Nat Chem Biol_7_834-853_2011

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NATURE CHEMICAL BIOLOGY

| VOL 7 | NOVEMBER 2011 | www.nature.com/naturechemicalbiology

843

ARTICLEPUBLISHED ONLINE: 9 OCTOBER 2011 |DOI: 10.1038/NCHEMBIO.671

Aminoglycosides act by binding to the bacterial ribosome,thereby inhibiting protein synthesis and generating errors inthe translation of the genetic code1. his class of antibiotics

has been widely used to treat severe bacterial infections for decades2,beginning with the use of the first effective antituberculosis agent,streptomycin3. Aminoglycosides are defined by two structuralclasses: kanamycins (Fig. 1), isolated from Streptomyces kanamy-ceticus4, are representative 4,6-disubstituted 2-deoxystreptamine(1)-containing aminoglycoside antibiotics along with the genta-micins5 and tobramycin6, whereas the neomycins7and butirosins8exemplify the 4,5-disubstituted aminoglycosides (Supplementary

Results, Supplementary Fig. 1). As with other antibiotics, theemergence of aminoglycoside-resistant pathogens raises seriousproblems1,9. he issue of resistance has been addressed by the semi-synthetic modification of natural aminoglycosides such as amikacin,dibekacin or arbekacin (Supplementary Fig. 1)10. A detailed under-standing of the biosynthetic pathway of aminoglycosides is a pre-requisite for not only the generation of more robust antibiotic agentsbut also the direct fermentative production of clinically importantantibiotics. However, although the biochemistry and geneticsunderlying the biosynthesis of 4,5-disubstituted aminoglycosideshave been relatively well characterized11, those of 4,6-disubstitutedaminoglycosides, including kanamycin, still remain unclear, mainlybecause of difficulties in genetically manipulating the producingactinomycetes and obtaining soluble functional enzymes.

he core moiety 1 and the pseudodisaccharides 2-N-acetylparomamine (2), paromamine (3) and neamine (4) are com-mon biosynthetic intermediates of most aminoglycosides, includingkanamycin. he biosynthetic route to 4via 1and 3from D-glucose-6-phosphate has been elucidated in the context of butirosin and neomycinbiosynthesis using purified recombinant enzymes, with the exceptionof the glycosylation step11, which has been partially characterized viaeither in vivogene disruption or the study of cell-free extracts (CFEs)(Fig. 1). In particular,1is biosynthesized from D-glucose-6-phosphate

by 2-deoxy-scyllo-inosose (2-DOI) synthase12, a dual-functionalL-glutamine:2-DOI aminotransferase13, and 2-deoxy-scyllo-inosamine(2-DOIA) dehydrogenase14. Next, N-acetylglucosaminyltransferasetransfers D-2-N-acetylglucosamine (GlcNAc) onto 1 to give 2(refs. 1517), which is deacetylated to 3by 2-N-acetylparomaminedeacetylase18. We have also shown that the recombinant KanA acts asa 2-DOI synthase19, that kanBencodes a dual-functional aminotrans-ferase by interspecies complementation in the neomycin producerStreptomyces fradiae20, and that kanA-kanB-kanK-kanF and kacAconstitute a minimal gene set for the biosynthesis of 3by heterolo-gous expression in Streptomyces lividans21. Most recently, tests of the

CFE of Escherichia coliexpressing kanE22

indicated that this glyco-syltransferase (otherwise known as KanM2) catalyzes the glucosyla-tion of 3to give 3-deamino-3-hydroxykanamycin C (5), suggestingthat 5is a precursor for kanamycin C (6). Other steps in the pathwayhave been predicted totally on the basis of the metabolites isolatedfrom culture extracts and isotope-labeled precursor incorporationstudies23,24. For example, 6 and kanamycin B (7) are supposed tobe formed by the transfer of a kanosamine (D-3-glucosamine, Kns)moiety onto 3and 4, respectively, by the glycosyltransferase KanE25.Additionally, kanamycin A (8) has been long believed to be pro-duced by the deamination of the 2position of 7(refs. 2527) (Fig. 1).However, as this proposed biosynthetic route lacks substantial geneticand biochemical evidence15,2527, the true biosynthetic pathway tokanamycin products still awaits discovery.

Recently, we isolated the kanamycin biosynthetic gene clusterfrom S. kanamyceticus28. he production of 8by expression of thisentire cluster in a nonaminoglycoside-producing S. venezuelaeindicated that it contains all of the genes sufficient for the biosynthe-sis of the kanamycin complex29. We now report further explorationof this gene cluster, which allowed us to completely decipher thebiosynthetic pathway of the kanamycins, revealing both previouslyundescribed kanamycin biosynthetic intermediates and a remark-able parallel pathway leading to kanamycins A and B independently.

1Department of Chemistry and Nanoscience, Ewha Womans University, Seoul, Republic of Korea. 2Department of Pharmaceutical Engineering, Institute of

Biomolecule Reconstruction, Sun Moon University, Chungnam, Republic of Korea. 3Interdisciplinary Program of Biochemical Engineering and Biotechnology,

Seoul National University, Seoul, Republic of Korea. 4Systems Microbiology Research Center, Korea Research Institute of Bioscience and Biotechnology,

Daejeon, Republic of Korea.

5

These authors contributed equally to this work. *e-mail: [email protected] or [email protected].

Discovery of parallel pathways of kanamycin

biosynthesis allows antibiotic manipulation

Je Won Park1,2,5, Sung Ryeol Park1,5, Keshav Kumar Nepal2, Ah Reum Han3, Yeon Hee Ban1,

Young Ji Yoo1, Eun Ji Kim1, Eui Min Kim2, Dooil Kim4, Jae Kyung Sohng2* & Yeo Joon Yoon1*

Kanamycin is one of the most widely used antibiotics, yet its biosynthetic pathway remains unclear. Current proposals suggestthat the kanamycin biosynthetic products are linearly related via single enzymatic transformations. To explore this system, wehave reconstructed the entire biosynthetic pathway through the heterologous expression of combinations of putative biosyn-thetic genes from Streptomyces kanamyceticusin the nonaminoglycoside-producing Streptomyces venezuelae. Unexpectedly,we discovered that the biosynthetic pathway contains an early branch point, governed by the substrate promiscuity of aglycosyltransferase, that leads to the formation of two parallel pathways in which early intermediates are further modified.Glycosyltransferase exchange can alter flux through these two parallel pathways, and the addition of other biosyntheticenzymes can be used to synthesize known and new highly active antibiotics. These results complete our understanding ofkanamycin biosynthesis and demonstrate the potential of pathway engineering for direct in vivoproduction of clinically useful

antibiotics and more robust aminoglycosides.
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845

ARTICLENATURE CHEMICAL BIOLOGYDOI: 10.1038/NCHEMBIO.671

as high as for the addition of UDP-Glc to the same acceptor 1(Table 1; Supplementary Fig. 23). herefore, it is evident that KanFaccepts both UDP-Glc and UDP-GlcNAc as cosubstrates but prefer-

entially transfers Glc to 1.

The parallel pseudodisaccharide C6amination pathwayshe conversion of paromamine (3) to neamine (4) may berequired for kanamycin biosynthesis as it is in the case of neo-mycin biosynthesis31. Based on the sequence analysis of the1-containing aminoglycoside gene clusters, there are two sets ofgenes predicted to be involved in the amination of the hydroxylgroups of kanamycins. kanI and kacL are suggested to encode a6-paromamine dehydrogenase and a 6-oxoparomamine amin-otransferase, respectively25, which are involved in the step from3to 4. On the other hand, kanCand kanDproducts are proposedto function in the C3 amination of UDP-Glc, yielding UDP-Kns25.Alternatively, KanC and KanD could be responsible for the forma-

tion of 6 from 5 (refs. 2527). o assign functional roles to thegene product pairs proposed, we first expressed kanI-kacLin thestrain PAR, which normally produces 3and 9; the resulting strain

PARil produced 4(2.2 M) and 2-deamino-2-hydroxyneamine(10; 4.3 M) along with 3and 9(Fig. 2c). 10has been known tobe a natural kanamycin intermediate for over 40 years 32, but its

precursor and the gene products involved in its formation havenever been characterized. Moreover, when the CFE of the recom-binant strain expressing kanI-kacLwas incubated in vitrowith 3and 9, the above 6-aminated pseudodisaccharides 4and 10wereproduced, respectively (Fig. 2d), establishing their roles in theC6amination of both 3and 9. he kanI-kacLproducts seem toprefer 3 as a substrate over 9, based on the conversion yields ofthe reactions. However, that expression of kanC-kanDin the strainPAR (strain PARcd) produced only 3and 9(Fig. 2c) indicates thatKanC-KanD does not participate in amination of the pseudodi-saccharides and instead may be involved in the subsequent stepsin pseudotrisaccharide biosynthesis.

The second glycosyltransferase adds UDP-Kns and UDP-Glc

A second glycosyltransferase activity is required for the biosynthe-sis of kanamycin complex. Expression of kanEencoding a secondglycosyltransferase along with the genes for biosynthesis of 3and 9

b 1

1

1

1

9 2

100

0 5 10 15

+ 1+ UDP-GlcNKanF

+ 1+ UDP-GlcNAcKanF

+ 1+ UDP-GlcKanF

+ 1KanF

e

d 3

3

4

100

10 15

+ 3Kanl-KacL

+ 3Kanl-KacL

99

10 + 9Kanl-KacL

+ 9Kanl-KacL

100

5 10

c

9

3

9

1

10

3 4

100

0 5 10 15 20

PARil

kanA-B-K kanF kacA kanl-kacL

PARcd

kanA-B-K kanF kacA kanC-kanD

1

1

1

2

9

9

3

100

0 5

Relative

abundance

Relative

abundance

Relative

abundance

Relative

abundance

Relative

abundance

10

Tret

(min)

Tret

(min)

Tret

(min)

Tret

(min) Tret

(min)

15 20

PAR

kanA-B-K kanF kacA

DOSf

kanA-B-K kanF

DOS

kanA-B-K

a

O

O

OH

HO

HO

H2N

NH2

OH

OHHO

2-Deamino-2-hydroxyparomamine (9)

O

O

OH

HO

HO

H2N

NH2

NH2

OHHO

2-Deamino-2-hydroxyneamine (10)

6 6

Figure 2 | HPLC-ESI-MS/MS analysis of kanamycin pseudodisaccharide intermediates obtained from the in vivoexpression of candidate kanamycin

gene sets in recombinant S. venezuelaehosts and in vitroreactions using cell-free extracts of recombinants.(a) Chromatograms of culture extractsfrom recombinants (DOS, DOSf and PAR) expressing kanA-kanB-kanK, kanA-kanB-kanK-kanF(the latter gene encoding the first glycosyltransferase)

and kanA-kanB-kanK-kanF-kacA (the latter gene encoding deacetylase). Each colored rectangle represents the gene(s) expressed in the corresponding

recombinant strains. (b) Chromatograms of the first glycosyltransferase KanF-catalyzed production of 2-N-acetylparomamine (2) and 2-deamino-2-

hydroxyparomamine (9) supplemented with1and three different uridine 5-diphospho-(UDP) sugars: UDP-glucose (Glc), UDP-2-N-acetylglucosamine

(GlcNAc) and UDP-2-glucosamine (GlcN). Filled boxes represent the CFEs of recombinant host expressing the corresponding gene(s) used for in vitro

reactions, and black-framed boxes indicate boiled CFEs used as controls. (c) Chromatograms of culture extracts from recombinants (PARcd and PARil)

expressing kanA-kanB-kanK-kanF-kacA together with kanC-kanDand kanA-kanB-kanK-kanF-kacA together with kanI-kacL. (d) Chromatograms of the

KanI-KacLcatalyzed production of neamine (4) and 2-deamino-2-hydroxyneamine (10) supplemented with paromamine (3) and 9, respectively.

(e) Structures of kanamycin pseudodisaccharide intermediates. Tret, retention time.

Table 1 | Kinetic parameters for KanF and KanE with donors and acceptors

Enzyme Acceptor (A) Donor (D) kcat(min1) Km(A) (mM) Km(D) (mM)

kcat/Km(D)

(min1mM1)

KanF 1 UDP-Glc 17.41 2.40 0.28 0.03 0.44 0.06 39.54

UDP-GlcNAc 19.82 2.42 0.32 0.04 2.54 0.24 7.79

KanE 3 UDP-Glc 3.18 0.37 0.25 0.04 0.26 0.05 12.23

UDP-Kns 3.36 0.39 0.05 0.01 0.03 0.01 112.02

Shown are kinetic parameters for KanF with two donors (UDP-Glc and UDP-GlcNAc) and an acceptor 2-deoxystreptamine ( 1) and for KanE with two donors (UDP-Glc and UDP-Kns) and an acceptor

paromamine (3). All kinetic data represent mean s.d. (n= 3), derived from the Michaelis-Menten equation using GraphPad Prism (GraphPad, version 5).
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848



ARTICLE NATURE CHEMICAL BIOLOGYDOI: 10.1038/NCHEMBIO.671

Modulation of kanamycin biosynthetic flux to kanamycin BKanamycin A (8) is the major fermentation product in both thewild-type kanamycin producer and the heterologous host becauseKanF glycosyltransferase and KanI-KacL prefer UDP-Glc overUDP-GlcNAc and 12 over 6, respectively (Table 1 and Fig. 3d).With the knowledge that the biosynthetic routes to kanamycins Aand B are separated by the initial KanF reaction, we were curious tosee whether we could bias the flux to produce kanamycin B ( 7) asa main product as it is a more valuable congener for the chemical

synthesis of dibekacin and arbekacin (Supplementary Fig. 1).Initially, this was addressed by separately substituting kanFin theDOSf strain with other glycosyltransferase-encoding genes includ-ing nemD(otherwise known as neoM or neo8), tobM1and gtmGfrom the neomycin16,37, tobramycin38and gentamicin17biosyntheticclusters, respectively. A control strain DOSf expressing kanFgener-ated 2 (1.4 M) and 9 (7.1 M), amounts corresponding to 16%and 84% of the total pseudodisaccharides produced, respectively. Incontrast, the production of 2(4.4 M; 57%) and 9(3.4 M; 43%)

by the recombinant expressing nemD(strain DOSn) indicates thatNemD is flexible toward the UDP sugars but seems to use UDP-GlcNAc preferentially over UDP-Glc in contrast to KanF. It waspreviously reported that the CFE from E. coliexpressing nemDcat-alyzed the glycosylation of 1with UDP-GlcNAc to give 2, whereasUDP-Glc was not accepted as a glycosyl donor37. However, in thisstudy, NemD was found to be able to transfer Glc to 1in addition toGlcNAc, which was still the more favorable substrate. When tobM1andgtmGreplaced kanF(DOSt1and DOSg, respectively), there wasno considerable change in the ratio of 2to 9produced (Fig. 5a).

In additional studies, these three glycosyltransferase-encodinggenes were separately substituted for kanFin the strain KCX, whichnormally produces 1.6 M 6and 6.0 M 12as 16% and 60% of totalpseudotrisaccharides, respectively. However, in the recombinant

expressing nemD(strain KCXfn), the ratio of pseudotrisaccharideyields was inverted (6: 5.0 M, 65%; 12: 0.4 M, 7%). Recombinantsexpressing either tobM1 (KCXft1) or gtmG (KCXfg) had unal-tered ratios of pseudotrisaccharide products (Fig. 5b). Finally, theyields of 7and 8generated by the strain KAB were 21% (2.3 M)and 61% (6.8 M) of the total amount of pseudotrisaccharides,respectively. Substitution of kanF with nemD in the strain KAB(KABfn) resulted in an increase of the yield of 7to 46% (3.9 M)(Fig. 5c), a result demonstrating the feasibility of biasing biosyn-thetic flux toward the kanamycin B (7) and C (6) pathways at theexpense of kanamycins A (8) and X (12). he yield of 7produced inthe strain KABfn was increased by approximately two-fold, whichwas less than expected considering the production of 6-hydroxykanamycins (Fig. 5) and was probably caused by the preference of

KanI and KacL for 12over 6(Fig. 3d).A noticeable difference in the substrate preferences of the glyco-syltransferases KanF and NemD prompted us to examine molecularmodels of the glycosyltransferase-substrate complex (SupplementaryMethods). Quantification of hydrogen bonds between glycosyldonors and glycosyltransferases and differences in binding free ener-gies of the various complexes supported the notion that the KanFUDP-Glc and NemDUDP-GlcNAc complexes are preferred overthe KanFUDP-GlcNAc and NemDUDP-Glc complexes, respec-tively (Supplementary Figs. 26,27; Supplementary Table 19).

Direct fermentative production of modified kanamycinsWe attempted to engineer the kanamycin pathway further for the directin vivoproduction of 1-N-acylated kanamycins containing AHBA such

as amikacin (15) and 3-deoxykanamycins such as tobramycin (16)(Fig. 6). he AHBA pharmacophore was originally observed in thenatural product butirosin8. Recently, it was revealed, through the useof in vitrochemoenzymatic reactions, that a set of seven genes (btrG-btrH-btrI-btrJ-btrK-btrO-btrV) is responsible for the biosynthesisand incorporation of the AHBA side chain into the amino group atthe C1 of 1in butirosin39. Introduction of these genes into the strainKCX (creating strain KCXb) led to the in vivogeneration of 0.6 mg l1of a new aminoglycoside, 1-N-AHBA-kanamycin X (17; 1.0 M).Furthermore, coexpression of the btrgene set in the stain KAB (strainKABb) led to the successful production 0.5 mg l1 of 1-N-AHBA-kanamycin A, known as amikacin (15; 0.8 M) (Fig. 6a,c).

obramycin (16) from S. tenebrarius6is known to be less proneto deactivation by aminoglycoside-modifying enzymes because itlacks a susceptible 3-hydroxy group10. Based on the bioinformatics

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Figure 5 | HPLC-ESI-MS/MS analysis of kanamycin biosynthetic

intermediates from recombinant S. venezuelaehosts in which the

first glycosyltransferase-encoding gene has been swapped.

(a) Chromatograms of culture extracts from recombinants (DOSf, DOSn,

DOSt1and DOSg) expressing kanA-kanB-kanKtogether with kanFfrom

S. kanamyceticus, nemDfrom neomycin-producing S. fradiae, tobM1fromtobramycin-producing S. tenebrariusor gtmGfrom gentamicin-producing

Micromonospora echinospora. Each colored rectangle represents the gene(s)

expressed in the corresponding recombinant strains. The bar graph

at right indicates the yield of 2-N-acetylparomamine (2) and

2-deamino-2-hydroxyparomamine (9) produced by each recombinant

host. (b) Chromatograms of culture extracts from recombinants

(KCX, KCXfn, KCXft1and KCXfg) expressing kanA-kanB-kanK-kacA-

kanE-kanC-kanD together with kanF, nemD, tobM1or gtmG. The bar

graph summarizes the yield of kanamycin pseudotrisaccharides

such as 3-deamino-3-hydroxykanamycin C (5), kanamycin C (6),

3-deamino-3-hydroxykanamycin X (11) or kanamycin X (12), produced

by each recombinant host. (c) Chromatograms of culture extracts from

recombinants (KAB and KABfn) expressing kanA-kanB-kanK-kacA-kanE-

kanC-kanD-kanI-kacLtogether with kanFor nemD. The bar graph indicatesthe yield of kanamycins such as 6, kanamycin B (7), kanamycin A (8) and

12, produced by each recombinant host. Data represent mean s.d. (n= 3).
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in 3and 10. Besides, the second glycosyltransferase, KanE, seemsto selectively transfer Kns to 3 and 10 but not to 18. o choosea glycosyltransferase that can transfer Kns to 18 to yield 16,CFEs from S. kanamyceticusand S. tenebrariusproducing 16wereprepared and incubated with 18. he CFE from S. tenebrariuswith tobM2as a second glycosyltransferase-encoding gene con-verted 18 into 16, whereas the CFE from S. kanamyceticus didnot (Supplementary Fig. 28). herefore, we constructed a newstrain (KABfnet2a) expressing aprD3-aprD4, nemDand tobM2

encoding glycosyltransferases and genes for 7 biosynthesis toproduce 16(0.4 mg l1; 0.8 M), thus demonstrating that obM2is able to use 18as a glycosyl acceptor together with the 3-deoxyanalogs of 3and 10(Fig. 6b,c). he CFE containing AprD3-AprD4converted 4to 18but did not transform 7 into 16, a result indi-cating that these enzymes are active only for pseudodisaccharides(Supplementary Fig. 29).

Improved antibacterial activity of 1-N-AHBA-kanamycin Xhe antibiotic activity of kanamycin biosynthetic intermediatesand derivatives obtained in this study were tested against twoGram-negative bacteria (E. coli and Pseudomonas aeruginosa)(Supplementary Table 20). he pseudodisaccharides were notas active as the major pseudotrisaccharide kanamycin congeners

6, 7and 8 against kanamycin-sensitive (KanS

) test strains. Of thepseudodisaccharides, the 6-amino compounds4and 10were moreactive than the 6-hydroxy derivatives 3and 9against KanSE. colistrains. he 6-amino counterparts7and 8of the 6-hydroxy pseudo-trisaccharide derivatives 6and 12were slightly more active againstKanStest strains. In addition, the 3-amino derivatives 6, 7, 8and 12had increased activity against the test strains compared with the cor-responding 3-hydroxy derivatives5, 13, 14and 11. herefore, ami-nation of the C6- and/or C3-hydroxyl groups of kanamycins seemsto increase their antibacterial activity, in agreement with the resultsof an earlier study32. All kanamycin biosynthetic intermediates wereinactive (minimal inhibitory concentration (MIC) >128 g ml1)against kanamycin-resistant (KanR) strains. When tested against anamikacin-sensitive (AmkS) P. aeruginosaclinical isolate, most kana-

mycin intermediates had very low activity, with the exception of themain kanamycin congeners 6, 7, 8and 12. Remarkably, comparedwith amikacin (15), the new compound 17had enhanced activityagainst all the test strains and 17, in particular, retained potency(MIC ~64 g ml1) against an amikacin-resistant (AmkR) clinicalisolate of P. aeruginosathat was insensitive to 15.

DISCUSSIONIt has long been believed that 6and 7would be the direct biosyn-thetic precursors of 8and that all kanamycin congeners might bederived from3(refs. 2527), predictions based on metabolic profilesfrom the culture extracts and the precursor feeding study23, whichdemonstrated the exclusive incorporation of [1-14C]GlcN into theGlcN moiety of 3and the 6-amino-6-deoxy Glc moiety of 8during

fermentation of S. kanamyceticus. However, our results clearly dem-onstrate that there are two independent biosynthetic routes to 7and8, and we show that 3is a precursor of 6and 7, whereas 9and 12are the true precursors of 8. he kanamycin biosynthetic pathwayrevealed herein is consistent with the results of another previousprecursor feeding study24in which radiolabeled 3was not incorpo-rated into 8, but it is contradictory to all previous suggestions aboutthe nature of the pathway.

Chemoenzymatic attachment of AHBA side chain onto several1-containing aminoglycosides, including 8, was reported39.However, such synthesis requires two steps in vitro even whenusing a synthetic substrate: the acyltransferase BtrH first transfersa synthetic acyl-N-acetylcysteamine thioester (acyl-SNAC) sub-strate, -L-glutamyl-AHBA-SNAC, onto the aminoglycosides, andthe resulting acyl-aminoglycoside products are treated with the

-L-glutamylcyclotransferase BtrG to give AHBA-aminoglycosidespecies. In contrast, we were able to produce AHBA-conjugated kana-mycins 15and 17directly in recombinants. o our knowledge, this isthe first report of the direct fermentative production of the semisyn-thetic amikacin (15). he new aminoglycoside17has more potent anti-bacterial activity than15against both the KanRand AmkRtest strains(Supplementary Table 20). he sole difference in structure between15and 17 is in the functional group attached to the C6(Fig. 5c),indicating that the removal of the 6-amino group in 15, which serves

as a target for aminoglycoside 6-acetyltransferase10, confers an effectagainst a 15-resistant pathogen. Another expected advantage of 17would be an improved toxicity profile because the side effects ofaminoglycoside therapeutics decrease with a decreasing number ofattached amino groups42. It is quite likely that this molecule can bea candidate for further development of next-generation aminogly-cosides on the basis of its structural resemblance to amikacin andreduced number of amino groups. In addition, the direct biosyn-thesis of tobramycin (16) via an engineered pathway would be moreeconomical than conventional procedures, which currently involvehydrolysis of 6-O-carbamoyltobramycin that constitutes only 9% ofthe total aminoglycosides produced by wild-type S. tenebrarius43.

Although the results presented herein were obtained in a hetero-logous host, application of the same strategy in industrial strains should

enable the practical mass production of clinically valuable antibioticsdirectly by fermentation. In addition, a detailed understanding of thebiosynthetic pathways of kanamycins is sure to open new opportuni-ties to assist in comprehending the biosynthesis of other related amin-oglycosides and to form the basis for pathway engineering toward thenext generation of antibiotics (exemplified by 17).

METHODSGeneral information.Bacterial strains, culture conditions, expression and purifica-tion of proteins, and materials are described in Supplementary Methods.

Construction of expression plasmids and mutants.Details regarding DNAisolation and manipulation as well as the construction of expression plasmids(Supplementary Tables 1,2) and mutants are described in Supplementary Methods.Genes used in this study are from kanamycin (AJ582817), neomycin (AJ786317 andAJ629247), gentamicin (AJ575934), tobramycin (AJ810851), butirosin (AJ494863

and AB097196) and apramycin (AJ629123) biosynthetic gene clusters.

Analysis of kanamycin biosynthetic intermediates and their analogs.Kanamycinbiosynthetic intermediates and their analogs produced by recombinant strains ofS. venezuelaeharboring diverse combinations of aminoglycoside biosynthetic genesets were extracted from the fermentation broth using the OASIS MCX (Waters)solid-phase extraction cleanup procedure (described in Supplementary Methods)and were then analyzed by HPLC-ESI-MS/MS; samples were separated on an XerraMS C18column (50 2.1 mm, 3.5 m, Waters) interfaced with a Waters/MicromassQuattro micro/MS instrument tracing by MS/MS using a gradient of acetonitrile and10 mM heptafluorobutyric acid (Fluka) over 45 min. racing was done by MS/MSoperated in multiple-reaction monitoring mode. Quantification was conducted bychoosing mass pairs specific for the selected analytes to detect the transition fromparent ion to product ion: 163 > 84 for 1;366 > 163 for 2;324 > 163 for 3and 10;323 > 163 for 4; 486 > 163 for 5,12and14; 485 > 163 for 6, 8, and 13; 484 > 163 for7; 325 > 163 for 9; 487 > 163 for 11; 586 > 264 for15; 468 > 163 for 16; 587 > 264 for17;307 > 163 for 18; and 469 > 163 for 19and 20. hree separate cultivations andindependent extractions were performed.

Isolation and structural identification of kanamycin biosynthetic intermediates andtheir analogs.Details regarding the isolation and characterization of products obtainedfrom in vivosamples are described in Supplementary Methods. he retention behav-iors of products on HPLC-ESI-MS/MS and data including MS/MS and NMR spectraare described in Supplementary Figures 221and Supplementary Tables 318.

In vitroreactions using cell-free extracts.CFEs of S. venezuelaewere prepared byglass-bead homogenization (described in Supplementary Methods). he resultingCFEs were suspended in ris buffer (pH 7.5) containing 100 mM ris-HCl (pH 7.6),10 mM MgCl2, 6 mM 2-mercaptoethanol and 1 mM phenylmethylsulfonyl fluoride(Sigma), and their protein concentrations were corrected. KanF glycosyltransferreactions were initiated by adding 100 M 1to the CFE obtained from a recom-binant host expressing only kanFand 200 M UDP-Glc (Sigma), UDP-GlcNAc(GeneChem) or UDP-GlcN (described in Supplementary Methods) as the cosub-strate. Reactions to determine the activity of KanI-KacL on 6 -amination were carriedout by supplementing CFE from the recombinant host expressing kanI-kacLwith100 M of the 6-hydroxy pseudodisaccharides or pseudotrisaccharides 3, 6, 9or 12.
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ARTICLENATURE CHEMICAL BIOLOGYDOI: 10.1038/NCHEMBIO.671

After incubation for 2 h at 30 C, the reaction was quenched with ice-cold phenol/chloroform/isoamyl alcohol (25:24:1; Sigma) and centrifuged at 18,000gfor 5 min.he supernatant containing the product of interest was extracted using OASIS MCXSPE cleanup (described in Supplementary Methods), reconstituted with 100 l ofwater, and was then analyzed by HPLC-ESI-MS/MS as described above.

Reactions to examine the activity of KanC-KanD were sequentially conductedusing CFE from two different recombinant strains of S. venezuelaeharboring eitherkanEor kanC-kanD. CFE containing KanE (or KanC-KanD) was incubated with200 M UDP-Glc plus 100 M 3or 4under the same conditions described above,and then the reaction was quenched. Supernatants containing products obtained fromthe first reaction were incubated for 2 h at 30 C with CFE containing KanC-KanD

(or KanE), and then the reaction was quenched again. he resulting supernatant wassubjected to the same cleanup procedure described above and was then analyzed byHPLC-ESI-MS. Sequential reactions using both CFEs were also carried out using thepseudodisaccharides9and 10.Independent experiments were performed in duplicate.

Kinetic analysis of KanF and KanE.KanF (6 M) was incubated with 1and UDP-Glc or UDP-GlcNAc, respectively, in 100 l reaction buffer (75 mM ris-HCl,10 mM MgCl2and 1 mg ml

1BSA) at 37 C for 1 h. For the measurement of theKmvalue for 1, the concentration of UDP-Glc and UDP-GlcNAc was fixed at 3 mM,while the concentration of 1was varied from 0.05 mM to 1.5 mM. In addition, todetermine the kinetic parameters of UDP-Glc or UDP-GlcNAc, the concentrationof 1was maintained at 1.5 mM, while the concentration of UDP-Glc and UDP-GlcNAc was varied from 1.0 mM to 6.0 mM. On the other hand, KanE (30 M)was incubated with3and UDP-Glc or UDP-Kns (described in SupplementaryMethods), respectively, in the same buffer as described above. For the determina-tion of the Kmfor 3, the concentration of UDP-Glc and UDP-Kns was kept at1.0 mM, while the concentration of 3was varied from 0.2 mM to 1.0 mM. he

parameters for UDP-Glc or UDP-Kns were determined by varying their concentra-tions from 0.2 to 2.0 mM, while the concentration of 3was fixed at 1.0 mM. Allreactions were quenched, extracted using SPE and finally subjected to HPLC-ESI-MSanalysis as described above. Independent experiments were performed in triplicate.

Measurement of MIC of kanamycin biosynthetic intermediates and theiranalogs.MICs of various kanamycin-related pseudodisaccharides and pseudo-trisaccharides as well as AHBA-conjugated analogs (except16, 18, 19and20)were determined using the microdilution method of the Clinical and LaboratoryStandard Institute44. Gram-negative E. coliand P. aeruginosatype strains and clini-cal isolates were grown at 30 C in Mueller-Hinton broth. Serial two-fold dilutionsof aminoglycosides were carried out to give final concentrations between 0.25 g ml1and 128 g ml1, and an aliquot of water was used as a negative control. he growthof test strains was monitored at 600 nm using a Labsystems Bioscreen C reader,and the MIC was determined as the lowest concentration of the aminoglycosidediluted in broth medium that inhibited the growth of the test bacterium.

Computational methods.Details regarding homology modeling, docking and

molecular dynamics simulation are described in Supplementary Methods.

Received 24 June 2010; accepted 28 July 2011;published online 9 October 2011

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(R0A-2008-000-20030-0) and Global Frontier Program for Intelligent Synthetic

Biology through the National Research Foundation of Korea (NRF), NRF grants

(2010-0001487 and 2010-0028193) funded by the Ministry of Education, Science &

echnology and the Marine and Extreme Genome Research Center Program of t he

Ministry of Land, ransportation and Maritime Affairs, Republic of Korea. J.W.P.

gratefully acknowledges a grant (20100623) from the echnology Development

Program for Agriculture and Forestry, Ministry for Food, Agriculture and Fisheries,

Republic of Korea.

Author contributions

J.W.P., S.R.P., J.K.S. and Y.J.Y. designed research and wrote the paper; J.W.P., S.R.P.,K.K.N., A.R.H., Y.H.B., Y.J.Y., E.J.K., E.M.K. and D.K. performed research; and J.W.P.,

S.R.P. and Y.J.Y. analyzed data.

Competing financial interestshe authors declare competing financial interests: details accompany the fu ll-text HML

version of the paper at http://www.nature.com/naturechemicalbiology/.

Additional informationSupplementary information and chemical compound information is available online at

http://www.nature.com/naturechemicalbiology/. Reprints and permissions information

is available online at http://www.nature.com/reprints/index.html. Correspondence and

requests for materials should be addressed to J.K.S. or Y.J.Y.

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