Letter Cite This: Org. Lett. 2018, 20, 7996−7999
pubs.acs.org/OrgLett
Biosynthesis of Quinolidomicin, the Largest Known Macrolide of Terrestrial Origin: Identification and Heterologous Expression of a Biosynthetic Gene Cluster over 200 kb Takuya Hashimoto,† Junko Hashimoto,‡ Ikuko Kozone,‡ Keita Amagai,§,∥ Teppei Kawahara,‡ Shunji Takahashi,∥ Haruo Ikeda,⊥ and Kazuo Shin-ya*,†,# †
National Institute of Advanced Industrial Science and Technology, 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan Japan Biological Informatics Consortium, 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan § Technology Research Association for Next Generation Natural Products Chemistry, 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan ∥ RIKEN Center for Sustainable Resource Science, Natural Product Biosynthesis Research Unit, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan ⊥ Kitasato Institute for Life Sciences, Kitasato University, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0373, Japan # The Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
Org. Lett. 2018.20:7996-7999. Downloaded from pubs.acs.org by UNIV OF WINNIPEG on 12/21/18. For personal use only.
‡
S Supporting Information *
ABSTRACT: Quinolidomicin A1 is the largest macrolide compound from terrestrial sources, consisting of a 60membered ring, and its biosynthetic gene cluster was revealed to be over 200 kb. The gene cluster for quinolidomicin was cloned and heterologously expressed. Confirmation of the product led to a structural revision, in which the hydroxy group in the chromophore moiety of the reported structure was replaced by an amine group.
M
the heterologous expression of biosynthetic gene clusters over 100 kb in size have been reported4,5 because it is extremely difficult to clone and introduce such giant biosynthetic gene clusters into the host strain. During the course of our focused efforts to establish a technique for the heterologous expression of large type-I PKS biosynthetic gene clusters, we succeeded in the heterologous production of mediomycin and neomediomycin,6 whose biosynthetic gene clusters are 161 and 183 kb long, respectively, using bacterial artificial chromosome (BAC) vectors. This success challenged us to further develop our technology and take full advantage of the capacity of BAC vectors (up to 300 kb genome libraries can be prepared with BAC vectors) for cloning larger size biosynthetic gene clusters. Quinolidomicin A1 (1) is a 60-membered macrocyclic compound and is the largest macrolide of terrestrial origin identified to date.7,8 Compound 1 was first isolated from Micromonospora sp. JY16 as a potent cytotoxic compound against HT-29, MKN28, K562, and KB. We identified Micromonospora chalcea AK-AN57 as another producer strain of 1 from our in-house library during the course of our drugscreening programs. The structure of 1 suggests that at least 33
acrocyclic polyketides, including so-called macrolides, are representative microbial secondary metabolites used as clinical drugs1 such as erythromycin, rapamycin, avermectin, amphotericin B, and FK506. Macrocyclic polyketides are typically biosynthesized by type-I polyketide synthases (PKSs). The biosynthesis machinery of type-I PKS has many similarities to that of fatty acid synthase (FAS) in terms of utilizing common precursors and similar catalytic units. Decarboxylative condensations for single-chain elongation during biosynthesis are catalyzed by three domains: βketosynthase (KS), acyl transferase (AT), and acyl carrier protein (ACP). In addition to this elongation scheme, the three modification domains, dehydratase (DH), enoylreductase (ER), and β-ketoreductase (KR), participate in the formation of structural variations. These catalytic domains are covalently linked, and a set of domains is called a “module”. Finally, the elongated acyclic intermediate is excised by thioesterase (TE) at a final module to form macrocycles.2 In the case of type-I PKS, the size of the macrocyclic polyketide compounds is determined by the number of modules, and many clinical drug macrolides consist of a large number of modules.3 Despite the many heterologous expression experiments for the production of microbial secondary metabolites performed over the past two decades,4 only a few examples of © 2018 American Chemical Society
Received: November 8, 2018 Published: December 13, 2018 7996
DOI: 10.1021/acs.orglett.8b03570 Org. Lett. 2018, 20, 7996−7999
Letter
Organic Letters
and downstream regions of the biosynthetic gene cluster, and the corresponding insert DNA involving the entire biosynthetic gene cluster for 1 was confirmed by analyzing the terminal sequence of the BamHI site of pKU518 vector.12 The obtained BAC clone, pKU518quiP9-L5, was introduced into S. lividans TK23ΔredDX strain,6 and the obtained transformant S. lividans TK23ΔredDX::pKU518quiP9-L5 was cultured in producing media. However, the production of 1 was not detected (Figure 2A).
modules participate in its biosynthesis, making 1 the ideal target for demonstrating our BAC technology because its biosynthetic gene cluster is expected to be larger than 200 kb. In this paper, we describe the identification, cloning, and heterologous expression of quinolidomicin biosynthetic genes, together with a structural revision of quinolidomicin A1 (1). We determined the biosynthetic gene cluster for 1 by determining the de novo draft genome sequence of M. chalcea AK-AN57 and obtained a 6.9 Mb genome sequence divided into seven scaffolds. We skimmed the entire draft genome and identified three candidate biosynthetic gene clusters containing type-I PKS genes. One of the clusters including 13 ORF of type-I pks genes was selected as a putative biosynthetic region of quinolidomicin A1 (1) as the other two candidates both encode only three pks genes. The 13 pks genes harboring 34 modules, including a loading module (Figures 1 and Figures S1 and S2).
Figure 2. Detection of quinolidomicin A1. UPLC analysis of culture extracts from (A) S. lividans TK23ΔredDX::pKU518quiP9-L5, (B) S. lividans TK23ΔredDX::pKU518quiP9-L5::pKU460-sav2794pqnmRIRII, and (C) AK-AN57 strains. The chromatograms at 270 nm were extracted. (D) Positive- and negative-ion mode HR-ESI-MS data of 1 are shown in the upper and lower panels, respectively.
We therefore reanalyzed the biosynthetic gene cluster of quinolidomicin A1 (1). In the biosynthesis of many polyketides derived from type-I PKS, it is well-known that cluster-specific large ATP-binding LuxR type regulator (LAL regulator) is encoded in the biosynthetic gene cluster which up-regulates the transcription level of the entire genes in the gene cluster.13−15 In the case of quinolidomicin A1 (1), a database search revealed two LAL regulator genes, qnmRI and qnmRII, tandemly encoded in the biosynthetic gene cluster. We therefore introduced these genes into S. lividans TK23ΔredDX::pKU518quiP9-L5 to activate the transcription level of the biosynthetic genes. We cloned these genes into another integrative vector, pKU460-sav2794,16 by Gibson’s assembly12,17 (Figure S3) and introduced the vector into S. lividans TK23ΔredDX::pKU518quiP9-L5. The obtained clone, S. lividans TK23ΔredDX::pKU518quiP9-L5::pKU460sav2794p-qnmRIRII, was cultured in producing media and the culture extract was analyzed by UPLC-TOF-MS (Figure 2B). As a result, 1 was detected from the culture extract of the transformant, thereby unequivocally identifying the biosynthetic gene cluster for 1 as the 213.7 kb region by heterologous expression. The yield of 1 was approximately 0.1 mg L−1 in the culture broth. The HR-ESI-MS data of the produced compound from the transformant and M. chalcea AK-AN57 was analyzed to be 1550.8944 (C83H133NO22SNa+, [M + Na]+ +1.2 mmu) and 1526.8956 (C83H132NO22S−, [M−H]− −1.1 mmu), respectively (Figure 2D). However, to our surprise, the molecular formula deduced by these values was inconsistent with the reported formula (C83H132O23S, [M + Na]+ 1551.8771). We suspected that quinolidomicin A1 produced by M. chalcea AKAN57 is different from that obtained from Micromonospora sp. JY16. To confirm this hypothesis, we isolated the original quinolidomicin A1 from the culture media of Micromonospora
Figure 1. Domain organization of PKS’s for the biosynthesis of quinolidomicin A1 (1).
Another characteristic feature of the biosynthetic gene cluster for 1 is the presence of genes related to the 3-amino-5hydroxybenzoic acid (AHBA) biosynthetic pathway, involved in the biosynthesis of the starter unit of 1. A comparison of these genes with the genes for AHBA in rifamycin is shown in Table 1.9,10 The entire biosynthetic gene cluster spans about 215 kb (Figure S1B). Table 1. Comparison between Rifamycin and 1 of the Biosynthetic Genes Related to the AHBA Pathway biosynthetic enzymes for AHBA
identity/similarity (%)
homologous protein
RifN RifJ RifK RifL RifM RifG RifH
54/67 72/79 69/79 58/68 64/74 66/73
QnmS2 QnmS3 QnmS4 QnmS5 QnmS6 QnmS7 not found
In order to clarify whether this biosynthetic gene cluster is responsible for the production of 1, we prepared a genome library to clone the entire biosynthetic gene cluster using the BAC vector pKU518.11 The genomic DNA of the producer strain was partially digested with BamHI and separated by Contour-clamped homogeneous electric field (CHEF) electrophoresis. Digested DNA fragments around 250 kb in length (Figure S1) were excised and ligated into the BamHI site of the pKU518 vector to construct a huge BAC library. Desired clones were screened by PCR amplification of the upstream 7997
DOI: 10.1021/acs.orglett.8b03570 Org. Lett. 2018, 20, 7996−7999
Letter
Organic Letters
intermediate is shown in Figure S3, and the stereochemistry of the fully elongated intermediate is proposed as 5L, 6D, 7D, 8L, 9D, 14D, 15D, 16L, 17D, 19D, 20L, 23L, 25L, 27L, 33L, 37L, 45L, 47L, 50L, 55L, 57L, 59D, 64L, and 67D (Figure S5). The predicted stereochemistry of 1 is shown in Figure 3. This information will be helpful for the future determination of the absolute configuration of 1. The stereochemistry at the C-3 and C-10 positions of 1 could not be predicted from the amino acid sequence of PKS due to conversions of the nascent product released from PKS: the formation of an acetal function between a ketone carbonyl C-3 and a hydroxy group at C-7 and double-bond migration27,28 at C-10 and C-11, respectively. The deduced functions of the genes encoded in the quinolidomicin A1 biosynthetic gene cluster are shown in Table S4. AHBA biosynthetic pathways are conserved and utilized in the synthesis of many macrocyclic polyketides containing AHBA units.9,10 The six genes qnmS2, S3, S4, S5, S6, and S7 show high sequence similarities to the AHBA biosynthetic genes rif N, J, K, L, M, and G, respectively, involved in the biosynthesis of rifamycin. These genes may be involved in the biosynthetic pathway of their respective starter units, in which case the amine group of the starter unit of 1 would be incorporated in a similar manner in the biosynthesis of AHBA. However, aminoDAHP synthase (rif H), which is required for the incorporation of phosphoenolpyruvate into 1deoxy-1-iminoerythrose 4-phosphate in the biosynthesis of AHBA, is absent from the quinolidomicin cluster (Table 1 and Figure S6).29 Other genes annotated as hypothetical proteins in the gene cluster might be involved in this step to complete the pathway leading to the putative starter, the 2-(methylthio)5-aminobenzoquinone 1-carboxylic acid moiety. However, few examples of biosynthetic mechanisms for incorporating sulfur atoms into natural products have been reported,30 and thus, further experimental verification is required to elucidate the source of the sulfur atom and the mechanism of reaction for building this functional group. To our knowledge, the biosynthetic gene cluster for 1 is the largest biosynthetic gene cluster of secondary metabolites identified to date. This is the first report of heterologous expression of a gene cluster larger than 200 kb. Few successful heterologous expressions of biosynthetic gene clusters larger than 150 kb have been reported.6 For the heterologous expression of 1, the additional introduction of pKU460sav2794p-qnmRIRII encoding the LAL regulator was essential, perhaps due to the incompatibility of the promoter of the Micromonospora strain located upstream of the LAL regulator genes, possibly resulting in the promoter sequence not being transcribed in S. lividans TK23ΔredDX. This could be addressed by introducing the sav2794 promoter upstream of the regulator gene to induce expression of the genes. We experimentally demonstrated heterologous expression of the biosynthetic gene cluster for quinolidomicin A1, which is over 200 kb long. During the course of our heterologous expression experiments, we have obtained a BAC clone containing a 260 kb insert (data not shown). This is therefore a promising heterologous expression technique applicable for the expression of most biosynthetic gene clusters of microbial secondary metabolites.
sp. JY16. The HR-MS data of purified quinolidomicin A1 from Micromonospora sp. JY16 indicated the same molecular formula as C83H133NO22S [M − H]− calculated for 1526.8965 (Δ −0.2 mmu). The preferred structure of 1 is that in which a hydroxy functional group at C-70 in the reported structure is substituted by an amine group (Figure 3). This revised
Figure 3. Detected 1H−15N-HMBC correlation of the structure of 1. The original reported structure is shown in the upper right. The predicted stereochemistry of 1 is shown.
structure is also consistent with the structure of the putative starter unit derived from AHBA. The structure cannot be assigned by the 13C NMR chemical shift value at C-70 (δC 150.7)18−20 and thus we verified the presence of an amine group at the C-70 position by measuring the 1H−15N-HMBC spectrum of 1. A 1H−15N long-range coupling from the aromatic proton 71-H (δH 5.66) to 70-N (δN −306) was observed. This confirmed the presence of an amine group at C70 (Figure 3, S6),21 and thus, we revised the structure of this quinone moiety in 1 to be a 2-(methylthio)-5-aminobenzoquinone. We subjected the biosynthetic gene cluster of 1 to a BLAST search by employing ClusterBlast in antiSMASH22 and identified two related gene clusters in M. aurantiaca ATCC 27029 and Micromonospora sp. L5. A comparison of these three gene clusters is shown in Figure S2B. We annotated the function of each domain included in the 13 pks ORFs in detail (Figures S2A, S4, and S5). The predicted substrate of each AT domain showed good agreement with the reported structure of 1 (Figure S4). However, two acyltransferase domains in modules 19 and 21 are distinctly different from the canonical AT domains. These ATs are categorized in the AT0 domain because catalytic serine residue in GHSxG motif is absent in these domains.23 Although the function of these AT0 domains remains unclear, these domains can accept malonyl-CoA as substrate deduced from the structure of 1 (Figures S4B and S5). The stereochemistry of the ACP-bound intermediate can be predicted from the sequence alignment of each domain: the LDD motif and W motif in the KR domain are markers for the stereochemistry of the β-hydroxyl product,24 and the YxP motif may correlate with the stereochemistry of the methyl residue at the α-position.25 According to these criteria, we distinguished all the KR domains as being of the A1, A2, B1, and B2 types, which correspond to the four combinations of methyl and hydroxy stereochemistry ((D, L), (L, L), (D, D), and (L, D),26 respectively, Figure S4D). Sequence alignment also suggested that the DH domain in module 1 and the KR domains in modules 17 and 33 are inactive because the catalytic residues in these domains are not conserved (Figures S4C and S4D). The resulting predicted structure of the ACP-bound 7998
DOI: 10.1021/acs.orglett.8b03570 Org. Lett. 2018, 20, 7996−7999
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(19) Kwak, J. H.; Schmitz, F. J.; Kelly, M. J. Nat. Prod. 2000, 63 (8), 1153−1156. (20) Takao, K. I.; Nemoto, R.; Mori, K.; Namba, A.; Yoshida, K.; Ogura, A. Chem. - Eur. J. 2017, 23 (16), 3828−3831. (21) Hesse, M.; Meier, H.; Zeeh, B. Spectroscopic Methods in Organic Chemistry, 2nd ed.; Thieme, 2008. (22) Weber, T.; Blin, K.; Duddela, S.; Krug, D.; Kim, H. U.; Bruccoleri, R.; Lee, S. Y.; Fischbach, M. A.; Muller, R.; Wohlleben, W.; Breitling, R.; Takano, E.; Medema, M. H. Nucleic Acids Res. 2015, 43 (W1), W237−W243. (23) Keatinge-Clay, A. T. Chem. Rev. 2017, 117 (8), 5334−5366. (24) Keatinge-Clay, A. T. Chem. Biol. 2007, 14 (8), 898−908. (25) Caffrey, P. ChemBioChem 2003, 4 (7), 654−657. (26) Keatinge-Clay, A. T. Nat. Prod. Rep. 2016, 33 (2), 141−149. (27) Lohr, F.; Jenniches, I.; Frizler, M.; Meehan, M. J.; Sylvester, M.; Schmitz, A.; Gutschow, M.; Dorrestein, P. C.; Konig, G. M.; Schaberle, T. F. Chem. Sci. 2013, 4 (11), 4175−4180. (28) Berkhan, G.; Merten, C.; Holec, C.; Hahn, F. Angew. Chem., Int. Ed. 2016, 55 (43), 13589−13592. (29) Guo, J. T.; Frost, J. W. J. Am. Chem. Soc. 2002, 124 (4), 528− 529. (30) Dunbar, K. L.; Scharf, D. H.; Litomska, A.; Hertweck, C. Chem. Rev. 2017, 117 (8), 5521−5577.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.8b03570. Experimental procedures, supplemental figures (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Keita Amagai: 0000-0001-5804-4047 Kazuo Shin-ya: 0000-0002-4702-0661 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the Japan Agency for Medical Research and Development (AMED) under Grant No. JP17ae0101002 for K.S. and JSPS Grant-in-Aid for Young Scientists for T.H. under Grant No. 18K14349.
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DOI: 10.1021/acs.orglett.8b03570 Org. Lett. 2018, 20, 7996−7999
