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2 12/ 1696 Al (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date k i ft - 9 February 2012 (09.02.2012) 2 12/ 1696 Al (51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every C07K 14/365 (2006.01) C12P 19/26 (2006.01) kind of national protection available): AE, AG, AL, AM, C12N 15/11 (2006.01) AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, (21) Number: International Application DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, PCT/EP201 1/063243 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, 1 August 201 1 (01 .08.201 1) ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD, (25) Filing Language: English SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, (26) Publication Language: English TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (30) Priority Data: (84) Designated States (unless otherwise indicated, for every 1017 183 1.0 4 August 2010 (04.08.2010) EP kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, (71) Applicant (for all designated States except US): BAYER ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, PHARMA AKTIENGESELLSCHAFT [DE/DE]; TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, Muller Strasse 178, 13353 Berlin (DE). EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, (72) Inventors; and SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, (75) Inventors/Applicants (for US only): SELBER, Klaus GW, ML, MR, NE, SN, TD, TG). [DE/DE]; Bachstr. 32, 42781 Haan (DE). WEINGART- NER, Bernhard [DE/DE]; Birkenweg 4, 42489 Wulfrath Published: (DE). WEHLMANN, Hermann [DE/DE]; Mastweg 3A, — with international search report (Art. 21(3)) 42349 Wuppertal (DE). WINFRIED, Rosen [DE/DE]; Kremenholl 27a, 42857 Remscheid (DE). — before the expiration of the time limit for amending the claims and to be republished in the event of receipt of (74) Common Representative: BAYER PHARMA AK¬ amendments (Rule 48.2(h)) TIENGESELLSCHAFT; Law and Patents, Patents and Licensing, 51368 Leverkusen (DE). — with sequence listing part of description (Rule 5.2(a)) © (54) Title: GENOMICS OF ACTINOPLANES UTAHENSIS © (57) Abstract: The present invention describes the DNA-sequence of the wild type genome as well as all genetic modifications which were introduced into the wild type-and further developed strains, based thereon. Thereby the first genotypic characteriza- tion of the developed strains, including the latest production strain, has been accomplished, accounting for the major part of the invention. Furthermore, on the basis of the determined DNA-sequences, potential genes were identified and account, combined with their functional annotation, for another part of the invention. In particular, the gene-and DNA-sequences, as well as protein- sequences derived there out, contribute to the invention which were affected by mutagenic modifications throughout the strain de velopment process, potentially contributing to the increased production yield. GENOM ICS O F ACTI NOPLAN ES UTAH ENSIS DESCRI PTION O F T HE INVENTION The gram-positive prokaryote Actinoplanes utahensis was described for the first time by John Couch in 1963 (Couch, J. N., Elisha Mitchell Sci. Soc, 1963, 79:53-70). Thereafter, in the year 1977, acarbose and its homologues were first found in the supernatant of an Actinoplanes utahensis culture (Schmidt e al., Naturwissenschaften, 1977, 64:535-536). Two years later, the medical effect of acarbose as an a-glucosidase-inhibitor within the human intestine was discovered (Caspary et al., Res. Exp. Med., 1979, 175:1-6) and within the same year, its potential application for the treatment of type-2 diabetes mellitus was propagated (Frommer et al., J. Med. Plant Res., 1979, 35:195-217). Since 1990 the a-glucosidase-inhibitor acarbose is produced and marketed for the treatment of type-2 diabetes mellitus. Starting from the A . utahensis wild type strain the production has been continuously improved with regard to an ever increasing acarbose yield by optimization of the fermentation process as well as the production strain itself. The strain development has been driven by a multitude of mutagenesis experiments, which are primarily responsible for the raising acarbose production. The genetic modifications in the organism, triggered by the mutagenesis experiments have so far only been recognizable by phenotypic characteristics (e.g. the increase of acarbose yield). More precisely, the genetic bases for the raising production yields have, until now, been completely unknown. However, this knowledge is of fundamental interest for the understanding of the mechanisms, leading to the rise in production. Furthermore it forms the most important prerequisite for the process of further, targeted genetic modification of the organism, optimizing A . utahensis to an even greater extend. The present invention describes the DNA-sequence of the wild type genome as well as all genetic modifications which were introduced into the wild type- and further developed strains, based thereon. Thereby the first genotypic characterization of the developed strains, including the latest production strain, has been accomplished, accounting for the major part of the invention. Furthermore, on the basis of the determined DNA-sequences, potential genes were identified and account, combined with their functional annotation, for another part of the invention. In particular, the gene- and DNA-sequences, as well as protein- sequences derived there out which were affected by mutagenic modifications throughout the strain development process, potentially contributing to the increased production yield, contribute to the invention. Material and Methods As briefly described above, a series of mutagenesis experiments has been performed on the Actinoplanes utahensis wild type strain SE50-100, originally isolated from a soil sample. These experiments were aimed at the identification of mutants with an improved production of acarbose as well as other parameters, relevant for industrial production by fermentation such as high growth rate, optimized nutrient needs and consumption as well as low formation of cumbersome byproducts. Initially based on the wild type strain, further mutagenesis experiments were continuously performed on the mutant strains selected from the previous experiments. During the course of the strain development, several mutants with outstanding attributes were selected as new production strains and transferred into large scale production. Of these, seven strains were selected, including the latest production strain as well as the wild type strain, to be sequenced by Bielefeld University's Center for Biotechnology (CeBiTec) Universitatsstrasse 27, 33615 Bielefeld, Germany. Table 1 lists all seven strains that have been used during this project in the chronological order of their development. Table 1 list all A . utahensis strains used in this study in their chronological order. Strain Symbol Development Order Remark SE50-100 (1) wild type strain SN223-29-47 2 C445-P47 3 SN12755-48 4 SC3687-18-43 5 SC7 177-40- 17 6 SN1991 0-37-21 7 latest production strain Strain Cultivation Cultivation of strains in order to check their acarbose productivity was done as described previously (Schmidt e ai, Naturwissenschaften, 1977, 64:535-536). In order to isolate DNA, the Actinoplanes strains were cultivated in a two-step shake flask system. Beside inorganic salts the medium contained starch hydrolysate as carbon source and yeast extract as nitrogen source. Preculture and main culture were run for 3 days and 4 days, respectively, on a rotary shaker at 28°C. Then the biomass was collected by centrifugation. Strain Mutagenesis The strain development of the Acarbose producer was performed by the method of stepwise selection of higher producing strains. This method uses the process of random mutation by chemical or physical means. Chemicals used to induce mutations were either alkylating agents or intercalating dyes that serve as frameshift mutagens. Physical treatment of cells to induce mutagenesis was done with UV light of 365 nm. Fragments of the mycelium were used for mutagenesis treatment in appropriate buffer systems. After the treatment the biological material was grown for a short period in liquid medium to allow phenotypic expression of the induced alterations and then plated on agar plates. A random selection of clones that survived the mutagenesis treatment was checked for their acarbose productivity in small scale shake flask experiments. The best mutant clones obtained during a mutation cycle of this kind were chosen for the next mutation step. Several such steps of mutation and selection resulted in a gradual increase of productivity. Preparation of Genomic DNA The preparation of genomic DNA of A . utahensis strain SE50-1 10 was performed by a modification of the general described procedure (Maniatis T., Fritsch E.F., Sambrook J., Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Press, 1982). The mycel of 50 mL of freshly grown culture was harvested by centrifugation (10 min., 4.000 rpm, 4 °C) in a Christ centrifuge. The pellet was washed 4 times in a buffer containing 15 % sucrose (Merck KGaA, Darmstadt, Germany, cat. 7651), 25 mM TrisHCI pH 7.2 (Merck KGaA, Darmstadt, Germany, cat. 1.08382.1000), and 25 mM EDTA (Merck KGaA, Darmstadt, Germany, cat. 8418) under the same conditions. Finally the pellet was resuspended in 4.5 mL of the same buffer and lysozyme (Merck KGaA, Darmstadt, Germany, cat. 1.05281 .0010) and RNAse (Qiagen, Hilden, Germany, cat.
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