CHEMBIOCHEM FULL PAPERS DOI: 10.1002/cbic.201402384
Uncovering a Glycosyltransferase Provides Insights into the Glycosylation Step during Macrolactin and Bacillaene Biosynthesis Wen Qin,[a] Yang Liu,[a] Pengfei Ren,[a] Jun Zhang,[a] Huayue Li,[a] Li Tian,[b, c] and Wenli Li*[a] Macrolactins (MLNs) have unique structural patterns containing a 24-membered ring lactone and diverse bioactivities. The MLN skeleton is biosynthesized via a trans-acyl transferase (AT) type I polyketide synthase (PKS) pathway, but the tailoring steps are still unknown. Herein, we report the identification of a glycosyltransferase (GT) gene bmmGT1, which is located at different locus from the MLN gene cluster in the genome of
marine-derived Bacillus marinus B-9987, and its functional characterization as an MLN GT, thus affording five novel MLNs analogues. Surprisingly, this GT is also capable of catalyzing the glycosylation of bacillaenes (BAEs), which are the prototypes of trans-AT polyketides, thus suggesting broad substrate flexibility. These results provide the first significant insights into the glycosylation step in MLN and BAE biosynthetic pathways.
Introduction Many bioactive natural products are equipped with sugar moieties, which not only increase water solubility thus improving bioavailability but also decrease toxicity.[1] Attachment of sugar moieties to aglycone scaffolds is usually performed by glycosyltransferases (GTs) that catalyze the transfer of sugar moieties from activated donor molecules to specific acceptor molecules by forming glycosidic bonds.[1] Almost all natural-product GTs belong to the GT-B type superfamily; they are functionally diverse but share a highly conserved basic architecture.[1c, d] The reversibility of the reaction and substrate flexibility of GTs make them powerful tools for the glycodiversification of natural products.[2] Most bacterial natural-product GTs that have been functionally characterized are from Actinomyces strains;[1d, 3] in contrast, only a couple of Bacillus natural-product GTs have been used for biocatalytic applications.[4] Macrolactins (MLNs), first isolated from a deep-sea bacterium in 1989 by Fenical and coworkers,[5] have unique structural patterns with a 24-membered ring lactone (Scheme 1). MLNs exhibit a wide range of biological activities, such as antibacterial,[6] antiviral,[5] anticancer,[5] anti-inflammatory,[7] and antiangiogenic.[8] Notably, MLNs can inhibit Staphylococcus aureus peptide deformylase (PDF),[9] which is considered an attractive [a] W. Qin,+ Y. Liu,+ P. Ren, J. Zhang, Dr. H. Li, Prof. Dr. W. Li Key Laboratory of Marine Drugs, Ministry of Education School of Medicine and Pharmacy, Ocean University of China No. 5 Yushan Road Qingdao, SD 266003 (China) E-mail: [email protected] [b] Prof. Dr. L. Tian First Institute of Oceanography, State Oceanic Administration No. 6 Xianxialing Road Qingdao, SD 266061 (China) [c] Prof. Dr. L. Tian Qingdao University of Science and Technology No. 53 Zhen zhou Road Qingdao, SD 266042 (China) [+] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201402384.
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
drug target in view of its wide distribution in bacteria and absence in eukaryotes.[10] At least 30 MLNs have been identified from various Bacillus strains, and several are glycosylated, such as MLNs B–D, O–R, and W.[5, 11] The MLN backbone is assembled by a special family of polyketide synthetases (PKSs) termed trans-acyltransferase (AT) PKSs.[12] Contrary to the general rule that bacterial natural-product GTs genes are clustered with other biosynthetic genes,[3] MLN GT genes are neither in a cluster nor in proximity to one.[13] Interestingly, the same was found for the precursor of trans-AT polyketide bacillaenes (BAEs).[14] Although glycosylated BAE B (Scheme 1) was identified in the fermentation broth of Bacillus amyloliquefaciens FZB42, no glycosyltransferase gene is present in the BAE biosynthetic gene cluster. In a previous study, six MLNs (MLN A, B, D, O, S, and T) were identified in the fermentation broth of Bacillus marinus B-9987, which was isolated from the rhizosphere of Suaeda salsa collected in the intertidal zone of Bohai Bay of Eastern China.[15] In an effort to address the glycosylation mechanism of MLNs, here we report the identification of the bmmGT1 gene in B. marinus B-9987 and characterization of BmmGT1 as both an MLN GT and a BAE GT, as well as its substrate flexibility.
Results Bioinformatics analysis of bmmGT1 In order to locate the GT gene(s) involved in glycosylation of MLNs, genome alignments of the MLN-producing strains B. marinus B-9987 (unpublished) and B. amyloliquefaciens FZB42 (CP000560.1) were carried out with non-MLN-producing strains Bacillus subtilis 168 (NC_000964.3), Bacillus licheniformis 9945A (NC_021362.1), and Bacillus cereus ATCC 14579 (NC_ 004721.2). No obvious unique GT genes were found in the genomes of the MLN-producing strains (compared with the nonChemBioChem 0000, 00, 1 – 8
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www.chembiochem.org revealed that it is phylogenetically close to macrolide GTs, AveBI (avermectin), and OleD/OleI (oleandomycin), thus indicating that BmmGT1 would likely function as an MLN GT.
Effects of bmmGT1 inactivation To probe the function of bmmGT1, we first carried out gene inactivation to detect the production of glycosylated MLNs. Plasmid pWLI204 (harboring a 527 bp internal bmmGT1 fragment) was introduced into B. marinus B-9987. Single-crossover mutants were obtained (Experimental Section), and, after confirmation by Southern blotting (Figure S2), fermentations of wildtype and mutant strains were carried out. HPLC analysis revealed that the production of the glycosylated MLN compound 1 b was completely abolished in the DbmmGT1 mutant (Figure 1, trace ii); non-glycosylated MLN compounds 1 a, 2 a and 3 a accumulated (trace ii), and the production of 1 a
Figure 1. HPLC traces of the fermentation products of B. marinus B-9987 strains: i) wild-type; ii) DbmmGT1 mutant; iii) bmmGT1 overexpression.
Scheme 1. Macrolactins 1 a–c, 2 a--c, and 3 a--c and bacillaenes 4 a, 4 b, 5 a, and 5 b.
MLNs-producing strains). Nevertheless, a conserved putative macrolide GT gene (bmmGT1) was found in all the analyzed genomes. bmmGT1 is in a two-gene operon that contains a putative cytochrome P450 gene bm1409 upstream of bmmGT1. BLASTP queries of the NCBI database revealed that BmmGT1 displays homology to Bacillus cereus BcGT3 (AAS41737.1, 71 % identity), B. subtilis YjiC (WP_015252306, 61 % identity), and B. licheniformis BL-C (AAU40842, 53 % identity). BcGT3 was previously found to be able to catalyze the glycosylation of flavonoids;[4c] BL-C was recently determined to glycosylate at different hydroxyl positions of geldanamycin analogues,[4b] flavonoids,[4a, d] and isoflavonoids;[16] YjiC has not been characterized. A highly conserved domain of the glycosyltransferase GT-B type superfamily was found in BmmGT1. Phylogenetic analysis of BmmGT1 with selected functionally characterized bacterial natural-product GTs (Figure S1 in the Supporting Information) 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
reached 105 mg L 1, which is about six times higher than for the wild-type strain (18 mg L 1, trace i). To our surprise, the production of another type of trans-AT polyketide compound BAE changed as well in the DbmmGT1 mutants. The yields of BAEs 4 a and 5 a clearly increased, while the productions of the glycosylated BAEs 4 b and 5 b were abolished in the DbmmGT1 mutant (trace ii). The identities of 1 a, 1 b, 2 a, and 3 a were confirmed as MLN A, MLN B, 7-O-malonyl-macrolactin A (MMA), and 7-O-succinylmacrolactin A (SMA) (Scheme 1), respectively, by comparison and analysis of the UV spectra, and HRMS and 1H NMR data with those previously reported (Figures S3–S7).[5] BAEs compounds are extremely unstable and very sensitive to light and temperature during isolation.[17] Compounds 4 a, 4 b, and 5 a were deduced to be BAE, BAE B, and dihydro-BAE (Scheme 1), respectively, by UV spectrum, HRMS, liquid chromatography tandem mass spectrometry (LC-MS/MS), and 1H NMR data analysis of enriched BAE mixtures (Figures S8–S9). HRMS measurement of 5 b suggested the molecular formula C40H60N2O11 (m/z 745.4275 [M+H] + , calcd 745.4230), which is six carbon and five oxygen atoms (162 mass units) more than for dihydrobacillaene (5 a: C34H50N2O6, m/z 583.3737 [M+H] + , calcd 583.3702). In addition, the LC-MS/MS spectrum of 5 b showed two fragChemBioChem 0000, 00, 1 – 8
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mentation ions at m/z 384.2 and 434.3, consistent with those of 5 a; the corresponding fragmentation ions for 4 a and 4 b were at m/z 382.2 and 432.2 (Figure S7), thus suggesting the presence of a double bond. Therefore, 5 b was tentatively assigned as dihydro-BAE B (Scheme 1), which would be a new glycosylated BAE compound. These results suggested BmmGT1 is involved in the glycosylation of MLNs in vivo, and probably of BAEs as well. Effects of bmmGT1 overexpression To further demonstrate its function, bmmGT1 was overexpressed under the control of the gapDH promoter (glyceraldehyde-3-phosphate dehydrogenase, gapDHp) in B. marinus B9987.[18] Unmethylated pWLI205 was introduced into B. marinus B-9987 to generate the bmmGT1 overexpression strains (B9987/bmmGT1), and confirmed by plasmid isolation and subsequent restriction analysis (data not shown). HPLC analysis of the fermentation broths showed that production of the nonglycosylated compounds 1 a–5 a was significantly reduced in B-9987/bmmGT1, as expected; to our delight, two novel glycosylated MLN analogues (3 b and 3 c) were generated, whereas the production of 1 b, 4 b, and 5 b decreased (Figure 1, trace iii). Subsequently, an 18 L fermentation of B-9987/bmmGT1 led to the isolation of 3 b and 3 c (Scheme 1). The chemical formula for both compounds was determined to be C34H48O13 by HRMS (m/z 687.3040 [M+Na] + , calcd 687.2993), 162 mass units greater than that of 3 a. Both compounds exhibited spectroscopic data similar to those of 3 a. Eventually, the structures of 3 b and 3 c were elucidated on the basis of extensive NMR analysis (1H,13C COSY, HSQC, HMBC, and NOESY; Table S1, Figures S10–S11). The HMBC correlations from H-15 to C-1’ in 3 b and H-13 to C-1’ in 3 c unambiguously demonstrated that the b-glucopyranosyl moiety was attached to C-15 of 3 b and C-13 of 3 c. These results further demonstrate that bmmGT1 functions as an MLN GT in vivo. In vitro characterization of BmmGT1 In order to characterize its biochemical function, bmmGT1 was overexpressed as an N-terminally His6-tagged soluble protein in Escherichia coli BL21(DE3) carrying pWLI206 (Table S2). After purification to near homogeneity by Ni2 + -NTA agarose affinity chromatography, His6-tagged BmmGT1 was detected by SDSPAGE, which revealed a protein with molecular weight consistent with the calculated value (47.9 kDa; Figure S12). We first tested UDP-d-glucose as the donor sugar substrate, and generated the glycosylated MLN compound 1 b, as expected (Figure 2, trace ii). Then we examined its activity at different pH and temperature values, and in the presence of different cations (Figure S13). BmmGT1 exhibited activity over 10–40 8C (optimum turnover at 30 8C). Without cations, BmmGT1 exhibited over half the best turnover value, but this clearly improved upon addition of Mg2 + (2 and 10 mm), Mn2 + (2 mm), or Ca2 + (2 and 10 mm); optimum activity was with 10 mm Mg2 + , and activity was inhibited by the presence of Ni2 + , 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 2. Sugar donor specificity of BmmGT1: i) 1 a; ii) 1 a + UDP-d-glucose + BmmGT1; iii) 1 a + TDP-d-glucose + BmmGT1; iv) 1 a + UDP-d-glucuronic acid + BmmGT1; v) 1 a + UDP-d-N-acetylglucosamine + BmmGT1.
Cu2 + , and Fe2 + . Interestingly, EDTA enhanced the activity of BmmGT1. The optimum Mg2 + concentration for turnover (tested over 0–30 mm) was 10 mm. Sugar donor specificity of BmmGT1 Under optimized conditions (50 mm Tris·HCl (pH 8.9), 10 mm MgCl2, 30 8C), the sugar nucleotide specificity of BmmGT1 was tested with TDP-d-glucose, UDP-d-N-acetylglucosamine, and UDP-d-glucuronic acid (instead of UDP-d-glucose). BmmGT1 was able to recognize TDP-d-glucose, but only a minor amount of 1 b was detected (Figure 2 trace iii); to our delight, BmmGT1 was also able to use UDP-d-N-acetylglucosamine as the sugar donor, as 1 a was converted into a novel MLN derivative 1 c with the expected molecular weight (Figure 2 trace v); no activity was observed with UDP-d-glucuronic acid (Figure 2 trace iv). Subsequently, 1 c was isolated from a 10 mL enzymatic reaction mixture (Experimental Section). HR MS analysis indicated that 1 c had the molecular formula C32H47NO10 (m/z 606.3320 [M+H] + , calcd 606.3234), which is 203 mass units greater than that of 1 a. A full set of 1D and 2D NMR spectra of 1 c was acquired, thereby allowing us to assign its 1H and 13C signals (Table S1, Figure S14). The spectroscopic data for 1 c were similar to those for 1 a, except for the presence of an N-acetyl glucosamine group at C-7, as determined by HMBC correlations from H-7 to C-2’, C-3’ and C-8’, and H-7 to C-1’. Acceptor specificity of BmmGT1 Given that 2 a–5 a accumulated in the DbmmGT1 mutant in addition to 1 a (Figure 1, trace ii), the acceptor substrate specificity of BmmGT1 was probed with MLN compounds 2 a and 3 a, and BAE compounds 4 a and 5 a. All the compounds tested were recognized by BmmGT1: 3 a transformed into 3 b and 3 c (Figure 3 A, trace vi), 4 a transformed into 4 b (Figure 3 B, trace ii), and 5 a transformed into 5 b (Figure 3 B, trace iv) as expected. All the products had the expected molecular masses according to LC-HRMS analysis (Figures S9–S11). In addition, two novel MLN compounds were generated from 2 a (2 b and 2 c; ChemBioChem 0000, 00, 1 – 8
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Glycosylation alters the stability, solubility, reactivity, and biological activity of aglycons, hence GTs are ideal for structural diversification of natural products.[1d] In general, bacterial GT genes are within the corresponding biosynthetic gene clusters;[3] however, MLN and BAE gene clusters harbor no GT genes, in spite of the production of the glycosylated MLNs and BAEs in Bacillus species.[13, 17c] Given the critical effects of sugar moieties in natural products,[1] we identified the GT gene bmmGT1 in the genome of B. marinus B9987, and functionally characterized it in vivo and in vitro. Firstly, GT mining by comparative genome analysis revealed that there is no specific GT gene in the geFigure 3. Aglycon specificity of BmmGT1: i) 1 a + UDP-d-glucose; ii) 1 a + UDP-d-glucose + BmmGT1; iii) 2 a + UDP-d-glucose; iv) 2 a + UDP-d-glucose + BmmGT1; v) 3 a + UDPnomes of the MLN-producing Bacillus strains (comd-glucose; vi) 3 a + UDP-d-glucose + BmmGT1. B) 4 a + UDP-d-glucose; ii) 4 a + UDP-d-glupared with non-MLN-producing strains). Phylogenetic cose + BmmGT1; iii) 5 a + UDP-d-glucose; iv) 5 a + UDP-d-glucose + BmmGT1. analysis with other functionally characterized bacterial natural product GTs revealed that BmmGT1 is a YjiC homologue and conserved among the genomes anaFigure 3 A, trace iv). These results demonstrate that BmmGT1 lyzed; it is closely related to macrolide GTs (Figure S1), thus incan catalyze the glycosylation of both MLNs and BAEs in dicating that it could function as an MLN GT. Disruption of vitro—broad substrate specificity regarding aglycons. bmmGT1 led to abolishment of the glycosylated MLN (1 b) and, Compounds 2 b and 2 c were isolated from a 10 mL enzysimultaneously, clear accumulation of non-glycosylated MLNs matic reaction mixture (Experimental Section). The chemical (1 a, 2 a, and 3 a; Figure 1, trace ii). To our surprise, glycosylaformula for both was C33H46O13, as determined by HRMS (m/z 673.2881 [M+Na] + , calcd 673.2791), 162 mass units greater tion of BAEs was also blocked in the DbmmGT1 mutant (Figure 1, trace ii), thus indicating involvement of BmmGT1 in than that of 2 a. 1D and 2D NMR spectra led to full assignments the glycosylation of both MLNs and BAEs. Subsequent in vitro of their 1H and 13C signals (Table S1, Figures S15–S16). The studies demonstrated that BmmGT1 is capable of catalyzing compounds exhibited spectroscopic data similar to those of the glycosylation of MLNs as well as BAEs, with broad flexibility 2 a, except for the substitutions at C-13 and C-15. The HMBC regarding both sugar donor (Figure 2) and acceptor (Figure 3). correlations from H-13 to C-1’ (2 c) and H-15 to C-1’ (2 b) sugAs a result, five novel glycosylated MLN compounds were gengested b-glucopyranosyl moieties at C-13 of 2 c and C-15 of erated; bioactivity assays with these are ongoing. 2 b. In contrast to actinomycete GTs, which have been extensiveNext, we performed steady-state kinetics characterizations of ly studied both in vivo and in vitro,[1d, 3] only a couple of Bacilthe BmmGT1-catalyzed reactions. BmmGT1 showed KM values of 1.85 and 17.23 mm for 1 a and UDP-d-glucose, respectively lus GTs have been examined biochemically.[4] The biocatalytic applications of the YjiC-homologous glycosyltransferases from (Table 1), and very close KM values for 2 a (4.43 mm) and 3 a B. cereus (BcGT3) and B. licheniformis DSM-13 (BL-C) have been (4.36 mm; about twice that of 1 a); kcat/KM for 1 a (16.4 min 1 mm 1) was significantly higher than those for 2 a demonstrated in the glycosylation of flavonoids,[4c] geldanamycin analogues,[4b] and isoflavonoids,[16] thus indicating robust yet flexible glycosyltransferase activity towards aglycons. HowTable 1. Kinetic parameters of BmmGT1 with different substrates. ever, neither has been functionally characterized in vivo. As BcGT3 and BL-C are homologous to BmmGT1, they might also 1 1 1 KM [mm] kcat [min ] kcat/KM [min mm ] be involved in the glycosylation of secondary metabolites proAcceptor duced by B. cereus and B. licheniformis DM13, respectively. 1a 1.85 0.36 30.34 0.49 16.4 Our results indicate that BmmGT1 prefers to catalyze glyco2a 4.43 1.03 1.93 0.09 0.44 sylation at 7-OH when it is available (Figure 2, trace ii; Fig3a 4.36 0.61 2.04 0.07 0.47 Donor ure 3 A, trace ii); when malonyl- (2 a) or succinyl-group (3 a) is UDP-d-glucose 17.23 1.93 39.80 1.08 2.31 at 7-OH, BmmGT1 tends to catalyze the attachment of the sugar moiety at 13-OH or 15-OH in vitro, thus affording four novel glycosylated MLN analogues (2 b, 2 c, 3 b and 3 c; Figure 3 A, traces iv and vi). Surprisingly, overexpression of (0.44 min 1 mm 1) and 3 a (0.47 min 1 mm 1). These results dembmmGT1 in vivo led to accumulation of 3 b and 3 c instead of onstrate that acceptor 1 a is clearly favored over 2 a and 3 a for 1 b (Figure 1, trace iii); kinetic parameters revealed that 1 a is BmmGT1 in vitro (Table 1, Figure S17). As BAEs are extremely the most favored substrate for BmmGT1 (Table 1), thus sugunstable,[17] kinetics parameters were not obtained for 4 a and 5 a. 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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CHEMBIOCHEM FULL PAPERS gesting large substrate discrepancy of BmmGT1 between in vivo and in vitro. The fact that both MLNs and BAEs are glycosylated by BmmGT1 in vivo demonstrates that they share a common GT gene; this might explain why the GT gene is located within neither the MLN nor the BAE biosynthetic gene cluster. Genome analysis revealed that the biosynthetic gene clusters for secondary metabolites in Bacillus strains usually do not contain specific GT genes,[13] in contrast to the situation for Streptomyces species.[3] bmmGT1 homologues are conserved among Bacillus species, whereas the MLN and BAE gene clusters are present in only some Bacillus strains. Therefore, the bmmGT1 gene likely evolved at different rates with the MLN and BAE gene clusters, and its homologues from different Bacillus strains probably evolved diversified functions under evolutionary forces. As BAEs are too unstable for isolation,[17] no pure NMR data were obtained. Nevertheless, the identities of BAEs were deduced from UV spectra and HRMS, LC-MS/MS, and 1H NMR analyses of enriched BAE mixtures (Figures S8–S9). For in vitro assays, small amounts of 4 a and 4 b were quickly prepared (to avoid exposure to light and oxygen). However, kinetic characterization of BmmGT1 with 4 a and 4 b could not be performed due to their extreme instability. Given that BAEs are linear compounds (quite different from MLNs), they might adopt a conformation similar to that of MLNs when binding at the active site of the enzyme; the catalytic pocket of BmmGT1 is probably large, thus allowing large substrates like BAEs to fit. Mutagenesis and crystallographic studies will be helpful to understand the substrate flexibility of BmmGT1.
Conclusion We identified and characterized, for the first time, a GT gene in B. marinus B-9987; it is involved in the glycosylation of both MLNs and BAEs. Inactivation of bmmGT1 led to abolishment of the glycosylated MLN (1 b) and BAEs (4 b and 5 b), and to accumulation of non-glycosylated MLNs (1 a, 2 a, and 3 a) and BAEs (4 a and 5 a). Overexpression of bmmGT1 resulted in the production of two novel glycosylated MLNs (3 b and 3 c). We demonstrated that BmmGT1 exhibits broad substrate specificity with regards to aglycon and sugar donor, and five novel glycosylated MLNs were generated. With the benefit of the sugar moiety bestowed on the compounds, we would expect improvements in solubility and bioactivity. Moreover, our results demonstrate the involvement of BmmGT1 in structure diversity of natural products.
Experimental Section Bacterial strains, plasmids, and culture conditions: All strains and plasmids are listed in Table S2. E. coli DH5a was used for general cloning. E. coli ET12567 (dam, dcm, and hsdRMS deficient)[19] was used to prepare the unmethylated E. coli–Bacillus shuttle plasmids. E. coli BL21(DE3) was used for protein expression. B. marinus B9987 (CGMCC No. 2095)[15] has been described previously. E. coli and Bacillus strains were routinely cultured in liquid lysogeny broth 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org (LB; 37 8C, 200 rpm) or on LB agar plates (37 8C). When appropriate, ampicillin (Amp; 100 mg mL 1 for E. coli), chloramphenicol (Chl; 5 mg mL 1 for Bacillus), and erythromycin (Erm; 5 mg mL 1 for Bacillus) was added to the medium. DNA isolation and manipulation: Plasmid extractions and DNA purifications were carried with commercial kits (Omega Bio-Tek, Guangzhou, China). PCR reactions used Pfu DNA polymerase. Oligonucleotide synthesis and DNA sequencing were performed by Shanghai Sunny Biotech Co. (Shanghai, China). Restriction endonucleases and T4 DNA ligase were purchased from Thermo Scientific (Shenzhen, China). A digoxigenin-11-dUTP labeling and detection kit (Roche Diagnostics) was used for the preparation of DNA probes and detection, with Southern hybridization carried out according to the manufacturer’s protocols. Bioinformatics analysis: Mauve software[20] was used to compare the genomes of Bacillus strains. Sequence comparisons and database searches were accomplished with BLAST programs (http:// blast.ncbi.nlm.nih.gov/Blast.cgi). Phylogenetic analysis and neighbor-joining tree construction of BmmGT1 and other natural products GTs were performed with Molecular Evolutionary Genetics Analysis (MEGA) software,[21] and the substitution model was Poisson correction. The amino acid sequences of 38 BLAST-related proteins were exported from the NCBI database and aligned with ClustalW.[22] Gene inactivation: The target gene was inactivated by singlecrossover recombination. An internal DNA fragment of bmmGT1 was inserted into pKSV7 (harboring the temperature-sensitive B. subtilis origin of replication; Table S3).[23] The resulting plasmid (pWLI204) was transformed into E. coli ET12567 to obtain an unmethylated plasmid, and then introduced into B. marinus B-9987 by electroporation according to an established procedure.[18] The transformants were selected on LB plates supplemented with Chl (5 mg mL 1) at 30 8C. After verification by plasmid extraction and restriction enzyme digestion, a single colony was inoculated into LB liquid medium containing Chl (5 mg mL 1) and incubated at 37 8C for 2 h. Serial dilutions of this culture were spread onto LB plates supplemented with Chl (5 mg mL 1), and incubated at 37 8C overnight. The generated single-crossover mutant (DbmmGT1) was confirmed by Southern blot analysis (Table S4 and Figure S2). Gene overexpression in B. marinus B-9987: To construct the plasmid for overexpression in B. marinus B-9987, the bmmGT1 gene was PCR amplified by Pfu DNA polymerase with primer pair bmmGT1OFP/ORP (Table S3), digested with SalI and XbaI, and then cloned into the same sites of pHT3101GFP[18] to yield the expression construct pWLI205. Introduction of pWLI205 into B. marinus B9987 by electroporation afforded B-9987/bmmGT1, in which the bmmGT1 gene was overexpressed from the constitutive gapDHp promoter. Heterologous expression and purification of BmmGT1: The bmmGT1 gene was amplified from B. marinus B-9987 by PCR with primer pair bmmGT1EFP/ERP (Table S3). The PCR product was digested with NdeI and BamHI, purified, and cloned into the same sites of pET28a to generate pWLI206. After confirmation by sequencing, pWLI206 was introduced into E. coli BL21(DE3). Expression of the recombinant protein was induced at OD600 0.6 by addition of isopropyl-b-d-thiogalactopyranoside (IPTG, 0.4 mm), and cultivation was continued for 16 h at 16 8C. Cells were harvested by centrifugation (10 000 g), washed twice, and resuspended in Tris·HCl (50 mm, pH 7.5). The resuspended cells were lysed by sonication in an ice/water bath with a VCX750 ultrasonic processor (Sonics & Materials Inc, PA), then centrifuged (11 000 g, 30 min, ChemBioChem 0000, 00, 1 – 8
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CHEMBIOCHEM FULL PAPERS 4 8C). The supernatant was applied to a HisTrap HP column (1 mL, GE Healthcare) and the N-His6-tagged BmmGT1 protein was eluted with a linear gradient of imidazole (10–500 mm) in binding buffer by using an KTA Purifier system (GE Healthcare). The purified protein was desalted in an Amicon Ultra-15 Centrifugal Filter Unit (Merck Millipore), and stored in Tris·HCl (50 mm, pH 8.0) containing glycerol (10 %) at 80 8C. Isolation of compounds from B. marinus B-9987 strains: For the isolation of 1 a, 2 a, and 3 a, fermentations were performed with the DbmmGT1 mutant strain as previously described.[18] The fermentation broth (30 L) was extracted three times with EtOAc (30 L for each extraction). The combined extracts were concentrated in vacuo to afford a brown residue (12.3 g), which was applied to silica gel column chromatography with elution in a stepped gradient of petroleum ether/EtOAc (4:1, 2:1, 1:1, 1:2, 1:4) to obtain five fractions (FA–FE). Fraction FD (330 mg) was found to contain 1 a, and FE (240 mg) was found to contain 2 a and 3 a; these were purified by semi-preparative HPLC (YMC-Pack ODS-A C18 column, 12 nm, 250 10 mm i.d., 5 mm; YMC Co., Kyoto, Japan) with gradient elution (phase A: formic acid (0.1 % in H2O); phase B: formic acid (0.1 % in acetonitrile) ; 2.5 mL min 1; UV detection at 260 nm) to obtain 1 a (27 mg), 2 a (13 mg), and 3 a (10 mg). For 1 b, 3 a, and 3 b isolation, a fermentation broth (20 L) of the bmmGT1 overexpression strain B-9987/bmmGT1 was extracted with EtOAc as above to afford a brown residue (8.9 g), which was applied to a silica gel column chromatography with gradient elution (petroleum ether/EtOAc (4:1, 1:1, 1:4) and EtOAc/MeOH (4:1, 1:4)) to obtain five fractions (FA–FE). Fraction FD (300 mg) was found to contain 1 b, 3 a, and 3 b, which were subjected to semi-preparative HPLC with elution gradient solvents to obtain purified 1 b (7.5 mg), 3 a (17.5 mg), and 3 b (15 mg). Because of the extreme instability of BAEs, enriched BAE mixtures were obtained as previously described.[17] For in vitro assays, small amounts of 4 a and 5 a were purified rapidly by using an analytical HPLC column (C18, YMC pack ODS-AQ, 5 mm, 150 4.6 mm; YMC Co.) with the exclusion of light and oxygen. To analyze the structures of the compounds, 1D and 2D NMR spectra were recorded with an Avance 600 spectrometer (Bruker). HRMS was carried out with an LTQ-XL mass spectrometer (Thermo Scientific). LC-MS/MS was performed with a model 6430 Triple Quadrupole LC mass spectrometer (Agilent Technologies). In vitro assays of BmmGT1: The concentration of purified protein was determined by the Bradford method with bovine serum albumin (BSA) as the standard. For aglycon flexibility experiments, a typical reaction (50 mL) consisted of aglycon (100 mm), UDP-d-glucose (1 mm), BmmGT1 (2 mm), and MgCl2 (10 mm) in Tris·HCl (50 mm, pH 8.9). The reaction mixtures were incubated at 30 8C for 30 min, and quenched by addition of CH3CN (50 mL), then denatured protein was removed by centrifugation. For sugar donor flexibility tests, the reaction mixtures contained 1 a (100 mm), sugar donor (2 mm), BmmGT1 (10 mm), and MgCl2 (10 mm) in Tris·HCl (50 mm, pH 8.9), and were incubated at 30 8C overnight. The assays were monitored by HPLC analysis with a YMC pack ODS-AQ C18 column (5 mm, 150 4.6 mm; YMC Co.) with UV detection at 260 nm (gradient: 37 % B (0–5 min), 37–55 % B (5–15 min), 100 % B (15–25 min); 1 mL min 1). The in vitro assays of BmmGT1 with BAEs were monitored with UV detection at 345 nm (strong absorbance at this wavelength). Methods for optimizing BmmGT1 reactions are described in the legend of Figure S13. Kinetic parameters were determined according to the Figure S17. Reactions in a volume of 10 mL were performed for isolation of the reaction products, which were purified by an semi-preparative HPLC YMC-Pack ODS-A C18
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org column (12 nm, 250 10 mm i.d., 5 mm; YMC Co.) with isocratic elution (phase A: formic acid (0.1 % in H2O); phase B: CH3CN; 2.5 mL min 1; UV detection 260 nm) to obtain 1 c (5.0 mg), 2 b (2.5 mg), and 2 c (2.0 mg). Structure analysis of the compounds was performed by the procedure described above. Nucleotide sequence accession number: The nucleotide sequence reported in this paper has been deposited in the GenBank database under accession number KJ639107.
Acknowledgements This work was mainly supported by grants from the National Natural Science Foundation of China (31070072 and 31171201), the National High Technology Research and Development Program of China (2012AA092104), the Program for New Century Excellent Talents in University (NCET-0900717) and the State Key Laboratory of Microbial Resources Program, Institute of Microbiology, CAS (No. SKLMR-20110601). We are grateful to Prof. Jçrn Piel (Institute of Microbiology, ETH Zrich, Switzerland) for his valuable suggestion for identification of bacillaenes, and Prof. Gang Liu (Institute of Microbiology, Chinese Academy of Sciences, China) for kindly providing pHT3101 and pKSV7. Keywords: bacillaenes (BAEs) · Bacillus marinus · biosynthesis · glycosylation · glycosyltransferases · macrolactins
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CHEMBIOCHEM FULL PAPERS [11] a) M. A. Mojid Mondol, J. H. Kim, H.-S. Lee, Y.-J. Lee, H. J. Shin, Bioorg. Med. Chem. Lett. 2011, 21, 3832 – 3835; b) C.-J. Zheng, S. Lee, C.-H. Lee, W.-G. Kim, J. Nat. Prod. 2007, 70, 1632 – 1635. [12] T. Nguyen, K. Ishida, H. Jenke-Kodama, E. Dittmann, C. Gurgui, T. Hochmuth, S. Taudien, M. Platzer, C. Hertweck, J. Piel, Nat. Biotechnol. 2008, 26, 225 – 233. [13] a) X. H. Chen, A. Koumoutsi, R. Scholz, A. Eisenreich, K. Schneider, I. Heinemeyer, B. Morgenstern, B. Voss, W. R. Hess, O. Reva, H. Junge, B. Voigt, P. R. Jungblut, J. Vater, R. Sssmuth, H. Liesegang, A. Strittmatter, G. Gottschalk, R. Borriss, Nat. Biotechnol. 2007, 25, 1007 – 1014; b) K. Schneider, X.-H. Chen, J. Vater, P. Franke, G. Nicholson, R. Borriss, R. D. Sssmuth, J. Nat. Prod. 2007, 70, 1417 – 1423; c) Y. Liu, H. Zheng, T.-t. Yao, L. Tian, W.-l. Li, Chin. J. Mar. Drugs 2014, 3, 69 – 75. [14] J. Moldenhauer, D. C. G. Gçtz, C. R. Albert, S. K. Bischof, K. Schneider, R. D. Sssmuth, M. Engeser, H. Gross, G. Bringmann, J. Piel, Angew. Chem. Int. Ed. 2010, 49, 1465 – 1467; Angew. Chem. 2010, 122, 1507 – 1509. [15] C. Xue, L. Tian, M. Xu, Z. Deng, W. Lin, J. Antibiot. 2008, 61, 668 – 674. [16] R. P. Pandey, P. Parajuli, N. Koirala, J. H. Lee, Y. I. Park, J. K. Sohng, Mol. Cells 2014, 37, 172 – 177.
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www.chembiochem.org [17] a) R. A. Butcher, F. C. Schroeder, M. A. Fischbach, P. D. Straight, R. Kolter, C. T. Walsh, J. Clardy, Proc. Natl. Acad. Sci. USA 2007, 104, 1506 – 1509; b) J. Moldenhauer, X.-H. Chen, R. Borriss, J. Piel, Angew. Chem. Int. Ed. 2007, 46, 8195 – 8197; Angew. Chem. 2007, 119, 8343 – 8345; c) J. Moldenhauer, D. C. G. Gçtz, C. R. Albert, S. K. Bischof, K. Schneider, R. D. Sssmuth, M. Engeser, H. Gross, G. Bringmann, J. Piel, Angew. Chem. Int. Ed. 2010, 49, 1465 – 1467; Angew. Chem. 2010, 122, 1507 – 1509. [18] Y. Liu, H. Zheng, G. Zhan, W. Qin, L. Tian, W. Li, Sci. China Life Sci. 2014, 57, 627 – 635. [19] D. J. MacNeil, K. M. Gewain, C. L. Ruby, G. Dezeny, P. H. Gibbons, T. MacNeil, Gene 1992, 111, 61 – 68. [20] A. C. E. Darling, B. Mau, F. R. Blattner, N. T. Perna, Genome Res. 2004, 14, 1394 – 1403. [21] K. Tamura, D. Peterson, N. Peterson, G. Stecher, M. Nei, S. Kumar, Mol. Biol. Evol. 2011, 28, 2731 – 2739. [22] K.-B. Li, Bioinformatics 2003, 19, 1585 – 1586. [23] H. Yakhnin, P. Babitzke, Appl. Microbiol. Biotechnol. 2004, 64, 382 – 386. Received: July 16, 2014 Published online on && &&, 0000
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FULL PAPERS W. Qin, Y. Liu, P. Ren, J. Zhang, H. Li, L. Tian, W. Li* && – && Uncovering a Glycosyltransferase Provides Insights into the Glycosylation Step during Macrolactin and Bacillaene Biosynthesis
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Glycosylation in macrolactin (MLN) and bacillaene (BAE) biosynthesis: BmmGT1, located in neither the MLN nor the BAE biosynthetic gene cluster, was identified in the genome of Bacillus marinus B-9987. Inactivation of bmmGT1 abolished glycosylated MLN and BAE compounds. However, BmmGT1 exhibited broad substrate specificity, and five novel glycosylated MLNs were generated.
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Uncovering a Glycosyltransferase Provides Insights into the Glycosylation Step during Macrolactin and Bacillaene Biosynthesis Wen Qin,[a] Yang Liu,[a] Pengfei Ren,[a] Jun Zhang,[a] Huayue Li,[a] Li Tian,[b, c] and Wenli Li*[a] Macrolactins (MLNs) have unique structural patterns containing a 24-membered ring lactone and diverse bioactivities. The MLN skeleton is biosynthesized via a trans-acyl transferase (AT) type I polyketide synthase (PKS) pathway, but the tailoring steps are still unknown. Herein, we report the identification of a glycosyltransferase (GT) gene bmmGT1, which is located at different locus from the MLN gene cluster in the genome of
marine-derived Bacillus marinus B-9987, and its functional characterization as an MLN GT, thus affording five novel MLNs analogues. Surprisingly, this GT is also capable of catalyzing the glycosylation of bacillaenes (BAEs), which are the prototypes of trans-AT polyketides, thus suggesting broad substrate flexibility. These results provide the first significant insights into the glycosylation step in MLN and BAE biosynthetic pathways.
Introduction Many bioactive natural products are equipped with sugar moieties, which not only increase water solubility thus improving bioavailability but also decrease toxicity.[1] Attachment of sugar moieties to aglycone scaffolds is usually performed by glycosyltransferases (GTs) that catalyze the transfer of sugar moieties from activated donor molecules to specific acceptor molecules by forming glycosidic bonds.[1] Almost all natural-product GTs belong to the GT-B type superfamily; they are functionally diverse but share a highly conserved basic architecture.[1c, d] The reversibility of the reaction and substrate flexibility of GTs make them powerful tools for the glycodiversification of natural products.[2] Most bacterial natural-product GTs that have been functionally characterized are from Actinomyces strains;[1d, 3] in contrast, only a couple of Bacillus natural-product GTs have been used for biocatalytic applications.[4] Macrolactins (MLNs), first isolated from a deep-sea bacterium in 1989 by Fenical and coworkers,[5] have unique structural patterns with a 24-membered ring lactone (Scheme 1). MLNs exhibit a wide range of biological activities, such as antibacterial,[6] antiviral,[5] anticancer,[5] anti-inflammatory,[7] and antiangiogenic.[8] Notably, MLNs can inhibit Staphylococcus aureus peptide deformylase (PDF),[9] which is considered an attractive [a] W. Qin,+ Y. Liu,+ P. Ren, J. Zhang, Dr. H. Li, Prof. Dr. W. Li Key Laboratory of Marine Drugs, Ministry of Education School of Medicine and Pharmacy, Ocean University of China No. 5 Yushan Road Qingdao, SD 266003 (China) E-mail: [email protected] [b] Prof. Dr. L. Tian First Institute of Oceanography, State Oceanic Administration No. 6 Xianxialing Road Qingdao, SD 266061 (China) [c] Prof. Dr. L. Tian Qingdao University of Science and Technology No. 53 Zhen zhou Road Qingdao, SD 266042 (China) [+] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201402384.
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
drug target in view of its wide distribution in bacteria and absence in eukaryotes.[10] At least 30 MLNs have been identified from various Bacillus strains, and several are glycosylated, such as MLNs B–D, O–R, and W.[5, 11] The MLN backbone is assembled by a special family of polyketide synthetases (PKSs) termed trans-acyltransferase (AT) PKSs.[12] Contrary to the general rule that bacterial natural-product GTs genes are clustered with other biosynthetic genes,[3] MLN GT genes are neither in a cluster nor in proximity to one.[13] Interestingly, the same was found for the precursor of trans-AT polyketide bacillaenes (BAEs).[14] Although glycosylated BAE B (Scheme 1) was identified in the fermentation broth of Bacillus amyloliquefaciens FZB42, no glycosyltransferase gene is present in the BAE biosynthetic gene cluster. In a previous study, six MLNs (MLN A, B, D, O, S, and T) were identified in the fermentation broth of Bacillus marinus B-9987, which was isolated from the rhizosphere of Suaeda salsa collected in the intertidal zone of Bohai Bay of Eastern China.[15] In an effort to address the glycosylation mechanism of MLNs, here we report the identification of the bmmGT1 gene in B. marinus B-9987 and characterization of BmmGT1 as both an MLN GT and a BAE GT, as well as its substrate flexibility.
Results Bioinformatics analysis of bmmGT1 In order to locate the GT gene(s) involved in glycosylation of MLNs, genome alignments of the MLN-producing strains B. marinus B-9987 (unpublished) and B. amyloliquefaciens FZB42 (CP000560.1) were carried out with non-MLN-producing strains Bacillus subtilis 168 (NC_000964.3), Bacillus licheniformis 9945A (NC_021362.1), and Bacillus cereus ATCC 14579 (NC_ 004721.2). No obvious unique GT genes were found in the genomes of the MLN-producing strains (compared with the nonChemBioChem 0000, 00, 1 – 8
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www.chembiochem.org revealed that it is phylogenetically close to macrolide GTs, AveBI (avermectin), and OleD/OleI (oleandomycin), thus indicating that BmmGT1 would likely function as an MLN GT.
Effects of bmmGT1 inactivation To probe the function of bmmGT1, we first carried out gene inactivation to detect the production of glycosylated MLNs. Plasmid pWLI204 (harboring a 527 bp internal bmmGT1 fragment) was introduced into B. marinus B-9987. Single-crossover mutants were obtained (Experimental Section), and, after confirmation by Southern blotting (Figure S2), fermentations of wildtype and mutant strains were carried out. HPLC analysis revealed that the production of the glycosylated MLN compound 1 b was completely abolished in the DbmmGT1 mutant (Figure 1, trace ii); non-glycosylated MLN compounds 1 a, 2 a and 3 a accumulated (trace ii), and the production of 1 a
Figure 1. HPLC traces of the fermentation products of B. marinus B-9987 strains: i) wild-type; ii) DbmmGT1 mutant; iii) bmmGT1 overexpression.
Scheme 1. Macrolactins 1 a–c, 2 a--c, and 3 a--c and bacillaenes 4 a, 4 b, 5 a, and 5 b.
MLNs-producing strains). Nevertheless, a conserved putative macrolide GT gene (bmmGT1) was found in all the analyzed genomes. bmmGT1 is in a two-gene operon that contains a putative cytochrome P450 gene bm1409 upstream of bmmGT1. BLASTP queries of the NCBI database revealed that BmmGT1 displays homology to Bacillus cereus BcGT3 (AAS41737.1, 71 % identity), B. subtilis YjiC (WP_015252306, 61 % identity), and B. licheniformis BL-C (AAU40842, 53 % identity). BcGT3 was previously found to be able to catalyze the glycosylation of flavonoids;[4c] BL-C was recently determined to glycosylate at different hydroxyl positions of geldanamycin analogues,[4b] flavonoids,[4a, d] and isoflavonoids;[16] YjiC has not been characterized. A highly conserved domain of the glycosyltransferase GT-B type superfamily was found in BmmGT1. Phylogenetic analysis of BmmGT1 with selected functionally characterized bacterial natural-product GTs (Figure S1 in the Supporting Information) 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
reached 105 mg L 1, which is about six times higher than for the wild-type strain (18 mg L 1, trace i). To our surprise, the production of another type of trans-AT polyketide compound BAE changed as well in the DbmmGT1 mutants. The yields of BAEs 4 a and 5 a clearly increased, while the productions of the glycosylated BAEs 4 b and 5 b were abolished in the DbmmGT1 mutant (trace ii). The identities of 1 a, 1 b, 2 a, and 3 a were confirmed as MLN A, MLN B, 7-O-malonyl-macrolactin A (MMA), and 7-O-succinylmacrolactin A (SMA) (Scheme 1), respectively, by comparison and analysis of the UV spectra, and HRMS and 1H NMR data with those previously reported (Figures S3–S7).[5] BAEs compounds are extremely unstable and very sensitive to light and temperature during isolation.[17] Compounds 4 a, 4 b, and 5 a were deduced to be BAE, BAE B, and dihydro-BAE (Scheme 1), respectively, by UV spectrum, HRMS, liquid chromatography tandem mass spectrometry (LC-MS/MS), and 1H NMR data analysis of enriched BAE mixtures (Figures S8–S9). HRMS measurement of 5 b suggested the molecular formula C40H60N2O11 (m/z 745.4275 [M+H] + , calcd 745.4230), which is six carbon and five oxygen atoms (162 mass units) more than for dihydrobacillaene (5 a: C34H50N2O6, m/z 583.3737 [M+H] + , calcd 583.3702). In addition, the LC-MS/MS spectrum of 5 b showed two fragChemBioChem 0000, 00, 1 – 8
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mentation ions at m/z 384.2 and 434.3, consistent with those of 5 a; the corresponding fragmentation ions for 4 a and 4 b were at m/z 382.2 and 432.2 (Figure S7), thus suggesting the presence of a double bond. Therefore, 5 b was tentatively assigned as dihydro-BAE B (Scheme 1), which would be a new glycosylated BAE compound. These results suggested BmmGT1 is involved in the glycosylation of MLNs in vivo, and probably of BAEs as well. Effects of bmmGT1 overexpression To further demonstrate its function, bmmGT1 was overexpressed under the control of the gapDH promoter (glyceraldehyde-3-phosphate dehydrogenase, gapDHp) in B. marinus B9987.[18] Unmethylated pWLI205 was introduced into B. marinus B-9987 to generate the bmmGT1 overexpression strains (B9987/bmmGT1), and confirmed by plasmid isolation and subsequent restriction analysis (data not shown). HPLC analysis of the fermentation broths showed that production of the nonglycosylated compounds 1 a–5 a was significantly reduced in B-9987/bmmGT1, as expected; to our delight, two novel glycosylated MLN analogues (3 b and 3 c) were generated, whereas the production of 1 b, 4 b, and 5 b decreased (Figure 1, trace iii). Subsequently, an 18 L fermentation of B-9987/bmmGT1 led to the isolation of 3 b and 3 c (Scheme 1). The chemical formula for both compounds was determined to be C34H48O13 by HRMS (m/z 687.3040 [M+Na] + , calcd 687.2993), 162 mass units greater than that of 3 a. Both compounds exhibited spectroscopic data similar to those of 3 a. Eventually, the structures of 3 b and 3 c were elucidated on the basis of extensive NMR analysis (1H,13C COSY, HSQC, HMBC, and NOESY; Table S1, Figures S10–S11). The HMBC correlations from H-15 to C-1’ in 3 b and H-13 to C-1’ in 3 c unambiguously demonstrated that the b-glucopyranosyl moiety was attached to C-15 of 3 b and C-13 of 3 c. These results further demonstrate that bmmGT1 functions as an MLN GT in vivo. In vitro characterization of BmmGT1 In order to characterize its biochemical function, bmmGT1 was overexpressed as an N-terminally His6-tagged soluble protein in Escherichia coli BL21(DE3) carrying pWLI206 (Table S2). After purification to near homogeneity by Ni2 + -NTA agarose affinity chromatography, His6-tagged BmmGT1 was detected by SDSPAGE, which revealed a protein with molecular weight consistent with the calculated value (47.9 kDa; Figure S12). We first tested UDP-d-glucose as the donor sugar substrate, and generated the glycosylated MLN compound 1 b, as expected (Figure 2, trace ii). Then we examined its activity at different pH and temperature values, and in the presence of different cations (Figure S13). BmmGT1 exhibited activity over 10–40 8C (optimum turnover at 30 8C). Without cations, BmmGT1 exhibited over half the best turnover value, but this clearly improved upon addition of Mg2 + (2 and 10 mm), Mn2 + (2 mm), or Ca2 + (2 and 10 mm); optimum activity was with 10 mm Mg2 + , and activity was inhibited by the presence of Ni2 + , 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 2. Sugar donor specificity of BmmGT1: i) 1 a; ii) 1 a + UDP-d-glucose + BmmGT1; iii) 1 a + TDP-d-glucose + BmmGT1; iv) 1 a + UDP-d-glucuronic acid + BmmGT1; v) 1 a + UDP-d-N-acetylglucosamine + BmmGT1.
Cu2 + , and Fe2 + . Interestingly, EDTA enhanced the activity of BmmGT1. The optimum Mg2 + concentration for turnover (tested over 0–30 mm) was 10 mm. Sugar donor specificity of BmmGT1 Under optimized conditions (50 mm Tris·HCl (pH 8.9), 10 mm MgCl2, 30 8C), the sugar nucleotide specificity of BmmGT1 was tested with TDP-d-glucose, UDP-d-N-acetylglucosamine, and UDP-d-glucuronic acid (instead of UDP-d-glucose). BmmGT1 was able to recognize TDP-d-glucose, but only a minor amount of 1 b was detected (Figure 2 trace iii); to our delight, BmmGT1 was also able to use UDP-d-N-acetylglucosamine as the sugar donor, as 1 a was converted into a novel MLN derivative 1 c with the expected molecular weight (Figure 2 trace v); no activity was observed with UDP-d-glucuronic acid (Figure 2 trace iv). Subsequently, 1 c was isolated from a 10 mL enzymatic reaction mixture (Experimental Section). HR MS analysis indicated that 1 c had the molecular formula C32H47NO10 (m/z 606.3320 [M+H] + , calcd 606.3234), which is 203 mass units greater than that of 1 a. A full set of 1D and 2D NMR spectra of 1 c was acquired, thereby allowing us to assign its 1H and 13C signals (Table S1, Figure S14). The spectroscopic data for 1 c were similar to those for 1 a, except for the presence of an N-acetyl glucosamine group at C-7, as determined by HMBC correlations from H-7 to C-2’, C-3’ and C-8’, and H-7 to C-1’. Acceptor specificity of BmmGT1 Given that 2 a–5 a accumulated in the DbmmGT1 mutant in addition to 1 a (Figure 1, trace ii), the acceptor substrate specificity of BmmGT1 was probed with MLN compounds 2 a and 3 a, and BAE compounds 4 a and 5 a. All the compounds tested were recognized by BmmGT1: 3 a transformed into 3 b and 3 c (Figure 3 A, trace vi), 4 a transformed into 4 b (Figure 3 B, trace ii), and 5 a transformed into 5 b (Figure 3 B, trace iv) as expected. All the products had the expected molecular masses according to LC-HRMS analysis (Figures S9–S11). In addition, two novel MLN compounds were generated from 2 a (2 b and 2 c; ChemBioChem 0000, 00, 1 – 8
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Glycosylation alters the stability, solubility, reactivity, and biological activity of aglycons, hence GTs are ideal for structural diversification of natural products.[1d] In general, bacterial GT genes are within the corresponding biosynthetic gene clusters;[3] however, MLN and BAE gene clusters harbor no GT genes, in spite of the production of the glycosylated MLNs and BAEs in Bacillus species.[13, 17c] Given the critical effects of sugar moieties in natural products,[1] we identified the GT gene bmmGT1 in the genome of B. marinus B9987, and functionally characterized it in vivo and in vitro. Firstly, GT mining by comparative genome analysis revealed that there is no specific GT gene in the geFigure 3. Aglycon specificity of BmmGT1: i) 1 a + UDP-d-glucose; ii) 1 a + UDP-d-glucose + BmmGT1; iii) 2 a + UDP-d-glucose; iv) 2 a + UDP-d-glucose + BmmGT1; v) 3 a + UDPnomes of the MLN-producing Bacillus strains (comd-glucose; vi) 3 a + UDP-d-glucose + BmmGT1. B) 4 a + UDP-d-glucose; ii) 4 a + UDP-d-glupared with non-MLN-producing strains). Phylogenetic cose + BmmGT1; iii) 5 a + UDP-d-glucose; iv) 5 a + UDP-d-glucose + BmmGT1. analysis with other functionally characterized bacterial natural product GTs revealed that BmmGT1 is a YjiC homologue and conserved among the genomes anaFigure 3 A, trace iv). These results demonstrate that BmmGT1 lyzed; it is closely related to macrolide GTs (Figure S1), thus incan catalyze the glycosylation of both MLNs and BAEs in dicating that it could function as an MLN GT. Disruption of vitro—broad substrate specificity regarding aglycons. bmmGT1 led to abolishment of the glycosylated MLN (1 b) and, Compounds 2 b and 2 c were isolated from a 10 mL enzysimultaneously, clear accumulation of non-glycosylated MLNs matic reaction mixture (Experimental Section). The chemical (1 a, 2 a, and 3 a; Figure 1, trace ii). To our surprise, glycosylaformula for both was C33H46O13, as determined by HRMS (m/z 673.2881 [M+Na] + , calcd 673.2791), 162 mass units greater tion of BAEs was also blocked in the DbmmGT1 mutant (Figure 1, trace ii), thus indicating involvement of BmmGT1 in than that of 2 a. 1D and 2D NMR spectra led to full assignments the glycosylation of both MLNs and BAEs. Subsequent in vitro of their 1H and 13C signals (Table S1, Figures S15–S16). The studies demonstrated that BmmGT1 is capable of catalyzing compounds exhibited spectroscopic data similar to those of the glycosylation of MLNs as well as BAEs, with broad flexibility 2 a, except for the substitutions at C-13 and C-15. The HMBC regarding both sugar donor (Figure 2) and acceptor (Figure 3). correlations from H-13 to C-1’ (2 c) and H-15 to C-1’ (2 b) sugAs a result, five novel glycosylated MLN compounds were gengested b-glucopyranosyl moieties at C-13 of 2 c and C-15 of erated; bioactivity assays with these are ongoing. 2 b. In contrast to actinomycete GTs, which have been extensiveNext, we performed steady-state kinetics characterizations of ly studied both in vivo and in vitro,[1d, 3] only a couple of Bacilthe BmmGT1-catalyzed reactions. BmmGT1 showed KM values of 1.85 and 17.23 mm for 1 a and UDP-d-glucose, respectively lus GTs have been examined biochemically.[4] The biocatalytic applications of the YjiC-homologous glycosyltransferases from (Table 1), and very close KM values for 2 a (4.43 mm) and 3 a B. cereus (BcGT3) and B. licheniformis DSM-13 (BL-C) have been (4.36 mm; about twice that of 1 a); kcat/KM for 1 a (16.4 min 1 mm 1) was significantly higher than those for 2 a demonstrated in the glycosylation of flavonoids,[4c] geldanamycin analogues,[4b] and isoflavonoids,[16] thus indicating robust yet flexible glycosyltransferase activity towards aglycons. HowTable 1. Kinetic parameters of BmmGT1 with different substrates. ever, neither has been functionally characterized in vivo. As BcGT3 and BL-C are homologous to BmmGT1, they might also 1 1 1 KM [mm] kcat [min ] kcat/KM [min mm ] be involved in the glycosylation of secondary metabolites proAcceptor duced by B. cereus and B. licheniformis DM13, respectively. 1a 1.85 0.36 30.34 0.49 16.4 Our results indicate that BmmGT1 prefers to catalyze glyco2a 4.43 1.03 1.93 0.09 0.44 sylation at 7-OH when it is available (Figure 2, trace ii; Fig3a 4.36 0.61 2.04 0.07 0.47 Donor ure 3 A, trace ii); when malonyl- (2 a) or succinyl-group (3 a) is UDP-d-glucose 17.23 1.93 39.80 1.08 2.31 at 7-OH, BmmGT1 tends to catalyze the attachment of the sugar moiety at 13-OH or 15-OH in vitro, thus affording four novel glycosylated MLN analogues (2 b, 2 c, 3 b and 3 c; Figure 3 A, traces iv and vi). Surprisingly, overexpression of (0.44 min 1 mm 1) and 3 a (0.47 min 1 mm 1). These results dembmmGT1 in vivo led to accumulation of 3 b and 3 c instead of onstrate that acceptor 1 a is clearly favored over 2 a and 3 a for 1 b (Figure 1, trace iii); kinetic parameters revealed that 1 a is BmmGT1 in vitro (Table 1, Figure S17). As BAEs are extremely the most favored substrate for BmmGT1 (Table 1), thus sugunstable,[17] kinetics parameters were not obtained for 4 a and 5 a. 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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CHEMBIOCHEM FULL PAPERS gesting large substrate discrepancy of BmmGT1 between in vivo and in vitro. The fact that both MLNs and BAEs are glycosylated by BmmGT1 in vivo demonstrates that they share a common GT gene; this might explain why the GT gene is located within neither the MLN nor the BAE biosynthetic gene cluster. Genome analysis revealed that the biosynthetic gene clusters for secondary metabolites in Bacillus strains usually do not contain specific GT genes,[13] in contrast to the situation for Streptomyces species.[3] bmmGT1 homologues are conserved among Bacillus species, whereas the MLN and BAE gene clusters are present in only some Bacillus strains. Therefore, the bmmGT1 gene likely evolved at different rates with the MLN and BAE gene clusters, and its homologues from different Bacillus strains probably evolved diversified functions under evolutionary forces. As BAEs are too unstable for isolation,[17] no pure NMR data were obtained. Nevertheless, the identities of BAEs were deduced from UV spectra and HRMS, LC-MS/MS, and 1H NMR analyses of enriched BAE mixtures (Figures S8–S9). For in vitro assays, small amounts of 4 a and 4 b were quickly prepared (to avoid exposure to light and oxygen). However, kinetic characterization of BmmGT1 with 4 a and 4 b could not be performed due to their extreme instability. Given that BAEs are linear compounds (quite different from MLNs), they might adopt a conformation similar to that of MLNs when binding at the active site of the enzyme; the catalytic pocket of BmmGT1 is probably large, thus allowing large substrates like BAEs to fit. Mutagenesis and crystallographic studies will be helpful to understand the substrate flexibility of BmmGT1.
Conclusion We identified and characterized, for the first time, a GT gene in B. marinus B-9987; it is involved in the glycosylation of both MLNs and BAEs. Inactivation of bmmGT1 led to abolishment of the glycosylated MLN (1 b) and BAEs (4 b and 5 b), and to accumulation of non-glycosylated MLNs (1 a, 2 a, and 3 a) and BAEs (4 a and 5 a). Overexpression of bmmGT1 resulted in the production of two novel glycosylated MLNs (3 b and 3 c). We demonstrated that BmmGT1 exhibits broad substrate specificity with regards to aglycon and sugar donor, and five novel glycosylated MLNs were generated. With the benefit of the sugar moiety bestowed on the compounds, we would expect improvements in solubility and bioactivity. Moreover, our results demonstrate the involvement of BmmGT1 in structure diversity of natural products.
Experimental Section Bacterial strains, plasmids, and culture conditions: All strains and plasmids are listed in Table S2. E. coli DH5a was used for general cloning. E. coli ET12567 (dam, dcm, and hsdRMS deficient)[19] was used to prepare the unmethylated E. coli–Bacillus shuttle plasmids. E. coli BL21(DE3) was used for protein expression. B. marinus B9987 (CGMCC No. 2095)[15] has been described previously. E. coli and Bacillus strains were routinely cultured in liquid lysogeny broth 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org (LB; 37 8C, 200 rpm) or on LB agar plates (37 8C). When appropriate, ampicillin (Amp; 100 mg mL 1 for E. coli), chloramphenicol (Chl; 5 mg mL 1 for Bacillus), and erythromycin (Erm; 5 mg mL 1 for Bacillus) was added to the medium. DNA isolation and manipulation: Plasmid extractions and DNA purifications were carried with commercial kits (Omega Bio-Tek, Guangzhou, China). PCR reactions used Pfu DNA polymerase. Oligonucleotide synthesis and DNA sequencing were performed by Shanghai Sunny Biotech Co. (Shanghai, China). Restriction endonucleases and T4 DNA ligase were purchased from Thermo Scientific (Shenzhen, China). A digoxigenin-11-dUTP labeling and detection kit (Roche Diagnostics) was used for the preparation of DNA probes and detection, with Southern hybridization carried out according to the manufacturer’s protocols. Bioinformatics analysis: Mauve software[20] was used to compare the genomes of Bacillus strains. Sequence comparisons and database searches were accomplished with BLAST programs (http:// blast.ncbi.nlm.nih.gov/Blast.cgi). Phylogenetic analysis and neighbor-joining tree construction of BmmGT1 and other natural products GTs were performed with Molecular Evolutionary Genetics Analysis (MEGA) software,[21] and the substitution model was Poisson correction. The amino acid sequences of 38 BLAST-related proteins were exported from the NCBI database and aligned with ClustalW.[22] Gene inactivation: The target gene was inactivated by singlecrossover recombination. An internal DNA fragment of bmmGT1 was inserted into pKSV7 (harboring the temperature-sensitive B. subtilis origin of replication; Table S3).[23] The resulting plasmid (pWLI204) was transformed into E. coli ET12567 to obtain an unmethylated plasmid, and then introduced into B. marinus B-9987 by electroporation according to an established procedure.[18] The transformants were selected on LB plates supplemented with Chl (5 mg mL 1) at 30 8C. After verification by plasmid extraction and restriction enzyme digestion, a single colony was inoculated into LB liquid medium containing Chl (5 mg mL 1) and incubated at 37 8C for 2 h. Serial dilutions of this culture were spread onto LB plates supplemented with Chl (5 mg mL 1), and incubated at 37 8C overnight. The generated single-crossover mutant (DbmmGT1) was confirmed by Southern blot analysis (Table S4 and Figure S2). Gene overexpression in B. marinus B-9987: To construct the plasmid for overexpression in B. marinus B-9987, the bmmGT1 gene was PCR amplified by Pfu DNA polymerase with primer pair bmmGT1OFP/ORP (Table S3), digested with SalI and XbaI, and then cloned into the same sites of pHT3101GFP[18] to yield the expression construct pWLI205. Introduction of pWLI205 into B. marinus B9987 by electroporation afforded B-9987/bmmGT1, in which the bmmGT1 gene was overexpressed from the constitutive gapDHp promoter. Heterologous expression and purification of BmmGT1: The bmmGT1 gene was amplified from B. marinus B-9987 by PCR with primer pair bmmGT1EFP/ERP (Table S3). The PCR product was digested with NdeI and BamHI, purified, and cloned into the same sites of pET28a to generate pWLI206. After confirmation by sequencing, pWLI206 was introduced into E. coli BL21(DE3). Expression of the recombinant protein was induced at OD600 0.6 by addition of isopropyl-b-d-thiogalactopyranoside (IPTG, 0.4 mm), and cultivation was continued for 16 h at 16 8C. Cells were harvested by centrifugation (10 000 g), washed twice, and resuspended in Tris·HCl (50 mm, pH 7.5). The resuspended cells were lysed by sonication in an ice/water bath with a VCX750 ultrasonic processor (Sonics & Materials Inc, PA), then centrifuged (11 000 g, 30 min, ChemBioChem 0000, 00, 1 – 8
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CHEMBIOCHEM FULL PAPERS 4 8C). The supernatant was applied to a HisTrap HP column (1 mL, GE Healthcare) and the N-His6-tagged BmmGT1 protein was eluted with a linear gradient of imidazole (10–500 mm) in binding buffer by using an KTA Purifier system (GE Healthcare). The purified protein was desalted in an Amicon Ultra-15 Centrifugal Filter Unit (Merck Millipore), and stored in Tris·HCl (50 mm, pH 8.0) containing glycerol (10 %) at 80 8C. Isolation of compounds from B. marinus B-9987 strains: For the isolation of 1 a, 2 a, and 3 a, fermentations were performed with the DbmmGT1 mutant strain as previously described.[18] The fermentation broth (30 L) was extracted three times with EtOAc (30 L for each extraction). The combined extracts were concentrated in vacuo to afford a brown residue (12.3 g), which was applied to silica gel column chromatography with elution in a stepped gradient of petroleum ether/EtOAc (4:1, 2:1, 1:1, 1:2, 1:4) to obtain five fractions (FA–FE). Fraction FD (330 mg) was found to contain 1 a, and FE (240 mg) was found to contain 2 a and 3 a; these were purified by semi-preparative HPLC (YMC-Pack ODS-A C18 column, 12 nm, 250 10 mm i.d., 5 mm; YMC Co., Kyoto, Japan) with gradient elution (phase A: formic acid (0.1 % in H2O); phase B: formic acid (0.1 % in acetonitrile) ; 2.5 mL min 1; UV detection at 260 nm) to obtain 1 a (27 mg), 2 a (13 mg), and 3 a (10 mg). For 1 b, 3 a, and 3 b isolation, a fermentation broth (20 L) of the bmmGT1 overexpression strain B-9987/bmmGT1 was extracted with EtOAc as above to afford a brown residue (8.9 g), which was applied to a silica gel column chromatography with gradient elution (petroleum ether/EtOAc (4:1, 1:1, 1:4) and EtOAc/MeOH (4:1, 1:4)) to obtain five fractions (FA–FE). Fraction FD (300 mg) was found to contain 1 b, 3 a, and 3 b, which were subjected to semi-preparative HPLC with elution gradient solvents to obtain purified 1 b (7.5 mg), 3 a (17.5 mg), and 3 b (15 mg). Because of the extreme instability of BAEs, enriched BAE mixtures were obtained as previously described.[17] For in vitro assays, small amounts of 4 a and 5 a were purified rapidly by using an analytical HPLC column (C18, YMC pack ODS-AQ, 5 mm, 150 4.6 mm; YMC Co.) with the exclusion of light and oxygen. To analyze the structures of the compounds, 1D and 2D NMR spectra were recorded with an Avance 600 spectrometer (Bruker). HRMS was carried out with an LTQ-XL mass spectrometer (Thermo Scientific). LC-MS/MS was performed with a model 6430 Triple Quadrupole LC mass spectrometer (Agilent Technologies). In vitro assays of BmmGT1: The concentration of purified protein was determined by the Bradford method with bovine serum albumin (BSA) as the standard. For aglycon flexibility experiments, a typical reaction (50 mL) consisted of aglycon (100 mm), UDP-d-glucose (1 mm), BmmGT1 (2 mm), and MgCl2 (10 mm) in Tris·HCl (50 mm, pH 8.9). The reaction mixtures were incubated at 30 8C for 30 min, and quenched by addition of CH3CN (50 mL), then denatured protein was removed by centrifugation. For sugar donor flexibility tests, the reaction mixtures contained 1 a (100 mm), sugar donor (2 mm), BmmGT1 (10 mm), and MgCl2 (10 mm) in Tris·HCl (50 mm, pH 8.9), and were incubated at 30 8C overnight. The assays were monitored by HPLC analysis with a YMC pack ODS-AQ C18 column (5 mm, 150 4.6 mm; YMC Co.) with UV detection at 260 nm (gradient: 37 % B (0–5 min), 37–55 % B (5–15 min), 100 % B (15–25 min); 1 mL min 1). The in vitro assays of BmmGT1 with BAEs were monitored with UV detection at 345 nm (strong absorbance at this wavelength). Methods for optimizing BmmGT1 reactions are described in the legend of Figure S13. Kinetic parameters were determined according to the Figure S17. Reactions in a volume of 10 mL were performed for isolation of the reaction products, which were purified by an semi-preparative HPLC YMC-Pack ODS-A C18
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org column (12 nm, 250 10 mm i.d., 5 mm; YMC Co.) with isocratic elution (phase A: formic acid (0.1 % in H2O); phase B: CH3CN; 2.5 mL min 1; UV detection 260 nm) to obtain 1 c (5.0 mg), 2 b (2.5 mg), and 2 c (2.0 mg). Structure analysis of the compounds was performed by the procedure described above. Nucleotide sequence accession number: The nucleotide sequence reported in this paper has been deposited in the GenBank database under accession number KJ639107.
Acknowledgements This work was mainly supported by grants from the National Natural Science Foundation of China (31070072 and 31171201), the National High Technology Research and Development Program of China (2012AA092104), the Program for New Century Excellent Talents in University (NCET-0900717) and the State Key Laboratory of Microbial Resources Program, Institute of Microbiology, CAS (No. SKLMR-20110601). We are grateful to Prof. Jçrn Piel (Institute of Microbiology, ETH Zrich, Switzerland) for his valuable suggestion for identification of bacillaenes, and Prof. Gang Liu (Institute of Microbiology, Chinese Academy of Sciences, China) for kindly providing pHT3101 and pKSV7. Keywords: bacillaenes (BAEs) · Bacillus marinus · biosynthesis · glycosylation · glycosyltransferases · macrolactins
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FULL PAPERS W. Qin, Y. Liu, P. Ren, J. Zhang, H. Li, L. Tian, W. Li* && – && Uncovering a Glycosyltransferase Provides Insights into the Glycosylation Step during Macrolactin and Bacillaene Biosynthesis
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Glycosylation in macrolactin (MLN) and bacillaene (BAE) biosynthesis: BmmGT1, located in neither the MLN nor the BAE biosynthetic gene cluster, was identified in the genome of Bacillus marinus B-9987. Inactivation of bmmGT1 abolished glycosylated MLN and BAE compounds. However, BmmGT1 exhibited broad substrate specificity, and five novel glycosylated MLNs were generated.
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