Identification and characterization of enzymes involved in the biosynthesis of pyrimidine nucleoside antibiotics.

Covering: up to September 2020 Hundreds of nucleoside-based natural products have been isolated from various microorganisms, several of which have been utilized in agriculture as pesticides and herbicides, in medicine as therapeutics for cancer and infectious disease, and as molecular probes to study biological processes. Natural products consisting of structural modifications of each of the canonical nucleosides have been discovered, ranging from simple modifications such as single-step alkylations or acylations to highly elaborate modifications that dramatically alter the nucleoside scaffold and require multiple enzyme-catalyzed reactions. A vast amount of genomic information has been uncovered the past two decades, which has subsequently allowed the first opportunity to interrogate the chemically intriguing enzymatic transformations for the latter type of modifications. This review highlights (i) the discovery and potential applications of structurally complex pyrimidine nucleoside antibiotics for which genetic information is known, (ii) the established reactions that convert the canonical pyrimidine into a new nucleoside scaffold, and (iii) the important tailoring reactions that impart further structural complexity to these molecules.

Cryptic phosphorylation in nucleoside natural product biosynthesis.

Kinases are annotated in many nucleoside biosynthetic gene clusters but generally are considered responsible only for self-resistance. Here, we report an unexpected 2'-phosphorylation of nucleoside biosynthetic intermediates in the nikkomycin and polyoxin pathways. This phosphorylation is a unique cryptic modification as it is introduced in the third of seven steps during aminohexuronic acid (AHA) nucleoside biosynthesis, retained throughout the pathway's duration, and is removed in the last step of the pathway. Bioinformatic analysis of reported nucleoside biosynthetic gene clusters indicates the presence of cryptic phosphorylation in other pathways and the importance of functional characterization of kinases in nucleoside biosynthetic pathways in general. This study also functionally characterized all of the enzymes responsible for AHA biosynthesis and revealed that AHA is constructed via a unique oxidative C-C bond cleavage reaction. The results indicate a divergent biosynthetic mechanism for three classes of antifungal nucleoside natural products.

Recent advances in the biosynthesis of nucleoside antibiotics.

Nucleoside antibiotics are a diverse class of natural products with promising biomedical activities. These compounds contain a saccharide core and a nucleobase. Despite the large number of nucleoside antibiotics that have been reported, biosynthetic studies on these compounds have been limited compared with those on other types of natural products such as polyketides, peptides, and terpenoids. Due to recent advances in genome sequencing technology, the biosynthesis of nucleoside antibiotics has rapidly been clarified. This review covering 2009-2019 focuses on recent advances in the biosynthesis of nucleoside antibiotics.

Suppression of pyrimidine biosynthesis by targeting DHODH enzyme robustly inhibits rotavirus replication.

Rotavirus infection remains a great health burden worldwide especially in some developing countries. It causes severe dehydrating diarrhea in infants, young children, as well as immunocompromised and organ transplanted patients. Viral replication heavily relies on the host to supply nucleosides. Thus, host enzymes involved in nucleotide biosynthesis represent potential targets for antiviral development. Dihydroorotate dehydrogenase (DHODH) is the rate-limiting enzyme in the de novo biosynthesis pathway of pyrimidines. In this study, we demonstrated that two specific DHODH enzyme inhibitors, brequinar (BQR) and leflunomide (LFM) robustly inhibited rotavirus replication in conventional human intestinal Caco2 cell line as well as in human primary intestinal organoids. The antiviral effect is conserved in both laboratory strain SA11 and rotavirus strain 2011K isolated from clinical sample. Mechanistic study indicated that BQR and LFM exerted their anti-rotavirus effect through targeting DHODH to deplete pyrimidine nucleotide pool. Therefore, targeting pyrimidine biosynthesis represents a potential approach for developing antiviral strategies against rotavirus.

An ATP-Dependent Ligase with Substrate Flexibility Involved in Assembly of the Peptidyl Nucleoside Antibiotic Polyoxin.

Polyoxin (POL) is an unusual peptidyl nucleoside antibiotic, in which the peptidyl moiety and nucleoside skeleton are linked by an amide bond. However, their biosynthesis remains poorly understood. Here, we report the deciphering of PolG as an ATP-dependent ligase responsible for the assembly of POL. A polG mutant is capable of accumulating multiple intermediates, including the peptidyl moiety (carbamoylpolyoxamic acid [CPOAA]) and the nucleoside skeletons (POL-C and the previously overlooked thymine POL-C). We further demonstrate that PolG employs an ATP-dependent mechanism for amide bond formation and that the generation of the hybrid nucleoside antibiotic POL-N is also governed by PolG. Finally, we determined that the deduced ATP-binding sites are functionally essential for PolG and that they are highly conserved in a number of related ATP-dependent ligases. These insights have allowed us to propose a catalytic mechanism for the assembly of peptidyl nucleoside antibiotic via an acyl-phosphate intermediate and have opened the way for the combinatorial biosynthesis/pathway engineering of this group of nucleoside antibiotics.IMPORTANCE POL is well known for its remarkable antifungal bioactivities and unusual structural features. Actually, elucidation of the POL assembly logic not only provides the enzymatic basis for further biosynthetic understanding of related peptidyl nucleoside antibiotics but also contributes to the rational generation of more hybrid nucleoside antibiotics via synthetic biology strategy.

A Pressure Test to Make 10 Molecules in 90 Days: External Evaluation of Methods to Engineer Biology.

Centralized facilities for genetic engineering, or "biofoundries", offer the potential to design organisms to address emerging needs in medicine, agriculture, industry, and defense. The field has seen rapid advances in technology, but it is difficult to gauge current capabilities or identify gaps across projects. To this end, our foundry was assessed via a timed "pressure test", in which 3 months were given to build organisms to produce 10 molecules unknown to us in advance. By applying a diversity of new approaches, we produced the desired molecule or a closely related one for six out of 10 targets during the performance period and made advances toward production of the others as well. Specifically, we increased the titers of 1-hexadecanol, pyrrolnitrin, and pacidamycin D, found novel routes to the enediyne warhead underlying powerful antimicrobials, established a cell-free system for monoterpene production, produced an intermediate toward vincristine biosynthesis, and encoded 7802 individually retrievable pathways to 540 bisindoles in a DNA pool. Pathways to tetrahydrofuran and barbamide were designed and constructed, but toxicity or analytical tools inhibited further progress. In sum, we constructed 1.2 Mb DNA, built 215 strains spanning five species ( Saccharomyces cerevisiae, Escherichia coli, Streptomyces albidoflavus, Streptomyces coelicolor, and Streptomyces albovinaceus), established two cell-free systems, and performed 690 assays developed in-house for the molecules.

[Functional characterization of gouC and gouD in gougerotin biosynthesis].

OBJECTIVE: To determine the functions of gouC and gouD in gougerotin biosynthesis, disruption of these two genes was performed. As gougerotin producing strain Streptomyces graminearus lacks efficient genetic manipulation system, the gene cluster for gougerotin biosynthesis was heterologously expressed in Streptomyces coelicolor M1146 to facilitate genetic manipulations of gouC and gouD. METHODS: By using fosmid D6-4H containing the complete gougerotin biosynthetic gene cluster, gouC and gouD were disrupted by PCR-targeting method to generate pGOUe-ΔC and pGOUe-ΔD. Both pGOUe-ΔC and pGOUe-ΔD were introduced into Streptomyces coelicolor M1146 by intergeneric conjugation, thus gouC and gouD disrpution mutants (Ml146-GOUe-AC and M1146-GOUe-ΔD) were obtained. The gougerotin production of M1146-GOUe-ΔC and M1146-GOUe-ΔD were assayed by HPLC analysis. The intermediates accumulated in these mutants were purified and subjected to MS and NMR analyses for structure determinations. Bioassay of these intermediates against tumor cell line were also carried out. RESULTS: Disruption mutants of gouC and gouD failed to produce gougerotin and the mutants accumulated different gougerotin intermediates, which lost their ability to inhibit cancer cell proliferation. CONCLUSION: gouC and gouD are key structual genes in the biosynthesis of gougerotin peptidyl moieties. This study will pave the way for the elucidation of gougerotin biosynthetic pathway.

An unusual UMP C-5 methylase in nucleoside antibiotic polyoxin biosynthesis.

Polyoxin is a group of structurally-related peptidyl nucleoside antibiotics bearing C-5 modifications on the nucleoside skeleton. Although the structural diversity and bioactivity preference of polyoxin are, to some extent, affected by such modifications, the biosynthetic logic for their occurence remains obscure. Here we report the identification of PolB in polyoxin pathway as an unusual UMP C-5 methylase with thymidylate synthase activity which is responsible for the C-5 methylation of the nucleoside skeleton. To probe its molecular mechanism, we determined the crystal structures of PolB alone and in complexes with 5-Br UMP and 5-Br dUMP at 2.15 Å, 1.76 Å and 2.28 Å resolutions, respectively. Loop 1 (residues 117-131), Loop 2 (residues 192-201) and the substrate recognition peptide (residues 94-102) of PolB exhibit considerable conformational flexibility and adopt distinct structures upon binding to different substrate analogs. Consistent with the structural findings, a PolB homolog that harbors an identical function from Streptomyces viridochromogenes DSM 40736 was identified. The discovery of UMP C5-methylase opens the way to rational pathway engineering for polyoxin component optimization, and will also enrich the toolbox for natural nucleotide chemistry.

Comparative Proteomic Analysis of Streptomyces aureochromogenes Under Different Carbon Sources and Insights into Polyoxin Production.

In this paper, fermentation dynamics process of Streptomyces aureochromogenes was researched. The production of polyoxins in culture medium with moderate concentration sucrose was at a relative high level. And, when the carbon source changed to glucose, the mycelium grew in a better condition, while no poloxins was produced. This phenomenon also happened when increasing the initial concentration of sucrose to three times. To evaluate the regulatory mechanism of polyoxin production, comparative proteomic analysis of S. aureochromogenes was conducted. The result showed that tricarboxylic acid cycle (TCA) cycle, amino metabolism, and fatty acid metabolism were active and glycolysis was repressed. And, the fact that stress and stress-related protein like superoxide dismutase (SOD), Clp protease, and catalase were highly expressed indicated a higher regulator level in synthesis of polyoxins. In addition, enzymes related to transcription or translation processes also revealed a correlation between specific cell growth rate and polyoxin production. This study will be helpful in better understanding the biosynthesis mechanism of polyoxins and metabolism regulation in this process.

Nucleoside antibiotics: biosynthesis, regulation, and biotechnology.

The alarming rise in antibiotic-resistant pathogens has coincided with a decline in the supply of new antibiotics. It is therefore of great importance to find and create new antibiotics. Nucleoside antibiotics are a large family of natural products with diverse biological functions. Their biosynthesis is a complex process through multistep enzymatic reactions and is subject to hierarchical regulation. Genetic and biochemical studies of the biosynthetic machinery have provided the basis for pathway engineering and combinatorial biosynthesis to create new or hybrid nucleoside antibiotics. Dissection of regulatory mechanisms is leading to strategies to increase the titer of bioactive nucleoside antibiotics.

Rheb protein binds CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase) protein in a GTP- and effector domain-dependent manner and influences its cellular localization and carbamoyl-phosphate synthetase (CPSase) activity.

Rheb small GTPases, which consist of Rheb1 and Rheb2 (also known as RhebL1) in mammalian cells, are unique members of the Ras superfamily and play central roles in regulating protein synthesis and cell growth by activating mTOR. To gain further insight into the function of Rheb, we carried out a search for Rheb-binding proteins and found that Rheb binds to CAD protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase), a multifunctional enzyme required for the de novo synthesis of pyrimidine nucleotides. CAD binding is more pronounced with Rheb2 than with Rheb1. Rheb binds CAD in a GTP- and effector domain-dependent manner. The region of CAD where Rheb binds is located at the C-terminal region of the carbamoyl-phosphate synthetase domain and not in the dihydroorotase and aspartate transcarbamoylase domains. Rheb stimulated carbamoyl-phosphate synthetase activity of CAD in vitro. In addition, an elevated level of intracellular UTP pyrimidine nucleotide was observed in Tsc2-deficient cells, which was attenuated by knocking down of Rheb. Immunostaining analysis showed that expression of Rheb leads to increased accumulation of CAD on lysosomes. Both a farnesyltransferase inhibitor that blocks membrane association of Rheb and knockdown of Rheb mislocalized CAD. These results establish CAD as a downstream effector of Rheb and suggest a possible role of Rheb in regulating de novo pyrimidine nucleotide synthesis.

GouR, a TetR family transcriptional regulator, coordinates the biosynthesis and export of gougerotin in Streptomyces graminearus.

Gougerotin is a peptidyl nucleoside antibiotic. It functions as a specific inhibitor of protein synthesis by binding ribosomal peptidyl transferase and exhibits a broad spectrum of biological activities. gouR, situated in the gougerotin biosynthetic gene cluster, encodes a TetR family transcriptional regulatory protein. Gene disruption and genetic complementation revealed that gouR plays an important role in the biosynthesis of gougerotin. Transcriptional analysis suggested that GouR represses the transcription of the gouL-to-gouB operon consisting of 11 structural genes and activates the transcription of the major facilitator superfamily (MFS) transporter gene (gouM). Electrophoresis mobility shift assays (EMSAs) and DNase I footprinting experiments showed that GouR has specific DNA-binding activity for the promoter regions of gouL, gouM, and gouR. Our data suggested that GouR modulates gougerotin production by coordinating its biosynthesis and export in Streptomyces graminearus.

Disruption of rimP-SC, encoding a ribosome assembly cofactor, markedly enhances the production of several antibiotics in Streptomyces coelicolor.

BACKGROUND: Ribosome assembly cofactor RimP is one of the auxiliary proteins required for maturation of the 30S subunit in Escherichia coli. Although RimP in protein synthesis is important, its role in secondary metabolites biosynthesis has not been reported so far. Considering the close relationship between protein synthesis and the production of secondary metabolites, the function of ribosome assembly cofactor RimP on antibiotics production was studied in Streptomyces coelicolor and Streptomyces venezuelae. RESULTS: In this study, the rimP homologue rimP-SC was identified and cloned from Streptomyces coelicolor. Disruption of rimP-SC led to enhanced production of actinorhodin and calcium-dependent antibiotics by promoting the transcription of actII-ORF4 and cdaR. Further experiments demonstrated that MetK was one of the reasons for the increment of antibiotics production. In addition, rimP-SC disruption mutant could be used as a host to produce more peptidyl nucleoside antibiotics (polyoxin or nikkomycin) than the wild-type strain. Likewise, disruption of rimP-SV of Streptomyces venezuelae also significantly stimulated jadomycin production, suggesting that enhanced antibiotics production might be widespread in many other Streptomyces species. CONCLUSION: These results established an important relationship between ribosome assembly cofactor and secondary metabolites biosynthesis and provided an approach for yield improvement of secondary metabolites in Streptomyces.

Improvement of gougerotin and nikkomycin production by engineering their biosynthetic gene clusters.

Nikkomycins and gougerotin are peptidyl nucleoside antibiotics with broad biological activities. The nikkomycin biosynthetic gene cluster comprises one pathway-specific regulatory gene (sanG) and 21 structural genes, whereas the gene cluster for gougerotin biosynthesis includes one putative regulatory gene, one major facilitator superfamily transporter gene, and 13 structural genes. In the present study, we introduced sanG driven by six different promoters into Streptomyces ansochromogenes TH322. Nikkomycin production was increased significantly with the highest increase in engineered strain harboring hrdB promoter-driven sanG. In the meantime, we replaced the native promoter of key structural genes in the gougerotin (gou) gene cluster with the hrdB promoters. The heterologous producer Streptomyces coelicolor M1146 harboring the modified gene cluster produced gougerotin up to 10-fold more than strains carrying the unmodified cluster. Therefore, genetic manipulations of genes involved in antibiotics biosynthesis with the constitutive hrdB promoter present a robust, easy-to-use system generally useful for the improvement of antibiotics production in Streptomyces.

Novel polyoxins generated by heterologously expressing polyoxin biosynthetic gene cluster in the sanN inactivated mutant of Streptomyces ansochromogenes.

BACKGROUND: Polyoxins are potent inhibitors of chitin synthetases in fungi and insects. The gene cluster responsible for biosynthesis of polyoxins has been cloned and sequenced from Streptomyces cacaoi and tens of polyoxin analogs have been identified already. RESULTS: The polyoxin biosynthetic gene cluster from Streptomyces cacaoi was heterologously expressed in the sanN inactivated mutant of Streptomyces ansochromogenes as a nikkomycin producer. Besides hybrid antibiotics (polynik A and polyoxin N) and some known polyoxins, two novel polyoxin analogs were accumulated. One of them is polyoxin P that has 5-aminohexuronic acid with N-glycosidically bound thymine as the nucleoside moiety and dehydroxyl-carbamoylpolyoxic acid as the peptidyl moiety. The other analog is polyoxin O that contains 5-aminohexuronic acid bound thymine as the nucleoside moiety, but recruits polyoximic acid as the sole peptidyl moiety. Bioassay against phytopathogenic fungi showed that polyoxin P displayed comparatively strong inhibitory activity, whereas the inhibitory activity of polyoxin O was weak under the same testing conditions. CONCLUSION: Two novel polyoxin analogs (polyoxin P and O) were generated by the heterologous expression of polyoxin biosynthetic gene cluster in the sanN inactivated mutant of Streptomyces ansochromogenes. Polyoxin P showed potent antifungal activity,while the activity of polyoxin O was weak. The strategy presented here may be available for other antibiotics producers.

Introduction of a fluorine atom at C3 of 3-deazauridine shifts its antimetabolic activity from inhibition of CTP synthetase to inhibition of orotidylate decarboxylase, an early event in the de novo pyrimidine nucleotide biosynthesis pathway.

The antimetabolite prodrug 3-deazauridine (3DUrd) inhibits CTP synthetase upon intracellular conversion to its triphosphate, which selectively depletes the intracellular CTP pools. Introduction of a fluorine atom at C3 of 3DUrd shifts its antimetabolic action to inhibition of the orotidylate decarboxylase (ODC) activity of the UMP synthase enzyme complex that catalyzes an early event in pyrimidine nucleotide biosynthesis. This results in concomitant depletion of the intracellular UTP and CTP pools. The new prodrug (designated 3F-3DUrd) exerts its inhibitory activity because its monophosphate is not further converted intracellularly to its triphosphate derivative to a detectable extent. Combinations with hypoxanthine and adenine markedly potentiate the cytostatic activity of 3F-3DUrd. This is likely because of depletion of 5-phosphoribosyl-1-pyrophosphate (consumed in the hypoxanthine phosphoribosyl transferase/adenine phosphoribosyl transferase reaction) and subsequent slowing of the 5-phosphoribosyl-1-pyrophosphate-dependent orotate phosphoribosyl transferase reaction, which depletes orotidylate, the substrate for ODC. Further efficient anabolism by nucleotide kinases is compromised apparently because of the decrease in pK(a) brought about by the fluorine atom, which affects the ionization state of the new prodrug. The 3F-3DUrd monophosphate exhibits new inhibitory properties against a different enzyme of the pyrimidine nucleotide metabolism, namely the ODC activity of UMP synthase.

[Cloning, expression and characterization of the nucleotidylyltransferase gene-amiE in amicetin biosynthesis].

OBJECTIVE: The aim of this study is to clone and express the nucleotidylytransferase encoding gene-amiE from the biosynthetic gene cluster of amicetin, a disaccharide nucleoside antibiotic, and to characterize AmiE in vitro. METHODS: The amiE, encoding a nucleotidylytransferase of 257 amino acid, was PCR amplified and cloned into pET28a, resulting in the plasmid pCSG4001, which was transformed into E. coli BL21(DE3) for expressing N-(His)6-tag AmiE. The recombinant AmiE was purified by affinity chromatography via AKTA Purifier 10 system. The AmiE-catalyzed reactions were performed using TTP (or UTP) and glucose-1-phosphate as substrates. The enzyme assays were analyzed by HPLC; the substrate flexibility of AmiE was probed with three unnatural sugars-1-phosphate, including galactose-1-phosphate, galactosamine-1-phosphate and mannos-1-phosphate. RESULTS: The N-(His)6-tag AmiE was expressed in E. coli in soluble form and was successfully purified via Ni2+ mediated affinity chromatography; in vitro biochemical experiments showed that AmiE could convert glucose-1-phosphate into TDP-glucose (or UDP-glucose) in the presence of TTP (or UTP). However, galactose-1-phosphate, galactosamine-1-phosphate and mannos-1-phosphate were not substrates of AmiE. CONCLUSION: The amiE was successfully cloned and expressed in E. coli, and the purified AmiE was biochemically confirmed to be a nucleotylyltransferase in amicetin biosynthesis pathway.

Engineering of an industrial polyoxin producer for the rational production of hybrid peptidyl nucleoside antibiotics.

Polyoxins and nikkomycins are potent antifungal peptidyl nucleoside antibiotics, which inhibit fungal cell wall biosynthesis. They consist of a nucleoside core and one or two independent peptidyl moieties attached to the core at different sites. Making mutations and introducing heterologous genes into an industrial Streptomyces aureochromogenes polyoxin producer, resulted in the production of four polyoxin-nikkomycin hybrid antibiotics designated as polyoxin N and nikkoxin B-D, whose structures were confirmed using high resolution MS and NMR. Two of the hybrid antibiotics, polyoxin N and nikkoxin D, were significantly more potent against some human or plant fungal pathogens than their parents. The data provides an example for rational generation of novel peptidyl nucleoside antibiotics in an industrial producer.

Characterization of the amicetin biosynthesis gene cluster from Streptomyces vinaceusdrappus NRRL 2363 implicates two alternative strategies for amide bond formation.

Amicetin, an antibacterial and antiviral agent, belongs to a group of disaccharide nucleoside antibiotics featuring an α-(1→4)-glycoside bond in the disaccharide moiety. In this study, the amicetin biosynthesis gene cluster was cloned from Streptomyces vinaceusdrappus NRRL 2363 and localized on a 37-kb contiguous DNA region. Heterologous expression of the amicetin biosynthesis gene cluster in Streptomyces lividans TK64 resulted in the production of amicetin and its analogues, thereby confirming the identity of the ami gene cluster. In silico sequence analysis revealed that 21 genes were putatively involved in amicetin biosynthesis, including 3 for regulation and transportation, 10 for disaccharide biosynthesis, and 8 for the formation of the amicetin skeleton by the linkage of cytosine, p-aminobenzoic acid (PABA), and the terminal (+)-α-methylserine moieties. The inactivation of the benzoate coenzyme A (benzoate-CoA) ligase gene amiL and the N-acetyltransferase gene amiF led to two mutants that accumulated the same two compounds, cytosamine and 4-acetamido-3-hydroxybenzoic acid. These data indicated that AmiF functioned as an amide synthethase to link cytosine and PABA. The inactivation of amiR, encoding an acyl-CoA-acyl carrier protein transacylase, resulted in the production of plicacetin and norplicacetin, indicating AmiR to be responsible for attachment of the terminal methylserine moiety to form another amide bond. These findings implicated two alternative strategies for amide bond formation in amicetin biosynthesis.

Revealing the first uridyl peptide antibiotic biosynthetic gene cluster and probing pacidamycin biosynthesis.

There is an urgent need for new antibiotics with resistance continuing to emerge toward existing classes. The pacidamycin antibiotics possess a novel scaffold and exhibit unexploited bioactivity rendering them attractive research targets. We recently reported the first identification of a biosynthetic cluster encoding uridyl peptide antibiotic assembly and the engineering of pacidamycin biosynthesis into a heterologous host. We report here our methods toward identifying the biosynthetic cluster. Our initial experiments employed conventional methods of probing a cosmid library using PCR and Southern blotting, however it became necessary to adopt a state-of-the-art genome scanning  and in silico hybridization approach  to pin point the cluster. Here we describe our "real" and "virtual" probing methods and contrast the benefits and pitfalls of each approach.