- Open Access
Discovery and characterization of the tubercidin biosynthetic pathway from Streptomyces tubercidicus NBRC 13090
© The Author(s) 2018
- Received: 2 July 2018
- Accepted: 14 August 2018
- Published: 28 August 2018
Tubercidin (TBN), an adenosine analog with potent antimycobacteria and antitumor bioactivities, highlights an intriguing structure, in which a 7-deazapurine core is linked to the ribose moiety by an N-glycosidic bond. However, the molecular logic underlying the biosynthesis of this antibiotic has remained poorly understood.
Here, we report the discovery and characterization of the TBN biosynthetic pathway from Streptomyces tubercidicus NBRC 13090 via reconstitution of its production in a heterologous host. We demonstrated that TubE specifically utilizes phosphoribosylpyrophosphate and 7-carboxy-7-deazaguanine for the precise construction of the deazapurine nucleoside scaffold. Moreover, we provided biochemical evidence that TubD functions as an NADPH-dependent reductase, catalyzing irreversible reductive deamination. Finally, we verified that TubG acts as a Nudix hydrolase, preferring Co2+ for the maintenance of maximal activity, and is responsible for the tailoring hydrolysis step leading to TBN.
These findings lay a foundation for the rational generation of TBN analogs through synthetic biology strategy, and also open the way for the target-directed search of TBN-related antibiotics.
- NADPH-dependent reductase
- Nudix hydrolase
- Synthetic biology
Tubercidin is susceptible to phosphorylation by adenosine kinase to mono-, di-, and tri-phosphorylated forms accounting for its structural resemblance to adenosine [5, 6]. Accordingly, it is capable of acting as a powerful inhibitor of RNA and DNA polymerase, and shows a relatively-broad spectrum of bioactivities . TBN is bioactive against Candida albicans, Mycobacterium tuberculosis, and Streptococcus faecalis, however, it exhibits no inhibition of other Gram positive bacteria and fungi [2, 5]. In addition, TBN shows antiviral and antitumor activities , and more interestingly, it can also kill trypanosomes by targeting glycolysis, especially by inhibition of phosphoglycerate kinase .
TBN features an unusual deazapurine core, in which N-7 is substituted for C-7 (Fig. 1a) . Previous metabolic labeling experiments indicated that the deazapurine core is derived from a purine precursor, likely GTP, and C-1′, 2′, and 3′ of ribose are utilized to construct the pyrrole ring with elimination of C-8 in GTP [8, 9]. Subsequently, the enzymatic logic for the construction of deazapurine core has been deciphered by independent studies, which were focused on the identification of the biosynthetic pathway of queuosine and toyocamycin/sangivamycin (Fig. 1b) [2, 10]. A four-enzyme cascade has been demonstrated to be responsible for the biosynthetic steps leading to PreQ0 (7-cyano-7-deazaguanine) (Fig. 1b). Among them, GTP cyclohydrolase I (GCH I, ToyD) catalyzes the first step from GTP to H2NTP (7, 8-dihydroneopterin-3′-triphosphate) with opening the ribose ring, followed by a series of Amadori rearrangement and subsequent recyclization. The intermediate H2NTP is then sequentially recognized by CPH4 (6-carboxy-5, 6, 7, 8-tetrahydropterin) synthase (QueD/ToyB) and CDG synthase (QueE/ToyC) [2, 10]. The enzymatic mechanism of QueE/ToyC, a member of the radical S-adenosyl-l-methionine (SAM) protein, has been elucidated by elegant studies of the Bandarian group. This enzyme catalyzes a key SAM- and Mg2+-dependent radical-mediated ring contraction step [11, 12], which is common to the biosynthetic pathways of all deazapurine-containing compounds. More recently, QueC/ToyM has been verified as an amide synthetase, as well as a nitrile synthetase, which could accept both the acid and amide forms of CDG enabling sequential amidation and dehydration to the nitrile [13, 14].
Although the distinguished activities and unusual structure of TBN are well known, nature’s strategy for the building of this molecule has as yet remained poorly understood. In the present study, we have identified the TBN biosynthetic gene cluster from S. tubercidicus NBRC 13090 (S. tubercidicus hereafter) by engineered production of TBN in a heterologous host, and have further elucidated that TBN biosynthesis involves a PRPP-dependent assembly logic associated with tailoring reduction and phosphohydrolysis steps. Our deciphering of the TBN biosynthetic pathway provides a solid basis for the further combinatorial biosynthesis of this group of nucleoside antibiotics towards improved features, and opens the way for the target-directed genome mining of novel TBN-related antibiotics from the available microbial genome reservoirs.
Identification of the TBN biosynthetic gene cluster
Deduced functions of the open reading frames in the tub gene cluster
Identity, similarity (%)
AWI43_15300, Streptomyces sp. WAC04657
ToyB, Streptomyces rimosus ATCC 14673
Radical SAM family protein
SPAR_33961, Streptomyces sparsogenes DSM 40356
GTP cyclohydrolase I (GCH I)
ToyD, Streptomyces rimosus ATCC 14673
ToyE, Streptomyces rimosus ATCC 14673
ToyH, Streptomyces rimosus ATCC 14673
UbiD family decarboxylase
ASE41_09730, Streptomyces sp. Root264
ASE41_09725, Streptomyces sp. Root264
Carbohydrate kinase family protein
ASE41_09720, Streptomyces sp. Root264
ASE41_09715, Streptomyces sp. Root264
ASE41_09630, Streptomyces sp. Root264
Engineered production of TBN in the heterologous host S. coelicolor M1154
To correlate the tub gene cluster to TBN production, a cosmid 12G4 (with appr. 30.0-kb insertion DNA, Additional file 1: Table S2) harboring the TBN gene cluster was screened from the pJTU2463b-derived genomic library of S. tubercidicus (Additional file 1: Table S2), and then it was introduced into the heterologous host S. coelicolor M1154  via conjugation. The validated conjugants were then fermented for further metabolite analysis, and LC–MS analysis indicated that the sample (M1154/12G4) is capable of producing the distinctive [M+H]+ ion of TBN at m/z 267.1080 (Fig. 2a). In addition, MS/MS analysis showed that the main fragments were generated at 134.8841, 248.9537, 231.1378, and so forth, fully consistent with those of the TBN authentic standard (Additional file 1: Figure S1). These combined data unambiguously demonstrate that the cosmid 12G4 confers the heterologous host S. coelicolor M1154 with the capability of TBN production.
In silico analysis of the TBN biosynthetic gene cluster
In silico analysis showed that the TBN gene cluster spans appr. 8.0-kb continuous chromosomal region, and consists of 9 genes (tubA-I) (Fig. 2a, Table 1). Genes tubCBA are proposed to be responsible for the initial biosynthetic steps to CDG. The gene tubC codes for GTP cyclohydrolase I which shows 64% identity to ToyD of toyocamycin biosynthesis, and the product of tubB (radical SAM enzyme) exhibits 71% identity to SPAR_33961 of S. sparsogenes DSM 40356. tubA encodes CPH4 synthase that indicates significant similarity (65% identity) to ToyB in toyocamycin biosynthetic pathway. tubD encodes a GMP reductase (61% identity to ToyE) that is proposed to be responsible for IMP (Inosine-5′-monophosphate) biosynthesis, whereas TubE has 39% identity to ToyH, an enzyme hypothetically responsible for the assembly step during toyocamycin biosynthesis. TubF exhibits high similarity (90% identity to ASE41_09730 from Streptomyces sp. Root264) to UbiD family decaboxylases, which bear a recently identified cofactor prFMN (prenylated-FMN) [16–19], but the roles of most UbiD family decaboxylases are functionally unassigned. TubG is annotated as a Nudix hydrolase, and this family of enzymes usually plays “house-cleaning” role to sanitize nucleotide pool in primary metabolism . tubHI individually code for carbohydrate kinase family protein (82% identity to ASE41_09720) and MFS (Major facilitator superfamily) transporter.
TubE functions as a CDG-PRPP phosphoribosyltransferase
Biochemical characterization of TubD as an NADPH-dependent reductase
Biochemical characterization of TubG as a tailoring Nudix hydrolase
Earlier bioinformatic analysis speculated that the 7-deazapurine core (7-cyano-7-deazaguanine/PreQ0) is biosynthesized preceding its assembly [2, 10]. In the present study, it seems to be logic and feasible in the TBN pathway. Two enzymes, TubE (PRPP transferase), and TubF (UbiD family decarboxylase), are demonstrated to be involved in the described enzymatic steps (Fig. 6). We initially deduced that CDG is likely to undergo decarboxylation prior to its condensation with PRPP for the accomplishment of assembly step, while TubE is not able to accept 7-deazaguanosine as the substrate. We therefore propose that CDG is first converted by TubE to form compound 1, which is then decarboxylated to compound 2 by TubF, an unusual decarboxylase likely using prenylated-FMN (prFMN) as cofactor (Fig. 6).
For the tailoring steps during TBN biosynthesis, TubD (NADPH-dependent reductase) and TubG (Nudix hydrolase), which were characterized in the present study using natural substrate analogs, are established to participate in the enzymatic reactions, compound 2 (GMP analog) is converted to 3 (IMP analog). We could imagine that these two enzymes definitely show the preference to the natural substrates, however, it is also commonly acceptable to explore the catalytic mechanisms of certain enzymes with substrate analogs . Indeed, enzymes are often rationally evolved to accept artificial substrate analogs for industrial biocatalysis purposes . It is very interesting to ask why the enzymes for the sequential transformation of 3 to 4, and to 5 are missing in the TBN pathway. Compound 3 is structurally similar to IMP; as a result, it is reasonable to propose that the missing two enzymes, adenylosuccinate synthetase (PurA), and adenylosuccinate lyase (PurB), could be certainly “borrowed” from the primary purine metabolic pathway (Fig. 6). As for compound 5, it is confirmed to be hydrolyzed by TubG with removal of a phosphate for the accomplishment of TBN biosynthesis (Fig. 6).
There are two other enzymes, TubH (kinase), and TubI (MFS transporter), also present in the TBN pathway. It is more reasonable to propose that TubI is responsible for transporting TBN out of the cell once synthesized. With respect to TubH, the assignment of its functional role in TBN biosynthesis is highly challenging. Previous studies showed that TBN is toxic to M. tuberculosis, and accordingly, it is acceptable to hypothesize that this compound is also potentially toxic to the host cell. Microbes have developed several intricate strategies for the self-resistance of its secondary metabolites during the long-term evolution, undoubtedly, targeted modification (e.g. phosphorylation) of the target antibiotic should be a more effective and convenient tactic, and a similar case for antibiotic self-resistance by host cell has been previously reported in capuramycin biosynthesis . From this perspective, we tentatively propose that TubH is likely to play a self-resistance role by phosphorylating TBN to relieve its potential toxicity to host cells, and related research is now underway.
Notably, the advent of rapid and affordable next-generation DNA sequencing has revolutionized and accelerated the traditional programs for the discovery of chemical diversities . Therefore, TBN could be used as a promising template for the target-directed genome mining of related antibiotics, and we have already been rewarded with uncovering several potential TBN-related antibiotics pathways from the currently-available reservoir of the microbial genomes (Additional file 1: Figure S10). As a consequence, it would be of great interest in the future to hunt for novel 7-deazapurine-containing antibiotics by targeted genome mining approach.
In summary, we report the discovery and functional analysis of the TBN biosynthetic pathway. We have determined that TBN biosynthesis employs a PRPP dependent strategy for the deazanucleoside scaffold assembly, and we have also provided the biochemical evidence that TubD and TubG are involved in the tailoring reduction and phosphohydrolysis steps. We anticipate that the deciphering of the biosynthetic puzzle of TBN will open the way for future combinatorial biosynthesis of this family of antibiotics for the rational generation of novel analogs with enhanced features.
Materials and general methods
Strains, plasmids used in this study are described in Additional file 1: Table S2, and PCR primers are listed in Additional file 1: Table S3. All of the enzymes (except for the DNA polymerase) were the products of New England Biolabs. The TBN standard was purchased from Medchem Express (MCE). Chemicals were purchased from Sigma-Aldrich, Thermo Scientific, J&K Scientific, or Sinopharm unless otherwise indicated. Standard protocols were employed to manipulate E. coli or Streptomyces according to those of Green et al.  or Kieser et al. .
Sequencing analysis of the genome of S. tubercidicus
Genomic DNA of S. tubercidicus was isolated on the basis of the standard protocol , and the genome sequencing was performed using the Illumina Hiseq4000 sequencing system, and the assembled sequence data was then annotated using the Glimmer 3.02 software. The online programs FramePlot 4.0beta (http://nocardia.nih.go.jp/fp4/) and 2ndFind (http://biosyn.nih.go.jp/2ndfind/) were utilized for the accurate analysis of the TBN gene cluster.
The DNA sequence of the tub gene cluster is deposited in the GenBank database under accession number MG706975.
Genomic library construction and screening for S. tubercidicus
For the construction of pJTU2463b-derived genomic library for S. tubercidicus, standard method was adopted, using the EPI300-T1R as suitable host cells, and a narrow-down PCR screening strategy  with primers TubidF/R were employed to screen the positive cosmid 12G4 from the genomic library.
Fermentation of related Streptomyces strains for TBN production
For production of TBN, Streptomyces strains were inoculated in TSB medium and cultivated for 2 days, after that, the cultures (2%, V/V) were transferred to fermentation medium (including 20 g glucose, 30 g soluble starch, 10 g corn steep liquor, 10 g soybean meal, 5 g peptone, 2 g NaCl, 5 g CaCO3, pH 7.0) and fermented (180 r/min, 28 °C) for 5 days. Subsequently, the fermentation beer was processed (adding oxalic acid till pH 3.0) for LC–MS analysis.
Overexpression and purification of TubE, TubD, and TubG in E. coli Rosetta (DE3)/pLysS
For overexpression of these proteins, their structural genes were amplified by KOD-plus DNA polymerase (TOYOBO) using related primers listed in Additional file 1: Table S3. Then the NdeI–EcoRI engineered DNA fragments were individually cloned into the counterpart sites of pET28a. After confirmation by sequencing, the related constructs were subsequently transformed into E. coli Rosetta (DE3)/pLysS cells according to the standard protocols . Expression and purification for the His6-tagged proteins were conducted according to the method by Wu et al. .
Enzymatic assays of TubE
For TubE activity, the reaction mixture (100 mM PBS buffer, pH 7.5; 1 mM CDG; 2 mM PRPP; 5 mM MgCl2; 1 mM DTT and 20 μg protein) was incubated at 30 °C for 8 h, and then terminated by the immediate addition of an equivalent volume methanol. After centrifugation to remove protein, the supernatant was filtrated by 0.22 μm filter. HPLC (Shimadzu LC-20A) analysis was performed on a reverse phase C18 column (Inertsil ODS-3, 4.6 × 250 mm, 5 µm) with the elution gradient of 5%–30% methanol:0.15% TFA over 35 min at a flow rate of 0.5 ml/min, and the elution was monitored at UV295 nm by a DAD detector.
Enzymatic assays of TubD
For TubD activity, the reaction containing 100 mM Tris–HCl buffer (pH 7.5), 1 mM GMP, 1 mM NADPH/NADH, 1 mM DTT, 50 mM KCl, 2 mM EDTA, and 20 μg protein, incubated at 30 °C for 4 h, then the supernatant was analyzed by LC–HRMS with the elution gradient of 5%–25% methanol:0.15% TFA over 20 min at a flow rate of 0.4 ml/min.
Enzymatic assays of TubG
The complete TubG reaction including 100 mM Tris–HCl buffer (pH 7.0), 1 mM AMP, 100 mM KCl, 5 mM divalent ion (Mg2+, Ni2+, Mn2+, Co2+, Ca2+, Fe2+, Cu2+, and Zn2+), and 20 μg protein, was incubated at 30 °C for 4 h. The protein in reaction was removed by adding an equal volume of methanol, then the supernatant was analyzed by HPLC (Shimadzu LC-20A) equipped with C-18 reversed-phase column (Inertsil ODS-3, 4.6 × 250 mm, 5 µm) with 5%–30% methanol:0.15% TFA over 15 min at a flow rate of 0.4 ml/min, the condition (30% methanol:0.15% TFA) was maintained for another 10 min.
The conditions for LC–MS analysis
LC–MS analysis was carried out on a Thermo Fisher Scientific ESI-LTQ Orbitrap (Scientific Inc.) equipped with a C-18 reversed-phase column (Inertsil ODS-3, 4.6 × 250 mm, 5 µm) in positive mode with an elution gradient of 5%–30% Methanol:0.15% TFA over 30 min at 0.5 ml/min, and the parameters for the LC–MS analysis are as follows: Dry gas at 275 °C, 10 l/ml, and nebulizer pressure of 30 psi.
YL, RG, XL, and PZ carried out experiments, QZ and YSC analyzed the primary data. YL, JW, and WC wrote the manuscript. WC and JW conceived the project and supervised the research. MW and ZD help with the critical reading of the manuscript. All authors read and approved the final manuscript.
We are very grateful to Prof. Guo-Qiang Chen (Tsinghua University) and Prof. Lili Zhang (Tarim University) for kindly providing us with the strains containing the potential TBN biosynthetic gene cluster.
The authors declare that they have no competing interests.
Availability of data and materials
The dataset(s) supporting the conclusions of this article is (are) included within the article [and its additional file(s)].
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This work was supported by grants of the National Natural Science Foundation of China (31770041).
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