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(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization I International Bureau (10) International Publication Number (43) International Publication Date WO 2013/041969 A2 28 March 2013 (28.03.2013) P O P C T (51) International Patent Classification: Not classified AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, (21) International Application Number: DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, PCT/IB2012/002361 HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, (22) International Filing Date: KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, 2 1 September 2012 (21 .09.2012) ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, (25) Filing Language: English RW, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, (26) Publication Language: English TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (30) Priority Data: 61/537,416 2 1 September 201 1 (21.09.201 1) US (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, (71) Applicant: KING ABDULLAH UNIVERSITY OF SCI¬ GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, ENCE AND TECHNOLOGY [SA/SA]; P.O. Box 55455, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, Jeddah, 21534 (SA). TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, (72) Inventors: QIAN, Pei-yuan; Hong Kong University Of MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, Science And Technology, Divison Of Life Science, Room TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, 5443, Clear Water Bay, Kowloon, Hong Kong (CN). XU, ML, MR, NE, SN, TD, TG). Ying, Sharon; Hong Kong University Of Science And Technology, Divison Of Life Science, Room 6321, Clear Published: Water Bay, Kowloon, Hong Kong (CN). LAI, Pok-yui; — without international search report and to be republished Hong Kong University Of Science And Technology, Divis upon receipt of that report (Rule 48.2(g)) on Of Life Science, Room 6321, Clear Water Bay, Kowloon, Hong Kong (CN). — with sequence listing part of description (Rule 5.2(a)) (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, (54) Title: DIDEMNIN BIOSYNTHETIC GENE CLUSTER IN TISTRELLA MOBILIS 8 * ¾ γ' 1 < -V © » ,-> (57) Abstract: The present invention includes compositions and methods related to a novel Tistrella bacterium. In particular, the o compositions and methods encompass didemnin precursors, didemnins, and didemnin derivatives. In specific embodiments, the bac - terium is Tistrella mobilis. In certain aspects, all or part of the didemnin synthesis pathway is incorporated into another host cell, such as E. coli. DIDEMNIN BIOSYNTHETIC GENE CLUSTER IN TISTRELLA MOBILIS This application claims priority to U.S. Provisional Patent Application Serial No. 61/537,416, filed September 21, 2011, which application is incorporated by reference herein in its entirety. TECHNICAL FIELD [0001] The present invention generally concerns the fields of cell biology, molecular biology, bacteriology, and medicine. In particular aspects, the invention concerns direct or indirect production of anti-cancer compounds in bacteria. BACKGROUND OF THE INVENTION [0002] The didemnins (FIG. 1) are a group of cyclic depsipeptides with extraordinary biological activities, including antitumor, antivirus and immunosuppressive activities (Vera and Joullie, 2002; Rawat et al., 2006). Rinehart and his coworkers (1981) initially isolated didemnins A, B and C from a Caribbean tunicate of the family Didemnidae in 1981. Since then, more than 20 other didemnins have been isolated from tunicates collected from different geographical locations. Owing to its potent antitumor activity, didemnin B was the first marine natural product selected to enter clinical trials. Although didemnin B failed in mid-1990s, a closely related didemnin compound-dehydrodidemnin B (Aplidin) is currently in human phase II clinic trials for solid and haematological malignant neoplasias like T cell lymphoma and myelofibrosis and in phase III clinical trials for multiple myeloma (Soto-Matos et al., 2011). Until now, didemnins are exclusively isolated from the eukaryotic marine tunicates. However, since cyclic peptides are usually synthesized by non-ribosomal peptide synthetases (NRPS) from microorganisms, a microorganism associated with the tunicates would be a useful source for producing didemnins. Until the present invention, careful scrutiny of tunicate symbiotic microorganisms has not been successful to locate such a microorganism and chemical synthesis remains to be the only route to obtain sufficient amount of didemnins. The chemical reactions to synthesize didemnin A, the simplest compound of the didemnin family, are too complicated to be finished within 10 steps (Jou et al, 1997), and consequently the yield is undoubtedly very low. Therefore, the discovery of didemnin biosynthetic gene cluster from a microbe will be beneficial for producing didemnins more economically. More importantly, novel didemnin analogues can be generated through genetic engineering of the biosynthetic pathways and be investigated for their biological activities, which may in turn provide more drug leads. BRIEF SUMMARY OF THE INVENTION [0003] The present invention is directed to a system, method, and compositions related to production of anti-cancer compounds or precursors or derivatives thereto from a bacterium. In particular aspects, the bacterium is from the Tistrella genus, and in specific aspects the bacterium is Tistrella mobilis. [0004] This invention relates to the production of didemnins or didemnin derivatives through the use of novel didemnin-producing bacterium and/or didemnins or didemnin derivatives produced by these species; it also relates to polynucleotide and amino acids from the novel bacterium producing the didemnins. Furthermore, the invention relates to therapeutic compositions containing the didemnins and to uses of the therapeutic compositions. [0005] It is therefore an object of the present invention to provide novel species of didemnin-producing Tistrella bacteria. It is also an object of the invention to provide a novel Tistrella mobilis bacterium or one or more strains thereof. In specific cases, the novel didemnin- producing Tistrella mobilis bacterium was deposited on July 27, 2011 as Accession No. NRRL B- 50531 with the depository Agricultural Research Service Culture Collection, National Center for Agricultural Utilization Research Agricultural Research Service, U.S. Department of Agriculture, 1815 North University Street, Peoria, Illinois 61604, U.S.A. [0006] An isolated or biologically pure culture of the bacterium is encompassed in the invention. A further object of the present invention is to provide didemnins, didemnin precursors, and/or didemnin derivatives produced by novel strains of Tistrella. In additional aspects of the present invention, there are novel amino acid and polynucleotide sequences of Tistrella mobilis, and exemplary sequences include SEQ ID NOS: 10-61. [0007] Any Tistrella species may be utilized in the invention. In specific embodiments, the following bacteria are utilized: Tistrella mobilis (Shi et al., 2002); Tistrella bauzanensis (Zhang et al., 2010), Tistrella sp. BZ78, Tistrella sp. Dl-34, Tistrella sp. Dl-36, Tistrella sp. D6-30, Tistrella sp. f-1-2, Tistrella sp. JW16.1a, Tistrella sp. MARC2PPND, Tistrella sp. PhS5A, Tistrella sp. S67-5, Tistrella sp. S73-3, and Tistrella sp. Zp5. [0008] Specific embodiments of the invention include the isolation of didemnin B and nordidemnin B from a Gram-negative marine-derived bacterium Tistrella mobilis. The complete genome sequence of this bacterium revealed the biosynthetic gene cluster responsible for the didemnin synthesis. Bioinformatic analysis was used to predict the function of the genes in the cluster. Culture conditions were optimized in order to increase the yield of the target didemnin compounds. [0009] In particular aspects, there is a process for producing a didemnin, which comprises the steps of: a) culturing at least one Tistrella strain in growth-supporting nutrient medium capable of promoting growth and reproduction of said bacteria, wherein said culturing is effected for a time sufficient to allow production of a didemnin; and b) recovering the didemnin from said bacteria or medium of step a). [0010] One embodiment of the present invention is to provide a plurality of bacteria for the mass production of a didemnin or precursor or derivative thereof. [0011] A particular embodiment of the present invention is to provide a novel process for the production of didemnins, didemnin precursors, or didemnin derivatives from bacteria. The industrial application of this process would provide renewable sources of didemnins, didemnin precursors, or didemnin derivatives for the pharmaceutical industry. In certain aspects, there is a biotransformation process in which bacteria-derived didemnins, didemnin precursors, or didemnin derivatives are converted into substances that are useful as therapeutic compounds or for the production of other therapeutic compounds. In certain aspects of the invention, didemnin B and/or nordidemnin B (for example) are produced endogenously by Tistrella mobilis and derivatives are made therefrom one or the other; however, in other cases other didemnins are produced endogenously by other Tistrella
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  • Complete Genome Sequencing and Antibiotics Biosynthesis Pathways

    Complete Genome Sequencing and Antibiotics Biosynthesis Pathways

    www.nature.com/scientificreports OPEN Complete genome sequencing and antibiotics biosynthesis pathways analysis of Streptomyces lydicus Received: 14 December 2016 Accepted: 13 February 2017 103 Published: 20 March 2017 Nan Jia1,2, Ming-Zhu Ding1,2, Hao Luo1,2,3, Feng Gao1,2,3 & Ying-Jin Yuan1,2 More and more new natural products have been found in Streptomyces species, which become the significant resource for antibiotics production. Among them,Streptomyces lydicus has been known as its ability of streptolydigin biosynthesis. Herein, we present the genome analysis of S. lydicus based on the complete genome sequencing. The circular chromosome of S. lydicus 103 comprises 8,201,357 base pairs with average GC content 72.22%. With the aid of KEGG analysis, we found that S. lydicus 103 can transfer propanoate to succinate, glutamine or glutamate to 2-oxoglutarate, CO2 and L-glutamate to ammonia, which are conducive to the the supply of amino acids. S. lydicus 103 encodes acyl-CoA thioesterase II that takes part in biosynthesis of unsaturated fatty acids, and harbors the complete biosynthesis pathways of lysine, valine, leucine, phenylalanine, tyrosine and isoleucine. Furthermore, a total of 27 putative gene clusters have been predicted to be involved in secondary metabolism, including biosynthesis of streptolydigin, erythromycin, mannopeptimycin, ectoine and desferrioxamine B. Comparative genome analysis of S. lydicus 103 will help us deeply understand its metabolic pathways, which is essential for enhancing the antibiotic production through metabolic engineering. Streptomyces species are high-GC Gram-positive bacteria found predominantly in soil1. Through a complex pro- cess of morphological and physiological differentiation, Streptomyces species could produce many specialized metabolites used for agricultural antibiotics2.