Monasone Naphthoquinone Biosynthesis and Resistance in Monascus Fungi

ABSTRACT Despite the important biological activities of natural product naphthoquinones, the biosynthetic pathways of and resistance mechanisms against such compounds remain poorly understood in fungi. Here, we report that the genes responsible for the biosynthesis of Monascus naphthoquinones (monasones) reside within the gene cluster for Monascus azaphilone pigments (MonAzPs). We elucidate the biosynthetic pathway of monasones by a combination of comparative genome analysis, gene knockouts, heterologous coexpression, and in vivo and in vitro enzymatic reactions to show that this pathway branches from the first polyketide intermediate of MonAzPs. Furthermore, we propose that the monasone subset of biosynthetic genes also encodes a two-tiered resistance strategy in which an inducible monasone-specific exporter expels monasones from the mycelia, while residual intracellular monasones may be rendered nontoxic through a multistep reduction cascade. IMPORTANCE The genes for Monascus naphthoquinone (monasone) biosynthesis are embedded in and form a composite supercluster with the Monascus azaphilone pigment biosynthetic gene cluster. Early biosynthetic intermediates are shared by the two pathways. Some enzymes encoded by the supercluster play double duty in contributing to both pathways, while others are specific for one or the other pathway. The monasone subcluster is independently regulated and inducible by elicitation with competing microorganisms. This study illustrates genomic and biosynthetic parsimony in fungi and proposes a potential path for the evolution of the mosaic-like azaphilone-naphthoquinone supercluster. The monasone subcluster also encodes a two-tiered self-resistance mechanism that models resistance determinants that may transfer to target microorganisms or emerge in cancer cells in case of naphthoquinone-type cytotoxic agents.

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Study of bicyclomycin biosynthesis in Streptomyces cinnamoneus by genetic and biochemical approaches

Scientific Reports ◽

10.1038/s41598-019-56747-7 ◽

2019 ◽

Vol 9 (1) ◽

Author(s):

Jerzy Witwinowski ◽

Mireille Moutiez ◽

Matthieu Coupet ◽

Isabelle Correia ◽

Pascal Belin ◽

...

Keyword(s):

Gene Cluster ◽

Biological Activities ◽

Large Family ◽

Biosynthetic Gene Cluster ◽

Biosynthetic Gene ◽

Bacterial Transcription ◽

Bicyclic Structure ◽

Transcription Termination Factor

AbstractThe 2,5-Diketopiperazines (DKPs) constitute a large family of natural products with important biological activities. Bicyclomycin is a clinically-relevant DKP antibiotic that is the first and only member in a class known to target the bacterial transcription termination factor Rho. It derives from cyclo-(l-isoleucyl-l-leucyl) and has an unusual and highly oxidized bicyclic structure that is formed by an ether bridge between the hydroxylated terminal carbon atom of the isoleucine lateral chain and the alpha carbon of the leucine in the diketopiperazine ring. Here, we paired in vivo and in vitro studies to complete the characterization of the bicyclomycin biosynthetic gene cluster. The construction of in-frame deletion mutants in the biosynthetic gene cluster allowed for the accumulation and identification of biosynthetic intermediates. The identity of the intermediates, which were reproduced in vitro using purified enzymes, allowed us to characterize the pathway and corroborate previous reports. Finally, we show that the putative antibiotic transporter was dispensable for the producing strain.

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Advanced Resistance Studies Identify Two Discrete Mechanisms in Staphylococcus aureus to Overcome Antibacterial Compounds that Target Biotin Protein Ligase

Antibiotics ◽

10.3390/antibiotics9040165 ◽

2020 ◽

Vol 9 (4) ◽

pp. 165 ◽

Cited By ~ 1

Author(s):

Andrew J. Hayes ◽

Jiulia Satiaputra ◽

Louise M. Sternicki ◽

Ashleigh S. Paparella ◽

Zikai Feng ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Transcriptional Repression ◽

Molecular Mechanisms ◽

Resistance Mechanism ◽

Resistance Mechanisms ◽

Resistance Rate ◽

Biotin Protein Ligase ◽

Essential Enzyme

Biotin protein ligase (BPL) inhibitors are a novel class of antibacterial that target clinically important methicillin-resistant Staphylococcus aureus (S. aureus). In S. aureus, BPL is a bifunctional protein responsible for enzymatic biotinylation of two biotin-dependent enzymes, as well as serving as a transcriptional repressor that controls biotin synthesis and import. In this report, we investigate the mechanisms of action and resistance for a potent anti-BPL, an antibacterial compound, biotinyl-acylsulfamide adenosine (BASA). We show that BASA acts by both inhibiting the enzymatic activity of BPL in vitro, as well as functioning as a transcription co-repressor. A low spontaneous resistance rate was measured for the compound (<10−9) and whole-genome sequencing of strains evolved during serial passaging in the presence of BASA identified two discrete resistance mechanisms. In the first, deletion of the biotin-dependent enzyme pyruvate carboxylase is proposed to prioritize the utilization of bioavailable biotin for the essential enzyme acetyl-CoA carboxylase. In the second, a D200E missense mutation in BPL reduced DNA binding in vitro and transcriptional repression in vivo. We propose that this second resistance mechanism promotes bioavailability of biotin by derepressing its synthesis and import, such that free biotin may outcompete the inhibitor for binding BPL. This study provides new insights into the molecular mechanisms governing antibacterial activity and resistance of BPL inhibitors in S. aureus.

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A Two-Step Mechanism for the Activation of Actinorhodin Export and Resistance in Streptomyces coelicolor

mBio ◽

10.1128/mbio.00191-12 ◽

2012 ◽

Vol 3 (5) ◽

Cited By ~ 28

Author(s):

Ye Xu ◽

Andrew Willems ◽

Catherine Au-yeung ◽

Kapil Tahlan ◽

Justin R. Nodwell

Keyword(s):

Secondary Metabolites ◽

Pathogenic Bacteria ◽

Streptomyces Coelicolor ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Resistance Mechanisms ◽

Biosynthetic Gene ◽

Content Type

ABSTRACT Many microorganisms produce secondary metabolites that have antibiotic activity. To avoid self-inhibition, the producing cells often encode cognate export and/or resistance mechanisms in the biosynthetic gene clusters for these molecules. Actinorhodin is a blue-pigmented antibiotic produced by Streptomyces coelicolor. The actAB operon, carried in the actinorhodin biosynthetic gene cluster, encodes two putative export pumps and is regulated by the transcriptional repressor protein ActR. In this work, we show that normal actinorhodin yields require actAB expression. Consistent with previous in vitro work, we show that both actinorhodin and its 3-ring biosynthetic intermediates [e.g., (S)-DNPA] can relieve repression of actAB by ActR in vivo. Importantly, an ActR mutant that interacts productively with (S)-DNPA but not with actinorhodin responds to the actinorhodin biosynthetic pathway with the induction of actAB and normal yields of actinorhodin. This suggests that the intermediates are sufficient to trigger the export genes in actinorhodin-producing cells. We further show that actinorhodin-producing cells can induce actAB expression in nonproducing cells; however, in this case actinorhodin is the most important signal. Finally, while the “intermediate-only” ActR mutant permits sufficient actAB expression for normal actinorhodin yields, this expression is short-lived. Sustained culture-wide expression requires a subsequent actinorhodin-mediated signaling step, and the defect in this response causes widespread cell death. These results are consistent with a two-step model for actinorhodin export and resistance where intermediates trigger initial expression for export from producing cells and actinorhodin then triggers sustained export gene expression that confers culture-wide resistance. IMPORTANCE Understanding the links between antibiotic resistance and biosynthesis is important for our efforts to manipulate secondary metabolism. For example, many secondary metabolites are produced at low levels; our work suggests that manipulating export might be one way to enhance yields of these molecules. It also suggests that understanding resistance will be relevant to the generation of novel secondary metabolites through the creation of synthetic secondary metabolic gene clusters. Finally, these cognate resistance mechanisms are related to mechanisms that arise in pathogenic bacteria, and understanding them is relevant to our ability to control microbial infections clinically.

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Heterologous Expression of Pseudouridimycin and Description of the Corresponding Minimal Biosynthetic Gene Cluster

Molecules ◽

10.3390/molecules26020510 ◽

2021 ◽

Vol 26 (2) ◽

pp. 510

Author(s):

Nils Böhringer ◽

Maria A. Patras ◽

Till F. Schäberle

Keyword(s):

Heterologous Expression ◽

Gene Cluster ◽

Streptomyces Coelicolor ◽

Biosynthetic Gene Cluster ◽

Infection Model ◽

Biosynthetic Gene ◽

Mouse Infection Model ◽

Production Platform

Pseudouridimycin (PUM) was recently discovered from Streptomyces sp. DSM26212 as a novel bacterial nucleoside analog that competes with UTP for access to the RNA polymerase (RNAP) active site, thereby inhibiting bacterial RNAP by blocking transcription. This represents a novel antibacterial mode of action and it is known that PUM inhibits bacterial RNAP in vitro, inhibits bacterial growth in vitro, and was active in vivo in a mouse infection model of Streptococcus pyogenes peritonitis. The biosynthetic gene cluster (BGC) was previously identified and characterized by knockout experiments. However, the minimal set of genes necessary for PUM production was not proposed. To identify the minimal BGC and to create a plug-and-play production platform for PUM and its biosynthetic precursors, several versions of a redesigned PUM BGC were generated and expressed in the heterologous host Streptomyces coelicolor M1146 under control of strong promotors. Heterologous expression allowed identification of the putative serine/threonine kinase PumF as an enzyme essential for heterologous PUM production and thus corroboration of the PUM minimal BGC.

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Cloning and Characterization of the Tetrocarcin A Gene Cluster from Micromonospora chalcea NRRL 11289 Reveals a Highly Conserved Strategy for Tetronate Biosynthesis in Spirotetronate Antibiotics

Journal of Bacteriology ◽

10.1128/jb.00533-08 ◽

2008 ◽

Vol 190 (17) ◽

pp. 6014-6025 ◽

Cited By ~ 65

Author(s):

Jie Fang ◽

Yiping Zhang ◽

Lijuan Huang ◽

Xinying Jia ◽

Qi Zhang ◽

...

Keyword(s):

Gene Cluster ◽

Structural Diversity ◽

Biosynthetic Gene Cluster ◽

Combinatorial Biosynthesis ◽

In Vitro Characterization ◽

Fused Gene ◽

Micromonospora Chalcea

ABSTRACT Tetrocarcin A (TCA), produced by Micromonospora chalcea NRRL 11289, is a spirotetronate antibiotic with potent antitumor activity and versatile modes of action. In this study, the biosynthetic gene cluster of TCA was cloned and localized to a 108-kb contiguous DNA region. In silico sequence analysis revealed 36 putative genes that constitute this cluster (including 11 for unusual sugar biosynthesis, 13 for aglycone formation, and 4 for glycosylations) and allowed us to propose the biosynthetic pathway of TCA. The formation of d-tetronitrose, l-amicetose, and l-digitoxose may begin with d-glucose-1-phosphate, share early enzymatic steps, and branch into different pathways by competitive actions of specific enzymes. Tetronolide biosynthesis involves the incorporation of a 3-C unit with a polyketide intermediate to form the characteristic spirotetronate moiety and trans-decalin system. Further substitution of tetronolide with five deoxysugars (one being a deoxynitrosugar) was likely due to the activities of four glycosyltransferases. In vitro characterization of the first enzymatic step by utilization of 1,3-biphosphoglycerate as the substrate and in vivo cross-complementation of the bifunctional fused gene tcaD3 (with the functions of chlD3 and chlD4) to ΔchlD3 and ΔchlD4 in chlorothricin biosynthesis supported the highly conserved tetronate biosynthetic strategy in the spirotetronate family. Deletion of a large DNA fragment encoding polyketide synthases resulted in a non-TCA-producing strain, providing a clear background for the identification of novel analogs. These findings provide insights into spirotetronate biosynthesis and demonstrate that combinatorial-biosynthesis methods can be applied to the TCA biosynthetic machinery to generate structural diversity.

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Structural evolution of a DNA repair self-resistance mechanism targeting genotoxic secondary metabolites

Nature Communications ◽

10.1038/s41467-021-27284-7 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Elwood A. Mullins ◽

Jonathan Dorival ◽

Gong-Li Tang ◽

Dale L. Boger ◽

Brandt F. Eichman

Keyword(s):

Natural Products ◽

Selective Pressure ◽

Alkylating Agents ◽

Resistance Mechanism ◽

Structural Evolution ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Structural Basis

AbstractMicrobes produce a broad spectrum of antibiotic natural products, including many DNA-damaging genotoxins. Among the most potent of these are DNA alkylating agents in the spirocyclopropylcyclohexadienone (SCPCHD) family, which includes the duocarmycins, CC-1065, gilvusmycin, and yatakemycin. The yatakemycin biosynthesis cluster in Streptomyces sp. TP-A0356 contains an AlkD-related DNA glycosylase, YtkR2, that serves as a self-resistance mechanism against yatakemycin toxicity. We previously reported that AlkD, which is not present in an SCPCHD producer, provides only limited resistance against yatakemycin. We now show that YtkR2 and C10R5, a previously uncharacterized homolog found in the CC-1065 biosynthetic gene cluster of Streptomyces zelensis, confer far greater resistance against their respective SCPCHD natural products. We identify a structural basis for substrate specificity across gene clusters and show a correlation between in vivo resistance and in vitro enzymatic activity indicating that reduced product affinity—not enhanced substrate recognition—is the evolutionary outcome of selective pressure to provide self-resistance against yatakemycin and CC-1065.

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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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Streptomyces lividans Blasticidin S Deaminase and Its Application in Engineering a Blasticidin S-Producing Strain for Ease of Genetic Manipulation

Applied and Environmental Microbiology ◽

10.1128/aem.03254-12 ◽

2013 ◽

Vol 79 (7) ◽

pp. 2349-2357 ◽

Cited By ~ 9

Author(s):

Li Li ◽

Jun Wu ◽

Zixin Deng ◽

T. Mark Zabriskie ◽

Xinyi He

Keyword(s):

Gene Cluster ◽

Genetic Manipulation ◽

Biosynthetic Gene Cluster ◽

Streptomyces Lividans ◽

Dna Uptake ◽

Biosynthetic Gene ◽

Content Type ◽

Blasticidin S

ABSTRACTBlasticidin S is a peptidyl nucleoside antibiotic produced byStreptomyces griseochromogenesthat exhibits strong fungicidal activity. To circumvent an effective DNA uptake barrier system in the native producer and investigate its biosynthesisin vivo, the blasticidin S biosynthetic gene cluster (bls) was engrafted to the chromosome ofStreptomyces lividans. However, the resulting mutant, LL2, produced the inactive deaminohydroxyblasticidin S instead of blasticidin S. Subsequently, a blasticidin S deaminase (SLBSD, forS. lividansblasticidin S deaminase) was identified inS. lividansand shown to govern thisin vivoconversion. Purified SLBSD was found to be capable of transforming blasticidin S to deaminohydroxyblasticidin Sin vitro. It also catalyzed deamination of the cytosine moiety of cytosylglucuronic acid, an intermediate in blasticidin S biosynthesis. Disruption of the SLBSD gene inS. lividansLL2 led to successful production of active blasticidin S in the resultant mutant,S. lividansWJ2. To demonstrate the easy manipulation of the blasticidin S biosynthetic gene cluster,blsE,blsF, andblsL, encoding a predicted radicalS-adenosylmethionine (SAM) protein, an unknown protein, and a guanidino methyltransferase, were individually inactivated to access their role in blasticidin S biosynthesis.

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The brlA Gene Deletion Reveals That Patulin Biosynthesis Is Not Related to Conidiation in Penicillium expansum

International Journal of Molecular Sciences ◽

10.3390/ijms21186660 ◽

2020 ◽

Vol 21 (18) ◽

pp. 6660

Author(s):

Chrystian Zetina-Serrano ◽

Ophélie Rocher ◽

Claire Naylies ◽

Yannick Lippi ◽

Isabelle P. Oswald ◽

...

Keyword(s):

Gene Cluster ◽

Gene Deletion ◽

Biosynthetic Gene Cluster ◽

Temporal Organization ◽

Penicillium Expansum ◽

Wild Type ◽

Metabolome Analysis ◽

Brla Gene

Dissemination and survival of ascomycetes is through asexual spores. The brlA gene encodes a C2H2-type zinc-finger transcription factor, which is essential for asexual development. Penicillium expansum causes blue mold disease and is the main source of patulin, a mycotoxin that contaminates apple-based food. A P. expansum PeΔbrlA deficient strain was generated by homologous recombination. In vivo, suppression of brlA completely blocked the development of conidiophores that takes place after the formation of coremia/synnemata, a required step for the perforation of the apple epicarp. Metabolome analysis displayed that patulin production was enhanced by brlA suppression, explaining a higher in vivo aggressiveness compared to the wild type (WT) strain. No patulin was detected in the synnemata, suggesting that patulin biosynthesis stopped when the fungus exited the apple. In vitro transcriptome analysis of PeΔbrlA unveiled an up-regulated biosynthetic gene cluster (PEXP_073960-PEXP_074060) that shares high similarity with the chaetoglobosin gene cluster of Chaetomium globosum. Metabolome analysis of PeΔbrlA confirmed these observations by unveiling a greater diversity of chaetoglobosin derivatives. We observed that chaetoglobosins A and C were found only in the synnemata, located outside of the apple, whereas other chaetoglobosins were detected in apple flesh, suggesting a spatial-temporal organization of the chaetoglobosin biosynthesis pathway.

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BET bromodomain inhibitor HMBA synergizes with MEK inhibition in treatment of malignant glioma

10.1101/2020.01.01.891739 ◽

2020 ◽

Author(s):

Elisa Funck-Brentano ◽

Dzeneta Vizlin-Hodzic ◽

Jonas A. Nilsson ◽

Lisa M. Nilsson

Keyword(s):

Biological Activities ◽

Glioma Cells ◽

Resistance Mechanisms ◽

Transcriptional Elongation ◽

Mek Inhibitors ◽

Bet Inhibitors ◽

Bromodomain Proteins ◽

Cancer Multiple

Abstract(1)BackgroundBET bromodomain proteins regulate transcription by binding acetylated histones and attracting key factors for e.g. transcriptional elongation. BET inhibitors have been developed to block pathogenic processes such as cancer and inflammation. Despite having potent biological activities, BET inhibitors have still not made a breakthrough in clinical use for treating cancer. Multiple resistance mechanisms have been proposed but thus far no attempts to block this in glioma has been made.(2)MethodsHere, we have conducted a pharmacological synergy screen in glioma cells to search for possible combination treatments augmenting the apoptotic response to BET inhibitors. We first used HMBA, a compound that was developed as a differentiation therapy four decades ago but more recently was shown to primarily inhibit BET bromodomain proteins. Data was also generated using other BET inhibitors.(3)ResultsIn the synergy screen, we discovered that several MEK inhibitors can enhance apoptosis in response to HMBA in rat and human glioma cells in vitro as well as in vivo xenografts. The combination is not unique to HMBA but also other BET inhibitors such as JQ1 and I-BET-762 can synergize with MEK inhibitors.(4)ConclusionsOur findings validate a combination therapy previously demonstrated to exhibit anti-cancer activities in multiple other tumor types but which appears to have been lost in translation to the clinic.

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