AU2003288826A1 - Polynucleotides and polypeptides isolated from lactobacillus rhamnosus hn001 materials incorporating them and methods for using them - Google Patents

Polynucleotides and polypeptides isolated from lactobacillus rhamnosus hn001 materials incorporating them and methods for using them Download PDF

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AU2003288826A1
AU2003288826A1 AU2003288826A AU2003288826A AU2003288826A1 AU 2003288826 A1 AU2003288826 A1 AU 2003288826A1 AU 2003288826 A AU2003288826 A AU 2003288826A AU 2003288826 A AU2003288826 A AU 2003288826A AU 2003288826 A1 AU2003288826 A1 AU 2003288826A1
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James Dekker
Matthew Glenn
Ilkka Havukkala
Mark William Lubbers
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Genesis Research and Development Corp Ltd
Fonterra Cooperative Group Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/335—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Lactobacillus (G)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

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Description

WO 2005/056801 PCT/NZ2003/000278 POLYNUCLEOTIDES AND POLYPEPTIDES ISOLATED FROM LACTOBA CILL US RHAMNOSUS HN001, MATERIALS INCORPORATING THEM AND METHODS FOR USING THEM 5 Technical Field of the Invention This invention relates to polynucleotides isolated from lactic acid bacteria as well as to probes and primers specific to the polynucleotides; genetic constructs comprising the polynucleotides; biological materials, including plants, microorganisms and multicellular 10 organisms, incorporating the polynucleotides; polypeptides expressed by the polynucleotides; and methods for using the polynucleotides and polypeptides. Background of the Invention The present invention relates to polynucleotides isolated from a specific strain of lactic 15 acid bacteria, namely Lactobacillus rhamnosus HN001 (L. rhamnosus HN001). Lactic acid bacteria, and their enzymes, are the major determinants of flavor and fermentation characteristics in fermented dairy products, such as cheese and yogurt. Flavors are produced through the action of bacteria and their enzymes on proteins, carbohydrates and lipids. Lactobacillus rhamnosus strain HN001 are heterofermentative bacteria that are Gram 20 positive, non-motile, non-spore forming, catalase negative, facultative anaerobic rods exhibiting an optimal growth temperature of 37±1*C and an optimum pH of 6.0 - 6.5. Experimental studies demonstrated that dietary supplementation with Lactobacillus rhannosus strain HN001 induced a sustained enhancement in several aspects of both natural and acquired immunity (See PCT International Publication No. WO 99/10476). In addition, L. 25 rhamnosus HN00 1, and certain other Gram-positive bacteria can specifically and directly modulate human and animal health (See, for example, Tannock et al., Applied Environ. Microbiol. 66:2578-2588, 2000; Gill et al., Brit. J. Nutrition 83:167-176; Quan Shu et al., Food and Chem. Toxicol. 38:153-161, 2000; Quan Shu et al., Intl. J Food Microbiol. 56:87 96, 2000; Quan Shu et al., Intl. Dairy J. 9:831-836, 1999; Prasad et al., Intl. Dairy J. 8:993 30 1002, 1998; Sanders and Huis in't Veld, Antonie van Leeuwenhoek 76:293-315, 1999; Salminen et al., 1998. In: Lactic Acid Bacteria, Salminen S and von Wright A (eds)., Marcel Dekker Inc, New York, Basel, Hong Kong, pp. 211-253; Delcour et al., Antonie van Leeuwenhoek 76:159-184, 1999; Blum et al., Antonie van Leeuwenhoek 76:199-205, 1999; 1 WO 2005/056801 PCT/NZ2003/000278 Yasui et al., Antonie van Leeuwenhoek 76:383-389, 1999; Hirayama and Rafter, Antonie van Leeuwenhoek 76:391-394, 1999; Ouwehand, 1998. In: Lactic Acid Bacteria, Salminen S and von Wright A (eds)., Marcel Dekker Inc, New York, Basel, Hong Kong, pp. 139-159; Isolauri et al., S 1998. In: Lactic Acid Bacteria, Salminen S and von Wright A (eds)., Marcel Dekker 5 Inc, New York, Basel, Hong Kong, pp. 255-268; Lichtenstein and Goldin, 1998. In: Lactic Acid Bacteria, Sahninen S and von Wright A (eds)., Marcel Dekker Inc, New York, Basel, Hong Kong, pp. 269-277; El-Nezami and Ahokas, 1998. In: Lactic Acid Bacteria, Salminen S and von Wright A (eds)., Marcel Dekker Inc, New York, Basel, Hong Kong, pp. 359-367; Nousianen et al., 1998. In: Lactic Acid Bacteria, Salminen S and von Wright A (eds)., Marcel 10 Dekker Inc, New York, Basel, Hong Kong, pp. 437-473; Meisel and Bockelmann, Antonie van Leeuwenhoek 76:207-215, 1999; Christensen et al., Antonie van Leeuwenhoek 76:217-246, 1999; Dunne et al., Antonie van Leeuwenhoek 76:279-292, 1999). Beneficial health effects attributed to these bacteria include the following: 15 Increased resistance to enteric pathogens and anti-infection activity, including treatment of rotavirus infection and infantile diarrhea - due to increases in antibody production caused by an adjuvant effect, increased resistance to pathogen colonization; alteration of intestinal conditions, such as pH; and the presence of specific antibacterial substances, such as bacteriocins and organic acids. 20 Aid in lactose digestion - due to lactose degradation by bacterial lactase enzymes (such as beta-galactosidase) that act in the small intestine. Anti-cancer (in particular anti-colon cancer) and anti-mutagenesis activities - due to anti 25 mutagenic activity; alteration of procancerous enzymatic activity of colonic microbes; reduction of the carcinogenic enzymes azoreductase, beta-glucuronidase and nitroreductase in the gut and/or faeces; stimulation of immune function; positive influence on bile salt concentration; and antioxidant effects. 30 Liver cancer reduction - due to aflatoxin detoxification and inhibition of mould growth. Reduction of small bowel bacterial overgrowth - due to antibacterial activity; and decrease in toxic metabolite production from overgrowth flora. 2 WO 2005/056801 PCT/NZ2003/000278 Immune system modulation and treatment of autoimmune disorders and allergies - due to enhancement of non-specific and antigen-specific defence against infection and tumors; enhanced mucosal immunity; adjuvant effect in antigen-specific immune responses; and 5 regulation of Thl/Th2 cells and production of cytokines. Treatment of allergic responses to foods- due to prevention of antigen translocation into blood stream and modulation of allergenic factors in food. 10 Reduction of blood lipids and prevention of heart disease - due to assimilation of cholesterol by bacteria; hydrolysis of bile salts; and antioxidative effects. Antihypertensive effect - bacterial protease or peptidase action on milk peptides produces antihypertensive peptides. Cell wall components act as ACE inhibitors 15 Prevention and treatment of urogenital infections - due to adhesion to urinary and vaginal tract cells resulting in competitive exclusion; and production of antibacterial substances (acids, hydrogen peroxide and biosurfactants). 20 Treatment of inflammatory bowel disorder and irritable bowel syndrome - due to immuno-modulation; increased resistance to pathogen colonization; alteration of intestinal conditions such as pH; production of specific antibacterial substances such as bacteriocins, organic acids and hydrogen peroxide and biosurfactants; and competitive exclusion. 25 Modulation of infective endocarditis - due to fibronectin receptor-mediated platelet aggregation associated with Lactobacillus sepsis. Prevention and treatment of Helicobacterpylori infection - due to competitive colonization and antibacterial effect. 30 Prevention and treatment of hepatic encephalopathy - due to inhibition and/or exclusion of urease-producing gut flora. 3 WO 2005/056801 PCT/NZ2003/000278 Improved protein and carbohydrate utilisation and conversion - due to production of beneficial products by bacterial action on proteins and carbohydrates. Other beneficial health effects associated with L. rhamnosus include: improved 5 nutrition; regulation of colonocyte proliferation and differentiation; improved lignan and isoflavone metabolism; reduced mucosal permeability; detoxification of carcinogens and other harmful compounds; relief of constipation and diarrhea; and vitamin synthesis, in particular folate. Peptidases are enzymes that break the peptide bonds linking the amino group of one 10 amino acid with the carboxy group (acid group) of an adjacent amino acid in a peptide chain. The bonds are broken in a hydrolytic reaction. There is a large family of peptidase enzymes that are defined by their specificity for the particular peptides bonds that they cleave (Barrett A J, Rawlings N D and Woessner J F (Eds.) 1998. Handbook ofproteolytic enzymes. Academic Press, London, UK). The two main families are exopeptidases and endopeptidases. 15 Exopeptidases cleave amino acids from the N- or C- terminus of a peptide chain, releasing free amino acids or short (di- and tri-) peptides. Different types of exopeptidases include: e Aminopeptidases - release a free amino acid from the N-terminus of a peptide chain; 20 . dipeptidyl-peptidase (also known as dipeptidyl-aminopeptidases) - release a dipeptide from the N-terminus of a peptide chain; * tripeptidyl-peptidases (also known as tripeptidyl-aminopeptidases) - release a tripeptide from the N-terminus of a peptide chain); * carboxypeptidases - release a free amino acid from the C-terminus of a peptide 25 chain; e peptidyl-dipeptidase - release a dipeptide from the C-terminus of a peptide chain; e dipeptidases -release two free amino acids from a dipeptide; and * tripeptidases - release a free amino acid and a dipeptide from a tripeptide. 30 Peptidases are important enzymes in the process of cheese ripening and the development of cheese flavor. The hydrolysis of milk caseins in cheese results in textural changes and the development of cheese flavors. The raft of proteolytic enzymes that cause this hydrolysis come from the lactic acid bacteria that are bound up in the cheese - either 4 WO 2005/056801 PCT/NZ2003/000278 starter cultures that grow up during the manufacture of the cheese, or adventitious and adjunct non-starter lactic acid bacteria that grow in the cheese as it ripens (Law Haandrikman, Int. Dairy J. 7:1-11, 1997). Many other enzymes can also influence dairy product flavor, and functional and 5 textural characteristics, as well as influencing the fermentation characteristics of the bacteria, such as speed of growth, acid production and survival (Urbach, Int. Dairy J 5:877-890, 1995; Johnson and Somkuti, Biotech. AppL. Biochem. 13:196-204, 1991; El Soda and Pandian, J Dairy Sci. 74:2317-2335, 1991; Fox et al,. In Cheese: chemistry, physics and microbiology. Volume 1, General aspects, 2 "d edition, P Fox (ed) Chapman and Hall, London; Christensen et 10 al., Antonie van Leeuwenhoek 76:217-246, 1999; Stingle et al., J Bacteriol. 20:6354-6360, 1999; Stingle et al., Mol. Microbiol. 32:1287-1295, 1999; Lemoine et al., Appl. Environ. Microbiol. 63:1512-3518, 1997). Enzymes influencing specific characteristics and/or functions include the following: = Lysis of cells. These enzymes are mostly cell wall hydrolases, including amidases; 15 muranidases; lysozymes, including N-acetyl muramidase; muramidase; N acetylglucosaminidase; and N-acetylmuramoyl-L-alanine amidase. DEAD-box helicase proteins also influence autolysis. - Carbohydrate utilization. Lactose, citrate and diacetyl metabolism, and alcohol 20 metabolism are particularly important. The enzymes involved include beta-galactosidase, lactate dehydrogenase, citrate lyase, citrate permease, 2,3 butanediol dehydrogenase (acetoin reductase), acetolactate decarboxylase, acetolactate synthase, pyruvate decarboxylase, pyruvate formate lyase, diacetyl synthase, diacetyl reductase, alcohol decarboxylase, lactate dehydrogenase, pyruvate dehydrogenase, and aldehyde 25 dehydrogenase. a Lipid degradation, modification or synthesis. Enzymes involved include lipases, esterases, phospholipases, serine hydrolases, desaturases, and linoleate isomerase. 30 - Polysaccharide synthesis. Polysaccharides are important not only for potential immune enhancement and adhesion activity but are important for the texture of fermented dairy products. The enzymes involved are a series of glucosyl transferases, including beta-(1-3) glucosyl transferase, alpha-N acetylgalactosaminyl transferase, phosphogalactosyl 5 WO 2005/056801 PCT/NZ20031000278 transferase, alpha-glycosyl transferase, UDP-N-acetylglucosanine C4 epimerase and UDP-N-acetylglucosamine transferase. - Amino acid degradation. Enzymes include glutamate dehydrogenase, aminotransferases, 5 amino acid decarboxylases, and enzymes involved in sulphur amino acid degradation including cystathione beta-lyase. Sequencing of the genomes, or portions of the genomes, of numerous organisms, including humans, animals, microorganisms and various plant varieties, has been and is being 1o carried out on a large scale. Polynucleotides identified using sequencing techniques may be partial or full-length genes, and may contain open reading frames, or portions of open reading frames, that encode polypeptides. Putative polypeptides may be identified based on polynucleotide sequences and further characterized. The sequencing data relating to polynucleotides thus represents valuable and useful information. 15 Polynucleotides and polypeptides may be analyzed for varying degrees of novelty by comparing identified sequences to sequences published in various public domain databases, such as EMBL. Newly identified polynucleotides and corresponding putative polypeptides may also be compared to polynucleotides and polypeptides contained in public domain information to ascertain homology to known polynucleotides and polypeptides. In this way, 20 the degree of similarity, identity or homology of polynucleotides and polypeptides having an unknown function may be determined relative to polynucleotides and polypeptides having known functions. Information relating to the sequences of isolated polynucleotides may be used in a variety of ways. Specified polynucleotides having a particular sequence may be isolated, or 25 synthesized, for use in in vivo or in vitro experimentation as probes or primers. Alternatively, collections of sequences of isolated polynucleotides may be stored using magnetic or optical storage medium and analyzed or manipulated using computer hardware and software, as well as other types of tools. 30 Summary of the Invention The present invention provides isolated polynucleotides comprising a sequence selected from the group consisting of: (a) sequences identified in the attached Sequence Listing as SEQ ID NOS: 1-80; (b) variants of those sequences; (c) extended sequences 6 WO 2005/056801 PCT/NZ20031000278 comprising the sequences set out in SEQ ID NOS: 1-80, and their variants; and (d) sequences comprising at least a specified number of contiguous residues of a sequence of SEQ ID NOS: 1-80 (x-mers). Oligonucleotide probes and primers corresponding to the sequences set out in SEQ ID NOS: 1-80, and their variants are also provided. All of these polynucleotides and 5 oligonucleotide probes and primers are collectively referred to herein, as "polynucleotides of the present invention." The polynucleotide sequences identified as SEQ ID NOS: 1-80 were derived from a microbial source, namely from fragmented genomic DNA of Lactobacillus rhamnosus, strain HN001, described in PCT International Publication No. WO 99/10476. Lactobacillus 10 rhamnosus strain HN001 are heterofermentative bacteria that are Gram positive, non-motile, non-spore forming, catalase negative, facultative anaerobic rods exhibiting an optimal growth temperature of 37±1'C and an optimum pH of 6.0 - 6.5. Experimental studies demonstrated that dietary supplementation with Lactobacillus rhamnosus strain HNOO 1 induced a sustained enhancement in several aspects of both natural and acquired immunity. A biologically pure 15 culture of Lactobacillus rhamnosus strain HN00 I was deposited at the Australian Government Analytical Laboratories (AGAL), The New South Wales Regional Laboratory, 1 Suakin Street, Pymble, NSW 2073, Australia, as Deposit No. NM97/09514, dated 18 August 1997. Certain of the polynucleotide sequences disclosed herein are "partial" sequences in that they do not represent a full-length gene encoding a full-length polypeptide. Such. partial 20 sequences may be extended by analyzing and sequencing various DNA libraries using primers and/or probes and well-known hybridization and/or PCR techniques. The partial sequences disclosed herein may thus be extended until an open reading frame encoding a polypeptide, a full-length polynucleotide and/or gene capable of expressing a polypeptide, or another useful portion of the genome is identified. Such extended sequences, including full-length 25 polynucleotides and genes, are described as "corresponding to" a sequence identified as one of the sequences of SEQ ID NOS: 1-80 or a variant thereof, or a portion of one of the sequences of SEQ ID NOS:. 1-80 or a variant thereof, when the extended polynucleotide comprises an identified sequence or its variant, or an identified contiguous portion (x-mer) of one of the sequences of SEQ ID NOS: 1-80 or a variant thereof. 30 The polynucleotides identified as SEQ ID NOS: 1-80 were isolated from Lactobacillus rhamnosus genomic DNA clones and represent sequences that are present in the cells from which the DNA was prepared. The sequence information may be used to identify and isolate, or synthesize, DNA molecules such as promoters, DNA-binding elements, open reading 7 WO 2005/056801 PCT/NZ2003/000278 frames or full-length genes, that then can be used as expressible or otherwise functional DNA in transgenic organisms. Similarly, RNA sequences, reverse sequences, complementary sequences, antisense sequences and the like, corresponding to the polynucleotides of the present invention, may be routinely ascertained and obtained using the polynucleotides 5 identified as SEQ ID NOS: 1-80. The present invention further provides isolated polypeptides encoded, or partially encoded, by the polynucleotides disclosed herein. In certain specific embodiments, the polypeptides of the present invention comprise a sequence selected from the group consisting of sequences identified as SEQ ID NO: 81-183, and variants thereof. Polypeptides encoded by 10 the polynucleotides of the present invention may be expressed and used in various assays to determine their biological activity. Such polypeptides may be used to raise antibodies, to isolate corresponding interacting proteins or other compounds, and to quantitatively determine levels of interacting proteins or other compounds. Genetic constructs comprising the inventive polynucleotides are also provided, 15 together with transgenic host cells comprising such constructs and transgenic organisms, such as microbes, comprising such cells. The present invention also contemplates methods for modulating the polynucleotide and/or polypeptide content and composition of an organism, such methods involving stably incorporating into the genome of the organism a genetic construct comprising a polynucleotide 20 of the present invention. In one embodiment, the target organism is a microbe, preferably a microbe used in fermentation, more preferably a microbe of the genus Lactobacillus, and most preferably Lactobacillus rhamnosus, or other closely microbial related species used in the dairy industry. In a related aspect, methods for producing a microbe having an altered genotype and/or phenotype is provided, such methods comprising transforming a microbial 25 cell with a genetic construct of the present invention to provide a transgenic cell, and cultivating the transgenic cell under conditions conducive to growth and multiplication. Organisms having an altered genotype or phenotype as a result of modulation of the level or content of a polynucleotide or polypeptide of the present invention compared to a wild-type organism, as well as components and progeny of such organisms, are contemplated by and 30 encompassed within the present invention. The isolated polynucleotides of the present invention may be usefully employed for the detection of lactic acid bacteria, preferably L. rhamnosus, in a sample material, using 8 WO 2005/056801 PCT/NZ2003/000278 techniques well known in the art, such as polymerase chain reaction (PCR) and DNA hybridization, as detailed below. The inventive polynucleotides and polypeptides may also be employed in methods for the selection and production of more effective probiotic bacteria; as bioactivee" (health 5 promoting) ingredients and health supplements for immune function enhancement; for reduction of blood lipids such as cholesterol; for production of bioactive material from genetically modified bacteria; as adjuvants; for wound healing; in vaccine development, particularly mucosal vaccines; as animal probiotics for improved animal health and productivity; in selection and production of genetically modified rumen microorganisms for 10 improved animal nutrition and productivity, better flavor and improved milk composition; in methods for the selection and production of better natural food bacteria for improved flavor, faster flavor development, better fermentation characteristics, vitamin synthesis and improved textural characteristics; for the production of improved food bacteria through genetic modification; and for the identification of novel enzymes for the production of, for example, 15 flavors or aroma concentrates. The isolated polynucleotides of the present invention also have utility in genome mapping, in physical mapping, and in positional cloning of genes of more or less related microbes. Additionally, the polynucleotide sequences identified as SEQ ID NOS: 1-80, and their variants, may be used to design oligonucleotide probes and primers. Such 20 oligonucleotide probes and primers have sequences that are substantially complementary to the polynucleotide of interest over a certain portion of the polynucleotide. Oligonucleotide probes designed using the polynucleotides of the present invention may be used to detect the presence and examine the expression patterns of genes in any organism having sufficiently similar DNA and RNA sequences in their cells, using techniques that are well known in the 25 art, such as slot blot DNA hybridization techniques. Oligonucleotide primers designed using the polynucleotides of the present invention may be used for polymerase chain reaction (PCR) amplifications. Oligonucleotide probes and primers designed using the polynucleotides of the present invention may also be used in connection with various microarray technologies, including the microarray technology of Affymetrix (Santa Clara, CA). 30 The polynucleotides of the present invention may also be used to tag or identify an organism or derived material or product therefrom. Such tagging may be accomplished, for example, by stably introducing a non-disruptive non-functional heterologous polynucleotide 9 WO 2005/056801 PCT/NZ2003/000278 identifier into an organism, the polynucleotide comprising at least a portion of a polynucleotide of the present invention. The polynucleotides of the present invention may also be used as promoters, gene regulators, origins of DNA replication, secretion signals, cell wall or membrane anchors for 5 genetic tools (such as expression or integration vectors). All references cited herein, including patent references and non-patent publications, are hereby incorporated by reference in their entireties. Brief Description of the Drawings 10 Fig. 1 shows the nucleotide sequence for L. rhamnosus strain HN001 deoD purine nucleoside phosphorylase AQ1 (SEQ ID NO: 78), showing ATG initiation and translation stop codons (boxed). Fig. 2 shows the amino acid sequence for L. rhamnosus strain HN001 deoD purine nucleoside phosphorylase AQ1 (SEQ ID NO: 181). 15 Fig. 3 shows the results of UV light exposure assay measuring relative viability in response to increasing doses of UV light forAQ1I HN001 strain (+) and wild-type HN001 (m). Results indicate that the AQ1- HN001 mutant strain showed enhanced survival to exposure to UV light compared to wild-type HNOO1. Fig. 4 shows the nucleotide sequence for L. rhamnosus strain HN001 relA GTP 20 pyrophosphokinase gene AM] (SEQ ID NO: 79) showing ATG initiation and translation stop codons (boxed). Fig. 5 shows the amino acid sequence of L. rhamnosus strain HN001 relA GTP pyrophosphokinase gene AM] (SEQ ID NO: 182). Fig. 6 shows the results of UV light exposure assay measuring relative viability in 25 response to increasing doses of UV light inAM1- HN001 strain (+) and wild-type HN001 (m). Results indicate that the AM - HN1OO mutant strain showed enhanced survival to exposure to UV light compared to wild-type HN001. 10 WO 2005/056801 PCT/NZ2003/000278 Detailed Description The polynucleotides disclosed herein were isolated by high throughput sequencing of DNA libraries from the lactic acid bacteria Lactobacillus rhamnosus as described below in Example 1. Cell wall, cell surface and secreted components of lactic acid bacteria are known 5 to mediate immune modulation, cell adhesion and antibacterial activities, resulting in many beneficial effects including: resistance to enteric pathogens; modulation of cancer, including colon cancer; anti-mutagenesis effects; reduction of small bowel bacterial overgrowth; modulation of auto-immune disorders; reduction in allergic disorders; modulation of urogenital infections, inflammatory bowel disorder, irritable bowel syndrome, Helicobacter 10 pylori infection and hepatic encephalopathy; reduction of infection with pathogens; regulation of colonocyte proliferation and differentiation; reduction of mucosal permeability; and relief of constipation and diarrhea. These cell components include, but are not limited to, peptidoglycans, teichoic acids, lipoteichoic acids, polysaccharides, adhesion proteins, secreted proteins, surface layer or S-layer proteins, collagen binding proteins and other cell surface 15 proteins, and antibacterial substances such as bacteriocins and organic acids produced by these bacteria. Polynucleotides involved in the synthesis of these proteins and in the synthesis, modification, regulation, transport, synthesis and/or accumulation of precursor molecules for these proteins can be used to modulate the immune effects, antibacterial, cell adhesion and competitive exclusion effects of the bacteria or of components that might be produced by these 20 bacteria. In order to function effectively as probiotic bacteria, L. rhamnosus HN001 must survive environmental stress conditions in the gastrointestinal tract, as well as commercial and industrial processes. Modification of particular polynucleotides or regulatory processes has been shown to be effective against a number of stresses including oxidative stress, pH, 25 osmotic stress, dehydration, carbon starvation, phosphate starvation, nitrogen starvation, amino acid starvation, heat or cold shock and mutagenic stress. Polynucleotides involved in stress resistance often confer multistress resistance, i.e., when exposed to one stress, surviving cells are resistant to several non-related stresses. Bacterial genes and/or processes shown to be involved in multistress resistance include: 30 Intracellular phosphate pools - inorganic phosphate starvation leads to the induction ofpho regulon genes, and is linked to the bacterial stringent response. Gene knockouts involving phosphate receptor genes appear to lead to multistress resistance. 11 WO 2005/056801 PCT/NZ2003/000278 Intracellular guanosine pools - purine biosynthesis and scavenger pathways involve the production of phosphate-guanosine compounds that act as signal molecules in the bacterial stringent response. Gene knockouts involving purine scavenger pathway genes appear to 5 confer multistress resistance. Osmoregulatory molecules - small choline-based molecules, such as glycine-betaine, and sugars, such as trehalose, are protective against osmotic shock and are rapidly imported and/or synthesized in response to increasing osmolarity. 10 Acid resistance - lactobacilli naturally acidify their environment through the excretion of lactic acid, mainly through the cit operon genes responsible for citrate uptake and utilization. Stress response genes - a number of genes appear to be induced or repressed by heat shock, 15 cold shock, and increasing salt through the action of specific promoters. The isolated polynucleotides of the present invention, and genetic constructs comprising such polynucleotides, may be employed to produce bacteria having desired phenotypes, including increased resistance to stress and improved fermentation properties. 20 Many enzymes are known to influence dairy product flavor, functional and textural characteristics as well as general fermentation characteristics such as speed of growth, acid production and survival. These enzymes include those involved in the metabolism of lipids, polysaccharides, amino acids and carbohydrates as well as those involved in the lysis of the bacterial cells. 25 The isolated polynucleotides and polypeptides of the present invention have demonstrated similarity to polynucleotides and/or polypeptides of known function. The identity and functions of the inventive polynucleotides based on such similarities are shown below in Table 1. 12 WO 2005/056801 PCT/NZ20031000278 TABLE 1 S EQ SEQI]D IDNO: NO: Utility Description DNA Polypeptide 1, 8, 53 81, 88, 145 Removal of undesirable flavor Homologue of lacG that encodes 6 characteristics. phsh-ea-galactosidase (EC Production of desirable flavors. 3.2.1.85). LacG is part of the lactose odified flavor, aroma, or etabolism, and hydrolyzes exture attributes. hospholactose, the product of a Construction of genetic vectors hosphor-enolpyruvate-dependent or controlled expression of RNA hosphotransferase system. It belongs d/or protein, fusion protein o the glycosidase family 1 and roduction, genetic modification, :ontributes to flavor, including bitter utagenesis amplification of flavor. enetic material or for other enetic or protein manipulations. Itered survival characteristics: survival of industrial processes, owth or storage in product ormats, persistence in gut nvironment). odified carbohydrate levels or ctional properties. tered metabolic properties. odified lactose metabolism. tered probiotic attributes. proved fermentation properties r other industrially useful rocesses. Organisms or materials with improved health properties (including immunoregulatory, ticancer, gut health, lactose olerance) 2 82 Production of bioactive or Homologue of pepS, encoding an fctional polypeptides. opeptidase (EC 3.4.11.-). PepS removal of undesirable flavor catalyzes the release of free amino characteristics. cids from peptides. Production of desirable flavors. Aminopeptidases are exopeptidases modified flavor, aroma and/or d ubiquitous enzymes, frequently texture attributes. bserved in animals, plants and Construction of genetic vectors mcroorganisms. They are involved in or controlled expression of RNA any different functions in the cell, d/or protein, fusion protein uch as protein maturation, protein reduction, genetic modification, over, hydrolysis of regulatory utagenesis amplification of eptides, nitrogen nutrition, genetic material or for other adulation of gene expression etc. 13 WO 2005/056801 PCT/NZ2003/000278 SEQ SEQID NO: NO: Utility Description DNA Polypeptide enetic or protein manipulations. d, consequently, are considered tered survival characteristics: ssential enzymes. The proteolytic survival of industrial processes, ystem of lactic acid bacteria is owth or storage in product essential for bacterial growth in milk ormats, persistence in gut ut also for the development of the environment. rganoleptic properties of dairy tered metabolic properties or products. PepS is involved both in egulation of metabolic bacterial growth by supplying amino athways. cids, and in the development of tered probiotic attributes. favor in dairy products, by Organisms or materials with ydrolyzing peptides (including bitter proved health properties peptides) and liberating aromatic (including immunoregulatory, o acids which are important ticancer, gut health). precursors of aroma compounds tered resistance to antibiotics. (Femandez-Espla and Rul, Eur. J iochein. 263:502-510, 1999). 3 83 reduction of bioactive or foolo e of PepC, encoding ctional polypeptides. op idase C (C 3.4.22.40). removal of undesirable flavor epc is also known as bleomycin characteristics. yrolase, which inactivates reduction of desirable flavors. leomycin B2 (a cytotoxic odified flavor, aroma and/or lycometayopeptide) by hydrolysis o exture attributes. carboxyamnide bond of b Construction of genetic vectors oalanine. It also has general or controlled expression of RNA opeptidase activity. PepC daor protein, fusion protein belongs to peptidase family Cl; also roduction, genetic modification, own as the papain family of thiol utagenesis amplification of roteases and is involved in flavor enetic material or for other roduction. The proteolytic system of enetic or protein manipulations. actic acid bacteria is essential for tered survival characteristics: bacterial. growth in milk but also for urvival of industrial processes, e development of the organoleptic owth or storage in product properties of dairy products. ormats, persistence in gut nvronent. tered metabolic properties or egulation of metabolic athways. tered probiotic attributes. Organisms or materials with proved health properties (including immunoregulatory, ticancer, gut health). tered resistance to antibiotics. 4ueremio acidfmetabolism. omolo e of isC odin foavor 14 WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID NO: NO: Utility Description DNA Polypeptide emoval of undesirable flavor stidinol phosphate aminotransferase characteristics. (EC 2.6.1.9). The HisC roduction of desirable flavors. otransfrase is pyridoxal-5 odified flavor, aroma and/or osphate LP)-dependent and is exture attributes. in e synthesis of histidine Construction of genetic vectors euatra HiFsC also has tyrosine or controlled expression of RNA dphyi anine aminotransferase dor protein, fusion protein i the biosynthesis of histidine roduction, genetic modification, s a central metabolic process in utagenesis amplification of organisms ranging from bacteria to enetic material or for other yeast and plants. enetic or protein manipulations. tered survival characteristics: urvival of industrial processes, owth or storage in product ormats, persistence in gut nvironment. tered metabolic properties. tered probiotic attributes. odified health properties (including immunoregulatory, ticancer, gut health). odified antibiotic resistance. proved fermentation properties r other industrially useful rocesses. 5 85 production of desirable flavors. Homologue of mvaD coding for odified flavor, aroma and/or evalonate pyrophosphate exture attributes. ecarboxylase (EC 4.1.1.33). MvaD onstruction of genetic vectors *s part of the mevalonate pathway for or controlled expression of RNA e biosynthesis of the central ' d/or protein, fusion protein soprenoid precursor, isopentenyl roduction, genetic modification, phosphate by catalyzing the utagenesis amplification of eaction of mevalonate 5-diphosphate enetic material or for other *th ATP to produce inorganic enetic or protein manipulations. hosphate, ADP, CO 2 and tered survival characteristics: sopentenyl diphosphate, the building survival of industrial processes, lock of sterol and isoprenoid growth or storage in product iosynthesis. formats, persistence in gut environment. ltered metabolic properties. odified carbohydrate levels or ctional properties. tered probiotic attributes. |Organisms or materials with 15 WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID ID NO: NO: Utility Description DNA Polypeptide proved health properties (including immunoregulatory, aticancer, gut health). 6 86 production of desirable flavors. omologue of protein p60, encoded odified flavor, aroma and/or y the gene termed iap. The p60 exture attributes. rotein is a major extracellular Construction of genetic vectors roduct secreted by all isolates of L. or controlled expression of RNA onocytogenes. This protein has d/or protein, fusion protein eptidoglycan hydrolase activity but roduction, genetic modification, so influences the uptake of L. utagenesis amplification of onocytogenes by non-phagocytic enetic material or for other ells. Proteins related to p60 are also enetic or protein manipulations. ound in all other Listeria species. It tered survival characteristics: as been shown that p60 protein is urvival of industrial processes, ong the strongest antigens in owth or storage in product isteriae for B- and T-cell responses. onnats, persistence in gut e protein p60 belongs to the E cli nvironment. pc listeria p60 family. tered metabolic properties. termed probiotic attributes. odified adhesion to human or lemal cells or cell lines. Organisms or materials with Atroved health properties (including inmunoregulatory, ticancer, gut health) tered resistance to antibiotics. proved antimicrobial roperties. 7 87 Construction of genetic vectors Homologue of elongation factors Tu or controlled expression of RNA (EF-Tu) and 1 alpha (EF- 1 alpha) that d/or protein, fusion protein ae homologous proteins essential to roduction, genetic modification, anslation in bacteria and eukaryotes, utagenesis amplification of respectively. EF-Tu and EF-lalpha enetic material or for other e GTPases that catalyze the binding enetic or protein manipulations. faminoacyl-tRNAs to the A-site of roduction of desirable flavors. e ribosome. As they are among the odifled flavor, aroma and/or lowest evolving proteins known, EFs exture attributes. e used to study cellular functions tered survival characteristics: d to root the universal tree of life urvival of industrial processes, (Gaucher et al. Proc. Nat. A cad. Sci. owth or storage in product USA 98:548-552, 2001), and are ormats, persistence in gut erefore an excellent genetic tool. environment. tered metabolic pro erties or 16 WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID ID NO: NO: Utility Description DNA Polypeptide regulation of metabolic pathways. tered probiotic attributes. 9 89 Construction of genetic vectors omologue of ribonuclease HII (EC or controlled expression of RNA 3.1.26.4), an RNAse that specifically d/or protein, fusion protein degrades the RNA moiety in roduction, genetic modification, A/DNA hybrids. Endogenous utagenesis amplification of ase H activity plays an essential enetic material or for other ole in biological effects mediated by enetic or protein manipulations. tisense oligonucleotides, molecules roduction of desirable flavors. considered as potential agents- against odified flavor, aroma and/or ectious diseases and pathologies exture attributes. resulting from dysfunctional genes. Itered survival characteristics: e prokaryotic RNAse HII is the urvival of industrial processes, evolutionary counterpart of the major owth or storage in product mammalian RNase H (Frank et aL, ormats, persistence in gut ro. NatI. Acad Sci. USA 95:12872 environment. 12877, 1998) and is necessary for cell tered metabolic properties or survival. egulation of metabolic athways. termed probiotic attributes. 10 90 rodution of desirable flavors. ipase homologue. Lipases are odi1led flavor, aroma and/or enzymes that catalyze hydrolysis of exture attributes. ty i etr bonds in of genetic vectors a ro (TAG) and releasing vr controlled expression of RNA oee fat acids. The reaction is mammia R s Fan ta. d/or protein, fusion protein. vrible and therefore the enzyme ductPion, genetic modification, can. catalyze esterification of glycerol utagenesis amplification of rm mono, di and triglycerides. genetic material or for other ree fatty acids are important in e netic or protein manipulations. roviding flavor-bearing compounds Itered survival characteristics: or dairy products such as cheese, and survival of industrial processes, ave a significant role in both flavor Cowth or storage in product and texture. Used. extensively in wide formats, persistence in gut range of convenience foods. Short environment. hain fatty acids are known to have a prtered metabolic properties. ariety of health impacts. Modified lipid, glycolipid or free gn aid levels or functional modified production of short m t chain fatty acids. tered lipid metabolism. tered probiotic attributes. 17 WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID ID NO: NO: Utility Description DNA Pol peptide rganisms or materials with improved health properties (including immunoregulatory, ticancer, gut health). 91 Altered amino acid metabolism. omo of hisD, encoding removal of undesirable flavor td ehydrogenase (HDH, EC characteristic. 1.1.1.23). HisD catalyzes the last two Production of desirable flavors. steps in the biosynthesis of L odified flavor, aroma and/or sdine: sequential NAD-dependent exture attributes. oxidations of L-histidinol to L Construction of genetic vectors histidinaldehyde and then to L or controlled expression of RNA histidine. Because hisD is absent in d/or protein, fusion protein mammals, it is a target for inhibition roduction, genetic modification, part of herbicide development utagenesis amplification of (Barbosa et al., Proc. Nat. Acad Sci. enetic material or for other USA 99:1859-1864, 2002). enetic or protein manipulations. tered survival characteristics: survival of industrial processes, owth or storage in product ormats, persistence in gut nvironment. tered metabolic properties. tered probiotic attributes. odified health properties (including immunoregulatory, ticancer, gut health). odified antibiotic resistance. 11 92 Altered amino acid metabolism. omologue of hisZ, coding for an removal of undesirable flavor oacyl-tRNA synthetase. HisZ is characteristic. essent opponent of the first reduction of desirable flavors. en e in tidine biosynthesis with modified flavor, aroma and/or TP ph oribosyltransferase texture attributes. C 2f..17) but lacks Construction of genetic vectors I(ainoacylation activity. HisZ is also or controlled expression of RNA caedn p dAor protein, fusion protein hosphoribosyltransferase regulatory roduction, genetic modification, subunit. HisZ is required for histidine utagenesis amplification of rototrophy and directly involved in genetic material or for other the transferase function. Both HisG enetic or protein manipulations. d HisZ are required for catalyzing tered survival characteristics: e ATP phosphoribosyltransferase urvival of industrial processes, eaction. Aminoacyl-tRNA owth or storage in product ynthetases have an essential catalytic formats, persistence in gut ole in protein biosynthesis, but also 18 WO 2005/056801 PCT/NZ20031000278 SEQ SEQ ID NO: NO: Utility Description DNA Polypeptide environment. articipate in numerous other egulation of amino acid ons, lauding regulation of etabolism. ene expression and amino acid tered metabolic properties. iosynthesis via transamidation tered probiotic attributes. pathways (Sissler et al., Proc. Nati. odified health properties cad Sci. USA 96:8985-8990,1999). (including immunoregulatory, because HisD is absent in mammals, ticancer, gut health). t is a target for inhibition as part of modified antibiotic resistance. erbicide development (Barbosa et ., Proc. Nat. Acad. Sci. USA 9:1859-1864, 2002). 12 93 Itered amino acid metabolism. o l e of proA, coding for a removal of undesirable flavor utme-5-seiialdehyde characteristics. ehydrogenase (EC 1.2.1.41). ProA is roduction of desirable flavors. so known as gamma odified flavor, aroma and/or lutamylphosphate reductase, and exture attributes. atalyzes the second step of proline Construction of genetic vectors iosynthesis, the NADPH-dependent or controlled expression of RNA eduction of L-gamma-glutamyl 5 d/or protein, fusion protein hosphate into L-glutamate 5 roduction, genetic modification, emialdehyde and phosphate. utagenesis amplification of tracellular accumulation of the enetic material or for other o acid proline has been linked to enetic or protein manipulations. alt tolerance and virulence potential tered survival characteristics: f a number of bacteria. Proline survival of industrial processes, iosynthesis plays an important role owth or storage in product survival in osmolyte-depleted ormats, persistence in gut nvironments of elevated osmolarity. environment. e survival of the food-borne tered metabolic properties. pathogen L. monocytogenes in tered probiotic attributes. ypersalie environments is odified health properties attributed mainly to the accumulation including immunoregulatory, f organic compounds termed ticancer, gut health). smolytes. Osnolytes, often referred odified antibiotic resistance. a as compatible salutes owing to improved antimicrobial eir compatibility with cellular properties. etabolism at high internal concentrations, can be either ansported into the cell or synthesized de novo and act by ounterbalancing the external osmotic trength, thus preventing water loss d plasmolysis. As well as its role as osmoprotectant, proline may ction as a virulence factor for certain pathoenic bacteria Sleator et 19 WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID NO: NO: Utility Description DNA Poly e tide I., Appl. Environ. Microbiol. 67:2571-2577, 2001). 12 94 tered amino acid metabolism. omologue of proB, coding for emoval of undesirable flavor amma-glutamyl kinase (EC characteristics. .7.2.11) also known as glutamate 5 roduction of desirable flavors. ' ase 1. ProB catalyzes the first step odified flavor, aroma and/or f praline biosynthesis, the transfer o texture attributes. phosphate group to glutamate to Construction of genetic vectors orm glutamate 5-phosphate which or controlled expression of RNA apidly cyclizes to 5-oxoproline. d/or protein, fusion protein tracellular accumulation of the roduction, genetic modification, . o acid proline has been linked to utagenesis amplification of e salt tolerance and virulence enetic material or for other otential of a number of bacteria. enetic or protein manipulations. roline biosynthesis plays an Itered survival characteristics: portant role in survival in survival of industrial processes, smolyte-depleted environments of owth or storage in product levated osmolarity. The survival of ormats, persistence in gut e food-borne pathogen L. environment. onocytogenes in hypersaline tered metabolic properties. nvironments is attributed mainly to tered probiotic attributes. e accumulation of organic odifled health properties ompounds termed osmolytes. (including immunoregulatory, Osmolytes, often referred to as ticancer, gut health). ompatible salutes owing to their modified antibiotic resistance. ompatibility with cellular proved antimicrobial etabolism at high internal roperties. oncentrations, can be either ansported into the cell or ynthesized de novo and act by ounterbalancing the external osmotic trength, thus preventing water loss d plasmolysis. As well as its role as osmoprotectant, praline may action as a virulence factor for ertain pathogenic bacteria (Sleator et L, AppL Environ. Microbiol. 67:2571-2577, 2001I). 13 95 tered cell wall or cell surface onlgeo aH rLH characteristics, structures or cdnaD-ctedhyrgas ctions.(D-LDH, EC 1.1.1.28). D-LD modified adhesion to human or reduces pyruvate to D-lactate and is ' al cells or cell lines. valved in bacterial cell wall reduction of desirable flavors. structure and function. Van} plays an odifled flavor, aroma and/or sential role in bacterial resistance to 20 WO 2005/056801 PCT/NZ20031000278 SEQ SEQ ID NO: NO: Utility Description DNA Polypeptide I I exture attributes. the antibiotic vancomycin. Construction of genetic vectors or controlled expression of RNA d/or protein, fusion protein roduction, genetic modification, utagenesis amplification of enetic material or for other enetic or protein manipulations. tered survival characteristics: urvival of industrial processes, owth or storage in product ormats, persistence in gut nvironment. tered metabolic properties. tered probiotic attributes. modified health properties (including inimunoregulatory, ticancer, gut health). odified antibiotic resistance. proved antimicrobial roperties. 14 96 Altered amino acid metabolism. omologue of metA that encodes Removal of undesirable flavor omoserine O-transsuccinylase (EC characteristics. .3.1.46). MetA catalyzes the first Production of desirable flavors. que step in bacterial and plant Modified flavor, aroma and/or ethionine biosynthesis involving the exture attributes. I tivation of the gamma-bydroxyl of construction of genetic vectors omoserine. The activity of this Or controlled expression of RNA e is closely regulated in vivo uor protein, fusion protein uerefore represents a critical roducion, genetic modification, control point for cell growth and utagenesis amplification of iability. enetic material or for other enetic or protein manipulations. tered survival characteristics: urvival of industrial processes, growth or storage in product formats, persistence in gut environment. Altered metabolic properties. Altered probiotic attributes. odified health properties (including immunoregulatory, nticancer gut health). odified antibiotic resistance. 21 WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID ID NO: NO: Utility Description DNA Polypeptide 15 97 Production of desirable flavors. Homologue of plnG, encoding an modified flavor, aroma and/or ATP binding cassette (ABC) texture attributes. transporter for the antimicrobial onstruction of genetic vectors compound (bacteriocin) plantaricin or controlled expression of RNA . PlnG displays strong similarities d/or protein, fusion protein to the proposed transport proteins of reduction, genetic modification, several other bacteriocins and to utagenesis amplification of proteins implicated in the signal enetic material or for other sequence-independent export of enetic or protein manipulations. scherichia coli hemolysin, PlnH is tered survival characteristics: its accessory protein (Huhne et al., urvival of industrial processes, icrobiol. 142:1437-1448, 1996). owth or storage in product ormats, persistence in gut environment. tered metabolic properties. tered probiotic attributes. odified health properties (including immunoregulatory, ticancer gut health). odified antibiotic resistance. proved antimicrobial roperties. 16 98 Construction of genetic vectors Homologue of hemN that encodes an or controlled expression of RNA oxygen-independent ad/or protein, fusion protein coproporphyrinogen III production, genetic modification, dehydrogenase (EC 1.3.3.3). HemN mutagenesis amplification of analyzes the oxidative enetic material or for other ecarboxylation of enetic or protein manipulations. coproporphyrinogen III to yield roduction of desirable flavors. rotoporphyrinogen IX and requires odified flavor, aroma and/or ADP+, ATP, Mg2+, and L texture attributes. ethionine. In association with Altered survival characteristics: specific apoproteins, it serves a wide survival of industrial processes, ange of important functions growth or storage in product excluding electron transport (e.g., formats, persistence in gut ytochromes), binding and transport environment. f 02 (e.g., hemoglobin), and Altered metabolic properties or xidative catalysis (e.g., peroxidases) regulation of metabolic (Fischer et al., J Bacteriol. pathways. 183:1300-1311, 2001). tered probiotic attributes. tered antimicrobial properties. odified health properties (including immunoregulatory, ofanticancer gut health)( 22 WO 2005/056801 PCT/NZ20031000278 SEQ SEQ ID ID NO: NO: Utility Description DNA Polypeptide 17 99 emoval of undesirable flavor Homologue of lacD, encoding characteristic. gatose- 1,6-bisphosphate aldolase odified flavor, aroma, texture 'EC 4.1.2.40). LacD is responsible ttributes. for the aldol cleavage of tagatose-1,6 Construction of genetic vectors isphosphate to form glycerone-P and or controlled expression of RNA lyceraldehyde 3-phosphate in the d/or protein, fusion protein agatose 6-phosphate pathway of roduction, genetic modification, actose catabolism in bacteria. The utagenesis amplification of enzyme activity is stimulated by enetic material or for other certain divalent cations. enetic or protein manipulations. tered survival characteristics: survival of industrial processes, owth or storage in product ormats, persistence in gut environment . odified carbohydrate levels or ctional properties. tered metabolic properties. odified lactose metabolism. tered probiotic attributes. rganisms or materials with mproved health properties (including immunoregulatory, anticancer, gut health, lactose olerance). 18 100 tered amino acid metabolism. omologue of asnH, encoding emoval of undesirable flavor paragine syntheiase [glutamine haracteristics. ydrolyzing] 2 (EC 6.3.5.4). AsnH tered cell wall or cell surface ansamidates asparagine, glutamate, haracteristics, structures or and diphosphate from aspartate, actions. lutamine and ATP as part of the roduction of desirable flavors. paragine biosynthesis pathway. modified flavor, aroma and/or sH gene is also involved in cell texture attributes. surface organization. Construction of genetic vectors or controlled expression of RNA aa/or protein, fusion protein reduction, genetic modification, utagenesis amplification of enepric material or for other enetic or protein manipulations. tered survival characteristics: survival of industrial processes, owth or stora e in product 23 WO 2005/056801 PCT/NZ20031000278 SEQ SEQ ID NO: NO: Utility Description DNA Polypeptide ormats, persistence in gut environment. Itered metabolic properties. Watered probiotic attributes. Modified health properties (including immunoregulatory, aticancer, gut health). proved fermentation properties r other industrially useful Descrptio processes. 19 101 Removal of undesirable flavor omogefbuBendig23 characteristics. uneodhrgae. Production of desirable flavors. 1114.Bt aaye h A+ modified flavor, aroma and/or texture attributes.u eiaowl 1.1.1.4). ButB catlyzsttehND+ provependentnoxidationoofr2,3 Construction of genetic vectors corresponding reverse reactions. It for controlled expression of RNA man also reduce diacetyl to acetoin. dor protein, fusion protein iacetyl is an important flavor production, genetic modification, compound in dairy products. utagenesis amplification of genetic material or for other genetic or protein manipulations. Altered survival characteristics: survival of industrial processes, growth or storage in product formats, persistence in gut environment. Watered metabolic properties. tered probiotic attributes. Modified health properties including ing imunoregulatory, anticancer, gut health). modified antibiotic resistance. proved fermentation properties or other industrially useful processes. 20 102 Altered cell wall or cell surface characteristic, structures or fctions.aneaids.Nacylrmo Modified adhesion to human or animal cells or cell lines. production of desirable flavors. Modified flavor, aroma and/ortel exture attributes. 20 12_ ' cel wal or ell urf Homo logue of a peptidoglycan stiesstructrhydrrolase (N-acetylmuramoyl-L t'onsalanine amidase). N-acetylmuramoyl mlcifie adesio tohuma or -alanine amidase is an autolysin jani e,11s o cel liesnvolved in degrading the cell wall rodi of esirbleflavrs. uring cell growth or programmed odifed favor aroa an/or ell death and is involved in cell rextre atribtesrowth and important for releasing Construction of genetic vectors enzymes important for flavor. 24 WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID ID NO: NO: Utility Description DNA Polypeptide for controlled expression of RNA and/or protein, fusion protein roduction, genetic modification, utagenesis amplification of enetic material or for other enetic or protein manipulations. tered survival characteristics: urvival of industrial processes, owth or storage in product ormats, persistence in gut nvironment. tered metabolic properties. tered probiotic attributes. odified health properties (including immunoregulatory, ticancer, gut health). odified antibiotic resistance. proved fermentation properties r other industrially useful rocesses. 21, 76 103, 175 Altered cell wall or cell surface omologe ofgalC, encoding characteristics, structures or eramidase (EC 3.2.1.46). functions. GalC hydrolyzes galactose ester Modified adhesion to human or onds ofgaactosylceramide, aial cells or cell lines. a ac sphingosine, Production of desirable flavors. onogalactosyldiglyceride and Modified flavor, aroma and/or a sylceranide. It is involved in exture attributes. e catabolism of galactosylceramide, Constrcton of genetic vectors major lipid in myelin, kidney-and or controlled expression of RNA epithelial cells of small intestine and dgor protein, fusion protein olot Wile bacteria may use ga C production, genetic modification, o- relIeasesugars for metabolism, the utagensis amplification of by-products, including ceramide, asct tor for other signalling molecules in eukaryotic mooglatoydilyerd andria enetic or protein manipulations. ys can lead to apoptosis or tered survival characteristics: iferentiation. Therefore, glaC plays survival of industrial processes, role in probiotic effects and survival growth or storage in product s, the gut environment. formats, persistence in gut environment. Altered metabolic properties. tered probiotic attributes. Modified health properties (including immunoregulatory, anticancer, gut health, apoptosis). odified antibiotic resistance. 25 WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID ED NO: NO: Utility Description DNA Polypeptide proved antimicrobial properties. Proved fermentation properties r other industrially useful recesses. 22 104 Production of bioactive or omologue of pepN, encoding functional polypeptides. embra alanyl aminopeptidase removal of undesirable flavor C3.4.11.2), also called lysyl characteristics. opeptidase and aminopeptidase Production of desirable flavors. PepN releases the N-terminal odified flavor, aroma and/or o acid, Xaa-I-Xbb- from a exture attributes. peptide, amide or arylamide. Construction of genetic vectors opeptidases are involved in or controlled expression of A any different functions in the cell, dlor protein, fusion protein uch as protein maturation, protein roduction, genetic modificaton, over, hydrolysis of regulatory utagenesis amplification of peptides, nitrogen nutrition, enetic material or for other odulation of gene expression etc. enetic or protein manipulations. d, consequently, are considered tered survival characteristics: ssential enzymes. The proteolytic survival of industrial processes, system of lactic acid bacteria is owth or storage in product ssential for bacterial growth in milk ormats, persistence in gut ut also for the development of the environment. rganoleptic properties of dairy tered metabolic properties or products. PepN is involved both in egulation of metabolic material growth by supplying amino athways. cids, and in the development of termed probiotic attributes. avor in dairy products, by Organisms or materials with ydrolyzing peptides (including bitter proved health properties peptides) and liberating aromatic (including immunoregulatory, o acids which are important ticancer, gut health). recursors of aroma compounds tered resistance to antibiotics. (Fernandez-Espla and Rul, Eur. J Biochem. 263:502-510, 1999). 23 105 reduction of desirable flavors. omologue of mvaB, encoding 3 rodild flavor, aroma and/or ydroxy-3-methylglutaryl coenzyme xtbe attributes. synt he MvaB catalyzes the instruction of genetic vectors odeatn of acetyl-CoA ith controlled expression of RNA acetoacetyl.-CoA to form 3-hydroxy dor protein, fusion protein P-methylglutaryl-CoA and CoA. roducd genetic modification, vaB is involved in evalonic acid utagenesis amplification of etabolisna as well as in biosynthesis genetic material or for other f cholesterol arnd ubiquinone genetic or protein manipulations. rogenitors. Terpenoids or tered survival characteristics: (enodconstitute a vast family o 26 WO 2005/056801 PCT/NZ20031000278 SEQ SEQ ID ID NO: NO: Utility Description DNA Polypeptide survival of industrial processes, organic compounds that includes owth or storage in product terols and carotenoids that have ormats, persistence in gut favor, color, texture and other environment. ensory impacts on food products. tered metabolic properties. e terpenoids in many organisms Modified carbohydrate levels or hare early steps in their biosynthesis, ctional properties. eluding the synthesis of 3-hydroxy tered probiotic attributes. -methylglutaryl-coenzyme A and its Organisms or materials with conversion to mevalonate. improved health properties (including immunoregulatory oticancer, gut health). 24 106 Construction of genetic vectors rolo andof hexB, one of two or controlled expression of RNA rois cvol ved in DNA mismatch d/or protein, fusion protein epair. The hex mismatch repair roduction, genetic modification, ystem of Streptococcus pneumoniae utagenesis amplification of cts both during transformation (a enetic material or for other combination process that directly genetic or protein manipulations. produces heteroduplex DNA) to reduction of desirable flavors, correct donor strands and afterDNA modified flavor, aroma an or replication to remove exture attributes. sincorporated nucleotides. The tered survival characteristics: exB is one of at least two proteins survival of industrial processes, equired for mismatch repair. HexB is growth or storage in product omologous to the mutL protein, ormats, persistence in gut ich is required for methyl-directed environment. ismatch repair in Salmonella Watered viability in response to phimurium and E. coli, and to the -tress conditions. MSI gene product, which is likely tered metabolic properties or o be involved in a mismatch regulation of metabolic orrection system in Saccharomyces athways. erevisiae (Prudhomme et a., J tered probiotic attributes. acteriol. 171:5332-5338, 1989). 25 107 Altered amino acid metabolism. omologue of araT, encoding an Removal of undesirable flavor omnatic amino acid aminotransferase haracteristics. EC 2.6.1.57). Aminotransferases reduction of desirable flavors. ave been widely applied in the Vodified flavor, aroma and/or Iarge-sclebiosynthesis of amino for at, esit nc n u tre aibutes. ds h are in increasing Construction of genetic vectors emand by the pharmaceutical or controlled expression of RNA dustry. AraT plays a major role in d/or protein, fusion protein e conversion of aromatic amino reduction, genetic modification, cids to aroma compounds. AraT also Ieutagenesis amplification of as a major physiological role in the genetic material or for other iocthesis of henylalanine and 27 WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID NO: NO: Utility Description DNA Pol e tide enetic or protein manipulations. sine. The enzymatic degradation tered survival characteristics: f amino acids in cheese plays a survival of industrial processes, major role in cheese flavor owth or storage in product development. Indeed, degradation ormats, persistence in gut d environment. ham, and sulfurous amino acids tered metabolic properties. ave been identified in various tered probiotic attributes. cheeses and highly contribute to their odified health properties favor or to off-flavors (Rijnen et al., (including immunoregulatory, . Environ. Microbial. 65:4873 ticancer, gut health). 880, 1999). odified antibiotic resistance. proved fermentation properties or other industrially useful rocesses. 26 108 tered cell wall or cell surface omologue of tagE, encoding characteristics, structures or Oly(glycerolphosphate) alpha etions. lucosyltransferase (BC 2.4.1.52) modified adhesion to human or so called uridine diphosphate e al cells or cell lines. glucose Poly-(glycerol phosphate) roduction of desirable flavors. pha-glucosyl transferase. TagE is odified flavor, aroma and/or volved in techoic acid synthesis. texture attributes, echoic acid is one component of the Construction of genetic vectors ick peptidoglycan layers in the cell or controlled expression of IRNA all of Gram-positive bacteria and is d/or protein, fusion protein usceptible to the enzyme lysozyme roduction, genetic modification, d to penicillin. utagenesis amplification of enetic material or for other genetic or protein manipulations. termed survival characteristics: survival of industrial processes, owth or storage in product ormats, persistence in gut environment. tered metabolic properties. tered probiotic attributes. odified health properties (nclucg immunoregulatory, ticancer, gut health). odifed antibiotic resistance. proved fermentation properties r other industrially useful rocesses. 27 109 tered amino acid metabolism. omolo e to hisB, encoding a 28 WO 2005/056801 PCT/NZ20031000278 SEQ SEQ ID ID NO: NO: Utility Description DNA Polypeptide removal of undesirable flavor biosynthesis bifunctional characteristics. r includes: inidazoleglycerol roduction of desirable flavors. phosphate dehydratase (EC 4.2.1.19) odified flavor, aroma and/or hi an d idio-phosphatase (EC exture attributes. .1.3.15). HisB rearranges the Construction of genetic vectors imidazole glycerol phosphate by a or controlled expression of RNA redox-neutral dehydrative reaction to d/or protein, fusion protein idazole acetol phosphate and roduction, genetic modification, atalyzes the dephosphorylation of utagenesis amplification of bisidinol phosphate to histidinol, the enetic material or for other irect precursor of histidine. Because enetic or protein manipulations. isB is absent from mammals, it has tered survival characteristics: . ecome a target for inhibition as part survival of industrial processes, of herbicide development (Barbosa et owth or storage in product l., Proc. Natl. Acad. Sci. USA ormats, persistence in gut 9:1859-1864,2002). Amino acid nvironment. degradation products in various tered metabolic properties. cheeses have been shown to greatly tered probiotic attributes. contribute to flavor or to off-flavors odified health properties (Rijnen et al, Appi Environ. including immunoregulatory, icrobiol 65:4873-4880, 1999). ticancer, gut health). odifled antibiotic resistance. 28 110 9tered amino acid metabolism. oologue of cysK, encoding emval of undesirable flavor cysteine synthase (BC 4.2.99.8), also rcterisiCS. own as -acetyl-L-serine acetate roduction of desirable flavors. yase (EC 4.2.99.8). CysK catalyzes od flavor, aroma and/or e formation of L-cysteine, the last exture attributes. tep of L-cysteine biosynthesis, from Construction of genetic vectors O-acetyl-L-serine and hydrogen or controlled expression of RNA ulfide. Cysteine synthase is involved d/or protein, fusion protein the assimilatory sulfate reduction roduction, genetic modification, athway and in the selenium utagenesis amplification of corporation into proteins, which enetic material or for other cours mainly as selenocysteine, in enetic or protein manipulations. bacteria. Sulphur-containing amino tered survival characteristics: cid metabolism is important for urvival of industrial processes, development of aroma and flavor owth or storage in product ompounds. ormats, persistence in gut environment. tered metabolic properties. tered probiotic attributes. odified health properties (including immunoregulatory, ticancer, gut health). 29 WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID ID NO: NO: Utility Description DNA Polypeptide Modified antibiotic resistance. 29 111 termed cell wall or cell surface of enn protein. Erin has characteristics, structures or que Ig-binding characteristics as it fnctions. acts preferentially with human modified adhesion to human or g13= t pC gene. Ern is a aial cells or cell lines. enae protein with similarity to roduction of desirable flavors. ethyl-accepting chemotaxis proteins odified flavor, aroma and/or d the streptococcal M proteins exture attributes. omologous with immunoglobulin Construction of genetic vectors finding factors. The M proteins have or controlled expression of RINA een studied because of their duor protein, fusion protein tiphagocytic function. reduction, genetic modification, utagenesis amplification of enetic material or for other genetic or protein manipulations. tered survival characteristics: urvival of industrial processes, owth or storage in product ormats, persistence in gut pvirounent. *etered metabolic properties. Altered probiotic attributes. modified health properties (including imunoregulatory, ticancer, gut health). modified antibiotic resistance. proved antimicrobial roperties. 30 112 tered amino acid metabolism. omologue of hisE, encoding a emoval of undesirable flavor stidine biosynthesis protein that haracteristics. lays a role in histidine biosynthesis. roduction of desirable flavors. ecause hisE is absent in mammals, odified flavor, aroma and/or it is a target for inhibition as part of exture attributes. erbicide development (Barbosa et Construction of genetic vectors L., Proc. Natl. Acad. Sci. USA or controlled expression of RNA 99:1859-1864, 2002). Amino acid d/or protein, fusion protein degradation products in various roduction, genetic modification, cheeses have been shown to greatly utagenesis amplification of ontribute to flavor or to off-flavors enetic material or for other (Rijnen et al., AppL. Environ. enetic or protein manipulations. Microbiol. 65:4873-4880, 1999). tered survival characteristics: urvival of industrial processes, owth or storage in product 30 WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID ID NO: NO: Utility Description DNA Polypeptide formats, persistence in gut environment. tered metabolic properties. tered probiotic attributes. odified health properties (including immunoregulatory, ticancer, gut health). odified antibiotic resistance. 30 113 A1ltered amino acid metabolism. omologue of hisI, encoding a emoval of undesirable flavor hstidine biosynthesis protein that haracteristics. plays a role in histidine biosynthesis. roduction of desirable flavors. because hisI is absent in mammals, it modified flavor, aroma and/or is a target for inhibition as part of exture attributes. erbicide development (Barbosa et Construction of genetic vectors L., Proc. Nat. Acad. Sci. USA or controlled expression of RNA 9:1859-1864, 2002). Amino acid d/or protein, fusion protein egradation products in various production, genetic modification, heeses have been shown to greatly utagenesis amplification of ontribute to flavor or to off-flavors enetic material or for other (Rijnen et al., Appl. Environ. genetic or protein manipulations. Microbiol. 65:4873-4880, 1999). Altered survival characteristics: survival of industrial processes, growth or storage in product ormats, persistence in gut environment. tered metabolic properties. tered probiotic attributes. odified health properties (including immunoregulatory, aticancer, gut health). Modified antibiotic resistance. 31 114 Production of desirable flavors. Homologue of estA, encoding a Modified flavor, aroma and/or serine-dependent arylesterase (EC texture attributes. 3.1.1.2). EstA hydrolyzes a variety of construction of genetic vectors ester compounds and prefers those or controlled expression of RNA with substituted phenyl alcohol or dor protein, fusion protein short-chain fatty acid groups. reduction, genetic modification, lsesterases are responsible for the utagenesis amplification of reduction of important flavor enetic material or for other ompounds and intermediates. enetic or protein manipulations. tered survival characteristics: survival of industrial processes, owth or storage in product 31 WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID UD NO: NO: Utility Description DNA Polypeptide formats, persistence in gut environment. Altered metabolic properties. Modified lipid, glycolipid or free atty acid levels or functional roperties. odified production of short chain fatty acids. tered lipid metabolism. tered probiotic attributes. Organisms or materials with proved health properties (including immunoregulatory, ticancer, gut health). 32 115 Altered amino acid metabolism. omologue of gInA that encodes a removal of undesirable flavor lua e synthetase (EC 6.3.1.2), characteristics. so cle glutamate Production of desirable flavors. igase catalyzes the first step in odified flavor, aroma and/or e onrsion of inorganic nitrogen exture attributes. Ammonium) into its organic form Construction of genetic vectors glutanaine (Gin). Bacterial glutamnine or controlled expression of RNA ynthetase export is associated with dlor protein, fusion protein pathogenicity and with the formation reduction, genetic modification, fa poly-L-glutamate/glutamine cell utagenesis amplification of all structure. Glutaine synthetase genetic material or for other s an enzyme that plays a central role genetic or protein manipulations. the nitrogen metabolism. The tered survival characteristics: nzyme and its products have roles in srpalo of industrial processes, avor and growth. t or storage in product formats, persistence in gut nvironment. Altered metabolic properties. Altered probiotic attributes. Modified health properties (including immunoregulatory, anticancer, gut health). tered cell wall or cell surface haracteristics, structures or ctions. 32 116 termed cell wall or cell surface omologue of Lipopolysaccharide characteristics, structures or synthesis protein yohJ. YohJ is functions. involved in techoic acid synthesis and Modified adhesion to human or important for cell wall functions nial cells or cell lines. includingg adhesion, immune cell 32 WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID ED NO: NO: Utility Description DNA Polypeptide Production of desirable flavors. interaction and product texture. odified flavor, aroma and/or texture attributes. Construction of genetic vectors for controlled expression of RNA d/or protein, fusion protein reduction, genetic modification, utagenesis amplification of enetic material or for other enetic or protein manipulations. tered survival characteristics: urvival of industrial processes, owth or storage in product ormats, persistence in gut environment. tered metabolic properties. tered probiotic attributes. odified health properties including immunoregulatory, ticancer, gut health). odified antibiotic resistance. proved antimicrobial roperties. 33 117 Production of desirable flavors. Homologue of sorA. SorA encodes Modified flavor, aroma and/or e first protein of the . texture attributes. hosphoenolpyruvate-dependent L Construction of genetic vectors sorbose-specific phosphotransferase or controlled expression of RNA system (PTS). The ketose L-sorbose d/or protein, fusion protein is transported and phosphorylated production, genetic modification, ough PTS. The enzyme is useful in utagenesis amplification of arbohydrate-specific regulation of enetic material or for other gene expression and flavor enetic or protein manipulations. evelopment. tered survival characteristics: urvival of industrial processes, owth or storage in product ormats, persistence in gut nvironment. tered metabolic properties. odified carbohydrate levels or ctional properties. tered probiotic attributes. Organisms or materials with proved health properties (including immunoregulatory, anticancer, gut health). 33 WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID ID NO: NO: Utility Description DNA Polypeptide 33 118 Production of desirable flavors. Homologue of sorF, encoding D odified flavor, aroma and/or orbitol-6-phosphate dehydrogenase, texture attributes. enzyme of the L-sorbose onstruction of genetic vectors etabolism. The ketose L-sorbose is or controlled expression of RNA ansported and phosphorylated d/or protein, fusion protein ough the phosphoenolpyruvate roduction, genetic modification, ependent L-sorbose-specific utagenesis amplification of hosphotransferase system (PTS). enetic material or for other seful for carbohydrate-specific enetic or protein manipulations. egulation of gene expression (Yebra tered survival characteristics: t al., J Bacteriol. 182:155-163, urvival of industrial processes, 000). owth or storage in product ormats, persistence in gut nvironment. tered metabolic properties. odified carbohydrate levels or ctional properties. tered probiotic attributes. Organisms or materials with improved health properties (including immunoregulatory, anticancer, gut health). 34 119 Production of bioactive or Homologue of pepA, encoding a ctional polypeptides. lutamyl aminopeptidase, which Removal of undesirable flavor belongs to peptidase family M42 and characteristic. s required for optimal growth of Production of desirable flavors. actococcus lactis MG1363 in milk. modified flavor, aroma and/or PepA has DNA-binding activity that exture attributes. functions in transcription control and Construction of genetic vectors plasmid dimer resolution. for controlled expression of RNA A opeptidases are involved in d/or protein, fusion protein any different functions in the cell, reduction, genetic modification, uch as protein maturation, protein utagenesis amplification of turnover, hydrolysis of regulatory genetic material or for other peptides, nitrogen nutrition, genetic or protein manipulations. modulation of gene expression etc. Altered survival characteristics: and, consequently, are considered survival of industrial processes, essential enzymes. The proteolytic gowth or storage in product system of lactic acid bacteria is ormats, persistence in gut essential for bacterial growth in milk environment. ut also for the development of the tered metabolic properties or rganoleptic properties of dairy egulation of metabolic products. PepA is involved both in pathways. bacterial growth by supplying amino 34 WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID ID NO: NO: Utility Description DNA Polypeptide Altered probiotic attributes. cids, and in the development of O'rganm or m awith avor in dairy products, by improved healthproperties ydrolyzing peptides (including bitter (including immunoregulatory, eptides) and liberating aromatic anticancer, gut health). o acids which are important recursors of aroma compounds Fermandez-Espla and Rul, Eur. J iochem. 263:502-510, 1999). 35 120 Atered amino acid metabolism. omologue of hom, encoding a removal of undesirable flavor omoserine dehydrogenase (EC characteristic. 1.1.1.3), an enzyme of the threonine roduction of desirable flavors. iosynthesis pathway. Threonine is Modified flavor, aroma and/or erived from aspartic acid. texture attributes. Conversion of aspartate to construction of genetic vectors omoserine proceeds with ATP for controlled expression of RNA ctivation of the B-carboxyl group as d/or protein, fusion protein mixed phosphoric anhydride production, genetic modification, ollowed by two sequential NADPH mutagenesis amplification of ependent reductions to homoserine. genetic material or for other Phosphorylation of homoserine genetic or protein manipulations. provides the substrate Altered survival characteristics: phosphohomoserine which suffers a survival of industrial processes, stereospecific 1,2-transposition owth or storage in product reaction to give threonine. The formats, persistence in gut enzymatic degradation of amino acids nvironment. cheese plays a major role in cheese Altered metabolic properties. favor development. Amino acid Altered probiotic attributes. degradation products greatly Modified health properties contribute to flavor or to off-flavors (including immunoregulatory, Tujnen et a., App. Environ. cancer, gut health). icrobiol. 65:4873-4880, 1999). 35 121 altered cell wall or cell surface oegue of flotillin. Flotillins characteristics, structures or hfav as resident integral membrane auctions. protein components of caveolae Modified adhesion to human or which are plasmalemmal aial cells or cell lines. mcrodomains and are involved in reduction of desirable flavors. esicular trafficking and signal modified flavor, aroma and/or ansducton Huang e al., Mo. exture attributes. icrobiol. 31:361-371, 1999). construction of genetic vectors lotillins (also known as epidermal or controlled expression of RNA race antigens (ESAs)) belong to d/or protein, fusion protein e family of caveolae-associated reduction, genetic modification, i tegral membrane proteins and may utagenesis amplification of ct as a scaffolding protein within genetic material or for other aveolar membranes. 35 WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID ID NO: NO: Utility Description DNA Polypeptide genetic or protein manipulations. Altered survival characteristics: survival of industrial processes, owth or storage in product ormats, persistence in gut environment. Altered metabolic properties. tered probiotic attributes. odified health properties (including immunoregulatory, ticancer, gut health). odified antibiotic resistance. proved antimicrobial roperties. 36 122 Removal of undesirable flavor omologue of areB, encoding benzyl characteristic. ohol dehydrogenase (EC 1.1.1.90). roduction of desirable flavors. eB catalyzes the oxidation of an odified flavor, aroma and/or omatic alcohol to an aromatic exture attributes. dehyde. This enzyme enables Construction of genetic vectors acteria to grow on a range of esters or controlled expression of RNA f aromatic alcohols and plays a role and/or protein, fusion protein in flavor development. production, genetic modification, mutagenesis amplification of genetic material or for other genetic or protein manipulations. Altered survival characteristics: survival of industrial processes, owth or storage in product formats, persistence in gut environment. tered metabolic properties. Altered probiotic attributes. Modified health properties (including immunoregulatory, ticancer, gut health). proved fermentation properties or other industrially useful. recesses. 37 123 tered amino acid metabolism. mologue of codB, encoding a emoval of undesirable flavor cosine pernease. CodB mediates characteristics. ptake of exogenously supplied reduction of desirable flavors. ytosine. It belongs to the ABC modified flavor, aroma and/or ansporter family. The cytosine texture attributes. ermease is an integral cytoplasmic 36 WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID ID NO: NO: Utility Description DNA Polypeptide Construction of genetic vectors membrane protein possessing several for controlled expression of RNA transmembrane-spanning domains. nd/or protein, fusion protein e enzymatic degradation of amino reduction, genetic modification, cids in cheese plays a major role in utagenesis amplification of heese flavor development. Amino enetic material or for other cid degradation products greatly enetic or protein manipulations. ontribute to flavor or to off-flavors tered survival characteristics: (Rijnen et al., Appl. Environ. survival of industrial processes, 4icrobiol. 65:4873-4880, 1999). owth or storage in product ormats, persistence in gut environment. Altered response to stress conditions. Altered metabolic properties. Watered probiotic attributes. Modified health properties (including immunoregulatory, ticancer, gut health). Modified antibiotic resistance. proved antimicrobial roperties. proved fermentation properties or other industrially useful processes. 38 124 Construction of genetic vectors . omologue ofhspl8, encoding a for controlled expression of RNA low-molecular-weight protein and/or protein, fusion protein belonging into a family of small heat production, genetic modification, shock proteins. Hsp18 is induced not mutagenesis amplification of only by heat shock but also at the enetic material or for other onset of solventogenesis. Small heat enetic or protein manipulations. shock proteins (sHsps) are a diverse roduction of desirable flavors. oup of heat-induced proteins that odified flavor, aroma and/or are conserved in prokaryotes and exture attributes. ukaryotes and are especially Altered survival characteristics: abundant in plants. Recent in vitro survival of industrial processes, ata indicate that sHsps act as owth or storage in product molecular chaperones to prevent ormats, persistence in gut ermal aggregation of proteins by environment. finding non-native intermediates, tered viability in response to hich can then be refolded in an stress conditions. TP-dependent fashion by other tered metabolic properties or chaperones (Lee and Vierling, Plant regulation of metabolic. hysiol. 122:189-198, 2000). pathways. aretered robotic atcnibutes. 37 WO 2005/056801 PCT/NZ2003/000278 SEQ SEQID NO: NO: Utility Description DNA Polypeptide 39 125 Altered cell wall or cell surface omologue to mccF, encoding a haracteristics, structures or er membrane associated protein of ctions. acteria. MccF determines resistance roduction of desirable flavors. o exogenous microcin. Possible odified flavor, aroma and/or action by preventing the reentering of exture attributes. e cell by exported translation Construction of genetic vectors ibitor microcin C7 (Gonzalez or controlled expression of RNA astor et al., J Bacteriol. 177:7131 d/or protein, fusion protein 140, 1995). production, genetic modification, utagenesis amplification of enetic material or for other enetic or protein manipulations. tered survival characteristics: survival of industrial processes, owth or storage in product ormats, persistence in gut nvironment Itered metabolic properties. tered probiotic attributes. odified health properties including immunoregulatory, ticancer, gut health). odified antibiotic resistance. provedd antimicrobial properties. 40 126 Production of desirable flavors. Homologue of sorE, encoding an L Modified flavor, aroma and/or sorbose- I-phosphate reductase. SorF exture attributes. s, together with D-glucitol-6 Construction of genetic vectors hosphate dehydrogenase, involved or controlled expression of RNAi the conversion of L-sorbose-1 and/or protein, fusion protein hosphate to D-fructose-6-phosphate. roduction, genetic modification, SorE is involved in flavor utagenesis amplification of evelopment and carbohydrate enetic material or for other etabolism. enetic or protein manipulations. tered survival characteristics: survival of industrial processes, owth or storage in product ormats, persistence in gut nvironment. tered metabolic properties. odified carbohydrate levels or ctional properties. tered probiotic attributes. Or anisms or materials with 38 WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID ED NO: NO: Utility Description DNA Polypeptide improved health properties (including immunoregulatory, ticancer, gut health). 41 127 Production of desirable flavors. omologue of the iolB gene, modified flavor, aroma and/or encoding a cis-acting catabolite exture attributes. esponsive element (cre) protein Construction of genetic vectors volved in the iol operon of the myo for controlled expression of RNA ositol catabolism pathway. Myo and/or protein, fusion protein ositol is abundant in nature, production, genetic modification, specially in soil. Various mutagenesis amplification of microorganisms are able to grow on genetic material or for other yo-inositol as the sole carbon genetic or protein manipulations. ource. The expression of the iol Altered survival characteristics: peron is under glucose repression survival of industrial processes, (Miwa and Fujita, J Bacteriol. owth or storage in product 183:5877-5884, 2001). ormats, persistence in gut nvironment. tered metabolic properties. odified carbohydrate levels or functional properties. Altered cell wall or cell surface characteristics, structures or functions. odified adhesion to human or animal cells or cell lines. Altered probiotic attributes. Organisms or materials with improved health properties (including immunoregulatory, anticancer, gut health). 41 128 Production of desirable flavors. Homologue of the iolC gene, Modified flavor, aroma and/or coding 2-dehydro-3 texture attributes. -eoxygluconokinase. IoIC Construction of genetic vectors phosphorylates the 2-deoxy-5-keto for controlled expression of RNA D-gluconic acid to 2-deoxy-5-keto-D and/or protein, fusion protein gluconic acid 6-phosphate and is part production, genetic modification, of the iol operon of the myo-inositol utagenesis amplification of katabolism pathway (Yoshida et aL, genetic material or for other I BacterioL 179:4591-4598, 1997). genetic or protein manipulations. yo-inositol is abundant in nature, tered survival characteristics: :specially in soil. Various survival of industrial processes, microorganisms are able to grow on owth or storage in product nyo-inositol as the sole carbon formats, ersistence in gut source. The expression of the iol 39 WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID NO: NO: Utility Description DNA Polypeptide_ nvironment. peron is under glucose repression tered metabolic properties. iwa and Fujita, J Bacteriol. odified carbohydrate levels or 183:5877-5884, 2001). .ctional properties. tered cell wall or cell surface haracteristics, structures or ctions. odified adhesion to human or aial cells or cell lines. tered probiotic attributes. Organisms or materials with mproved health properties (including immunoregulatory, ticancer, gut health). 41 129 Production of desirable flavors. Homologue of the iolE gene, modified flavor, aroma and/or ncoding a protein involved in the iol exture attributes. peron of the myo-inositol Construction of genetic vectors atabolism pathway. Myo-inositol is or controlled expression of RNA bundant in nature, especially in soil. d/or protein, fusion protein arious microorganisms are able to roduction, genetic modification, ow on myo-inositol as the sole utagenesis amplification of arbon source. The expression of the enetic material or for other ol operon is under glucose repression enetic or protein manipulations. (Miwa and Fujita, J Bacteriol. tered survival characteristics: 183:5877-5884, 2001). urvival of industrial processes, owth or storage in product ormats, persistence in gut environment. tered metabolic properties. odified carbohydrate levels or ctional properties. tered cell wall or cell surface haracteristics, structures or ctions. odified adhesion to human or nial cells or cell lines. tered probiotic attributes. Organisms or materials with proved health properties (including immunoregulatory, ticancer, gut health). 41 130 roduction of desirable flavors. omologue of the iolG gene, odified flavor, aroma and/or ncoding inositol dehydrogenase (EC exture attributes. 1.1.1.18). IolG catalyzes the first An WO 2005/056801 PCT/NZ20031000278 SEQ SEQ ID NO: NO: Utility Description DNA Polypeptide Construction of genetic vectors reaction of the inositol catabolism, for controlled expression of RNA e dehydrogenation of myo-inositol d/or protein, fusion protein to 2-keto-myo-inositol (2-inosose) reduction, genetic modification, Yoshida et al., J Bacteriol. utagenesis amplification of 179:4591-4598, 1997). IolG is part of enetic material or for other e iol operon of the myo-inositol enetic or protein manipulations. atabolism pathway. Myo-inositol is tered survival characteristics: abundant in nature, especially in soil. urvival of industrial processes, various microorganisms are able to owth or storage in product ow on myo-inositol as the sole ormats, persistence in gut carbon source. The expression of the environment. o1 operon is under glucose repression tered metabolic properties. (Miwa and Fujita, J Bacteriol. odified carbohydrate levels or 183:5877-5884, 2001). ctional properties. termed cell wall or cell surface characteristics, structures or actions. odified adhesion to human or amal cells or cell lines. tered probiotic attributes. Organisms or materials with improved health properties (including immunoregulatory, anticancer, gut health). 41 131 Production of desirable flavors. omologue of the iolJ gene, Modified flavor, aroma and/or ncoding-fructose-bisphosphate texture attributes. dolase (EC 4.1.2.13) or tagatose Construction of genetic vectors isphosphate aldolase (4.1.2.40). IolJ for controlled expression of RNA leaves 2-deoxy-5-keto-D-gluconic and/or protein, fusion protein cid 6-phosphate to yield production, genetic modification, dihydroxyacetone phosphate and utagenesis amplification of alonic senialdehyde and is part of enetic material or for other e iol operon of the myo-inositol enetic or protein manipulations. atabolism pathway (Yoshida et al., tered survival characteristics:. Bacteriol. 179:4591-4598, 1997). urvival of industrial processes, yo-inositol is abundant in nature, owth or storage in product especially in soil. Various ormats, persistence in gut .croorganisms are able to grow on environment. yo-mositol as the sole carbon tered metabolic properties. source. The expression of the iol odified carbohydrate levels or peron is under glucose repression ctional properties. (Miwa and Fujita, . Bacteriol tered cell wall or cell surface 183:5877-5884, 2001). haracteristics, structures or actions. 41 WO 2005/056801 PCT/NZ20031000278 SEQ |SEQ ID EDNO: NO: utility Description DNA Polypeptide Modified adhesion to human or imle l a , aror cell lines. tered probiotic attributes. Organisms or materials with imnroved health properties (including immunoregulatory, ticancer, gut health). 41 132 tered amino acid metabolism. orc. Te efpres of tol Removal of undesirable flavor ero i er gloe terecharacteristics. (Miwa an dFui, aeio reduction of desirable flavors. 18n87-84 2001). The nm modified flavor, aroma and/or s a o u d for th onalie texture attributes. i gdisolegneatisn al Cntion ofgnt ti vectone hact onvrt both91458 propan. or ontoled xprsson f A omIoolou ofmsA orndn iolat, e rtein, ea o proerti. ndiethylmoxoroaoate t dified crhdt levicatios ialro eaydeo The hdoenmat Iol tionaesproperties.oegraation of vert aonic s nle heese ere ctr all expression of yo-Inaiol ibnd i lavor genetic or protein manipulations. operon is under glucose repression Alrcteristics: (Miwa and Fujita, J Bacteriol. survival of industrial processes, 183:5877-5884, 2001). The enzyme rowth or storage in product s also required for growth on valine formats, persistence in gut d isoleucine as it is an acylating environment. enzyme that converts both propanal tered metabolic properties. nd 2-Methyl-3-oxopropanoate to. modified carbohydrate levels or ropanoyl-CoA. The enzymatic optional properties. degradation of amino acids in cheese tered cell wall or cell surface l ao rol phsa haracteristics, structures or y e subnt Aino (c4. factions. gaainpoutgrtl Modified adhesion to human or o calle to slase o yclas aial cells or cell lines.d/ r ubunit His l hin tered probiotic attributes. prbio s and-is9a Organisms or materials with proved health proper-ties (including immunoregulatory, playsner au maorroelntheseflvo 42 13 Vtec d minoacid etablis devenlopment. Ai noig n 0 al desidegradationglyprouctspraty rodctonofdeiraleflvos. sctibue toP flavor ortoof-laos 42 133 Alteredi a acdttboi sm.es Eomoloue boyheis endiga Construction of genetic vectors member of the glutamine 42 WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID NO: NO: Utility Description DNA Pol e tide or controlled expression of RNA idotransferase family. d/or protein, fusion protein e enzymatic degradation of amino roduction, genetic modification, cids in cheese plays a major role in utagenesis amplification of heese flavor development. Amino enetic material or for other cid degradation products greatly enetic or protein manipulations. ontribute to flavor or to off-flavors itered survival characteristics: 'jnen et al., AppL. Environ, survival of industrial processes, icrobiol. 65:4873-4880, 1999). owth or storage in product ormats, persistence in gut environment. tered metabolic properties, tered probiotic attributes. odified health properties (including immunoregulatory, anticancer, gut health). odified antibiotic resistance. 43 134 Construction of genetic vectors omologue of yqcM, encoding or controlled expression of RNA senate reductase (EC 1.97.1.5). d/or protein, fusion protein qcM reduces the arsenate ion roduction, genetic modification, (H 2 AsO) to arsenite ion (AsO). utagenesis amplification of senate is an abundant oxyanion enetic material or for other at, because of its ability to mimic enetic or protein manipulations. e phosphate group, is toxic to cells. roduction of desirable flavors. senate reductase participates to odified flavor, aroma and/or chieve arsenate resistance in both exture attributes. rokaryotes and yeast by reducing tered survival characteristics: senate to arsenite; the arsenite is survival of industrial processes, en exported by a specific owth or storage in product ansporter. Arsenite reductase is ormats, persistence in gut oupled to the glutathione and environment. lutaredoxin system for its enzyme tered viability in response to activity (Bennett et al., Proc. NatI. tress conditions cad. Sci. USA 98:13577-13582, tered metabolic properties or 001). egulation of metabolic athways. tered probiotic attributes. mproved fermentation properties r other industrially useful rocesses. odified health properties (including immunoregulatory, ticancer, gut health). 44 135 tered amino acid metabolism. omologue of cna, encoding a 43 WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID D NO: NO: Utility Description DNA Polypeptide Removal of undesirable flavor ollagen adhesion. Cna mediates characteristics. t haracerisics.ttachment of bacterial cells to roduction of desirable flavors. a o s odified flavor, aroma and/or ched to the cell wall exture attributes. eptidoglycan by an amide bond. Construction of genetic vectors or controlled expression of RNA d/or protein, fusion protein reduction, genetic modification, utagenesis amplification of genetic material or for other genetic or protein manipulations. tered Survival characteristics: urvival of industrial processes, owth or storage in product ormats, persistence in gut environment. tered metabolic properties. tered probiotic attributes. odified health properties including imunoregulatory, ticancer, gut health). modified antibiotic resistance. proved antimicrobial properties. 45, 65 136, 159 Production of bioactive or Homologue of pepQ, encoding a fctional polypeptides. Xaa-Pro dipeptidase (EC 3.4.13.9). Removal of undesirable flavor PepQ hydrolyzes Xaa-Pro dipeptides haracteristics. ut not Pro-Pro) and also acts on roduction of desirable flavors. aioacyl-hydroxyproline analogs odified flavor, aroma and/or s peptidase belongs to peptidase exture attributes. family M24A (methionyl Construction of genetic vectors opeptidase family). It has a or controlled expression of RNA otential use in the dairy industry as a d/or protein, fusion protein heese-ripening agent since proline roduction, genetic modification, elease from proline-containing utagenesis amplification of eptides in cheese reduces bitterness. enetic material or for other e proteolytic system of lactic acid enetic or protein manipulations. acteria is essential for bacterial tered survival characteristics: owth in milk but also for the urvival of industrial processes, evelopment of the organoleptic owth or storage in product roperties of dairy products. PepQ is ormats, persistence in gut volved both in bacterial growth by environment. upplying amino acids, and in the tered metabolic properties or evelopment of flavor in dairy egulation of metabolic roducts, b hydrolyzing petides 44 WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID D NO: NO: Utility Description DNA Polypeptide pathways. (including bitter peptides) and tered probiotic attributes. berating aromatic amino acids Organisms or materials with are important precursors of mpovehelhpoets whicharimotnprcrosf ed health properties ma compounds (Femandez-Espla (including immunoregulatory) d Rul, Eur. J Biochem. 263:502-' 510, 1999). 46 137 Altered amino acid metabolism. omologue of argG, encoding a Removal of undesirable flavor gininosuccinate synthase (EC characteristics. 6.3.4.5). ArgG catalyzes the Production of desirable flavors. penultimate step of the arginine modified flavor, aroma and/or iosynthesis. It belongs to the extureaattributes. gininosuccinate synthase family. Construction of genetic vectors he enzymatic degradation of amino or controlled expression of RNA cids in cheese plays a major role in d/or protein, fusion protein heese flavor development. Amino roduction, genetic modification, cid degradation products greatly utagenesis amplification of ontribute to flavor or to off-flavors enetic material or for other (Rijnen et al., AppL. Environ. enetic or protein manipulations. Microbiol. 65:4873-4880, 1999). tered survival characteristics: urvival of industrial processes, owth or storage in product ormats, persistence in gut nvironment. tered metabolic properties. tered probiotic attributes. odified health properties including immunoregulatory, aticancer, gut health). odified antibiotic resistance. 46 138 tered amino acid metabolism. Homologue of argH, encoding emoval of undesirable flavor gininosuccinate lyase (EC 4.3.2.1). haracteristics. gH catalyzes the last step in the roduction of desirable flavors. ginine biosynthesis. odified flavor, aroma and/or Argininosuccinate lyase also exture attributes. articipates in the urea cycle, the Construction of genetic vectors ajor pathway for the detoxification or controlled expression of RNA of ammonia, where it catalyzes the d/or protein, fusion protein eversible breakdown of roduction, genetic modification, gininosuccinic acid into arginine utagenesis amplification of d fumarate. The enzymatic enetic material or for other graduation of amino acids in cheese enetic or protein manipulations. lays a major role in cheese flavor tered survival characteristics: development. Amino acid urvival of industrial processes, egradation products greatly WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID I NO: NO: Utility Description DNA Polypeptide I owth or storage in product ontute to flavor or to off-flavors formats, persistence in gut t al., AppE environment. Watered metabolic properties. Watered probiotic attributes. Modified health properties (including immunoregulatory, aticancer, gut health). Modified antibiotic resistance. proved fermentation properties or other industrially useful processes. 47 139 Construction of genetic vectors Homologue of purL, encoding a for controlled expression of RNA phosphoribosylformylglycinamidine ad/or protein, fusion protein (FGAM) synthetase (EC 6.3.5.3). production, genetic modification, PurL catalyzes the fourth step in the mutagenesis amplification of biosynthesis of purines. It is involved genetic material or for other inmultistress resistance. Purines play genetic or protein manipulations. essential roles in many cellular Production of desirable flavors. cinicuigDArpiain Modified flavor, aroma and/or asrpin nr-adetaclua texture attributes.igaienrymtblsnda Watered survival characteristics: oez esfrmnbicmca survival of industrial processes, ectns gowth or storage in product formats, persistence in gut environment. Watered viability in response to stress conditions. Watered metabolic properties or regulation of metabolic pathways. Watered probiotic attributes. 48 140 Atered amino acid metabolism. Homologue of hisH, encoding a Removal of undesirable flavor imdazole glycerol phosphate characteristics. synthase subunit that is also known as Production of desirable flavors. [GP synthase glutamine Modified flavor, aroma and/or amdotransferase subunit. HisH texture attributes. catalyzes the fifth step of the histidine Construction of genetic vectors biosynthesis. The hisH subunit for controlled expression of RNA provides the glutamine ad/or protein, fusion protein amdotransferase activity that production, genetic modification, produces the ammonia necessary to mutagenesis amplification of hisF for the synthesis of IGP and genetic material or for other ,ACAR. The enzyatic degradation 46 WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID NO: NO: Utility Description DNA Polypeptide_ enetic or protein manipulations. f amino acids in cheese plays a tered survival characteristics: ajor role in cheese flavor survival of industrial processes, development. Amino acid owth or storage in product degradation products greatly formats, persistence in gut contribute to flavor or to off-flavors environment. nen et a., App. Environ tered metabolic properties. IcrobioL 65:4873-4880, 1999). tered probiotic attributes. odified health properties (including immunoregulatory, aticancer, gut health). 49 141 Co nstruction of genetic vectors romologue of ndK. encoding r controlled expression of RNAo cleoside-diphosphate kinase (EC d/or protein, fusion protein 7.4.6), which converts reduction, genetic modification, Amoxy)ribonucleoside diphosphates utagenesis amplification of dto their corresponding genetic material or for other phobte a s Micrbio. 65483480 1999).biq enetic or protein manipulation specific enzyme but is an production of desirable flavors. ptt lular enzyme that odifled flavor, aroma and/or nitr= n anaisnctd for ~ ~ ~ ~ ~ ~ ~r cotole exrsinomNAacesd-ihspainaset(eC texture attributes. ools and has been implicated in a tered survival characteristics: um ber o regulatory processes, survival of industrial processes, iludin signal transduction, owth or storage in product development and cell surface ormats, persistence in gut olysacharide synthesis. nvironment tered viability in response to tress conditions. tered metabolic properties or egulation of metabolic tered probiotic attributes. 50 142 tered cell wall or cell surface roilogue of PrtB, a P1-type haracteristcs, structures or as precursor (Lactocepin) formaso cled cell wall-associated serine odifled adhesion to huan or teinase (EC 3.4.21.96). PrtB mal cells or cell lines. reaks down milk proteins during the roduction of desirable flavors. owth of the bacteria on milk and modified flavor, aroma and/or at provides the peptides essential exture attributes, or cell growth. It has endopeptidase onstruction of genetic vectors activity with very broad specificity. It or controlled expression of RNA is best known for its action on dlor protein, fusion protein caseins, although it has been shown roduction, genetic modification, o hydrolze hemoglobin and WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID ID NO: NO: utility Description DNA Polypeptide mutagenesis amplification of oxidized insulin b-chain. Lactocepin enetic material or for other *s a type I membrane protein, located enetic or protein manipulations. *n the cell wall and belongs to tered survival characteristics: peptidase family S8; also known as survival of industrial processes, e Subtilase Family. Lactocepin is owth or storage in product responsible for the hydrolysis of ormats, persistence in gut ilk and specificity environment. ifferces between lactocepins from tered metabolic properties. ir strains may be partly tered probiotic attributes. sponsieor imparting different odified health properties, favor qualities to cheese (Broadbent (including immunoregulatory, t al., App!. Environ. Microbiol. ticancer, gut health). 68:1778-1785, 2002). modified antibiotic resistance. proved fermentation properties or other industrially useful rocesses. 51 143 [Atered amino acid metabolism. omologue of aspB, encoding an removal of undesirable flavor aspartate aminotransferase (EC: characteristics. 6.1.1), also called aspartate reduction of desirable fl . ansam ase. AspB catalyzes the odified flavor, aroma an/r o group transfer between amino exture attributes. cids and 2-oxo acids and that plays a Construction of genetic vectors entral role in amino acid metabolism or controlled expression of RNA organisms. The transferase is and/or protein, fusion protein portant for the metabolism of roduction, genetic modification, noacids and Krebs cycle related utagenesis amplification of rgac acids. It plays a role in the enetic material or for other production of important flavor enetic or protein manipulations. eterininants. The enzymatic termed survival characteristics: degradation of amino acids in cheese survival of industrial processes, lays a major role in cheese flavor owth or storage in product evelopment. Amino acid ormats, persistence in gut egradation products greatly environment. contribute to flavor or to off-flavors tered metabolic properties. (Rijnen et a., App!. Environ. tered probiotic attributes. icrobiol 65:4873-4880, 1999). odified health properties (i nc ludg eonunoregulatory, ticancer, gut health). modified antibiotic resistance. proved fermentation properties r other industrially usefu processes. 52 144 roduction of desirable flavors. omolo ae of iolF, encoding anlt WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID ID NO: NO: Utility Description DNA Polypeptide Modified flavor, aroma and/or inositol transporter (TC#: texture attributes. .A. 1.1.27). IolF transports myo Construction of genetic vectors inositol into the bacterial cell. IoIF is for controlled expression of RNA part of the ol operon of the myo and/or protein, fusion protein ositol catabolism pathway. Myo production, genetic modification, ositol is abundant in nature, mutagenesis amplification of specially in soil. Various enetic material or for other croorganisms are able to grow on genetic or protein manipulations. yo-inositol as the sole carbon Altered survival characteristics: source. The expression of the iol survival of industrial processes, >peron is under glucose repression growth or storage in product (Miwa and Fujita, J Bacteriol. formats, persistence in gut 183:5877-5884, 2001). environment. Altered metabolic properties. Modified carbohydrate levels or ctional properties. Altered cell wall or cell surface characteristics, structures or functions. Modified adhesion to human or animal cells or cell lines. Altered probiotic attributes. Organisms or materials with improved health properties (including immunoregulatory, anticancer, gut health). 54 146 Altered cell wall or cell surface omologue of mga4, a positive characteristics, structures or regulatory protein that acts as a functions. opponent of a signal transducing Modified adhesion to human or system. Positive regulatory proteins animal cells or cell lines. r activator proteins bind in their Production of desirable flavors. active state to DNA in the promoter Modified flavor, aroma and/or region and help RNA polymerase to texture attributes. ind and transcribe that gene. Mga4 Construction of genetic vectors states transcription of surface or controlled expression of RNA sociated/virulence factors. ocdror protein, fusion protein production, genetic modification, rhpgenesis amplification of enetic material or for other enetic or protein manipulations. ftered survival characteristics: survival of industrial processes, growth or storage in product geormats, persistence in gut WO 2005/056801 PCT/NZ2003/000278 SEQ- SEQ ID ED NO: NO: Utility Description DNA Polypeptide environment. tered metabolic properties. tered probiotic attributes. odified health properties including immunoregulatory, ticancer, gut health). odified antibiotic resistance. proved antimicrobial roperties. Regulation of polysaccharide reduction, adhesion, immune odulation. 55 147 Altered amino acid metabolism. omologue of BH3554, encoding a emoval of undesirable flavor :arboxylesterase (3.1.1.1). BH3554 characteristic. ydrolyzes carboxylic ester bonds Production of desirable flavors. wth relatively broad substrate odified flavor, aroma and/or specificity. It is involved in amino exture attributes. cid metabolism and flavor. The onstruction of genetic vectors enzymatic degradation of amino acids or controlled expression of RNA cheese plays a major role in cheese cl/or protein, fusion protein favor development. Amino acid roduction, genetic modification, degradation products greatly utagenesis amplification of ontribute to flavor or to off-flavors enetic material or for other (Rijnen et al., App. Environ. enetic or protein manipulations. Microbiol. 65:4873-4880, 1999). tered survival characteristics: survival of industrial processes, owth or storage in product ormats, persistence in gut nvironment. tered metabolic properties. tered probiotic attributes. odified health properties (including immunoregulatory, ticancer, gut health). odified antibiotic resistance. proved fermentation properties or other industrially useful rocesses. 56 148 Altered amino acid metabolism. oooe ofAmdI, encoding an emoval of undesirable flavor oayase (EC 3.5.1.14). Amdl haracteristics. eacetylates acylated amino acids. It roduction of desirable flavors. lays a role in the production of odified flavor, aroma and/or portent flavor determinants. The exture attributes. nzymatic degradation of amino acids S0 WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID ID NO: NO: Utility Description DNA Polypeptide Construction of genetic vectors cheese plays a major role in- cheese for controlled expression of RNA favor development. Amino. acid d/or protein, fusion protein egradation products greatly reduction, genetic modification, ontribute to flavor or to off-flavors utagenesis amplification of (Rijnen et al., Appl. Environ. enetic material or for other icrobioL 65:4873-4880, 1999). genetic or protein manipulations. tered survival characteristics: urvival of industrial processes, owth or storage in product ormats, persistence in gut nvironment. tered metabolic properties. tered probiotic attributes. odified health properties (including immunoregulatory, ticancer, gut health). odified antibiotic resistance. proved fermentation properties or other industrially useful processes. 57 149 Altered cell wall or cell surface omologue of tmpA, encoding a characteristics, structures or putative transiembrane protein. functions. mpA plays a role in adhesion and is Modified adhesion to human or art of an operon containing the animal cells or cell lines. apA gene. Production of desirable flavors. Modified flavor, aroma and/or texture attributes. Construction of genetic vectors for controlled expression of RNA d/or protein, fusion protein reduction, genetic modification, mutagenesis amplification of genetic material or for other genetic or protein manipulations. Altered survival characteristics: survival of industrial processes, owth or storage in product formats, persistence in gut environment. Itered metabolic properties. tered probiotic attributes. odified health properties (including immunoregulatory, anticancer, gut health).
WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID ED NO: NO: Utility Description DNA Polypeptide Modified antibiotic resistance. Improved fermentation properties processes. 58 150 Construction of genetic vectors omologue of npr, encoding a for controlled expression of RNA NADH peroxidase (EC 1.11.1.-1). Npr and/or protein, fusion protein utilizes hydrogen peroxide to create roduction, genetic modification, ater and nicotinamide adenine utagenesis amplification of dinucleotide (NADH) from its enetic material or for other xidized form (NAD). Metabolism of enetic or protein manipulations. zo-factors such as NADH can greatly roduction of desirable flavors. influence the speed and type of Modified flavor, aroma and/or etabolic pathway utilized under exture attributes. iferent redox conditions, and can Altered survival characteristics: terefore influence flavor and/or survival of industrial processes, functionality. owth or storage in product ormats, persistence in gut nvironment. tered viability in response to stress conditions. tered metabolic properties or regulation of metabolic pathways. Altered probiotic attributes. Improved fermentation properties or other industrially useful processes. 59, 60 151, 152 tered amino acid metabolism. omologue of nifS. NifS is involved emoval of undesirable flavor cysteine metabolism and haracteristics. development of flavor compounds. roduction of desirable flavors. e enzymatic degradation of amino odified flavor, aroma and/or cids in cheese plays a major role in texture attributes. cheese flavor development. Amino Construction of genetic vectors cid degradation products greatly for controlled expression of RNA contribute to flavor or to off-flavors d/or protein, fusion protein cen et a l, ispp Environ. roduction, gnetic modification, icrobiol. 65:4873-4880, 1999). utagenesis amplification of enhetic material or for other enetic or protein manipulations. tered survival characteristics: survival of industrial processes, growth or storage in product ormats, persistence in gutions.
WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID ID NO: NO: Utility Description DNA Polypeptide environment. Altered metabolic properties. tered probiotic attributes. odified health properties (including immunoregulatory, ticancer, gut health). odified antibiotic resistance. proved fermentation properties r other industrially useful rocesses. 61 153 Production of desirable flavors. Romologue of fabA, encoding a 3 Modified flavor, aroma and/or ydroxydecanoyl-ACP dehydratase. texture attributes. abA introduces cis unsaturation into Construction of genetic vectors atty acids during saturated fatty acid for controlled expression of RNA iosynthesis. The dehydratase and/or protein, fusion protein elongs to the thioester dehydratase reduction, genetic modification, family. Free fatty acids are important utagenesis amplification of providing flavor-bearing enetic material or for other compounds for dairy products such as enetic or protein manipulations. cheese, and have a significant role in tered survival characteristics: both flavor and texture. Used survival of industrial processes, extensively in wide range of owth or storage in product convenience foods. Short chain fatty formats, persistence in gut acids are known to have a variety of environment. health impacts. Altered metabolic properties. Modified lipid, glycolipid or free fatty acid levels or functional properties. modified production of short chain fatty acids. tered lipid metabolism. Altered probiotic attributes. Organisms or materials with improved health properties (including immunoregulatory, anticancer, gut health) 62 154 Altered cell wall or cell surface ornlogue of aggH, encoding an characteristics, structures or utoaggregation mediating protein. Actions. ggH contains a region of similarity odified adhesion to human or o ATP-dependent DEAD-box aial cells or cell lines. elicase. The protein is involved in roduction of desirable flavors. genetic exchange, pathogen exclusion Hodifoed flavor, aroma and/or d persistence in the gut texture attributes. nvirorment by promoting WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID ID NO: NO: Utility Description DNA Polypeptide Construction of genetic vectors aggregation between bacteria. for controlled expression of RNA d/or protein, fusion protein reduction, genetic modification, utagenesis amplification of enetic material or for other enetic or protein manipulations. tered survival characteristics: urvival of industrial processes, growth or storage in product formats, persistence in gut environment. Altered metabolic properties. Altered probiotic attributes. modified health properties including immunoregulatory, ticancer, gut health). odified antibiotic resistance. proved antimicrobial properties. Improved fermentation properties or other industrially useful processes. 63 155 Production of desirable flavors. Homologue of the iolH gene, Modified flavor, aroma and/or encoding a protein involved in the iol texture attributes. operon of the myo-inositol Construction of genetic vectors 'atabolism pathway. Myo-inositol is or controlled expression of RNA abundant in nature, especially in soil. d/or protein, fusion protein Various microorganisms are able to production, genetic modification, grow on myo-inositol as the sole utagenesis amplification of carbon source. The expression of the enetic material or for other iol operon is under glucose repression genetic or protein manipulations. (Miwa and Fujita, J Bacteriol. Altered survival characteristics: 183:5877-5884, 2001) survival of industrial processes, growth or storage in product formats, persistence in gut environment. Altered metabolic properties. Modified carbohydrate levels or ctional properties. tered cell wall or cell surface characteristics, structures or functions. Modified adhesion to human or animal cells or cell lines.
WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID ID NO: NO: Utility Description DNA Polypeptide Altered probiotic attributes. rganisms or materials with proved health properties (including immunoregulatory, ticancer, gut health). 63 156 roduction of desirable flavors. Homologue of the iolL gene, odified flavor, aroma and/or encoding a protein involved in the iol texture attributes. peron of the myo-inositol Construction of genetic vectors atabolism pathway. Myo--nositol is or controlled expression of RNA abundant in nature, especially in soil. d/or protein, fusion protein arious microorganisms are able to roduction, genetic modification, ow on myo-inositol as the sole utagenesis amplification of carbon source. The expression of the genetic material or for other ol operon is under glucose repression genetic or protein manipulations. (Miwa and Fujita, J Bacteriol. tered survival characteristics: 183:5877-5884, 2001) urvival of industrial processes, owth or storage in product formats, persistence in gut environment. Altered metabolic properties. Modified carbohydrate levels or functional properties. Altered cell wall or cell surface characteristics, structures or functions. odified adhesion to human or al cells or cell lines. altered probiotic attributes. Organisms or materials with proved health properties (including immunoregulatory, anticancer, gut health). 64 157 Removal of undesirable flavor omologue of citX, encoding apo characteristics. citrate lyase phosphoribosyl Modified flavor, aroma, texture dephospho-CoA transferase (Apo ttributes. ACP nucleodityltransferase; EC Construction of genetic vectors 2.8.3.10). The transferase belongs to or controlled expression of RNA e citX family (Schneider et al., ad/or protein, fusion protein Bochem. 39:9438-9450, 2000). production, genetic modification, ''arbohydrate metabolism impacts on utagenesis amplification of favor, functionality and survival as genetic material or for other well as growth. genetic or protein manipulations. altered survival characteristics: WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID NO: NO: Utility Description DNA Polypeptide (survival of industrial processes, growth or storage in product ormats, persistence in gut environment). odified carbohydrate levels or ctional properties. tered metabolic properties. odified citrate metabolism. tered probiotic attributes. Organisms or materials with improved health properties (including immunoregulatory, ticancer, gut health). proved fermentation properties r other industrially useful rocesses. 64 158 emoval of undesirable flavor omologuc of pycB, encoding haractristics. yruvate carboxylase, which Modified flavor, aroma, texture atalyzes a two-step reaction, involving the ATP-dependent Construction of genetic vectors arboxylation of the covalently or controlled expression of RNA attached biotin in the first step and d/or protein, fusion protein e transfer of the carboxyl group to reduction, genetic modification, yruvate to generate oxaloacetate in utagenesis amplification of e second. The enzyme is involved genetic material or for other gluconeogenesis and amino acid enetic or protein manipulations. iotransformations. Carbohydrate tered survival characteristics: etabolism impacts on flavor, (survival of industrial processes, ctionality and survival as well as owth or storage in product owth. ormats, persistence in gut environment . Modified carbohydrate levels or functional properties. Altered metabolic properties. Altered probiotic attributes. Organisms or materials with improved health properties (including immunoregulatory, aticancer, gut health) improved fermentation properties r other industrially useful processes. 66 160 roduction of desirable flavors. Homologue of fox3, encoding a 3 Modified flavor, aroma and/or oxoacyl-coenzyme A thiolase (EC WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID ID NO: NO: Utility Description DNA Polypeptide exture attributes. .3.1.16) also called acetyl-CoA C Construction of genetic vectors acyltransferase, which participates in or controlled expression of RNA e beta-oxidation of fatty acids. d/or protein, fusion protein xpression of the FOX3 gene can be roduction, genetic modification, induced by oleate and repressed by utagenesis amplification of glucose (Einerhand et al., Mol. Cell. enetic material or for other Biol. 15:3405-3414, 1995). Free fatty enetic or protein manipulations. cids are important in providing tered survival characteristics: avor-bearing compounds for dairy survival of industrial processes, roducts such as cheese, and have a owth or storage in product significant role in both flavor and ormats, persistence in gut exture. Used extensively in wide environment. ange of convenience foods. Short tered metabolic properties. hain fatty acids are known to have a odified lipid, glycolipid or free ariety of health impacts. atty acid levels or functional roperties. odified production of short chain fatty acids. tered lipid metabolism. tered probiotic attributes. Organisms or materials with proved health properties (including immunoregulatory, ticancer, gut health). proved fermentation properties r other industrially useful rocesses. 67 161 Construction of genetic vectors Eomologue of YchH, encoding an for controlled expression of RNA acetyltransferase. Transfer of acetyl d/or protein, fusion protein oups are important in regulation of reduction, genetic modification, metabolic pathways was well as co utagenesis amplification of actor production and can influence genetic material or for other avor and/or functionality. genetic or protein manipulations. roduction of desirable flavors. Modified flavor, aroma and/or texture attributes. altered survival characteristics: survival of industrial processes, owth or storage in product ormats, persistence in gut environment. tered viability in response to tress conditions. tered metabolic properties or WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID ID NO: NO: Utility Description DNA Polypeptide regulation of metabolic pathways. tered probiotic attributes. 68 162 Production of desirable flavors. Homologue of SC6F7, encoding a odified flavor, aroma and/or ipase. Lipases are involved in the exture attributes. breakdown of triglycerides, Construction of genetic vectors metabolism, growth, production of or controlled expression of RNA avor compounds, and the release of d/or protein, fusion protein free fatty acids. Can also catalyze roduction, genetic modification, esterification of glycerol to form utagenesis amplification of mono, di- and triglycerides. Free fatty enetic material or for other acids are important in providing enetic or protein manipulations. favor-bearing compounds for dairy tered survival characteristics: products such as cheese, and have a urvival of industrial processes, significant role in both flavor and owth or storage in product texture. Used extensively in wide ormats, persistence in gut age of convenience foods. Short nvironment. ha atty acids are known to have a tered metabolic properties. variety of health impacts. Modified lipid, glycolipid or free fatty acid levels or functional properties. Modified production of short hai n fatty acids. Altered lipid metabolism. Altered probiotic attributes. Organisms or materials with improved health properties (including immunoregulatory, anticancer, gut health). proved fermentation properties r other industrially useful rocesses. 69 163 Altered cell wall or cell surface Homologue of yohH, a characteristics, structures or ipopolysaccharide synthesis protein. functions. YohI is involved in techoic acid Modified adhesion to human or synthesis, and important for cell wall animal cells or cell lines. actions including adhesion, Production of desirable flavors. immune cell interaction, product Modified flavor, aroma and/or texture. texture attributes. Construction of genetic vectors or controlled expression of RNA and/or protein, fusion protein production, gerietic modification, WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID ID NO: NO: Utility Description DNA Polypeptide utagenesis amplification of enetic material or for other enetic or protein manipulations. tered survival characteristics: survival of industrial processes, owth or storage in product ormats, persistence in gut environment. Watered metabolic properties. Watered probiotic attributes. Modified health properties (including immnunoregulatory, aticancer, gut health). Modified antibiotic resistance. proved antimicrobial properties. Proved fermentation properties or other industrially useful processes. 69 164 Atered cell wall or cell surface ooou fyhecdn characteristics, structures or iooyacaieboytei fctions.roenYoJiinovdntchc Modified adhesion to human or cisytesadmprntfrel animal cells or cell lines,.l ucin nldn dein Production of desirable flavors. uecl neato n rdc Modified flavor, aroma and/or etu. Constrlogionoffyoeneticcodctors oripopolysaccharideessosynthesis proteiproteins fusiond proechoi acidgsyneesisandplmortantiorncel walltfuncatesrinclurigfodhotien immuneccellpinteractionipndatroduc tered urvivl chaacterstics teuralo atinutril. ese, Constrctorofgeinetiovector oamats, prstence uin protei nrocneneticmdfiain ereeticoli promnpuatins. Watered srbiovac caribtersis odrivafied stri process,. (incudin irmstorgulatoroduc formatse, gu e lth). n u odtered antbiotic atreiutae. _______ Improved antimicrobial 59 WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID UD NO: NO: Utility Description DNA Polypeptide roperties. proved fermentation properties r other industrially useful rocesses. 70 165 Construction of genetic vectors omologue of pstS, encoding a or controlled expression of RNA hosphate-binding protein that is part d/or protein, fusion protein f the phosphate specific transporter roduction, genetic modification, (Pst) in bacteria. Pst is a multisubunit utagenesis amplification of ystem and belongs to the ABC enetic material or for other uperfamily of transporters (Novak et enetic or protein manipulations. 1., JBacteriol. 181:1126-1133, roduction of desirable flavors. 1999). Intracellular phosphate levels odified flavor, aroma and/or inuence survival of bacteria in exture attributes. environmental stress conditions, and tered survival characteristics: e involved in the stringent response. urvival of industrial processes, owth or storage in product ormats, persistence in gut nvironment. tered viability in response to tress conditions. Altered metabolic properties or regulation of metabolic pathways. Altered probiotic attributes. 71 166 Altered cell wall or cell surface omologue of oppA, encoding an characteristics, structures or tegral membrane protein of the functions. ligopeptide tansportsystem (Opp) roduction of bioactive or f Lactococcus lactis. Opp proteins ctional polypeptides. d the proteinase FrtP are important odified adhesion to human or opponents of the proteolytic system. al cells or cell lines. e Opp system belongs to the tered amino acid metabolism. uperfanily of ABC transporters and emoval of undesirable flavor consists of five proteins: the integral haracteristics. ebrane proteins OppB and OppC, roduction of desirable flavors. e ATP-binding proteins OppD and odified flavor, aroma and/or OppF, and the OppA a receptor texture attributes. protein (Detmers et al., Proc. Natl Construction of genetic vectors cad Sc.l USA 97:12487-12492, or controlled expression of RNA 000). Important for the uptake and dior protein, fusion protein supply of amino acids to bacteria, and production, genetic modification, e resultant production of flavorful utagenesis amplification of r functional amino-acid degradation genetic material or for other roducts. genetic or protein manipulations.
WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID ID NO: NO: Utility Description DNA Polypeptide Watered survival characteristics: survival of industrial processes, growth or storage in product formats, persistence in gut environment. tered metabolic properties. Altered probiotic attributes. odified health properties (including immunoregulatory, ticancer, gut health). Fnproved fermentation properties r other industrially useful rocesses. 71 167 Altered cell wall or cell surface omologue of oppB, encoding an characteristics, structures or ctions.ligopeptide transport system (Opp) reduction of bioactive or fLactococcus lactis. Opp proteins functional polypeptides. d the proteinase PAP are important Modified adhesion to human or components of the proteolytic system nial cells or cell lines. e Opp system belongs to the tered amino acid metabolism. uperfamily of ABC transporters and emoval of undesirable flavor consists of five proteins: the integral haracteristics. membrane proteins OppB and OppC, roduction of desirable flavors. e ATP-binding proteins OppD and odified flavor, aroma and/or OppF, and the OppA a receptor exture attributes. protein etmers et a., Proc. Na. Construction of genetic vectors cad . 97:12487 or controlled expression of RNA 000). Important for the uptake and l/or protein, fusion protein up y of amino acids to bacteria, and production, genetic modification, e resultant production of flavorful utagenesis amplification of or functional amino-acid degradation enetic material or for other products. enetic or protein manipulations. termed survival characteristics: survival of industrial processes, owth or storage in product formats, persistence in gut environment. tered metabolic properties. termed probiotic attributes. odified health properties (Including immunoregulatory, anticancer, gut health). proved fermentation properties r other industrially useful processes.
WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID ID NO: NO: Utility Description DNA Polypeptide 71 168 tered cell wall or cell surface omologue of OppC, encoding an characteristics, structures or teral membrane protein of the ctions. peptide transport system (Opp) roduction of bioactive or fLactococcus lactis. Opp proteins ctional polypeptides. d e roteinase PrP are important odified adhesion to human or components of the proteolytic system. aial cells or cell lines. e Opp system belongs to the tered amino acid metabolism. uperfamily of ABC transporters and emoval of undesirable flavor consists of five proteins: the integral haracteristics. membrane proteins OppB and OppC, roduction of desirable flavors. e ATP-binding proteins OppD and odified flavor, aroma and/or OppF, and the OppA a receptor exture, attributes. rotein (Detmners et al., Proc.. Nati. Construction of genetic vectors cad ScH USA 97:12487-12492, or controlled expression of RNA 000). Important for the uptake and d/or protein, fusion protein supply of ammo acids to bacteria, and production, genetic modification, e resultant production of flavorful utagenesis amplification of or functional amino-acid degradation enetic material or for other products. enetic or protein manipulations. termed survival characteristics: survival of industrial processes, owth or storage in product formats, persistence in gut nvironment. tered metabolic properties. tered probiotic attributes. odified health properties including immunoregulatory, ticancer, gut health). proved fermentation properties r other industrially useful recesses. 71 169 tered cell. wall orcell surface oologue of OppF, encoding an foaracterstics, structures or tegral membrane protein of the Mctions. ligopeptide transport system (Opp) reduction of bioactive or f Lactococcus lactis. Opp proteins ectional polypeptides. d the proteinase PrtP are important Modified adhesion to human or components of the proteolytic system. mal cel or cell. lines. e Opp system belongs to the termed o acid metabolism. uperfamily of ABC transporters and emoval of undesirable flavor onsists of five proteins: the integral characteristic. membrane proteins OppB and OppC, roduction of desirable flavors. e AT-binding proteins OppD and odified flavor, aroma and/or 0F, and the 0A a receptor WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID ID NO: NO: Utility Description DNA Polypeptide exture attributes. protein (Detmers et al., Proc. NatI. onstruction of genetic vectors cad Sci. USA 97:12487-12492, or controlled expression of RNA 2000). Important for the uptake and d/or protein, fusion protein supply of amino acids to bacteria, and roduction, genetic modification, te resultant production of flavorful utagenesis amplification of r functional amino-acid degradation enetic material or for other roducts. enetic or protein manipulations. tered survival characteristics: urvival of industrial processes, owth or storage in product ormats, persistence in gut environment. tered metabolic properties. tered probiotic attributes. odified health properties (including imnmunoregulatory, ticancer, gut health). proved fermentation properties r other industrially useful rocesses. 72 170 Removal of undesirable flavor omologue of thdF, which is characteristics. evolved in the oxidation of odified flavor, aroma, texture erivatives of the sulphur-containing ttributes. eterocycle thiophene and is induced onstruction of genetic vectors uring stationary phase. The thdF or controlled expression of RNA ene is subject to substantial d/or protein, fusion protein atabolite repression by glucose and roduction, genetic modification, its expression is also greatly utagenesis amplification of ecreased in the absence of oxygen enetic material or for other Zabel et al., Microbios. 101:89-103, enetic or protein manipulations. 2000). tered survival characteristics: (survival of industrial processes, owth or storage in product ormats, persistence in gut environment . tered metabolic properties. odified metabolism of sulphur ontaining compounds. tered probiotic attributes. rganisms or materials with mproved health properties including immunoregulatory, ticancer, gut health).
WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID ED NO: NO: Utility Description DNA Polypeptide 72 171 construction of genetic vectors omologue of gidA, encoding a or controlled expression of RNA lucose-inhibited division protein A, d/or protein, fusion protein which is involved in cell division and roduction, genetic modification, in moderating translational fidelity utagenesis amplification of (Kinscherf and Willis, J Bacteriol. enetic material or for other 184:2281-2286, 2002). Affects enetic or protein manipulations. owth and viability in different roduction of desirable flavors. owth environments. odified flavor, aroma and/or exture attributes. tered survival characteristics: survival of industrial processes, owth or storage in product ormats, persistence in gut environment. tered viability in response to ess conditions. tered metabolic properties or egulation of metabolic athways. tered probiotic attributes. 73 172 Construction of genetic vectors Homologue of poxB, encoding a or controlled expression of RNA yruvate oxidase (EC 1.2.3.3), which ad/or protein, fusion protein decarboxylates pyruvate. The enzyme reduction, genetic modification, is a flavoprotein (FAD) requiring mutagenesis amplification of tiamine diphosphate and is genetic material or for other portant for aerobic growth and genetic or protein manipulations. survival in aerobic conditions. Production of desirable flavors. Carbohydrate metabolism impacts on odified flavor, aroma and/or avor, functionality and survival as texture attributes. ell as growth. Altered survival characteristics: survival of industrial processes, growth or storage in product ormats, persistence in gut environment. Altered viability in response to stress conditions. Altered metabolic properties or regulation of metabolic pathways. tered probiotic attributes. 74 173 Altered amino acid metabolism. omologue of gltD, encoding a Removal of undesirable flavor glutamate synthase (EC 1.4.1.13), characteristics. - vhich catalyzes the reductive transfer WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID ED NO: NO: Utility Description DNA Polypeptide Production of desirable flavors. of the amide group of glutamine to Modified flavor, aroma and/or the keto position of 2-oxoglutarate to texure attributes. yield two molecules of glutamate. Construction of genetic vectors e resulting glutamine and for controlled expression of RNA lutamate serve as nitrogen donors in nd/or protein, fusion protein Oe biosynthesis of various nitrogen roduction, genetic modification, ontaining compounds. This pathway utagenesis amplification of s involved in the integration of enetic material or for other carbon and nitrogen assimilations. enetic or protein manipulations. io acid degradation products tered survival characteristics: eatly contribute to flavor or to off survival of industrial processes, avors (Rijnen et al., App. Environ owth or storage in product icrobiol. 65:4873-4880, 1999). ormats, persistence in gut environment. tered metabolic properties. tered probiotic attributes. odified health properties (including immunoregulatory, ticancer, gut health). odified antibiotic resistance. 75 174 tered amino acid metabolism. Homologue of dhfR, encoding a emoval of undesirable flavor ydrofolate reductase (EC 1.5.1.3), haracteristics. wich catalyzes the essential step for roduction of desirable flavors. e novo glycine and purine synthesis, odified flavor, aroma and/or NA precursor synthesis, and for the texture attributes. onversion of dUMP to dTMP. Construction of genetic vectors volved in folate production, which or controlled expression of RNA as major health impacts and also d/or protein, fusion protein detoxifies some chemotherapeutic roduction, genetic modification, gs and other cytotoxic utagenesis amplification of ompounds. enetic material or for other enetic or protein manipulations. tered survival characteristics: survival of industrial processes, growth or storage in product formats, persistence in gut nvironment. tered metabolic properties. increased folate production. tered probiotic attributes. odified health properties (including immunoregulatory, anticancer, gut health). Protection of intestinal cells from WO 2005/056801 PCT/NZ20031000278 SEQ SEQ ID ID NO: NO: Utility Description DNA Polypeptide . toxic compounds. Modified antibiotic resistance. proved fermentation properties r other industrially useful processes. 77 176 Altered amino acid metabolism. nomologue of trpA, encoding the Removal of undesirable flavor tryptophan synthase alpha chain (EC characteristics. 4.2.1.20). TrpA catalyzes the Production of desirable flavors. formation of indole from the cleavage Modified flavor, aroma and/or -f 3-indolyl-D-glyceraldehyde 3' exture attributes. phosphate. Seven structural genes are Construction of genetic vectors required for tryptophan biosynthesis: for controlled expression of RNA tpABCDEFG. TrpA encodes the and/or protein, fusion protein .ryptophan synthase alpha chain (EC roduction, genetic modification, 4.2.1.20) Tryptophan is important for utagenesis amplification of favor development. Amino acid enetic material or for other degradation products greatly genetic or protein manipulations. contribute to flavor or to off-flavors tered survival characteristics: 'Rijnen et a., AppL Environ. survival of industrial processes, Microbiol. 65:4873-4880, 1999). growth or storage in product formats, persistence in gut environment. Altered metabolic properties. Altered probiotic attributes. Modified health properties (including immunoregulatory, anticancer, gut health). Modified antibiotic resistance. 77 177 Altered amino acid metabolism. Homologue of trpB, encoding Removal of undesirable flavor tryptophan synthase beta chain (EC characteristics. 4.2.1.20). TrpB catalyzes the Production of desirable flavors. condensation of indole to a serine Modified flavor, aroma and/or derived amninoacrylate moiety bound exture attributes. to pyridoxal phosphate. Seven onstruction of genetic vectors structural genes are required for or controlled expression of RNA typtophan biosynthesis: d/or protein, fusion protein trpABCDEFG. TrpB encodes the roduction, genetic modification, typtophan synthase beta chain (EC utagenesis amplification of 4.2.1.20). Tryptophan is important for genetic material or for other flavorr development. Amino acid genetic or protein manipulations. degradation products greatly tered survival characteristics: contribute to flavor or to off-flavors urvival of industrial processes, (Rijnen et a, AppL. Environ. growth or storage in product kicrobiol. 65:4873-4880, 1999).
WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID ID NO: NO: Utility Description DNA Polypeptide formats, persistence in gut environment. Altered metabolic properties. Altered probiotic attributes. Modified health properties (including immunoregulatory, ticancer, gut health). Modified antibiotic resistance. 77 178 Altered amino acid metabolism. Homologue of trpC, encoding the Removal of undesirable flavor bifunctional enzyme characteristics. phosphoribosylanthranilate isomerase Production of desirable flavors. (EC 5.3.1.24) -indoleglycerol Modified flavor, aroma and/or phosphate synthetase (EC 4.1.1.48). texture attributes. TrpC catalyzes the ring closure of 1 Construction of genetic vectors (2-carboxyphenylamino)-1 for controlled expression of RNA deoxyribulose 5-phosphate to' d/or protein, fusion protein ndoleglycerol phosphate, the fifth production, genetic modification, tep in the pathway of tryptophan mutagenesis amplification of biosynthesis from chorismate. Seven genetic material or for other structural genes are required for enetic or protein manipulations. tryptophan biosynthesis: Altered survival characteristics: trpABCDEFG. Tryptophan is survival of industrial processes, portent for flavor development. growth or storage in product o acid degradation products formats, persistence in gut neatly contribute to flavor or to off environment. favors (Rijnen et aL, App. Environ. Altered metabolic properties. icrobioL 65:4873-4880, 1999). Altered probiotic attributes. Modified health properties (including immunoregulatory, anticancer, gut health). Modified antibiotic resistance. 77 179 Altered amino acid metabolism. omologue of trpD, encoding Removal of undesirable flavor hosphoribosyl anthranilate characteristics. ansferase (BC 2.4.2.18),. TrpD Production of desirable flavors. analyzes the reaction N-(5-phospho Modified flavor, aroma and/or -ribosyl)-anthranilate + diphosphate texture attributes. = anthranilate + 5-phospho-a-D Construction of genetic vectors ribose 1-diphosphate. Seven for controlled expression of RNA structural genes are required for and/or protein, fusion protein tryptophan biosynthesis: production, genetic modification, trpABCDEFG. Tryptophan is mutagenesis amplification of important for flavor development. enetic material or for other Aio acid degradation products genetic or protein manipulations. E y contribute to flavor or to off 67 WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID D NO: NO: Utility Description DNA Polypeptide Altered survival characteristics: avors(Pijneneta.,App.Environ. survival of industrial processes, crobiol 65:4873-4880, 1999). owth or storage in product ormats, persistence in gut environment. tered metabolic properties. tered probiotic attributes. odified health properties (including immunoregulatory, ticancer, gut health). odified antibiotic resistance. 77 180 Altered am-ino acid metabolism. Hoolge of trpF, encoding Removalo undeial flvo phshrbsylanthranilate isomerase characteristics. (EC 5.31.2n4). TrpF catalyzes the Production of desirable flavors. con version of N-(5' Modified flavor, aroma and/or dipbosphoribosyl)anthranilate to 1 exture attributes. (o-carboxyphenylamino)-1 Construction of genetic vectors eoxyribulose 5 phosphate. Seven for controlled expression of RNA tructural genes are required for d/or protein, fusion protein tophan biosynthesis: reduction, genetic modification, ABCDEFG. Tryptophan is utagenesis amplification of portant for flavor development. enetic material or for other Aio acid degradation products enetic or protein manipulations. eatly contribute to flavor or to off ltered survival characteristics: flavors (Rijnen et al., Appl. Environ. survival of industrial processes, Microbiol. 65:4873-4880, 1999). owth or storage in product ormats, persistence in gut environment. tered metabolic properties. Itered probiotic attributes. odified health properties (including immunoregulatory, ticancer, gut health). odified antibiotic resistance. 78 181 Construction of genetic vectors Homologue of purine nucleoside or controlled expression of RNA hosphorylase (PNP) (EC 2.4.2.1). and/or protein, fusion protein NP catalyzes the reversible production, genetic modification, hosphorolysis of (2'-deoxy)purine utagenesis amplification of rbonucleosides to free base and (2' enetic material or for other eoxy)ribose- I-phosphate and has a enetic or protein manipulations. etabolic role in purine salvage. roduction of desirable flavors. tracellular phosphate levels odified flavor, aroma and/or uence survival of bacteria in exture attributes. nvironmental stress conditions, and 68 WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID ED NO: NO: Utility Description DNA Polypeptide Altered survival characteristics: e involved in the stringent response. survival of industrial processes, growth or storage in product formats, persistence in gut environment. Altered viability in response to tress conditions. Altered metabolic properties or regulation of metabolic pathways. Altered probiotic attributes. 79 182 Construction of genetic vectors Homologue to relA. RelA plays a role for controlled expression of RNA in synthesis and degradation of the and/or protein, fusion protein highly phosphorylated guanosine production, genetic modification, nucleotides (p)ppGp. Intracellular mutagenesis amplification of phosphate levels influence survival of genetic material or for other bacteria in environmental stress genetic or protein manipulations. conditions, and are involved in the Production of desirable flavors. stringent response. Modified flavor, aroma and/or texture attributes. Altered survival characteristics: survival of industrial processes, growth or storage in product formats, persistence in gut environment. Altered viability in response to tress conditions. Altered metabolic properties or regulation of metabolic pathways. Altered probiotic attributes. 80 183 Altered cell wall or cell surface omologue of lysostaphin, an characteristics, structures or timicrobial immunity factor of functions. taphylococcus simulans biovar Improved antimicrobial taphylolyticus active against roperties staphylococcus areas. Lysostaphin Modified adhesion to human or s currently being investigated for use animal cells or cell lines. against mastitis in dairy cattle, caused Production of desirable flavors. y Staphylococcus aureus (Kerr et Modified flavor, aroma and/or a., Nat. Biotechnol. 19:66-70, 2001). texture attributes. THe gene contains the conserved Construction of genetic vectors sotif GPh LF, which is also present or controlled expression of RNA n several secreted peptidases. fdlor protein, fusion protein sostalbin has utility as an 69 WO 2005/056801 PCT/NZ2003/000278 SEQ SEQ ID ED NO: NO: Utility Description DNA Polypeptide I reduction, genetic modification, atimicrobial for human and utagenesis amplification of veterinary use. genetic material or for other enetic or protein manipulations. Altered survival characteristics: survival of industrial processes, owth or storage in product ormats, persistence in gut environment. tered metabolic properties. Altered probiotic attributes. Modified health properties (including immunoregulatory, ticancer, gut health). Modified antibiotic resistance. proved fermentation properties or other industrially useful recesses. Isolated polynucleotides of the present invention include the polynucleotides identified herein as SEQ ID NOS: 1-80; isolated polynucleotides comprising a polynucleotide sequence 5 selected from the group consisting of SEQ ID NOS: 1-80; isolated polynucleotides comprising at least a specified number of contiguous residues (x-mers) of any of the polynucleotides identified as SEQ ID NOS: 1-80; isolated polynucleotides comprising a polynucleotide sequence that is complementary to any of the above polynucleotides; isolated polynucleotides comprising a polynucleotide sequence that is a reverse sequence or a reverse complement of 10 any of the above polynucleotides; antisense sequences corresponding to any of the above polynucleotides; and variants of any of the above polynucleotides, as that term is described in this specification. The word "polynucleotide(s)," as used herein, means a single or double stranded polymer of deoxyribonucleotide or ribonucleotide bases and includes DNA and corresponding 15 RNA molecules, including mRNA molecules, both sense and antisense strands of DNA and RNA molecules, and comprehends cDNA, genomic DNA and recombinant DNA, as well as wholly or partially synthesized polynucleotides. A polynucleotide of the present invention may be an entire gene, or any portion thereof. A gene is a DNA sequence which codes for a functional protein or RNA molecule. Operable antisense polynucleotides may comprise a 70 WO 2005/056801 PCT/NZ2003/000278 fragment of the corresponding polynucleotide, and the definition of "polynucleotide" therefore includes all operable antisense fragments. Antisense polynucleotides and techniques involving antisense polynucleotides are well known in the art and are described, for example, in Robinson-Benion, et al., "Antisense techniques," Methods in Enzymol. 254(23): 363-375, 5 1995; and Kawasaki, et al., Artific. Organs 20 (8): 836-848, 1996. The definitions of the terms "complement," "reverse complement," and "reverse sequence," as used herein, are best illustrated by the following examples. For the sequence 5' AGGACC 3', the complement, reverse complement, and reverse sequences are as follows: complement 3' TCCTGG 5' 10 reverse complement 3' GGTCCT 5' reverse sequence 5' CCAGGA 3' Identification of genomic DNA and heterologous species DNA can be accomplished by standard DNA/DNA hybridization techniques, under appropriately stringent conditions, using all or part of a DNA sequence as a probe to screen an appropriate library. Alternatively, 15 PCR techniques using oligonucleotide primers that are designed based on known DNA and protein sequences can be used to amplify and identify other identical or similar DNA sequences. Synthetic DNA corresponding to the identified sequences or variants thereof may be produced by conventional synthesis methods. All of the polynucleotides described herein are isolated and purified, as those terms are commonly used in the art. 20 The polynucleotides identified as SEQ ID NOS: 1-80 contain open reading frames ("ORFs"), or partial open reading frames, encoding polypeptides. Additionally, polynucleotides identified as SEQ ID NOS: 1-80 may contain non-coding sequences such as promoters and terminators that may be useful as control elements. Additionally, open reading frames encoding polypeptides may be identified in extended or full-length sequences 25 corresponding to the sequences set out as SEQ ID NOS: 81-183. Open reading frames may be identified using techniques that are well known in the art. These techniques include, for example, analysis for the location of known start and stop codons, most likely reading frame identification based on codon frequencies, similarity to known bacterial expressed genes, etc. Tools and software suitable for ORF analysis include GeneWise (The Sanger Center, 30 Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 ISA, United Kingdom), Diogenes (Computational Biology Centers, University of Minnesota, Academic Health Center, UMHG Box 43 Minneapolis MN 55455), and GRAIL (Informatics Group, Oak Ridge National Laboratories, Oak Ridge, Tennessee, TN). Open reading frames and portions of open 71 WO 2005/056801 PCT/NZ20031000278 reading frames may be identified in the polynucleotides of the present invention. Once a partial open reading frame is identified, the polynucleotide may be extended in the area of the partial open reading frame using techniques that are well known in the art until the polynucleotide for the full open reading frame is identified. Thus, polynucleotides and open 5 reading frames encoding polypeptides may be identified using the polynucleotides of the present invention. Once open reading frames are identified in the polynucleotides of the present invention, the open reading frames may be isolated and/or synthesized. Expressible genetic constructs comprising the open reading frames and suitable promoters, initiators, terminators, 10 etc., which are well known in the art, may then be constructed. Such genetic constructs may be introduced into a host cell to express the polypeptide encoded by the open reading frame. Suitable host cells may include various prokaryotic and eukaryotic cells. In vitro expression of polypeptides is also possible, as well known in the art. As used herein, the term "oligonucleotide" refers to a relatively short segment of a 15 polynucleotide sequence, generally comprising- between 6 and 60 nucleotides, and comprehends both probes for use in hybridization assays and primers for use in the amplification of DNA by polymerase chain reaction. As used herein, the term "x-mer," with reference to a specific value of "x," refers to a polynucleotide comprising at least a specified number ("x") of contiguous residues of any of 20 the polynucleotides identified as SEQ ID NOS: 1-80. The value of x may be from about 20 to about 600, depending upon the specific sequence. In another aspect, the present invention provides isolated polypeptides encoded, or partially encoded, by the above polynucleotides. In specific embodiments, such polypeptides comprise a sequence selected from the group consisting of SEQ ID NO: 81-183, and variants 25 thereof. As used herein, the term "polypeptide" encompasses amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds. The term "polypeptide encoded by a polynucleotide" as used herein, includes polypeptides encoded by a polynucleotide which comprises an isolated polynucleotide sequence or variant provided herein. Polypeptides of the present invention may be naturally 30 purified products, or may be produced partially or wholly using recombinant techniques. Such polypeptides may be glycosylated with bacterial, fungal, mammalian or other eukaryotic carbohydrates or may be non-glycosylated. 72 WO 2005/056801 PCT/NZ2003/000278 Polypeptides of the present invention may be produced recombinantly by inserting a polynucleotide that encodes the polypeptide into an expression vector and expressing the polypeptide in an appropriate host. Any of a variety of expression vectors known to those of ordinary skill in the art may be employed. Expression may be achieved in any appropriate 5 host cell that has been transformed or transfected with an expression vector containing a polypeptide encoding a recombinant polypeptide. Suitable host cells include prokaryotes, yeast and higher eukaryotic cells. Preferably, the host cells employed are Escherichia coli, Lactococcus lactis, Lactobacillus, insect, yeast or a mammalian cell line such as COS or CHO. The polynucleotide(s) expressed in this manner may encode naturally occurring polypeptides, 10 portions of naturally occurring polypeptides, or other variants thereof. In a related aspect, polypeptides are provided that comprise at least a functional portion of a polypeptide having an amino acid sequence encoded by a polynucleotide of the present invention. As used herein, a "functional portion" of a polypeptide is that portion which contains the active site essential for affecting the function of the polypeptide, for example, the 15 portion of the molecule that is capable of binding one or more reactants. The active site may be made up of separate portions present on one or more polypeptide chains and will generally exhibit high binding affinity. Functional portions of a polypeptide may be identified by first preparing fragments of the polypeptide by either chemical or enzymatic digestion of the polypeptide, or by mutation 20 analysis of the -polynucleotide that encodes the polypeptide and subsequent expression of the resulting mutant polypeptides. The polypeptide fragments or mutant polypeptides are then tested to determine which portions retain biological activity, using, for example, the representative assays provided below. Portions and other variants of the inventive polypeptides may be generated by 25 synthetic or recombinant means. Synthetic polypeptides having fewer than about 100 amino acids, and generally fewer than about 50 amino acids, may be generated using techniques that are well known to those of ordinary skill in the art. For example, such polypeptides may be synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a 30 growing amino acid chain (See Merrifield, J Am. Chem. Soc. 85:2149-2154, 1963). Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer/Applied Biosystems, Inc. (Foster City, CA), and may be operated according to the manufacturer's instructions. Variants of a native polypeptide may be prepared 73 WO 2005/056801 PCT/NZ2003/000278 using standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis (Kunkel, Proc. Nati. Acad. Sci. USA 82: 488-492, 1985). Sections of DNA sequences may also be removed using standard techniques to permit preparation of truncated polypeptides. 5 In general, the polypeptides disclosed herein are prepared in an isolated, substantially pure form. Preferably, the polypeptides are at least about 80% pure; more preferably at least about 90% pure; and most preferably at least about 99% pure. As used herein, the term "variant" comprehends polynucleotide or polypeptide sequences different from the specifically identified sequences, wherein one or more 10 nucleotides or amino acid residues is deleted, substituted, or added. Variants may be naturally occurring allelic variants, or non-naturally occurring variants: Variant polynucleotide sequences preferably exhibit at least 60%, more preferably at least 75%, more preferably yet at least 90%, and most preferably at least 95% identity to a sequence of the present invention. Variant polypeptide sequences preferably exhibit at least 60%, more preferably at least 75%, 15 more preferably yet at least 90%, and most preferably at least 95% identity to a sequence of the present invention. The percentage identity is determined by aligning the two sequences to be compared as described below, determining the number of identical residues in the aligned portion, dividing that number by the total number of residues in the inventive (queried) sequence, and multiplying the result by 100. 20 Polynucleotide and polypeptide sequences may be aligned, and the percentage of identical residues in a specified region may be determined against another polynucleotide or polypeptide, using computer algorithms that are publicly available. Two exemplary algorithms for aligning and identifying the similarity of polynucleotide sequences are the BLASTN and FASTA algorithms. Polynucleotides may also be analyzed using the BLASTX 25 algorithm, which compares the six-frame conceptual translation products of a nucleotide query sequence (both strands) against a protein sequence database. The percentage identity of polypeptide sequences may be examined using the BLASTP algorithm. The BLASTN, BLASTX and BLASTP programs are available on the NCBI anonymous FTP server and from the National Center for Biotechnology Information (NCBI), National Library of Medicine, 30 Building 38A, Room 8N805, Bethesda, MD 20894, USA. The BLASTN algorithm Version 2.0.4 [Feb-24-1998], Version 2.0.6 [Sept-16-1998] and Version 2.0.11 [Jan-20-2000], set to the parameters described below, is preferred for use in the determination of polynucleotide variants according to the present invention. The BLASTP algorithm, set to the 74 WO 2005/056801 PCT/NZ2003/000278 parameters described below, is preferred for use in the determination of polypeptide variants according to the present invention. The use of the BLAST family of algorithms, including BLASTN, BLASTP and BLASTX, is described in the publication of Altschul et al., Nucleic Acids Res. 25: 3389-3402, 1997. 5 The computer algorithm FASTA is available on the Internet and from the University of Virginia by contacting David Hudson, Vice Provost for Research, University of Virginia, P.O. Box 9025, Charlottesville, VA 22906-9025, USA. FASTA Version 2.0u4 [February 1996], set to the default parameters described in the documentation and distributed with the algorithm, may be used in the determination of variants according to the present invention. 10 The use of the FASTA algorithm is described in Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444-2448, 1988; and Pearson , Methods in Enzymol. 183: 63-98, 1990. The following running parameters are preferred for determination of alignments and similarities using BLASTN that contribute to the E values and percentage identity for polynucleotide sequences: Unix running command: blastall -p blastn -d embldb -e 10 -GO -EO 15 -r 1 -v 30 -b 30 -i queryseq -o results; the parameters are: -p Program Name [String]; -d Database [String]; -e Expectation value (E) [Real]; -G Cost to open a gap (zero invokes default behavior) [Integer]; -E Cost to extend a gap (zero invokes default behavior) [Integer]; -r Reward for a nucleotide match (BLASTN only) [Integer]; -v Number of one-line descriptions (V) [Integer]; -b Number of alignments to show (B) [Integer]; -i Query File [File 20 In]; and -o BLAST report Output File [File Out] Optional. The following running parameters are preferred for determination of alignments and similarities using BLASTP that contribute to the E values and percentage identity of polypeptide sequences: blastall -p blastp -d swissprottrembledb -e 10 -G 0 -E 0 -v 30 -b 30 -i queryseq -o results; the parameters are: -p Program Name [String]; -d Database [String]; -e 25 Expectation value (E) [Real]; -G Cost to open a gap (zero invokes default behavior) [Integer]; -E Cost to extend a gap (zero invokes default behavior) [Integer]; -v Number of one-line descriptions (v) [Integer]; -b Number of alignments to show (b) [Integer]; -I Query File [File In]; -o BLAST report Output File [File Out] Optional. The "hits" to one or more database sequences by a queried sequence produced by BLASTN, FASTA, BLASTP or a similar 30 algorithm, align and identify similar portions of sequences. The hits are arranged in order of the degree of similarity and the length of sequence overlap. Hits to a database sequence generally represent an overlap over only a fraction of the sequence length of the queried sequence. 75 WO 2005/056801 PCT/NZ20031000278 The BLASTN, FASTA, and BLASTP algorithms also produce "Expect" values for alignments. The Expect value (E) indicates the number of hits one can "expect" to see over a certain number of contiguous sequences by chance when searching a database of a certain size. The Expect value is used as a significance threshold for determining whether the hit to a 5 database, such as the preferred EMBL database, indicates true similarity. For example, an E value of 0.1 assigned to a polynucleotide hit is interpreted as meaning that in a database of the size of the EMBL database, one might expect to see 0.1 matches over the aligned portion of the sequence with a similar score simply by chance. By this criterion, the aligned and matched portions of the polynucleotide sequences then have a probability of 90% of being the 10 same. For sequences having an E value of 0.01 or less over aligned and matched portions, the probability of finding a match by chance in the EMBL database is 1% or less using the BLASTN or FASTA algorithm. According to one embodiment, "variant" polynucleotides and polypeptides, with reference to each of the polynucleotides and polypeptides of the present invention, preferably 15 comprise sequences producing an E value of 0.01 or less when compared to the polynucleotide or polypeptide of the present invention. That is, a variant polynucleotide or polypeptide is any sequence that has at least a 99% probability of being the same as the polynucleotide or polypeptide of the present invention, measured as having an E value of 0.01 or less using the BLASTN, FASTA, or BLASTP algorithms set at parameters described above. According to a 20 preferred embodiment, a variant polynucleotide is a sequence having the same number or fewer nucleic acids than a polynucleotide of the present invention that has at least a 99% probability of being the same as the polynucleotide of the present invention, measured as having an E value of 0.01 or less using the BLASTN or FASTA algorithms set at parameters described above. Similarly, according to a preferred embodiment, a variant polypeptide is a 25 sequence having the same number or fewer amino acids than a polypeptide of the present invention that has at least a 99% probability of being the same as a polypeptide of the present invention, measured as having an E value of 0.01 or less using the BLASTP algorithm set at the parameters described above. As noted above, the percentage identity is determined by aligning sequences using one 30 of the BLASTN, FASTA, or BLASTP algorithms, set at the running parameters described above, and identifying the number of identical nucleic or amino acids over the aligned portions; dividing the number of identical nucleic or amino acids by the total number of nucleic or amino acids of the polynucleotide or polypeptide sequence of the present invention; 76 WO 2005/056801 PCT/NZ2003/000278 and then multiplying by 100 to determine the percentage identity. For example, a polynucleotide of the present invention having 220 nucleic acids has a hit to a polynucleotide sequence in the EMBL database having 520 nucleic acids over a stretch of 23 nucleotides in the alignment produced by the BLASTN algorithm using the parameters described above. The 5 23 nucleotide hit includes 21 identical nucleotides, one gap and one different nucleotide. The percentage identity of the polynucleotide of the present invention to the hit in the EMBL library is thus 21/220 times 100, or 9.5%. The polynucleotide sequence in the EMBL database is thus not a variant of a polynucleotide of the present invention. In addition to having a specified percentage identity to an inventive polynucleotide or 10 polypeptide sequence, variant polynucleotides and polypeptides preferably have additional structure and/or functional features in common with the inventive polynucleotide or polypeptide. Polypeptides having a specified degree of identity to a polypeptide of the. present invention share a high degree of similarity in their primary structure and have substantially similar functional properties. In addition to sharing a high degree of similarity in their primary 15 structure to polynucleotides of the present invention, polynucleotides having a specified degree of identity to, or capable of hybridizing to an inventive polynucleotide preferably have at least one of the following features: (i) they contain an open reading frame or partial open reading frame encoding a polypeptide having substantially the same functional properties as the polypeptide encoded by the inventive polynucleotide; or (ii) they contain identifiable 20 domains in common. Alternatively, variant polynucleotides of the present invention hybridize to the polynucleotide sequences recited in SEQ ID NOS: 1-80, or complements, reverse sequences, or reverse complements of those sequences under stringent conditions. As used herein, "stringent conditions" refers to prewashing in a solution of 6X SSC, 0.2% SDS; hybridizing at 25 65 0 C, 6X SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in IX SSC, 0.1% SDS at 65'C and two washes of 30 minutes each in 0.2X SSC, 0.1% SDS at 65 0 C. The present invention also encompasses polynucleotides that differ from the disclosed sequences but that, as a consequence of the discrepancy of the genetic code, encode a polypeptide having similar enzymatic activity as a polypeptide encoded by a polynucleotide of 30 the present invention. Thus, polynucleotides comprising sequences that differ from the polynucleotide sequences recited in SEQ ID NOS: 1-80, or complements, reverse sequences, or reverse complements of those sequences as a result of conservative substitutions are encompassed within the present invention. Additionally, polynucleotides comprising 77 WO 2005/056801 PCT/NZ2003/000278 sequences that differ from the inventive polynucleotide sequences or complements, reverse complements, or reverse sequences as a result of deletions. and/or insertions totaling less than 10% of the total sequence length are also contemplated by and encompassed within the present invention. Similarly, polypeptides comprising sequences that differ from the inventive 5 polypeptide sequences as a result of amino acid substitutions, insertions, and/or deletions totaling less than 10% of the total sequence length are contemplated by and encompassed within the present invention, provided the variant polypeptide has similar activity to the inventive polypeptide. The polynucleotides of the present invention may be isolated from various libraries, or 10 may be synthesized using techniques that are well known in the art. The polynucleotides may be synthesized, for example, using automated oligonucleotide synthesizers (e.g., Beckman Oligo 1000M DNA Synthesizer) to obtain polynucleotide segments of up to 50 or more nucleic acids. A plurality of such polynucleotide segments may then be ligated using standard DNA manipulation techniques that are well known in the art of molecular biology. One 15 conventional and exemplary polynucleotide synthesis technique involves synthesis of a single stranded polynucleotide segment having, for example, 80 nucleic acids, and hybridizing that segment to a synthesized complementary 85 nucleic acid segment to produce a 5-nucleotide overhang. The next segment may then be synthesized in a similar fashion, with a 5-nucleotide overhang on the opposite strand. The "sticky" ends ensure proper ligation when the two 20 portions are hybridized. In this way, a complete polynucleotide of the present invention may be synthesized entirely in vitro. Certain of the polynucleotides identified as SEQ ID NOS: 1-80 are generally referred to as "partial" sequences, in that they may not represent the full coding portion of a gene encoding a naturally occurring polypeptide. The partial polynucleotide sequences disclosed 25 herein may be employed to obtain the corresponding full-length genes for various species and organisms by, for example, screening DNA expression libraries using hybridization probes based on the polynucleotides of the present invention, or using PCR amplification with primers based upon the polynucleotides of the present invention. In this way one can, using methods well known in the art, extend a polynucleotide of the present invention upstream and 30 downstream of the corresponding DNA, as well as identify the corresponding mRNA and genomic DNA, including the promoter and enhancer regions, of the complete gene. The present invention thus comprehends isolated polynucleotides comprising a sequence identified in SEQ ID NOS: 1-80, or a variant of one of the specified sequences, that encode a functional 78 WO 2005/056801 PCT/NZ20031000278 polypeptide, including full length genes. Such extended polynucleotides may have a length of from about 50 to about 4,000 nucleic acids or base pairs, and preferably have a length of less than about 4,000 nucleic acids or base pairs, more preferably yet a length of less than about 3,000 nucleic acids or base pairs, more preferably yet a length of less than about 2,000 nucleic 5 acids or base pairs. Under some circumstances, extended polynucleotides of the present invention may have a length of less than about 1,800 nucleic acids or base pairs, preferably less than about 1,600 nucleic acids or base pairs, more preferably less than about 1,400 nucleic acids or base pairs, more preferably yet less than about 1,200 nucleic acids or base pairs, and most preferably less than about 1,000 nucleic acids or base pairs. 10 Polynucleotides of the present invention comprehend polynucleotides comprising at least a specified number of contiguous residues (x-mers) of any of the polynucleotides identified as SEQ ID NOS: 1-80 or their variants. According to preferred embodiments, the value of x is preferably at least 20, more preferably at least 40, more preferably yet at least 60, and most preferably at least 80. Thus, polynucleotides of the present invention include 15 polynucleotides comprising a 20-mer, a 40-mer, a 60-mer, an 80-mer, a 100-mer, a 120-mer, a 150-mer, a 180-mer, a 220-mer a 250-mer, or a 300-mer, 400-mer, 500-mer or 600-mer of a polynucleotide identified as SEQ IID NOS: 1-80 or a variant of one of the polynucleotides identified as SEQ ID NOS: 1-80. Oligonucleotide probes and primers complementary to and/or corresponding to SEQ 20 ID NOS: 1-80, and variants of those sequences, are also comprehended by the present invention. Such oligonucleotide probes and primers are substantially complementary to the polynucleotide of interest. An oligonucleotide probe or primer is described as "corresponding to" a polynucleotide of the present invention, including one of the sequences set out as SEQ ID NOS: 1-80 or a variant, if the oligonucleotide probe or primer, or its complement, is 25 contained within one of the sequences set out as SEQ ID NOS: 1-80 or a variant of one of the specified sequences. Two single stranded sequences are said to be substantially complementary when the nucleotides of one strand, optimally aligned and compared, with the appropriate nucleotide insertions and/or deletions, pair with at least 80%, preferably at least 90% to 95%, and more 30 preferably at least 98% to 100%, of the nucleotides of the other strand. Alternatively, substantial complementarity exists when a first DNA strand will selectively hybridize to a second DNA strand under stringent hybridization conditions. Stringent hybridization conditions for determining complementarity include salt conditions of less than about 1 M, 79 WO 2005/056801 PCT/NZ2003/000278 more usually less than about 500 mM and preferably less than about 200 mM. Hybridization temperatures can be as low as 5 0 C, but are generally greater than about 22 0 C, more preferably greater than about 30'C and most preferably greater than about 37"C. Longer DNA fragments may require higher hybridization temperatures for specific hybridization. Since the stringency 5 of hybridization may be affected by other factors such as probe composition, presence of organic solvents and extent of base mismatching, the combination of parameters is more important than the absolute measure of any one alone. DNA-DNA hybridization studies may performed using either genomic DNA or DNA derived by preparing cDNA from the RNA present in a sample to be tested. 10 In addition to DNA-DNA hybridization, DNA-RNA or RNA-RNA hybridization assays are also possible. In the first case, the mRNA from expressed genes would then be detected instead of genomic DNA or cDNA derived from mRNA of the sample. In the second case, RNA probes could be used. In addition, artificial analogs of DNA hybridizing specifically to target sequences could also be used. 15 In specific embodiments, the oligonucleotide probes and/or primers comprise at least about 6 contiguous residues, more preferably at least about 10 contiguous residues, and most preferably at least about 20 contiguous residues complementary to a polynucleotide sequence of the present invention. Probes and primers of the present invention may be from about 8 to 100 base pairs in length or, preferably from about 10 to 50 base pairs in length or, more 20 preferably from about 15 to 40 base pairs in length. The primers and probes may be readily selected using procedures well known in the art, taking into account DNA-DNA hybridization stringencies, annealing and melting temperatures, potential for formation of loops and other factors, which are well known in the art. Tools and software suitable for designing probes, and especially for designing PCR primers, are available from Premier Biosoft International, 25 3786 Corina Way, Palo Alto, CA 94303-4504. Preferred techniques for designing PCR primers are also disclosed in Dieffenbach and Dyksler, PCR primer: a laboratory manual, CSHL Press: Cold Spring Harbor, NY, 1995. A plurality of oligonucleotide probes or primers corresponding to a polynucleotide of the present invention may be provided in a kit form. Such kits generally comprise multiple 30 DNA or oligonucleotide probes, each probe being specific for a polynucleotide sequence. Kits of the present invention may comprise one or more probes or primers corresponding to a polynucleotide of the present invention, including a polynucleotide sequence identified in SEQ ID NOS: 1-80. 80 WO 2005/056801 PCT/NZ2003/000278 In one embodiment useful for high-throughput assays, the oligonucleotide probe kits of the present invention comprise multiple probes in an array format, wherein each probe is immobilized in a predefined, spatially addressable location on the surface of a solid substrate. Array formats which may be usefully employed in the present invention are disclosed, for 5 example, in U.S. Patents No. 5,412,087, 5,545,531, and PCT Publication No. WO 95/00530, the disclosures of which are hereby incorporated by reference. Oligonucleotide probes for use in the present invention may be constructed synthetically prior to immobilization on an array, using techniques well known in the art (See, for example, Gait, ed., Oligonucleotide synthesis a practical approach, IRL Press: Oxford, 10 England, 1984). Automated equipment for the synthesis of oligonucleotides is available commercially from such companies as Perkin Elmer/Applied Biosystems .Division (Foster City, CA) and may be operated according to the manufacturer's instructions. Alternatively, the probes may be constructed directly on the surface of the array using techniques taught, for example, in PCT Publication No. WO 95/00530. 15 The solid substrate and the surface thereof preferably form a rigid support and are generally formed from the same material. Examples of materials from. which the solid substrate may be constructed include polymers, plastics, resins, membranes, polysaccharides, silica or silica-based materials, carbon, metals and inorganic glasses. Synthetically prepared probes may be immobilized on the surface of the solid substrate using techniques well known 20 in the art, such as those disclosed in U.S. Patent No. 5,412,087. In one such technique, compounds having protected functional groups, such as thiols protected with photochemically removable protecting groups, are attached to the surface of the substrate. Selected regions of the surface are then irradiated with a light source, preferably a laser, to provide reactive thiol groups. This irradiation step is generally performed using a 25 mask having apertures at predefined locations using photolithographic techniques well known in the art of semiconductors. The reactive thiol groups are then incubated with the oligonucleotide probe to be immobilized. The precise conditions for incubation, such as temperature, time and pH, depend on the specific probe and can be easily determined. by one of skill in the art. The surface of the substrate is washed free of unbound probe and the 30 irradiation step is repeated using a second mask having a different pattern of apertures. The surface is subsequently incubated with a second, different, probe. Each oligonucleotide probe is typically immobilized in a discrete area of less than about I mm 2 . Preferably each discrete area is less than about 10,000 mm 2 , more preferably less than about 100 mm 2 . In this manner, 81 WO 2005/056801 PCT/NZ2003/000278 a multitude of oligonucleotide probes may be immobilized at predefined locations on the array. The resulting array may be employed to screen for differences in organisms or samples or products containing genetic material as follows. Genomic or cDNA libraries are prepared 5 using techniques well known in the art. The resulting target DNA is then labeled with a suitable marker, such as a radiolabel, chromophore, fluorophore or chemiluminescent agent, using protocols well known for those skilled in the art. A solution of the labeled target DNA is contacted with the surface of the array and incubated for a suitable period of time. The surface of the array is then washed free of unbound target DNA and the probes to 1o which the target DNA hybridized are determined by identifying those regions of the array to which the markers are attached. When the marker is a radiolabel, such as 32 P, autoradiography is employed as the detection method. In one embodiment, the marker is a fluorophore, such as fluorescein, and the location of bound target DNA is determined by means of fluorescence spectroscopy. Automated equipment for use in fluorescence scanning of oligonucleotide 15 probe arrays is available from Affymetrix, Inc. (Santa Clara, CA) and may be operated according to the manufacturer's instructions. Such equipment may be employed to determine the intensity of fluorescence at each predefined location on the array, thereby providing a measure of the amount of target DNA bound at each location. Such an assay would be able to indicate not only the absence and presence of the marker probe in the target, but also the 20 quantitative amount as well. The significance of such high-throughput screening system is apparent for applications such as microbial selection and quality control operations in which there is a need to identify large numbers of samples or products for unwanted materials, to identify microbes or samples or products containing microbial material for quarantine purposes, etc., or to ascertain the true 25 origin of samples or products containing microbes. Screening for the presence or absence of polynucleotides of the present invention used as identifiers for tagging microbes and microbial products can be valuable for later detecting the genetic composition of food, fermentation and industrial microbes or microbes in human or animal digestive system after consumption of probiotics, etc. 30 In this manner, oligonucleotide probe kits of the present invention may be employed to examine the presence/absence (or relative amounts in case of mixtures) of polynucleotides in different samples or products containing different materials rapidly and in a cost-effective 82 WO 2005/056801 PCT/NZ2003/000278 manner. Examples of microbial species which may be examined using the present invention, include lactic acid bacteria, such as Lactobacillus rhamnosus, and other microbial species. Another aspect of the present invention involves collections of a plurality of polynucleotides of the present invention. A collection of a plurality of the polynucleotides of 5 the present invention, particularly the polynucleotides identified as SEQ ID NOS: 1-80, may be recorded and/or stored on a storage medium and subsequently accessed for purposes of analysis, comparison, etc. Suitable storage media include magnetic media such as magnetic diskettes, magnetic tapes, CD-ROM storage media, optical storage media, and the like. Suitable storage media and methods for recording and storing information, as well as 10 accessing information such as polynucleotide sequences recorded on such- media, are well known in the art. The polynucleotide information stored on the storage medium is preferably computer-readable and may be used for analysis and comparison of the polynucleotide information. Another aspect of the present invention thus involves storage medium on which are 15 recorded a collection of the polynucleotides of the present invention, particularly a collection of the polynucleotides identified as SEQ ID NOS: 1-80. According to one embodiment, the storage medium includes a collection of at least 20, preferably at least 50, more preferably at least 100, and most preferably at least 200 of the polynucleotides of the present invention, preferably the polynucleotides identified as SEQ ID NOS: 1-80, including variants of those 20 polynucleotides. Another aspect of the present invention involves a combination of polynucleotides, the combination containing at least 5, preferably at least 10, more preferably at least 20, and most preferably at least 50 different polynucleotides of the present invention, including polynucleotides selected from SEQ ID NOS: 1-80, and variants of these polynucleotides. 25 In another aspect, the present invention provides genetic constructs comprising, in the 5'-3' direction, a gene promoter sequence and an open reading frame coding for at least a functional portion of a polypeptide encoded by a polynucleotide of the present invention. In certain embodiments, the genetic constructs of the present invention also comprise a gene termination sequence. The open reading frame may be oriented in either a sense or antisense 30 direction. Genetic constructs comprising a non-coding region of a gene coding for a polypeptide encoded by an inventive polynucleotide or a nucleotide sequence complementary to a non-coding region, together with a gene promoter sequence, are also provided. A terminator sequence may form part of this construct. Preferably, the gene promoter and 83 WO 2005/056801 PCT/NZ2003/000278 termination sequences are fictional in a host organism. More preferably, the gene promoter and termination sequences are common to those of the polynucleotide being introduced. The genetic construct may further include a marker for the identification of transformed cells. Techniques for operatively linking the components of the genetic constructs are well 5 known in the art and include the use of synthetic linkers containing one or more restriction endonuclease sites as described, for example, by Sambrook et al., in Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratories Press: Cold Spring Harbor, NY, 1989. The genetic constructs of the present invention may be linked to a vector having at least one replication system, for example, E. coli, whereby after each manipulation, the resulting 10 construct can be cloned and sequenced and the correctness of the manipulation determined. Transgenic microbial cells comprising the genetic constructs of the present invention are also provided by the present invention, together with microbes comprising such transgenic cells, products and progeny of such microbes, and materials including such microbes. Techniques for stably incorporating genetic constructs into the genome of target microbes, 15 such as Lactobacillus species, Lactococcus lactis or E coli, are well known in the art of bacterial transformation and are exemplified by the transformation of E coli for sequencing described in Example 1. Transgenic non-microbial cells comprising the genetic constructs of the present invention are also provided, together with organisms comprising such transgenic cells, and 20 products and progeny of such organisms. Genetic constructs of the present invention may be stably incorporated into the genomes of non-microbial target organisms, such as fungi, using techniques well known in the art. In preferred embodiments, the genetic constructs of the present invention are employed to transform microbes used in the production of food products, ingredients, processing aids, 25 additives or supplements and for the production of microbial products for pharmaceutical uses, particularly for modulating immune system function and immunological effects, and in the production of chemoprotectants providing beneficial effects, probiotics and health supplements. The inventive genetic constructs may also be employed to transform bacteria that are used to produce enzymes or substances such as polysaccharides, flavor compounds and 30 bioactive substances, and to enhance resistance to industrial processes such as drying and to adverse stimuli in the human digestive system. The genes involved in antibiotic production, and phage uptake and resistance in Lactobacillus rhamnosus are considered to be especially useful. The target microbe to be used for transformation with one or more polynucleotides or 84 WO 2005/056801 PCT/NZ2003/000278 genetic constructs of the present invention is preferably selected from the group consisting of bacterial genera Lactococcus, Lactobacillus, Streptococcus, Oenococcus, Lactosphaera, Trichococcus, Pediococcus and others potentially useful in various fermentation industries and is most preferably selected from the group consisting of the following Lactobacillus species: 5 Lactobacillus acetotolerans, Lactobacillus acidophilus, Lactobacillus agilis, Lactobacillus alimentarius, Lactobacillus amylolyticus, Lactobacillus amylophilus, Lactobacillus amylovorus, Lactobacillus animalis, Lactobacillus arizonae, Lactobacillus aviarius, Lactobacillus bavaricus, Lactobacillus bifermentans, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus collinoides, 10 Lactobacillus corynformis, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus farciminis, Lactobacillus fermentum, Lactobacillus fructivorans, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus graminis, Lactobacillus hamsteri, Lactobacillus helveticus, Lactobacillus helveticus subsp. jugurti, Lactobacillus 15 hetero, Lactobacillus hilgardii, Lactobacillus homohiochii, Lactobacillus japonicus, Lactobacillus johnsonii, Lactobacillus kefiri, Lactobacillus lactis, Lactobacillus leichmannii, Lactobacillus lindneri, Lactobacillus mali, Lactobacillus maltaromicus, Lactobacillus manihotivorans, Lactobacillus mucosae, Lactobacillus murinus, Lactobacillus oris, Lactobacillus panis, Lactobacillus paracasei, Lactobacillus paracasei subsp. 20 pseudoplantarum, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus ruminis, Lactobacillus sake, Lactobacillus salivarius, Lactobacillus salivarius subsp. salicinius, Lactobacillus salivarius subsp. salivarius, Lactobacillus sanfranciscensis, Lactobacillus sharpeae, Lactobacillus thermophilus, Lactobacillus vaginalis, Lactobacillus 25 vermiforme, and Lactobacillus zeae. In yet a further aspect, the present invention provides methods for modifying the concentration, composition and/or activity of a polypeptide in a host organism, such as a microbe, comprising stably incorporating a genetic construct of the present invention into the genome of the host organism by transforming the host organism with such a genetic construct. 30 The genetic constructs of the present invention may be used to transform a variety of .organisms including plants, such as monocotyledonous angiosperms (e.g., grasses, corn, grains, oat, wheat and barley); dicotyledonous angiosperms (e.g., Arabidopsis, tobacco, legumes, alfalfa, oaks, eucalyptus, maple); gymnosperms, (e.g., Scots pine (Aronen, Finnish 85 WO 2005/056801 PCT/NZ2003/000278 Forest Res. Papers, Vol. 595, 1996); white spruce (Ellis et al., Biotechnology 11:84-89, 1993); larch (Huang, et al., In Vitro Cell 27:201-207, 1991); and any kind of plant amenable to genetic engineering. Thus, in yet another aspect, transgenic plant cells comprising the genetic constructs of 5 the present invention are provided, together with plants comprising such transgenic cells, and fruits, seeds, products and progeny of such plants. Techniques for stably incorporating genetic constructs into the genome of target organisms, such as plants, are well known in the art and include Agrobacterium tumefaciens mediated introduction, electroporation, protoplast fusion, injection into reproductive organs, injection into immature embryos, high velocity projectile 10 introduction and the like. The choice of technique will depend upon the target plant to be transformed. For example, dicotyledonous plants, and certain monocots and gymnosperms, may be transformed by Agrobacterium Ti plasmid technology, as described, for example by Bevan, Nucleic Acids Res. 12:8711-8721, 1984. Targets for the introduction of the genetic constructs include tissues, such as leaf tissue, disseminated cells, protoplasts, seeds, embryos, 15 meristematic regions, cotyledons, hypocotyls, and the like. Once the cells are transformed, cells having the genetic construct incorporated in their genome are selected. Transgenic cells may then be cultured in an appropriate medium, using techniques well known in the art. In the case of protoplasts, the cell wall is allowed to reform under appropriate osmotic conditions. In the case of seeds or embryos, an appropriate 20 germination or callus initiation medium is employed. For explants, an appropriate regeneration medium is used. Regeneration of plants is well established for many species. For a review of regeneration of forest trees, see Dunstan et al., "Somatic embryogenesis in woody plants," in Thorpe, T.A., ed., In vitro embryogenesis ofplants, (Current Plant Science and Biotechnology in Agriculture), 20(12):471-540, 1995. Specific protocols for the 25 regeneration of spruce are discussed by Roberts et al. ("Somatic embryogenesis of Spruce," in Redenbaugh K., ed., Synseed: applications of synthetic seed to crop improvement, CRC Press: Ch.23:427-449, 1993). The resulting transformed plants may be reproduced sexually or asexually, using methods well known in the art, to give successive generations of transgenic plants and practically unlimited amounts of tagged plant-derived products. 30 .The polynucleotides of the present invention may be further employed as non disruptive tags for marking organisms, particularly microbes. Other organisms may, however, be tagged with the polynucleotides of the present invention, including commercially valuable plants, animals, fish, fungi and yeasts. Genetic constructs comprising polynucleotides of the 86 WO 2005/056801 PCT/NZ2003/000278 present invention may be stably introduced into an organism as heterologous, non-functional, non-disruptive tags. It is then possible to identify the origin or source of the organism at a later date by determining the presence or absence of the tag(s) in a sample of material. Detection of the tag(s) may be accomplished using a variety of conventional techniques, and 5 will generally involve the use of nucleic acid probes. Sensitivity in assaying the presence of probe can be usefully increased by using branched oligonucleotides, as described by Horn et al., Nucleic Acids Res. 25(23):4842-4849, 1997, enabling detection of as few as 50 DNA molecules in the sample. Polynucleotides of the present invention may also be used to specifically suppress gene 10 expression by methods that operate post-transcriptionally to block the synthesis of products of targeted genes, such as RNA interference (RNAi), and quelling. Briefly, traditional methods of gene suppression, employing anti-sense RNA or DNA, operate by binding to the reverse sequence of a gene of interest such that binding interferes with subsequent cellular processes and therefore blocks synthesis of the corresponding protein. RNAi also operates on a post 15 translational level and is sequence specific, but suppresses gene expression far more efficiently. Exemplary methods for controlling or modifying gene expression using RNAi are provided in WO 99/49029 and WO 99/53050. In these methods, post-transcriptional gene silencing is brought about by a sequence-specific RNA degradation process which results in the rapid degradation of transcripts of sequence-related genes. Studies have shown that 20 double-stranded RNA may act as a mediator of sequence-specific gene silencing (see, for example, Montgomery and Fire, Trends in Genetics, 14:255-258, 1998). Gene constructs that produce transcripts with self-complementary regions are particularly efficient at gene silencing. A unique feature of this post-transcriptional gene silencing pathway is that silencing is not limited to the cells where it is initiated. The gene-silencing effects may be 25 disseminated to other parts of an organism and even transmitted through the germ line to several generations. The polynucleotides of the present invention may thus be employed to generate gene silencing constructs and/or gene-specific self-complementary RNA sequences that can be delivered by conventional art-known methods to cells, such as microbial cells. Within genetic 30 constructs, sense and antisense sequences can be placed in regions flanking an intron sequence in proper splicing orientation with donor and acceptor splicing sites, such that intron sequences are removed during processing of the transcript and sense and antisense sequences, as well as splice junction sequences, bind together to form double-stranded RNA. 87 WO 2005/056801 PCT/NZ2003/000278 Alternatively, spacer sequences of various lengths may be employed to separate self complementary regions of sequence in the construct. During processing of the gene construct transcript, intron sequences are spliced-out, allowing sense and anti-sense sequences, as well as splice junction sequences, to bind forming double-stranded RNA. Select ribonucleases then 5 bind to and cleave the double-stranded RNA, thereby initiating the cascade of events leading to degradation of specific mRNA gene sequences, and silencing specific genes. Alternatively, rather than using a gene construct to express the self-complementary RNA sequences, the gene-specific double-stranded RNA segments are delivered to one or more targeted areas to be internalized into the cell cytoplasm to exert a gene silencing effect. The double-stranded RNA 10 must have sufficient homology to the targeted gene to mediate RNAi and is preferably at least 25 nucleotides in length. Preferably, the double-stranded RNA corresponds specifically to a polynucleotide of the present invention. Gene silencing RNA sequences comprising the polynucleotides of the present invention are useful for creating genetically modified organisms, such as microbes, with desired phenotypes as well as for characterizing genes (for 15 example, in high-throughput screening of sequences), and studying their functions in intact organisms. In another aspect, the present invention provides methods for using one or more of the inventive polypeptides or polynucleotides to treat disorders in a mammal, such as a human. In this aspect, the polypeptide or polynucleotide is generally present within a 20 composition, such as a pharmaceutical or immunogenic composition. Pharmaceutical compositions may comprise one or more polypeptides, each of which may contain one or more of the above sequences (or variants thereof), and a physiologically acceptable carrier. Immunogenic compositions may comprise one or more of the above polypeptides and an immunostimulant, such as an adjuvant or a liposome, into which the polypeptide is 25 incorporated. Alternatively, a composition of the present invention may contain DNA encoding one or more polypeptides described herein, such that the polypeptide is generated in situ. In such compositions, the DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, and bacterial and 30 viral expression systems. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter and terminator signal). Bacterial delivery systems involve the administration of a bacterium (such as Bacillus Calmette-Guerin) that expresses an immunogenic portion of the polypeptide on its cell surface. 88 WO 2005/056801 PCT/NZ2003/000278 In a preferred embodiment, the DNA may be introduced using a viral expression system (e.g., vaccinia or other poxvirus, retrovirus, or adenovirus), which may involve the use of a non pathogenic, or defective, replication competent virus. Techniques for incorporating DNA into such expression systems are well known in the art. The DNA may also be "naked," as 5 described, for example, in Ulmer et al., Science 259:1745-1749, 1993 and reviewed by Cohen, Science 259:1691-1692, 1993. The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells. While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of this invention, the type of carrier will vary depending 10 on the mode of administration. For parenteral administration, sach- as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, a lipid, a wax or a buffer. For oral administration, any of the above carriers or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed. Biodegradable microspheres (e.g., polylactic galactide) may 15 also be employed as carriers for the pharmaceutical compositions of this invention. Suitable biodegradable microspheres are disclosed, for example, in U.S. Patent Nos. 4,897,268 and 5,075,109. Any of a variety of adjuvants may be employed in the immunogenic compositions of the present invention to non-specifically enhance an immune response. Most adjuvants 20 contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a non-specific stimulator of immune responses, such as lipid A, Bordetella pertussis or M tuberculosis. Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Freund's Complete Adjuvant (Difco Laboratories, Detroit, MI), and Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ). Other 25 suitable adjuvants include alum, biodegradable microspheres, monophosphoryl lipid A and Quil A. Routes and frequency of administration, as well as dosage, vary from individual to individual. In general, the inventive compositions may be administered by injection (e.g., intradermal, intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration) or 30 orally. In general, the amount of polypeptide present in a dose (or produced in situ by the DNA in a dose) ranges from about 1 pg to about 100 mg per kg of host, typically from about 10 pg to about 1 mg per kg of host, and preferably from about 100 pg to about 1 ptg per kg of 89 WO 2005/056801 PCT/NZ2003/000278 host. Suitable dose sizes will vary with the size of the patient, but will typically range from about 0.1 ml to about 2 ml. The following examples are offered by way of illustration and not by way of limitation. 5 Example 1 ISOLATION AND CHARACTERIZATION OF DNA SEQUENCES FROM LACTOBACILLUS RHAMVOSUS STRAIN HNOO 10 Lactobacillus rhamnosus strain HN001 DNA libraries were constructed and screened as follows. DNA was prepared in large scale by cultivating the bacteria in 2 x 100 ml cultures with 100 ml MRS broth (Difco Laboratories, Detroit MI) and 1 ml Lactobacillus glycerol stock as inoculum, placed into 500 ml culture flasks and incubated at 37 'C for approx. 16 hours with 15 shaking (220 rpm). The cultures were centrifuged at 3500 rpm for 10 min to pellet the cells. The supernatant was removed and the cell pellet resuspended in 40 ml fresh MRS broth and transferred to clean 500 ml culture flasks. Fresh MRS broth (60 ml) was added to bring the volume back to 100 ml and flasks were incubated for a further 2 hrs at 3 7'C with shaking (220 20 rpm). The cells were pelleted by centrifugation (3500 rpm for 10 min) and supernatant removed. Cell pellets were washed twice in 20 ml buffer A (50 mM NaCl, 30 mM Tris pH 8.0, 0.5 mM EDTA). Cells were resuspended in 2.5 ml buffer B (25% sucrose (w/v), 50 mM Tris pH 8.0, 1 mM EDTA, 20 mg/ml lysozyme, 20 ig/ml mutanolysin) and incubated at 37 'C for 45 min. 25 Equal volumes of EDTA (0.25 Mv) was added to each tube and allowed to incubate at room temperature for 5 min. 20% SDS (1 ml) solution was added, mixed and incubated at 65 'C for 90 min. 50 pl Proteinase K (Gibco BRL, Gaithersburg, MD) from a stock solution of 20 mg/ml was added and tubes incubated at 65 'C for 15 min. DNA was extracted with equal volumes of phenol:chloroform:isoamylalcohol 30 (25:24:1). Tubes were centrifuged at 3500 rpm for 40 min. The aqueous phase was removed to clean sterile Oak Ridge centrifuge tubes (30 ml). Crude DNA was precipitated with an equal volume of cold isopropanol and incubated at -20 *C overnight. 90 WO 2005/056801 PCT/NZ2003/000278 After resuspension in 500 pLl TE buffer, DNase-free RNase was added to a final concentration of 100 pg/ml and incubated at 37 *C for 30 min. The incubation was extended for a further 30 min after adding 100 pl Proteinase K from a stock solution of 20 mg/ml. DNA was precipitated with ethanol after a phenol:chloroform:isoamylalcohol (25:24:1) and a 5 chloroform:isoamylalcohol (24:1) extraction and dissolved in 250 pl TE buffer. DNA was digested with Sau3AI at a concentration of 0.004 U/jig in a total volume of 1480 pl, with 996 tl DNA, 138.75 pLd loX REACT 4 buffer and 252.75 pl H 2 0. Following incubation for 1 hour at 37 'C, DNA was divided into two tubes. 31 p1l 0.5 M EDTA was added to stop the digestion and 17 pil samples were taken for agarose gel analysis. Samples 10 were put into 15 ml Falcon tubes and diluted to 3 ml for loading onto sucrose gradient tubes. Sucrose gradient size fractionation was conducted as follows. 100 ml of 50% sucrose (w/v) was made in TEN buffer (lM NaCl, 20 mM Tris pH 8.0, 5 mM EDTA) and sterile filtered. Dilutions of 5, 10, 15, 20, 25, 30, 35 and 40% sucrose were prepared and overlaid carefully in Beckman Polyallomer tubes, and kept overnight at 4'C. TEN buffer (4 ml) was 15 loaded onto the gradient, with 3 ml of DNA solution on top. The gradients were centrifuged at 26K for 18 hours at 40C in a Centricon T-2060 centrifuge using a Kontron TST 28-38 rotor. After deceleration without braking (approx. I hour), the gradients were removed and fractions collected using an auto Densi-Flow (Haake-Buchler Instruments). Agarose gel was used to analyze the fractions. The best two pairs of fractions were pooled and diluted to contain less 20 than 10% sucrose. TEN buffer (4 ml) was added and DNA precipitated with 2 volumes of 100% ice cold ethanol and an overnight incubation at -200C. DNA pellets were resuspended in 300 pl TE buffer and re-precipitated for approx. 6 hours at -20 *C after adding 1/10 volume 3 M NaOAC pH 5.2 and 2 volumes of ethanol. DNA was pelleted at top speed in a microcentrifuge for 15 min, washed with 70% ethanol and 25 pelleted again, dried and resuspended in 10 pl TE buffer. DNA was ligated into dephosphorylated BamHI-digested pBluescript SK 1T and dephosphorylated Baml--digested lambda ZAP Express using standard protocols. Packaging of the DNA was done using Gigapack III Gold packaging extract (Stratagene, La Jolla, CA) following the manufacturer's protocols. Packaged libraries were stored at 4 C. 30 Mass excision from the primary packaged phage library was done using XL1-Blue MRF' cells and ExAssist Helper Phage (Stratagene). The excised phagemids were diluted with NZY broth (Gibco BRL, Gaithersburg, MD) and plated out onto LB-kanamycin agar plates containing 5-bromo-4-chloro-3-indolyl-3-D-galactoside (X-gal) and isopropylthio-beta 91 WO 2005/056801 PCT/NZ2003/000278 galactoside (IPTG). After incubation, single colonies were picked for PCR size determination before the most suitable libraries were selected for sequencing. Of the colonies picked for DNA minipreps and subsequent sequencing, the large majority contained an insert suitable for sequencing. Positive colonies were cultured in LB 5 broth with kanamycin or ampicillin depending on the vector used, and DNA was purified by means of rapid alkaline lysis minipreps (solutions: Qiagen, Venlo, The Netherlands; clearing plates, Millipore, Bedford, MA). Agarose gels at 1% were used to screen sequencing templates for chromosomal contamination and concentration. Dye terminator sequencing reactions were prepared using a Biomek 2000 robot (Beckman Coulter, Inc., Fullerton, CA) and Hydra 96 10 (Robbins Scientific, Sunnyvale, CA) for liquid handling. DNA amplification was done in a 9700 PCR machine (Perkin Elmer/Applied Biosystems, Foster City, CA) according to the manufacturer's protocol. The sequence of the genomic DNA fragments was determined using a Perkin Elmer/Applied Biosystems Division Prism 377 sequencer. The DNA clones were sequenced 15 from the 5' and/or 3 end, and are identified as SEQ ID NOS: 1-80 disclosed herein. This example not only shows how the sequences were obtained, but also that a bacterium (E. coli) can be stably transformed with any desired DNA fragment of the present invention for permanent marking for stable inheritance. 20 BLASTNPolvnucleotide Analysis The determined DNA sequences were compared to and aligned with known sequences in the public databases. Specifically, the polynucleotides identified in SEQ ID NO: 1-80 were compared to polynucleotides in the EMBL database as of August 12, 2002, using BLASTN algorithm Version 2.0.11 [Jan-20-2000], set to the following running parameters: Unix 25 running command: blastall -p blastn -d embldb -e 10 -G 0 -E 0 -r 1 -v 30 -b 30 -i queryseq -o results. Multiple alignments of redundant sequences were used to build up reliable consensus sequences. The cDNA sequences of SEQ ID NOS: 1-32 and 34-80 were determined to have less than 60% identity, determined as described above, to sequences in the EMBL database using 30 the computer algorithm BLASTN, as described above. The cDNA sequence of SEQ ID NO: 33 was determined to have less than 90% identity, determined as described above, to sequences in the EMBL database using BLASTN, as described above. 92 WO 2005/056801 PCT/NZ2003/000278 BLASTP Amino AcidAnalysis The polypeptide sequences were compared to sequences in the SwissProt-TrEMBLE protein databases using the computer algorithm BLASTP. Comparisons of amino acid sequences provided in SEQ ID NOS: 81-183 to sequences in the SwissProt-TrEMBLE protein 5 databases (using BLASTP) were made as of August 12, 2002 using BLASTN algorithm Version 2.0.11 [Jan-20-2000], and the following Unix running command: blastall -p blastp -d swissprottrembledb -e 10 -GO -EO -v 30 -b 30 -i queryseq -o. The predicted amino acid sequences of SEQ ID NOS: 84-86, 89, 90, 92, 95, 96, 101 103, 108, 111, 114, 116, 119-122, 124, 125, 130, 134-136, 140, 146, 147, 152, 156, 159, 162, 10 164, 166, 168, 175 and 183 were determined to have less than 50% identity, determined as described above, to sequences in the SWISSPROT-TrEMBLE database using the BLASTP computer algorithm as described above. The predicted amino acid sequences of SEQ ID NOS: 81-83, 88, 91, 93, 94, 97-100, 104-107, 109, 110, 112, 113, 115, 123, 127-129, 131-133, 137, 138, 141-145, 148-151, 153-155, 157, 158, 160, 161, 163, 165, 167, 169-173 and 180-182 15 were determined to have less than 75% identity, determined as described above, to sequences in the SWISSPROT-TrEMBLE database using the computer algorithm BLASTP, as described above. The predicted amino acid sequences of SEQ ID NOS: 87, 139 and 176-179 were determined to have less than 90% identity, determined as described above, to sequences in the SWISSPROT-TrEMBLE database using the computer algorithm BLASTP, as described 20 above. The predicted amino acid sequences of SEQ ID NOS: 117, 118 and 126 were determined to have less than 98% identity, determined as described above, to sequences in the SWISSPROT-TrEMBLE database using the computer algorithm BLASTP, as described above. 25 BLASTXPolvnucleotide Analysis The isolated cDNA sequences were compared to sequences in the SwissProt TrEMBLE protein databases using the computer algorithm BLASTX. Comparisons of DNA sequences provided in SEQ ID NOS: 1-80, to sequences in the SwissProt-TrEMBLE database (using BLASTX) were made as of August 12, 2002 using BLAST algorithm Version 2.0.11 30 [Jan-20-2000], and the following Unix running command: blastall -p blastx -d swissprottrembleldb -e 10 -GO -E -v 30 -b 30 -i queryseq -o. The cDNA sequences of SEQ ID NOS: 1-14, 16-49, 52-58, 60-72, 74-78 and 80 were determined to have less than 50% identity, determined as described above, to sequences in the 93 WO 2005/056801 PCT/NZ2003/000278 SWISSPROT-TrEMBLE database using the computer algorithm BLASTX, as described above. The cDNA sequences of SEQ ID NOS: 15, 50, 51, 59, 73 and 79 were determined to have less than 75% identity, determined as described above, to sequences in the SWISSPROT TrEMBLE database using BLASTX, as described above. 5 Based on similarity to known sequences, the isolated polynucleotides of the present invention identified as SEQ ID NOS: 1-80 were putatively identified as encoding polypeptides having similarity to the polypeptides shown above in Table 1. The amino acid sequences encoded by the DNA sequences of SEQ ID NO: 1-80 are provided in SEQ ID NO: 81-183, 10 respectively. Several of the sequences provided in SEQ ID NO: 1-80 were found to be full-length and to contain open reading frames (ORFs). These full-length sequences, the location of ORFs (by nucleotide position) contained within these sequences, and the corresponding amino acid sequences are provided in Table 2 below. 15 94 WO 2005/056801 PCT/NZ2003/000278 TABLE 2 Polynucleotide Polypeptide SEQ ID NO: ORF SEQ ID NO 1 5831-7288 81 2 4395-5630 82 3 1445-2791 83 4 316-1413 84 5 1392-2444 85 6 1-1083 86 7 2881-4071 87 8 1859-3295 88 9265-1023 89 10 1160-2005 90 11 2324-3604 91 11 548-1696 92 12 1102-2358 93 12 188-1020 94 13 140-1138 95 14 5612-6413 96 15 84-2276 97 16 1130-2275 98 17 1644-2645 99 18 500-2404 100 19 110-1153 101 20 167-718 102 21 1-3669 103 22 2327-4951 104 23 522-1694 105 24 973-2928 106 25 133-1296 107 26 1938-3497 108 27 69-716 109 28 125-1054 110 29 84-3377 111 30 555-887 112 30 226-558 113 31 77-862 114 32 3135-4673 115 32 6384-7877 116 33 412-828 117 33 863-1663 118 34 1642-2682 119 35 814-2037 120 35 3510-5084 121 36 101-1222 122 37 113-760 123 38 1-477 124 95 WO 2005/056801 PCT/NZ2003/000278 Polynucleotide Polypeptide SEQ ID NO: ORF SEQ ID NO: 39 12447-13400 125 40 1186-2439 126 41 7973-8812 127 41 6950-7924 128 41 1925-2773 129 41 3916-4956 130 41 1023-1895 131 41 8822-10489 132 42 102-860 133 43 3759-4343 134 44 622-1113 135 45 1129-1722 136 46 1216-2439 137 46 2345-3835 138 47 6155-8361 139 48 550-1176 140 49 455-901 141 51 31-1179 143 52 766-2142 144 53 2336-3817 145 54 7567-9057 146 55 3713-4447 147 56 426-1625 148 57 138-851 149 58 3066-4769 150 59 31-1188 151 60 132-1328 152 61 94-534 153 62 652-1998 154 63 1033-1905 155 63 1902-2789 156 64 951-1646 157 64 1824-3227 158 65 179-1030 159 66 1244-2425 160 67 6885-7589 161 68 333-1121 162 69 2638-4251 163 69 1312-2622 164 70 1587-2441 165 71 4642-6489 166 71 7554-8516 167 71 6625-7536 168 71 8518-9469 169 72 2344-3732 170 72 3755-5674 171 96 WO 2005/056801 PCT/NZ2003/000278 Polynucleotide Polypeptide SEQ ID NO: ORF SEQ ID NO: 73 102-1874 172 74 2993-4429 173 75 1008-1499 174 76 3565-5967 175 77 759-1708 176 77 1488-2837 177 77 3295-4074 178 77 4071-5096 179 77 2692-3291 180 78 165-842 181 79 66-2291 182 80 269-1006 183 Example 2 ISOLATION AND CHARACTERIZATION OF PURINE NUCLEOSIDE PHOSPHORYLASE 5 FROM L. RHAMNOSUS STRAIN HNOOI The full-length polynucleotide sequence of the deoD purine nucleoside phosphorylase gene AQ1 from L. rhamnosus HN001 is given in SEQ ID NO: 78 and shown in Fig. I (with ATG initiation and translation stop codons boxed). The polypeptide sequence ofAQ1 is given 10 in SEQ ID NO: 181 and shown in Fig. 2. A 634 bp internal AQ1 fragment was amplified by PCR using standard laboratory protocols. The nucleotide sequences of the oligonucleotide primers are given in SEQ ID NOS: 184 and 185. The fragments were cloned into the pBEryl vector cut with SmaI. The 3.6 kb pBEryl vector was constructed using the replicon and multiple cloning site (MCS) from the 15 phagemid pBlueScript (pBS-SK+) (Stratagene, La Jolla CA, USA). The ampicillin resistance gene in pBS-SK+ was removed by digestion with Rcal (Roche, Auckland, New Zealand), and the 1,953 bp fragment containing the ColE1 origin and multiple cloning site purified and treated with Klenow enzyme (Roche) to give a blunt-ended fragment. A gene encoding resistance to erythromycin (Em) was isolated on a 1.6 kb fragment obtained after cutting 20 pVA891 (Macrina et al., Gene 25:145-50, 1983) with ClaI and HindIII and treatment with Klenow to give blunt ends. The 1.6 kb Em fragment was ligated to the 1,953 bp pBS-SK+ fragment, transformed into E. coli TG1 (Gibson TJ, Studies on the Epstein-Barr virus genome. Ph.D. Thesis, University of Cambridge, Cambridge, England, 1984), and plated on LB agar plates containing 200 .tg/ml Em. Maintenance of ca-complementation for blue/white color 97 WO 2005/056801 PCT/NZ2003/000278 selection of recombinant pBEryl clones was confirmed by growing E. coli colonies on agar plates containing IPTG and X-gal. The resulting pBEry1 construct encoding the HN001 deoD purine nucleoside phosphorylase AQ1 gene was transformed into competent HN0OI cells and grown 5 anaerobically for 48 hrs at 37 *C on MRS lactobacilli agar (Difco, Detroit MI) containing 2.5 pg/ml Em. Em-resistant HNOO 1 were checked for integration of the plasmid construct into the deoD gene by PCR using vector-specific (T3 or T7) and AQI internal fragment-specific primers. Colonies giving PCR patterns consistent with the insertional inactivation of the 10 endogenous HN001 deoD purine nucleoside phosphorylase AQI gene were assessed for increased resistance to UV irradiation. Briefly, single colonies of wild-type of AQl -knockout HNOOl strains were inoculated into 5 ml MRS, incubated aerobically overnight at 37 'C, and 0,1 ml then used to inoculate a 10 ml MRS culture. Cultures were grown to log phase (i.e. an
OD
6 oo of approximately 0.3) at 37 *C, and then 5 ml of culture taken and cells collected by 15 centrifugation. Cells were resuspended in 1 ml normal saline (0.9% NaCl), and 20 pl aliquots placed on sterile petri dishes. Petri dishes were then placed uncovered and inverted onto a standard laboratory transilluminator and exposed to UV light for 0, 20 or 30 seconds. Samples were transferred to 1 ml MRS media and grown for 2 hours at 37 *C in the dark. Following culture, samples were appropriately diluted and duplicate samples plated onto MRS plates, 20 incubated anaerobically for 48 hours at 37 *C and colonies counted. Fig. 3 shows the results of UV light exposure assay measuring relative viability in response to increasing doses of LTV light forAQ1' HNOO1 strain (*) and wild-type HN001 (=). Results indicate that the AQ1 HNOO mutant strain showed enhanced survival to exposure to UV light compared to- wild-type HNOOI. UV light exposure of 20 seconds appeared to have 25 no effect on AQ1- viability while the viability of wild-type HN001 cells had dropped to.34.7%. After 30 seconds UV light exposure, 86.9% of AQ1' cells survived compared to only 27.9% for wild type. Therefore, removal of AQ1 gene expression led to enhanced survival of UV light exposure, indicating that AQ1 encodes the HN001 deoD purine nucleoside phosphorylase. 30 Purine nucleoside phosphorylase (EC 2.4.2.1) is involved in the purine biosynthesis and salvage pathways. Its role in maintaining intracellular guanosine pools suggests that it may be involved in resistance to a number of stress conditions including UV light exposure, as 98 WO 2005/056801 PCT/NZ2003/000278 well as high salt, pH and temperature (Duwat et al, Int J Food Microbiol. 55:83-6, 2000). Applications for HNOO purine nucleoside phosphorylase AQI include: " methods of enhanced survival of industrial processes; e improved colonization of human intestinal environment; and 5 * improved survival of multiple stress conditions. Example 3 ISOLATION AND CHARACTERIZATION OF GTP PYROPHOSPHOKINASE FROML. RHAmNOSUS HN001 10 The full-length polynucleotide sequence of the relA GTP pyrophosphokinase gene AM] from L. rhamnosus HN001 is given in SEQ ID NO: 79 and shown in Fig. 4 (with ATG initiation and translation stop codons boxed). The polypeptide sequence of AMI is given in SEQ ID NO: 182 and shown in Fig, 5. 15 A 798 bp internal AM] fragment was amplified by PCR using standard laboratory protocols. The nucleotide sequences of the oligonucleotide primers are given in SEQ ID NOS: 186 and 187. The fragments were cloned into the pBEryl vector cut with SmaI, as described in Example 2. The resulting pBEryl construct encoding the HN001 relA GTP pyrophosphokinase AM1 gene was transformed into competent HN001 cells and grown 20 anaerobically for 48 hrs at 37 *C on MRS lactobacilli agar (Difco, Detroit MI) containing 2.5 ig/ml Em. Em-resistant HN001 were checked for integration of the plasmid construct into the relA gene by PCR using vector-specific (T3 or T7) and AMI internal fragment-specific primers. Colonies giving PCR patterns consistent with the insertional inactivation of the 25 endogenous HN001 relA GTP pyrophosphokinase AM] gene, were assessed for increased resistance to UV irradiation as described in Example 2. Fig. 6 shows the results of a UV light exposure assay measuring relative viability in response to increasing doses of UV light in AM1' HN001 (+) and wild-type HN001 (a) strains. Results indicate that the AM]~ HN001 mutant strain showed enhanced survival to 30 exposure to UV light compared to wild-type HN001. UV light exposure of 20 seconds appeared to have little effect on AM]- cell viability (91.7%) while the viability of wild-type HNOO cells had dropped to 34.7%. After 30 seconds UV light exposure, 61.1% ofAM1- cells survived compared to only 27.9% for wild type. Therefore, removal of AM] gene expression 99 WO 2005/056801 PCT/NZ2003/000278 led to enhanced survival of UV light exposure, indicating that AM] encodes the HN001 relA GTP pyrophosphokinase. GTP pyrophosphokinase or (EC 2.7.6.5) produces guanosine 3-diphosphate 5' triphosphate, a marker of the "stringent response", a regulatory state induced in bacteria by 5 nutrient starvation and other environmental stresses (reviewed in Chatterji and Ojha, Curr Opin Microbiol. 4:160-5, 2001). Studies have indicated that suppression of GTP pyrophosphokinase relA gene expression improved the resistance to a number of stress conditions including UV light exposure, as well as high salt, pH and temperature, in Lactococcus lactis (Duwat et al, Int J Food Microbiol. 55:83-6, 2000). Applications for 10 HN001 GTP pyrophosphokinase AM1 include: " methods of enhanced survival of industrial processes; " improved colonization of human intestinal environment; and " improved survival of Lactobacilli to multiple stress conditions. 15 SEQ ID NOS: 1-187 are set out in the attached Sequence Listing. The codes for nucleotide sequences used in the attached Sequence Listing, including the symbol "n," conform to WIPO Standard ST.25 (1998), Appendix 2, Table 1. All references cited herein, including patent references and non-patent publications, are hereby incorporated by reference in their entireties. 20 While in the foregoing specification this invention has been described in relation to certain preferred embodiments, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that-the. invention is susceptible to additional embodiments and that certain of the details described herein may be varied considerably without departing from the basic principles of the invention. 25 100

Claims (10)

1. An isolated polynucleotide comprising a sequence selected from the group consisting of: SEQ ID NO: 1-80.
2. An isolated polynucleotide comprising a sequence selected from the group consisting of: (a) complements of SEQ ID NO: 1-80; (b) reverse complements of SEQ ID NO: 1-80; and (c) reverse sequences of SEQ ID NO: 1-80.
3. An isolated polynucleotide comprising a sequence selected from the group consisting of: (a) ~ sequences having at least 75%, identity to a sequence of SEQ ID NO: 1-80; (b) sequences having at least 90% identity to a sequence of SEQ ID NO: 1-80; and (c) sequences having at least 95% identity to a sequence of SEQ ID NO: 1-80.
4. An isolated polynucleotide comprising a sequence selected from the group consisting of: (a) nucleotide sequences that are 200-mers of a sequence of SEQ ID NO: 1-80; (b) nucleotide sequences that are I 00-mers of a sequence of SEQ ID NO: 1-80; (c) nucleotide sequences that are 40-mers of a sequence of SEQ ID NO: 1-80; and (d) nucleotide sequences that are 20-mers of a sequence of SEQ ID NO: 1-80.
5. An isolated oligonucleotide probe or primer comprising at least 10 contiguous residues complementary to 10 contiguous residues of a nucleotide sequence recited in any one of claims 1-3.
6. A kit comprising a plurality of oligonucleotide probes or primers of claim 5.
7. A genetic construct comprising a polynucleotide of any one of claims 1-4.
8. A transgenic host cell comprising a genetic construct according to claim 7. 101 WO 2005/056801 PCT/NZ2003/000278
9. A genetic construct comprising, in the 5'-3' direction: (a) a gene promoter sequence; and (b) a polynucleotide sequence comprising at least one of the following: (1) a polynucleotide coding for at least a functional portion of a polypeptide of SEQ ID NO:
81-183; and (2) a polynucleotide comprising a non-coding region of a polynucleotide of any one of claims 1-3. 10. The genetic construct of claim 9, wherein the polynucleotide sequence is in a sense orientation. 11. The genetic construct of claim 9, wherein the polynucleotide sequence is in an anti-sense orientation. 12. The genetic construct of claim 9, wherein the gene promoter sequence is functional in a prokaryote or eukaryote. 13. A transgenic host cell comprising a construct of claim 9. 14. A transgenic organism comprising a transgenic host cell according to claim 13, or progeny thereof. 15. The transgenic organism of claim 14, wherein the organism is selected from the group consisting of Lactobacillus species. 16. A method for modulating the activity of a polypeptide in an organism, comprising stably incorporating into the genome of the organism a polynucleotide of any one of claims 1-3. 17. The method of claim 16, wherein the organism is a microbe. 102 WO 2005/056801 PCT/NZ2003/000278 18. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 81-183. 19. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of: (a) sequences having at least 75% identity to a sequence of SEQ ID NO: 81-183; (b) sequences having at least 90% identity to a sequence of SEQ ID NO: 81-183; and (c) sequences having at least 95% identity to a sequence of SEQ ID NO: 81-183. 20. An isolated polynucleotide that encodes a polypeptide of any one of claims 18 and 19. 21. An isolated polypeptide encoded by a polynucleotide of any one of claims 1-3. 22. A fusion protein comprising at least one polypeptide according to any one of claims 18 and 19. 23. A composition comprising a polypeptide according to any one of claims 18 and 19 and at least one component selected from the group consisting of: physiologically acceptable carriers and immunostimulants. 24. A composition comprising a polynucleotide according to any one of claims 1-3 and at least one component selected from the group consisting of: physiologically acceptable carriers and immunostimulants. 25. A composition comprising a fusion protein according to claim 22 and at least one component selected from the group consisting of: physiologically acceptable carriers and immunostimulants. 26. A method for treating a disorder in a mammal, comprising administering a composition according to claim 23. 103 WO 2005/056801 PCT/NZ20031000278 27. A method for treating a disorder in a mammal, comprising administering a composition according to claim 24. 28. A method of treating a disorder in a mammal, comprising administering a composition according to claim 25. 104
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GB9313586D0 (en) * 1993-07-01 1993-08-18 Europ Economic Community Lys-aminopeptidase pepn from lactobacillus delbrukii ssp.lactis,nucleic acids coding for it,and its use in fermentation processes
US7026463B2 (en) * 1999-08-13 2006-04-11 Matthew Glenn Polynucleotides and polypeptides, materials incorporating them and methods for using them
US6436703B1 (en) * 2000-03-31 2002-08-20 Hyseq, Inc. Nucleic acids and polypeptides
FR2807446B1 (en) * 2000-04-11 2005-05-06 Agronomique Inst Nat Rech GENES OF LACTOCOCCUS LACTIS, POLYPEPTIDES AND USES

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