RESUMEN
The anti-Clostridium difficile infection (CDI) drug fidaxomicin is a natural polyketide metabolite mainly produced by Micromonosporaceae, such as Actinoplanes deccanensis, Dactylosporangium aurantiacum, and Micromonospora echinospora. In the present study, we employed a stepwise strategy by combining heterologous expression, chassis construction, promoter engineering, activator and transporters overexpression, and optimization of fermentation media for high-level production of fidaxomicin. The maximum yield of 384 mg/L fidaxomicin was achieved with engineered Streptomyces albus D7-VHb in 5 L-tank bioreactor, and it was approximately 15-fold higher than the native strain Actinoplanes deccanensis YP-1 with higher strain stability and growth rate. This study developed an enhanced chassis strain, and for the first time, achieved the heterologous synthesis of fidaxomicin through a combinatorial metabolic engineering strategy.
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Structural motifs containing nitrogen-nitrogen (N-N) bonds are prevalent in a large number of clinical drugs and bioactive natural products. Hydrazine (N2H4) serves as a widely utilized building block for the preparation of these N-N-containing molecules in organic synthesis. Despite its common use in chemical processes, no enzyme has been identified to catalyze the incorporation of free hydrazine in natural product biosynthesis. Here, we report that a hydrazine transferase catalyzes the condensation of N2H4 and an aromatic polyketide pathway intermediate, leading to the formation of a rare N-aminolactam pharmacophore in the biosynthesis of broad-spectrum antibiotic albofungin. These results expand the current knowledge on the biosynthetic mechanism for natural products with N-N units and should facilitate future development of biocatalysts for the production of N-N-containing chemicals.
Asunto(s)
Hidrazinas , Hidrazinas/química , Hidrazinas/metabolismo , Antibacterianos/química , Antibacterianos/biosíntesis , Antibacterianos/farmacología , Streptomyces/enzimología , Streptomyces/metabolismo , Lactamas/química , Lactamas/metabolismo , FarmacóforoRESUMEN
The biological signaling molecule nitric oxide (NO) has recently emerged as a metabolic precursor for the creation of microbial natural products with diversified structures and biological activities. Within the biosynthetic gene clusters (BGCs) of these compounds, genes associated with NO production pathways have been pinpointed. In this study, we employ a nitric oxide synthase (NOS)-guided genome mining strategy for the targeted discovery of NO-derived bacterial natural products and NO-utilizing biocatalysts. We show that a conserved NOS-containing BGC, distributed across several actinobacterial genomes, is responsible for the biosynthesis of lajollamycin, a unique nitro-tetraene-containing antibiotic whose biosynthetic mechanism remains elusive. Through a combination of in vivo and in vitro studies, we unveil the first cytochrome P450 enzyme capable of catalyzing olefin nitration in natural product biosynthesis. These results not only expand the current knowledge about biosynthetic nitration processes but also offer an efficient way for targeted identification of NO-utilizing metabolic pathways and novel nitrating biocatalysts.
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IMPORTANCE: Cascade regulation networks are almost present in various kinds of microorganisms, but locating and systematically elucidating specific pleiotropic regulators related to a certain gene cluster can be a tricky problem. Here, based on the promoter of the fidaxomicin pathway-specific regulator FadR1, we utilized a "DNA to Proteins" affinity purification method and captured a global regulator MtrA, which positively regulates fidaxomicin biosynthesis. In the mtrA overexpressed strain, the production of fidaxomicin was improved by 37% compared to the native strain. Then, we combined the "Protein to DNAs" affinity purification method (DAP-seq) with the results of RNA-seq and systematically elucidated the primary and secondary metabolic processes in which MtrA directly or indirectly participates. Thus, our work brought up a new way to improve fidaxomicin production from the perspective of global regulation and analyzed the regulatory mechanism of MtrA. Meanwhile, we provided a novel methodology for the research of cascade regulation networks and vital secondary metabolites.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Regulación Bacteriana de la Expresión Génica , Fidaxomicina , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Familia de Multigenes , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismoRESUMEN
Azoxy compounds exhibit a wide array of biological activities and possess distinctive chemical properties. Although there has been considerable interest in the biosynthetic mechanisms of azoxy metabolites, the enzymatic basis responsible for azoxy bond formation has remained largely enigmatic. In this study, we unveil the enzyme cascade that constructs the azoxy bond in valanimycin biosynthesis. Our research demonstrates that a pair of metalloenzymes, comprising a membrane-bound hydrazine synthase and a nonheme diiron azoxy synthase, collaborate to convert an unstable pathway intermediate to an azoxy product through a hydrazine-azo-azoxy pathway. Additionally, by characterizing homologues of this enzyme pair from other azoxy metabolite pathways, we propose that this two-enzyme cascade could represent a conserved enzymatic strategy for azoxy bond formation in bacteria. These findings provide significant mechanistic insights into biological N-N bond formation and should facilitate the targeted isolation of bioactive azoxy compounds through genome mining.
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Bacterias , HidrazinasRESUMEN
Microbial natural products have been one of the most important sources for drug development. In the current postgenomic era, sequence-driven approaches for natural product discovery are becoming increasingly popular. Here, we develop an effective genome mining strategy for the targeted discovery of microbial metabolites with antitumor activities. Our method employs uvrA-like genes as genetic markers, which have been identified in the biosynthetic gene clusters (BGCs) of several chemotherapeutic drugs of microbial origin and confer self-resistance to the corresponding producers. Through systematic genomic analysis of gifted actinobacteria genera, identification of uvrA-like gene-containing BGCs, and targeted isolation of products from a BGC prioritized for metabolic analysis, we identified a new tetracycline-type DNA intercalator timmycins. Our results thus provide a new genome mining strategy for the efficient discovery of antitumor agents acting through DNA intercalation.
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Hydroxytryptophan serves as a chemical precursor to a variety of bioactive specialized metabolites, including the human neurotransmitter serotonin and the hormone melatonin. Although the human and animal routes to hydroxytryptophan have been known for decades, how bacteria catalyze tryptophan indole hydroxylation remains a mystery. Here we report a class of tryptophan hydroxylases that are involved in various bacterial metabolic pathways. These enzymes utilize a histidine-ligated heme cofactor and molecular oxygen or hydrogen peroxide to catalyze regioselective hydroxylation on the tryptophan indole moiety, which is mechanistically distinct from their animal counterparts from the nonheme iron enzyme family. Through genome mining, we also identify members that can hydroxylate the tryptophan indole ring at alternative positions. Our results not only reveal a conserved way to synthesize hydroxytryptophans in bacteria but also provide a valuable enzyme toolbox for biocatalysis. As proof of concept, we assemble a highly efficient pathway for melatonin in a bacterial host.
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5-Hidroxitriptófano , Melatonina , Animales , Humanos , Triptófano/metabolismo , Hemo/química , Bacterias/metabolismoRESUMEN
Bacterial azapteridine-containing phytotoxin toxoflavin is a causal agent of rice grain rot. Here, we heterologously reconstitute Bukholderia toxoflavin biosynthesis in Escherichia coli and identify key pathway intermediates, including the hitherto unknown ribityl-dedimethyl-toxoflavin. Furthermore, we characterized a cofactorless oxidase that converts ribityl-dedimethyl-toxoflavin to ribose and dedimethyl-toxoflavin, the latter of which then undergoes stepwise methylations to form toxoflavin. These findings provide new insights into the biosynthetic pathways of toxoflavin and related triazine metabolites.
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Oxidorreductasas , Pirimidinonas , Triazinas , Escherichia coli/genética , Escherichia coli/metabolismoRESUMEN
The nitrogen-nitrogen bond is a core feature of diverse functional groups like hydrazines, nitrosamines, diazos, and pyrazoles. Such functional groups are found in >300 known natural products. Such N-N bond-containing functional groups are also found in significant percentage of clinical drugs. Therefore, there is wide interest in synthetic and enzymatic methods to form nitrogen-nitrogen bonds. In this review, we summarize synthetic and biosynthetic approaches to diverse nitrogen-nitrogen-bond-containing functional groups, with a focus on biosynthetic pathways and enzymes.
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Productos Biológicos , Nitrógeno , Productos Biológicos/química , Vías Biosintéticas , Hidrazinas/química , Hidrazinas/metabolismo , Nitrógeno/químicaRESUMEN
Molecules with a nitrogen-nitrogen (N-N) bond in their structures exhibit various biological activities and other unique properties. A few microbial proteins are recently emerging as dedicated N-N bond forming enzymes in natural product biosynthesis. However, the details of these biochemical processes remain largely unknown. Here, through in vitro biochemical characterization and computational studies, we report the molecular basis of hydrazine bond formation by a family of di-domain enzymes. These enzymes are widespread in bacteria and sometimes naturally exist as two standalone enzymes. We reveal that the methionyl-tRNA synthase-like domain/protein catalyzes ATP-dependent condensation of two amino acids substrates to form a highly unstable ester intermediate, which is subsequently captured by the zinc-binding cupin domain/protein and undergoes redox-neutral intramolecular rearrangement to give the N-N bond containing product. These results provide important mechanistic insights into enzymatic N-N bond formation and should facilitate future development of novel N-N forming biocatalyst.
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Proteínas Portadoras/química , Enzimas/química , Nitrógeno/química , Zinc/química , Aminoácidos , Bacterias/enzimología , Bacterias/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Productos Biológicos/metabolismo , Catálisis , Hidrazinas , Estructura Molecular , ARN de Transferencia , RhodococcusRESUMEN
Luzopeptins and related decadepsipeptides are bisintercalator nonribosomal peptides featuring rare acyl-substituted tetrahydropyridazine-3-carboxylic acid (Thp) subunits that are critical to their biological activities. Herein, we reconstitute the biosynthetic tailoring pathway in luzopeptinâ A biosynthesis through in vivo genetic and in vitro biochemical approaches. Significantly, we revealed a multitasking cytochromeâ P450 enzyme that catalyzes four consecutive oxidations including the highly unusual carbon-nitrogen bond desaturation, forming the hydrazone-bearing 4-OH-Thp residues. Moreover, we identified a membrane-bound acyltransferase that likely mediates the subsequent O-acetylation extracellularly, as a potential self-protective strategy for the producer strain. Further genome mining of novel decadepsipeptides and an associated P450 enzyme have provided mechanistic insights into the P450-mediated carbon-nitrogen bond desaturation. Our results not only reveal the molecular basis of pharmacophore formation in bisintercalator decadepsipeptides, but also expand the catalytic versatility of P450 family enzymes.
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Carbono/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Hidrazonas/metabolismo , Nitrógeno/metabolismo , Carbono/química , Hidrazonas/química , Hidroxiquinolinas/química , Hidroxiquinolinas/metabolismo , Estructura Molecular , Nitrógeno/químicaRESUMEN
Microbial fermentation is currently still the major way to produce structural complicated clinical drugs. Yet, the low productivity and genetic instability of producing strains remain the bottlenecks in microbial pharmaceutical industry. Fidaxomicin is a microbial drug against the Clostridium difficile infection. Here, a genome-based combinatorial engineering strategy was established to improve both fidaxomicin production and the genetic stability of Actinoplanes deccanensis YP-1. Guided by genomic analysis, several genetic instability-associated elements were cumulatively deleted, generating a more genetically stable mutant. Further rational engineering approaches including elimination of a pigment pathway, duplication of the fidaxomicin gene cluster, overexpression of a positive regulator and optimization of the fermentation medium, led to an overall 27-folds improvement in fidaxomicin production. Taken together, the genome-based rational combinatorial engineering strategy was efficient to enhance the fidaxomicin production and ameliorate the genetic stability of YP-1, it can also be widely used in other industrial actinomycetes for strain improvement.
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Actinoplanes , Clostridioides difficile , Aminoglicósidos , Antibacterianos , FidaxomicinaRESUMEN
Bacterial hormones, such as the iconic gamma-butyrolactone A-factor, are essential signaling molecules that regulate diverse physiological processes, including specialized metabolism. These low molecular weight compounds are common in Streptomyces species and display species-specific structural differences. Recently, unusual gamma-butyrolactone natural products called salinipostins were isolated from the marine actinomycete genus Salinispora based on their antimalarial properties. As the salinipostins possess a rare phosphotriester motif of unknown biosynthetic origin, we set out to explore its construction by the widely conserved 9-gene spt operon in Salinispora species. We show through a series of in vivo and in vitro studies that the spt gene cluster dually encodes the salinipostins and newly identified natural A-factor-like gamma-butyrolactones (Sal-GBLs). Remarkably, homologous biosynthetic gene clusters are widely distributed among many actinomycete genera, including Streptomyces, suggesting the significance of this operon in bacteria.
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4-Butirolactona/metabolismo , Productos Biológicos/metabolismo , Ésteres/metabolismo , Transducción de Señal , Compuestos Bicíclicos Heterocíclicos con Puentes/metabolismo , Genes Bacterianos , Streptomyces/genética , Streptomyces/metabolismoRESUMEN
The heterocycle 1,2,3-triazole is among the most versatile chemical scaffolds and has been widely used in diverse fields. However, how nature creates this nitrogen-rich ring system remains unknown. Here, we report the biosynthetic route to the triazole-bearing antimetabolite 8-azaguanine. We reveal that its triazole moiety can be assembled through an enzymatic and non-enzymatic cascade, in which nitric oxide is used as a building block. These results expand our knowledge of the physiological role of nitric oxide synthase in building natural products with a nitrogen-nitrogen bond, and should also inspire the development of synthetic biology approaches for triazole production.
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Bacterias/metabolismo , Óxido Nítrico/metabolismo , Triazoles/metabolismo , Azaguanina/metabolismo , Bacterias/enzimología , Bacterias/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Productos Biológicos , Vías Biosintéticas/genética , Genes Bacterianos/genética , Óxido Nítrico Sintasa/metabolismo , Nitrógeno , Streptomyces/enzimología , Streptomyces/genética , Streptomyces/metabolismo , Biología SintéticaRESUMEN
The introduction of multi-gene metabolic pathways is generally the first step for the construction of microbial cell factories and plays an essential role in metabolic engineering and synthetic biology. Here, we developed a "PCR & Go" system for facile integration and assembly of multi-gene pathways into the chromosome of Saccharomyces cerevisiae. The core component of the "PCR & Go" system was an expression chassis, where eight promoter/terminator pairs were pre-installed into the yeast chromosome and PCR amplified gene fragments could be inserted directly for functional expression. In combination with the CRISPR/Cas9 system and a gRNA plasmid library, the ß-carotene (three genes), zeaxanthin (four genes), and astaxanthin (five genes) biosynthetic pathways were integrated and assembled into the yeast genome with an efficiency of ~93, ~85, and 69%, respectively, using PCR amplified gene fragments with ~40 bp homology arms in a single step. Therefore, the "PCR & Go" system can be used for fast construction of yeast cell factories harboring multi-gene pathways with high efficiency and flexibility.
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Pyrazomycin is a rare C-nucleoside antibiotic containing a naturally occurring pyrazole ring, the biosynthetic origin of which has remained obscure for decades. In this study we report the identification of the gene cluster responsible for pyrazomycin biosynthesis in Streptomyces candidus NRRLâ 3601, revealing that the StrR-family regulator PyrR is the cluster-situated transcriptional activator governing pyrazomycin biosynthesis. Furthermore, our results from in vivo reconstitution and stable-isotope feeding experiments provide support for the hypothesis that PyrN is a new nitrogen-nitrogen bond-forming enzyme that catalyzes the linkage of the ϵ-NH2 nitrogen atom of l-N6 -OH-lysine and the α-NH2 nitrogen atom of l-glutamic acid. This study lays the foundation for further genetic and biochemical characterization of pyrazomycin pathway enzymes involved in constructing the characteristic pyrazole ring.
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Antibacterianos/biosíntesis , Ribosa/biosíntesis , Streptomyces/metabolismo , Amidas , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Familia de Multigenes , Pentosiltransferasa/genética , Pentosiltransferasa/metabolismo , Pirazoles , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Streptomyces/genéticaRESUMEN
Azoxy bond is an important chemical bond and plays a crucial role in high energy density materials. However, the biosynthetic mechanism of azoxy bond remains enigmatic. Here we report that the azoxy bond biosynthesis of azoxymycins is an enzymatic and non-enzymatic coupling cascade reaction. In the first step, nonheme diiron N-oxygenase AzoC catalyzes the oxidization of amine to its nitroso analogue. Redox coenzyme pairs then facilitate the mutual conversion between nitroso group and hydroxylamine via the radical transient intermediates, which efficiently dimerize to azoxy bond. The deficiency of nucleophilic reactivity in AzoC is proposed to account for the enzyme's non-canonical oxidization of amine to nitroso product. Free nitrogen radicals induced by coenzyme pairs are proposed to be responsible for the efficient non-enzymatic azoxy bond formation. This mechanism study will provide molecular basis for the biosynthesis of azoxy high energy density materials and other valuable azoxy chemicals.
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Compuestos Azo/química , Modelos Químicos , Compuestos Azo/metabolismo , Catálisis , Simulación por Computador , Oxidación-ReducciónRESUMEN
Microbes produce specialized metabolites to thrive in their natural habitats. However, it is rare that a given specialized metabolite is biosynthesized via pathways with distinct intermediates and enzymes. Here, we show that the core assembly mechanism of the antibiotic indolmycin in marine gram-negative Pseudoalteromonas luteoviolacea is distinct from its counterpart in terrestrial gram-positive Streptomyces species, with a molecule that is a shunt product in the Streptomyces pathway employed as a biosynthetic substrate for a novel metal-independent N-demethylindolmycin synthase in the P. luteoviolacea pathway. To provide insight into this reaction, we solved the 1.5 Å resolution structure in complex with product and identified the active site residues. Guided by our biosynthetic insights, we then engineered the Streptomyces indolmycin producer for titer improvement. This study provides a paradigm for understanding how two unique routes to a microbial specialized metabolite can emerge from convergent biosynthetic transformations.
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Bacterias/metabolismo , Vías Biosintéticas , Bacterias/genética , Biocatálisis , Familia de MultigenesRESUMEN
Nitric oxide (NO) has wide-ranging roles in biology, but less is known about its role in building chemical diversity. Here we report a new route to NO from the biosynthetic pathway to the N-nitroso compound streptozocin. We show that the N-nitroso group of streptozocin comes from the biosynthetic reassembly of l-arginine, with the guanidino nitrogens forming a nitrogen-nitrogen bond. To understand this biosynthetic process, we identify the biosynthetic gene cluster of streptozocin and demonstrate that free l-arginine is N-methylated by StzE to give Nω-monomethyl-l-arginine. We show that this product is then oxidized by StzF, a nonheme iron-dependent enzyme unrelated to known nitric oxide synthases, generating a urea compound and NO. Our work implies that formation and capture of NO is the likely route to N-nitroso formation in vivo. Altogether, our work unveils a new enzyme pair for the production of NO from l-arginine and sets the stage for understanding biosynthetic routes to N-nitroso natural products.
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Arginina/metabolismo , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico/metabolismo , Compuestos Nitrosos/metabolismo , Proteínas de Hierro no Heme/metabolismo , Arginina/química , Estructura Molecular , Óxido Nítrico/química , Compuestos Nitrosos/químicaRESUMEN
Covering: up to mid-2018 Pyridoxal 5'-phosphate (PLP) is a versatile organic cofactor used to catalyze diverse reactions on amino acid, oxoacid, and amine substrates. Here we review the reactions catalyzed by PLP-dependent enzymes, highlighting enzymes reported in the natural product biosynthetic literature. We describe enzymes that catalyze transaminations, Claisen-like condensations, and ß- and γ-eliminations and substitutions, along with epimerizations, decarboxylations, and transaldolations. Finally, we describe a newly reported group of O2-, PLP-dependent enzymes. Altogether, natural product biosynthesis showcases the incredible versatility of PLP-dependent transformations for building chemical complexity.