US 201402961 61A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0296.161 A1 Qian et al. (43) Pub. Date: Oct. 2, 2014 (54) DIDEMININ BIOSYNTHETIC GENE CLUSTER Publication Classification INTISTRELLA MOBILIS (51) Int. Cl. (71) Applicant: King Abdullah University of Science C07K II/02 (2006.01) and Technology, Thuwal (SA) C07K I4/95 (2006.01) (72) Inventors: Pei-Yuan Qian, Hong Kong (CN); Ying (52) U.S. Cl. Sharon Xu, Hong Kong (CN); Pok-Yui CPC ............... C07K II/02 (2013.01); C07K 14/195 Lai, Hong Kong (CN) (2013.01) (73) Assignee: King Abdullah University of Science USPC ......... 514/21.1:536/23.1; 53.59. 530/324; and Technology, Thuwal (SA) 435/252.3; 435/69.1 (21) Appl. No.: 14/346,068 (57) ABSTRACT (22) PCT Filed: Sep. 21, 2012 (86). PCT No.: PCT/B2O12/OO2361 A novel Tistrella mobilis strain having Accession Deposit S371 (c)(1), Number NRRL B-50531 is provided. A method of producing (2), (4) Date: Mar. 20, 2014 a didemnin precursor, didemnin or didemnin derivative by O O using the Tistrella mobilis strain, and the therapeutic compo Related U.S. Application Data sition comprising at least one didemnin or didemnin deriva (60) Provisional application No. 61/537.416, filed on Sep. tive produced from the strain or modified strain thereof are 21, 2011. also provided. Patent Application Publication Oct. 2, 2014 Sheet 1 of 16 US 2014/0296.161 A1 OMe M-0 N.0-Melyr H II0 OMe HO s O HO Thr'III O Me OH O). NY"N . Cy 0^ M s OH LaC Q Me NH H0O y HO, If M ) 0 H, Isf N-Me-D-leu PO Didemnin Band its mOnOmerS OMe s' N O Me 0 is 0 O Me OH'-N."N O. NH, 0 H, S-NH H 00sI N - Q H - 0 OH O N-N-N- C.His 0 H O H Didemnin X 01NH, FIG. A Patent Application Publication Oct. 2, 2014 Sheet 2 of 16 US 2014/0296.161 A1 OMe O Me X)",N (...) OH NOrdidemnin B OMe Dehydrodidemnin B (Aplidine) FIG. 1A (Cont'd) Patent Application Publication Oct. 2, 2014 Sheet 3 of 16 US 2014/0296.161 A1 HC H 3 OH O' HCO -- HC CH, Ni CH OCH, OH Didemnin A R = H W Didemnin B. R = GorOH Didemnin C R = O O HC O FIG. 1B Patent Application Publication Oct. 2, 2014 Sheet 4 of 16 US 2014/0296.161 A1 1-----=*----------kA.} T----------------- s w were s gas Patent Application Publication Oct. 2, 2014 Sheet 6 of 16 US 2014/0296.161 A1 ººººººººººººººººo@@@@@@ Patent Application Publication Oct. 2, 2014 Sheet 7 of 16 US 2014/0296.161 A1 Patent Application Publication Oct. 2, 2014 Sheet 8 of 16 US 2014/0296.161 A1 Patent Application Publication Oct. 2, 2014 Sheet 9 of 16 US 2014/0296.161 A1 Patent Application Publication Oct. 2, 2014 Sheet 10 of 16 US 2014/0296.161 A1 Patent Application Publication Oct. 2, 2014 Sheet 11 of 16 US 2014/0296.161 A1 ?WO Patent Application Publication Oct. 2, 2014 Sheet 12 of 16 US 2014/0296.161 A1 Patent Application Publication Oct. 2, 2014 Sheet 13 of 16 US 2014/0296.161 A1 Patent Application Publication Oct. 2, 2014 Sheet 14 of 16 US 2014/0296.161 A1 r ---- -1 Patent Application Publication Oct. 2, 2014 Sheet 15 of 16 US 2014/0296.161 A1 [X]p3/0£TIÚT?!?-1779-MÊMuni1000$| 7.TEMYSE?NETVÆRSVEIFON Patent Application Publication Oct. 2, 2014 Sheet 16 of 16 US 2014/0296.161 A1 -T-t-t-t-t-t-t-t-t-t-t-t- ges Y ———————————————————————————————————————————??[?]/[]] US 2014/0296.161 A1 Oct. 2, 2014 DDEMININ BIOSYNTHETIC GENE CLUSTER producing the didemnins. Furthermore, the invention relates INTISTRELLA MOBILS to therapeutic compositions containing the didemnins and to uses of the therapeutic compositions. 0001. This application claims priority to U.S. Provisional Patent Application Ser. No. 61/537,416, filed Sep. 21, 2011, 0006. It is therefore an object of the present invention to which application is incorporated by reference herein in its provide novel species of didemnin-producing Tistrella bacte entirety. ria. It is also an object of the invention to provide a novel Tistrella mobilis bacterium or one or more strains thereof. In TECHNICAL FIELD specific cases, the novel didemnin-producing Tistrella mobi lis bacterium was deposited on Jul. 27, 2011 as Accession No. 0002 The present invention generally concerns the fields NRRL B-50531 with the depository Agricultural Research of cell biology, molecular biology, bacteriology, and medi Service Culture Collection, National Center for Agricultural cine. In particular aspects, the invention concerns direct or Utilization Research Agricultural Research Service, U.S. indirect production of anti-cancer compounds in bacteria. Department of Agriculture, 1815 North University Street, Peoria, Ill. 61604, U.S.A. BACKGROUND OF THE INVENTION 0007 An isolated or biologically pure culture of the bac 0003. The didemnins (FIG. 1) are a group of cyclic dep terium is encompassed in the invention. A further object of the sipeptides with extraordinary biological activities, including present invention is to provide didemnins, didemnin precur antitumor, antivirus and immunosuppressive activities (Vera sors, and/or didemnin derivatives produced by novel strains and Joullié, 2002; Rawat et al., 2006). Rinehart and his of Tistrella. In additional aspects of the present invention, coworkers (1981) initially isolated didemnins A, B and C there are novel amino acid and polynucleotide sequences of from a Caribbean tunicate of the family Didemnidae in 1981. Tistrella mobilis, and exemplary sequences include SEQ ID Since then, more than 20 other didemnins have been isolated NOS:10-61. from tunicates collected from different geographical loca 0008 Any Tistrella species may be utilized in the inven tions. Owing to its potent antitumor activity, didemnin B was tion. In specific embodiments, the following bacteria are uti the first marine natural product selected to enterclinical trials. lized: Tistrella mobilis (Shi et al., 2002); Tistrella bauzanen Although didemnin B failed in mid-1990s, a closely related sis (Zhang et al., 2010), Tistrella sp. BZ78, Tistrella sp. didemnin compound-dehydrodidemnin B (Aplidin) is cur D1-34, Tistrella sp. D1-36, Tistrella sp. D6-30, Tistrella sp. rently in human phase II clinic trials for Solid and haemato f-1-2, Tistrella sp. JW16.1a, Tistrella sp. MARC2PPND, logical malignant neoplasias like T cell lymphoma and Tistrella sp. PhS5A, Tistrella sp. S67-5, Tistrella sp. S73-3, myelofibrosis and in phase III clinical trials for multiple and Tistrella sp. Zp5. myeloma (Soto-Matos et al., 2011). Until now, didemnins are exclusively isolated from the eukaryotic marine tunicates. 0009 Specific embodiments of the invention include the However, since cyclic peptides are usually synthesized by isolation of didemnin B and nordidemnin B from a Gram non-ribosomal peptide synthetases (NRPS) from microor negative marine-derived bacterium Tistrella mobilis. The ganisms, a microorganism associated with the tunicates complete genome sequence of this bacterium revealed the would be a useful source for producing didemnins. Until the biosynthetic gene cluster responsible for the didemnin Syn present invention, careful scrutiny of tunicate symbiotic thesis. Bioinformatic analysis was used to predict the func microorganisms has not been Successful to locate Such a tion of the genes in the cluster. Culture conditions were opti microorganism and chemical synthesis remains to be the only mized in order to increase the yield of the target didemnin route to obtain sufficient amount of didemnins. The chemical compounds. reactions to synthesize didemnin A, the simplest compound 0010. In particular aspects, there is a process for producing of the didemnin family, are too complicated to be finished a didemnin, which comprises the steps of: a) culturing at least within 10 steps (Jou et al., 1997), and consequently the yield is one Tistrella strain in growth-Supporting nutrient medium undoubtedly very low. Therefore, the discovery of didemnin capable of promoting growth and reproduction of said bacte biosynthetic gene cluster from a microbe will be beneficial ria, wherein said culturing is effected for a time sufficient to for producing didemnins more economically. More impor allow production of a didemnin; and b) recovering the didem tantly, novel didemnin analogues can be generated through nin from said bacteria or medium of step a). genetic engineering of the biosynthetic pathways and be 0011. One embodiment of the present invention is to pro investigated for their biological activities, which may in turn vide a plurality of bacteria for the mass production of a provide more drug leads. didemnin or precursor or derivative thereof. BRIEF SUMMARY OF THE INVENTION 0012 A particular embodiment of the present invention is to provide a novel process for the production of didemnins, 0004. The present invention is directed to a system, didemnin precursors, or didemnin derivatives from bacteria. method, and compositions related to production of anti-can The industrial application of this process would provide cer compounds or precursors or derivatives thereto from a renewable sources of didemnins, didemnin precursors, or bacterium. In particular aspects, the bacterium is from the didemnin derivatives for the pharmaceutical industry. In cer Tistrella genus, and in specific aspects the bacterium is tain aspects, there is a biotransformation process in which Tistrella mobilis. bacteria-derived didemnins, didemnin precursors, or didem 0005. This invention relates to the production of nin derivatives are converted into Substances that are useful as didemnins or didemnin derivatives through the use of novel therapeutic compounds or for the production of other thera didemnin-producing bacterium and/or didemnins or didem peutic compounds. In certain aspects of the invention, didem nin derivatives produced by these species; it also relates to nin B and/or nordidemnin B (for example) are produced polynucleotide and amino acids from the novel bacterium endogenously by Tistrella mobilis and derivatives are made US 2014/0296.161 A1 Oct.