genera as a Resource for Natural Product Discovery

Microbiology Resource Announcements ◽

10.1128/mra.00485-20 ◽

2020 ◽

Vol 9 (42) ◽

Author(s):

Alex J. Mullins ◽

Cerith Jones ◽

Matthew J. Bull ◽

Gordon Webster ◽

Julian Parkhill ◽

...

Keyword(s):

Natural Product ◽

Genome Mining ◽

Genomic Analysis ◽

Gene Clusters ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Natural Product Discovery

ABSTRACT The genomes of 450 members of Burkholderiaceae, isolated from clinical and environmental sources, were sequenced and assembled as a resource for genome mining. Genomic analysis of the collection has enabled the identification of multiple metabolites and their biosynthetic gene clusters, including the antibiotics gladiolin, icosalide A, enacyloxin, and cepacin A.

Download Full-text

Genomic analysis of siderophore β-hydroxylases reveals divergent stereocontrol and expands the condensation domain family

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1903161116 ◽

2019 ◽

Vol 116 (40) ◽

pp. 19805-19814 ◽

Cited By ~ 8

Author(s):

Zachary L. Reitz ◽

Clifford D. Hardy ◽

Jaewon Suk ◽

Jean Bouvet ◽

Alison Butler

Keyword(s):

Predictive Power ◽

Genome Mining ◽

Genomic Analysis ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Peptide Synthetase ◽

Condensation Domain

Genome mining of biosynthetic pathways streamlines discovery of secondary metabolites but can leave ambiguities in the predicted structures, which must be rectified experimentally. Through coupling the reactivity predicted by biosynthetic gene clusters with verified structures, the origin of the β-hydroxyaspartic acid diastereomers in siderophores is reported herein. Two functional subtypes of nonheme Fe(II)/α-ketoglutarate–dependent aspartyl β-hydroxylases are identified in siderophore biosynthetic gene clusters, which differ in genomic organization—existing either as fused domains (IβHAsp) at the carboxyl terminus of a nonribosomal peptide synthetase (NRPS) or as stand-alone enzymes (TβHAsp)—and each directs opposite stereoselectivity of Asp β-hydroxylation. The predictive power of this subtype delineation is confirmed by the stereochemical characterization of β-OHAsp residues in pyoverdine GB-1, delftibactin, histicorrugatin, and cupriachelin. The l-threo (2S, 3S) β-OHAsp residues of alterobactin arise from hydroxylation by the β-hydroxylase domain integrated into NRPS AltH, while l-erythro (2S, 3R) β-OHAsp in delftibactin arises from the stand-alone β-hydroxylase DelD. Cupriachelin contains both l-threo and l-erythro β-OHAsp, consistent with the presence of both types of β-hydroxylases in the biosynthetic gene cluster. A third subtype of nonheme Fe(II)/α-ketoglutarate–dependent enzymes (IβHHis) hydroxylates histidyl residues with l-threo stereospecificity. A previously undescribed, noncanonical member of the NRPS condensation domain superfamily is identified, named the interface domain, which is proposed to position the β-hydroxylase and the NRPS-bound amino acid prior to hydroxylation. Through mapping characterized β-OHAsp diastereomers to the phylogenetic tree of siderophore β-hydroxylases, methods to predict β-OHAsp stereochemistry in silico are realized.

Download Full-text

Recent advances in the genome mining of Aspergillus secondary metabolites (covering 2012–2018)

MedChemComm ◽

10.1039/c9md00054b ◽

2019 ◽

Vol 10 (6) ◽

pp. 840-866 ◽

Cited By ~ 16

Author(s):

Jillian Romsdahl ◽

Clay C. C. Wang

Keyword(s):

Aspergillus Terreus ◽

Genome Mining ◽

Gene Clusters ◽

Genetic Identification ◽

Biosynthetic Pathways ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

The Past ◽

Made In

This review covers advances made in genome mining SMs produced by Aspergillus nidulans, Aspergillus fumigatus, Aspergillus niger, and Aspergillus terreus in the past six years (2012–2018). Genetic identification and molecular characterization of SM biosynthetic gene clusters, along with proposed biosynthetic pathways, is discussed in depth.

Download Full-text