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Identification and Interrogation of the Herbicidin Biosynthetic Gene Cluster:
First Insight into the Biosynthesis of a Rare Undecose Nucleoside Antibiotic
Geng-Min Lin, Anthony Romo, Priscilla Liem, Zhang Chen, and Hung-wen Liu
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b08985 • Publication Date (Web): 07 Nov 2017
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1
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7 Identification and Interrogation of the Herbicidin Biosynthetic
8
9
Gene Cluster: First Insight into the Biosynthesis of a Rare
10 Undecose Nucleoside Antibiotic
11
12 Geng-Min Lin,1,§ Anthony J. Romo,2,§,† Priscilla H. Liem,3 Zhang Chen,1 Hung-wen Liu1,2,3,*
13
14 1 2
Department of Chemistry; Division of Chemical Biology and Medicinal Chemistry, College of Pharmacy; and
15 3
Department of Biochemistry, University of Texas at Austin, Austin, TX 78712, USA
16
17
18
Supporting Information Placeholder
19
20 while a hemiketal linkage between pyran and furan subunits
ABSTRACT: Herbicidins are adenosine-based nucleoside
21 has been reported in several polyketide-derived natural
antibiotics with an unusual tricyclic undecose core decorated
22 7
products such as the ionophore monensin A, spliceostatin
with a (5-hydroxy)tiglyl moiety. Feeding studies are herein
23 reported demonstrating that the tricyclic core is derived
8 9
FR901464 and phorboxazole A, a similar linkage has also
24 from D-glucose and D-ribose, whereas the tiglyl moiety is been reported in the carbohydrate-based metabolite spec-
10
25 derived from an intermediate of L-isoleucine catabolism. tinomycin. The potential for a carbohydrate-based origin of
26 Identification of the gene cluster for herbicidin A biosynthe- the herbicidins is of particular interest, because undecosyl
27 sis in Streptomyces sp. L-9-10 as well as its verification by skeletons (highlighted in Figure 1) are rare among carbohy-
11,12 13
28 heterologous expression in a non-producing host are de- drate-based natural products, with the tunicamycins and
14
29 scribed, and the results of in vitro characterization of a car- hikizimycin being the only other known examples. Moreo-
30 boxyl methyltransferase encoded in the cluster, Her8, are ver, a carbohydrate-based origin would raise additional ques-
31 presented. Based on these observations, a biosynthetic path- tions as to how the methylene-bridged linkage between the
32 way is proposed for herbicidins. A and C rings might be introduced.
33
34
35
Herbicidins (1a through 1k, Figure 1) are adenosine-
36
derived nucleoside antibiotics that have been isolated from
37 1
Streptomyces saganonensis, Streptomyces sp. L-9-10, and
2
38 3
Streptomyces scopuliridis RB72. Herbicidin variants differ
39 with respect to methylation at the C2′-OH and C11′-COOH
40 functionalities as well as the short, branched-chain fatty acid
41 attached at C8′ via an ester linkage. The most extensively-
42 decorated herbicidin reported to date is herbicidin A (1a),
43 which features a 5-hydroxytiglyl group (2b) at C8′. Herbi-
44 cidins show selective herbicidal activity toward dicotyle-
45 donous plants and inhibit the germination of Chinese cab-
1b
46 bage and rice seeds. Furthermore, they can protect rice
1b 1c
47 plants from leaf blight and also exhibit antialgal and anti-
1d 4
48 fungal activities. However, their biological mode of action
49 and biosynthetic origins remain unclear.
50 The characteristic tricyclic core of herbicidins is composed
51 of a pyran (ring-C) connected to a furan (ring-A) via a meth-
52 ylene bridge and a hemiketal linkage to form a third six-
Figure 1. Structures of herbicidins.
53 membered heterocycle (ring-B). This tricyclic furano-pyrano-
54 pyran structure locks all of the ring-C substituents into an The tiglyl and 5-hydroxytiglyl (2a and 2b) esters present in
1 5
55 axial conformation (similar to C4), and its reconstruction some herbicidin variants are also atypical substituents
6
has drawn the interest of synthetic chemists. While the among bacterial natural products. Tiglyl (2a) along with the
56
ether and hemiketal linkages in the herbicidin core structure isomeric angelyl (2d) groups are more commonly found in
57 7
are suggestive of a ladderether polyketide, an alternative plant metabolites such as 3β-tigloyloxytropane, where they
58 hypothesis is that the furano-pyrano-pyran heterocycle is are derived from the corresponding CoA esters produced via
59 constructed from carbohydrate precursors. For example, 15
the catabolism of L-isoleucine. However, the only known
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angelyl ester substituent in bacterial metabolites is found in that closely matched these predictions was identified using
18,19
1 SF2575 and heliosupine, where it is hypothesized to be of the antiSMASH server. An attempt to locate genes encod-
16 17 15b
2 polyketide origin. Likewise, there has been little work ing potential O-tiglyl transferases in the genome was simi-
3 done to determine whether 2a and 2b originate from the larly unsuccessful.
4 catabolism of L-isoleucine or arise though polyketide- methyltransferase
unknown enzyme
5 medicated biosynthetic pathways.
6 To investigate the biosynthesis of herbicidins, conditions O
H 5' H
11' 10' O O 1' N
7 were first optimized for the production and isolation of MeO 6' 4'
3' 2' N
NH2
9' 7'
8 herbicidin A (1a). Ha and coworkers previously identified HO
8'
O N
OH H OMe N
9 growth conditions that optimized the production of 1a from O O
3b
10 Streptomyces scopuliridis. Building on this, it was found methyltransferase
that reducing the culture time and simplifying the chroma- epimerase
11 Me OH
oxidoreductase
12 tographic steps further improved the isolable yield of 1a from acyltransferase
13 cultures of Streptomyces sp. L-9-10 to 50–100 mg/L compared monooxygenase
2
14 to 0.135 mg/L as previously reported (see Section S2). Using
these conditions, cultures of Streptomyces sp. L-9-10 were fed Figure 3. Enzyme activities initially predicted to be associat-
15 13 15
with [U- C, N]isoleucine, and herbicidin A was isolated pri- ed with the herbicidin A biosynthetic pathway given D-
16 13
or to C NMR analysis. As shown in Figure 2, isoleucine is glucose and D-ribose building blocks for the herbicidin core
17 incorporated into 2b as an intact entity. In contrast, no sig- structure.
18 13
nificant C enrichment of 2b was observed when the same
19 13 A partial match for the predicted gene cluster was identi-
experiment was performed with sodium [2- C]acetate. These
20 fied, however, when a single locus across the entire genome
results indicated that biosynthesis of the 2b moieties in
21 was found to include two genes annotated as encoding me-
herbicidin A follows a path similar to what is observed in
22 13 thyltransferases. As these methyltransferases may be respon-
plant metabolites. Likewise, analysis of C NMR of herbicidin
23 sible for catalyzing methylation of the C2′-OH and C11′-
A isolated from the Streptomyces sp. L-9-10 culture fed with
13 COOH, the surrounding genomic context was analyzed to
24 [5- C]ribose suggested a ribosyl origin of the C5′ carbon of
reveal approximately twelve clustered genes (denoted her)
25 ring-B in herbicidin A. Additional feeding experiments with
13 that loosely matched the original predictions (see Figure 5).
26 uniquely-labeled glucose, [U- C]ribose, and [Me-
13 Homologous clusters were also found in two other species of
27 C]methionine also revealed that ring-A in herbicidin A is
Streptomyces, including the herbicidin-producing strain S.
28 derived from D-ribose, the carbons of ring-C are from D-
scopuliridis RB72 (see Section S4), suggesting that the her
29 glucose, and the two methyl groups are from L-methionine
cluster may indeed be responsible for herbicidin biosynthe-
30 (see Section S3).
sis. Interestingly, O-methyltransferase-encoding genes have
31 been used to locate other natural product biosynthetic gene
32 clusters. For example, the identification of three genes in
33 close proximity to each other encoding predicted O-
34 methyltransferases helped to locate the griseofulvin gene
20
35 cluster. In another case, degenerate primers based on O-
36 methyltransferases led to the cloning of the caprazamycin
21
37 gene cluster.
38 0.50
39
0.40
40
1a
41 0.30 1b
42 13 AU D: B + C
43 Figure 2. Selected C NMR spectra of herbicidin A isolated 0.20
C: standard
from cultures of Streptomyces sp. L-9-10 fed with (A) no ad-
44 13 13 0.10 B: Sa-L8B17
ditive, (B) sodium [2- C]acetate, and (C) [U- C,
45 15
N]isoleucine. Carbon signals from 2b are labeled with their A: Sa-pOJ446
46 numberings.
0.00
0 5 10 15 20 25
47 retention time (minutes)
48 Based on the results from the feeding studies, glucuronic
49 acid and adenosine derivatives were predicted to be building Figure 4. HPLC traces of (A) 10-day extract of Sa-pOJ446, (B)
50 blocks for the herbicidin core. If correct, then the herbicidin 10-day extract of Sa-L8B17, (C) herbicidin standard, and (D)
51 gene cluster was expected to encode at least one epimerase co-injection of the samples in traces B and C.
52 for C3 inversion of the adenosine derivative (C3′ in herbi-
cidins), an oxidoreductase for C2 oxidation of the glucuronic
53 To determine the biological function of the her cluster, the
acid derivative (C7′ in herbicidins), one or more enzymes for
54 cosmid L8B17, which included the her cluster, was isolated
concatenation of the two units, an acyltransferase for instal-
55 lation of the tiglyl moiety, a monooxygenase for the oxida-
22
from a pOJ446-based cosmid library of Streptomyces sp. L-
56 tion of the tiglyl moiety, and two methyltransferases as 9-10 (see Section S5). Intergeneric conjugal transfer of cos-
57 shown in Figure 3. However, when the genome of Streptomy- mid L8B17 from Escherichia coli S17-1 to the non-producing
58 ces sp. L-9-10 was sequenced (see Section S1.4), no cluster heterologous host Streptomyces albus J1074 was carried out
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to generate strain Sa-L8B17. The same conjugal transfer was Decoration of the core is proposed to be catalyzed by the
1 also performed in parallel using the empty pOJ446 vector to ketosynthase homolog Her3 (see Section S6), the two puta-
2 yield the strain Sa-pOJ446 as a negative control. The positive tive methyltransferases Her8 and Her10, and the annotated
3 exoconjugants were cultured under producing conditions, P450 hydroxylase Her11. Herbicidin A (1a) is proposed to be
4 and the production of herbicidins A (1a) and B (1b) was only hydrolyzed to herbicidin B (1b) by the serine hydrolase hom-
5 observed in the culture of Sa-L8B17 as shown in Figure 4. The olog Her9, because 1b accumulates later than 1a in cultures
6 identities of the observed products were established by co- of Streptomyces sp. L-9-10 (see Section S2). Alternatively,
7 elution with herbicidin standards on HPLC (Figure 4, trace substrate promiscuity of the decorating enzymes may explain
8 D) and electrospray ionization-mass spectrometry (ESI-MS) the production of 1b and other herbicidins. Consequently,
9 of the collected HPLC peaks (see Section S5). These results the unassigned gene product Her2 may be responsible for
imply that the her cluster encodes the enzymes responsible generating 1i through 1k that are only produced by Strepto-
10
for the biosynthesis of herbicidins. myces sp. L-9-10, since no homolog of Her2 was identified in
11
the corresponding clusters of other producing strains (see
12 8
Section S4). Finally, the absence of polyketide modules in the
13 1 2 3 4 5 6 7 9 10 11 12
her cluster is consistent with the feeding results indicating
14 her
that the tiglyl moiety is derived from the catabolism of L-
15 1 kb isoleucine.
16 core assembly regulator/transporter
It was hypothesized that one of the two methyltransferases,
17 peripheral modification resistance gene?
Her8 or Her10, is responsible for catalyzing the methylation
18 1 XRE transcriptional regulator 7 Inositol 2-dehydrogenase of the C11′-carboxylate of the herbicidins. To test this hy-
19 2 NAD-dependent epimerase 8 methyltransferase pothesis, the putative substrates 9 and 10 were prepared by
20 3 ketoacyl-ACP synthase III 9 serine hydrolase
hydrolyzing herbicidin A using LiOH. Her8 and Her10 were
4 SAH hydrolase 10 methyltransferase
21 5 ABC transporter 11 P450 overexpressed in E. coli and purified as N-His6-tagged and
22 6 B12-radical SAM 12 LuxR transcriptional regulator maltose-binding protein (MBP)-fusion constructs, respec-
23 tively. The MBP fused to Her10 was subsequently cleaved
24 using TEV protease (see Section S7.2). A solution of 100 μM
25 of 9 or 10 was incubated with 1 μM Her8 or Her10 in the
26 presence of 0.5 mM S-adenosyl-L-methionine (SAM) in po-
27 tassium phosphate buffer (pH 6.5, 50 mM) at room tempera-
28 ture for 24 h. Among all the combinations, only Her8 was
29 able to convert 9 to herbicidin A (1a) as shown in Figure 6,
30 which was confirmed by co-injection with a standard and
LC-ESI-MS analysis (see Section S7.3). All assays excluding
31
Her8 showed no consumption of 9 or 10. These observations
32
suggested that Her8 is the C11′-O-methyltransferase. Fur-
33 thermore, 10 was not methylated in the presence of Her8 and
34 SAM suggesting that esterification of C8′ occurs prior to
35 methylation of C11′. The observation of equilibrium between
36 the hemiketal and free carbonyl forms of 9 and 10. (see Sec-
37 tion S7.1) is consistent with the proposed nonenzymatic cy-
Figure 5. Annotated her gene cluster responsible for the bio-
38 clization.
synthesis of herbicidin A. A biosynthetic pathway consistent
39 with the gene annotations is proposed beginning with UDP-
40 glucuronic acid and adenosine; however, other immediate
41 precursors are possible.
42
43 A possible biosynthetic pathway for herbicidins based on
44 sequence analysis of the genes in the her cluster is shown in
45 Figure 5. The tricyclic core is proposed to form by the cou-
pling of a D-ribose derivative such as adenosine (4) and a D-
46
glucose derivative such as UDP-glucuronic acid (3) in a reac- 0.50 SAM / SAH
47
tion catalyzed by Her6, which is annotated as a B12- 9
1a
48 dependent radical SAM enzyme. This hypothesis is similar to
0.40
* F :9 + Her8
49 the proposed reaction for the putative radical-SAM enzyme 0.30 E: 9 + Her10
50 TunB, which may catalyze the addition of a 5′-uridyl radical
AU D: 9 + buffer
0.20 10
51 23
to an exo-glycal during biosynthesis of tunicamycins. Sub- C: 10 + Her8
52 0.10 B: 10 + Her10
sequent C3′-epimerization may be mediated by the SAH hy-
53 A: 10 + buffer
drolase homolog Her4 (see Section S6). Oxidation of the 0.00
0 10 20
54 resulting undecose (7 → 8) may then be catalyzed by the
retention time (minutes)
55 putative dehydrogenase Her7 predisposing the resulting in-
56 termediate 8 to non-enzyme-catalyzed hemiketal formation Figure 6. Assays for the methyltransferase activity of Her8
57 and generation of the tricyclic herbicidin core. and Her10 showing HPLC chromatograms following incuba-
58 tion of substrate with the gene product and SAM as indicated
59
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next to each trace. The peak labeled with an asterisk is 5′- (3) (a) Choi, C. W.; Choi, J.-S.; Ko, Y. K.; Kim, C.-J.; Kim, Y. H.;
1 deoxy-5′-methylthioadenosine from the degradation of Oh, J. S.; Ryu, S. Y.; Yon, G. H. Bull. Korean Chem. Soc. 2014,
2 SAM.
24 35, 1215–1217. (b) Ha, S.; Lee, K. J.; Lee, S. I.; Gwak, H. J.; Lee,
J.-H.; Kim, T.-W.; Choi, H.-J.; Jang, J.-Y.; Choi, J.-S.; Kim, C.-
3
In summary, feeding results demonstrate that the precur- J.; Kim, J.-C.; Kim, H. H.; Park, H. W. J. Microbiol. Biotechnol.
4 2017, 27, 947–955.
sors for herbicidin biosynthesis are D-glucose, D-ribose, L-
5 isoleucine, and L-methionine. This establishes a carbohy- (4) Won, O. J.; Kim, Y. T.; Choi, J. S.; Oh, T.-K.; Shinoki, Y.; Park,
6 drate origin for the herbicidin core and an amino acid origin K.W. J. Fac. Agr., Kyushu Univ. 2016, 61, 47–51.
7 (5) Terahara, A.; Haneishi, T.; Arai, M.; Hata, T.; Kuwano, H.;
for the tiglyl and 5-hydroxytiglyl functionalities. The biosyn- Tamura, C. J. Antibiot. (Tokyo) 1982, 35, 1711–1714.
8 thetic gene cluster was identified in the draft genome and (6) (a) Ichikawa, S.; Shuto, S.; Matsuda, A. J. Am. Chem. Soc.
9 subsequently verified by production of herbicidins in a non- 1999, 121, 10270–10280. (b) Hager, D.; Mayer, P.; Paulitz, C.;
10 producing host expressing this cluster. This was further sub- Ti´ebes, J.; Trauner, D. Angew. Chem. Int. Ed. 2012, 51, 6525–
11 stantiated by the successful reconstitution of Her8 activity in 6528.
12 vitro. To the best of our knowledge, this is the first example (7) Gallimore, A. R. Nat. Prod. Rep. 2009, 26, 266–280.
of a bacterial pathway that may borrow tiglyl-CoA (5) from (8) Eustáquio, A. S.; Janso, J. E.; Ratnayake, A. S.; O’Donnell, C.
13
J.; Koehn, F. E. Proc. Natl. Acad. Sci. U.S.A. 2014, 111, E3376–
14 the catabolism of L-isoleucine to modify a secondary metabo-
E3385.
15 lite. Only a small number of undecoses have been discovered (9) Searle, P. A.; Molinski, T. F. J. Am. Chem. Soc. 1995, 117, 8126–
16 to date, and our understanding of their biosynthesis is still in 8131.
17 its infancy. Nevertheless, our work not only represents a ma- (10) Wiley, P. F.; Argoudelis, A. D.; Hoeksema, H. J. Am. Chem.
18 jor advancement towards future studies characterizing herbi- Soc. 1963, 85, 2652–2659.
cidin biosynthesis but also lays the foundation for identifica- (11) Lin, C.-I.; McCarty, R. M.; Liu, H.-w. Chem. Soc. Rev. 2013, 42,
19 4377–4407.
tion of related nucleoside natural product gene clusters in
20 (12) Elshahawi, S. I.; Shaaban, K. A.; Kharel, M. K.; Thorson, J. S.
hitherto unexplored microbial genomes.
21 Chem. Soc. Rev. 2015, 44, 7591–7697.
22 (13) (a) Takatsuki, A.; Arima, K.; Tamura, G. J. Antibiot. (Tokyo)
ASSOCIATED CONTENT 1971, 24, 215–223. (b) Takatsuki, A.; Tamura, G. J. Antibiot.
23
Supporting Information (Tokyo) 1971, 24, 224–231.
24 (14) Uchida, K.; Ichikawa, T.; Shimauchi, Y.; Ishikura, T.; Ozaki,
25 Details regarding the experimental procedures, molecular cloning, A. J. Antibiot. (Tokyo) 1971, 24, 259–262.
26 enzyme assays, and spectroscopic characterization of new com- (15) (a) Leete, E. Planta Med. 1979, 36, 97–112. (b) Rabot, S.; Peer-
27 pounds. This material is available free of charge via the Internet at less, A. C.; Robins, R. J. Phytochemistry 1995, 39, 315–322.
http://pubs.acs.org. (16) (a) Pickens, L. B.; Kim, W.; Wang, P.; Zhou, H.;Watanabe, K.;
28
Gomi, S.; Tang, Y. J. Am. Chem. Soc. 2009, 131, 17677–17689.
29 AUTHOR INFORMATION (b) Inahashi, Y.; Shiraishi, T.; Palm, K.; Takahashi, Y.; Ōmu-
30 ra, S.; Kuzuyama, T.; Nakashima, T. Chembiochem 2016, 17,
31 Corresponding Author 1442–1447.
32 *h.w.liu@mail.utexas.edu (17) Yoshikawa, H.; Takiguchi, Y.; Terao, M. J. Antibiot. (Tokyo)
33 1983, 36, 30–35.
Present Addresses (18) Weber, T.; Blin, K.; Duddela, S.; Krug, D.; Kim, H. U.; Bruc-
34 † coleri, R.; Lee, S. Y.; Fischbach, M. A.; Mller, R.; Wohlleben,
Present address: University of Texas M.D. Anderson Cancer
35 W.; Breitling, R.; Takano, E.; Medema, M. H. Nucleic Acids
Center, Investigational Pharmacy Services, Houston, TX 77030.
36 Res. 2015, 43, W237–W243.
37 Author Contributions (19) The cluster was not identified by antiSMASH 3.0. However, a
38 §
G.-M.L. and A.J.R. contributed equally. recent version of antiSMASH 4.0 did find the cluster dis-
cussed below as a putative fatty-acid type gene cluster using
39
ClusterFinder algorithm.
40 Notes
(20) Chooi, Y.-H.; Cacho, R.; Tang, Y. Chem. Biol. 2010, 17, 483–
41 The authors declare no competing financial interests. 494.
42 (21) Kaysser, L.; Lutsch, L.; Siebenberg, S.; Wemaker, E.; Kam-
ACKNOWLEDGMENT merer, B.; Gust, B. J. Biol. Chem. 2009, 284, 14987–14996.
43
(22) Bierman, M.; Logan, R.; O’Brien, K.; Seno, E. T.; Rao, R. N.;
44 We thank Prof. Julian Davies at The University of British Colum-
Schoner, B. E. Gene 1992, 116, 43–49.
45 bia for generously providing Streptomyces sp. L-9-10 used in this
(23) Wyszynski, F. J.; Lee, S. S.; Yabe, T.; Wang, H.; Gomez-
46 study. This work was supported by grants from the National Insti-
Escribano, J. P.; Bibb, M. J.; Lee, S. J.; Davies, G. J.; Davis, B.
tutes of Health (GM035906) and the Welch Foundation (F-1511).
47 G. Nat. Chem. 2012, 4, 539–546.
48 (24) Iwig, D. F.; Booker, S. J. Biochemistry, 2004, 43, 13496–13509.
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17 her
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Herbicidin Gene Cluster
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21 NH2 HO O
22 OH OH
HO
23 O OH

24 NH2 O OH
HO
25 S
OH
HO OH
26 O
OH
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