Characterization of Three Tailoring Enzymes in Dutomycin

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Characterization of three tailoring enzymes in dutomycin biosynthesis and generation of a potent antibacterial analogue Lei Sun, Siyuan Wang, Shuwei Zhang, Lei Shao, Qian Zhang, Chad Skidmore, Cheng-Wei Tom Chang, Dayu Yu, and Jixun Zhan ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00245 • Publication Date (Web): 11 May 2016 Downloaded from http://pubs.acs.org on May 17, 2016

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Characterization of three tailoring enzymes in dutomycin biosynthesis and generation of a potent antibacterial analogue

Lei Sun,†,1 Siyuan Wang,† ,1 Shuwei Zhang,† Lei Shao,† Qian Zhang,‡ Chad Skidmore,† ChengWei Tom Chang,‡ Dayu Yu,†,║ Jixun Zhan†,§,*

†

Department of Biological Engineering, Utah State University, 4105 Old Main Hill, Logan, UT

84322-4105, USA ‡

Department of Chemistry and Biochemistry, Utah State University, 0300 Old Main Hill, Logan,

UT 84322-0300, USA ║

Department of Applied Chemistry and Biological Engineering, College of Chemical

Engineering, Northeast Dianli University, Jilin, Jilin 132012, China §

TCM and Ethnomedicine Innovation & Development Laboratory, School of Pharmacy, Hunan

University of Chinese Medicine, Changsha, Hunan 410208, China

1

These authors contributed to this work equally.


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ABSTRACT: The anthracycline natural product dutomycin and its precursor POK-MD1 were isolated from Streptomyces minoensis NRRL B-5482. The dutomycin biosynthetic gene cluster was identified by genome sequencing and disruption of the ketosynthase gene. Two polyketide synthase (PKS) systems are present in the gene cluster, including a type II PKS and a rare highly reducing iterative type I PKS. The type I PKS DutG repeatedly uses its active sites to create a 9carbon triketide chain that is subsequently transferred to the α-L-axenose moiety of POK-MD1 at 4"-OH to yield dutomycin. Using a heterologous recombination approach, we disrupted a putative methyltransferase gene (dutMT1) and two glycosyltransferase genes (dutGT1 and dutGT2). Analysis of the metabolites of these mutants revealed the functions of these genes and yielded three dutomycin analogues SW140, SW91 and SW75. The major product SW91 in Streptomyces minoensis NRRL B-5482-∆DutMT1 was identified as 12-desmethyl-dutomycin, suggesting that DutMT1 is the dedicated 12-methyltransferase. This was confirmed by the in vitro enzymatic assay. DutGT1 and DutGT2 were found to be responsible for the introduction of β-D-amicetose and α-L-axenose, respectively. Dutomycin and SW91 showed strong antibacterial activity against Staphylococcus aureus and methicillin-resistant S. aureus, whereas POK-MD1 and SW75 had no obvious inhibition, which revealed the essential role of the C-4" triketide chain in antibacterial activity. The minimal inhibitory concentration of SW91 against the two strains was 0.125 µg mL-1, lower than that of dutomycin (0.25 µg mL-1), indicating that the antibacterial activity of dutomycin can be improved through biosynthetic structural modification.



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INTRODUCTION

Infectious diseases remain a leading cause of death worldwide. Antibiotics play a critical role in treating bacterial infections and have saved numerous lives. Since the discovery of penicillin in 1928, more natural compounds with promising antibacterial activities, such as tetracycline and kanamycin, have been identified and used as anti-infectious drugs. However, extensive use of antibiotics also led to the emergence of drug resistance in microorganisms. For example, methicillin-resistant Staphylococcus aureus is considered a "superbug" because its ability to fight

off treatment, including the antibiotic methicillin. Spread of these antibiotic-resistant bacterial pathogens poses a serious threat to public health. Therefore, there remains an urgent need for new-generation antibiotics that can directly combat methicillin-resistant S. aureus and other pathogenic bacteria. Natural products are an important source of new drugs.1 In fact, many clinically used antibiotics are natural products, including tetracycline, penicillin, and vancomycin. Actinomycetes are a major group of producers of antibiotics. Continuous search for new antimicrobial molecules from nature is critical for development of new antibiotics to combat drug resistance. On the other hand, engineered biosynthesis can complement the time-consuming and resource-intensive discovery process, and represents an attractive approach to creating molecular diversity for drug discovery. Novel “unnatural” natural products can be obtained through direct genetic modification of the biosynthetic pathway in the producing strains or combinatorial biosynthesis in a heterologous host. Polyketides are a large group of medicinally important natural products, exemplified by lovastatin (anti-cholesterol),2 oxytetracycline (antibacterial),3 pradimicin A (antifungal and antiviral),4–8 and erythromycin (antibacterial).9 These molecules are assembled by polyketide synthases (PKSs) and associated tailoring enzymes


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through stepwise condensations of simple carboxylic acid precursors and further structural decorations.10 PKSs are classified into three types, including type I, II and III.11–13 PKSs represent a well-established system for engineered biosynthesis because of their ability to produce structurally and functionally diverse molecules.14 The purpose of this work is to discover and engineer the biosynthesis of new antibacterial agents. Here, we report the isolation and identification of the anthracycline antibiotic dutomycin (1, Figure 1a) and its precursor POK-MD1 (2, Figure 1a) from Streptomyces minoensis NRRL B5482. 1 was previously isolated from a strain of Streptomyces as an anticancer agent.15 In this work, the culture of S. minoensis NRRL B-5482 showed antibacterial activity and 1 was found to be a major metabolite responsible for this activity. The dutomycin biosynthetic gene cluster was then discovered by genome sequencing and confirmed by disrupting the ketosynthase gene. Functions of three tailoring enzymes were identified by targeted gene disruption and three dutomycin analogues (3–5, Figure 1a) were generated. Compounds 1–4 were tested for their antibacterial activity and 3 has better activity against S. aureus and methicillin-resistant S. aureus than the natural product 1 (Figure 1b). This work not only provides useful information about the formation of dutomycin and the structure-activity relationship (SAR), but also yields a potent antibacterial agent for further structure and activity optimization.

RESULTS AND DISCUSSION Isolation and Identification of Two Anthracyclines from S. minoensis NRRL B-5482. In

our initial screening for antibacterial microbial extracts, the ethyl acetate extract of S. minoensis NRRL B-5482 was tested against three bacterial strains including the Gram-negative bacterium Escherichia coli ATCC 25922 as well as the Gram-positive bacterial strains S. aureus ATCC 25923 and S. aureus ATCC 33591 (methicillin-resistant). This extract was found to be active



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against S. aureus and methicillin-resistant S. aureus, with the minimal inhibitory concentrations (MICs) of 500 and 1000 µg mL-1, respectively (Figure 1b). In contrast, it did not show any activity to E. coli (Figure 1B). HPLC analysis of the ethyl acetate extract of the culture of S. minoensis on YM agar at 460 nm showed a major product at 27.5 min (1) and a minor metabolite at 11.3 min (2), respectively (trace i, Figure 2). Both compounds shared the typical UV absorptions of anthracycline antibiotics (Supplementary Figure S1), suggesting that they are structurally related analogues. ESI-MS spectrum of 1 showed the [M-H]- ion peak at m/z 853.2 (Supplementary Figure S2), indicating that it has a molecular weight of 854, which is the same as that of dutomycin, an anticancer agent previously isolated from Streptomyces sp. 1725.15 The 1H and

13

C NMR data (Supplementary Figures S3a–S3d, Tables 1 and 2) were consistent with the

reported data, which confirmed that 1 is dutomycin (Figure 1A). ESI-MS analysis revealed that the molecular weight of 2 is 716 based on the [M-H]- peak at m/z 715.1 (Supplementary Figure S2), suggesting that 2 is a dutomycin derivative that lacks the C-4" side chain. This compound was previously found in the ∆pokMT1 mutant of the polyketomycin-producing strain Streptomyces diastatochromogenes Tü 6028, and was named POK-MD1. However, this compound was not structurally characterized. Thus, we conducted a NMR analysis for 2 (Supplementary Figures S3e and S3f). The

13

C NMR spectrum of 2 showed 35 carbon signals,

which are 9 carbons less than 1. A comparison of the spectra with those of 1 revealed that the carbon signals of the 9-carbon side chain at C-4" were missing. Accordingly, 2 was characterized as POK-MD1 shown in Figure 1a. The 1H and 13C NMR signals were assigned by a comparison with those of 1 and are shown in Tables 1 and 2, respectively. 1 and 2 are two anthracyclines that feature a tetracyclic quinone core structure and two sugar moieties, β-D-amicetose and α-L-axenose. The structures of 1 and 2 differ in the C-4" side chain.


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We propose that 2 serves as a biosynthetic intermediate in dutomycin biosynthesis. A time course analysis of the production of 1 and 2 was conducted (Supplementary Figure S4). The titer of 2 reached the highest (10.0 ± 0.7 mg L-1) at 4 days and then decreased with longer culture time, while the production of 1 kept increasing over the time. After 10 days, the production of 2 was not detected. This observation supports that 2 is a precursor of 1. The titer of 1 reached 82.5 ± 1.8 mg L-1 at 10 days. Identification of a Ketosynthase Gene Involved in Dutomycin Biosynthesis. The structures of 1 and 2 indicated that they are assembled through a type II polyketide biosynthetic pathway. We used a pair of degenerated primers, KSα-F and KSα-R,16 to amplify the KS gene fragment from the genome of S. minoensis NRRL B-5482. These primers were designed to amplify the conserved region of KS genes. A 0.6-kb fragment was obtained and ligated to pJET1.2. Sequencing of ten correct clones revealed three different KS genes that are homologous to reported type II KS genes in GenBank. These gene fragments were named ks1, ks2 and ks3, indicating that there are multiple type II polyketide biosynthetic gene clusters in this strain. To identify which ks gene is involved in dutomycin biosynthesis, we ligated these three fragments into temperature-sensitive E. coli-Streptomyces shuttle vector pKC1139 to yield the corresponding plasmids pLS54, pLS55 and pLS56 (Supplementary Table S1). These plasmids were transferred into S. minoensis NRRL B-5482 through conjugation. Analysis of the products of the mutants revealed that the production of 1 and 2 was abolished in S. minoensis NRRL B5482/pLS54 (trace ii, Figure 2), while the other two mutants still produced the two compounds (data not shown). Thus, ks1 was identified to be involved in dutomycin biosynthesis. Genome Sequencing and Discovery of the Dutomycin Biosynthetic Gene Cluster. To better understand this producing strain and locate the dutomycin (dut) biosynthetic gene cluster,



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the genomic DNA of S. minoensis NRRL B-5482 was extracted and sequenced, yielding approximately 10.4 Mb of gene sequence data (GC content of 70.1%). We then searched the sequencing data using ks1 that was found to be involved in dutomycin biosynthesis. A 47.5-kb region, which has 39 open reading frames (ORFs) (Figure 3a), was identified as the dut biosynthetic gene cluster. It was deposited into GenBank under accession number KP710956. The putative functions of these ORFs are given in Table 3. Many of the genes are homologous to the previously reported polyketomycin biosynthetic genes.17 A dutomycin biosynthetic pathway is proposed in Figure 3b. DutA, DutB and DutC are the ketosynthase (KS), chain length factor (CLF), and acyl carrier protein (ACP), respectively. They form the minimal PKS that generates the nascent poly-β-ketone chain from 10 units of malonylCoA. The ks1 fragment used for the gene disruption above is a part of dutA. Specific primers including DutA-Check1 and DutA-Check2 were then designed (Supplementary Table S2). Together with the vector-specific primers M13-47 and RM-V, the disruption of ks1 gene in S. minoensis NRRL B-5482/pLS54 was confirmed by PCR (Supplementary Figure S5), further supporting that ks1/dutA is involved in dutomycin biosynthesis. The dut biosynthetic gene cluster contains a variety of tailoring enzymes. The decaketide chain synthesized by the dut minimal PKS is cyclized by the immediate tailoring enzymes including DutD (aromatase), DutE (cyclase) and DutF (cyclase) to form the tetracyclic structure. Further decorations are done by additional tailoring enzymes such as oxygenases (DutO1–O4) and methyltransferases (DutMT1 and DutMT2) to afford the aglycone 5. Nine deoxysugar biosynthetic genes were found in the dut gene cluster and named dutS1–S9. These genes are proposed to synthesize the two NDP-deoxysugars, including NDP-D-amicetose and NDP-L-


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axenose from glucose-1-phosphate.18 Two glycosyltransferases (GTs), DutGT1 and DutGT2, then transfer the sugar moieties to 10-OH of 5 to yield 2. BLAST analysis of the amino acid sequence of DutG revealed that it is a highly reducing iterative type I PKS. Fungal PKS pathways such as those for macrolactones often contain a highly reducing iterative type I PKS that synthesizes a polyketide chain as a starter unit for its partner nonreducing PKS.19–22 To our best knowledge, this is the first time that a highly reducing iterative type I PKS was found in a bacterial PKS pathway. While polyketomycin shares a similar pathway with 1, there is a nonreducing iterative type I PKS named PokM1 in its pathway that synthesizes the tetraketide-derived 3,6-dimethylsalicylic acid moiety.17 This leads to the structural difference at C-4" between polyketomycin (Figure 1) and 1. DutG consists of six domains, including KS, AT (acyltransferase), DH (dehydratase), ER (enoyl reductase), KR (ketoreductase) and ACP. Analysis of the sequence of the AT domain showed the conserved GHSXG motif. Previous research reported that “X” in malonyl-specific ATs is usually a branched hydrophobic amino acid such as valine (V) or isoleucine (I), while ATs that have a less bulky residue such as glutamine (Q) or methionine (M) at this position select other substrates including methylmalonate.11,

23–25

ATDutG has a GHSMG motif, suggesting that it selects a

substrate other than malonyl-CoA. Based on the structure of the C-4" side chain of 1, it is proposed that this AT takes methylmalonyl-CoA as the substrate. This was confirmed by the presence of the motif of VASH. It has been previously reported that a conserved [Y/V/W]ASH motif is present in methylmalonyl-specific ATs, while a [H/T/V/Y]AFH motif is found in malonyl-specific ATs.26 DutG selectively and repeatedly uses its reductive domains to generate a triketide chain from three units of methylmalonyl-CoA. Specifically, it uses the complete set of reductive domains to conduct ketoreduction, dehydration and enoyl reduction to yield a saturated



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C-C bond in the first elongation step. In contrast, it only uses KS, AT, ACP, KR and DH to form a C-C double bond in the second step. An ACP shuttle-type acyltransferase (DutH) then transfers the triketide chain to the 4"-OH of 2 to yield the final structure of 1. Similar to 1, several other bioactive natural products such as cervimycins, SF2575 and polyketomycin also have a functional group attached to 4"-OH of a terminal deoxysugar moiety. A KS III homologue from Streptomyces tendae, CerJ, was identified as an ACP shuttle–type AT responsible for attaching a malonyl unit to cervimycins.27 Similar genes were also found in the SF2575 (ssfG)28 and polyketomycin (pokM2).17 Two SARP family regulator genes are found in the dut gene cluster, dutK and dutV. The gene cluster also contains two ABC transporter genes dutT and dutU, which are likely involved in transporting the final product out of the cells. Inactivation of a Putative MT (DutMT1) and Production of a New Demethylated Derivative of Dutomycin. The structure of 1 has two methyl groups, 13-CH3 and 16-CH3 that are introduced by dedicated MTs. There are two putative MT genes in the dut gene cluster. Based on the sequence homology, DutMT1 was deduced to be a C- or O-MT (Table 3). To understand its role in dutomycin biosynthesis and create new analogues, we disrupted dutMT1 in S. minoensis using the same approach for ks1–ks3 described above. To this end, a fragment of dutMT1 (Supplementary Figure S6) was amplified and ligated to pKC1139 to yield the corresponding disruption plasmid pSW91 (Supplementary Table S1). Integration of pSW91 into the genome of S. minoensis was screened by the apramycin resistance and verified by PCR analysis. As shown in Supplementary Figure S6, ~0.9 kb gene fragments were amplified from the mutant using primer pairs M13-47/DutMT1-Check1 (lane 3) and RM-V/DutMT1-Check2 (lane 4). By contrast, no PCR products were obtained from wild type S. minoensis using the same primers (lanes 1 and 2). This result confirmed that pSW91 was successfully inserted into


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dutMT1 to yield the desired ∆DutMT1 mutant. The products of this mutant were analyzed by LC-MS. As shown in Figure 2, the production of 1 in S. minoensis NRRL B-5482-∆DutMT1 was not detected (trace iii), while a major product 3 at 26.8 min was produced. We next isolated 3 from a scaled-up culture of this mutant for structural characterization. ESI-MS analysis revealed that the molecular weight of 3 is 840 according to the [M-H]- ion peak at m/z 839.1 (Supplementary Figure S2), which is 14 mass units smaller than 1. The molecular formula of 3 was determined to be C43H52O17 based on the [M+Na]+ ion peak at m/z 863.3133 (calcd. 863.3097) in the HR-ESI/APCI-MS spectrum. The 13C NMR spectrum of 3 showed 43 carbons, one carbon less than 1. A comparison of the NMR spectra of 1 and 3 (Supplementary Figures S3g and S3h) confirmed that a methyl group was missing in 3, while a new aromatic proton signal was found. The only methyl group attached to an aromatic ring is 13-CH3. Therefore, 3 is likely the 12-demethylated derivative of 1. This was supported by the HMBC correlations of 12H at δ 7.53 to C-1 (δ 179.1), C-11 (δ 36.8), C-4a (δ 113.6) and C-5a (δ 123.3) (Figure 4). Thus, the structure of 3 was determined to be 12-desmethyl-dutomycin. It is a new compound and was named as SW91. The carbon and proton signals were assigned based on the 2D NMR (Supplementary Figures S3i–S3k) and a comparison with 1, and are shown in Tables 1 and 2, respectively. The titer of 3 was determined to be 66.7 ± 1.0 mg L-1. Identification of 3 revealed that DutMT1 is a C-MT that attaches a methyl group to C-12. In vitro functional characterization of DutMT1. To further confirm the function of DutMT1, the corresponding gene was ligated to pET28a and overexpressed in E. coli BL21(DE3). The His6-tagged DutMT1 was purified to near homogeneity (Figure 5a) and reacted with 3. HPLC analysis (Figure 5b) showed that incubation of DutMT1 with 3 yielded a product at 27.5 min, which has the same retention time and UV spectra as 1, suggesting that 3 was



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methylated to yield 1. This was further confirmed by the ESI-MS spectrum of this product (Figure 5b), which indicated that its molecular weight is 854 based on the [M-H]- ion peak at m/z 853.2. Thus, this in vitro reaction allowed unambiguous functional characterization of DutMT1 as the 12-methyltransferase in dutomycin biosynthesis. The conversion rate in this reaction was low even though the concentration of DutMT1 was relatively high. One possible reason is that 12-desmethyl-dutomycin might not be a favored substrate for DutMT1. In other words, it is likely that DutMT1 functions in an earlier step in dutomycin biosynthesis rather than the last biosynthetic step. In this case, other late tailoring enzymes can accept the 12-demethylated biosynthetic intermediates to form 12-desmethyl-dutomycin when dutMT1 was disrupted. Inactivation of DutGT1 and DutGT2 and Characterization of Two New Dutomycin Analogues. Deoxysugars are frequently present in bioactive molecules such as erythromycin, doxorubicin and pradimicin A. Some of these sugar moieties are found to be essential in the biological activities. Thus, identification of enzymes responsible for transferring these sugars is of interest. There are two putative GTs in the dut biosynthetic gene cluster, likely involved in the introduction of the two sugar moieties to the aglycone 5. In order to characterize the functions of these two GTs, we first disrupted the dutGT2 gene. The correct ∆DutGT2 mutant was verified by PCR (Supplementary Figure S7). LC-MS analysis revealed that 1 was not produced, but a new product 4 accumulated as the major metabolite at 8.2 min (trace iv, Figure 2). ESI-MS spectrum of 4 showed a [M-H]- ion peak at m/z 571.1, indicating that its molecular weight is 572. The molecular formula was determined to be C28H28O13 based on the [M+Na]+ ion peak at m/z 595.1440 (calcd. 595.1422) in the HR-MS spectrum. This corresponds to the structure of the aglycone with D-amicetose, which is the first sugar moiety in 1. To confirm the structure, 4 was isolated from 1 L of the culture. The purified compound was subjected to NMR analysis


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(Supplementary Figures S3l–S3o). A comparison of the NMR data of 4 and 1 revealed that the second sugar moiety and the C-4" side chain were missing in this new compound, confirming that its structure only has the D-amicetose moiety. The spin system of H-1'/H2-2'/H2-3'/H-4'/H5'/H3-6' of this sugar moiety was observed in the 1H-1H COSY spectrum (Figure 4). Accordingly, the structure of 4 can be established as shown in Figure 1a and was named as SW75. The proton and carbon signals were assigned based on the 1D and 2D NMR spectra. The assigned signals of 4 are listed in Tables 1 and 2, respectively. The titer of 4 was determined to be 34.8 ± 0.2 mg L-1. Identification of 4 indicated that DutGT2 is the GT responsible for transferring the second sugar (L-axenose) moiety. DutGT1 is thus proposed to introduce the first sugar moiety to the aglycone. The dutGT1 gene was disrupted using the same single crossover approach. The correct ∆DutGT1 mutant was verified by PCR (Supplementary Figure S8). LC-MS analysis revealed that the production of 1 was abolished in this mutant, and a major product 5 at 7.0 min was produced (trace v, Figure 2) instead. The ESI-MS spectrum of 5 showed a [M-H]- ion peak at m/z 457.0 (Supplementary Figure S2), suggesting that the molecular weight of this compound is 458, which corresponds to the aglycone intermediate in dutomycin biosynthesis (Figure 3b). The identity of 5 was confirmed by a comparison of the methanolysis product of 1 (Supplementary Figure S9).15 DutGT1 was thus confirmed to be responsible for introducing the first sugar moiety to the aglycone. Antibacterial activity test for dutomycin and new analogues. Compounds 1–4 were tested against E. coli ATCC 25922, S. aureus ATCC 25923 and S. aureus ATCC 33591 (methicillinresistant). 1 showed significant antibacterial activity against the Gram-positive strains, but was not active against the Gram-negative E. coli strain (Figure 1b). This indicated that 1 specifically



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targets Gram-positive bacteria. The MIC values of 1 for both S. aureus and methicillin-resistant S. aureus were determined to be 0.25 µg mL-1 (Figure 1b), indicating that this compound is a highly active antibacterial agent. Anthracyclines are ranked among the most effective anticancer therapeutics ever developed.29 1 was previously discovered as an antitumor compound

15

and

showed strong in vitro cytotoxicity against leukemia P388 cells.15 This is the first report of the antibacterial activity of 1. Its natural analogue polyketomycin from other actinomycetes has also shown similar antibacterial activity against Gram-positive bacteria.30 This suggests that this family of anthracyclines represents promising lead compounds for the development of newgeneration antibiotics. The activity of 2–4 was also tested. However, 2 and 4 had no obvious antibacterial activity and the MICs were found to be 250 µg mL-1, which is nearly 1,000-fold higher than 1 (Figure 1b). In contrast, 3 showed strong antibacterial activity against S. aureus ATCC 25923 and methicillin-resistant S. aureus ATCC 33591. The MICs of 3 against these two strains were determined to be 0.125 µg mL-1 (Figure 1b), which are much lower than those of 1. The antibacterial testing results of these four compounds revealed some important SAR information. The presence of the C-4" side chain synthesized by the type I PKS DutG in 1 is essential, as removal of this side chain led to almost complete loss of the antibacterial activity. The same phenomenon was observed for 4, which lacks both the L-axenose moiety and the attached C-4" polyketide chain, further confirming that the important role of this functional group. 3 is a demethylated derivative of 1. Its MICs were only 50% of those of 1, indicating that removal of the 13-CH3 can significantly increase the antibacterial activity. The activity of 5 was not tested as it lacks the sugar moieties and C-4" side chain. It is expected that this compound has no antibacterial activity.


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In summary, dutomycin and its precursor POK-MD1 was isolated from S. minoensis and structurally characterized in this work. The dutomycin biosynthetic gene cluster was identified through genome sequencing and gene disruption. The functions of DutMT1, DutGT1 and DutGT2 were identified and three dutomycin analogues were generated through gene disruption. Antibacterial studies on these molecules revealed that the C-4" side chain plays an essential role in the biological activity of dutomycin. Removal of the CH3 group at C-12 significantly improved the antibacterial activity, suggesting that it is possible to create new dutomycin analogues through engineered biosynthesis and the antibacterial activity may be further optimized by additional structural modifications to yield novel antibiotics to combat Grampositive pathogens including those developed with drug-resistance such as methicillin-resistant S. aureus.

METHODS General. Products from S. minoensis NRRL B-5482 or mutants were analyzed and purified

on an Agilent 1200 HPLC instrument. Compounds were detected at 460 nm. ESI-MS spectra were obtained on an Agilent 6130 quadrupole LC-MS. 1D and 2D NMR spectra were recorded on a JEOL ECX-300 or a Bruker AvanceIII HD Ascend-500 NMR instrument. The chemical shift (δ) values and coupling constants (J values) are reported in parts per million (ppm) and hertz (Hz), respectively. Strains and Vectors. S. minoensis NRRL B-5482 was obtained from the USDA Agricultural Research Service Culture Collection. E. coli ATCC 25922, S. aureus ATCC 25923 and S. aureus ATCC 33591 (methicillin-resistant) used in the antibacterial assay were purchased from the American Type Culture Collection. E. coli XL-1 Blue (Stratagene) and the pJET1.2 cloning



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vector (Fermentas) were used for routine subcloning. E. coli ET12567 (pUZ8002) and pKC1139 vector were used for the gene disruption experiments.31 Media and Culture Conditions. S. minoensis NRRL B-5482 and its mutants were maintained on YM (yeast extract-malt extract) agar plate at 28°C.32 S. aureus and E. coli were cultured in Luria-Bertani (LB) broth at 37°C. MS, ISP4, and TSB media were used in the conjugation experiments. Ampicillin and apramycin were added at 50 µg mL-1 to the media appropriately for cloning and conjugation, respectively. Extraction and Sequencing of the Genomic DNA. S. minoensis NRRL B-5482 was grown in 50 mL of YM medium at 28°C with shaking at 250 rpm for 5 days. The genomic DNA was extracted as previously reported.32 The genome was sequenced using an Illumina MiSeq desktop sequencer and the obtained data were assembled with Velvet. The genome sequence was annotated with RAST.33 The gene information of the dutomycin gene cluster was deposited in GenBank under accession number KP710956. Construction of Plasmids. To find the type II KS involved in dutomycin biosynthesis, a ~0.6-kb fragment was amplified from the genomic DNA of S. minoensis using KSα-F and KSαR (Supplementary Table S2) with a 30-cycle PCR program (15 s at 98°C, 15 s at 55°C, and 30 s at 72°C). The PCR product was ligated into the cloning vector pJET1.2 to yield three distinct plasmids including pLS51, pLS52 and pLS53 based on the sequencing results. The three different ks fragments (ks1–ks3) in these plasmids were excised and ligated into pKC1139 between BglII and BamHI to afford pLS54, pLS55 and pLS56, respectively. For inactivation of dutMT1, dutGT1 and dutGT2, 0.6-kb fragments of these genes were amplified from the genomic DNA with primer pairs DutMT1-F1/DutMT1-R1, DutGT2-F/DutGT2-R, and DutGT1F/DutGT1-R (Supplementary Table S2) using PCR and ligated to pJET1.2 to yield pSW70,


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pSW82, and pSW138, respectively. These gene fragments were introduced to pKC1139 between HindIII and XbaI to form pSW75, pSW91 and pSW140, respectively. To obtain DutMT1 for in vitro reactions, the gene was amplified with DutMT1-F2/DutMT1R2 (Supplementary Table S2) and was ligated to pJET1.2 to yield pSUN151. The dutMT1 gene was excised with NdeI and XhoI and ligated to pET28a between the same sites to yield pSUN168. All the plasmids constructed in this study are listed in Supplementary Table S1. Conjugation and Confirmation of the Mutants. Spores of S. minoensis NRRL B-5482 were obtained by growing it on ISP4 medium. The gene disruption plasmids were introduced to E. coli ET12567 (pUZ8002) through chemical transformation. Plasmids were introduced into S. minoensis NRRL B-5482 by E. coli–Streptomyces conjugation, following the previously reported procedure.24 Positive colonies from MS plates were picked and grown in TSB medium with 50 µg mL-1 of apramycin at 28°C and 250 rpm. After 5 days, 50 µL of these cultures were spread on ISP4 plates with 50 µg mL-1 of apramycin. Colonies showing up at 37°C were considered as recombination strains. The mutants were confirmed by PCR analysis using genome- and pKC1139-specific primers (Supplementary Table S2). The PCR verification results are shown in Figures S5–S8. Extraction, Analysis and Purification of Compounds. S. minoensis and mutants were grown on YM agar plates (with or without apramycin) at 28°C. The cultures were chopped and extracted three times with an equal volume of ethyl acetate. The resulting extracts were dried in vacuo and the residues were re-dissolved in methanol for LC-MS analysis. To isolate 1 and 2, 1 L of culture of wild type S. minoensis was extracted with ethyl acetate and separated on a silica gel 60 column, successively eluted with 100:0, 75:25, 50:50, 25:75, 0:100 (v/v) chloroformmethanol to yield 5 fractions. The 75:25 fraction was found to contain 1. It was further separated



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by HPLC with an Agilent Eclipse XDB-C18 column (5 µm, 250 mm × 4.6 mm) and eluted with a gradient of acetonitrile-water (70:30, v/v to 75:25, v/v over 20 min) at 1 mL min-1. The peak at 17 min was collected to yield 48.7 mg of 1 in pure form. Similarly, the 50:50 fraction containing 2 was separated by the same HPLC instrument, eluted with acetonitrile-water from 30:70, v/v to 35:65, v/v over 15 min at 1 mL min-1, to yield 6.2 mg of 2. A total of 38.9 mg of 3 was isolated from 1 L of S. minoensis-∆DutMT1 culture using the same procedure as for 1. For the purification of 4, the extract of 1 L of S. minoensis-∆DutGT2 culture was separated on a MCI column, successively eluted with 0:100, 25:75, 50:50, 75:25, 100:0% methanol-water (v/v) to yield 5 fractions. The 100% methanol fraction contained 4, which was further separated by HPLC. The sample was eluted with acetonitrile-water 30:70 (v/v) at 1 mL min-1 for 15 min, and the peak with a retention time of 12 min was collected to yield 15.4 mg of 4. NMR and MS spectra of these purified compounds were collected for structure elucidation. The major product 5 in S. minoensis-∆DutGT1 was identified by MS and a comparison with the methanolysis product of 1. Expression and purification of His6-tagged DutMT1. To produce His6-tagged DutMT1, E. coli BL21(DE3) harboring pSUN168 was grown at 37°C and 250 rpm in LB broth that was supplemented with kanamycin at 50 µg mL-1. Isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added at 200 µM to induce the expression of DutMT1 when the OD600 value of the culture reached 0.6. The induced broth was then incubated at 18°C and 250 rpm for an additional 16 h. After that, the culture was centrifuged at 3,500 rpm for 10 min to harvest the cells. The cell pellet was re-suspended in the lysis buffer (0.5 M NaCl, 20 mM Tris–HCl, pH 7.9). The cell suspension was sonicated to disrupt the cells. The lysate was centrifuged at 13,000 rpm and 4°C for 15 min to remove cell debris. The N-His6-tagged protein was purified using the Ni-NTA


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(nickel-nitrilotriacetic acid) resin. Purified DutMT1 was desalted against buffer A that contained 50 mM Tris–HCl (pH 7.9), 2 mM ethylenediaminetetraacetic acid and 1 mM dithiothreitol and concentrated with a centrifugal filter device. In vitro reaction of DutMT1. The reaction was conducted at 30°C in a total volume of 100 µL that contained 1 mM SAM, 10 µM 3, and 10 µg of DutMT1 in 100 mM phosphate buffer (pH 7.0). After 60 min, the reaction was quenched by adding 50 µL of methanol and analyzed on an Agilent 1200 HPLC that was equipped with an Agilent Eclipse Plus C18 column. The sample was eluted with a linear gradient of acetonitrile-water from 30 to 95% (v/v) over 30 min at 1 mL min-1. The substrate and product were detected at 460 nm. The methylation product was characterized by a comparison of the retention time and UV spectrum with those of 1. Its identity was further confirmed by the ESI-MS spectrum of this product collected on an Agilent 6130 quadrupole LC-MS. A reaction with inactivated DutMT1 was used as negative control. The reaction was conducted in triplicate and a representative one was shown in Figure 5b. Minimum inhibition concentration test. S. aureus ATCC 25923, methicillin-resistant S. aureus ATCC 33591 and E. coli ATCC 25922 were used for the antibacterial assay. These strains were incubated in LB broth with the test samples in 96-well plates at different concentrations including 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, and 0.064 µg mL-1 at 37°C for 20 h to determine the MICs.34

ASSOCIATED CONTENT

Supporting Information

Supporting Information Available: Tables S1–S2 and Figures S1–S9. This material is available free

of charge via the Internet at http://pubs.acs.org.



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AUTHOR INFORMATION

Corresponding Author * Phone: 435-797-8774. Fax: 435-797-1248. Email: [email protected]. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS

This research was supported by the National Institutes of Health grants AI065357 RM DP 008

and AI089347 (to J. Zhan) and grants from the National Natural Science Foundation of China (31470787 and 31170763 to D. Yu). The Bruker AvanceIII HD Ascend-500 NMR instrument used in this work was funded by the National Science Foundation Award CHE-1429195.

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Table 1. 13C NMR data (75 MHz) for 1-4 (1 and 3 in CDCl3, 2 in CD3OD, and 4 in DMSO-d6). Position 1a 1 2 3 4 4a 5 5a 6 6a 7 8 9 10 11a 11 12a 12 13 14 15 16 1' 2' 3' 4' 5' 6' 1'' 2'' 3'' 4'' 5'' 6'' 7'' 1''' 2''' 3''' 4''' 5''' 6''' 7''' 8''' 9'''

1 132.9 (C) 181.5 (C) 161.3 (C) 108.8 (CH) 190.4 (C) 113.8 (C) 162.5 (C) 123.5 (C) 192.6 (C) 81.2 (C) 195.2 (C) 110.8 (C) 191.2 (C) 73.9 (CH) 75.7 (C) 35.0 (CH2) 150.9 (C) 132.4 (C) 16.8 (CH3) 200.9 (C) 26.7 (CH3) 57.1 (CH3) 102.2 (CH) 29.5 (CH2) 30.3 (CH2) 80.1 (CH) 74.5 (CH) 17.4 (CH3) 100.3 (CH) 37.0 (CH2) 68.9 (C) 74.2 (CH) 62.8 (CH) 16.8 (CH3) 25.9 (CH3) 167.4 (C) 125.2 (C) 21.1 (CH3) 150.1 (CH) 33.5 (CH) 20.7 (CH3) 39.8 (CH2) 20.8 (CH2) 14.4 (CH3)

2 133.9 (C) 182.8 (C) 162.7 (C) 109.6 (CH) 189.5 (C) 114.9 (C) 163.3 (C) 124.5 (C) 192.2 (C) 83.4 (C) 195.3 (C) 112.6 (C) 192.2 (C) 73.2 (CH) 75.6 (C) 35.5 (CH2) 152.8 (C) 133.8 (C) 17.0 (CH3) 200.5 (C) 27.7 (CH3) 57.7 (CH3) 103.5 (CH) 30.6 (CH2) 31.3 (CH2) 81.3 (CH) 74.7 (CH) 17.9 (CH3) 101.3 (CH) 37.1 (CH2) 70.6 (C) 75.2 (CH) 64.5 (CH) 17.2 (CH3) 27.0 (CH3)

3 133.9 (C) 179.1 (C) 160.7 (C) 110.6 (CH) 189.8 (C) 113.6 (C) 163.7 (C) 123.3 (C) 190.5 (C) 81.5 (C) 195.4 (C) 110.7 (C) 191.7 (C) 73.5 (CH) 75.7 (C) 36.8 (CH2) 150.1 (C) 120.1 (CH) 201.3 (C) 25.9 (CH3) 57.1 (CH3) 102.2 (CH) 29.5 (CH2) 30.3 (CH2) 80.1 (CH) 74.6 (CH) 18.1 (CH3) 100.4 (CH) 37.1 (CH2) 69.0 (C) 74.2 (CH) 62.9 (CH) 16.8 (CH3) 25.9 (CH3) 167.5 (C) 125.3 (C) 21.1 (CH3) 150.1 (CH) 33.5 (CH) 20.8 (CH3) 39.8 (CH2) 20.9 (CH2) 14.4 (CH3)


4 132.5 (C) 181.4 (C) 162.2 (C) 108.9 (CH) 191.2 (C) 111.7 (C) 161.1 (C) 123.6 (C) 194.2 (C) 82.2 (C) 194.2 (C) 113.3 (C) 191.2 (C) 74.9 (CH) 75.6 (C) 34.5 (CH2) 151.4 (C) 131.9 (C) 16.7 (CH3) 199.9 (C) 26.8 (CH3) 57.6 (CH3) 101.9 (CH) 30.7 (CH2) 31.2 (CH2) 70.7 (CH) 75.7 (CH) 17.4 (CH3)


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Table 2. 1H NMR data (300 MHz) for 1-4 (1 and 3 in CDCl3, 2 in CD3OD, and 4 in DMSO-d6.) Position 3 5-OH 10 11 12 13 15 16 1' 2' 3' 4' 5' 6' 1'' 2'' 4'' 5'' 6'' 7'' 3''' 4''' 5''' 6''' 7''' 8''' 9'''

1 6.11 (1H, s) 14.17 (1H, s) 4.43 (1H, brs) 3.78 (1H, d, 17.5) 3.01 (1H, m) 2.57 (3H, s) 2.70 (3H, s) 3.93 (3H, s) 4.66 (1H, d, 8.2) 1.57 (1H, m) 2.18 (1H, m) 2.11 (1H, m) 1.63 (1H, m) 3.11 (1H, m) 3.02 (1H, m) 0.58 (3H, d, 5.5) 4.97 (1H, d, 3.1) 1.88 (1H, m) 1.60 (1H, m) 4.76 (1H, s) 4.38 (1H, q, 6.5) 1.07 (3H, d, 6.9) 1.07 (3H, s) 1.91 (3H, s) 5.70 (1H, dd, 1.0, 11.3) 3.07 (1H, m) 0.96 (3H, d, 6.5) 1.25 (2H, m) 1.25 (2H, m) 0.86 (3H, t, 6.5)

2 6.26 (1H, s) 4.46 (1H, brs) 3.78 (1H, d, 17.5) 3.03 (1H, m) 2.60 (3H, s) 2.65 (3H, s) 3.93 (3H, s) 4.67 (1H, brs) 1.57 (1H, m) 2.12 (1H, m) 2.12 (1H, m) 1.61 (1H, m) 3.10 (1H, m) 2.95 (1H, m) 0.56 (3H, m) 4.95 (1H, overlapped) 1.85 (1H, m) 1.60 (1H, m) 4.86 (1H, overlapped) 4.30 (1H, q, 6.3) 1.19 (3H, d, 6.9) 1.14 (3H, s)

3 6.17 (1H, s) 13.50 (1H, s) 4.46 (1H, brs) 3.75 (1H, d, 17.5) 3.05 (1H, m) 7.53 (1H, s) 2.70 (3H, s) 3.95 (3H, s) 4.71 (1H, d, 8.2) 1.56 (1H, m) 2.18 (1H, m) 2.12 (1H, m) 1.66 (1H, m) 3.13 (1H, m) 3.04 (1H, m) 0.68 (3H, d, 5.5) 4.99 (1H, d, 2.3) 1.86 (1H, m) 1.60 (1H, m) 4.76 (1H, s) 4.38 (1H, q, 6.5) 1.07 (3H, d, 6.9) 1.08 (3H, s) 1.92 (3H, s) 5.71 (1H, d, 10.3) 3.10 (1H, m) 0.97 (3H, d, 6.6) 1.26 (2H, m) 1.26 (2H, m) 0.87 (3H, t, 6.5)


4 6.37 (1H, s) 14.10 (1H, s) 4.43 (1H, brs) 3.58 (1H, d, 17.5) 2.95 (1H, d, 17.8) 2.50 (3H, s) 2.62 (3H, s) 3.91 (3H, s) 4.57 (1H, d, 8.2) 1.45 (1H, m) 1.91 (1H, m) 1.85 (1H, m) 1.24 (1H, m) 2.84 (1H, overlapped) 2.84 (1H, overlapped) 0.44 (3H, d, 4.5)

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Table 3. Deduced functions of the genes in the dutomycin (dut) biosynthetic gene cluster. ORF

aa

DutS9 DutS7 DutO4 DutO2 DutJ DutD DutL DutO3 DutE DutMT2 DutF DutO1 DutI DutMT1

319 204 507 540 251 314 495 103 150 350 261 407 259 345

DutK DutS2 DutS1 DutP DutN DutM DutC DutB DutA DutS3 DutS4 DutQ DotR DutS5 DutS6 DutGT1 DutG DutH DutT DutU DutGT2 DutV DutS8 DutW DutX

270 326 354 470 175 565 85 407 422 471 332 200 384 431 316 396 2176 345 309 268 382 1064 410 326 402

a

Putative function/Homologous protein and GenBank accession No. NDP-hexose 4-ketoreductase/PokS9 from SDa, ACN64856 NDP-hexose-3,5-epimerase/PokS7 from SD, ACN64855 Monooxygenase/PokO4 from SD, ACN64854 Monooxygenase/PokO2 from SD, ACN64853 Reductase/PokT2 from SD, ACN64852 Aromatase/PokC3 from SD, ACN6485 Acyl CoA ligase/PokL from SD, ACN64850 Monooxygenase/PokO3 from SD, ACN64849 Cyclase/PokC1 from SD, ACN64848 O-Methyltransferase/PokMT3 from SD, ACN64847 Cyclase/PokC2 from SD, ACN64846 Monooxygenase/PokO1 from SD, ACN64845 Ketoreductase/PokT1 from SD, ACN64844 O-Methyltransferase or C-methyltransferase/PokMT2 from SD, ACN64843 SARP family regulator/PokR2 from SD, ACN64842 NDP-glucose 4,6-dehydratase/PokS2 from SD, ACN64841 NDP-glucose synthase/PokS1 from SD, ACN64840 Biotin carboxylase/PokAC2 from SD, ACN64839 Biotin carboxylase carrier protein//PokAC3 from SD, ACN64838 Acetyl CoA carboxylase/PokAC1 from SD, ACN64837 Acyl carrier protein/PokP3 from SD, ACN64836 Chain length factor/PokP2 from SD, ACN64835 Ketoacyl synthase/PokP1 from SD, ACN64834 NDP-hexose 2,3-dehydratase/PokS3 from SD, ACN64829 NDP-hexose 3-ketoreductase/PokS4 from SD, ACN64828 Flavin reductase or dimerase/PokU2 from SD, ACN64827 Acyl-CoA dehydrogenase/PokU1 from SD, ACN64826 NDP-hexose 3,4-dehydratase/PokS5 from SD, ACN64825 NDP-4-keto-6-deoxyhexose reductase/PokS6 from SD, ACN64824 Glycosyltransferase/PokGT1 from SD, ACN64823 Modular polyketide synthase from S. lividans, WP_016325502 ACP shuttle–type acyltransferase/CerJ from S. tendae, AEI91069 ABC transporter/PokABC1 from SD, ACN64822 ABC transporter/PokABC2 from SD, ACN64821 Glycosyltransferase/PokGT2 from SD, ACN64820 SARP family transcriptional regulator/PokR1 from SD, ACN64819 NDP-hexose 3-C-methyltransferase/PokS8 from SD, ACN64818 Putative carbohydrate kinase/PokR3 from SD, ACN64817 S-Adenosylmethionine synthase from S. somaliensis, WP_010470430

SD = S. diastatochromogenes Tü6028


%Identity/ Similarity 85/90 92/95 91/93 88/91 90/92 89/94 88/92 89/96 89/96 93/95 93/97 93/96 88/92 93/95

E value 4e-170 4e-133 0.0 0.0 5e-148 0.0 0.0 6e-61 4e-94 0.0 4e-174 0.0 7e-155 0.0

97/98 92/95 94/97 91/95 84/87 87/90 84/89 89/94 94/96 86/92 84/88 82/87 80/85 97/98 84/90 84/89 45/59 34/49 83/90 93/97 87/92 81/86 86/89 88/92 94/96

0.0 0.0 0.0 0.0 2e-97 0.0 7e-41 0.0 0.0 0.0 0.0 7e-83 0.0 0.0 1e-16 0.0 0.0 8e-52 7e-177 3e-159 0.0 0.0 0.0 0.0 4e-93


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Figure 1. Structures and antibacterial activity of dutomycin (1) and analogues. a) Structures of 1, polyketomycin, and the analogues 2–5. b) Antibacterial activity of the ethyl acetate extract of wild type S. minoensis and compounds 1–4. MICs (µg mL-1) against E. coli ATCC 25922, S. aureus ATCC 25923 and methicillin-resistant S. aureus ATCC 33591 are shown.

Figure 2. HPLC analysis of the metabolites of wild type S. minoensis NRRL-B5482 and four mutants at 460 nm. (i) wild type; (ii) S. minoensis NRRL-B5482/pLS54 (∆DutA); (iii) S. minoensis NRRL-B5482/pSW91 (∆DutMT1); (iv) S. minoensis NRRL-B5482/pSW75 (∆DutGT2); (v) S. minoensis NRRL-B5482/pSW140 (∆DutGT1).

Figure 3. The biosynthetic gene cluster of dutomycin (dut) (a) and the proposed biosynthetic pathway (b).

Figure 4. Selected 1H-1H COSY and HMBC spectra for 3 and 4.

Figure 5. In vitro methylation of 3 by DutMT1. a) SDS-PAGE analysis of the expression and purification of DutMT1 in E. coli. b) LC-MS analysis of the in vitro reaction of DutMT1 at 460 nm. (i) Incubation of inactivated DutMT1 with 3; (ii) Incubation of DutMT1 with 3; (iii) Purified standard of 1. The ESI-MS spectrum of the methylation product is shown.


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Figure 1a 182x103mm (300 x 300 DPI)



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Figure 1b 249x94mm (96 x 96 DPI)


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Figure 2 177x180mm (96 x 96 DPI)



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Figure 4 178x63mm (300 x 300 DPI)


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Characterization of Three Tailoring Enzymes in Dutomycin

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