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Discovery and Characterization of the Tubercidin Biosynthetic Pathway

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Discovery and Characterization of the Tubercidin Biosynthetic Pathway Liu et al. Microb Cell Fact (2018) 17:131 https://doi.org/10.1186/s12934-018-0978-8 Microbial Cell Factories RESEARCH Open Access Discovery and characterization of the tubercidin biosynthetic pathway from Streptomyces tubercidicus NBRC 13090 Yan Liu1, Rong Gong2, Xiaoqin Liu2, Peichao Zhang2, Qi Zhang1, You‑Sheng Cai2, Zixin Deng2, Margit Winkler3, Jianguo Wu1* and Wenqing Chen2* Abstract Background: Tubercidin (TBN), an adenosine analog with potent antimycobacteria and antitumor bioactivities, high‑ lights 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. Results: 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 specifcally utilizes phosphoribosylpyrophosphate and 7-carboxy-7-deazaguanine for the precise construction of the deazapurine nucleoside scafold. Moreover, we provided biochemical evidence that TubD functions as an NADPH- dependent reductase, catalyzing irreversible reductive deamination. Finally, we verifed that TubG acts as a Nudix 2 hydrolase, preferring ­Co + for the maintenance of maximal activity, and is responsible for the tailoring hydrolysis step leading to TBN. Conclusions: These fndings 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. Keywords: Biosynthesis, Tubercidin, 7-deazapurine, Phosphoribosylpyrophosphate, NADPH-dependent reductase, Nudix hydrolase, Synthetic biology Background activities [1, 2]. Previously, toyocamycin/sangivamycin Pyrrolopyrimidine (also known as 7-deazapurine) con- (produced by Streptomyces rimosus ATCC 14673) and taining compounds have been discovered to be widely tubercidin (TBN, produced by Streptomyces tubercidicus distributed in nature as microbial secondary metabo- NBRC 13090) served as prototypes for the deazapurines lites (SMs) and also as hypermodifed base (queuosine) (Fig. 1a) [2–4]. Since their discovery, a series of structur- in RNA [1, 2]. Usually, this family of SMs shows diverse ally-related antibiotics has been continuously isolated biological functions, ranging from cofactors (involved from terrestrial and marine sources with guidance of bio- in the biosynthesis of tetracycline antibiotics) as well as activity assays (Fig. 1a) [2]. DNA repair to antibiotics with herbicidal, antibacte- Tubercidin is susceptible to phosphorylation by adeno- rial, antiviral, antifungal, antitumor, and antineoplastic sine 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 *Correspondence: [email protected]; [email protected] 1 State Key Laboratory of Virology, and College of Life Sciences, Wuhan inhibitor of RNA and DNA polymerase, and shows a University, Wuhan 430072, China relatively-broad spectrum of bioactivities [2]. TBN is bio- 2 Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, active against Candida albicans, Mycobacterium tubercu- Ministry of Education, and School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China losis, and Streptococcus faecalis, however, it exhibits no Full list of author information is available at the end of the article inhibition of other Gram positive bacteria and fungi [2, © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/ publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Liu et al. Microb Cell Fact (2018) 17:131 Page 2 of 10 Fig. 1 Structures of TBN and related antibiotics and the confrmed biosynthetic pathway to PreQ 0. a Chemical structures of TBN and related purine nucleoside antibiotics. TBN is produced by S. tubercidicus NBRC 13090; 5′‑sulfamoyl TBN is produced by S. mirabilis; cadeguomycin is produced by S. hygroscopicus; sangivamycin/toyocamycin is produced by S. rimosus ATCC 14673; kanagawamicin is produced by Actinoplanes kanagawaensis; echiguanine A is produced by Streptomyces sp. MI698‑50F1; Ara‑A (arabinofuranosyladenine) is produced by S. antibioticus NRRL 3238. b The confrmed biosynthetic pathway leading to PreQ0. A four‑enzyme cascade for the synthesis of PreQ0 is identifed in both queuosine and sangivamycin/toyocamycin biosynthetic pathways. GCH I, GTP cyclohydrolase I (ToyD); QueD/ToyB, 6‑carboxy‑5,6,7,8‑tetrahydropterin (CPH4) synthase; H2NTP, 7, 8‑dihydroneopterin‑3′‑triphosphate; QueE/ToyC, 7‑carboxy‑7‑deazaguanine (CDG) synthase; QueC/ToyM, 7‑cyano‑7‑deazaguanine (PreQ0) synthetase 5]. In addition, TBN shows antiviral and antitumor activ- recognized by CPH4 (6-carboxy-5, 6, 7, 8-tetrahydrop- ities [5], and more interestingly, it can also kill trypano- terin) synthase (QueD/ToyB) and CDG synthase (QueE/ somes by targeting glycolysis, especially by inhibition of ToyC) [2, 10]. Te enzymatic mechanism of QueE/ToyC, phosphoglycerate kinase [7]. a member of the radical S-adenosyl-l-methionine (SAM) TBN features an unusual deazapurine core, in which protein, has been elucidated by elegant studies of the N-7 is substituted for C-7 (Fig. 1a) [4]. Previous metabolic Bandarian group. Tis enzyme catalyzes a key SAM- and labeling experiments indicated that the deazapurine core Mg2+-dependent radical-mediated ring contraction step is derived from a purine precursor, likely GTP, and C-1′, [11, 12], which is common to the biosynthetic pathways 2′, and 3′ of ribose are utilized to construct the pyrrole of all deazapurine-containing compounds. More recently, ring with elimination of C-8 in GTP [8, 9]. Subsequently, QueC/ToyM has been verifed as an amide synthetase, as the enzymatic logic for the construction of deazapurine well as a nitrile synthetase, which could accept both the core has been deciphered by independent studies, which acid and amide forms of CDG enabling sequential amida- were focused on the identifcation of the biosynthetic tion and dehydration to the nitrile [13, 14]. pathway of queuosine and toyocamycin/sangivamycin Although the distinguished activities and unusual (Fig. 1b) [2, 10]. A four-enzyme cascade has been demon- structure of TBN are well known, nature’s strategy for strated to be responsible for the biosynthetic steps lead- the building of this molecule has as yet remained poorly ing to PreQ0 (7-cyano-7-deazaguanine) (Fig. 1b). Among understood. In the present study, we have identifed them, GTP cyclohydrolase I (GCH I, ToyD) catalyzes the the TBN biosynthetic gene cluster from S. tubercidicus ′ frst step from GTP to H2NTP (7, 8-dihydroneopterin-3 - NBRC 13090 (S. tubercidicus hereafter) by engineered triphosphate) with opening the ribose ring, followed by production of TBN in a heterologous host, and have fur- a series of Amadori rearrangement and subsequent recy- ther elucidated that TBN biosynthesis involves a PRPP- clization. Te intermediate H2NTP is then sequentially dependent assembly logic associated with tailoring Liu et al. Microb Cell Fact (2018) 17:131 Page 3 of 10 reduction and phosphohydrolysis steps. Our deciphering also existed in toyocamycin, sangivamycin, and other of the TBN biosynthetic pathway provides a solid basis related antibiotics; we therefore deduced that the ini- for the further combinatorial biosynthesis of this group tial enzymatic steps for the building of the core of them of nucleoside antibiotics towards improved features, and follow an identical logic. We accordingly utilized ToyD opens the way for the target-directed genome mining of (GTP cyclohydrolase I) and ToyB (CPH 4 synthase) as novel TBN-related antibiotics from the available micro- the enzyme probes to conduct BlastP analysis against bial genome reservoirs. the genome of S. tubercidicus. As expected, a candidate gene cluster (tub) encoding enzymes involving TubC Results (64% identity to ToyD) and TubA (65% identity to ToyB) Identifcation of the TBN biosynthetic gene cluster was identifed from the genome (Fig. 2a, Table 1). Fur- To identify the gene cluster for TBN biosynthesis, ther looking through the surrounding region resulted in the genome of S. tubercidicus was sequenced by an the identifcation of the genes coding for a radical SAM Illumina Hiseq method, rendering appr. 7.88-Mb of enzyme (TubB) and a GMP reductase (TubD) (Fig. 2a, non-redundant bases after assembly of clean reads Table 1). Tese results suggest that the tub gene cluster (Additional fle 1: Table S1). Te genomic data was is very likely responsible for the biosynthesis of TBN. then annotated by Glimmer 3.02 software, afording 7263 open reading frames (ORFs) (Additional fle 1: Table S1). TBN features the deazapurine core, which is Fig. 2 Genetic organization and validation of the gene cluster (tub) for TBN biosynthesis. a Genetic organization of the tub gene cluster. b LC–MS analysis of the target metabolite produced by the recombinants of S. coelicolor M1154. TBN Std,
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