Chemical Science View Article Online EDGE ARTICLE View Journal | View Issue Biosynthesis of trioxacarcin revealing a different starter unit and complex tailoring steps for type II Cite this: Chem. Sci.,2015,6, 3440 polyketide synthase† Mei Zhang,‡a Xian-Feng Hou,‡a Li-Hua Qi,a Yue Yin,a Qing Li,a Hai-Xue Pan,a Xin-Ya Chena and Gong-Li Tang*ab Trioxacarcins (TXNs) are highly oxygenated, polycyclic aromatic natural products with remarkable biological activity and structural complexity. Evidence from 13C-labelled precursor feeding studies demonstrated that the scaffold was biosynthesized from one unit of L-isoleucine and nine units of malonyl-CoA, which suggested a different starter unit in the biosynthesis. Genetic analysis of the biosynthetic gene cluster revealed 56 genes encoding a type II polyketide synthase (PKS), combined with a large amount of tailoring enzymes. Inactivation of seven post-PKS modification enzymes resulted in the production of a Received 12th January 2015 series of new TXN analogues, intermediates, and shunt products, most of which show high anti-cancer Accepted 7th April 2015 Creative Commons Attribution-NonCommercial 3.0 Unported Licence. activity. Structural elucidation of these new compounds not only helps us to propose the biosynthetic DOI: 10.1039/c5sc00116a pathway, featuring a type II PKS using a novel starter unit, but also set the stage for further www.rsc.org/chemicalscience characterization of the enzymatic reactions and combinatorial biosynthesis. Introduction The high biological activities, especial anti-cancer activity, along with unusual and complex structural features of TXN-A Microorganisms can produce a large variety of biologically distinguish it from other aromatic polyketides, thus providing active secondary metabolites representing a vast diversity of an interesting but challenging target for total synthesis. This article is licensed under a fascinating molecular architecture, which usually attract Recently, Myers's group successfully established a multiply attention for chemical synthesis, mode of action, biosynthesis, convergent, component-based route to chemically synthesize and even drug discovery studies. As an example, trioxacarcin A TXN-A and its structural analogues.8,9 However, the biosynthetic (TXN-A, 1, Fig. 1) represents a special family of complex studies have never been explored to these structurally complex Open Access Article. Published on 07 April 2015. Downloaded 9/28/2021 11:58:41 AM. aromatic natural products, which was rst isolated from antibiotics. Herein, we describe (1) incorporation studies with Streptomyces bottropensis DO-45 (NRRL 12051) in 1981,1–3 and 13C-labelled precursors, which elucidated the biosynthetic subsequently re-isolated from a marine Streptomyces sp. B8652 origin of the scaffold for the TXN family of natural products; (2) with a series of analogues in 2004.4,5 It displays extraordinary the genetic characterization of txn gene cluster, which afforded anti-bacterial, anti-malarial, and anti-tumor activity with sub- four polyketide derivates and seven TXN analogues; and (3) a 1–5 ff nanomolar IC70 values in various cancer cell lines. Structur- proposed biosynthetic pathway, involving a di erent starter ally, TXN-A contains an unusual condensed polycyclic trisketal, unit for priming type II polyketide synthase (PKS) and complex bearing a fused spiro-epoxide, which is believed to be a tailoring steps. “warhead” to covalently bind to DNA, followed by cleavage of the resultant TXN–DNA complex, to yield another natural product gutingimycin (3, Fig. 1) through an abstraction of the guanine.6,7 In addition, it has unique glycosylation patterns, including a rare g-branched octose. aState Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032, China. E-mail: [email protected] bShanghai Collaborative Innovation Center for Biomanufacturing Technology, 130 Meilong Road, Shanghai 200237, China † Electronic supplementary information (ESI) available: The experimental procedures, strains, plasmids and PCR primers, and compound characterization. See DOI: 10.1039/c5sc00116a Fig. 1 Chemical structure of trioxacarcin (TXN) and relative natural ‡ M. Zhang and X.-F. Hou contributed equally to this work. products. 3440 | Chem. Sci.,2015,6, 3440–3447 This journal is © The Royal Society of Chemistry 2015 View Article Online Edge Article Chemical Science Results and discussion suggested the folding pattern of the polyketide chain in Fig. 2A, which was further supported by the [1,2-13C]-acetate feeding ff Biosynthetic origin of the polycyclic sca old of TXNs results (Fig. S1 and Table S1†). Obviously, the right ring TXN-A was originally isolated from S. bottropensis DO-45 with contains three malonate-derived intact acetate units (C-9a to the isolation of 20 mg from an 18 L fermentation broth;1,2 C-1, C-2 to C-3, and C-4 to C-4a), which suggested the folding however this titer was not efficient enough for biosynthetic pattern of the polyketide chain could be classied as a typical studies. In our early efforts to optimize the fermentation and Streptomyces mode.11 In addition, the incorporation of the isolation processes, we noticed that the yield of TXN-A could be [2-13C]-acetate to C-18 indicated that a decarboxylation step signicantly improved by a hundred times through the addition should be involved in the formation of a fused-ring skeleton. of the hydrophobic resin HP-20 into the fermentation medium, However, the labelled pattern of the ve-carbon fragment (C-13, À even up to titers of 100–200 mg L 1 in shaking asks.10 Under C-14, C-15, C-16, and C-17) remains confused, which hints that this optimized condition, the precursors [1-13C]-acetate, [2-13C]- this ve-carbon unit may be derived from another origin. acetate, and [1,2-13C]-acetate were added to a fermentation Moreover, the ve-carbon unit was likely employed by the type II À culture (a total of 0.7 g L 1) by pulse feeding aer 48, 56, 64, 72, PKS as a non-acetate starter unit to generate the polyketide in 80, 88 h of incubation in separate incorporation experiments, which the decarboxylation is usually performed on the last and the fermentation lasted 120 h. TXN-A isolated from the carbon of the fully elongated polyketide chain. feeding fermentations was subjected to 13C-NMR analysis to A ve-carbon unit (C-13 to C-17), most possibly from 2- conrm the polyketide extender units of the scaffold (ESI, Table methylbutyryl-CoA, serving as the starter unit of PKS, is seldom S1†). All the 13C abundance at each position of the TXN-A observed in natural product biosynthesis. The only exception is backbone could be sufficiently separated and identied (ESI, involved in the biosynthesis of avermectin “a” components, Fig. S1†). These incorporation results are summarized in Table which are 16-membered macrocyclic lactones generated by type S1† and Fig. 2A. Signicant enrichment was observed at C-1, C- I PKS through loading 2-methylbutyryl-CoA as the starter unit.12 13 Creative Commons Attribution-NonCommercial 3.0 Unported Licence. 3, C-4a, C-6, C-8, C-9, C-10a, and C-11 in the [1- C]-acetate For the type II PKS, although non-acetate starter units, labelled TXN-A, as well as C-2, C-4, C-5, C-7, C-8a, C-9a, C-10, C- including propionate, malonamate, polyketide or fatty acid, and 12, and C-18 in the [2-13C]-acetate labelled TXN-A; both even amino acid derivates have also been employed,13 2-methyl- butyryl-CoA has never been reported as a starter unit to generate aromatic polyketides. Given the fact that 2-methylbutyryl-CoA is usually derived from L-isoleucine (Ile) through deamination and decarboxylation by transaminase and branched-chain 2-oxo acid dehydrogenase in vivo, we performed the feeding experi- 13 ment with C6-L-Ile to validate this hypothesis (Fig. 2A). This article is licensed under a Remarkably, ESI-MS showed TXN-A from this feeding experi- ment was +5 m/z heavier than that without feeding (Fig. 2B), indicating the incorporation of a ve-carbon unit which arose Open Access Article. Published on 07 April 2015. Downloaded 9/28/2021 11:58:41 AM. from an intact Ile. Further speci c and signi cant signal enrichment at C-13 to C-17 (Fig. 2C) in the 13C-NMR spectra, and all the 13C–13C coupling data (Fig. 2D) are consistent with the same conclusion (JC-16/C-15 ¼ 62 Hz, JC-15/C-14 ¼ 58 Hz, JC-13/C-14 ¼ 54 Hz, and JC-17/C-14 ¼ 32 Hz), which are in agreement with this ve-carbon unit originating from Ile via an intact incorporation manner. Thus, these results unambiguously demonstrated that the missing ve-carbon unit, C-13 to C-17, is derived from L-Ile, which most likely follows a deamination and decarboxylation process similar to that of the avermectin “a” components biosynthesis.12 Cloning, sequencing, and identication of the biosynthetic gene cluster of TXNs The aromatic polycyclic skeleton of TXNs and the primary 13C- Fig. 2 Characterization of the biosynthetic origin of TXNs by labeled acetate feeding experiments suggest that a type-II PKS precursor feeding experiments. (A) Summary of feeding results with should be involved in the biosynthesis. Therefore, we cloned the 13 13 C-labeled sodium acetate and C6-L-isoleucine (Ile). (B) MS analysis gene cluster by the PCR approach specic for accessing the 13 of production of TXN by fermentation without (I) or with C6-L-Ile (II). genes encoding a ketosynthase (KS)-chain length factor (CLF) (C) 13C-NMR spectra of TXN-A with (II) and without (I) feeding of 13C - 6 heterodimer.14 By screening the genomic library and the L-Ile.