USOO81 68417B2 (12) United States Patent (10) Patent No.: US 8,168,417 B2 Berka et al. (45) Date of Patent: May 1, 2012 (54) BACILLUS LICHENIFORMIS CHROMOSOME OTHER PUBLICATIONS Kunst et al., 1997, PIR database Accession No. D69904, “butyrate (75) Inventors: Randy Berka, Davis, CA (US); Michael acetoacetate CoA-transferase (EC 2.8.3.9.) large chain homolog Rey, Davis, CA (US); Preethi Ramaiya, yodR-Bacillus subtilis”.* Walnut Creek, CA (US); Jens Tonne Claus, D. and Berkeley, R.C.W. (1986) in Bergey's Manual of Sys Andersen, Naerum (DK); Michael tematic Bacteriology, vol. 2., eds. Sneath, P.H.A. etal. (Williams and Dolberg Rasmussen, Vallensbaek (DK); Wilkins Co., Baltimore, MD.), pp. 1105-1139. Peter Bjarke Olsen, Copenhagen Ø Eveleigh, D.E. (1981) Scientific American 245, 155-178. (DK) Erickson, R.J. (1976) in Microbiology, ed. Schlesinger, D. (Am. Soc. Microbiol. Washington, DC), pp. 406-419. (73) Assignees: Novozymes A/S, Bagsvaerd (DK); Logan, N. A. and Berkeley, R.C.W. (1981), in The Aerobic Novozymes, Inc., Davis, CA (US) Endospore-Forming Bacteria. Classification and Identification, eds. Berkeley, R.C.W. and Goodfellow, M. (Academic Press, Inc., Lon (*) Notice: Subject to any disclaimer, the term of this don), pp. 106-140. patent is extended or adjusted under 35 O'Donnell, A.G., Norris, J.R., Berkeley, R.C.W., Claus, D., Kanero, U.S.C. 154(b) by 0 days. T., Logan, N. A., and Nozaki, R. (1980) Internat. J. Systematic Bacteriol. 30, 448-459. (21) Appl. No.: 12/972,306 Lapidus et al., (2002), Co-linear scaffold of the Bacillus licheniformis and Bacillus subtilis genomes and its use to compare (22) Filed: Dec. 17, 2010 their competence genes, FEMS Microbiology Letters, 209, pp. 23-30. (65) Prior Publication Data Kunst et al., Nature, 1997, vol. 390, pp. 249-266. NCBI submission of B. subtilis DNA, 180 kb region of repliation US 2011 FOO864O7 A1 Apr. 14, 2011 origin, ID BAC 180K, publicly available Jun. 2, 1999, at ncbi.nlm. nih.gov. Related U.S. Application Data Avita et al., Temporal Secretion of a multicellulolytic system in Division of application No. 12/322.974, filed on Feb. Myxobacter sp. AL-1 Molecular cloning and heterologous expres (62) sion of cel9 encoding a modular endocellulase clustered in an operon 9, 2009, now Pat. No. 7,863,032, which is a division of with ce148, an exocellobiohdrolase gene, Eur, J. Biochem., 2000, application No. 10/983,128, filedon Nov. 5, 2004, now v.267, 7058-7064. Pat. No. 7,494,798. Liu et al., 2004, Curr Microbiol 49, 234-238. (60) Provisional application No. 60/535.988, filed on Jan. Lloberas et al., 1991, Eur J. Biochem 197,337-343. Moriya et al., 2005, NCBI Access No. D26.185. 9, 2004, provisional application No. 60/561,059, filed O'Donnell et al., 2007, Geneseqp Access No. ADJ79377. on Apr. 8, 2004, provisional application No. Rey et al., 2004, Gen Biol 5(10), R77-1. 60/572,403, filed on May 18, 2004. Sanchez et al., 2003, Eur J Biochem 270(13), 2913-2919. Sneath et al., 1977, 1104-1139. (51) Int. C. Sook et al., 2002, J Microbiol Biotechnol 12(5), 773-779. CI2N 9/10 (2006.01) Veith et al., 2004, J Mol Microbiol Biotechnol 7(4), 204-211. CI2N 5/54 (2006.01) Xu et al., 2003, EMBL Access No. AF478085. (52) U.S. Cl. ....................................... 435/193: 536/23.2 Xu et al., 2003, Intl Sys Evo Micro 53(3), 695-704. (58) Field of Classification Search ........................ None Sinchaikul et al 2002, J Chromat 771,261-287. See application file for complete search history. * cited by examiner (56) References Cited Primary Examiner—Jon PWeber Assistant Examiner — William W. Moore U.S. PATENT DOCUMENTS (74) Attorney, Agent, or Firm — Eric J. Fechter; Robert L. 5,589,381 A 12/1996 Neyra et al. Starnes 5,665,354 A 9/1997 Neyra et al. 6,060.241 A * 5/2000 Corthesy-Theulaz ............ 435/6 6,506,581 B1* 1/2003 Fleischmann et al. ....... 435/69.1 (57) ABSTRACT 6,528,289 B1* 3/2003 Fleischmann et al. ..... 435,9141 The present invention relates to an isolated polynucleotide of 6,593,114 B1* 7/2003 Kunsch et al. ............. 435,9141 6,846,651 B2 * 1/2005 Fleischmann et al ... 435/69.1 the complete chromosome of Bacillus licheniformis. The 7,018,794 B2* 3/2006 Berka et al. ... ... 435/6 present invention also relates to isolated genes of the chro 7.691,574 B2 * 4/2010 Berka et al. ....................... 435/6 mosome of Bacillus licheniformis which encode biologically 2002fO146721 Al 10/2002 Berka et al. ....................... 435/6 active Substances and to nucleic acid constructs, vectors, and FOREIGN PATENT DOCUMENTS host cells comprising the genes as well as methods for pro ducing biologically active substances encoded by the genes WO WOO2,29113 A2 4/2002 WO WOO229113 * 4, 2002 and to methods of using the isolated genes of the complete WO WOO3,OOO941 1, 2003 chromosome of Bacillus licheniformis. WO WOO3/O54163 A2 T 2003 WO WOO3,087.149 10, 2003 2 Claims, No Drawings US 8,168,417 B2 1. 2 BACILLUS LICHENIFORMS CHROMOSOME in the environment. Unlike most other bacilli that are pre dominantly aerobic, Bacillus licheniformis is a facultative CROSS-REFERENCE TO RELATED anaerobe which may allow it to grow in additional ecological APPLICATIONS niches. This species produces a diverse assortment of extra cellular enzymes that are believed to contribute to the process This application is a divisional of U.S. application Ser. No. of nutrient cycling in nature (Claus, D. and Berkeley, R. C.W., 12/322,974, filed Feb. 9, 2009, now U.S. Pat. No. 7,863,032, 1986. In Bergey's Manual of Systematic Bacteriology, Vol. 2., which is a divisional of U.S. application Ser. No. 10/983,128, eds. Sneath, P. H. A. et al., Williams and Wilkins Co., Balti filed Nov. 5, 2004, now U.S. Pat. No. 7,494,798, which claims more, Md., pp. 1105-1139). Certain Bacillus licheniformis the benefit of U.S. Provisional Application No. 60/535.988, 10 isolates are capable of denitrification, however, the relevance filed Jan. 9, 2004, U.S. Provisional Application No. 60/561, of this characteristic to environmental denitrification may be 059, filed Apr. 8, 2004, and U.S. Provisional Application No. Small since the species generally persists in soil as endospores 60/572.403, filed May 18, 2004, which applications are incor porated herein by reference. (Alexander, M., 1977, Introduction to Soil Microbiology. 15 John Wiley and Sons, Inc., New York). There are numerous industrial and agricultural uses for REFERENCE TO ASEQUENCE LISTING Bacillus licheniformis and its extracellular products. The spe This application contains a Sequence Listing in computer cies has been used for decades in the manufacture of indus readable form, which is incorporated herein by reference. trial enzymes including several proteases, C.-amylase, peni cillinase, pentosanase, cycloglucosyltransferase, BACKGROUND OF THE INVENTION B-mannanase, and several pectinolytic enzymes, owing largely to its ability to secrete sizeable amounts of degradative 1. Field of the Invention enzymes. Bacillus licheniformis is also used to produce pep The present invention relates to an isolated polynucleotide tide antibiotics such as bacitracin and proticin, in addition to molecule comprising the complete chromosome of Bacillus 25 a number of specialty chemicals such as citric acid, inosine, licheniformis. The present invention also relates to features inosinic acid, and poly-Y-glutamic acid. The proteases from (genes) of the complete chromosomal DNA molecule of Bacillus licheniformis are used in the detergent industry as Bacillus licheniformis which encode biologically active sub well as for dehairing and batting of leather (Eveleigh, D. E., stances and to nucleic acid constructs, vectors, and host cells 1981, Scientific American 245, 155-178). Amylases from comprising the features as well as methods for producing 30 Bacillus licheniformis are deployed for the hydrolysis of biologically active Substances encoded by the features and to starch, desizing of textiles, and sizing of paper (Erickson, R. methods of using the isolated features derived from the com J., 1976. In Microbiology, ed. Schlesinger, D. (Am. Soc. plete chromosomal DNA molecule of Bacillus licheniformis. Microbiol. Washington, D.C.), pp. 406-419). Certain strains 2. Description of the Related Art of Bacillus licheniformis have shown efficacy to destroy fun Microbes, which make up most of the earth's biomass, 35 gal pathogens affecting maize, grasses, and vegetable crops have evolved for some 3.8 billion years. They are found in (U.S. Pat. No. 5,589,381; U.S. Pat. No. 5,665,354). As an virtually every environment, Surviving and thriving in endospore-forming bacterium, the ability of the organism to extremes of heat, cold, radiation, pressure, salt, acidity, and Survive under unfavorable environmental conditions may darkness. Often in these environments, no other forms of life enhance its potential as a natural control agent. are found and the only nutrients come from inorganic matter. 40 Bacillus licheniformis can be differentiated from other The diversity and range of their environmental adaptations bacillion the basis of metabolic and physiological tests (Lo indicate that microbes long ago 'solved’ many problems for gan, N. A. and Berkeley, R. C. W., 1981. In The Aerobic which scientists are still actively seeking Solutions. The value Endospore-Forming Bacteria Classification and Identifica in determining the complete genome sequence of microbes is tion, eds. Berkeley, R. C. W. and Goodfellow, M., Academic that it provides a detailed blueprint for the organism revealing 45 Press, Inc., London, pp. 106-140; O'Donnell, A.G., Norris, J. all of the biochemical pathways, Substrates, intermediates, R., Berkeley, R.