US 20110086407A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2011/0086407 A1 Berka et al. (43) Pub. Date: Apr. 14, 2011 (54) BACILLUS LCHENFORMS Publication Classification CHROMOSOME (51) Int. Cl. (75) Inventors: Randy Berka, Davis, CA (US); CI2N 9/12 (2006.01) Michael Rey, Davis, CA (US); CI2N 9/90 (2006.01) Preethi Ramaiya, Walnut Creek, C07K I4/32 (2006.01) CA (US); Jens Tonne Andersen, CI2N 9/00 (2006.01) Naerum (DK); Michael Dolberg CI2N 9/56 (2006.01) Rasmussen, Vallensbaek (DK); CI2N 9/16 (2006.01) Peter Bjarke Olsen, Copenhagen O CI2N 9/10 (2006.01) CI2N 9/88 (2006.01) (DK) CI2N 9/78 (2006.01) (73) Assignees: Novozymes A/S, Bagsvaerd (DK); C07K I4/95 (2006.01) Novozymes, Inc., Davis, CA (US) (52) U.S. Cl. ......... 435/194; 435/233; 530/350: 435/183; (21) Appl. No.: 12/972,306 435/222; 435/196; 435/193; 435/232:435/227 (22) Filed: Dec. 17, 2010 Related U.S. Application Data (57) ABSTRACT The present invention relates to an isolated polynucleotide of (62) Division of application No. 12/322.974, filed on Feb. the complete chromosome of Bacillus licheniformis. The 9, 2009, now Pat. No. 7,863,032, which is a division of present invention also relates to isolated genes of the chro application No. 10/983,128, filedon Nov. 5, 2004, now mosome of Bacillus licheniformis which encode biologically Pat. No. 7,494,798. active Substances and to nucleic acid constructs, vectors, and (60) Provisional application No. 60/535.988, filed on Jan. host cells comprising the genes as well as methods for pro 9, 2004, provisional application No. 60/561,059, filed ducing biologically active substances encoded by the genes on Apr. 8, 2004, provisional application No. 60/572, and to methods of using the isolated genes of the complete 403, filed on May 18, 2004. chromosome of Bacillus licheniformis. US 2011/0086407 A1 Apr. 14, 2011 BACILLUS LCHENFORMS or water, and understanding of specialized systems used by CHROMOSOME microbial cells to live in natural environments. 0008 Bacillus licheniformis is a gram positive spore CROSS-REFERENCE TO RELATED forming bacterium that is widely distributed as a saprophytic APPLICATIONS organism in the environment. Unlike most other bacilli that 0001. This application is a divisional of U.S. application are predominantly aerobic, Bacillus licheniformis is a facul Ser. No. 12/322,974, filed Feb. 9, 2009, which is a divisional tative anaerobe which may allow it to grow in additional of U.S. application Ser. No. 10/983,128, filed Nov. 5, 2004, ecological niches. This species produces a diverse assortment now U.S. Pat. No. 7,494,798, which claims the benefit of U.S. of extracellular enzymes that are believed to contribute to the Provisional Application No. 60/535.988, filed Jan. 9, 2004, process of nutrient cycling in nature (Claus, D. and Berkeley, U.S. Provisional Application No. 60/561,059, filed Apr. 8, R. C. W., 1986. In Bergey's Manual of Systematic Bacteriol 2004, and U.S. Provisional Application No. 60/572,403, filed ogy, Vol. 2., eds. Sneath, P. H. A. et al., Williams and Wilkins May 18, 2004, which applications are incorporated herein by Co., Baltimore, Md., pp. 1105-1139). Certain Bacillus reference. licheniformis isolates are capable of denitrification, however, the relevance of this characteristic to environmental denitri REFERENCE TO ASEQUENCE LISTING fication may be small since the species generally persists in soil as endospores (Alexander, M., 1977, Introduction to Soil 0002 This application contains a Sequence Listing in Microbiology. John Wiley and Sons, Inc., New York). computer readable form, which is incorporated herein by 0009. There are numerous industrial and agricultural uses reference. for Bacillus licheniformis and its extracellular products. The species has been used for decades in the manufacture of BACKGROUND OF THE INVENTION industrial enzymes including several proteases, C.-amylase, 0003 1. Field of the Invention penicillinase, pentosanase, cycloglucosyltransferase, B-man 0004. The present invention relates to an isolated poly nanase, and several pectinolytic enzymes, owing largely to its nucleotide molecule comprising the complete chromosome ability to secrete sizeable amounts of degradative enzymes. of Bacillus licheniformis. The present invention also relates to Bacillus licheniformis is also used to produce peptide antibi features (genes) of the complete chromosomal DNA mol otics such as bacitracin and proticin, in addition to a number ecule of Bacillus licheniformis which encode biologically of specialty chemicals such as citric acid, inosine, inosinic active substances and to nucleic acid constructs, vectors, and acid, and poly-Y-glutamic acid. The proteases from Bacillus host cells comprising the features as well as methods for licheniformis are used in the detergent industry as well as for producing biologically active Substances encoded by the fea dehairing and batting of leather (Eveleigh, D. E., 1981, Sci tures and to methods of using the isolated features derived entific American 245, 155-178). Amylases from Bacillus from the complete chromosomal DNA molecule of Bacillus licheniformis are deployed for the hydrolysis of starch, desiz licheniformis. ing of textiles, and sizing of paper (Erickson, R. J., 1976. In 0005 2. Description of the Related Art Microbiology, ed. Schlesinger, D. (Am. Soc. Microbiol. 0006 Microbes, which make up most of the earth's biom Washington, D.C.), pp. 406-419.). Certain strains of Bacillus ass, have evolved for some 3.8 billion years. They are found licheniformis have shown efficacy to destroy fungal patho in virtually every environment, Surviving and thriving in gens affecting maize, grasses, and vegetable crops (U.S. Pat. extremes of heat, cold, radiation, pressure, salt, acidity, and No. 5,589.381; U.S. Pat. No. 5,665,354). As an endospore darkness. Often in these environments, no other forms of life forming bacterium, the ability of the organism to Survive are found and the only nutrients come from inorganic matter. under unfavorable environmental conditions may enhance its The diversity and range of their environmental adaptations potential as a natural control agent. indicate that microbes long ago 'solved’ many problems for 0010 Bacillus licheniformis can be differentiated from which scientists are still actively seeking Solutions. The value other bacillion the basis of metabolic and physiological tests in determining the complete genome sequence of microbes is (Logan, N. A. and Berkeley, R. C. W., 1981. In The Aerobic that it provides a detailed blueprint for the organism revealing Endospore-Forming Bacteria Classification and Identifica all of the biochemical pathways, Substrates, intermediates, tion, eds. Berkeley, R. C. W. and Goodfellow, M., Academic and end products as well as regulatory networks, and evolu Press, Inc., London, pp. 106-140; O'Donnell, A.G., Norris, J. tionary relationships to other microbes. A complete manifest R., Berkeley, R. C. W., Claus, D., Kanero, T., Logan, N.A., of proteins, both structural and catalytic, is encoded as a list of and Nozaki, R., 1980, Internat. J. Systematic Bacteriol. 30: features in the DNA molecule comprising the genome, as well 448-459). However, biochemical and phenotypic character as their likely cellular location. istics may be ambiguous among closely related species. Lapi 0007 Knowledge about the enormous range of microbial dus et al. (Lapidus, A., Galleron, N., Andersen, J. T., Jor capacities has broad and far-reaching implications for envi gensen, P. L. Ehrlich, S. D., and Sorokin, A., 2002, FEMS ronmental, energy, health, and industrial applications, such as Microbiol. Lett. 209: 23-30) recently constructed a physical cleanup of toxic-waste, production of novel therapeutic and map of the Bacillus licheniformis chromosome using a PCR preventive agents (drugs and vaccines), energy generation approach, and established a number of regions of co-linearity and development of renewable energy sources, production of where gene content and organization were ostensibly con chemical catalysts, reagents, and enzymes to improve effi served with the Bacillus subtilis chromosome. ciency of industrial processes, management of environmental 0011. It would be advantageous to the art to have available carbon, nitrogen and nutrient cycling, detection of disease the complete primary structure of the chromosomal DNA causing organisms and monitoring of the safety of food and molecule of the Bacillus licheniformis type strain ATCC water Supplies, use of genetically altered bacteria as living 14580. With the complete chromosome data in hand, it should sensors (biosensors) to detect harmful chemicals in soil, air, be possible to do comparative genomics and proteomics stud US 2011/0086407 A1 Apr. 14, 2011 ies that can lead to improved industrial strains as well as to a without an inducing Substrate, and labeled second nucleic better understanding of genome evolution among closely acid probes, isolated from the microbial strain cultured on related bacilli in the subtilis-licheniformis group. medium with the inducing Substrate, to an array of Bacillus 0012. It is an object of the present invention to provide an licheniformis genes selected from the group consisting of isolated polynucleotide with the sequence of the complete nucleotides SEQID NOS: 2-4198, complementary strands of chromosome of Bacillus licheniformis. SEQID NOS: 2-4198, or fragments of SEQID NOS: 2-4198, under conditions where the labeled nucleic acid probes SUMMARY OF THE INVENTION hybridize to complementary