Characterization of the Two Methylation Steps Involved in the

Article pubs.acs.org/jnp

Characterization of the Two Methylation Steps Involved in the Biosynthesis of Mycinose in Tylosin Eunji Kim,† Myoung Chong Song,† Myoun Su Kim,† Ji Yoon Beom,† Eun Yeol Lee,‡ Dong-Myung Kim,§ Sang-Jip Nam,† and Yeo Joon Yoon*,† †

Department of Chemistry and Nanoscience, Ewha Womans University, Seoul 03760, Republic of Korea Department of Chemical Engineering, Kyung Hee University, Gyeonggi-do 17104, Republic of Korea § Department of Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon 34134, Republic of Korea ‡

S Supporting Information *

ABSTRACT: The S-adenosyl-L-methionine-dependent Omethyltransferases TylE and TylF catalyze the last two methylation reactions in the tylosin biosynthetic pathway of Streptomyces f radiae. It has long been known that the TylEcatalyzed C2‴-O-methylation of the 6-deoxy-D-allose bound to demethylmacrocin or demethyllactenocin precedes the TylFcatalyzed C3‴-O-methylation of the D-javose (C2‴-O-methylated 6-deoxy-D-allose) attached to macrocin or lactenocin. This study reveals the unexpected substrate promiscuity of TylE and TylF responsible for the biosynthesis of D-mycinose (C3‴-O-methylated D-javose) in tylosin through the identification of a new minor intermediate 2‴-O-demethyldesmycosin (2; 3‴-methyl-demethyllactenocin), which lacks a 2‴-O-methyl group on the mycinose moiety of desmycosin, along with 2‴-Odemethyltylosin (1; 3‴-methyl-demethylmacrocin) that was previously detected from the S. f radiae mutant containing a mutation in the tylE gene. These results unveil the unique substrate flexibility of TylE and TylF and demonstrate their potential for the engineered biosynthesis of novel glycosylated macrolide derivatives.

T

transferases TylE and TylF were purified from S. f radiae and shown to sequentially methylate the C2‴- and C3‴-hydroxy groups of the 6-deoxy-D-allose moiety of demethylmacrocin and demethyllactenocin, yielding tylosin and desmycosin via macrocin and lactenocin, respectively (Figure 1).13,14 All the Dmycinose biosynthetic genes of S. f radiae were also cloned and sequenced.15 It has been shown using purified enzymes that both TylE and TylF possess high substrate specificity and that the C2‴-O-methylation of the 6-deoxy-D-allose moiety catalyzed by TylE generating D-javose (C2‴-O-methylated 6deoxy-D-allose) is a prerequisite for the subsequent C3‴-Omethylation by TylF needed to generate D-mycinose (C3‴-Omethylated D-javose).13,14 This methylation order is consistent with the biosynthetic pathway of mycinamicin IV reported recently. The purified recombinant MycE, a TylE homologue, methylates the C2″-hydroxy group of 6-deoxy-D-allose, converting mycinamicin VI to mycinamicin III, and the TylF homologue MycF catalyzes the second C3″-O-methylation of 16 D-javose giving mycinamicin IV. However, trace amounts of 2‴-O-demethyltylosin (1; 3‴-methyl-demethylmacrocin), which lacks the C2‴-O-methyl group on the mycinose moiety of tylosin, were detected by thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC)17 from

ylosin is a 16-membered macrolide antibiotic composed of a polyketide lactone (tylactone) and three deoxyhexose sugars (D-mycaminose, L-mycarose, and D-mycinose) (Figure 1) and is produced by several Streptomyces species including Streptomyces f radiae,1 Streptomyces rimosus,2 and Streptomyces hygroscopicus.3 It has been used in veterinary medicine to treat Gram-positive bacterial infections and as an animal growth promoter in the swine industry. The tylosin biosynthetic gene cluster of S. f radiae was identified and sequenced,4,5 and its biosynthetic pathway was studied mainly by analysis of the compounds produced by blocked mutants of S. f radiae, cosynthesis, and bioconversion experiments.6−9 Tylactone, generated by the polyketide synthases TylG1−TylG5, is first glycosylated at the C5-hydroxy group with D-mycaminose by the glycosyltransferase TylM2.10 Hydroxylation/dehydrogenation of the C20-methyl group to a formyl group and the hydroxylation of the C23-methyl group are catalyzed by the cytochrome P450s TylI and TylH1, respectively, to generate Omycaminosyltylonolide.4 Subsequent addition of 6-deoxy-Dallose to the C23-oxymethylene group and L-mycarose to the hydroxy group on C4′ of D-mycaminose are respectively catalyzed by the glycosyltransferases TylN11 and TylC512 yielding their respective compounds demethyllactenocin and demethylmacrocin in a preferred but not obligatory order. In the final steps of tylosin biosynthesis, the 6-deoxy-D-allose moiety is converted to D-mycinose via bis-O-methylation. The two S-adenosyl-L-methionine (SAM)-dependent O-methyl© XXXX American Chemical Society and American Society of Pharmacognosy

Received: March 24, 2016

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DOI: 10.1021/acs.jnatprod.6b00267 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Figure 1. Biosynthetic pathway from tylactone to tylosin. The bold and normal lines represent the preferred and alternative pathways, respectively. The dashed arrows indicate the new biosynthetic pathways found in this study.

the S. f radiae mutant GS16 strain6 containing a mutation in the tylE gene. Furthermore, expression of an additional copy of the tylF gene in this mutant produced 2‴-O-demethyltylosin (1) in appreciable quantities.17 These results indicate that C3‴-Omethylation by TylF can occur prior to the C2‴-O-methylation by TylE although an efficient methylation by TylF requires a previous methylation of the C2‴-hydroxy group by TylE. The modification of sugar moieties can significantly alter the biological properties of a macrolide, so enzymatic or combinatorial biosynthetic approaches have been employed to generate structurally diverse glycosylated derivatives of macrolides.18,19 Particularly, the common O-methylation of hydroxy groups on deoxysugar moieties catalyzed by SAMdependent methyltransferases can protect the reactive hydroxy group from undesired modification and change the solubility or pharmacokinetic properties of the resulting molecules.20 Therefore, characterizing and exploiting substrate-flexible Omethyltransferases involved in the modification of deoxysugar moieties will increase the structural diversity of deoxysugarcontaining macrolides and allow for the generation of new bioactive molecules.

The present study was undertaken to detail the sequence of the O-methylation of the 6-deoxy-D-allose moiety and to generate new tylosin derivatives. Confirmation of the presence of a previously poorly characterized intermediate 2‴-Odemethyltylosin (1) and the identification of the previously undescribed 2‴-O-demethyldesmycosin (2; 3‴-methyl-demethyllactenocin) through bioconversion experiments using Streptomyces venezuelae mutant strains allowed us to uncover the remarkable substrate promiscuity of TylE and TylF catalyzing the biosynthesis of the D-mycinose moiety. The unique and substrate-flexible O-methyltransferases involved in these deoxysugar modifications can provide a potential tool for the combinatorial biosynthesis of novel macrolide derivatives containing diverse sugar moieties.

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RESULTS AND DISCUSSION It has long been known that TylE-catalyzed C2‴-O-methylation of the 6-deoxy-D-allose moiety normally precedes the TylFcatalyzed C3‴-O-methylation.8,13,14 However, to confirm whether TylF can catalyze C3‴-O-methylation of the 6deoxy-D-allose moiety of demethyllactenocin instead of the Djavose in lactenocin, we constructed S. venezuelae mutant strains B



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which can transfer TDP-6-deoxy-D-allose to the exogenously fed O-mycaminosyltylonolide and further modify either the 6deoxy-D-allose moiety via 2‴- or 3‴-O-methylation as well as both. S. venezuelae YJ028, in which the entire pikromycin biosynthetic gene cluster was deleted from the wild-type S. venezuelae ATCC 15439,21 was used for the construction of mutant strains. The high-copy-number Escherichia coli− Streptomyces shuttle vector pSE3422 containing the constitutive ermE* promoter23 and a thiostrepton resistance marker was used for expressing the genes involved in the biosynthesis of the tylosin biosynthetic intermediates. Plasmid pYJ646 was constructed to express the genes encoding the TDP-6-deoxyD-allose biosynthetic enzymes (Des3, Des4, TylJ, and TylD), allosyltransferase (TylN), and 2‴-O-methyltransferase of the 6deoxy-D-allose moiety (TylE). Plasmids pYJ647 and pYJ648 contain the same gene set except for the methyltransferase: pYJ647 contains 3‴-O-methyltransferase (TylF) instead of TylE and pYJ648 contains both TylE and TylF (Figure 1; Table S1). For the biosynthesis of the common deoxysugar biosynthetic intermediate TDP-4-keto-6-deoxy-D-glucose, the glucose-1-phosphate thymidylyl transferase gene des3 and glucose-4,6-dehydratase gene des4 of S. venezuelae ATCC 15439 were used24 instead of the corresponding genes tylA1 and tylA2, respectively, in the tylosin biosynthetic gene cluster of S. fradiae.15 These engineered replicative plasmids were separately introduced into S. venezuelae YJ028, to provide YJ028/pYJ646, YJ028/pYJ647, and YJ028/pYJ648, respectively. A list of the strains and plasmids used in this study is presented in the Table S1. To validate the efficiency of the bioconversion system, Omycaminosyltylonolide was fed to a culture of YJ028/pYJ646 at a concentration of 20 mg/L. As expected, a peak corresponding to lactenocin was observed at a retention time of 19.6 min with m/z = 758 (Figure 2A) in the HPLC-ESI-MS analysis. This peak was predicted to be lactenocin and upon MS/MS spectrometry fragmented into its characteristic ions at m/z = 598, corresponding to O-mycaminosyltylonolide, and m/z = 174, corresponding to the D-mycaminose moiety from the parent ion (Figure 2B). A small peak corresponding to demethyllactenocin was also observed at a retention time of 18.1 min with m/z = 744 and fragmented into characteristic ions at m/z = 598 and 174 (Figure S1). The respective retention times and MS/MS fragmentation patterns of standard lactenocin and demethyllactenocin were identical to those of lactenocin and demethyllactenocin produced by the mutant strain YJ028/pYJ646 (Figure S2E,F). Approximately 60% of Omycaminosyltylonolide was converted to lactenocin. These results proved that our bioconversion system biosynthesizes and transfers TDP-6-deoxy-D-allose to the fed O-mycaminosyltylonolide and efficiently methylates the 6-deoxy-D-allose moiety. The organic extracts obtained from the strain YJ028/pYJ647 fed with O-mycaminosyltylonolide were analyzed by HPLCESI-MS, and a peak with retention time 20.4 min and m/z = 758 was detected along with demethyllactenocin (Figures 2C and S1F−J). The fact that the m/z and MS/MS fragmentation pattern of this peak were the same as those of lactenocin (Figure 2D) suggested that this peak corresponded to the previously undescribed shunt metabolite of the tylosin biosynthetic pathway, 2‴-O-demethyldesmycosin (2). The conversion yield of O-mycaminosyltylonolide to compound 2 was approximately 25%. Relatively larger amounts of demethyllactenocin were detected compared to the case of

Figure 2. HPLC-ESI-MS/MS analysis of tylosin intermediates obtained from the bioconversion of O-mycaminosyltylonolide. (A) HPLC-ESI-MS chromatogram selected for m/z = 598 and 758 corresponding to O-mycaminosyltylonolide (▼) and lactenocin (■), respectively, of culture extracts from YJ028/pYJ646 supplemented with O-mycaminosyltylonolide. (B) MS/MS spectrum of lactenocin from YJ028/pYJ646 supplemented with O-mycaminosyltylonolide. (C) HPLC-ESI-MS chromatogram selected for m/z = 598 and 758 corresponding to O-mycaminosyltylonolide (▼) and 2‴-O-demethyldesmycosin (2) (◊), respectively, of culture extracts from YJ028/ pYJ647 supplemented with O-mycaminosyltylonolide. (D) MS/MS spectrum of 2‴-O-demethyldesmycosin (2) from YJ028/pYJ647 supplemented with O-mycaminosyltylonolide. (E) HPLC-ESI-MS chromatogram selected for m/z = 598 and 772 corresponding to Omycaminosyltylonolide (▼) and desmycosin (⧫), respectively, of culture extracts from YJ028/pYJ648 supplemented with O-mycaminosyltylonolide. (F) MS/MS spectrum of desmycosin from YJ028/ pYJ648 supplemented with O-mycaminosyltylonolide. OMT, Omycaminosyltylonolide; myc, mycaminose.

YJ028/pYJ646 (Figure S1). To isolate a sufficient quantity of this new metabolite 2 for NMR analysis, the supernatant of a 200 mL culture of S. venezuelae YJ028/pYJ647 supplemented with 4 mg of O-mycaminosyltylonolide was extracted with ethyl acetate. The obtained extract was evaporated, and the resultant brown residue was fractionated by semipreparative HPLC and then further purified by analytical HPLC to yield pure compound 2 (0.6 mg). The molecular formula for 2, C38H63NO14, was deduced from the interpretation of the protonated peak at m/z 758.4327 [M + H]+. The structure of 2 was determined from the analysis of 1D and 2D NMR spectroscopic data (Table 1; Figures S3−S8). The 1H NMR spectrum (Figure S3) showed characteristic signals for the Omycaminosyltylonolide skeleton: an aldehyde proton [δ 9.69 (1H, s)], the double bond protons [δ 7.32 (1H, d, J = 15.0 Hz, C



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Table 1. NMR Spectroscopic Data (600 MHz, CDCl3) for Compounds 1 and 2 2‴-O-demethyltylosin (1) position

δC, type

1

173.9, C

2

39.3, CH2

3 4 5 6

66.9, 41.0, 81.9, 31.2,

7

32.8, CH2

8 9 10 11 12 13 14 15

44.9, CH 203.2, C 118.5, CH 148.2, CH 135.0, C 142.2, CH 45.0, CH 75.2, CH

16

25.8, CH2

17 18

8.9, CH3 9.5, CH3

19

43.7, CH2

20 21 22

203.1, CH 17.7, CH3 13.1, CH3

23

69.0, CH2

1′ 2′ 3′ 4′ 5′ 6′ 7′ 1″

103.4, CH 71.7, CH 68.6, CH 74.9, CH 72.8, CH 19.0, CH3 41.9, CH3 95.4, CH

2″

40.8, CH2

3″ 4″ 5″ 6″ 7″ 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ 8‴

69.8, C 76.5, CH 65.9, CH 18.4, CH3 25.4, CH3 101.1, CH 76.6, CH 79.8, CH 73.0, CH 70.6, CH 17.8, CH3 61.8, CH3

CH CH CH CH

2‴-O-demethyldesmycosin (2)

δH (J in Hz)

δC, type

δH (J in Hz)

173.8, C 2.49, 1.95, 3.81, 1.62, 3.72, 1.09, 1.60, 1.19, 2.61,

brd (15.0) brd (15.0) m m brd (9.0) m m m m

39.4, CH2 66.3, 40.8, 81.4, 31.6,

CH CH CH CH

32.7, CH2

6.27, d (15.0) 7.32, d (15.0) 5.89, 2.93, 4.97, 1.87, 1.59, 0.92, 0.98, 2.95, 2.39, 9.63, 1.19, 1.79, 3.99, 3.55, 4.23, 3.52, 2.40, 3.25, 3.24, 1.25, 2.02, 5.01, 2.01, 1.74,

d (10.2) m ddd (7.8, 7.8, 2.4) dqd (14.4, 7.2, 2.4) dqd (14.4, 7.2, 6.0) dd (7.2, 7.2) d (7.2) brdd (10.2, 6.6) m brs d (7.2) s dd (9.0, 4.0) dd (9.0, 6.0) d (7.2) dd (7.2, 8.4) dd(7.8, 8.4) dd (8.4, 7.8) qd (7.2, 8.4) d (7.2) s brs brd (13.8) dd (13.8, 3.6)

2.94, 4.05, 1.27, 1.22, 4.55, 3.41, 3.71, 3.21, 3.49, 1.25, 3.60,

d (8.4) qd (6.0, d (6.0) s d (7.2) dd (7.2, dd (3.0, dd (8.4, qd (7.2, d (7.2) s

44.7, CH 203.1, C 118.1, CH 148.0, CH 135.0, C 142.2, CH 45.0, CH 75.0, CH

2.50, 1.94, 3.83, 1.63, 3.72, 1.10, 1.59, 1.19, 2.61,

brd (15.0) brd (15.0) m m brd (9.0) m m m m

6.27, d (15.0) 7.32, d (15.0)

104.0, CH 71.1, CH 70.4, CH 71.0, CH 73.3, CH 17.7, CH3 41.7, CH3

5.91, 2.93, 4.98, 1.88, 1.60, 0.94, 1.01, 2.97, 2.42, 9.69, 1.21, 1.79, 3.99, 3.55, 4.24, 3.47, 2.39, 3.07, 3.27, 1.26, 2.52,

d (10.2) m ddd (7.8, 7.8, 2.4) dqd (14.4, 7.2, 2.4) dqd (14.4, 7.2, 6.0) dd (7.2, 7.2) d (7.2) brdd (10.2, 6.6) m brs d (7.2) s dd (9.0, 4.0) dd (9.0, 6.0) d (7.2) dd (7.2, 8.4) dd (8.4, 8.4) dd (9.0, 8.4) qd (7.2, 9.0) d (7.2) s

100.8, CH 77.0, CH 79.8, CH 73.0, CH 70.9, CH 17.9, CH3 61.7, CH3

4.54, 3.43, 3.73, 3.20, 3.51, 1.26, 3.61,

d (7.2) dd (7.2, dd (3.0, dd (8.4, qd (7.2, d (7.2) s

25.4, CH2 8.9, CH3 9.6, CH3 43.8, CH2 202.9, CH 17.3, CH3 12.9, CH3 69.1, CH2

8.4)

3.0) 3.0) 3.0) 8.4)

H-11), 6.27 (1H, d, J = 15.0 Hz, H-10), 5.91 (1H, d, J = 10.2 Hz, H-13)], an anomeric proton [δ 4.24 (1H, d, J = 7.2 Hz, H1′)], two nitrogenated methyl singlets [δ 2.52 (6H, s, H-7′)], and five methyl signals [δ 1.79 (3H, s, H-22), 1.26 (3H, d, J = 7.2 Hz, H-6′), 1.21 (3H, d, J = 7.2 Hz, H-21), 1.01 (3H, d, J = 7.2 Hz, H-18), 0.94 (3H, dd, J = 7.2, 7.2 Hz, H-17)] (Table 1). The 1H NMR spectrum of 2 also displayed an anomeric proton [δ 4.54 (1H, d, J = 7.2 Hz, H-1‴)], four oxygenated methine

3.0) 3.0) 3.0) 8.4)

protons [δ 3.73 (1H, dd, J = 3.0, 3.0, H-3‴), 3.51 (1H, dq, J = 8.4, 7.2, H-5‴), 3.43 (1H, dd, J = 7.2, 3.0, H-2‴), 3.20 (1H, dd, J = 8.4, 3.0, H-4‴)], one oxygenated methyl singlet [δ 3.61 (3H, s, H-8‴)], and one methyl doublet [δ 1.26 (3H, d, J = 7.2 Hz, H-6‴)]. The 13C NMR and HSQC spectroscopic data (Figures S4 and S6) indicated 38 signals including 9 methyl, 5 methylene, 21 methine, 1 dialkylated olefinic, and 2 carbonyl carbons. In particular, the 13C NMR and HSQC spectroscopic D



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MS/MS fragmentation pattern of this compound were identical to those of standard desmycosin (Figure S2D). Production of the new tylosin biosynthetic shunt metabolite 2‴-O-demethyldesmycosin (2) through the unexpectedly somewhat promiscuous substrate specificity of TylF prompted us to re-examine if TylF can also recognize demethylmacrocin as a substrate and generate 2‴-O-demethyltylosin (1), which was characterized previously only by TLC and HPLC.17 Because purified authentic demethylmacrocin was available, bioconversion of demethylmacrocin using the mutant strains YJ028/pYJ766 and YJ028/pYJ767 expressing TylE or TylF, respectively, was carried out. The organic extract of YJ028/pYJ766 fed with demethylmacrocin at a concentration of 20 mg/L was analyzed by HPLCESI-MS, and as expected, a peak with m/z = 902 corresponding to macrocin was observed at a retention time of 24.9 min (Figure 3A) in a conversion yield of approximately 93%. The

data [δ 202.9/9.69 (C-20/H-20), 203.1 (C-9), and 173.8 (C1)] suggested the presence of aldehyde, ketone, and ester functional groups, respectively. The 13C NMR and HSQC spectroscopic data [δ 148.0/7.32 (C-11/H-11), 142.2/5.91 (C13/H-13), 135.0 (C-12), 118.1/6.27 (C-10/H-10), 104.0/4.24 (C-1′/H-1′), and 100.8/4.54 (C-1‴/H-1‴)] also supported the presence of two double bonds and two sugar moieties, respectively. Further interpretation of 2D NMR spectroscopic data permitted the construction of two substructures: Omycaminosyltylonolide and O-methylated 6-deoxy-D-allose. The O-mycaminosyltylonolide was established from the analysis of COSY and HMBC correlations (Figures S5 and S7). COSY cross-peaks [H-17/H-16/H-15/H-14/H-13, H-14/ H-23, H-10/H-11, H-2/H-3/H-4/H-5/H-6/H-7/H-8, H-18/ H-4, H-8/H-21, H-6/H-19] allowed the construction of three fragments with 6, 2, and 10 carbon units, respectively. The connectivity of three units was secured from long-range HMBC correlations. HMBC correlations from methyl singlet H-22 to carbons C-11, C-12, and C-13 and from methyl doublet H-21 and olefinic proton H-10 to carbon C-9 provided the attachment of C-11/C-12/C-13 and C-8/C-9/C-10, respectively. The ester linkage between C-1 and C-15 was also established from the three-bond HMBC correlation of H-15 to C-1. COSY cross-peaks [H-1′/H-2′/H-3′/H-4′/H-5′/H-6′] and HMBC correlations from the two methyl singlets H-7′ to carbon C-3′ provided the assignment of the mycaminose moiety. These data allowed the establishment of Omycaminosyltylonolide. The anomeric proton showed a doublet at δ 4.54 with a coupling constant (J) of 7.2 Hz, indicating the β-configuration of O-methylated 6-deoxy-D-allose moiety. An O-methylated 6-deoxy-D-allose moiety was also constructed from the interpretation of COSY and HMBC correlations. COSY cross-peaks [H-1‴/H-2‴/H-3‴/H-4‴/H5‴/H-6‴] and a three-bond HMBC correlation from methyl singlet H-8‴ to carbon C-3‴ permitted the establishment of the O-methylated 6-deoxy-D-allose moiety. Lastly, observation of a three-bond correlation from anomeric proton H-1‴ to carbon C-23 completed the assignment of the structure for 2 as shown in Figure 1. These results unequivocally clarified the position of the methoxy group on C3‴ of 2‴-O-demethyldesmycosin (2), indicating that TylF-catalyzed C3‴-O-methylation of the 6deoxy-D-allose moiety can precede the C2‴-O-methylation catalyzed by TylE when TylF is overexpressed in the absence of TylE and that TylF possesses a certain degree of substrate flexibility allowing it to produce the previously unknown shunt metabolite 2‴-O-demethyldesmycosin (2) even though previous studies have shown that TylF has a narrow substrate specificity and is unable to methylate demethyllactenocin even under in vitro conditions.13 We did not evaluate the activity of 2‴-O-demethyldesmycosin (2) because while C20-modified derivatives of desmycosin such as tilmicosin (20-deoxo-20-(3,5dimethylpiperidin-1-yl)desmycosin) exhibit good efficacy in vivo25 and have been developed for use in veterinary medicine desmycosin and its C20-unmodified derivatives showed almost no activity in vivo and no improved activities compared to tylsoin.26 Thus, it seems unlikely that the C20-unmodified 2‴O-demethyldesmycosin (2) would be active. Recombinant YJ028/pYJ648 harboring both TylE and TylF and supplemented with O-mycaminosyltylonolide produced desmycosin at a conversion yield of approximately 80%. Desmycosin was detected at a retention time of 23.9 min with m/z = 772 (Figure 2E) and fragmented into characteristic ions at m/z = 598 and 174 (Figure 2F). The retention time and

Figure 3. HPLC-ESI-MS/MS analysis of tylosin intermediates obtained from the bioconversion of demethylmacrocin. (A) HPLCESI-MS chromatogram selected for m/z = 888 and 902 corresponding to demethylmacrocin (▽) and macrocin (●), respectively, of culture extracts from YJ028/pYJ766 supplemented with demethylmacrocin. (B) MS/MS spectrum of macrocin from YJ028/pYJ766 supplemented with demethylmacrocin. (C) HPLC-ESI-MS chromatogram selected for m/z = 888 and 902 corresponding to demethylmacrocin (▽) and 2‴-O-demethyltylosin (1) (○), respectively, of culture extracts from YJ028/pYJ767 supplemented with demethylmacrocin. (D) MS/MS spectrum of 2‴-O-demethyltylosin (1) from YJ028/pYJ767 supplemented with demethylmacrocin. mcr, mycarose; myc, mycaminose.

MS/MS spectrum of this ion corresponding to macrocin showed fragment ions at m/z = 758 for the loss of L-mycarose and at m/z = 174 corresponding to D-mycaminose (Figure 3B). Standard macrocin showed the same retention time and MS/ MS fragmentation patterns (Figure S2B). To investigate whether 2‴-O-demethyltylosin (1) can be synthesized by the action of TylF, demethylmacrocin was supplemented to the culture of YJ028/pYJ767. Following incubation, a compound with mass corresponding to 2‴-O-demethyltylosin (1) was detected at a retention time of 25.5 min in the HPLC-ESI-MS analysis (Figure 3C). This peak showed an m/z and MS/MS fragmentation pattern to identical those of macrocin (Figure 3D) but was eluted at a different retention time from macrocin, suggesting that this compound is the tylosin biosynthetic shunt metabolite 2‴-O-demethyltylosin (1). The conversion yield of E



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demethylmacrocin to 2‴-O-demethyltylosin (1) was approximately 18%. Compound 1 (0.9 mg) was isolated from the 300 mL culture of YJ028/pYJ767 fed with 6 mg of demethylmacrocin, and the structure elucidation of 1 was performed (Table 1; Figures S9−S14). The molecular formula for 1, C45H75NO17, was established from the interpretation of the protonated peak at m/z 902.5113 [M + H]+. The 1H and 13C NMR spectroscopic data (Figures S9 and S10) of 1 had features similar to those of 2 except that 1 displayed additional protons [δ 5.01 (1H, brs, H-1″), 4.05 (1H, qd, J = 6.0, 8.4 Hz, H-5″), 2.94 (1H, d, J = 8.4 Hz, H-4″), 2.01 (1H, brd, J = 13.8 Hz, H2″a), 1.74 (1H, dd, J = 13.8, 3.6 Hz, H-2″b), 1.27 (3H, d, J = 6.0 Hz, H-6″), 1.22 (3H, s, H-7″)] and carbon signals [δ 95.4 (C-1″), 76.5 (C-4″), 69.8 (C-3″), 65.9 (C-5″), 40.8 (C-2″), 25.4 (C-7″), 18.4 (C-6″)], respectively (Table 1). The observation of COSY cross-peaks [H-1″/H-2″, H-4″/H-5″/ H-6″] and HMBC correlations (Figures S11 and S13) from methyl singlet H-7″ to carbons C-2″, C-3″, and C-4″ indicated that 1 had one additional sugar, a mycarose moiety. The configuration of the α-mycarose anomeric carbon was confirmed with the broad singlet signal of the anomeric proton signal (H-1″) (Figure S12). The observation of a three-bond HMBC correlation from anomeric proton H-1″ to carbon C-4′ provided the ether linkage between C-4′ and C-1″. Interpretation of 2D NMR spectroscopic data (Figures S11− S13) allowed the structure of 1 to be assigned as shown in Figure 1. To investigate whether TylE also possesses some degree of substrate flexibility and is able to catalyze the C2‴-Omethylation reaction in the presence of the methoxy group on C-3‴ and because only small amounts of 2‴-Odemethyldesmycosin (2) were available as substrate, in vitro bioconversion of 2‴-O-demethyldesmycosin (2) to desmycosin was carried out with the cell-free extracts of YJ028/pYJ766. Incubation of lactenocin with cell-free extracts of YJ028/ pYJ767 expressing TylF produced desmycosin with a conversion yield of approximately 90%, verifying the functionality of the cell-free extract system (Figure 4A). In this reaction of cell-free extracts derived from YJ028/pYJ766, approximately 45% of the 2‴-O-demethyldesmycosin (2) was converted to desmycosin (Figure 4B). Similarly, 2‴-O-demethyltylosin (1) was converted to tylosin by YJ028/pYJ766 at a conversion yield of approximately 50% (Figure 4C). These results prove that TylE is able to catalyze the C2‴-O-methylation of 2‴-Odemethyldesmycosin (2) and 2‴-O-demethyltylosin (1) in contrast to the previous studies.14 Our results clearly demonstrate that the sequence of the two O-methylation reactions catalyzed by TylE and TylF is interchangeable in vitro or under the artificial conditions in which TylF is overexpressed in the absence of TylE. In addition, trace amounts of the shunt metabolites 2‴-Odemethyltylosin (1) and 2‴-O-demethyldesmycosin (2) were detected by HPLC-ESI-MS analysis of the culture extract of wild-type S. f radiae ATCC 19609 (Figure S15). Taken together, the previous report on the production of 2‴-Odemethyltylosin (1) from the tylE-mutated S. f radiae GS16 strain17 and our results suggest that there are two independent shunt biosynthetic routes to desmycosin and tylosin mediated by the substrate promiscuity of the two methyltransferases (Figure 1). While the conversion yields in this study are not quantitative, they demonstrate that TylE prefers demethylmacrocin/ demethyllactenocin as substrates over 2‴-O-demethyltylosin

Figure 4. HPLC-ESI-MS analysis of tylosin intermediates obtained from the bioconversion of lactenocin, 2‴-O-demethyldesmycosin (2), or 2‴-O-demethyltylosin (1). (A) HPLC-ESI-MS chromatogram selected for m/z = 758 and 772 corresponding to lactenocin (■) and desmycosin (⧫), respectively, of cell-free extracts from YJ028/ pYJ767 incubated with lactenocin. (B) HPLC-ESI-MS chromatogram selected for m/z = 758 and 772 corresponding to 2‴-Odemethyldesmycosin (2) (◊) and desmycosin (⧫), respectively, of cell-free extracts from YJ028/pYJ766 incubated with 2‴-O-demethyldesmycosin (2). (C) HPLC-ESI-MS chromatogram selected for m/z = 902 and 916 corresponding to 2‴-O-demethyltylosin (1) (○) and tylosin (◎), respectively, of cell-free extracts from YJ028/pYJ766 incubated with 2‴-O-demethyltylosin (1).

(1)/2‴-O-demethyldesmycosin (2) and that TylF has a preference for macrocin/lactenocin over demethylmacrocin/ demethyllactenocin. Therefore, it is likely that the newly found biosynthetic routes to desmycosin and tylosin via 2‴-Odemethyldesmycosin (2) and 2‴-O-demethyltylosin (1), respectively, are minor tylosin biosynthetic pathways. This is probably the reason why previous studies13,14 using the TylE and TylF purified from S. f radiae did not detect the production of these minor biosynthetic intermediates. In contrast to the highly homologous MycE and MycF involved in the biosynthesis of the D-mycinose moiety in mycinamicin, TylE and TylF exhibit unique substrate flexibility and would be useful for the combinatorial biosynthesis of novel macrolide derivatives containing diverse sugar moieties as exemplified by the production of 2‴-O-demethyltylosin (1) and the previously unknown 2‴-O-demethyldesmycosin (2).

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EXPERIMENTAL SECTION

General Experimental Procedures. NMR spectra were acquired using a Varian INOVA 600 spectrometer operating at 600 MHz for 1H and 150 MHz for 13C nuclei. Chemical shifts are given in ppm using tetramethylsilane (TMS) as an internal reference. All NMR data processing was done using Mnova software (Mestrelab Research S.L.). Samples for NMR analysis were prepared by dissolving each compound in 250 μL of CDCl3 (Sigma) and placing the solutions in 5 mm Shigemi advanced NMR microtubes (Sigma) matched to the solvent. HPLC-ESI-MS/MS spectra using the previously reported method for the analysis of tylosin derivatives21 were recorded on a Waters/Micromass Quattro micro MS interface consisting of a Waters 2695 separation module connected directly to a Micromass Quattro micro MS. HR-ESI-MS was recorded on a ZEVO G2-S qTOF (Waters, USA). HPLC purification was done using semipreparative Kromasil 1005C8 column (250 mm × 10 mm, 5 μm) and analytical Kromasil 1005C18 column (250 mm × 4.6 mm, 5 μm) on an YL9100 HPLC system (YL Instrument Co. Ltd., Korea) consisting of a YL9110 gradient pump coupled with a YL9160 photo diode array detector with 1024 channels. F



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Bacterial Strains, Culture Conditions, Genetic Manipulation, and Chemicals. S. venezuelae YJ02821 was used for the construction of mutant strains. The transformants of S. venezuelae were selected on R2YE agar plates27 supplemented with thiostrepton. The S. venezuelae strains were propagated on SPA medium.27 S. venezuelae ATCC 15439 grown in SGGP medium27 and S. f radiae ATCC 19609 grown in tryptic soy broth at 30 °C were used for the preparation of genomic DNA. Transformation of DNA into protoplasts of S. venezuelae was performed according to the Streptomyces standard protocols.27 Genetic manipulations of plasmids were carried out in E. coli DH10B according to standard procedures.28 Litmus 28 (New England Biolabs) was used for subcloning. The high-copy-number E. coli−Streptomyces shuttle vector pSE3422 was used for expressing the genes involved in the biosynthesis of the tylosin biosynthetic intermediates. The purified tylosin biosynthetic intermediates used in this study as substrates for bioconversion or as standard compounds for structural characterization, O-mycaminosyltylonolide, demethylmacrocin, desmycosin, macrocin, and lactenocin, were obtained from Professor Eric Cundliffe (University of Leicester, UK). Construction of Expression Plasmids and Mutant Strains. Plasmid pYJ646 was constructed for the biosynthesis of TDP-6-deoxyD-allose (des3, des4, tylJ, and tylD) as well as for its transfer (tylN) and 2‴-O-methylation (tylE). Plasmid pYJ647 carrying the genes encoding the TDP-6-deoxy-D-allose biosynthetic enzymes, allosyltransferase, and 3‴-O-methyltransferase (tylF) was constructed in an identical manner. Plasmid pYJ648 which contains the des3, des4, tylJ, tylD, tylN, tylE, and tylF genes was also constructed similarly. tylE or tylF was individually introduced into the pSE34 generating pYJ766 and pYJ767, respectively. Details regarding the construction of these plasmids are described in the Supporting Information. These plasmids were separately introduced into S. venezuelae YJ028 (Table S1). Bioconversion and HPLC-ESI-MS Analysis of the Tylosin Biosynthetic Intermediates. Mutant strains were cultured in 50 mL of SCM liquid medium (1.5% soluble starch, 2% soytone, 0.01% CaCl2, 0.15% yeast extract, and 1.05% MOPS, pH 7.2) at 30 °C for 24 h under appropriate antibiotic selection. One milligram of Omycaminosyltylonolide or demethylmacrocin was added into each culture and then incubated for an additional 48 h. The resulting culture broth was extracted using one volume of ethyl acetate and concentrated with methanol. The relative amount of each compound produced was compared using the peak intensity obtained from the LC-MS chromatogram. The production level of each compound was calculated by averaging the yield from five separate cultivations and extractions. In Vitro Assays of TylE/TylF Activity Using Cell-Free Extracts. Mycelium were cultivated in 200 mL of SCM medium for 3 days at 30 °C, harvested by centrifugation, washed twice with 0.1 M Tris-HCl (pH 7.6), and then resuspended in 5 mL of reaction buffer (50 mM Tris-HCl, 10 mM MgCl2, 6 mM 2-mercaptoethanol, 1 mM PMSF (phenylmethanesulfonyl fluoride), 0.2 mM SAM, pH 7.6) at 4 °C. Cell-free extracts of the S. venezuelae mutant strains were prepared by glass bead homogenization as previously described.29 The entire process was carried out at 4 °C and generated 3 mL of supernatant. The methyltransferase reaction was initiated by adding 0.1 mg of substrate [lactenocin, 2‴-O-demethyldesmycosin (2) or 2‴-Odemethyltylosin (1)] dissolved in 1 mL of reaction buffer to 1 mL of the cell-free extracts. Reaction mixtures were incubated at 30 °C for 2 h before quenching with 15 mL of phenol−chloroform−isoamyl alcohol (25:24:1, Sigma) that had been cooled on ice. After centrifugation, reaction mixtures were extracted and subjected to HPLC-ESI-MS analysis as described above. Independent experiments were performed in triplicate. Extraction, Isolation, and Identification. The whole culture broth of strain S. venezualae YJ028/pYJ647 or YJ028/pYJ767 grown in SCM medium supplemented with 20 mg/L of O-mycaminosyltylonolide or demethylmacrocin, respectively, was centrifuged, and the supernatant layer was extracted with ethyl acetate. The obtained extract was evaporated and the resultant brown residue was fractionated by semipreparative HPLC employing 45% aqueous acetonitrile as the mobile phase with a flow rate of 4 mL/min. The

fraction containing 2 or 1 was purified by analytical HPLC employing 45% aqueous acetonitrile (0.05% TFA) as the mobile phase with a flow rate of 1 mL/min to yield the pure compound. 2‴-O-Demethyldesmycosin (2). Pale yellow powder; IR (KBr) 3665, 3350, 2980, 1736, 1210, 1028; positive HR-ESI-(qTOF)-MS m/ z 758.4327 [M + H]+ (calcd for C38H64NO14+, 758.4321) 2‴-O-Demethyltylosin (1). Pale yellow powder; IR (KBr) 3670, 3380, 2930, 1742, 1210, 1026; positive HR-ESI-(qTOF)-MS m/z 902.5113 [M + H]+ (calcd for C45H76NO17+, 902.5108)

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00267. Experimental details; tables for bacterial strains, plasmids, and primers used in this study; and 1D and 2D NMR spectra of 1 and 2 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +82-2-3277-4446. Fax: +82-2-3277-3419. E-mail: [email protected]. Author Contributions

E.K. and M.C.S. contributed equally to this paper. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Prof. Eric Cundliffe for providing tylosin intermediates and Dr. Kris Rathwell for critically reading this manuscript. This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (MISP) (2016R1A2A1A05005078), the Intelligent Synthetic Biology Center of the Global Frontier Project funded by MISP (20110031961), Advanced Production Technology Development Program, Ministry of Agriculture, Food and Rural Affairs, and High Value-added Food Technology Development Program, Ministry of Agriculture, Food and Rural Affairs, Republic of Korea. M.C.S. was supported by RP-Grant 2016 from Ewha Womans University.

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DEDICATION Dedicated to the memory of Eun Ai Choi. REFERENCES

(1) McGuire, J. M.; Boniece, W. S.; Higgens, C. E.; Hoehn, M. M.; Stark, W. M.; Westhead, J.; Wolfe, R. N. Antibiot. Chemother. (Washington, D.C.) 1961, 11, 320−327. (2) Pape, H.; Brillinger, G. U. Arch. Microbiol. 1973, 88, 25−35. (3) Jensen, A. L.; Darken, M. A.; Schultz, J. S.; Shay, A. J. Antimicrob. Agents Chemother. (1961-70) 1963, 161, 49−53. (4) Cundliffe, E.; Bate, N.; Butler, A.; Fish, S.; Gandecha, A.; MersonDavies, L. Antonie van Leeuwenhoek 2001, 79, 229−234. (5) Fishman, S. E.; Cox, K.; Larson, J. L.; Reynolds, P. A.; Seno, E. T.; Yeh, W. K.; Van Frank, R.; Hershberger, C. L. Proc. Natl. Acad. Sci. U. S. A. 1987, 84, 8248−8252. (6) Baltz, R. H.; Seno, E. T. Antimicrob. Agents Chemother. 1981, 20, 214−225. (7) Baltz, R. H.; Seno, E. T. Annu. Rev. Microbiol. 1988, 42, 547−574. (8) Baltz, R. H.; Seno, E. T.; Stonesifer, J.; Wild, G. M. J. Antibiot. 1983, 36, 131−141.

G



Article

(9) (a) Omura, S.; Sadakane, N.; Matsubara, H. Chem. Pharm. Bull. 1982, 30, 223−229. (b) Omura, S.; Tanaka, H.; Tsukui, M. Biochem. Biophys. Res. Commun. 1982, 107, 554−560. (10) Melançon, C. E., 3rd; Takahashi, H.; Liu, H. W. J. Am. Chem. Soc. 2004, 126, 16726−16727. (11) Wilson, V. T.; Cundliffe, E. Gene 1998, 214, 95−100. (12) Min, Y.; Lv, H.; Zheng, Y. Process Biochem. 2007, 42, 729−733. (13) Bauer, N. J.; Kreuzman, A. J.; Dotzlaf, J. E.; Yeh, W. K. J. Biol. Chem. 1988, 263, 15619−15625. (14) Kreuzman, A. J.; Turner, J. R.; Yeh, W. K. J. Biol. Chem. 1988, 263, 15626−15633. (15) (a) Bate, N.; Cundliffe, E. J. Ind. Microbiol. Biotechnol. 1999, 23, 118−122. (b) Fouces, R.; Mellado, E.; Díez, B.; Barredo, J. L. Microbiology 1999, 145, 855−868. (16) Li, S.; Anzai, Y.; Kinoshita, K.; Kato, F.; Sherman, D. H. ChemBioChem 2009, 10, 1297−1301. (17) Cox, K. L.; Seno, E. T.; Wild, G. M. U.S. Patent 5,063,155, 1991. (18) Park, S. R.; Han, A. R.; Ban, Y. H.; Yoo, Y. J.; Kim, E. J.; Yoon, Y. J. Appl. Microbiol. Biotechnol. 2010, 85, 1227−1239. (19) Song, M. C.; Kim, E.; Ban, Y. H.; Yoo, Y. J.; Kim, E. J.; Park, S. R.; Pandey, R. P.; Sohng, J. K.; Yoon, Y. J. Appl. Microbiol. Biotechnol. 2013, 97, 5691−5704. (20) Zubieta, C.; He, X. Z.; Dixon, R. A.; Noel, J. P. Nat. Struct. Biol. 2001, 8, 271−279. (21) Jung, W. S.; Han, A. R.; Hong, J. S.; Park, S. R.; Choi, C. Y.; Park, J. W.; Yoon, Y. J. Appl. Microbiol. Biotechnol. 2007, 76, 1373− 1381. (22) Yoon, Y. J.; Beck, B. J.; Kim, B. S.; Kang, H. Y.; Reynolds, K. A.; Sherman, D. H. Chem. Biol. 2002, 9, 203−214. (23) Schmitt-John, T.; Engels, J. W. Appl. Microbiol. Biotechnol. 1992, 36, 493−498. (24) Han, A. R.; Park, J. W.; Lee, M. K.; Ban, Y. H.; Yoo, Y. J.; Kim, E. J.; Kim, E.; Kim, B. G.; Sohng, J. K.; Yoon, Y. J. Appl. Environ. Microbiol. 2011, 77, 4912−4923. (25) Debono, M.; Willard, K. E.; Kirst, H. A.; Wind, J. A.; Crouse, G. D.; Tao, E. V.; Vicenzi, J. T.; Counter, F. T.; Ott, J. L.; Ose, E. E.; Omura, S. J. Antibiot. 1989, 42, 1253−1267. (26) Mutak, S.; Maršić, N.; Kramarić, M. D.; Pavlović, D. J. Med. Chem. 2004, 47, 411−431. (27) Kieser, T.; Bibb, M. J.; Buttner, M. J.; Chater, K. F.; Hoopwood, D. A. In Practical Streptomyces Genetics; John Innes Centre: Norwich, U.K., 2000. (28) Sambrook, J.; Fritsch, E. F.; Maniatis, T. In Molecular cloning: a laboratory manual, 3rd ed.; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, 2001. (29) Jung, W. S.; Kim, E.; Yoo, Y. J.; Ban, Y. H.; Kim, E. J.; Yoon, Y. J. Appl. Microbiol. Biotechnol. 2014, 98, 3701−3713.

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Characterization of the Two Methylation Steps Involved in the

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