Coordinated Biosynthesis of the Purine Nucleoside AntibioticsAristeromycin and Coformycin in Actinomycetes

Gudan Xu,a Liyuan Kong,a,b Rong Gong,a Liudong Xu,c Yaojie Gao,c Ming Jiang,c You-Sheng Cai,a Kui Hong,a Youcai Hu,b

Peng Liu,a Zixin Deng,a,c Neil P. J. Price,d Wenqing Chena,b,c

aKey Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and School ofPharmaceutical Sciences, Wuhan University, Wuhan, China

bState Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica,Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China

cState Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao TongUniversity, Shanghai, China

dAgricultural Research Service, U.S. Department of Agriculture, National Center for Agricultural UtilizationResearch, Peoria, Illinois, USA

ABSTRACT Purine nucleoside antibiotic pairs, concomitantly produced by a singlestrain, are an important group of microbial natural products. Here, we report atarget-directed genome mining approach to elucidate the biosynthesis of the purinenucleoside antibiotic pair aristeromycin (ARM) and coformycin (COF) in Micromono-spora haikouensis DSM 45626 (a new producer for ARM and COF) and Streptomycescitricolor NBRC 13005 (a new COF producer). We also provide biochemical data thatMacI and MacT function as unusual phosphorylases, catalyzing an irreversible reac-tion for the tailoring assembly of neplanocin A (NEP-A) and ARM. Moreover, wedemonstrate that MacQ is shown to be an adenosine-specific deaminase, likely re-lieving the potential “excess adenosine” for producing cells. Finally, we report thatMacR, an annotated IMP dehydrogenase, is actually an NADPH-dependent GMP re-ductase, which potentially plays a salvage role for the efficient supply of the precur-sor pool. Hence, these findings illustrate a fine-tuned pathway for the biosynthesisof ARM and also open the way for the rational search for purine antibiotic pairs.

IMPORTANCE ARM and COF are well known for their prominent biological activitiesand unusual chemical structures; however, the logic of their biosynthesis has longbeen poorly understood. Actually, the new insights into the ARM and COF pathwaywill not only enrich the biochemical repertoire for interesting enzymatic reactionsbut may also lay a solid foundation for the combinatorial biosynthesis of this groupof antibiotics via a target-directed genome mining strategy.

KEYWORDS coordinated biosynthesis, aristeromycin, coformycin, actinomycetes,fine-tuned pathway

Nucleoside antibiotics constitute a large family of important microbial natural productsbearing diverse biological activities, such as antibacterial, antifungal, antiviral, and

antitumor activities (1–4). Structurally, this family of antibiotics highlights distinctive moi-eties, derived from simple building blocks, either nucleosides or nucleotides of primaryorigin (1). The biosynthesis of nucleoside antibiotics often follows a succinct logic ofsequential modifications of nucleosides or nucleotides and leads to complex structures (2,3, 5). Because of their unique chemical diversity, nucleoside antibiotics yield leads/analogswith novel structural features and diverse bioactivities (6, 7).

Purine-based nucleoside antibiotics, which include the pentostatin (PTN)-relatedcompounds 2=-Cl PTN, coformycin (COF), adecypenol, and carbocyclic COF (Fig. 1A andB) (8, 9), share an unusual heterocyclic 1,3-diazepine core in which an additional carbon

Received 29 July 2018 Accepted 4September 2018

Accepted manuscript posted online 14September 2018

Citation Xu G, Kong L, Gong R, Xu L, Gao Y, JiangM, Cai Y-S, Hong K, Hu Y, Liu P, Deng Z, Price NPJ,Chen W. 2018. Coordinated biosynthesis of thepurine nucleoside antibiotics aristeromycin andcoformycin in actinomycetes. Appl EnvironMicrobiol 84:e01860-18. https://doi.org/10.1128/AEM.01860-18.

Editor Marie A. Elliot, McMaster University

Copyright © 2018 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Wenqing Chen,wqchen@whu.edu.cn.

GENETICS AND MOLECULAR BIOLOGY

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is inserted between C-6 and N-1 of the purine ring. Metabolic labeling experimentsindicated that the “extra carbon” was derived from C-1 of D-ribose (10). Previous studieshave revealed that the biosynthesis of PTN and Ara-A (arabinofuranosyladenine) isgoverned by a single gene cluster but arises from independent pathways and employsan unusual protector-protégé strategy; i.e., PTN may protect Ara-A from deamination bythe housekeeping adenosine deaminase (8). More recently, a similar strategy has beenshown for 2=-Cl PTN and 2=-amino-2=-deoxyadenosine (produced by Actinomadura sp.strain ATCC 39365) (Fig. 1A) (11), as well as by the fungus-derived PTN and cordycepinpair (produced by Cordyceps militaris and other fungal strains) (12). In addition, we havepreviously shown that a short-chain dehydrogenase and a SAICAR (phosphoribosyl-aminoimidazolesuccinocarboxamide) synthetase are highly conserved for the biosyn-thesis of the heterocyclic 1,3-diazepine ring of the PTN-related compounds (8).

Neplanocin A (NEP-A), aristeromycin (ARM), adecypenol, and carbocyclic COF (Fig.1A and B), in the carbocyclic purine nucleoside group of antibiotics (9, 13), aredistinguished by a unique five-membered cyclitol moiety that is linked to adenine viaan N-glycosidic bond. ARM displays several interesting biological properties, includingthe inhibition of AMP synthesis in mammalian cells and the blocking of cell division andelongation in rice plants (14). NEP-A has notable antiviral and antitumor activities (15),but both NEP-A and ARM have a very limited spectrum of antibacterial activities (16,17). Mechanistically, these two antibiotics act as competitive inhibitors of S-adenosyl-L-homocysteine (SAH) hydrolase (14, 15). Metabolic feeding experiments by the Parrygroup showed that the origin of the cyclitol moiety is from D-glucose, from which theC-2 and C-6 carbons are utilized to construct the carbocyclic ring via a carbon-carbon

FIG 1 Chemical structures of relevant antibiotics. (A) Structure of the related (potential) antibiotic pairs. Except for ARM and carbocyclicCOF (blue dashed box), the antibiotic at the top together with the one shown below constitutes an antibiotic pair which is concomitantlyproduced by a single microorganism. The formycin A and COF pair is individually produced by Streptomyces kaniharenesis ATCC 21070and Nocardia interforma ATCC 21072. The Ara-A and PTN pair is produced by Streptomyces antibioticus NRRL 3238. The 2=-amino dA(2=-amino-2=-deoxyadenosine) and 2=-Cl PTN pair is produced by Actinomadura sp. ATCC 39365. Multiple nucleoside antibiotics, includingARM, NEP-A, NEP-D, Carbo-I, and COF, are produced by M. haikouensis and S. citricolor. Green dashed boxes highlight the antibiotics thatare produced by a single strain, either M. haikouensis or S. citricolor. (B) Chemical structures of NEP-A, Carbo-I, NEP-D, and adecypenol.

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bond and the adenine ring is directly from adenosine (14, 15). Subsequently, Hill et al.identified two key intermediates of the NEP-A and ARM biosynthetic pathway andtentatively proposed a concise pathway (18). Although the ARM gene cluster fromStreptomyces citricolor NBRC 13005 (S. citricolor herein) has been identified and aplausible biosynthetic pathway has been proposed (19), several aspects remained to bedetermined.

In the present study, we report that Micromonospora haikouensis DSM 45626 (M.haikouensis herein) (20) and S. citricolor are new producing strains of multiplenucleoside antibiotics and further show that the ARM and COF gene clusters featuregenetic organization diversity. We also demonstrate that NEP-A and ARM biosyn-thesis includes an irreversible catalytic step for the final assembly and is associatedwith a fine-tuned pathway. This will lay a solid foundation for the combinatorialbiosynthesis of this group of antibiotics and for the rational discovery of novelpurine nucleoside antibiotic pairs.

RESULTSDiscovery of M. haikouensis and S. citricolor as producers of multiple nucleo-

side antibiotics. The enzymes for the biosynthesis of the PTN-related purine nucleo-side antibiotics (Fig. 1A), including a short-chain dehydrogenase and a SAICAR synthe-tase, are highly conserved and widely distributed (8), and they are therefore potentialprobes for the discovery of new purine nucleoside antibiotic pathways from availablemicrobial genomes. We then took advantage of the target enzymes, PenB (short-chaindehydrogenase) and PenC (SAICAR synthetase) from the PTN pathway (8), to conducta BLASTP search against the NCBI database, leading to the discovery of multiplepotential gene clusters for the biosynthesis of PTN-related antibiotic pairs. Theseinclude the mac gene cluster from M. haikouensis (Fig. 2A; Table 1), in which two genescorrespondingly code for the candidate proteins GA0070558_12452 (designated MacM,60% identity to PenC) and GA0070558_12451 (designated MacN, 49% identity to PenB).Further examination of the surrounding region revealed genes whose products arehighly homologous and match those of the ARM biosynthetic pathway in S. citricolor(Fig. 2A), suggesting that the gene cluster (mac) is potentially involved in the biosyn-thesis of ARM- and PTN-related compounds.

To gain evidence whether the candidate mac gene cluster is active at the transcrip-tional level, we performed reverse transcription (RT)-PCR analysis of the target genesmacM and macN. As expected, the results indicated that the gene cluster is indeedtranscribed (see Fig. S1A in the supplemental material), implying that M. haikouensis iscapable of producing related nucleoside antibiotics. To confirm this, we analyzedmetabolites of this strain by liquid chromatography-mass spectrometry (LC-MS). Adistinctive [M�H]� ion for COF (m/z 285.1182) was observed (Table S1), rather than PTNor other related molecules (Fig. 1A and B). Tandem mass spectrometry (MS/MS) analysisindicated that the major fragment ions generated were at m/z 134.9773, 153.0337, and267.0721 (Table S1), fully consistent with the fragmentation pattern of the authenticCOF standard. In addition, characteristic UV peaks were detected for NEP-A and ARM(Fig. 2B; Fig. S1B and C). LC-MS analysis of these showed the distinctive [M�H]� ionsof NEP-A (m/z 264.1085) and ARM (m/z 266.1238), whose fragmentation patterns matchthose of the authentic standards of NEP-A and ARM, respectively (Table S1). NEP-A andARM are both adenosine analogs that are prone to deamination by host adenosinedeaminases, suggesting that the M. haikouensis strain is also likely to produce thedeaminated products of NEP-A and ARM, including NEP-D and Carbo-I (Fig. 1B). Weaccordingly reanalyzed the metabolites for the characteristic [M�H]� ions of NEP-D(m/z 265.0927) and Carbo-I (m/z 267.1082). MS/MS analysis showed that the [M�H]�

fragment ions arising from these were produced at m/z 136.9908 and 247.2916, as wellas m/z 136.9264 and 249.0855 (Table S1), correspondingly identical to those of NEP-Dand Carbo-I (Fig. 1B).

Similarly, trace amounts of NEP-D and Carbo-I were also detected from the metab-olites of S. citricolor (Table S1), a well-characterized ARM and NEP-A producer. Hence,

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this strain is also a potential producer of a PTN-related compound(s). In confirmation ofthis, LC-MS analysis of metabolites from this strain showed a distinctive [M�H]� ion ofCOF (m/z 285.1188) (Table S1) but not those for PTN or other related antibiotics (Fig. 1Aand B). This was also evident from the fragmentation pattern corresponding to that ofthe COF standard (Table S1). All these data verify that both M. haikouensis and S.citricolor are the producers of multiple nucleoside antibiotics.

Reconstitution of the ARM and COF pathways in a heterologous host. Toreconstitute the ARM and COF pathways in a heterologous host, 63B3, a cosmidcontaining the whole mac gene cluster, was screened from the genomic library of M.haikouensis. It was then introduced into Streptomyces aureochromogenes CXR14 (CXR14herein), which was previously shown to be an appropriate host for the heterologousproduction of PTN-related purine nucleoside antibiotic pairs (Table 2) (8). The resultantrecombinant was confirmed by PCR and fermented for further metabolite analysis.High-performance liquid chromatography (HPLC) analysis of the CXR14/63B3 recombi-nant showed target NEP-A and ARM peaks, which correspond to those of standards(Fig. 2B). Further LC-MS analysis of these peaks showed characteristic NEP-A and ARM[M�H]� ions at m/z 264.1105 and m/z 266.1254, respectively (Table S1). In addition, theMS/MS fragmentation patterns of the two ions were correspondingly consistent withthose of the authentic ARM and NEP-A standards (Table S1), but these ions were absentfrom the metabolites of CXR14/2463b, a control strain without the mac gene cluster.

FIG 2 Genetic organization and verification of the ARM and COF biosynthetic gene cluster. (A) Genetic organization of the ARM and COF gene cluster. The macgene cluster is responsible for ARM and COF biosynthesis in M. haikouensis, while two separate gene clusters (ari and com) independently govern thebiosynthesis of ARM and COF in S. citricolor. (B) Engineered production of ARM, NEP-A, and COF in S. aureochromogenes CXR14. (C) LC-MS analysis of the targetmetabolite of COF produced by related strains. Std, the authentic standards of ARM and NEP-A; haikou-WT, wild-type strain of M. haikouensis; CXR14/63B3, S.aureochromogenes CXR14 containing 63B3 cosmid; CXR14/pJTU2463b, S. aureochromogenes CXR14 containing pJTU2463b as a negative control.

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LC-MS also indicated that the CXR14/63B3 strain is able to produce COF, evident froma [M�H]� ion at m/z 285.1184 (Fig. 2C) and associated fragment ions at m/z 134.9610,152.9790, and 267.1675 and similar to those for COF produced by M. haikouensis (TableS1). All of this demonstrates that a single mac gene cluster spanning 25.3 kb isresponsible for the biosynthesis of ARM, NEP-A, and COF.

The ARM producer S. citricolor also harbors a COF gene cluster (com) which islocated at an unlinked locus. Although S. citricolor is shown to be a COF producer inthe present study, the genes for the biosynthesis of this antibiotic were missing in thesurrounding region of the ari gene cluster (Fig. 2A) (19), suggesting that they areprobably distributed over the S. citricolor genome. To locate the COF gene cluster, wesequenced the genome of S. citricolor and used the conserved enzymes, includingMacM (SAICAR synthetase) and MacN (short-chain dehydrogenase), as probes, allowingthe identification of a candidate gene cluster (com) (Fig. 2A).

To correlate the com gene cluster to COF biosynthesis, 12H2, a cosmid housing thecomplete com gene cluster, was screened from the S. citricolor genomic library andsubsequently conjugated into the heterologous host, S. aureochromogenes CXR14(Table 2). After validation by PCR, the recombinant (CXR14/12H2) was fermented formetabolite production and analyzed by LC-MS. The metabolites of CXR14/12H2 werecharacterized by a [M�H]� ion at m/z 285.1189 (Fig. 3A), plus fragment ions at m/z134.9322, 152.9562, and 266.9613, consistent with the fragmentation pattern of COFproduced by S. citricolor (Fig. 3B to D). Hence, these combined data demonstrate thatthe target com gene cluster is responsible for COF biosynthesis in S. citricolor.

Comparative analysis of the gene cluster diversity for ARM and COF biosyn-thesis in M. haikouensis and S. citricolor. Comparative analysis of the target geneclusters in M. haikouensis and S. citricolor indicated that they generally match (theidentity of the corresponding enzymes encoded by the gene clusters ranges from 35%to 81%) but also contain some particular genes (Fig. 2A). Of these, genes macWXRS areunique to the mac gene cluster (Fig. 2A; Table 1), and they may play additional roles in

TABLE 1 Deduced functions of the open reading frames in the mac gene cluster

Proteina

No. ofamino acids Protein function Homolog, origin

Identity,similarity (%) Accession no.

MacV 217 NAD-dependentepimerase/dehydratase

Ari15, S. citricolor 56, 64 BAV57070

MacW 478 SAH hydrolase ASC68_24850, Devosia sp. Root105 38, 53 KQU93055MacX 220 ATP phosphoribosyl-transferase Sare_0588, Salinispora arenicola CNS-205 58, 71 ABV96516MacA 334 Dehydrogenase Ari1, S. citricolor 42, 58 BAV57056MacB 364 Five-membered cyclitol-phosphate

synthaseAri2, S. citricolor 81, 88 BAV57057

MacC 216 Short-chain dehydrogenase Ari3, S. citricolor 50, 61 BAV57058MacD 249 Short-chain dehydrogenase Ari4, S. citricolor 62, 77 BAV57059MacE 139 Enamine deaminase Ari5, S. citricolor 64, 78 BAV57060MacF 344 Ribokinase Ari6, S. citricolor 55, 66 BAV57061MacG 415 SAH hydrolase Ari7, S. citricolor 81, 90 BAV57062MacH 474 Phosphoglucomutase Ari8, S. citricolor 53, 62 BAV57063MacI* 272 Adenosine phosphorylase Ari9, S. citricolor 76, 87 BAV57064MacJ 422 SAH hydrolase Ari10, S. citricolor 71, 80 BAV57065MacK 407 SAH hydrolase Ari11, S. citricolor 67, 77 BAV57066MacL 230 Phosphoglycerate mutases Ari12, S. citricolor 61, 71 BAV57066MacM 243 SAICAR synthetase PenC, S. antibioticus NRRL 3238 60, 74 AKA87338MacN 239 Short-chain dehydrogenase PenB, S. antibioticus NRRL 3238 49, 64 AKA87339MacO 430 HAD family hydrolase Krac_6662, Ktedonobacter racemifer DSM 44963 34, 50 EFH85441MacP 248 Phosphatase Ari14, S. citricolor 58, 74 BAV57069MacQ* 330 Adenosine deaminase Ari17, S. citricolor 35, 48 BAV57071MacR* 488 GMP reductase N864_01465, Intrasporangium chromatireducens Q5-1 58, 72 EWT05858MacS 325 Epimerase Amir_4843, Actinosynnema mirum DSM 43827 42, 57 ACU38670MacT* 272 Adenosine phosphorylase Ari9, S. citricolor 49, 64 BAV57064MacU 383 MFS transporter Ari13, S. citricolor 63, 75 BAV57068a*, function was confirmed in vitro. MacV corresponds to the locus tag GA0070558_12467 (NCBI accession number SCF06629.1), and likewise, MacU matches to that ofaccession no. GA0070558_12444 (GenBank: SCF06432.1).

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ARM or COF biosynthesis in M. haikouensis. The product of macX shows 58% identity toSare_0588 (ATP phosphoribosyltransferase, HisG homolog), and macR and macS areindividually annotated as an IMP dehydrogenase and epimerase. Likewise, three dis-pensable genes, including orf1 (encoding a transporter) (Fig. 2A; Table 1) and comEF(coding for a phosphohydrolase and an MFS transporter, respectively) (Fig. 2A, Table 3),are present only in the ari or com gene cluster.

The in silico analysis also showed that there are four genes, macWGJK, coding forS-adenosyl-L-homocysteine (SAH) hydrolases in the mac gene cluster, whereas thecorresponding ari gene cluster includes only three of these (ari7, ari10, and ari11) (Fig.2A; Table 1). Since the com gene cluster in S. citricolor is entirely responsible for COFbiosynthesis, the four genes macXMNO accordingly have a role in COF biosynthesis inM. haikouensis (Fig. 2A; Tables 1 and 3). Hence, this implies that the COF and ARMnatural products arise from independent biosynthetic pathways.

MacI and MacT function as unusual phosphorylases, catalyzing an irreversiblestep for the tailoring assembly of NEP-A and ARM. The mac gene cluster containstwo genes, macIT, that likely encode a deduced 5=-methylthioadenosine phosphorylase

TABLE 2 Strains, plasmids, and cosmids used in this study

Strain, plasmid, or cosmid Description Reference or source

StrainsM. haikouensis DSM 45626 Wild-type producer of ARM, NEP-A, and COF This studyS. citricolor NBRC 13005 Wild-type producer of ARM, NEP-A, and COF 19S. aureochromogenes

CXR14 An industrial polyoxin producer with the entire polyoxin gene cluster deleted 34CXR14/63B3 CXR14 strain containing 63B3 This studyCXR14/63B3�macI CXR14 strain containing 63B3�macI This studyCXR14/63B3�macT CXR14 strain containing 63B3�macT This studyCXR14/63B3�macQ CXR14 strain containing 63B3�macQ This studyCXR14/63B3�macW CXR14 strain containing 63B3�macW This studyCXR14/63B3�macG CXR14 strain containing 63B3�macG This studyCXR14/63B3�macJ CXR14 strain containing 63B3�macJ This studyCXR14/63B3�macK CXR14 strain containing 63B3�macK This studyCXR14/12H2 CXR14 strain containing 12H2 This studyCXR14/2463b CXR14 strain containing pJTU2463b This study

E. coliDH10B F� mcrA Δ(mrr-hsdRMS-mcrBC) �80d lacZΔM15 ΔlacX74 deoR recA endA1

araD139 Δ(ara, leu)7697 galU galK �� rpsL nupGGibco-BRL

BW25113/pIJ790 �RED (gam beta exo) cat araC rep101 21ET12567(pUZ8002) F� dam-13::Tn9 dcm-6 hsdM hsdR recF143 zjj-202::Tn10 galK2 galT22 ara-14

pacY1 xyl-5 leuB6 thi-1 pUZ800231

BL21(DE3)�rihC BL21(DE3) strain with rihC mutated This study

PlasmidspEASY-Blunt pUCori lacZ f1 ori neo Amp TransGen BiotechpOJ446 aac(3)IV SCP2 reppMB1* att�C31 oriT 35pJTU2463b The site of SCP2 in pOJ446 was replaced by int and attP 36pET28a neo reppMB1; T7 promoter NovagenpET26b neo reppMB1; T7 promoter NovagenpET28a/macI pET28a derivative carrying an NdeI-EcoRI fragment containing macI This studypET28a/macT pET28a derivative carrying an NdeI-EcoRI fragment containing macT This studypET26b/macQ pET26b derivative carrying an NdeI-HindIII fragment containing macQ This studypET28a/macR pET28a derivative carrying an NdeI-EcoRI fragment containing macR This studypET28a/udp pET28a derivative carrying an NdeI-EcoRI fragment containing udp This study

Cosmids63B3 The cosmid from M. haikouensis containing the entire mac gene cluster This study63B3�macI The cosmid 63B3 with macI in-frame deleted This study63B3�macT The cosmid 63B3 with macT in-frame deleted This study63B3�macW The cosmid 63B3 with macW in-frame deleted This study63B3�macG The cosmid 63B3 with macG in-frame deleted This study63B3�macJ The cosmid 63B3 with macJ in-frame deleted This study63B3�macK The cosmid 63B3 with macK in-frame deleted This study63B3�macQ The cosmid 63B3 with macQ in-frame deleted This study12H2 The cosmid from S. citricolor containing the entire com gene cluster This study

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(Table 1). To investigate their functional roles in vivo, both were independently mutatedon the 63B3 cosmid by a PCR-targeting technology (Fig. S2A and B) (21), and theresulting 63B3 variants were introduced into S. aureochromogenes CXR14, respectively.After verification, the recombinants CXR14/63B3�macI and CXR14/63B3�macT werefermented for metabolite analysis and were shown to have abolished ARM and NEP-Aproduction, whereas the recombinant CXR14/63B3 (positive control) is capable ofproducing both antibiotics (Fig. 4A). This demonstrates the essential roles of these twogenes for the biosynthesis of ARM and NEP-A.

Further investigation indicated that both MacI and MacT individually display 76% and49% identity, respectively, to Ari9, a member of purine nucleoside phosphorylase super-family proteins (Table 1). This superfamily of enzymes is widely distributed among allkingdoms of life, normally catalyzing a reversible reaction using purine (analog) andribose-1-phosphate as the substrates (22). We tentatively proposed that these two enzymesare involved in the tailoring assembly step during the biosynthesis of ARM and NEP-A. Tovalidate this, we overexpressed and purified the two proteins (MacI and MacT) (Fig. S2C)and tested their activities in vitro, initially using adenosine and phosphate as the substrates

FIG 3 Heterologous expression of the com gene cluster in S. aureochromogenes CXR14. (A) Extract ion chroma-tography (EIC) analysis of COF produced by S. citricolor and S. aureochromogenes CXR14/12H2. (B) Fragmentationpattern of COF. (C) LC-MS/MS analysis of COF produced by S. citricolor. (D) LC-MS/MS analysis of COF from thetarget metabolites produced by S. aureochromogenes CXR14/12H2. citri-WT, the sample of the S. citricolor wild-typestrain; CXR14/12H2, the sample of S. aureochromogenes CXR14 containing 12H2; CXR14/pJTU2463b, the sample ofS. aureochromogenes CXR14 containing pJTU2463b as a negative control; 12H2, a cosmid containing the whole comgene cluster.

TABLE 3 Deduced functions of open reading frames in the com gene cluster

ProteinNo. ofamino acids Protein function Homolog, origin

Identity,similarity (%) Accession no.

ComF 403 MFS transporter OV450_6573, Actinobacteria bacterium OV450 64, 75 KPI33463ComE 336 Metal-dependent phosphohydrolase PenF, S. antibioticus NRRL 3238 39, 49 AKA87335ComA 231 SAICAR synthetase MacM, M. haikouensis 59, 71 SCF06495ComB 229 Short-chain dehydrogenase MacN, M. haikouensis 57, 67 SCF06487ComC 140 Hypothetical protein MacO, M. haikouensis 34, 44 SCF06476ComD 224 Phosphatase MacO, M. haikouensis 33, 46 SCF06476

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(Fig. 4B). HPLC analysis of the products showed that the MacI or MacT reactions couldgenerate a distinctive peak for adenine, whose identity was verified by LC-MS analysis. Theenzyme-negative reaction could not generate this target peak, establishing that MacI andMacT could utilize adenosine and phosphate as the substrates to form adenine (Fig. 4C; Fig.S2D to F). To determine if the MacI and MacT reactions are reversible for ARM and NEP-A,as are most of this family of enzymes, we tested the activity of MacI and MacT using ARM,NEP-A, and phosphate as the substrates. The results showed that ARM and NEP-A are notrecognized as the substrates (Fig. S2G and H). Furthermore, to evaluate if MacI and MacTare responsible for the tailoring assembly, we used adenine and the compound 14 (see Fig.7B) analog ribose-1-phosphate generated by a coupled uridine phosphorylase reaction asthe substrates (Fig. 4B; Fig. S2C). The results indicated that the reactions of MacI and/orMacT could generate a characteristic peak for adenosine (Fig. 4D) under these reactionconditions. The identity of adenosine was further confirmed by LC-MS analysis (Fig. S3A toD). Taken together, these data suggest that MacI and MacT function as unusual phospho-rylases, catalyzing an irreversible reaction for the tailored assembly of NEP-A and ARM butharboring a reversible enzymatic activity of hydrolyzing adenosine to supply adenine asprecursor.

MacQ is just an adenosine-specific deaminase. The bioinformatic analysis indi-cated that MacQ possesses 35% identity to an annotated adenosine deaminase (ADA)Orf2 in the ari gene cluster (Table 1), but the functional role of the enzyme is as yetunassigned. To investigate the possible functional role of MacQ in vivo, we mutated

FIG 4 Functional investigation of MacI and MacT in the biosynthesis of ARM and NEP-A. (A) Extract ion chromatography (EIC) analysis of the metabolitesproduced by S. aureochromogenes CXR14/63B3 and its variants. 63B3, the sample from the strain of S. aureochromogenes CXR14 containing 63B3; 63B3�macI,the sample from the strain of S. aureochromogenes CXR14 containing 63B3�macI. The other two samples are correspondingly designated. (B) Scheme of theMacI- and MacT-catalyzed reaction. R-1-P, ribose-1-phosphate. (C) HPLC traces of the MacI- and MacT-catalyzed reactions using adenosine as the substrate. Std,the authentic standards of adenine and adenosine; MacI, the MacI-catalyzed reaction; MacT, the MacT-catalyzed reaction; N.C, the reaction without enzymeadded as a negative control. (D) HPLC analysis of the MacI- and/or MacT- and UDP-coupled reactions. UDP hydrolyzes uridine to provide ribose-1-phosphate,which is used as a substrate by MacI and MacT. Std, the authentic standards of related compounds; MacI, the MacI- and UDP-coupled reaction; MacT, the MacT-and UDP-coupled reaction; MacI�MacT, the MacI-, MacT-, and UDP-coupled reaction, with MacI and MacT added simultaneously; N.C, the UDP reaction withadenine added as a negative control. UDP, uridine phosphorylase (accession no. ACT45511). R-1-P, ribose-1-phosphate.

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macQ on the cosmid 63B3 via a PCR-targeting technology (Fig. S4A) (21); the resultantcosmid variant was conjugated into the CXR14 strain. After fermentation, metabolitesfrom the recombinant CXR14/63B3�macQ strain were submitted to LC-MS analysis.Unexpectedly, the results showed that the ARM production of the CXR14/63B3�macQrecombinant was dramatically decreased in comparison to that of CXR14/63B3 (Fig. S4Band C), suggesting that macQ plays a positive role for ARM production.

We also deduced that MacQ is likely responsible for self-resistance by deaminationof ARM and NEP-A to alleviate the toxicity to host cells, as proposed previously (19).MacQ was expressed and purified from Escherichia coli (Fig. S5A) and assayed in vitrousing either ARM or NEP-A as the substrate. However, and unexpectedly, MacQ was notcapable of catalyzing the deamination of ARM or NEP-A (Fig. S5B and C). This suggestedthat MacQ may function as an adenosine deaminase (Fig. 5A). Adenosine was thereforetested as the substrate and showed that the MacQ reaction gave rise to a distinctiveinosine peak (Fig. 5B), validated by LC-MS analysis, whereas this is absent for theMacQ-negative reaction (negative control) (Fig. S5D and E). More than that, the activityof MacQ can be inhibited by the COF analog PTN (Fig. 5B). These data demonstrate thatMacQ is an adenosine-specific deaminase and probably not directly involved in theself-resistance to ARM and NEP-A by the host cell, as has been recently proposed. An

FIG 5 In vitro characterization of MacQ as an adenosine-specific deaminase. (A) Scheme of the MacQ-catalyzed reaction. (B) HPLC analysis of the MacQ-catalyzed reaction. Std, the authentic standards of PTN,adenosine, and inosine. PTN (pentostatin) is a MacQ inhibitor. �1 mM PTN, the MacQ reaction usingadenosine and 1 mM PTN as the substrate; �0.1 mM PTN, the MacQ reaction using adenosine and 0.1mM PTN as the substrate; �0.01 mM PTN, the MacQ reaction using adenosine and 0.01 mM PTN as thesubstrate; no PTN, the MacQ reaction using adenosine as the substrate but without PTN added; N.C, thereaction without enzyme added as a negative control.

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alternative suggestion is that MacQ functions in a fine-tuned pathway to modulate thepotential “excess adenosine” stress on the producing cells (there are four SAH hydrolasegenes in the mac gene cluster).

Biochemical characterization of MacR as an NADPH-dependent GMP reductase.MacR shows 58% identity to N864_01465 of Intrasporangium chromatireducens Q5-1(Table 1), an annotated GuaB1 family IMP dehydrogenase-related protein that isgenerally involved in GMP biosynthesis (23). MacR (Fig. S6A) was therefore tested invitro using IMP as the substrate, although the expected product, XMP, was notdetected, suggesting that MacR is not an IMP dehydrogenase. However, closer exam-ination of the conserved domain of MacR showed a relationship to GMP reductasefamily proteins. An in vitro assay of MacR using GMP as the substrate and NADPH as acofactor generated a distinctive IMP [M�H]� ion at m/z 349.0535, with MS/MS frag-ment ions at m/z 136.8778, 232.9607, and 330.9223 (Fig. S6B to D), conforming to thefragmentation pattern of the IMP authentic standard. This IMP [M�H]� ion was absentfrom the MacR-negative reactions (negative control) (Fig. 6B). We subsequently evalu-ated the cofactor specificity for MacR, and LC-MS analysis confirmed the specificity forNADPH, so that NADH was not recognized as a cofactor to maintain the enzymaticactivity (Fig. 6B). Hence, these data establish that MacR functions as an NADPH-dependent reductase catalyzing GMP to IMP.

DISCUSSION

The advent of rapid next-generation DNA sequencing has revolutionized the pro-cess for the discovery of natural products from microorganisms (24, 25). Carbocyclicnatural products include many interesting chemotypes, including the antitumor anti-biotic pactamycin, the chitinase inhibitor allosamidin, and the carbocyclic purinenucleoside antibiotics (13). Generally, purine nucleoside natural products are synthe-sized by a nonmodular assembly line (2), which complicates the identification of theirbiosynthetic gene clusters. In this respect, the discovery of new ARM and COF producerstrains (see Fig. S7 in the supplemental material) opens the way for the rational searchfor newer purine antibiotic pairs.

The COF-related antibiotics, including PTN, 2=-Cl PTN, carbocyclic COF, and adecype-nol, share common enzymatic steps for the construction of the heterocyclic 1,3-diazepine ring (1, 9), and in this report we have confirmed these assignments. Threeenzymes, MacX (ATP phosphoribosyltransferase), MacM (SAICAR synthetase), and MacN(short-chain dehydrogenase), are needed for COF biosynthesis. MacX catalyzes theinitial condensation of ATP and PRPP (phosphoribosyl pyrophosphate) to producecompound 1, which is sequentially converted to compound 2 by HisE (phosphoribosyl-ATP pyrophosphatase), HisI (phosphoribosyl-AMP cyclohydrolase), and HisA (phospho-ribosylisomerase) enzymes from the histidine pathway (Fig. 7A). Compound 2 under-goes modification by MacM to form compound 3, which is then sequentiallydephosphorylated and dehydrogenated to yield the end product COF (Fig. 7A) (8, 11).

Previous labeling studies have established that ARM is derived from D-glucose (14),and more recently, it was shown to arise from D-fructose-6-phosphate (F6P) (19).Investigation of the mac gene cluster led to the identification of 20 genes (except formacXMNO) that are involved in ARM biosynthesis. We propose that ARM biosynthesisis initiated by MacB (Ari2 homolog), a MIPS (myo-inositol-1-phosphate synthase)-likeenzyme that catalyzes the conversion of F6P to form compound 7. This is followed byhydrolysis (catalyzed by MacP and MacL) to give compound 8 and continuous reduc-tions to generate compound 10 (Fig. 7B). Subsequently, compound 10 undergoessequential epimerization and reduction by the enzymes MacV/MacS and MacD to giverise to compound 12 (Fig. 7B). Compounds 11 and 12 have been previously isolatedfrom mutants of S. citricolor and confirmed to be the essential intermediates for ARMbiosynthesis (18). Once formed, compound 12 is activated by phosphorylation andserves as the primary substrate for the final assembly of NEP-A. Two enzymes, MacF(ribokinase) and MacH (phosphoglucomutase), are proposed to catalyze the sequentialreactions to form compounds 13 and then 14 (Fig. 7B).

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The adenine ring in both ARM and NEP-A originate from adenosine (14, 15). FourSAH hydrolases (MacW, MacG, MacJ, and MacK) and two phosphorylases (MacI andMacT) are found in the mac gene cluster as suitable candidates for the adenine supplyand final assembly of NEP-A (Fig. 7B; Fig. S8). SAH hydrolases catalyze the reversiblehydrolysis of SAH to produce adenosine through a well-characterized oxidoreductionmechanism (26). We therefore propose that SAH is first hydrolyzed to adenosine,followed by further hydrolysis to adenine under the catalysis of MacI and MacT (Fig. 7B).Subsequently, MacI and MacT catalyze an unusual irreversible synthesis of NEP-A by theassembly of adenine and compound 14 (Fig. 7B). Earlier work has shown that ARMbiosynthesis involves a final reduction step, with NEP-A as the substrate (27), and in thepresent study we therefore propose that MacA (or other related enzymes) is responsiblefor the final tailoring reduction (Fig. 7B).

FIG 6 In vitro characterization of MacR as an NADPH-dependent reductase. (A) Scheme of the MacR-catalyzed reaction. (B) HPLC analysis of the MacR-catalyzed reaction. Std, the authentic standards of IMPand GMP; NADPH�, the MacR-catalyzed reaction with NADPH as a cofactor and GMP as the substrate;NADH�, the MacR-catalyzed reaction with NADH as a cofactor and GMP as the substrate; no cofactor, theMacR-catalyzed reaction with GMP as the substrate but without a cofactor added; N.C, the reaction (withGMP and NADPH added) but without enzyme added as a negative control.

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It is very interesting to ask why the ARM pathway includes more SAH hydrolases inM. haikouensis than it seemingly needs. In this respect, it is notable that ARM and NEP-Aare both potent SAH hydrolase inhibitors that could reversely undermine the biosyn-thesis of ARM and NEP-A by feedback inhibition of the activities of SAH hydrolases (Fig.8) (13). The additional SAH hydrolases might therefore collaborate to maintain theefficient synthesis of ARM and NEP-A (Fig. S8).

The role of the adenosine deaminase (MacQ) in ARM and NEP-A biosynthesis is alsounclear (28). In the present study, MacQ is shown to be an adenosine-specific deami-nase. An excess of adenosine is generally recycled to produce dATP by the cells, whichwill exert a direct feedback inhibition of the ribonucleotide reductase under this stresscircumstance and hence lead to a termination of DNA synthesis (29). Thus, the role ofMacQ might be to fine-tune the pathway to prevent the buildup of the potential excess

FIG 7 Proposed biosynthetic pathways for COF (A) and for NEP-A and ARM (B).

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adenosine (Fig. 8). The inhibitory function of COF on MacQ may act as the safeguard forthe protection of ARM and NEP-A from deamination by host ADAs (Fig. 8). IMP, theenzymatic product of MacR (GMP reductase), could be further recycled to AMP andother related products required for ARM biosynthesis (Fig. 8). Hence, an intricate controlstrategy has developed to modulate the biosynthesis of ARM.

In summary, we report the discovery of new producers of multiple nucleosideantibiotics by a target-oriented genome mining strategy and have delineated the genecluster diversity of ARM and COF in Actinomycetes. We propose a fine-tuned pathwayassociated with the biosynthesis of these antibiotics, notably MacI and MacT catalyzingan irreversible reaction for the tailoring assembly of ARM and NEP-A, MacQ acting as anadenosine-specific deaminase, and MacR functioning as a dedicated GMP reductase forthe supply of IMP pool. We anticipate that the deciphering of the genetic andenzymatic diversities for ARM and COF biosynthesis will accelerate the discovery ofnovel purine-related nucleoside antibiotic pairs.

MATERIALS AND METHODSGeneral materials and methods. Strains, plasmids, and cosmids used in this study are described in

Table 2, and primers are listed in Table 4. General methods employed in this work are in accordance withthe standard protocols of Green and Sambrook (30) or Kieser et al. (31).

Enzymes, chemicals, and reagents. All of the restriction enzymes and T4 DNA ligase used in thisstudy were the products of New England Biolabs. The chemicals and reagents were purchased from

FIG 8 A proposed fine-tuned pathway associated with ARM and NEP-A biosynthesis. Blue arrows indicate that the reaction is found in the ARM pathway, andthe red line (the T-like inhibition symbol) signifies the specific enzymatic step capable of being inhibited by a certain end product. ADSS, adenylosuccinatesynthetase; ADSL, adenylosuccinate lyase; MAT, methionine adenosyltransferase; MT, methyltransferase.

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Sigma-Aldrich, Thermo Scientific, or J&K Scientific. The standards of ARM and NEP-A were individuallypurchased from Santa Cruz Biotechnology, Inc., and Cayman Chemical.

Sequence analysis of M. haikouensis and S. citricolor. The sequences of the M. haikouensis genome(accession no. FMCW01000024.1) and ARM biosynthetic gene cluster (accession no. LCO54541.1) weredownloaded from the GenBank database. Sequencing of the S. citricolor genome was performed on anIllumina HiSeq 2500 machine. The raw data were processed to render the resulting clean reads, whichwere assembled by Velvet software (v1.2.07) to obtain the scaffold. After that, Glimmer 3.0 software wasused for the genome annotation. Accurate bioinformatic analysis of the target DNA region was per-formed on the basis of the online programs FramePlot 4.0beta (http://nocardia.nih.go.jp/fp4/) and2ndFind (http://biosyn.nih.go.jp/2ndfind/).

Transcriptional analysis of the targeted genes (macM and macN) by RT-PCR. For RT-PCR analysisof the target gene cluster, the fermentation samples were individually collected at 1 day (24 h), 2 days(48 h), and 3 days (72 h), and the total RNA was extracted with TRIzol reagent (Life Technologies) (32)

TABLE 4 PCR primers used in this study

Primer Sequence (5=–3=)mac-hrdB F CGACTACACCAAGGGCTTCAmac-hrdB R TCAGCTCGATGACCTGGAAmacNidF AAGCGGGCTGTCGTATTGCmacNidR CCGGTCGTAACTCGATCTCAmacMidF CGAGGTCATCGTCAAGAACGmacMidR CAAGGCAGAAGTCCCACAGAHaikou upidF CGAGATCATCACGCACCCGHaikou upidR CTCCACGCCGAACCAGAACHaikou downidF GTCATCCCGACGTACAACCGHaikou downidR CCACGCCGACCGACTAAATcitri wk-id1F CCAGTTCCAGGGCACGATcitri wk-id1R GGTAGTTGTCCAGCAGGTTCcitri wk-id3F GACCTCTGGGACTTCTGCCcitri wk-id3R CGACTCCACGCTCATCTTGTmacI PCRtgtF CCGTCGGACGTGCCTTACCAGGCCAACCTGTGGGCGCTGTCTAGAGCTATTCCAGAAGTmacI PCRtgtR GTAGGAGAGATTCATGTAGCAGAGCTGGAGCTCGCGTGCACTAGTCTGGATGCCGACGmacT PCRtgtF CCGGCGAACGTGGCGGCACTCAAGGAGCTCGGCGCACGTTCTAGAGCTATTCCAGAAGTmacT PCRtgtR CAGGTCCTCCGAAATTACGCCGTAGTCGGTGCAGAAGGAACTAGTCTGGATGCCGACGmacW PCRtgtF ACAAGCACCGGGTTGTGGCGGCTGGGCAAGCTGCCTGACTCTAGAGCTATTCCAGAAGTmacW PCRtgtR TTGGATCGCGCCGACGTGTGCCGGCCGGGGCGGCGGCGAACTAGTCTGGATGCCGACGmacG PCRtgtF TACGGCTGCGACCGTGAGACCTTCTACCAGCAGGTGCAGTCTAGAGCTATTCCAGAAGTmacG PCRtgtR CATCACCTCGGCCGGATGTGCCTCCGCCGCGGACTGGCCACTAGTCTGGATGCCGACGmacJ PCRtgtF GTCTTCGGCCGGCGCGGAATGACCACCGCCGAGGTCGGTTCTAGAGCTATTCCAGAAGTmacJ PCRtgtR CTGCTCCGTCGACACGGTCCGCGCCTGAAGGTCGGCCAGACTAGTCTGGATGCCGACGmacK PCRtgtF GACCTGCGCAAGGGTGTCGAAGAGGTCCTGCTCACCTGGTCTAGAGCTATTCCAGAAGTmacK PCRtgtR GGTCAGCACCTCCGAACCGGGATGCGCTGCCGGCATTCGACTAGTCTGGATGCCGACGmacQ PCRtgt2F GAAGTGGCGCGAAACAACGACATCCGACTGCCTGCGGACTCTAGAGCTATTCCAGAAGTmacQ PCRtgt2R GATCCGGCGCGGCCCATACGGCAGGACTTCCCGGACAGAACTAGTCTGGATGCCGACGmacIexF GTCCATATGTCCAGAGCCGAGACGmacIexR GGAATTCTCACGTCCGGGCTCCCGTmacTexF GTCCATATGATCGACCTAGGAATCmacTexR GGAATTCCTACCGATGCCCCTCCGTmacRexF GTCCATATGGATCAGCAACGGTTTmacRexR GGAATTCTCATCGGATGGGCATCGTCmacQOP-eF GTCCATATGGTTGAGGGTAGCGCAmacQOP-eR CCCAAGCTTGCTGCGCACCAGATCUDPexF GTCCATATGTCCAAGTCTGATGTTTUDPexR CCAAGCTTACAGCAGACGACGCGCCmacI idF CGTCATCGACACTCCCTTCGmacI idR AAACGGCCTCCGCCTCTTCmacT idF GGATCGTCGAGACACCCTAmacT idR CTCCGTATTCAGCCAGCACmacW idF CTCAATTCGATGCCGGTGTTmacW idR CGGTGCGTTCGTGATGGTmacG idF CCGCTGTCCACGAAGGATmacG idR TGAGGTACTGCTGCTGACCCmacJ idtF AGCCAGATGCCGTTGCTCmacJ idtR TGACCTCGCTCGGATTGCmacK idtF ACCGAGCAGATGGTCGTGCmacK idR CCAGGTTGGCGATGTTGCmacQ idF CACGCTGCCGATGATGGAmacQ idR CGTCGCAGGTCGGTGTTGA

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from 1 ml biomass of the cells. The quantity and quality of the extracted RNAs were assessed by ananodrop spectrophotometer (Thermo Scientific), and 5 �g total RNA was treated with 1 �l RNA-freeDNase I (Thermo Scientific) at 37°C for 1 h. RNA (1 �g) was reverse-transcribed into cDNA using theRevertAid first-strand cDNA synthesis kit (Thermo Scientific) primed with a random hexamer primer perthe manufacturer’s instructions. The reverse transcription reaction product was directly used for PCRamplification with the related pair of primers (mac-hrdBF/mac-hrdBR, macNidF/macNidR, and macMidF/macMidR). The PCR products were then evaluated by gel electrophoresis.

Genomic library construction and screening for M. haikouensis and S. citricolor. The genomiclibraries of M. haikouensis and S. citricolor were constructed on the basis of the standard protocol usingEPI300-T1R as the host cell and pJTU2463b as the vector. For the screening of the positive cosmids fromthe genomic libraries of M. haikouensis, a narrow-down PCR approach was performed with the followingPCR primers: Haikou upidF/Haikou upidR, Haikou downidF/Haikou downidR, and macMidF/macMidR (33).Likewise, the cosmid 12H2 was screened from the library of S. citricolor NBRC 13005 with primers citriwk-id1F/citri wk-id1R and citri wk-id3F/citri wk-id3R.

In-frame deletion of the target mac genes by PCR-targeting technology. For in-frame deletion ofthe target genes, the kanamycin resistance cassette (neo) from SuperCos1 was amplified with primers (Table4) and then correspondingly recombined into the target gene in 63B3 by a PCR-targeting strategy (21).Subsequently, the neo cassette was removed in vitro from the cosmid variants by XbaI-SpeI double digestion,followed by religation to generate the in-frame deletion scar of the target genes (Fig. S2A and B, S4A, and S8A;Table S2). The in-frame deletions were finally confirmed by PCR and sequencing analysis with the counterpartprimers (Table 4).

Fermentation and detection of related nucleoside antibiotics. M. haikouensis and S. citricolor werecultivated on YS agar (including 2 g yeast extract, 10 g soluble starch, and 15 g agar per liter, pH 7.3) andan MS plate (31), respectively. For fermentation, a single clone was inoculated in TSB medium andcultivated for 3 days; after that, the cultures (2%, vol/vol) were transferred to fermentation medium (16)and fermented (180 rpm, 28°C) for 5 days. For HPLC and LC-MS analysis, the fermentation beer wasprocessed (with the addition of oxalic acid until pH 3.0 was reached). The HPLC analysis was performedusing a Shimadzu LC-20AT instrument equipped with a C18 column (Dikma Diamonsil Plus; 5 �m, 4.6 by250 mm) with an elution gradient of 5% to 20% methanol– 0.15% aqueous trifluoroacetic acid (TFA) over25 min at a flow rate 0.5 ml/min. LC-MS analysis was carried out on a Thermo Fisher Scientific ESI-LTQOrbitrap mass analyzer controlled by Xcalibur.

Expression and purification of protein in E. coli BL21(DE3)�rihC. For the overexpression andpurification of the target proteins (with MacI as an example), macI was PCR amplified using the primerslisted in Table 4 and then cloned into a pEASY-Blunt vector. After confirmation by DNA sequencing, theNdeI-EcoRI engineered fragment was cloned into pET28a at the corresponding sites. Finally, the resultingexpression construct was transformed into BL21(DE3)�rihC competent cells. Expression and purification of theHis6-tagged MacI were conducted according to the protocol of Wu et al. (8). Protein concentration wasquantified using a bicinchoninic acid protein assay kit.

Biochemical assays of MacI and MacT coupled with the UDP (uridine phosphorylase) reaction.For the hydrolysis activity assays, the MacI and MacT reaction mixtures (100 �l), consisting of 50 mM TrisHCl buffer (pH 7.5, 30°C), 2 mM EDTA, 20 mM phosphate buffer (pH 7.5), 1 mM substrate (Nep-A, ARM,or adenosine), and the proteins UDP and MacI or MacT (20 �g each), were incubated at 30°C for 4 h andthen immediately terminated by adding an equivalent volume of methanol. Following centrifugation(12,000 rpm, 5 min) to remove the protein pellet, the reaction mixtures were subsequently analyzed byHPLC (Shimadzu LC-20AT) and LC-MS (Thermo LTQ-Orbitrap). HPLC was performed with an elutiongradient of 5% to 20% methanol–15 mM TFA over 20 min at a flow rate of 0.5 ml/min. LC-MS/MS analysiswas conducted in an electrospray ionization (ESI) trap mass spectrometer in the positive-ion mode withthe following parameters: drying gas at 275°C, 10 liters/ml, and nebulizer pressure at 30 lb/in2.

In vitro assay of MacQ. For the MacQ activity assay, the reaction mixture (100 �l), consisting of 50 mMphosphate buffer (pH 7.5), 1 mM substrate (Nep-A, ARM, or adenosine), 1 mM, 0.1 mM, or 0.01 mM PTN(pentostatin), and 20 �g MacQ, was incubated at 30°C for 2 h and then inactivated by the immediate additionof an equivalent volume of methanol. After the protein pellet was removed by centrifugation (12,000 rpm, 5min), the reaction supernatant was assayed under the same conditions as described above.

Enzymatic assay of MacR. For the enzymatic assay of MacR, the reaction mixture (100 �l), consistingof 1 mM IMP, 2 mM NADPH or NADH, 100 mM Tris HCl buffer (pH 8.0), 50 mM KCl, 1 mM dithiothreitol,2 mM EDTA, and 20 �g MacR, was incubated at 30°C for 4 h, and then the reaction was terminated bythe prompt addition of the methanol (an equivalent volume). Subsequently, MacR was detected in thesame way as described above.

Accession number(s). The DNA sequence for the COF gene cluster from S. citricolor is deposited inthe GenBank database under accession number KY313601.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://doi.org/10.1128/AEM.01860-18.

SUPPLEMENTAL FILE 1, PDF file, 1.7 MB.

ACKNOWLEDGMENTSThis work was supported by grants from the National Natural Science Foundation of

China (31770041) and the Open Funding Projects of the State Key Laboratory of

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Bioactive Substance and Function of Natural Medicines (GTZK201701), as well as theState Key Laboratory of Microbial Metabolism (MMLKF16-03).

REFERENCES1. Isono K. 1988. Nucleoside antibiotics: structure, biological activity, and

biosynthesis. J Antibiot (Tokyo) 41:1711–1739. https://doi.org/10.7164/antibiotics.41.1711.

2. Chen W, Qi J, Wu P, Wan D, Liu J, Feng X, Deng Z. 2016. Natural andengineered biosynthesis of nucleoside antibiotics in Actinomycetes. J IndMicrobiol Biotechnol 43:401– 417. https://doi.org/10.1007/s10295-015-1636-3.

3. Winn M, Goss RJ, Kimura K, Bugg TD. 2010. Antimicrobial nucleosideantibiotics targeting cell wall assembly: recent advances in structure-function studies and nucleoside biosynthesis. Nat Prod Rep 27:279 –304.https://doi.org/10.1039/B816215H.

4. Chen S, Kinney WA, Van Lanen S. 2017. Nature’s combinatorial biosyn-thesis and recently engineered production of nucleoside antibiotics inStreptomyces. World J Microbiol Biotechnol 33:66. https://doi.org/10.1007/s11274-017-2233-6.

5. Niu G, Tan H. 2015. Nucleoside antibiotics: biosynthesis, regulation, andbiotechnology. Trends Microbiol 23:110 –119. https://doi.org/10.1016/j.tim.2014.10.007.

6. Shelton J, Lu X, Hollenbaugh JA, Cho JH, Amblard F, Schinazi RF. 2016.Metabolism, biochemical actions, and chemical synthesis of anticancernucleosides, nucleotides, and base analogs. Chem Rev 116:14379 –14455. https://doi.org/10.1021/acs.chemrev.6b00209.

7. Serpi M, Ferrari V, Pertusati F. 2016. Nucleoside derived antibiotics tofight microbial drug resistance: new utilities for an established classof drugs? J Med Chem 59:10343–10382. https://doi.org/10.1021/acs.jmedchem.6b00325.

8. Wu P, Wan D, Xu G, Wang G, Ma H, Wang T, Gao Y, Qi J, Chen X, Zhu J,Li YQ, Deng Z, Chen W. 2017. An unusual protector-protégé strategy forthe biosynthesis of purine nucleoside antibiotics. Cell Chem Biol 24:171–181. https://doi.org/10.1016/j.chembiol.2016.12.012.

9. Bush BD, Fitchett GV, Gates DA, Langley D. 1993. Carbocyclic nucleosidesfrom a species of Saccharothrix. Phytochemistry 32:737–739. https://doi.org/10.1016/S0031-9422(00)95163-X.

10. Hanvey JC, Hardman JK, Suhadolnik RJ, Baker DC. 1984. Evidence for theconversion of adenosine to 2=-deoxycoformycin by Streptomyces antibi-oticus. Biochemistry 23:904 –907. https://doi.org/10.1021/bi00300a017.

11. Gao Y, Xu G, Wu P, Liu J, Cai YS, Deng Z, Chen W. 2017. Biosynthesis of2=-chloropentostatin and 2=-amino-2=-deoxyadenosine highlights a sin-gle gene cluster responsible for two independent pathways in Actino-madura sp. strain ATCC 39365. Appl Environ Microbiol 83:e00078-17.https://doi.org/10.1128/AEM.00078-17.

12. Xia Y, Luo F, Shang Y, Chen P, Lu Y, Wang C. 2017. Fungal cordycepinbiosynthesis is coupled with the production of the safeguard moleculepentostatin. Cell Chem Biol 24:1479 –1489. https://doi.org/10.1016/j.chembiol.2017.09.001.

13. Jenkins GN, Turner NJ. 1995. The biosynthesis of carbocyclic nucleosides.Chem Soc Rev 24:169 –176. https://doi.org/10.1039/cs9952400169.

14. Parry RJ, Bornemann V. 1985. Biosynthesis of aristeromycin— elucidationof the origin of the adenine and cyclopentane rings. J Am Chem Soc107:6402– 6403. https://doi.org/10.1021/ja00308a047.

15. Parry RJ, Bornemann V, Subramanian R. 1989. Biosynthesis of the nucle-oside antibiotic aristeromycin. J Am Chem Soc 111:5819 –5824. https://doi.org/10.1021/ja00197a048.

16. Kusaka T, Yamamoto H, Shibata M, Muroi M, Kishi T. 1968. Streptomycescitricolor nov. sp. and a new antibiotic, aristeromycin. J Antibiot (Tokyo)21:255–263. https://doi.org/10.7164/antibiotics.21.255.

17. Yaginuma S, Muto N, Tsujino M, Sudate Y, Hayashi M, Otani M. 1981.Studies on neplanocin-A, new anti-tumor antibiotic. 1. Producing organ-ism, isolation and characterization. J Antibiot (Tokyo) 34:359 –366.

18. Hill JM, Jenkins GN, Rush CP, Turner NJ, Willetts AJ, Buss AD, Dawson MJ,Rudd BAM. 1995. Revised pathway for the biosynthesis of aristeromycinand neplanocin a from D-glucose in Streptomyces citricolor. J Am ChemSoc 117:5391–5392. https://doi.org/10.1021/ja00124a035.

19. Kudo F, Tsunoda T, Takashima M, Eguchi T. 2016. Five-memberedcyclitol phosphate formation by a myo-inositol phosphate synthaseorthologue in the biosynthesis of the carbocyclic nucleoside antibi-

otic aristeromycin. Chembiochem 17:2143–2148. https://doi.org/10.1002/cbic.201600348.

20. Xie QY, Qu Z, Lin HP, Li L, Hong K. 2012. Micromonospora haikouensis spnov., isolated from mangrove soil. Antonie Van Leeuwenhoek 101:649 – 655. https://doi.org/10.1007/s10482-011-9682-y.

21. Gust B, Challis GL, Fowler K, Kieser T, Chater KF. 2003. PCR-targetedStreptomyces gene replacement identifies a protein domain needed forbiosynthesis of the sesquiterpene soil odor geosmin. Proc Natl Acad SciU S A 100:1541–1546. https://doi.org/10.1073/pnas.0337542100.

22. Zang Y, Wang WH, Wu SW, Ealick SE, Wang CC. 2005. Identification ofa subversive substrate of Trichomonas vaginalis purine nucleosidephosphorylase and the crystal structure of the enzyme-substratecomplex. J Biol Chem 280:22318 –22325. https://doi.org/10.1074/jbc.M501843200.

23. Buey RM, Ledesma-Amaro R, Velazquez-Campoy A, Balsera M, Cha-goyen M, de Pereda JM, Revuelta JL. 2015. Guanine nucleotidebinding to the Bateman domain mediates the allosteric inhibition ofeukaryotic IMP dehydrogenases. Nat Commun 6:8923. https://doi.org/10.1038/ncomms9923.

24. Tao W, Yurkovich ME, Wen S, Lebe KE, Samborskyy M, Liu Y, Yang A, LiuY, Ju Y, Deng Z, Tosin M, Sun Y, Leadlay PF. 2016. A genomics-ledapproach to deciphering the mechanism of thiotetronate antibioticbiosynthesis. Chem Sci 7:376 –385. https://doi.org/10.1039/C5SC03059E.

25. Bachmann BO, Van Lanen SG, Baltz RH. 2014. Microbial genome miningfor accelerated natural products discovery: is a renaissance in the mak-ing? J Ind Microbiol Biotechnol 41:175–184. https://doi.org/10.1007/s10295-013-1389-9.

26. Reddy MC, Kuppan G, Shetty ND, Owen JL, Ioerger TR, Sacchettini JC.2008. Crystal structures of Mycobacterium tuberculosis S-adenosyl-L-homocysteine hydrolase in ternary complex with substrate and inhibi-tors. Protein Sci 17:2134 –2144. https://doi.org/10.1110/ps.038125.108.

27. Parry RJ, Jiang YJ. 1994. The biosynthesis of aristeromycin-conversion ofneplanocin-A to aristeromycin by a novel enzymatic reduction. Tetrahe-dron Lett 35:9665–9668. https://doi.org/10.1016/0040-4039(94)88354-8.

28. Nakamura H, Koyama G, Iitaka Y, Ono M, Yagiawa N. 1974. Structure ofcoformycin, an unusual nucleoside of microbial origin. J Am Chem Soc96:4327– 4328. https://doi.org/10.1021/ja00820a049.

29. Johnston JB. 2011. Mechanism of action of pentostatin and cladribine inhairy cell leukemia. Leuk Lymphoma 52(Suppl 2):S43–S45. https://doi.org/10.3109/10428194.2011.570394.

30. Green MR, Sambrook J. 2002. Molecular cloning: a laboratory manual,3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

31. Kieser T, Bibb MJ, Chater KF, Butter MJ, Hopwood DA. 2000. PracticalStreptomyces genetics, 2nd ed. John Innes Foundation, Norwich, UnitedKingdom.

32. Chomczynski P. 1993. A reagent for the single-step simultaneous isola-tion of RNA, DNA and proteins from cell and tissue samples. Biotech-niques 15:532.

33. Cheng L, Chen W, Zhai L, Xu D, Huang T, Lin S, Zhou X, Deng Z. 2011.Identification of the gene cluster involved in muraymycin biosynthesisfrom Streptomyces sp. NRRL 30471. Mol Biosyst 7:920 –927. https://doi.org/10.1039/C0MB00237B.

34. Qi J, Wan D, Ma H, Liu Y, Gong R, Qu X, Sun Y, Deng Z, Chen W. 2016.Deciphering carbamoylpolyoxamic acid biosynthesis reveals unusualacetylation cycle associated with tandem reduction and sequentialhydroxylation. Cell Chem Biol 23:935–944. https://doi.org/10.1016/j.chembiol.2016.07.011.

35. Bierman M, Logan R, O’Brien K, Seno ET, Rao RN, Schoner BE. 1992.Plasmid cloning vectors for the conjugal transfer of DNA from Esche-richia coli to Streptomyces spp. Gene 116:43– 49. https://doi.org/10.1016/0378-1119(92)90627-2.

36. Zhai L, Lin S, Qu D, Hong X, Bai L, Chen W, Deng Z. 2012. Engineering ofan industrial polyoxin producer for the rational production of hybridpeptidyl nucleoside antibiotics. Metab Eng 14:388. https://doi.org/10.1016/j.ymben.2012.03.006.

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