Heterologous Expression of the Unusual Terreazepine Biosynthetic Gene Cluster Reveals a Promising Approach for Identifying New Chemical Scaffolds

ABSTRACT Advances in genome sequencing have revitalized natural product discovery efforts, revealing the untapped biosynthetic potential of fungi. While the volume of genomic data continues to expand, discovery efforts are slowed due to the time-consuming nature of experiments required to characterize new molecules. To direct efforts toward uncharacterized biosynthetic gene clusters most likely to encode novel chemical scaffolds, we took advantage of comparative metabolomics and heterologous gene expression using fungal artificial chromosomes (FACs). By linking mass spectral profiles with structural clues provided by FAC-encoded gene clusters, we targeted a compound originating from an unusual gene cluster containing an indoleamine 2,3-dioxygenase (IDO). With this approach, we isolate and characterize R and S forms of the new molecule terreazepine, which contains a novel chemical scaffold resulting from cyclization of the IDO-supplied kynurenine. The discovery of terreazepine illustrates that FAC-based approaches targeting unusual biosynthetic machinery provide a promising avenue forward for targeted discovery of novel scaffolds and their biosynthetic enzymes, and it also represents another example of a biosynthetic gene cluster “repurposing” a primary metabolic enzyme to diversify its secondary metabolite arsenal. IMPORTANCE Here, we provide evidence that Aspergillus terreus encodes a biosynthetic gene cluster containing a repurposed indoleamine 2,3-dioxygenase (IDO) dedicated to secondary metabolite synthesis. The discovery of this neofunctionalized IDO not only enabled discovery of a new compound with an unusual chemical scaffold but also provided insight into the numerous strategies fungi employ for diversifying and protecting themselves against secondary metabolites. The observations in this study set the stage for further in-depth studies into the function of duplicated IDOs present in fungal biosynthetic gene clusters and presents a strategy for accessing the biosynthetic potential of gene clusters containing duplicated primary metabolic genes.

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Identification of the kinanthraquinone biosynthetic gene cluster by expression of an atypical response regulator

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbaa082 ◽

2021 ◽

Vol 85 (3) ◽

pp. 714-721

Author(s):

Risa Takao ◽

Katsuyuki Sakai ◽

Hiroyuki Koshino ◽

Hiroyuki Osada ◽

Shunji Takahashi

Keyword(s):

Heterologous Expression ◽

Gene Cluster ◽

Secondary Metabolite ◽

Response Regulator ◽

Bac Library ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Gene Organization ◽

Biosynthetic Gene ◽

Streptomyces Sp

ABSTRACT Recent advances in genome sequencing have revealed a variety of secondary metabolite biosynthetic gene clusters in actinomycetes. Understanding the biosynthetic mechanism controlling secondary metabolite production is important for utilizing these gene clusters. In this study, we focused on the kinanthraquinone biosynthetic gene cluster, which has not been identified yet in Streptomyces sp. SN-593. Based on chemical structure, 5 type II polyketide synthase gene clusters were listed from the genome sequence of Streptomyces sp. SN-593. Among them, a candidate gene cluster was selected by comparing the gene organization with grincamycin, which is synthesized through an intermediate similar to kinanthraquinone. We initially utilized a BAC library for subcloning the kiq gene cluster, performed heterologous expression in Streptomyces lividans TK23, and identified the production of kinanthraquinone and kinanthraquinone B. We also found that heterologous expression of kiqA, which belongs to the DNA-binding response regulator OmpR family, dramatically enhanced the production of kinanthraquinones.

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Identification of Putative Biosynthetic Gene Clusters for Tolyporphins in Multiple Filamentous Cyanobacteria

Life ◽

10.3390/life11080758 ◽

2021 ◽

Vol 11 (8) ◽

pp. 758

Author(s):

Xiaohe Jin ◽

Yunlong Zhang ◽

Ran Zhang ◽

Kathy-Uyen Nguyen ◽

Jonathan S. Lindsey ◽

...

Keyword(s):

Gene Cluster ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Filamentous Cyanobacterium ◽

Biosynthetic Gene ◽

Filamentous Cyanobacteria ◽

Biosynthetic Gene Clusters ◽

Common Component ◽

Homology Searching ◽

Similar Gene

Tolyporphins A–R are unusual tetrapyrrole macrocycles produced by the non-axenic filamentous cyanobacterium HT-58-2. A putative biosynthetic gene cluster for biosynthesis of tolyporphins (here termed BGC-1) was previously identified in the genome of HT-58-2. Here, homology searching of BGC-1 in HT-58-2 led to identification of similar BGCs in seven other filamentous cyanobacteria, including strains Nostoc sp. 106C, Nostoc sp. RF31YmG, Nostoc sp. FACHB-892, Brasilonema octagenarum UFV-OR1, Brasilonema octagenarum UFV-E1, Brasilonema sennae CENA114 and Oculatella sp. LEGE 06141, suggesting their potential for tolyporphins production. A similar gene cluster (BGC-2) also was identified unexpectedly in HT-58-2. Tolyporphins BGCs were not identified in unicellular cyanobacteria. Phylogenetic analysis based on 16S rRNA and a common component of the BGCs, TolD, points to a close evolutionary history between each strain and their respective tolyporphins BGC. Though identified with putative tolyporphins BGCs, examination of pigments extracted from three cyanobacteria has not revealed the presence of tolyporphins. Overall, the identification of BGCs and potential producers of tolyporphins presents a collection of candidate cyanobacteria for genetic and biochemical analysis pertaining to these unusual tetrapyrrole macrocycles.

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Biosynthetic Investigations of Lactonamycin and Lactonamycin Z: Cloning of the Biosynthetic Gene Clusters and Discovery of an Unusual Starter Unit

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00717-07 ◽

2007 ◽

Vol 52 (2) ◽

pp. 574-585 ◽

Cited By ~ 27

Author(s):

Xiujun Zhang ◽

Lawrence B. Alemany ◽

Hans-Peter Fiedler ◽

Michael Goodfellow ◽

Ronald J. Parry

Keyword(s):

Gene Cluster ◽

Genetic Locus ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Open Reading Frames ◽

Antibacterial Drug ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Starter Unit ◽

High Degree

ABSTRACT The antibiotics lactonamycin and lactonamycin Z provide attractive leads for antibacterial drug development. Both antibiotics contain a novel aglycone core called lactonamycinone. To gain insight into lactonamycinone biosynthesis, cloning and precursor incorporation experiments were undertaken. The lactonamycin gene cluster was initially cloned from Streptomyces rishiriensis. Sequencing of ca. 61 kb of S. rishiriensis DNA revealed the presence of 57 open reading frames. These included genes coding for the biosynthesis of l-rhodinose, the sugar found in lactonamycin, and genes similar to those in the tetracenomycin biosynthetic gene cluster. Since lactonamycin production by S. rishiriensis could not be sustained, additional proof for the identity of the S. rishiriensis cluster was obtained by cloning the lactonamycin Z gene cluster from Streptomyces sanglieri. Partial sequencing of the S. sanglieri cluster revealed 15 genes that exhibited a very high degree of similarity to genes within the lactonamycin cluster, as well as an identical organization. Double-crossover disruption of one gene in the S. sanglieri cluster abolished lactonamycin Z production, and production was restored by complementation. These results confirm the identity of the genetic locus cloned from S. sanglieri and indicate that the highly similar locus in S. rishiriensis encodes lactonamycin biosynthetic genes. Precursor incorporation experiments with S. sanglieri revealed that lactonamycinone is biosynthesized in an unusual manner whereby glycine or a glycine derivative serves as a starter unit that is extended by nine acetate units. Analysis of the gene clusters and of the precursor incorporation data suggested a hypothetical scheme for lactonamycinone biosynthesis.

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Insights into the Evolution of Macrolactam Biosynthesis through Cloning and Comparative Analysis of the Biosynthetic Gene Cluster for a Novel Macrocyclic Lactam, ML-449

Applied and Environmental Microbiology ◽

10.1128/aem.00744-09 ◽

2009 ◽

Vol 76 (1) ◽

pp. 283-293 ◽

Cited By ~ 51

Author(s):

Hanne Jørgensen ◽

Kristin F. Degnes ◽

Alexander Dikiy ◽

Espen Fjærvik ◽

Geir Klinkenberg ◽

...

Keyword(s):

Gene Cluster ◽

Evolutionary Relationship ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Side Chain ◽

Small Ring ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

The Difference ◽

High Level

ABSTRACT A new compound, designated ML-449, structurally similar to the known 20-membered macrolactam BE-14106, was isolated from a marine sediment-derived Streptomyces sp. Cloning and sequencing of the 83-kb ML-449 biosynthetic gene cluster revealed its high level of similarity to the BE-14106 gene cluster. Comparison of the respective biosynthetic pathways indicated that the difference in the compounds' structures stems from the incorporation of one extra acetate unit during the synthesis of the acyl side chain. A phylogenetic analysis of the β-ketosynthase (KS) domains from polyketide synthases involved in the biosynthesis of macrolactams pointed to a common ancestry for the two clusters. Furthermore, the analysis demonstrated the formation of a macrolactam-specific subclade for the majority of the KS domains from several macrolactam-biosynthetic gene clusters, indicating a closer relationship between macrolactam clusters than with the macrolactone clusters included in the analysis. Some KS domains from the ML-449, BE-14106, and salinilactam gene clusters did, however, show a closer relationship with KS domains from the polyene macrolide clusters, suggesting potential acquisition rather than duplication of certain PKS genes. Comparison of the ML-449, BE-14106, vicenistatin, and salinilactam biosynthetic gene clusters indicated an evolutionary relationship between them and provided new insights into the processes governing the evolution of small-ring macrolactam biosynthesis.

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Discovery of Gene Cluster for Mycosporine-Like Amino Acid Biosynthesis from Actinomycetales Microorganisms and Production of a Novel Mycosporine-Like Amino Acid by Heterologous Expression

Applied and Environmental Microbiology ◽

10.1128/aem.00727-14 ◽

2014 ◽

Vol 80 (16) ◽

pp. 5028-5036 ◽

Cited By ~ 39

Author(s):

Kiyoko T. Miyamoto ◽

Mamoru Komatsu ◽

Haruo Ikeda

Keyword(s):

Amino Acid ◽

Gene Cluster ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Dependent Manner ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Gram Positive ◽

Content Type ◽

Gram Positive Bacteria

ABSTRACTMycosporines and mycosporine-like amino acids (MAAs), including shinorine (mycosporine-glycine-serine) and porphyra-334 (mycosporine-glycine-threonine), are UV-absorbing compounds produced by cyanobacteria, fungi, and marine micro- and macroalgae. These MAAs have the ability to protect these organisms from damage by environmental UV radiation. Although no reports have described the production of MAAs and the corresponding genes involved in MAA biosynthesis from Gram-positive bacteria to date, genome mining of the Gram-positive bacterial database revealed that two microorganisms belonging to the orderActinomycetales,Actinosynnema mirumDSM 43827 andPseudonocardiasp. strain P1, possess a gene cluster homologous to the biosynthetic gene clusters identified from cyanobacteria. When the two strains were grown in liquid culture,Pseudonocardiasp. accumulated a very small amount of MAA-like compound in a medium-dependent manner, whereasA. mirumdid not produce MAAs under any culture conditions, indicating that the biosynthetic gene cluster ofA. mirumwas in a cryptic state in this microorganism. In order to characterize these biosynthetic gene clusters, each biosynthetic gene cluster was heterologously expressed in an engineered host,Streptomyces avermitilisSUKA22. Since the resultant transformants carrying the entire biosynthetic gene cluster controlled by an alternative promoter produced mainly shinorine, this is the first confirmation of a biosynthetic gene cluster for MAA from Gram-positive bacteria. Furthermore,S. avermitilisSUKA22 transformants carrying the biosynthetic gene cluster for MAA ofA. mirumaccumulated not only shinorine and porphyra-334 but also a novel MAA. Structure elucidation revealed that the novel MAA is mycosporine-glycine-alanine, which substitutesl-alanine for thel-serine of shinorine.

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Alternative Biosynthetic Starter Units Enhance the Structural Diversity of Cyanobacterial Lipopeptides

Applied and Environmental Microbiology ◽

10.1128/aem.02675-18 ◽

2018 ◽

Vol 85 (4) ◽

Cited By ~ 5

Author(s):

Jan Mareš ◽

Jan Hájek ◽

Petra Urajová ◽

Andreja Kust ◽

Jouni Jokela ◽

...

Keyword(s):

Fatty Acid ◽

Gene Cluster ◽

Plasma Membranes ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Published Data ◽

Biosynthetic Gene ◽

Structural Variants ◽

Biosynthetic Gene Clusters ◽

Content Type

ABSTRACT Puwainaphycins (PUWs) and minutissamides (MINs) are structurally analogous cyclic lipopeptides possessing cytotoxic activity. Both types of compound exhibit high structural variability, particularly in the fatty acid (FA) moiety. Although a biosynthetic gene cluster responsible for synthesis of several PUW variants has been proposed in a cyanobacterial strain, the genetic background for MINs remains unexplored. Herein, we report PUW/MIN biosynthetic gene clusters and structural variants from six cyanobacterial strains. Comparison of biosynthetic gene clusters indicates a common origin of the PUW/MIN hybrid nonribosomal peptide synthetase and polyketide synthase. Surprisingly, the biosynthetic gene clusters encode two alternative biosynthetic starter modules, and analysis of structural variants suggests that initiation by each of the starter modules results in lipopeptides of differing lengths and FA substitutions. Among additional modifications of the FA chain, chlorination of minutissamide D was explained by the presence of a putative halogenase gene in the PUW/MIN gene cluster of Anabaena minutissima strain UTEX B 1613. We detected PUW variants bearing an acetyl substitution in Symplocastrum muelleri strain NIVA-CYA 644, consistent with an O-acetyltransferase gene in its biosynthetic gene cluster. The major lipopeptide variants did not exhibit any significant antibacterial activity, and only the PUW F variant was moderately active against yeast, consistent with previously published data suggesting that PUWs/MINs interact preferentially with eukaryotic plasma membranes. IMPORTANCE Herein, we deciphered the most important biosynthetic traits of a prominent group of bioactive lipopeptides. We reveal evidence for initiation of biosynthesis by two alternative starter units hardwired directly in the same gene cluster, eventually resulting in the production of a remarkable range of lipopeptide variants. We identified several unusual tailoring genes potentially involved in modifying the fatty acid chain. Careful characterization of these biosynthetic gene clusters and their diverse products could provide important insight into lipopeptide biosynthesis in prokaryotes. Some of the variants identified exhibit cytotoxic and antifungal properties, and some are associated with a toxigenic biofilm-forming strain. The findings may prove valuable to researchers in the fields of natural product discovery and toxicology.

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Transcription Factor Repurposing Offers Insights into Evolution of Biosynthetic Gene Cluster Regulation

mBio ◽

10.1128/mbio.01399-21 ◽

2021 ◽

Author(s):

Wenjie Wang ◽

Milton Drott ◽

Claudio Greco ◽

Dianiris Luciano-Rosario ◽

Pinmei Wang ◽

...

Keyword(s):

Transcription Factor ◽

Secondary Metabolites ◽

Gene Cluster ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

The Other ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Metabolite Production ◽

Fungal Secondary Metabolites

Fungal secondary metabolites (SMs) are an important source of pharmaceuticals on one hand and toxins on the other. Efforts to identify the biosynthetic gene clusters (BGCs) that synthesize SMs have yielded significant insights into how variation in the genes that compose BGCs may impact subsequent metabolite production within and between species.

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Phylogenetic analysis of the salinipostin γ-butyrolactone gene cluster uncovers new potential for bacterial signaling-molecule diversity

10.1101/2020.10.16.342204 ◽

2020 ◽

Author(s):

Kaitlin E. Creamer ◽

Yuta Kudo ◽

Bradley S. Moore ◽

Paul R. Jensen

Keyword(s):

Gene Cluster ◽

Species Interactions ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Signaling Molecules ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Specialized Metabolism ◽

Bacterial Phyla ◽

Marine Actinomycete

AbstractBacteria communicate by small-molecule chemicals that facilitate intra- and inter-species interactions. These extracellular signaling molecules mediate diverse processes including virulence, bioluminescence, biofilm formation, motility, and specialized metabolism. The signaling molecules produced by members of the phylum Actinobacteria are generally comprised of γ-butyrolactones, γ-butenolides, and furans. The best known actinomycete γ-butyrolactone is A-factor, which triggers specialized metabolism and morphological differentiation in the genus Streptomyces. Salinipostins A-K are unique γ-butyrolactone molecules with rare phosphotriester moieties that were recently characterized from the marine actinomycete genus Salinispora. The production of these compounds has been linked to the 9-gene biosynthetic gene cluster spt. Critical to salinipostin assembly is the γ-butyrolactone synthase encoded by spt9. Here, we report the global distribution of spt9 among sequenced bacterial genomes, revealing a surprising diversity of gene homologs across 12 bacterial phyla, the majority of which are not known to produce γ-butyrolactones. Further analyses uncovered a large group of spt-like gene clusters outside of the genus Salinispora, suggesting the production of new salinipostin-like diversity. These gene clusters show evidence of horizontal transfer between many bacterial taxa and location specific homologous recombination exchange among Salinispora strains. The results suggest that γ-butyrolactone production may be more widespread than previously recognized. The identification of new γ-butyrolactone biosynthetic gene clusters is the first step towards understanding the regulatory roles of the encoded small molecules in Actinobacteria.ImportanceSignaling molecules orchestrate a wide variety of bacterial behaviors. Among Actinobacteria, γ-butyrolactones mediate morphological changes and regulate specialized metabolism. Despite their importance, few γ-butyrolactones have been linked to their cognate biosynthetic gene clusters. A new series of γ-butyrolactones called the salinipostins was recently identified from the marine actinomycete genus Salinispora and linked to the spt biosynthetic gene cluster. Here we report the detection of spt-like gene clusters in diverse bacterial families not known for the production of this class of compounds. This finding expands the taxonomic range of bacteria that may employ this class of compounds and provides opportunities to discover new compounds associated with chemical communication.

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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.

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Biosynthetic gene clusters and the evolution of fungal chemodiversity

Natural Product Reports ◽

10.1039/c9np00045c ◽

2020 ◽

Vol 37 (7) ◽

pp. 868-878 ◽

Cited By ~ 9

Author(s):

Antonis Rokas ◽

Matthew E. Mead ◽

Jacob L. Steenwyk ◽

Huzefa A. Raja ◽

Nicholas H. Oberlies

Keyword(s):

Gene Cluster ◽

Horizontal Transfer ◽

De Novo ◽

Functional Divergence ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Evolutionary Processes

This highlight synthesizes knowledge of the molecular evolutionary processes – functional divergence, horizontal transfer, and de novo assembly – that govern biosynthetic gene cluster diversification and the generation of chemodiversity in fungi.

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