Schafhauser Et Al Final

1 Classification: BIOLOGICAL SCIENCES: Microbiology; Plant Biology 2 3 Title: Antitumor astins originate from the fungal endophyte Cyanodermella asteris living 4 within the medicinal plant Aster tataricus 5 Short title: Astins originate from the fungus C. asteris 6 7 Authors: 8 T Schafhausera,b,d,1, L Jahnb,1, N Kirchnerc, A Kulika, L Flord, A Langd, T Caradece, DP Fewerf, K 9 Sivonenf, WJH van Berkelg, P Jacquese,h, T Weberi, H Grossc, KH van Péed, W Wohllebena,2,3 and 10 J Ludwig-Müllerb,2,3 11 1equal contribution 12 2these authors share senior authorship 13 3corresponding authors: [email protected], phone: +49-70712976944; 14 [email protected], phone: +49-35146333939 15 16 aMicrobiology and Biotechnology, Interfaculty Institute of Microbiology and Infection Medicine, Eberhard Karls 17 University Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany 18 bInstitute of Botany, TU Dresden, Zellescher Weg 20b, 01217 Dresden, Germany 19 cPharmaceutical Institute, Department of Pharmaceutical Biology, Eberhard Karls University Tübingen, Auf der 20 Morgenstelle 8, 72076 Tübingen, Germany 21 dGeneral Biochemistry, TU Dresden, 01062 Dresden, Germany 22 eUniversity Lille, INRA, ISA, University Artois, University Littoral Côte d’Opale, EA 7394 – ICV – Institut Charles 23 Viollette, Lille, France 24 fDepartment of Microbiology, University of Helsinki, Viikinkaari 9, FI-00014, Helsinki, Finland 25 gLaboratory of Biochemistry, Wageningen University & Research, Stippeneng 4, 6708 WE Wageningen, The 26 Netherlands 27 hTERRA Teaching and Research Centre, Microbial Processes and Interactions, Gembloux Agro-Bio Tech University 28 of Liege, Gembloux, Belgium 29 iThe Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet bygning 30 220, 2800 Kgs. Lyngby, Denmark 31 32 Keywords: astin, natural product, antitumor, cross-species biosynthesis, medicinal plant, NRPS 33 1 34 Abstract 35 Medicinal plants are a prolific source of natural products with remarkable 36 chemical and biological properties often constituting remedial benefits. 37 Numerous medicinal plants are suffering from wildcrafting and thus 38 biotechnological production processes of their products are urgently needed. 39 The plant Aster tataricus is widely used in traditional Chinese medicine and 40 contains unique active ingredients named astins. These are macrocyclic peptides 41 showing promising antitumor activities and usually containing the highly unusual 42 moiety 3,4-dichloroproline. Although astins have been studied for decades, their 43 biosynthetic origin is unknown. Here we show that astins are produced by the 44 recently discovered fungal endophyte Cyanodermella asteris. We were able to 45 produce astins in reasonable and reproducible amounts in axenic culture of the 46 endophyte. We identified the gene cluster responsible for astin biosynthesis in 47 the genome of C. asteris and propose a production pathway that is based on a 48 non-ribosomal peptide synthetase. Striking differences in the production profiles 49 of endophyte and host plant imply a symbiotic cross-species biosynthesis 50 pathway for astin C derivatives, in which plant enzymes or plant signals are 51 required to trigger the synthesis of plant-exclusive variants such as astin A. Our 52 findings lay the foundation for a sustainable biotechnological astin production 53 independent from aster plants. 54 2 55 Significance Statement 56 The natural products astins bind to a crucial human regulatory protein, the 57 stimulator of interferon genes (STING), which is a promising new therapeutic 58 target for cancer and immune disorders. Astins have long been regarded as 59 phytochemicals of a Chinese medicinal plant. However, we could show that they 60 are produced by the newly identified fungal endophyte Cyanodermella asteris 61 via a non-ribosomal biosynthetic pathway. These findings pave the way for a 62 cost-effective biotechnological astin production. Moreover, we provide strong 63 evidence that certain astin variants are further modified inside the host through 64 the involvement of a further biological partner. Such symbiotic interactions are 65 barely described for phytochemicals and might be more widespread than 66 previously expected. 67 3 68 /body 69 Introduction 70 Medicinal plants provide health-promoting or curative properties in versatile 71 ways and are used since prehistoric times across the world. It is estimated that 72 more than 70% of the world’s population rely on medicinal plants (1) – with 73 increasing tendency (2). As a result, some medicinal plants are facing existential 74 problems to get extinct by wildcrafting and over-collection (3) making alternative 75 approaches to exploit their beneficial properties desirable. 76 Medicinal plants contain numerous chemically highly diverse natural products 77 (NPs) with versatile biological functions. In traditional medicine, predominantly 78 multicomponent mixtures (e.g. tea, decoction, tincture) are applied even though 79 the active principle(s) largely are inadequately investigated (2). Moreover, plant 80 NPs are a rich source for successful single-molecule drugs in modern medicine. 81 Indeed, more than half of the small molecule pharmaceuticals approved in the 82 last decades were derived or inspired from nature (4) and a substantial number 83 of these compounds has been discovered in higher plants (5). Prominent 84 examples of plant-derived pharmaceuticals are paclitaxel, vincristine and 85 vinblastine, camptothecin, and artemisinin (5-7). 86 Plant NPs are generally characterized by low production yields and complex 87 chemical structures making it challenging to meet market demands by means of 88 plant cultivation or chemical synthesis(8). Instead, successful resupply of plant- 89 derived drugs on the pharmaceutical market relies on biotechnological 90 production that requires knowledge of the usually complex biosynthetic 91 pathways(9). Endophytic microorganisms, i.e. bacteria or fungi residing in the 92 interior of plants(10, 11), are increasingly reported to produce NPs originally 93 known from their host plants(12-16). Nevertheless, none of these endophytes 4 94 has proved suitable for industrial resupply of the desired compound mainly due 95 to low and unstable production titers(13, 17). 96 The flowering plant Aster tataricus L. f. (Compositae) has long found use in 97 traditional Chinese medicine for the treatment of cough and bronchial 98 diseases(18). Several studies performing chemical profiling of A. tataricus roots 99 revealed a large number of chemically diverse NPs(19-21). Of these, the 100 macrocyclic peptides astins show anticancer and immunosuppressive 101 activities(22-25) by binding to a crucial innate immune protein, the cytosolic DNA 102 sensor STING(26). In total, 24 astin variants (astins A-P(20, 27-32)) have been 103 elucidated including the structurally related branched (tataricins(33)) and linear 104 variants (asterinins(34, 35)). Quantitatively, the astin variants A-C dominate in A. 105 tataricus preparations(18, 31, 36). They consist of five amino acids including L-2- 106 aminobutyric acid (2Abu), β-(R)-phenylalanine (βPhe), and the unique 3,4- 107 dichlorinated L-proline (ProCl2) (Fig. 1a). Low natural availability(27, 31, 37) and 108 insufficient chemical synthesis approaches(38, 39) of astins prevent a thorough 109 exploration of their biological activity. 110 The biosynthetic origins of astins remained unclear but it was widely considered 111 that they are made by the plant A. tataricus(40), possibly via a non-ribosomal 112 peptide synthetase (NRPS)(33). NRPSs are large multi-domain enzymes 113 predominantly found in bacteria and fungi that catalyze a stepwise peptide 114 synthesis by a thiotemplate mechanism. NRPSs are able to utilize a large number 115 of non-proteinogenic amino acid substrates to generate non-ribosomal peptides 116 (NRPs)(41). The mycotoxin cyclochlorotine from the fungus Talaromyces 117 islandicus(42) is an NRP showing remarkable structural similarities to astins 118 suggesting that astins likewise might be produced by a fungus. 5 119 Here, we show that astins are in fact of fungal origin. The endophyte 120 Cyanodermella asteris that has been recently isolated from inflorescences of A. 121 tataricus(43) produces astin variants in pure culture including astin C, one of the 122 three main variants isolated from A. tataricus(25, 27, 31, 37). Plants free of C. 123 asteris did not contain any astins and re-infection with this fungus restored astin 124 production including plant-exclusive variants such as astin A. We identified the 125 astin biosynthetic gene cluster in the recently sequenced(44) fungal genome. C. 126 asteris is characterized by stable and sound production of astins, which is unlike 127 other endophytes that often only poorly and unreliably produce NPs originally 128 known from their host plants(45-48). Our findings pioneer prospective 129 biotechnological production of this valuable NP. 130 131 Results 132 Aster tataricus harbors an endophytic community including the astin producing 133 fungus Cyanodermella asteris 134 Astins are primarily reported from dried roots and rhizomes of A. tataricus(20, 135 28, 29, 31). Extracts from fresh plant roots clearly also contained astins according 136 to HPLC-MS analyses (Fig. 1b). Interestingly, some of the investigated A. tataricus 137 plants did not contain any traces of astins (Fig. 1c), despite cultivation under 138 identical conditions. This information

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