CA2549710A1 - Polynucleotides and polypeptides isolated from lactobacillus rhamnosus hn001 materials incorporating them and methods for using them - Google Patents

Abstract

Novel polynucleotides isolated from Lactobacillus rhamnosus, as well as oligonucleotide probes and primers, genetic constructs comprising the polynucleotides, biological materials, including plants, microorganisms and multicellular organisms incorporating the polynucleotides, polypeptides expressed by the polynucleotides, and methods for using the polynucleotides and polypeptides are disclosed.

Description

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POLYNUCLEOTIDES AND POLYPEPTIDES ISOLATED FROM LACTOBACILL US RAAMNOSUS
HN001, MATERIALS INCORPORATING THEM AND METHODS FOR USING THEM
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 l0 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 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 Grarn 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 HN001 induced a sustained enhancement in several aspects of both natural and acquired immunity (See PCT International Publication No. WO 99110476). In addition, L.
rhamnosus HN001, 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. 8:993-1002, 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;

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 Inc, New York, Basel, Hong Kong, pp. 255-268; Lichtenstein and Goldin, 1998..In: Lactic Acid Bacteria, Salminen 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
anti. von Wright A (eds)., Marcel Dekker Inc, New York, Basel, Hong Kong, pp.
359-367;
Nousianen et al., 1998. In: Lactic Acid Bacteria, Salininen S and von Wright A
(eds)., Marcel l0 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:
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.
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-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 ftmction; positive influence on bile salt concentration; and antioxidant effects.
3o 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.

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 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.
l0 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 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). -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.
Modulation of infective endocarditis - due to fibronectin receptor-mediated platelet aggregation associated with Lactobacillus sepsis.
Prevention and treatment of Helicobacter pylori infection - due to competitive colonization and antibacterial effect.
Prevention and treatment of hepatic encephalopathy - due to inhibition and/or exclusion of urease-producing gut flora.

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 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 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.
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:
~ Anlinopeptidases - release a free amino acid from the N-terminus of a peptide chain;
2o . ~ 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 chain;
~ peptidyl-dipeptidase - release a dipeptide from the C-terminus of a peptide chain;
~ dipeptidases - release two free amino acids from a dipeptide; and ~ trzpeptidases - release a free amino acid and a dipeptide from a tripeptide.
3o 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 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 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°a edition, P Fox (ed) Chapman and Hall, London; Christensen et to 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;
muramidases; 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 metabolism are particularly important. The enzymes involved include beta-galactosidase, lactate dehydrogenase, citrate lyase; citrate permease, 2,3 butanediol dehydrogenase (acetoiil reductase), acetolactate decarboxylase, acetolactate synthase, pyruvate decarboxylase, pyruvate formate lyase, diacetyl synthase, diacetyl reductase, alcohol decarboxylase, lactate dehydrogenase, pyruvate dehydrogenase, and aldehyde dehydrogenase.
~ Lipid degradation, modification or synthesis. Enzymes involved include lipases, esterases, phospholipases, serine hydrolases, desaturases, and linoleate isomerase.
~ 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 transferase, alpha-glycosyl transferase, UDP-N-acetylglucosamine C4 epimerase and UDP-N-acetylglucosamine transferase.
~ Amino acid degradation. Enzymes include glutamate dehydrogenase, aminotransferases, 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.
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, ' 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 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.
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 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 )D NOS:
1-80 (x-meis). 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 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 to 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 HN001 induced a sustained enhancement in several aspects of both natural and acquired immunity. A
biologically pure culture of Lactobacillus rhamnosus strain HN001 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 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 polynucleotides and genes, are described as "corresponding to" a sequence identified as one of the sequences of SEQ m 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 >D NOS: 1-80 or a variant thereof.
3o 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 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 identified as SEQ lD 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 1o 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, 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 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 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 3o 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. rhamriosus, in a sample material, using 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 "bioactive" (health y 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 to 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).
3o 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 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 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 Fig. 1 shows the nucleotide sequence for L. rhamnosus strain HN001 deoD purine nucleoside phosphorylase AQI (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 AQI (SEQ ID NO: 181 ).
Fig. 3 shows the results of LTV light exposure assay measuring relative viability in response to increasing doses of LTV light forAQl' HN001 strain (~) and wild-type HN001 (~).
Results indicate that the AQI' HN001 mutant strain showed enhanced survival to exposure to W light compared to wild-type HN001.
Fig. 4 shows the nucleotide sequence for L. rhamnosus strain HN001 relA GTP
pyrophospholcinase gene AMI (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 AMI (SEQ 117 NO: 182).
Fig. 6 shows the results of LTV light exposure assay measuring relative viability in response to increasing doses of UV light inAMl' HN001 strain (~) and wild-type HN001 (~).
Results indicate that theAM1' HN001 mutant strain showed enhanced survival to exposure to UV light compared to wild-type HN001.

Detailed Description The polynucleotides disclosed herein were isolated by high throughput sequencing of DNA libraries from the lactic acid bacteria Lactobacillus rhamr~osus as described below in Example 1. Cell wall, cell surface and secreted components of lactic acid bacteria are known 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 l0 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 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 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, 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:
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.

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 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.
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, 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.
2o 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.
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.

SEQ SEQ ID
NO : NO: Utility Description DNA Pol a e tid 1, 3 81, emoval of undesirableomologue of lace that 8, 88, flavor encodes 6-haracteristics. hospho-beta-galactosidase (EC

roduction of desirable.2.1.85). Lace is part flavors. of the lactose odified flavor, aroma,etabolism, and hydrolyzes or exture attributes. hospholactose, the product of a Construction of genetichosphor-enolpyruvate-dependent vectors or controlled expressionhosphotransferase system.
of RNA It belongs d/or protein, fusion o the glycosidase family protein 1 and roduction, genetic ontributes to flavor, modification, including bitter utagenesis amplificationavor.
of enetic material or for other _ enetic or protein manipulations.

ltered 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 proved health properties ( including immunoregulatory, ticancer, gut health, lactose olerance) 2 82 roduction of bioactiveomologue of peps, encoding or an ctional polypeptides.' opeptidase (EC 3.4.11.-).
PepS

emoval of undesirableatalyzes the release flavor of free amino haracteristics. cids from peptides.

roduction of desirable' opeptidases are exopeptidases flavors.

odified flavor, aromad ubiquitous enzymes, and/or frequently e xture attributes. bserved in animals, plants and onstruction of genetic'croorganisms. They are vectors involved ' o r controlled expressionany different functions of RNA in the cell, d/or protein, fusion uch as protein maturation, protein protein roduction, genetic over, hydrolysis of regulatory modification, utagenesis amplificationeptides, nitrogen nutrition, of enetic material or odulation of ene ex ression for other etc.

SEQ SEQ ID

NO : NO: Utility Description DNA Pol a e tid enetic or protein . d, consequently, are manipulations considered tered survival characteristics:ssential enzymes. The proteolytic survival of industrialystem of lactic acid bacteria processes, is owth or storage in ssential for bacterial product growth in milk ormats, persistence ut also for the development in gut of the environment. rganoleptic properties of dairy tered metabolic propertiesroducts. Peps is involved or both in egulation of metabolicacterial growth by supplying amino athways. cids, and in the 'development of tered probiotic attributes.avor in dairy products, by Organisms or materialsydrolyzing peptides (including with bitter proved health propertieseptides) and liberating aromatic (including immunoregulatory,' o acids which are important ticancer, gut health).recursors of aroma compounds tered resistance to (Fernandez-Espla and Rul, antibiotics. Eur. J.

iochem. 263:502-510, 1999).

3 83 roduction of bioactiveomologue of PepC, encoding or ctional polypeptides.' opeptidase C (EC 3.4.22.40).

emoval of undesirableepC is also known as bleomycin flavor characteristics. ydrolase, which inactivates roduction of desirableleomycin B2 (a cytotoxic flavors.

odified flavor, aromalycometallopeptide) by and/or hydrolysis o exture attributes. carboxyamide bond of b-Construction of genetic' oalanine. It also has vectors general or controlled expression' opeptidase activity.
of RNA PepC

d/orprotein, fusion elongs to peptidase family protein C1; also roduction, genetic own as the papain family modification, of thiol utagenesis amplificationroteases and is involved of in flavor enetic material or roduction. The proteolytic for other system of enetic or protein actic acid bacteria is manipulations. essential for tered survival characteristics:acterial growth in milk but also for urvival of industriala development of the organoleptic processes, owth or storage in roperties of dairy products.
product omlats, persistence in gut nvironment.

tered metabolic properties or egulation of metabolic athways.

tered probiotic attributes.

O rganisms or materials with proved health properties (i ncluding immunoregulatory, ticancer, gut health).

tered resistance to antibiotics.

4 84 tered amino acid metabolism.omolo a of hisC, coding for~a L-SEQ SEQ ID
NO : NO: Utility Description DNA Pol a e tid emoval of undesirable'stidinol phosphate aminotransferase flavor characteristics. (EC 2.6.1.9). The HisC

roduction of desirable' otransferase is pyridoxal-5'-flavors.

odified flavor, aromahosphate (PLP)-dependent and/or and is exture attributes. ' volved in the synthesis of histidine Construction of geneticeubacteria. HisC also vectors has tyrosine or controlled expressiond phenylalanine aminotransferase of RNA

d/or protein, fusion ctivity. The biosynthesis protein of histidine roduction, genetic 's a central metabolic modification, process in utagenesis amplificationorganisms ranging from of bacteria to enetic material or east and plants.
for other enetic or protein manipulations.

ltered 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.

85 roduction of desirableomologue of mvaD coding flavors. for odified flavor, aromaevalonate pyrophosphate and/or exture attributes. ecarboxylase (EC 4.1.1.33).
MvaD

onstruction of genetics part of the mevalonate vectors ' pathway for or controlled expressiona biosynthesis of the of RNA central d/or protein, fusion soprenoid precursor, protein ' isopentenyl .

roduction, genetic 'phosphate by catalyzing modification, the utagenesis amplificationeaction of mevalonate of 5-diphosphate enetic material or 'th ATP to produce inorganic for other enetic or protein hosphate, ADP, C02 and manipulations.

tered survival characteristics:sopentenyl diphosphate, ' the building s urvival of industriallock of sterol and isoprenoid processes, owth or storage in iosynthesis.
product ormats, persistence in gut nvironment.

tered metabolic properties.

odified carbohydrate levels or ctional properties.

tered probiotic attributes.

O r anisms or materials with SEQ SEQ ID

NO : NO: Utility Description DNA Pol a e tid proved health properties (including immunoregulatory, ticancer, gut health).

6 86 - reduction of desirableomologue of protein p60, flavors. encoded edified flavor, aromay the gene termed iap.
and/or The p60 exture attributes. rotein is a major extracellular Construction of geneticroduct secreted by all vectors isolates of L.

or controlled expressiononocytogenes. This protein of RNA has dJor protein, fusion eptidoglycan hydrolase protein activity but reduction, ,genetic so influences the uptake modification, of L.

utagenesis amplificationonocytogenes by non-phagocytic of enetic material or ells. Proteins related for other to p60 are also enetic or protein ound in all other Listeria manipulations. species. It tered survival characteristics:as been shown that p60 protein is urvival of industrialong the strongest antigens processes, in owth or storage in isteriae for B- and T-cell product responses.

ormats, persistence a protein p60 belongs in gut to.the E. coli nvironment. pc / listeria p60 family.

tered metabolic properties.

tered probiotic attributes.

edified adhesion to human or 'mal cells or cell lines.

Organisms or materials with proved health properties (including immunoregulatory, ticancer, gut health) tered resistance to antibiotics.

proved antimicrobial roperties.

7 87 Construction of geneticomologue of elongation vectors factors Tu or controlled expressionEF-Tu) and 1 alpha of RNA ( (EF-1 alpha) that .

d/or protein, fusion a homologous proteins protein essential to reduction, genetic anslation in bacteria modification, and eukaryotes, utagenesis amplificationespectively. EF-Tu and of EF-lalpha enetic material or a GTPases that catalyze for other the binding enetic or protein f aminoacyl-tRNAs to manipulations. the A-site of reduction of desirablea ribosome. As they are flavors. among the edified flavor, aromalowest evolving proteins and/or known, EFs exture attributes. a used to study cellular functions tered survival characteristics:d to root the universal tree of life urvival of industrialGaucher et al. Proc.
processes, ( Natl. Acad. Sci.

owth or storage in SA 98:548-552, 2001), product U and are o rmats, persistence erefore an excellent in gut genetic tool.

e nvironment.

tered metabolic ro erties or SEQ SEQ ID

NO : NO: Utility Description DNA Pol a e tid egulation of metabolic athways.

tered probiotic attributes.

9 89 Construction of geneticomologue of ribonuclease vectors HII (EC

or controlled expression3.1.26.4), an RNAse that of RNA specifically d/or protein, fusion egrades the RNA moiety protein in roduction, genetic A/DNA hybrids. Endogenous modification, utagenesis ampl~cationase H activity plays of an essential enetic material or ole in biological effects for other mediated by _enetic or protein tisense oligonucleotides, manipulations. molecules roduction of desirableonsidered as potential flavors. agents against odified flavor, aromaectious diseases and and/or pathologies exture attributes. esulting from dysfunctional genes.

tered survival characteristics:a prokaryotic RNAse HII
is the urvival of industrialvolutionary counterpart processes, of the major owth or storage in ammalian RNase H (Frank product et al., ormats, persistence roc. Natl. Acad. Sci.
in gut USA 95:12872-environment. 12877, 1998) and is necessary for cell tered metabolic propertiesurvival.
or egulation of metabolic athways.

tered probiotic attributes.

90 roduction of desirableipase homologue. Lipases flavors. are odified flavor, aromaenzymes that catalyze and/or hydrolysis of exture attributes. atty acid ester bonds in Construction of genetic'acylglycerol (TAG) and vectors releasing or controlled expressionee fatty acids. The reaction of RNA is d/or protein, fusion eversible and therefore protein ~ the enzyme roduction, genetic can catalyze esterification modification, of glycerol utagenesis amplificationo form mono, di and triglycerides.
of enetic material or ree fatty acids are important for other in enetic or protein roviding flavor-bearing manipulations. compounds ltered survival characteristics:or dairy products such as cheese, and urvival of industrialave a significant role processes, in both flavor owth or storage in nd texture. Used. extensively product a in wide ormats, persistence ange of convenience foods.
in gut Short nvironment. hain fatty acids are known to have a tered metabolic properties.ariety of health impacts.

odified lipid, glycolipid or free atty acid levels or functional roperties.

odified production of short hain fatty acids.

tered lipid metabolism.

tered robiotic attributes.

SEQ SEQ ID

NO : NO: Utility Description DNA Pol a e tid Organisms or materials with proved health properties including immunoregulatory, ticancer, gut health).

11 91 tered amino acid metabolism.omologue of hisD, encoding emoval of undesirable'stidinol dehydrogenase flavor (HDH, EC

haracteristics. 1.1.1.23). HisD catalyzes the last two reduction of desirableteps in the biosynthesis flavors. of L-odified flavor, aroma'stidine: sequential and/or NAD-dependent exture attributes. oxidations of L-histidinol to L-Construction of genetic'stidinaldehyde and then vectors to L-or controlled expression'stidine. Because hisD
of RNA is absent in d/or protein, fusion ammals, it is a target protein for inhibition reduction, genetic part of herbicide development modification, utagenesis amplification(Barbosa et al., Proc.
of Natl. Acad. Sci.

enetic material or USA 99:1859-1864, 2002).
for other 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.

edified health properties (including immunoregulatory, ticancer, gut health).

edified antibiotic resistance.

11 92 tered amino acid metabolism.omologue of hisZ, coding for an emoval of undesirable, ' oacyl-tRNA synthetase.
flavor HisZ is haracteristics. essential component of the first reduction of desirablenzyme in histidine biosynthesis flavors. e with edified flavor, aromaTP phosphoribosyltransferase and/or exture attributes. HisG, EC 2.4.2.17) but ( lacks C onstruction of genetic' oacylation activity.
vectors HisZ is also or controlled expressionalled an ATP
of RNA c d/or protein, fusion hosphoribosyltransferase protein regulatory reduction, genetic ubunit. HisZ is required modification, s for histidine utagenesis amplificationrototrophy and directly of involved in enetic material or a transferase function.
for other Both HisG

enetic or protein d HisZ are required for manipulations. catalyzing tered survival characteristics:a ATP phosphoribosyltransferase u rvival of industrial action. Aminoacyl-tRNA
processes, e owth or storage in nthetases have an essential product y catalytic o rmats, ersistence le in rotein bios thesis, in t o but also SEQ SEQ ID
NO: NO: Utility Description DNA Polvnentide ronment. anticipate in numerous other elation of aminoctions, including regulation acid of ~bolism. ene expression and amino acid red metabolic iosynthesis via transamidation properties.

red probiotic athways (Sissler et al., attributes. Proc. Natl.

!ified health cad. Sci. USA 96:8985-8990, properties 1999).

uding immunoregulatory,ecause HisD is absent in mammals, ;ancer, gut health).'t is a target for inhibition as part of ified antibioticerbicide development (Barbosa resistance. et l., Proc. Nat1 Acad. Sci.
USA

9:1859-1864, 2002).

1293 ltered amino acid omologue of proA, coding metabolism. for a emoval of undesirablelutamate-5-semialdehyde flavor characteristics. ehydrogenase (EC 1.2.1.41).
ProA i roduction of desirableso known as gamma-flavors.

odified flavor, aromalutamylphosphate reductase, and/or and exture attributes. atalyzes the second step of proline Construction of geneticiosynthesis, the NADPH-dependent vectors or controlled expressioneduction of L-gamma-glutamyl of RNA 5-d/or protein, fusionhosphate into L-glutamate protein 5-roduction, genetic emialdehyde and phosphate.
modification, utagenesis amplificationtracellular accumulation of of the enetic material or ' o acid proline has for other been linked to enetic or protein alt tolerance and virulence manipulations. potential tered survival characteristics:f a number of bacteria.
Proline survival of industrialiosynthesis plays an processes, important role owth or storage in survival in osmolyte-depleted product ormats, persistence nvironments of elevated in gut osmolarity.

e nvironment. a survival of the food-borne tered metabolic properties.athogen L. monocytogenes in tered probiotic attributes.ypersaline environments is odified health propertiesttributed mainly to the accumulation including immunoregulatory,f organic compounds termed ticancer, gut health).smolytes. Osmolytes, often referred odified antibiotic o as compatible solutes resistance. owing to mproved antimicrobialeir compatibility with cellular roperties. etabolism at high internal 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, proline may ction as a virulence factor for ertain pathogenic bacteria (Sleator et SEQ SEQ ID
NO: NO: ~ Utility Description DNA Polvnentide ., Appl. Environ. Microbiol.
':2571-2577, 2001).
12 94 _ tered amino acid metabolism. omologue of proB, coding for emoval of undesirable flavor gamma-glutamyl kinase (EC

characteristics. .7.2.11) also known as glutamate 5-roducoon of desirable flavors.~ ase 1. ProB catalyzes the first step odified flavor, aroma and/or f proline biosynthesis, the transfer o:

exture attributes. phosphate group to .glutamate to Construction of genetic vectorsorm glutamate 5-phosphate which br 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 a salt tolerance and virulence enetic material or for other otential of a number of bacteria.

enetic or protein manipulations.roline biosynthesis plays an ltered survival characteristics:portant role in survival in urvival of industrial processes,smolyte-depleted environments of owth or storage in product levated osmolarity. The survival of brmats, persistence in gut a food-borne pathogen L.

nvironment. onocytogenes in hypersaline tered metabolic properties. nvironments is attributed mainly to tered probiotic attributes. a accumulation of organic odified health properties ompounds termed osmolytes.

(including immunoregulatory, Osmolytes, often referred to as ticancer, gut health). ompaoble solutes owing to their odified antibiotic resistance.ompaobility 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, proline may ction as a virulence factor for ertain pathogenic bacteria (Sleator et l., Appl. Environ. Microbiol.

67:2571-2577, 2001).

13 95 tered cell wall or cell surfaceomologue of vanHE or LDHD

, haracterisocs, structures or ncoding a D-lactate dehydrogenase coons. (D-LDH, EC 1.1.1.28).
D-LDH

odified adhesion to human or educes pyruvate to D-lactate and is al cells or cell lines. ' volved in bacterial cell wall roducoon of desirable flavors. fracture and function.
Vanes plays an odified flavor, aroma and/or ssenoal role in bacterial resistance to SEQ SEQ ID
NO : NO: Utility Description DNA Pol a tide exture attributes. a 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..

odified health properties (including immunoregulatory, ticancer, gut health).

odified antibiotic resistance.

proved antimicrobial roperties.

14 96 tered amino acid metabolism.omologue of metA that encodes emoval of undesirableomoserine O-transsuccinylase flavor (EC

haracteristics. .3.1.46). MetA catalyzes the first roduction of desirable'que step in bacterial flavors. and plant odified flavor, aromaethionine biosynthesis and/or involving the exture attributes. ctivation of the gamma-hydroxyl of Construction of geneticomoserine. The activity vectors of this or controlled expressionxzyme is closely regulated of RNA in vivo .

d/or protein, fusion d therefore represents protein a critical roduction, genetic ontrol point for cell modification, growth and utagenesis amplificationiability.
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 e nvironment.

tered metabolic properties.

tered probiotic attributes.

odified health properties (i ncluding immunoregulatory, ticancer gut health).

odified antibiotic resistance.

SEQ SEQ ID
D : NO: Utility Description NO Pol a DNA tide 15 97 roduction of desirableomologue of plnG, encoding flavors. an odified flavor, aromaTP binding cassette (ABC) and/or exture attributes. ansporter for the antimicrobial onstruction of geneticcompound (bacteriocin) vectors plantaricin or controlled expression. PInG displays strong of RNA similarities - d/or protein, fusion o the proposed transport protein proteins of roduction, genetic everal other bacteriocins modification, and to utagenesis amplificationroteins implicated in of the signal-enetic material or sequence-independent for other export of enetic or protein scherichia coli hemolysin, manipulations. PlnH is tered survival characteristics:'ts accessory protein (Huhne et al., urvival of industrialicrobiol. 142:1437-1448, processes, 1996).

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 antimicrobial roperties.

16 98 Construction of geneticomologue of hemN that vectors encodes an or controlled expressionxygen-independent of RNA

d/or protein, fusion oproporphyrinogen III
protein roduction, genetic dehydrogenase (EC 1.3.3.3).
modification, HemN

utagenesis amplificationatalyzes the oxidative of enetic material or ecarboxylation of for other enetic or protein oproporphyrinogen III
manipulations. c to yield roduction of desirablerotoporphyrinogen IX
flavors. and requires odified flavor, aromaADP+, ATP, Mg2+, and and/or L-exture attributes. ethionine. In association with tered survival characteristics:pecific apoproteins, it serves a wide urvival of industrialange of important functions processes, owth or storage in cluding electron transport product ' (e.g., ormats, persistence ytochromes), binding in gut and transport nvironment. f O2 (e.g., hemoglobin), and tered metabolic propertiesxidative catalysis (e.g., or peroxidases) egulation of metabolicFischer et al., J. Bacteriol.
( athways. 1 83:1300-1311, 2001).

tered probiotic attributes.

tered antimicrobial properties.

odified health properties (i ncluding immunoregulatory, a nticancer gut health .

SEQ SEQ ID

NO : NO: Utility Description DNA Poly e a tid 17 99 emoval of undesirableomologue of lacD, encoding flavor haracteristics. gatose-1,6-bisphosphate aldolase edified flavor, aroma,(EC 4.1.2.40). LacD is texture responsible ttributes. or the aldol cleavage of tagatose-1,6-Construction of geneticisphosphate to form glycerone-P
vectors and or controlled expressionlyceraldehyde 3-phosphate of RNA in the d/or protein, fusion agatose 6-phosphate pathway protein of reduction, genetic actose catabolism in modification, bacteria. The utagenesis amplificationnzyme activity is stimulated of by enetic material or ertain divalent canons.
for other enetic or protein manipulations.

tered survival characteristics:

survival of industrial processes, gowkh or storage in product ormats, persistence in gut nvironment).

edified carbohydrate levels or ctional properties.

tered metabolic properties.

edified 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 undesirableparagine synthetase [glutamine-flavor haracteristics. ydrolyzing] 2 (EC 6.3.5.4).
AsnH

tered cell wall or ansamidates asparagine, cell surface glutamate, haracteristics, structuresand diphosphate from or aspartate, ctions. lutamine and ATP as part of the reduction of desirableparagine biosynthesis flavors. pathway.

edified flavor, aromasnH gene is also involved and/or in cell exture attributes. urface organization.

C onstruction of genetic vectors or controlled expression.
of RNA

d/or protein, fusion protein reduction, generic modification, utagenesis amplification of enenc material or for other enetic or protein manipulations.

tered survival characteristics:

s urvival of industrial processes, owth or stora a in roduct SEQ SEQ ID

NO : NO: Utility Description DNA Poly a e tid ormats, persistence in gut environment.

ltered metabolic properties.

tered probiotic attributes.

odified health properties (including immunoregulatory, ticancer, gut health).

proved fermentation properties r other industrially useful rocesses.

19 101 emoval of undesirableomologue of butB, encoding flavor . 2,3-characteristics. utanediol dehydrogenase (EC

roduction of desirable1.1.1.4). ButB catalyzes flavors. the NAD+-odified flavor, aromaependent oxidation of and/or 2,3-exture attributes. utanediol acetoin, as well as the Construction of geneticcorresponding reverse vectors reactions. It or controlled expressionan also reduce diacetyl of RNA to acetoin.

dlor protein, fusion iacetyl is an important protein flavor roduction, genetic ompound in dairy products.
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.

20 102. tered cell wall or omologue of a peptidoglycan cell surface haracteristics, structuresydrolase (N-acetylmuramoyl-L-or ctions. arsine amidase). N-acetylmuramoyl-odified adhesion to -alanine amidase is an human or autolysin mimal cells or cell volved in degrading the lines. ' cell wall roduction of desirablewring cell growth or flavors. programmed odified flavor, aromaell death and is involved and/or in cell e xture attributes. owth and important for releasing C onstruction of eneticnzymes im ortant for vectors e flavor.

SEQ SEQ ID
NO : NO: Utility Description DNA Pol a tide 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.

odified health properties (including immunoregulatory, ticancer, gut health).

odified antibiotic resistance.

proved fermentation properties r other industrially useful rocesses.

21, 103, 175 tered cell wall or omologue of galC, encoding 76 cell surface haracteristics, structuresalactosylceramidase (EC
or 3.2.1.46).

ctions. GaIC hydrolyzes galactose ester odified adhesion to onds of galactosylceramide, human or al cells or cell lines.alactosylsphingosine, roduction of desirableonogalactosyldiglyceride flavors. and odified flavor, aromaactosylceramide. It is and/or involved in exture attributes. a catabolism of galactosylceramide, Construction of geneticmajor lipid in myelin, vectors lcidneyand or controlled expressionpithelial cells of small of RNA intestine and d/or protein, fusion olon. While bacteria protein may use galC

roduction, genetic o release sugars for modification, metabolism, the utagenesis amplificationy-products, including of ceramide, acct enetic material or signalling molecules for other in eukaryotic enetic or protein ells and can lead to manipulations. apoptosis or tered survival characteristics:'fferentiation. Therefore, glaC plays s urvival of industrialrole in probiotic effects processes, and survival owth or storage in the gut environment.
product ormats, persistence in gut nvironment.

tered metabolic properties.

tered probiotic attributes.

odified health properties (i ncluding immunoregulatory, a nticancer, gut health, apoptosis).

odified antibiotic resistance.

SEQ SEQ ID

NO : NO: Utility Description DNA Pol a e tid proved antimicrobial roperties.

proved fermentation s propertie r other industrially useful rocesses.

22 104 roduction of bioactiveomologue of pepN, encoding or ctional polypeptides.embrane alanyl aminopeptidase emoval of undesirable(EC 3.4.11.2), also called flavor lysyl haracteristics. ' opeptidase and aminopeptidase roduction of desirable. PepN releases the N-terminal flavors.

odified flavor, aroma' o acid, Xaa-~-Xbb- from and/or a exture attributes. eptide, amide or arylamide.

Construction of genetic~ opeptidases are involved vectors in or controlled expressionany different functions of RNA in the cell, d/or protein, fusion uch as protein maturation, protein ~ protein roduction, genetic over, hydrolysis of regulatory modification, utagenesis amplificationeptides, nitrogen nutrition, of enetic material or odulation of gene expression for other etc.

enetic or protein d, consequently, are considered manipulations.

tered survival characteristics:essential enzymes. The proteolytic survival of industrialystem of lactic acid bacteria processes, is owth or storage in ssential for bacterial product growth in milk ormats, persistence ut also for the development in gut of the environment. rganoleptic properties of dairy tered metabolic propertiesroducts. PepN is involved or both in egulation of metabolicacterial growth by supplying amino athways. cids, and in the development of tered probiotic attributes.avor in dairy products, by Organisms or materialsydrolyzing peptides (including with bitter proved health propertieseptides) and liberating aromatic ( including immunoregulatory,' o acids which are important ticancer, gut health).recursors of aroma compounds tered resistance to Fernandez-Espla and Rul, antibiotics. ( Eur. ,I.

iochem. 263:502-510, 1999).

23 105 roduction of desirableomologue of mvaB, encoding flavors. 3-odified flavor, aromaydroxy-3-methylglutaryl and/or coenzyme e xtuie attributes. synthase. MvaB catalyzes the C onstruction of geneticondensation of acetyl-CoA
vectors with or controlled expressioncetoacetyl-CoA to form of RNA 3-hydroxy-d/or protein, fusion -methylglutaryl-CoA and protein CoA.

roduction, genetic vaB is involved in mevalonic modification, acid utagenesis amplificationetabolism as well as in of biosynthesis enetic material or f cholesterol and ubiquinone for other e netic or protein manipulations.ogenitors. Terpenoids r or tered survival characteristics:o renoids constitute a 's vast family o SEQ SEQ ID

NO : NO: Utility Description DNA Pol a e tid survival of industrialrganic compounds that processes, includes owth or storage in terols and carotenoids product that have ormats, persistence avor, color, texture in gut and other nvironment. ensory impacts on food products.

tered metabolic properties.a terpenoids in many organisms odified carbohydrate hare early steps in their levels or biosynthesis, ctional properties. ' cluding the synthesis . of 3-hydroxy-tered probiotic attributes.-methylglutaryl-coenzyme A and its Organisms or materialsonversion to mevalonate.
with proved health properties (including immunoregulatory ticancer, gut health).

24 106 Construction of geneticomologue of hexB, one vectors of two or controlled expressionroteins involved in DNA
of RNA mismatch d/or protein, fusion epair. The hex mismatch protein repair roduction, genetic ystem of Streptococcuspneumoniae modification, utagenesis amplificationcts both during transformation of (a enetic material or ecombination process for other that directly enetic or protein roduces heteroduplex manipulations. DNA) to roduction of desirablecorrect donor strands flavors. and after DNA

odified flavor, aromaeplication to remove and/or exture attributes. 'sincorporated nucleotides.
The tered survival characteristics:exB is one of at least two proteins urvival of industrialequired for mismatch processes, repair. HexB is owth or storage in omologous to the mutt product protein, ormats, persistence hich is required for in gut methyl-directed nvironment. ismatch repair in Salmonella tered viability in phimurium and E. coli, response to and to the r tress conditions. MSl gene product, which is likely tered metabolic propertieso be involved in a mismatch or egulation of metabolicorrection system in Saccharomyces athways. erevisiae (Prudhomme et al., J.

tered probiotic attributes.acteriol. 171:5332-5338, 1989).

25 107 ltered amino acid omologue of araT, encoding metabolism. an emoval of undesirableomatic amino acid aminotransferase flavor haracteristics. ( EC 2.6.1.57). Aminotransferases roduction of desirableave been widely applied flavors. in the odified flavor, aromaarge-scale biosynthesis and/or of amino exture attributes. cids, which are in increasing C onstruction of geneticerrand by the pharmaceutical vectors or controlled expressiondustry. AraT plays a of RNA ' major role in d/or protein, fusion a conversion of aromatic protein amino roduction, genetic cids to aroma compounds.
modification, AraT also utagenesis amplificationas a major physiological of role in the enetic material or os thesis of henylalanine for other i and SEQ SEQ ID
NO: NO: Utility Description DNA Polwentide enetic or protein _ osine. The enzymatic manipulations. degradation .ltered survival f amino acids in cheese characteristics: plays a .irvival of industrialajor role in cheese processes, flavor ~owth or storage evelopment. Indeed, in product degradation rrmats, persistenceroducts from aromatic, in gut branched-ivironment. hair, and sulfurous amino acids ltered metabolic ave been identified properties. in various ' ltered probiotic heeses and highly contribute attributes. to their edified health propertiesaver or to off flavors (Rijnen et al., ~cluding immunoregulatory,ppl. Environ. Microbiol.
65:4873-ticancer, gut health).880, 1999).

edified antibiotic resistance.

proved fermentation properties other industrially j useful 26 108 tered cell wall or cell omologue of tagE, encoding surface haracteristics, structures or oly(glycerol-phosphate) alpha-ctions. _lucosyltransferase (EC 2.4.1.52) edified adhesion to human or so called uridine diphosphate-' al cells or cell lines. _lucose poly-(glycerol phosphate) reduction of desirable flavors. pha-glucosyl transferase.
TagE is edified flavor, aroma and/or ' volved in techoic acid synthesis.

exture attributes. echoic acid is one component of the Construction of genetic vectors 'ck peptidoglycan layers in the cell br controlled expression of RNA all of Gram-positive bacteria and is d/or protein, fusion protein usceptible to the enzyme lysozyme reduction, genetic modification,d to penicillin.

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 ltered metabolic properties.
ltered probiotic attributes.
:edified health properties icluding immunoregulatory, ticancer, gut health).
edified antibiotic resistance.
proved fermentation properties other industrially useful 27 109 tered amino acid metabolism. omologue to hisB, encoding a SEQ SEQ ID
NO : NO: Utility Description DNA Pol a e tid emoval of undesirable'stidine biosynthesis flavor bifunctional characteristics. rotein -includes: imidazoleglycerol-roduction of desirablehosphate dehydratase (EC
flavors. 4.2.1.19) odified flavor, aromad histidinol-phosphatase and/or (EC

exture attributes. .1.3.15). HisB rearranges the Construction of genetic'dazole glycerol phosphate vectors by a or controlled expressionedox-neutral dehydrative of RNA reaction to d/or protein, fusion 'dazole acetol phosphate protein and roduction, genetic atalyzes the dephosphorylation modification, of utagenesis amplification'sidinol phosphate to of histidinol, the enetic material or irect precursor of histidine.
for other Because enetic or protein 'sB is absent from mammals, manipulations. it has tered survival characteristics:ecome a target for inhibition . as part survival of industrialof herbicide development processes, (Barbosa et owth or storage in 1., Proc. Natl. Acad.
product Sci. USA

ormats, persistence 9:1859-1864, 2002). Amino in gut acid nvironment. egradation products in various tered metabolic properties.heeses have been shown to greatly tered probiotic attributes.ontribute to flavor or to off flavors odified health properties(Rijnen et al., Appl.
Environ.

including immunoregulatory,icrobiol. 65:4873-4880, 1999).

ticancer, gut health).

odifled antibiotic resistance.

28 110 tered amino acid metabolism.omologue of cysK, encoding emoval of undesirablecysteine synthase (EC
flavor 4.2.99.8), also characteristics. own as O-acetyl-L-serine acetate-roduction of desirableyase (EC 4.2.99.8). CysK
flavors. catalyzes odifled flavor, aromaa formation of L-cysteine, and/or the last exture attributes. fep of L-cysteine biosynthesis, from Construction of geneticO-acetyl-L-serine and vectors hydrogen or controlled expressionulfide. Cysteine synthase of RNA is involved d/or protein, fusion the assimilatory sulfate protein reduction roduction, genetic athway and in the selenium modification, utagenesis amplificationcorporation into proteins, of ' which enetic material or ccurs mainly as selenocysteine, for other in enetic or protein acteria. Sulphur-containing manipulations. amino tered survival characteristics:cid metabolism is important for urvival of industrialevelopment of aroma and processes, flavor owth or storage in ompounds.
product ormats, persistence in gut nvironment.

tered metabolic properties.

tered probiotic attributes.

odified health properties (i ncluding immunoregulatory, ticancer, t health .

SEQ SEQ ID

NO : NO: Utility Description DNA Pol a tide odified antibiotic resistance.

29 111 tered cell wall or omologue of enn protein.
cell surface Enn has haracteristics, structures'que Ig-binding characteristics or as it ctions. eacts preferentially with human odified adhesion to gG3, the tlpC gene. Enn human or is a al cells or cell lines.embrane protein with similarity to roduction of desirableethyl-accepting chemotaxis flavors. proteins odified flavor, aromad the streptococcal M
and/or proteins exture attributes. omologous with immunoglobulin-Construction of geneticfinding factors. The vectors M proteins have or controlled expressioneen studied because of of RNA their d/or protein, fusion tiphagocytic function.
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 antimicrobial roperties.

30 112 tered amino acid metabolism.omologue of hisE, encoding a emoval of undesirable'stidine biosynthesis flavor protein that haracteristics. lays a role in histidine biosynthesis.

roduction of desirableecause hisE is absent flavors. in mammals, odified flavor, aromat is a target for inhibition and/or ' as part of exture attributes. erbicide development (Barbosa et C onstruction of genetic1., Proc. Natl. Acad.
vectors Sci. USA

or controlled expression9:1859-1864, 2002). Amino of RNA 9 acid d/or protein, fusion egradation products in protein d various roduction, genetic heeses have been shown modification, c to greatly utagenesis amplificationontribute to flavor or of to off flavors enetic material or Rijnen et al., Appl.
for other ( Environ.

enetic or protein icrobiol. 65:4873-4880, manipulations. 1999).

tered survival characteristics:

urvival of industrial processes, owth or stora a in roduct SEQ SEQ ID

NO : NO: Utility Description DNA Poly a e tid ormats, persistence in gut nvironment.

tered metabolic properties.

tered probiotic attributes.

edified health properties (including immunoregulatory, ticancer, gut health).

edified antibiotic resistance.

30 113 tered amino acid metabolism.omologue of hisI, encoding a emoval of undesirable'stidine biosynthesis flavor protein that haracteristics. lays a role in histidine biosynthesis.

reduction of desirableecause hisI is absent flavors. in mammals, it edified flavor, aroma's a target for inhibition and/or as part of exture attributes. erbicide development (Barbosa et Construction of genetic1., Proc. Natl. Acad.
vectors Sci. USA

or controlled expression9:1859-1864, 2002). Amino of RNA acid d/or protein, fusion egradation products in protein various reduction, genetic heeses have been shown modification, to greatly utagenesis amplificationontribute to flavor or of to off flavors enetic material or (Rijnen et al., Appl.
for other Environ.

enetic or protein icrobiol. 65:4873-4880, manipulations. 1999).

tered survival characteristics:

urvival of industrial processes, owth or storage in product ormats, persistence in gut nvironment.

tered metabolic properties.

tered probiotic attributes.

edified health properties ( including immunoregulatory, ticancer, gut health).

edified antibiotic resistance.

31 114 reduction of desirableomologue of estA, encoding flavors. a edified flavor, aromaerine-dependent arylesterase and/or s (EC

exture attributes. .1.1.2). EstA hydrolyzes a variety of Construction of geneticster compounds and prefers vectors those or controlled expression'th substituted phenyl of RNA alcohol or d/or protein, fusion hort-chain fatty acid protein groups.

reduction, genetic lsesterases are responsible modification, for the utagenesis amplificationreduction of important of flavor enetic material or ompounds and intermediates.
for other enetic or protein manipulations.

tered survival characteristics:

s urvival of industrial. , processes, owth or storage in roduct SEQ SEQ ID

NO : NO: Utility Description DNA Poly a tide ormats, persistence in gut nvironment.

tered metabolic properties.

odified 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 tered amino acid metabolism.omologue of gln.A that encodes a emoval of undesirablelutamine synthetase (EC
flavor 6.3.1.2), haracteristics. so called glutamate-ammonia roduction of desirableigase. GInA catalyzes flavors. the first step in odified flavor, aromaa conversioil of inorganic and/or nitrogen exture attributes. (ammonium) into its organic form Construction of geneticlutamine (Gln). Bacterial vectors glutamine or controlled expressionynthetase export is associated of RNA with d/or protein, fusion athogenicity and with protein the formation roduction, genetic f a poly-L-glutamate/glutamine modification, cell utagenesis amplificationall structure. Glutamine of synthetase enetic material or 's an enzyme that plays for other a central role enetic or protein ' the nitrogen metabolism.
manipulations. The tered survival characteristics:nzyme and its products have roles in s urvival of industrialavor and growth.
processes, owth or storage in .
product ormats, persistence in gut nvironment.

tered metabolic properties.

tered probiotic attributes.

odified health properties ( including immunoregulatory, a nticancer, gut health).

tered cell wall or cell surface haracteristics, structures or ctions.

32 116 tered cell wall or omologue of Lipopolysaccharide cell surface haracteristics, structuresynthesis protein yohJ.
or s YohJ is ctions. ' volved in techoic acid synthesis and odified adhesion to portant for cell wall human or functions al cells or cell lines.cludin adhesion, immune ' cell SEQ SEQ ID
NO : NO: Utility Description DNA Pol a e tid roduction of desirable'nteraction and product flavors. texture.

odified flavor, aroma and/or exture attributes.

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.

odified health properties including immunoregulatory, ticancer, gut health).

odified antibiotic resistance.

proved antimicrobial roperties.

33 117 roduction of desirableomologue of sorA. SorA
flavors., encodes odified flavor, aromaa first protein of the and/or exture attributes. hosphoenolpyruvate-dependent L-Construction of geneticorbose-specific phosphotransferase vectors or controlled expressionsystem (PTS). The ketose of RNA L-sorbose .

d/or protein, fusion s transported and phosphorylated protein ' roduction, genetic ough PTS. The enzyme modification, is useful in utagenesis amplificationarbohydrate-specific of regulation of enetic material or ene expression and flavor for other enetic or protein evelopment.
manipulations.

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.

O rganisms or materials with proved health properties (i ncluding immunoregulatory, a nticancer, t health).

SEQ SEQ ID

NO : NO: Utility Description DNA Pol a tide .

33 118 roduction of desirableomologue of sorF, encoding flavors. D-odified flavor, aromaorbitol-6-phosphate dehydrogenase, and/or exture attributes. enzyme of the L-sorbose onstruction of geneticetabolism. The ketose vectors L-sorbose is or controlled expressionansported and phosphorylated of RNA

d/or protein, fusionough the phosphoenolpyruvate-protein roduction, genetic ependent L-sorbose-specific modification, utagenesis amplificationhosphotransferase system of (PTS).

enetic material or seful for carbohydrate-specific for other enetic or protein egulation of gene expression manipulations. (Yebra tered survival characteristics:t al., J. Bacteriol. 182:155-163, urvival of industrial000).
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, ticancer, gut health).

34 119 roduction of bioactiveomologue of pepA, encoding or a ctional polypeptides.lutamyl aminopeptidase, which emoval of undesirableelongs to peptidase family flavor M42 and haracteristics. 's required for optimal growth of roduction of desirableactococcus lactis MG1363 flavors. in milk.

odified flavor, aromaepA has DNA-binding activity and/or that exture attributes. ctions in transcription control and Construction of geneticlasmid dimer resolution.
vectors or controlled expression' opeptidases are involved of RNA in d/or protein, fusionany different functions protein in the cell, roduction, genetic uch as protein maturation, modification, protein utagenesis amplificationover, hydrolysis of regulatory of enetic material or eptides, nitrogen nutrition, for other enetic or protein odulation of gene expression manipulations. etc.

tered survival characteristics:d, consequently, are considered urvival of industrialssential enzymes. The processes, proteolytic owth or storage in ystem of lactic acid bacteria product is ormats, persistence ssential for bacterial in gut e growth in milk nvironment. ut also for the development of the tered metabolic propertiesrganoleptic properties or of dairy egulation of metabolicroducts. PepA is involved both in athwa s. arterial owth by su lyin amino SEQ SEQ ID

NO : NO: Utility Description DNA Pol a tide tered probiotic attributes.cids, and in the development of Organisms or materialsavor in dairy products, with by 'mproved health propertiesydrolyzing peptides (including bitter (including immunoregulatory,eptides) and liberating aromatic ticancer, gut health).' o acids which are important recursors of aroma compounds (Fernandez-Espla and Rul, Eur. J.

iochem. 263:502-510, 1999).

35 120 tered amino acid metabolism.omologue of hom, encoding a emoval of undesirableomoserine dehydrogenase flavor (EC

haracteristics. 1.1.1.3), an enzyme of the threonine roduction of desirableiosynthesis pathway.
flavors. . Threonine is odified flavor, aromaerived from aspartic and/or acid.

exture attributes. Conversion of aspartate to onstruction of geneticomoserine proceeds with vectors ATP

or controlled expressionctivation of the B-carboxyl of RNA group as d/or protein, fusion mixed phosphoric anhydride protein roduction, genetic ollowed by two sequential modification, NADPH-utagenesis amplificationependent reductions to of homoserine.

enetic material or hosphorylation of homoserine for other enetic or protein rovides the substrate manipulations.

tered survival characteristics:hosphohomoserine which suffers a survival of industrialtereospecific 1,2-transposition processes, owth or storage in eaction to give threonine.
product The ormats, persistence enzymatic degradation in gut of amino acids nvironment. cheese plays a major role in cheese tered metabolic properties.avor development. Amino acid ltered probiotic attributes.egradation products greatly odified health propertiescontribute to flavor or to off flavors (including immunoregulatory,Rijnen et al., Appl.
( Environ.

ticancer, gut health).icrobiol. 65:4873-4880, 1999).

35 121 ltered cell wall or omologue of flotillin.
cell surface Flotillins haracteristics, structuresehave as resident integral or membrane ctions. rotein components of caveolae odified adhesion to hich are plasmalemmal human or al cells or cell lines.'crodomains and are involved in roduction of desirableesicular trafficking flavors. and signal odified flavor, aromaznsduction (Huang et and/or al., MoL

exture attributes. icrobioL 31:361-371, 1999).

onstruction of geneticlotillins (also known vectors as epidermal or controlled expressionurface antigens (ESAs)) of RNA belong to d/or protein, fusion a family of caveolae-associated protein roduction, genetic tegral membrane proteins modification, ' and may utagenesis amplificationct as a scaffolding protein of within enetic material or aveolar membranes.
for other SEQ SEQ ID

NO : NO: Utility Description DNA Poly a e tid 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.

36 122 emoval of undesirableomologue of area, encoding flavor benzyl haracteristics. cohol dehydrogenase (EC
1.1.1.90).

roduction of desirableeB catalyzes the oxidation flavors. of an odified flavor, aromaomatic alcohol to an and/or aroriiatic exture attributes. dehyde. This enzyme enables Construction of geneticacteria to grow on a vectors range of esters or controlled expressionf aromatic alcohols and of RNA plays a role znd/or protein, fusionflavor development.
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).

proved fermentation properties r other industrially , useful rocesses.

37 123 tered amino acid metabolism.omologue of coda, encoding a emoval of undesirableytosine permease. Coda flavor c mediates haracteristics. ptake of exogenously supplied roduction of desirableytosine. It belongs to flavors. the ABC

odified flavor, aromaansporter family. The and/or cytosine e xture attributes. ermease is an integral c ~ o lasmic SEQ SEQ ID
NO : NO: Utility Description DNA Pol a tide Construction of geneticembrane protein possessing vectors 'several or controlled expressionansmembrane-spanning of RNA domains.

d/or protein, fusion a enzymatic degradation protein of amino roduction, genetic cids in cheese plays modification, a major role in utagenesis amplificationheese flavor development.
of Amino enetic material or cid degradation products for other greatly enetic or protein ontribute to flavor or manipulations. to off flavors tered survival characteristics:(Rijnen et al., Appl.
Environ.

survival of industrialicrobiol. 65:4873-4880, processes, 1999).

owth or storage in product ormats, persistence in gut nvironment.

tered response to stress onditions.

tered metabolic properties.

tered probiotic attributes.

odified health properties (including immunoregulatory, ticancer, gut health).

odified antibiotic resistance.

proved antimicrobial . roperties.

proved fermentation properties r other industrially useful rocesses.

38 124 onstruction of geneticomologue of hspl8, encoding vectors a or controlled expressionow-molecular-weight protein of RNA

d/or protein, fusion elonging into a family protein of small heat roduction, genetic hock proteins. Hspl8 modification, is induced not utagenesis amplificationnly by heat shock but of also at the enetic material or nset of solventogenesis.
for other Small heat enetic or protein hock proteins (sHsps) manipulations. are a diverse roduction of desirableoup of heat-induced proteins flavors. that odified flavor, aromazre conserved in prokaryotes and/or and exture attributes. ukaryotes and are especially tered survival characteristics:bundant in plants. Recent in vitro s urvival of industrialata indicate that sHsps processes, act as owth or storage in olecular chaperones to product prevent ormats, persistence ermal ag~egation of proteins in gut by nvironment. finding non-native intermediates, tered viability in hich can then be refolded response to in an s tress conditions. TP-dependent fashion by other tered metabolic propertieshaperones (Lee and Vierling, or c Plant.

egulation of metabolichysiol. 122:189-198, . 2000).

athways.

tered ~ robiotic attributes.

SEQ SEQ ID
NO: NO: Utility ~ Description DNA Polwentide 39 125 tered cell wall or omologue to mccF, encoding cell surface a haracteristics, structureser membrane associated or protein o:

ctions. bacteria. MccF determines resistance roduction of desirableo exogenous microcin.
flavors. Possible odified flavor, aromaction by preventing and/or the reentering of exture attributes. a cell by exported translation Construction of genetic'bitor microcin C7 vectors (Gonzalez-br controlled expressionactor et al., J. Bacteriol.
of RNA 177:7131-d/or protein, fusion 140, 1995).
protein roduction, 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 metabolic properties.
probiotic attributes.
d health properties sg immunoregulatory, er, gut health).
3 antibiotic resistance.
d antimicrobial 40 126 roduction of desirableomologue of sorE, encoding flavors. an L-odified flavor, aromasorbose-1-phosphate and/or reductase. SorF

e xture attributes. s, together with D-glucitol-6-' Construction hosphate dehydrogenase, of genetic involved vectors or controlled the conversion of L-sorbose-1-expression of RNA ' and/or protein, hosphate to D-fructose-6-phosphate.
fusion protein roduction, genetic modification, SorE is involved in flavor utagenesis evelopment and carbohydrate amplification of enetic material etabolism.
or for other enetic or protein manipulations.

tered survival characteristics:

survival of industrial processes, owth or storage in product ormats, persistence in gut ~ed metabolic properties.
ified carbohydrate levels or Tonal properties.
ed probiotic attributes.
lisms or materials with SEQ SEQ ID

NO : NO: Utility Description DNA Pol a tide proved health properties (including immunoregulatory, ticancer, gut health).

41 127 roduction of desirableomologue of the iolB
flavors. gene, odified flavor, aromacncoding a cis-acting and/or catabolite-exture attributes. esponsive element (cre) protein Construction of genetic' volved in the iol operon vectors of the myo-or controlled expression' ositol catabolism pathway.
of RNA Myo-d/or protein, fusion' ositol is abundant protein in nature, roduction, genetic specially in soil. Various modification, utagenesis amplification'croorganisms are able of to grow on genetic material yo-inositol as-the sole or for other carbon enetic or protein ounce. The expression manipulations. of the iol tered survival characteristics:peron is under glucose repression urvival of industrial(Miwa and Fujita, J.
processes, Bacteriol.

owth or storage in 183:5877-5884, 2001).
product ormats, persistence in gut nvironment.

tered metabolic properties.

odified carbohydrate-levels or ctional properties.

tered cell wall or cell surface characteristics, structures or ctions.

odified adhesion to human or al cells or cell lines.

tered probiotic attributes.

Organisms or materials with proved health properties (including immunoregulatory, ticancer, gut health).

41 128 roduction of desirableomologue of the iolC
flavors. gene, .

odified flavor, aromancoding 2-dehydro-3-and/or exture attributes. eoxygluconokinase. IoIC

Construction of genetichosphorylates the 2-deoxy-5-keto-vectors or controlled expression-gluconic acid to 2-deoxy-5-keto-D-of RNA

d/or protein, fusionluconic acid 6-phosphate protein and is part roduction, genetic f the iol operon of the modification, myo-inositol utagenesis amplificationatabolism pathway (Yoshida of et al., enetic material or . Bacteriol. 179:4591-4598, for other 1997).

enetic or protein yo-inositol is abundant manipulations. in nature, tered survival characteristics:specially in soil. Various urvival of industrial'croorganisms are able processes, to grow on owth or storage in yo-inositol as the sole product carbon ormats, ersistence ounce. The ex ression in gut of the iol SEQ SEQ ID

NO : NO: Utility Description DNA Pol a e tid nvironment. peron is under glucose repression tered metabolic properties.iwa and Fujita, J. Bacteriol.

odified carbohydrate 183:5877-5884, 2001).
levels or ctional properties.

tered cell wall or cell surface haracteristics, structures or ctions.

odified adhesion to human or al cells or cell lines.

tered probiotic attributes.

Organisms or materials with 'mproved health properties (including immunoregulatory, ticancer, gut health).

41 129 roduction of desirableomologue of the iolE
flavors. gene, odified flavor, aromancoding a protein involved and/or in the iol exture attributes. peron of the myo-inositol Construction of geneticatabolism pathway. Myo-inositol vectors is or controlled expressionbundant in nature, especially of RNA in soil.

d/or protein, fusion arious microorganisms protein are able to roduction, genetic ow on myo-inositol as modification, the sole utagenesis amplificationarbon source. The expression of of the enetic material or 'ol operon is under glucose for other repression enetic or protein (Miwa and Fujita, J.
manipulations. Bacteriol.

tered survival characteristics:183:5877-5884, 2001).

urvival of industrial processes, owth or storage in product ormats, persistence in gut nvironment.

tered metabolic properties.

odified carbohydrate levels or ctional properties.

tered cell wall or cell surface haracteristics, structures or ctions.

odified adhesion to human or al cells or cell lines.

tered probiotic attributes.

O rganisms or materials with proved health properties (i ncluding immunoregulatory, ticancer, gut health).

41 130 roduction of desirableomologue of the iolG
flavors. gene, odified flavor, aromancoding inositol dehydrogenase and/or (EC

e xture attributes. 1.1.18 . IoIG catal zes 1. the first SEQ SEQ ID
NO : NO: Utility Description DNA Pol a e tid Construction of geneticeaction of the inositol vectors catabolism, or controlled expressiona dehydrogenation of of RNA myo-inositol d/or protein, fusion ' to 2-keto-myo-inositol protein (2-inosose) roduction, genetic (Yoshida et al., J. Bacteriol.
modification, utagenesis amplification179:4591-4598, 1997).
of IoIG is part of enetic material or a iol operon of the myo-inositol for other enetio or protein atabolism pathway. Myo-inositol manipulations. is tered survival characteristics:abundant in nature, especially in soil.

urvival of industrialarious microorganisms processes, are able to owth or storage in ow on myo-inositol as product the sole ormats, persistence arbon source. The expression in gut of the nvironment. 'ol operon is under glucose repression tered metabolic properties.(Miwa and Fuj.ita, J.
Bacteriol.

odified carbohydrate 183:587'7-5884, 2001).
levels or ctional properties.

tered cell wall or cell surface haracteristics, structures or ctions.

odified adhesion to human or 'mal cells or cell lines.

tered probiotic attributes.

Organisms or materials with proved health properties (including immunoregulatory, anticancer, gut health).

41 131 roduction of desirableomologue of the iolJ
flavors. gene, odified flavor, aromancoding~fructose-bisphosphate and/or exture attributes. dolase (EC 4.1.2.13) or tagatose-Construction of geneticisphosphate aldolase vectors (4.1.2.40). IoIJ

or controlled expressionleaves 2-deoxy-5-keto-D-gluconic of RNA

d/or protein, fusion cid 6-phosphate to yield protein roduction, genetic ihydroxyacetone phosphate modification, Z and utagenesis amplificationalonic semialdehyde and of is part of enetic material or a iol operon of the myo-inositol for other enetic or protein atabolism pathway (Yoshida manipulations. et al., tered survival characteristics:_. Bacteriol. 179:4591-4598, 1997).

urvival of industrialyo-inositol is abundant processes, in nature, owth or storage in specially in soil. Various product e ormats, persistence 'croorganisms are able in gut to grow on nvironment. yo-inositol as the sole carbon tered metabolic properties.ource. The expression s of the iol odified carbohydrate peron is under glucose levels or repression ctional properties. Miwa and Fujita, J. Bacteriol.
( tered cell wall or 83:5877-5884, 2001).
cell surface 1 haracteristics, structures or ctions.

SEQ SEQ ID

NO : NO: Utility Description DNA Poly e a tid odified adhesion to human or al cells or cell lines.

ltered probiotic attributes.

Organisms or materials with proved health properties (including immunoregulatory, ticancer, gut health).

41 132 tered amino acid metabolism.omologue of mmsA or iolA, emoval of undesirableencoding methylmalonate-flavor characteristics. semialdehyde dehydrogenase.
IoIA

roduction of desirableconverts malonic semialdehyde flavors. into odified flavor, aromacetyl CoA and CO2, the and/or final step of exture attributes. ' ositol degradation (Yoshida et al., Construction of genetic. Bacteriol. 179:4591-4598, vectors 1997).

or controlled expressionyo-Inositol is abundant of RNA in nature, d/or protein, fusion specially in soil. Various protein roduction, genetic 'croorganisms are able modification, to grow on utagenesis amplificationyo-inositol as the sole of carbon enetic material or ource. The expression for other of the iol enetic or protein peron is under glucose manipulations. repression tered survival characteristics:(Miwa and Fujita, J.
Bacteriol.

survival of industrial183:5877-5884, 2001).
processes, The enzyme owth or storage in 's also required for product growth on valine ormats, persistence d isoleucine as it is in gut an acylating nvironment. izyme that converts both propanal tered metabolic properties.d 2-Methyl-3-oxopropanoate to odified carbohydrate ropanoyl-CoA. The enzymatic levels or ctional properties. egradation of amino acids in cheese tered cell wall or lays a major role in cell surface cheese flavor haracteristics, structuresdevelopment. Amino acid or ctions. egradation products greatly odified adhesion to ontribute to flavor or human or to off flavors al cells or cell lines.Rijnen et al., App1 Environ.
( tered probiotic attributes.icrobiol. 65:4873-4880, 1999).

O rganisms or materials with proved health properties ( including immunoregulatory, a nticancer, gut health).

42 133 tered amino acid metabolism.omologue of hisF, encoding emoval of undesirable'dazole glycerol phosphate flavor haracteristics. ynthase subunit hisF
(EC 4.1.3.-) roduction of desirableso called IGP synthase flavors. cyclase odified flavor, aromaubunit. HisF links histidine and/or and de e xture attributes, ovo purine biosynthesis and is a C onstruction of geneticember of the glutamine vectors SEQ SEQ ID
NO: NO: Utility Description DNA Polwentide controlled expression of RNA idotransferase family.
/or protein, fusion protein he enzymatic degradation of amino auction, genetic modification, cads in cheese plays a major role in agenesis amplification of heese flavor development. Amino tic material or for other cad degradation products greatly tic or protein manipulations. ontribute to flavor or to off flavors red survival characteristics: ~nen et al, Appl. Environ.
ival of industrial processes, icrobiol. 65:4873-4880, 1999).
Jth or storage in product cats, persistence in gut !altered metabolic properties.
Altered probiotic attributes.
vlodified health properties including immunoregulatory, nticancer, gut health).
Modified antibiotic resistance.
43 134 Construction of genetic omologue of yqcM, encoding vectors br controlled expression of RNA arsenate reductase (EC
1.97.1.5).

d/or protein, fusion protein qcM reduces the arsenate ion reduction, genetic modification,(HZAsO) to arsenate ion (As0).

utagenesis amplification of senate is an abundant oxyanion enetic material or for other at, because of its ability to mimic enetic or protein manipulations.a phosphate group, is toxic to cells.

reduction of desirable flavors.senate reductase participates to edified flavor, aroma and/or chieve arsenate resistance in both exture attributes. rokaryotes and yeast by reducing tered survival characteristics:senate to arsenate;
the arsenate is survival of industrial processes,en exported by a specific owth or storage in product ansporter. Arsenate reductase is ormats, persistence in gut oupled to the glutathione and environment, lutaredoxin system for its enzyme tered viability in response ctivity (Bennett et to al., Proc. Natl.

tress conditions. cad. Sci. USA 98:13577-13582 , tered metabolic properties or 001).

egulation of metabolic tered probiotic attributes.
.proved fermentation properties other industrially useful health properties immunoregulatory, ', gut health).
44 135 tered amino acid metabolism. omologue of cna, encoding a SEQ SEQ ID
D NO: NO: Utility Description DNA Polvnentide moval of undesirablecollagen adhesin. Cna flavor mediates ~racteristics. ttachment of bacterial cells to >duction of desirableollagen-containing flavors. substrata and is edified flavor, ttached to the cell aroma and/or wall lure attributes. eptidoglycan by an amide bond.

lstruction of genetic vectors controlled expression of RNA

/or protein, fusion protein auction, genetic modification, agenesis amplification of etic material or for other tic or protein manipulations.

.red survival characteristics:

ival of industrial processes, with or storage in product tats, persistence in gut ~l.ltered metabolic properties.
filtered probiotic attributes.
vlodified health properties including immunoregulatory, ~.nticancer, gut health).
dodified antibiotic resistance.
mproved antimicrobial 45, 65 136, roduction of bioactiveIomologue of pepQ, encoding 159 or a ctional polypeptides.~aa-Pro dipeptidase (EC
3.4.13.9).

emoval of undesirableepQ hydrolyzes Xaa-Pro flavor dipeptides haracteristics. ut not Pro-Pro) and also acts on roduction of desirable' oacyl-hydroxyproline flavors. analogs odified flavor, 's peptidase belongs aroma and/or to peptidase exture attributes. amity M24A (methionyl Construction ' opeptidase family).
of genetic It has a vectors br controlled otential use in the dairy expression industry as of RNA

d/or protein, heese-ripening agent fusion protein since proline roduction, elease from proline-containing genetic modification, utagenesis eptides in cheese reduces amplification bitterness.
of enetic material a proteolytic system or for other of lactic acid enetic or acteria is essential protein manipulations. for bacterial tered survival _ owth in milk but also characteristics: for the urvival of evelopment of the organoleptic industrial processes, owth or storage roperties of dairy products.
in product PepQ is ormats, persistence volved both in bacterial in gut ' growth by nvironment. upplying amino acids, and in the tered metabolic evelopment of flavor properties in dairy or egulation roduct~_ by hvr~rnlv~ina of metabolic nant;rlae SEQ SEQ ID

NO : NO: Utility Description DNA Pol a e tid athways. (including bitter peptides) and tered probiotic attributes.iterating aromatic amino acids Organisms or materialshich are important precursors with of proved health propertiesoma compounds (Fernandez-Espla including immunoregulatory)d Rul, Eur. J. Biochem.
263:502-10, 1999).

46 137 tered amino acid metabolism.omologue of argG, encoding a emoval of undesirablegininosuccinate synthase flavor (EC

haracteristics. 6.3.4.5). ArgG catalyzes the roduction of desirableenultimate step of the flavors, arginine odified flavor, aromaiosynthesis. It belongs and/or to the exture attributes. gininosuccinate synthase family.

Construction of genetiche enzymatic degradation vectors of amino or controlled expressioncids in cheese plays of RNA a major role in d/or protein, fusion heese flavor development.
protein Amino roduction, genetic cid degradation products modification, greatly utagenesis amplificationontribute to flavor or of to off flavors enetic material or (Rijnen et al., Appl.
for other Environ.

enetic or protein icrobioL 65:4873-4880, manipulations. 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, ticancer, gut health).

odifled antibiotic resistance.

46 138 tered amino acid metabolism.omologue of argH, encoding emoval of undesirablegininosuccinate lyase flavor (EC 4.3.2.1).

haracteristics. gH catalyzes the last step in the roduction of desirableginine biosynthesis.
flavors.

odified flavor, aromagininosuccinate lyase and/or also exture attributes. anticipates in the urea cycle, the C onstruction of geneticajor pathway for the vectors detoxification or controlled expressionf ammonia, where it catalyzes of RNA o the d/or protein, fusion eversible breakdown of protein roduction, genetic gininosuccinic acid into modification, arginine utagenesis amplificationd fu.marate. The enzymatic of enetic material or egradation of amino acids for other in cheese enetic or protein lays a maj or role in manipulations. cheese flavor tered survival characteristics:evelopment. Amino acid urvival of industrialegradation roducts greatly rocesses, d SEQ SEQ ID

NO : NO: Utility Description DNA Pol a e tid owth or storage in ontribute to flavor or product to off=flavors ormats, persistence (Rijnen et al., Appl:
in gut Environ.

environment. icrobiol. 65:4873-4880, 1999).

tered metabolic properties.

tered probiotic attributes.

edified health properties (including immunoregulatory, ticancer, gut health).

edified antibiotic resistance.

proved fermentation properties r other industrially useful recesses.

47 139 Construction of geneticomologue of purl, encoding vectors a or controlled expressionhosphoribosylformylglycinamidine of RNA

d/or protein, fusion (FGAM) synthetase (EC
protein 6.3.5.3).

reduction, genetic urL catalyzes the fourth modification, step in the utagenesis amplificationiosynthesis of purines.
of It is involved enetic material or multistress resistance.
for other Purines play enetic or protein ssential roles in many manipulations. cellular reduction of desirablections, including DNA
flavors. replication, edified flavor, aromaanscription, infra- and and/or extra-cellular exture attributes. ignaling, energy metabolism, and as tered survival characteristics:coenzymes for many biochemical urvival of industrialeactions.
processes, owth or storage in product ormats, persistence in gut nvironment.

tered viability in response to tress conditions.

tered metabolic properties or egulation of metabolic athways.

tered probiotic attributes..

48 140 tered amino acid metabolism.omologue of hisH, encoding a emoval of undesirable'dazole glycerol phosphate flavor haracteristics. ynthase subunit that is also known as reduction of desirableGP synthase glutamine flavors.

edified flavor, aroma'dotransferase subunit.
and/or HisH

e xture attributes. atalyzes the fifth step of the histidine C onstruction of geneticiosynthesis. The hisH
vectors subunit o r controlled expressionrovides the glutamine of RNA

d/or protein, fusion 'dotransferase activity protein that reduction, genetic reduces the ammonia necessary modification, to utagenesis amplification'sF for the synthesis of of IGP and enetic material or CAR. The a atic de adation for other SEQ SEQ ID

NO : NO: Utility Description DNA Pol a e tid enetic or protein . f amino acids in cheese manipulations plays a tered survival characteristics:ajor role in cheese flavor urvival of industrialdevelopment. Amino acid processes, owth or storage in degradation products product greatly ormats, persistence ontribute to flavor or in gut to off flavors nvironment. (Rijnen et al., Appl.
Environ.

tered metabolic properties.icrobiol. 65:4873-4880, 1999).

tered probiotic attributes.

edified health properties (including immunoregulatory, ticancer, gut health).

edified antibiotic resistance.

49 141 Construction of geneticomologue of ndK, encoding vectors or controlled expressionucleoside-diphosphate of RNA kinase (EC

d/or protein, fusion .7.4.6), which converts protein reduction, genetic (deoxy)ribonucleoside modification, diphosphates utagenesis amplification' to their corresponding of enetic material or 'phosphates. NdK is an for other ubiquitous enetic or protein d nonspecific enzyme manipulations. but is an reduction of desirableportant cellular enzyme flavors. that edified flavor, aromaonitors and maintains and/or nucleotide exture attributes. eels and has been implicated in a tered survival characteristics:umber of regulatory processes, survival of industrial' cluding signal transduction, processes, owth or storage in evelopment and cell surface product ormats, persistence olysaccharide synthesis.
in gut nvironment.

tered viability in response to tress conditions.

tered metabolic properties or egulation of metabolic athways.

tered probiotic.attributes.

50 142 tered cell wall or omologue of PrtB, a PII-type cell surface haracteristics, structuresroteinase precursor (Lactocepin) or ctions. so called cell wall-associated serine odifled adhesion to roteinase (E.C 3.4.21.96).
human or PrtB

'mal cells or cell reaks down milk proteins lines. during the reduction of desirableowth of the bacteria flavors. _ on milk and edified flavor, aromaat provides the peptides and/or essential e xture attributes. r cell growth. It has o endopeptidase C onstruction of geneticctivity with very broad vectors a specificity. It o r controlled expressionbest known for its action of RNA 's on d/or protein, fusion seins, although it has protein ca been shown r eduction, enetic modification,hydrol ze hemoglobin o and SEQ SEQ ID

NO : NO: Utility Description DNA Pol a e tid utagenesis amplificationxidized insulin b-chain.
of Lactocepin enetic material or 's a type I membrane for other protein, located _enetic or protein . 'n the cell wall and manipulations belongs to .

tered survival characteristics:eptidase family S8; also known as survival of industriala Subtilase Family. Lactocepin processes, is owth or storage in esponsible for the hydrolysis product of ormats, persistence asein in milk and specificity in gut environment. ifferences between lactocepins from tered metabolic properties.ifferent starter strains may be partly tered probiotic attributes.esponsible for imparting different odified health propertiesavor qualities to cheese . (Broadbent (including immunoregulatory,t al., Appl. Environ.
Microbiol.

ticancer, gut health).68:1778-1785, 2002).

odified antibiotic resistance.

proved fermentation properties or other industrially useful rocesses.

51 143 tered amino acid metabolism.omologue of aspB, encoding an emoval of undesirableaspartate aminotransferase flavor (EC:

haracteristics. .6.1.1), also called aspartate roduction of desirableansaminase. AspB catalyzes flavors. the odified flavor, aroma' o group transfer between and/or amino exture attributes. cids and 2-oxo acids and that plays a Construction of geneticentral role in amino vectors acid metabolism or controlled expression' organisms. The transferase of RNA is znd/or protein, fusionportant for the metabolism protein of roduction, genetic 'no acids and Krebs cycle modification, related utagenesis amplificationrganic acids. It plays of a role in the enetic material or roduction of important for other flavor enetic or protein eterminants. The enzymatic manipulations.

tered survival characteristics:egradation of amino acids in cheese s urvival of industriallays a maj or role in processes, cheese flavor owth or storage in evelopment. Amino acid product ormats, persistence egradation products greatly in gut e nvironment. ontribute to flavor or to off flavors tered metabolic properties.lZijnen et al., AppL
( Environ.

tered probiotic attributes.icrobiol. 65:4873-4880, 1999).

odified health properties ( including immunoregulatory, ticancer, gut health).

odified antibiotic resistance.

proved fermentation properties r other industrially useful rocesses.

52 144 roduction of desirableomolo a of iolF, encoding flavors. an SEQ SEQ ID
NO: NO: Utility Description DNA Pol a tide odified flavor, aroma' ositol transporter and/or (TC#:

exture attributes. .A.1.1.27). IoIF transports myo-Construction of genetic' ositol into the bacterial vectors cell. IoIF is or controlled expressionart of the iol operon of RNA of the myo-and/or protein, fusion' ositol catabolism pathway.
protein Myo-roducoon, genetic ' ositol is abundant modification, in nature, utagenesis amplificationspecially in soil. Various of enetic material or 'croorganisms are able for other to grow on enetic or protein yo-inositol as the sole manipulations. carbon ltered survival characteristics:source. The expression of the iol urvival of industrialperon,is under glucose processes, repression owth or storage in (Miwa and Fujita, J.
product Bacteriol.

ormats, persistence 183:5877-5884, 2001).
in gut environment.

tered metabolic properties.

odified carbohydrate levels or coonal properties. .

tered cell wall or cell surface haracterisocs, structures or coons.

odified adhesion to human or al cells or cell lines.

tered probiooc attributes.

Organisms or materials with proved health properties (including immunoregulatory, ticancer, gut health).

54 146 tered cell wall or omologue of mga4, a positive cell surface haracterisocs, structuresegulatory protein that or acts as.a coons. omponent of a signal transducing odified adhesion to ystem. Positive regulatory human or proteins al cells or cell lines.r activator proteins bind in their roduction of desirablecove state to DNA in flavors. the promoter odified flavor, aromaegion and help RNA polymerase andlor to exture attributes. ind and transcribe that gene. Mga4 Construction of genetic'tiates transcription vectors of surface-or controlled expressionssociated/virulence factors.
of RNA

d/or protein, fusion protein roducoon, genetic modification, utagenesis amplification of eneoc material or for other eneoc or protein manipulations.

tered survival characteristics:

urvival of industrial processes, owth or storage in product ormats, ersistence in gut SEQ~ SEQ ID

NO : NO: Utility Description DNA Pol a e tid environment.

tered metabolic properties.

tered probiotic attributes.

edified health properties including immunoregulatory, ticancer, gut health).

edified antibiotic resistance.

proved antimicrobial roperties.

egulation of polysaccharide reduction, adhesion, immune odulation.

s5 147 tered amino acid metabolism.omologue of BH3554, encoding a emoval of undesirablearboxylesterase (3.1.1.1).
flavor BH35s4 haracteristics. ydrolyzes carboxylic ester bonds reduction of desirable'th relatively broad flavors. substrate edified flavor, aromapecificity. It is involved and/or in amino exture attributes. cid metabolism and flavor.
The onstruction of geneticnzymatic degradation vectors of amino acids or controlled expression' cheese plays a major of RNA role in cheese d/or protein, fusion aver development. Amino protein acid reduction, genetic egradation products greatly modification, utagenesis amplificationontribute to flavor or of to off=flavors enetic material or (Rijnen et al., Appl.
for other Environ.

enetic or protein icrobiol. 6s:4873-4880, manipulations. 1999).

tered survival characteristics:

survival of industrial processes, owth or storage in product ormats, persistence in gut nvironment.

tered metabolic properties.

tered probiotic attributes.

edified health properties ( including immunoregulatory, ticancer, gut health).

edified antibiotic resistance.

proved fermentation properties r other industrially useful recesses.

56 148 tered amino acid metabolism.omologue of Amdl, encoding an emoval of undesirable' oacylase (EC 3.5.1.14).
flavor Amdl haracteristics. eacetylates acylated amino acids. It reduction of desirablelays a role in the production flavors. of edified flavor, aromaportant flavor determinants.
andlor The e xture attributes. atic de adation of amino acids so SEQ SEQ ID
NO : NO: Utility Description DNA Pol a e tid Construction of geneticcheese plays a major vectors role in~ cheese or controlled expressionaver development. Amino.
of RNA acid d/or protein, fusion egradation products greatly protein reduction, genetic ontribute to flavor or modification, to off flavors utagenesis amplification(Rijnen et al., Appl.
of Environ.

enetic material or icrobiol. 65:4873-4880, for other 1999).

genetic or protein manipulations.

tered survival characteristics:

urvival of industrial processes, owth or storage ire product ormats, persistence in gut nvironment.

tered metabolic properties.

tered probiotic attributes.

edified health properties (including immunoregulatory, ticancer, gut health).

edified antibiotic resistance.

proved fermentation properties r other industrially useful recesses.

57 149 tered cell wall or omologue of tmpA, encoding cell surface a haracteristics, structuresutative transmembrane or protein.

ctions. mpA plays a role in adhesion and is edified adhesion to art of an operon containing human or the al cells or cell lines.apA gene.

reduction of desirable flavors.

edified flavor, aroma and/or exture attributes.

Construction of genetic vectors or 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 e nvironment.

ltered metabolic properties..

tered probiotic attributes.

edified health properties ( including immunoregulatory, ticancer, t health).

SEQ SEQ ID

NO : NO: Utility Description DNA Poly a tide odified antibiotic resistance.

proved fermentation properties or other industrially useful rocesses.

58 1 SO Construction of geneticomologue of npr, encoding vectors a or controlled expressionADH peroxidase (EC 1.11:1:1 of RNA ). Npr d/or protein, fusion tilizes hydrogen peroxide protein to create roduction, genetic ater and nicotinamide modification, adenine utagenesis amplificationdinucleotide (NADH) from of its enetic material or xidized form (NAD). Metabolism for other of enetic or protein o-factors such as NADH
manipulations. can greatly roduction of desirableuence the speed and type flavors. of odified flavor, aromaetabolic pathway utilized and/or under exture attributes. 'fferent redox conditions, and can tered survival characteristics:erefore influence flavor andlor urvival of industrialctionality.
processes, owth or storage in product ormats, persistence in gut ' nvironment.

tered viability in response to tress conditions.

tered metabolic properties or egulation of metabolic athways.

tered probiotic attributes.

proved fermentation properties or other industrially useful rocesses.

59, 151, 152 tered amino acid metabolism.omologue of nifS. NifS
60 is involved emoval of undesirablecysteine metabolism and flavor c haracteristics. evelopment of flavor compounds.

roduction of desirablea enzymatic degradation flavors. of amino odified flavor, aromacids in cheese plays and/or a major role in exture attributes. heese flavor development.
c Amino Construction of geneticcid degradation products vectors greatly or controlled expressionontribute to flavor or of RNA to off flavors d/or protein, fusion 'jnen et al., Appl. Environ.
protein roduction, genetic icrobiol. 65:4873-4880, modification, 1999).

utagenesis amplification of enetic material or for other enetic or protein manipulations.

tered survival characteristics:

urvival of industrial processes, g rowth or storage in product ormats, ersistence in t SEQ SEQ ID

NO : NO: Utility Description DNA Pol a e tid 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.

61 153 roduction of desirableomologue of fabA, encoding flavors. a 3-odified flavor, aromaydroxydecanoyl-ACP dehydratase.
and/or exture attributes. abA introduces cis unsaturation into Construction of geneticariy acids during saturated vectors fatty acid or controlled expressioniosynthesis. The dehydratase of RNA

d/or protein, fusion elongs to the thioester protein dehydratase roduction, genetic unify. Free fatty acids modification, are important utagenesis amplificationproviding flavor-bearing of enetic material or compounds for dairy products for other such as enetic or protein cheese, and have a significant manipulations. role in tered survival characteristics:oth flavor and texture.
Used survival of industrialxtensively in wide range processes, of owth or storage in onvenience foods. Short product chain fatty ormats, persistence cids are laiown to have in gut a variety of nvironment. ealth impacts.

tered metabolic properties.

odified lipid, glycolipid or free atty acid levels or functional roperties.

odified production of short hair fatty acids.

tered lipid metabolism.

tered probiotic attributes.

Organisms or materials with ' mproved health properties ( including immunoregulatory, a nticancer, gut health) 62 154 tered cell wall or omologue of aggH, encoding cell surface an haracteristics, structuresutoaggregation mediating or protein.

ctions. ggH contains a region of similarity odified adhesion to o ATP-dependent DEAD-box human or al cells or cell lines.elicase. The protein is involved in roduction of desirableenetic exchange, pathogen flavors. exclusion odified flavor, aromad persistence in the and/or gut e xture attributes. nvironment b romoting SEQ SEQ ID .
NO : NO: Utility Description DNA Poly a tide Construction of geneticggregation between bacteria.
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 nvironxnent.

tered metabolic properties.

tered probiotic attributes.

odified health properties including immunoregulatory, ticancer, gut health).

odified antibiotic resistance.

proved antimicrobial roperties.

proved fermentation properties r other industrially useful rocesses.

63 155 roduction of desirableomologue of the iolH
flavors. gene, odified flavor, aromancoding a protein involved and/or in the iol exture attributes. peron of the myo-inositol Construction of geneticatabolism pathway. Myo-inositol vectors is or controlled expressionbundant in nature, especially of RNA in soil.

d/or protein, fusionarious microorganisms protein are able to roduction, genetic ow on myo-inositol as modification, the sole utagenesis amplificationarbon source. The expression of of the enetic material or ol operon is under glucose for other ' repression enetic or protein Miwa and Fujita, J. Bacteriol.
manipulations. ( tered survival characteristics:183:5877-5884, 2001) urvival of industrial processes, owth or storage in product ormats, persistence in gut nvironment.

tered metabolic properties.

odified carbohydrate levels or ctional properties.

tered cell wall or cell surface haracteristics, structures or ctions.

odified adhesion to human or al cells or cell lines.

SEQ SEQ ID

NO : NO: Utility Description DNA Pol a e dd tered probiotic attributes., rganisms or materials with proved health properties (including immunoregulatory, ticancer, gut health).

63 156 roduction of desirableomologue of the iolL
flavors. gene, odified flavor, aromancoding a protein involved and/or in the iol exture attributes. peron of the myo-inositol Construction of geneticatabolism pathway. Myo--nositol vectors is or controlled expressionabundant in nature, especially of RNA in soil.

d/or protein, fusion arious microorganisms protein are able to roduction, genetic _ ow on myo-inositol modification, as the sole utagenesis amplificationarbon source. The expression of of the enetic material or 'ol operon is under glucose for other repression enetic or protein (Miwa and Fujita, J.
manipulations. Bacteriol.

tered survival characteristics:183:5877-5884, 2001) urvival of industrial processes, owth or storage in product ormats, persistence in gut nvironment.

tered metabolic properties.

odified carbohydrate levels or ctional properties.

tered cell wall or cell surface haracteristics, structures or ctions.

odified adhesion to human or al cells or cell lines.

tered probiotic attributes.

Organisms or materials with proved health properties ( including immunoregulatory, ticancer, gut health).

64 157 emoval of undesirableomologue of citX, encoding flavor apo-haracteristics. itrate lyase phosphoribosyl-odified flavor, aroma,ephospho-CoA transferase texture d (Apo-ttributes. CP nucleodityltransferase;
EC

C onstruction of genetic.8.3.10). The transferase vectors belongs to or controlled expressiona citX family (Schneider of RNA et al., d/or protein, fusion iochem. 39:9438-9450, protein 2000).

roduction, genetic arbohydrate metabolism modification, C impacts on utagenesis amplificationavor, functionality and of survival as enetic material or ell as growth.
for other enetic or protein manipulations.

tered survival characteristics:

SEQ SEQ ID

NO : NO: Utility Description DNA Pol a tide (survival of industrial processes, owth or storage in product ormats, persistence in gut nviromnent).

odified carbohydrate levels or ctional properties.

tered metabolic properties.

odified citrate metabolism.

tered probiotic attributes.

Organisms or materials with proved health properties (including immunoregulatory, ticancer, gut health).

proved fermentation properties r other industrially useful rocesses.

64 158 emoval of undesirableomologue of pycB, encoding flavor haracteristics. yruvate carboxylase, which odified flavor, aroma,atalyzes a two-step reaction, texture ttributes. ' volving the ATP-dependent Construction of geneticarboxylation of the covalently vectors or controlled expressionttached biotin in the of RNA first step and d/or protein, fusion a transfer of the carboxyl protein group to roduction, genetic yruvate to generate oxaloacetate modification, in utagenesis amplificationa second. The enzyme of is involved enetic material or gluconeogenesis and amino for other acid enetic or protein iotransformations. Carbohydrate manipulations.

tered survival characteristics:etabolism impacts on flavor, ( survival of industrialctionality and survival processes, as well as owth or storage in owth.
product ormats, persistence in gut nvironment).

odified carbohydrate levels or ctional properties.

tered metabolic properties..

tered probiotic attributes.

O rganisms or materials with proved health properties ( including immunoregulatory, ticancer, gut health) mproved fermentation properties o r other industrially useful rocesses.

66 160 roduction of desirableomologue of fox3, encoding flavors. a 3-odified flavor, aromaxoac 1-coe a A thiolase and/or C

SEQ SEQ ID
NO : NO: Utility Description DNA Pol a e tid exture attributes. .3.1.16) also called acetyl-CoA C-Construction of geneticacyltransferase, which vectors participates in or controlled expressiona beta-oxidation of fatty of RNA acids.

d/or protein, fusion xpression of the FOX3 protein gene can be reduction, genetic ' duced by oleate and modification, repressed by utagenesis amplificationlucose (Einerhand et of al., Mol. Cell.

enedc material or iol. 15:3405-3414, 1995).
for other Free fatty enetic or protein cids are important in manipulations. providing tered survival characteristics:aver-bearing compounds for dairy survival of industrialroducts such as cheese, processes, and have a owth or storage in significant role in both product flavor and ormats, persistence exture. Used extensively in gut in wide nvironment. ange of convenience foods.
Short tered metabolic properties.hair fatty acids are laiown to have a odifled lipid, glycolipidariety of health impacts.
or free atty acid levels of functional roperties.

edified 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 recesses.

67 161 Construction of geneticomologue of YchH, encoding vectors an or controlled expressioncetyltransferase. Transfer of RNA of acetyl d/or protein, fusion oups are important in protein regulation of reduction, genetic etabolic pathways was modification, well as co-utagenesis amplificationactor production and of can influence enetic material or aver and/or functionality.
for other enetic or protein manipulations.

reduction of desirable flavors.

edified flavor, aroma and/or exture attributes.

tered survival characteristics:

urvival of industrial processes, owth or storage in product ormats, persistence in gut nvironment.

tered viability in response to tress conditions.

tered metabolic ro erties or SEQ SEQ ID

NO : NO: Utility Description DNA Poly a tide egulation of metabolic athways.

tered probiotic attributes.

68 162 roduction of desirableomologue of SC6F7, encoding flavors. a odified flavor, aroma'pace. Lipases are involved and/or in the exture attributes. reakdown of triglycerides, Construction of geneticetabolism, growth, production vectors of or controlled expressionavor compounds, and the of RNA release of d/or protein, fusion ee fatty acids. Can also protein catalyze roduction, genetic sterification of glycerol modification, to form utagenesis amplificationono, di- and triglycerides.
of Free fatty enetic material or cids are important in for other providing enetic or protein avor-bearing compounds manipulations. for dairy tered survival characteristics:roducts such as cheese, and have a urvival of industrialsignificant role in both processes, flavor and owth or storage in exture. Used extensively product in wide ormats, persistence ange of convenience foods.
in gut Short nvironment. hair fatty acids are known to have a tered metabolic properties.ariety of health impacts.

odifled lipid, glycolipid or free atty acid levels or functional roperties.

odified production of short hairs fatty acids.

tered lipid metabolism.

tered probiotic attributes.

Organisms or materials with ' mproved health properties ( including immunoregulatory, ticancer, gut health).

proved fermentation properties r other industrially useful rocesses.

69 163 tered cell wall or omologue of yohH, a cell surface haracteristics, structures'popolysaccharide synthesis or protein.

ctions. ohH is involved in techoic acid odified adhesion to ynthesis, and. important human or for cell wall al cells or cell lines.ctions including adhesion, roduction of desirablearse cell interaction, flavors. product odified flavor, aromaexture.
and/or exture attributes.

onstruction of genetic vectors or controlled expression of RNA

d/or protein, fusion protein roduction, erietic modification, SEQ SEQ ID
NO : NO: Utility Description DNA Poly a tide 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 antimicrobial roperties.

proved fermentation properties or other industrially useful rocesses.

69 164 tered cell wall or omologue of yohJ, encoding cell surface a haracteristics, structuresipopolysaccharide biosynthesis or ctions. rotein. YohJ is involved in techoic odified adhesion to cid synthesis, and important human or for cell animal cells or cell all functions including lines. adhesion, roduction of desirableune cell interaction flavors. and product odified flavor, aromaexture.
and/or exture attributes.

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:

s urvival of industrial processes, .

owth or storage in product ormats, persistence in gut nvironment.

tered metabolic properties.

tered probiotic attributes.

odified health properties (i ncluding immunoregulatory, ticancer, gut health).

odified antibiotic resistance.

roved antimicrobial SEQ SEQ ID

NO : NO: Utility Description DNA Poly a tide roperties.

proved fermentation s propertie r other industrially useful rocesses.

70 165 Construction of geneticomologue of pstS, encoding vectors a or controlled expressionhosphate-binding protein of RNA that is part d/or protein, fusion f the phosphate specific protein transporter roduction, genetic (Pst) in bacteria. Pst modification, is a multisubunit utagenesis amplificationystem and belongs to of the ABC

enetic material or uperfamily of transporters for other (Novak et enetic or protein l., JBacteriol. 181:1126-1133, manipulations.

roduction of desirable1999). Intracellular flavors. phosphate levels odified flavor, aromauence survival of bacteria and/or in exture attributes. environmental stress conditions, and tered survival characteristics:a 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.

tered metabolic properties or egulation of metabolic athways.

tered probiotic attributes.

71 166 ~ tered cell wall omologue of oppA, encoding or cell surface an haracteristics, structures' tegral membrane protein or of the ctions. ligopeptide transport system (Opp) roduction of bioactivef Lactococcus dactis.
or Opp proteins ctional polypeptides.d the proteinase PrtP
are important odified adhesion to omponents of the proteolytic human or system.

al cells or cell lines.a Opp system belongs to the tered amino acid metabolism.uperfamily of ABC transporters and emoval of undesirableonsists of five proteins:
flavor the integral haracteristics. embrane proteins OppB
and OppC, roduction of desirablea ATP-binding proteins flavors. OppD and odified flavor, aromaOppF, and the OppA a and/or receptor exture attributes. rotein (Detmers et al., Proc. Natl.
.

C onstruction of geneticcad Sci. USA 97:12487-12492, vectors or controlled expression000). Important for the of RNA uptake and d/or protein, fusion upply of amino acids protein to bacteria, and roduction, genetic a resultant production modification, of flavorful utagenesis amplificationr functional amino-acid of degradation enetic material or roducts.
for other enetic or rotein mani ulations.

SEQ SEQ ID
NO : NO: Utility Description DNA Pol a e tid tered survival characteristics:

survival of industrial.
processes, growth or storage in product ormats, 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 rocesses.

71 167 tered cell wall or omologue of oppB, encoding cell surface an haracteristics, structures' tegral membrane protein or of the ctions. ligopeptide transport system (Opp) roduction of bioactivef Lactococcus lactis.
or Opp proteins ctional polypeptides.d the proteinase PrtP
are important odified adhesion to components of the proteolytic human or system.

al cells or cell lines.a Opp system belongs to the tered amino acid metabolism.uperfamily of ABC transporters and emoval of undesirableonsists of five proteins:
flavor the integral haracteristics. embrane proteins OppB
and OppC, roduction of desirablea ATP-binding proteins flavors. OppD and odified flavor, aromaOppF, and the OppA a and/or receptor exture attributes. rotein (Detmers et al., Proc. Natl.

Construction of geneticcad. Sci. USA 97:12487-12492, vectors or controlled expression000). Important for the of RNA uptake and d/or protein, fusion upply of amino acids protein to bacteria, and roduction, genetic a resultant production modification, of flavor~'ul utagenesis amplificationr functional amino-acid of degradation enetic material or roducts.
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 (i ncluding immunoregulatory, a nticancer, gut health).

proved fermentation properties r other industrially useful rocesses.

SEQ SEQ ID

NO : NO: Utility Description DNA Pol a e tid 71 168 tered cell wall or omologue of OppC, encoding cell surface an characteristics, structures' tegral membrane protein or of the ctions. ligopepode transport system (Opp) reduction of bioactivef Lactococcus lactis.
or Opp proteins ctional polypepodes. d the proteinase PrtP
are important edified adhesion to omponents of the proteolytic human or system.

al cells or cell lines.a Opp system belongs to the tered amino acid metabolism.uperfamily of ABC transporters and emoval of undesirableonsists of five proteins:
flavor the integral haracteristics. embrane proteins OppB
and OppC, reduction of desirablea ATP-binding proteins flavors. OppD and edified flavor, aromaOppF, and the OppA a and/or receptor exture attributes. rotein (Detmers et al., Proc.. NatL

Construction of geneticcad. Sci. USA 97:12487-12492, vectors or controlled expression000). Important for the of RNA uptake and d/or protein, fusion supply of amino acids protein to bacteria, and reduction, genetic a resultant production modification, of flavorful utagenesis amplificationor functional amino-acid of degradation enetic material or roducts.
for other eneoc 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.

edified health properties including immunoregulatory, ocancer, gut health).

proved fermentation properties n other industrially useful recesses.

71 169 tered cell wall or omologue of OppF, encoding cell surface an haracterisocs, structurestegral membrane protein or ' of the coons. ligopepode transport system (Opp) reduction of bioacovef Lactococcus lactis.
or o Opp proteins coonal polypeptides. d the proteinase PrtP
are important edified adhesion to omponents of the proteolyoc human or system.

'mal cells or cell a Opp system belongs lines. to the tered amino acid metabolism.uperfamily of ABC transporters and emoval of undesirableonsists of five proteins:
flavor the integral haracterisocs. embrane proteins OppB
and OppC, reduction of desirablea ATP-binding proteins flavors. OppD and edified flavor, aromaF, and the O A a rece and/or O for SEQ SEQ ID
D : NO: Utility Description NO Pol a DNA tide exture attributes. rotein (Detmers et al., Proc. Natl.

onstruction of geneticcad. Sci. USA 97:12487-12492, vectors or controlled expression000). Important for the of RNA uptake and d/or protein, fusion upply of amino acids protein to bacteria, and roduction, genetic a resultant production modification, of flavorful utagenesis amplificationr functional amino-acid of degradation enetic material or roducts.
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).

proved fermentation properties r other industrially useful rocesses.

72 170 emoval of undesirableomologue of thdF, which flavor is characteristics. ~ ' volved in the oxidation of odified flavor, aroma,erivatives of the sulphur-containing texture ttributes. eterocycle thiophene and is induced Construction of geneticwring stationary phase.
vectors The thdF

or controlled expressionene is subject to substantial of RNA

dlor protein, fusion atabolite repression protein by glucose and roduction, genetic ts expression is also modification, ' greatly utagenesis amplificationecreased in the absence of of oxygen enetic material or Zabel et al., Microblos.
for other 101:89-103, enetic or protein 000).
manipulations.

tered survival characteristics:

( survival of industrial processes, owth or storage in product ormats, persistence in gut nvironment).

tered metabolic properties.

odified metabolism of sulphur-ontaining compounds.

tered probiotic attributes.

rganisms or materials with ' mproved health properties i ncluding immunoregulatory, ticancer, gut health).

SEQ SEQ ID

NO : NO: Utility Description DNA Poly a tide 72 171 onstruction of geneticomologue of gidA, encoding vectors a or controlled expressionlucose-inhibited division of RNA protein A, d/or protein, fusion hich is involved in cell protein division and roduction, genetic ' moderating translational modification, fidelity utagenesis amplification(Kinscherf and Willis, of J. Bacteriol.

enetic material or 184:2281-2286, 2002).
for other Affects enetic or protein owth and viability in manipulations. different roduction of desirableowih environments.
flavors.

odified flavor, aroma and/or exture attributes.

tered survival characteristics:

survival of industrial processes, owth or storage in product ormats, persistence in gut nvironment.

tered viability in response to tress conditions.

tered metabolic properties or egulation of metabolic athways.

tered probiotic attributes.

73 172 Construction of geneticomologue of poxB, encoding vectors a or controlled expressionyruvate oxidase (EC 1.2.3.3), of RNA which d/or protein, fusion decarboxylates pyruvate.
protein The enzyme roduction, genetic s a flavoprotein (FAD) modification, ' requiring utagenesis amplification'amine diphosphate and of is enetic material or portant for aerobic growth for other and enetic or protein urvival in aerobic conditions.
manipulations. s roduction of desirableCarbohydrate metabolism flavors. impacts on odified flavor, aromaavor, functionality and and/or survival as exture attributes, ell as growth.

ltered survival characteristics:

urvival of industrial processes, owth or storage in product ormats, persistence in gut nvironment.

tered viability in response to tress conditions.

tered metabolic properties or egulation of metabolic athways.

tered probiotic attributes.

74 173 tered amino acid metabolism.omologue of gltD, encoding a emoval of undesirablelutamate synthase (EC
flavor 1.4.1.13), haracteristics, hick catal zes the reductive transfer SEQ SEQ ID , NO : NO: Utility Description DNA Poly e a tid reduction of desirableof the amide group of flavors. glutamine to edified flavor, aromaa keto position of 2-oxoglutarate and/or to exture attributes. 'eld two molecules of glutamate.

Construction of genetica resulting glutamine vectors and or controlled expressionlutamate serve as nitrogen of RNA donors in d/or protein, fusion a biosynthesis of various protein nitrogen-roduction, genetic ontaining compounds.
modification, This pathway utagenesis amplification's involved in the integration of of enetic material or carbon and nitrogen assimilations.
for other enetic or protein ' o acid degradation manipulations. products tered survival characteristics:eatly contribute to flavor or to off survival of industrialavers (Rijnen et al., processes, Appl. Environ.

owth or storage in icrobiol. 65:4873-4880, product 1999).

ormats, persistence in gut environment.

tered metabolic properties.

tered probiotic attributes.

edified health properties (including immunoregulatory, ticancer, gut health).

edified antibiotic resistance.

75 174 tered amino acid metabolism.omologue of dhfR, encoding a emoval of undesirable' ydrofolate reductase flavor (EC 1.5.1.3), haracteristics. hick catalyzes the essential step for reduction of desirablea novo glycine and purine flavors. synthesis, edified flavor, aromaNA precursor synthesis, and/or and for the exture attributes. onversion of dLTMP to dTIVIP.

Construction of geneticvolved in folate production, vectors which or controlled expressionas major health impacts of RNA and also d/or protein, fusion detoxifies some chemotherapeutic protein reduction, genetic gs and other cytotoxic modification, utagenesis amplificationompounds.
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.

ncreased folate production.

tered probiotic attributes.

edified health properties (i ncluding immunoregulatory, ticancer, gut health).

rotection of intestinal cells from SEQ SEQ ID

NO : NO: Utility Description DNA Pol a tide oxic compounds.

odified antibiotic resistance.

proved fermentation properties r other industrially useful rocesses. .

77 176 ltered amino acid omologue of trpA, encoding metabolism. the emoval of undesirabletophan synthase alpha flavor chain (EC

haracteristics. .2.1.20). TrpA catalyzes the roduction of desirableormation of indole from flavors. the cleavage odified flavor, aromaf 3-indolyl-D-glyceraldehyde and/or 3'-exture attributes. hosphate. Seven structural genes are Construction of geneticequired for tryptophan vectors biosynthesis:

or controlled expressionABCDEFG. TrpA encodes of RNA the d/or protein, fusiontophan synthase alpha protein chain (EC

roduction, genetic .2.1.20) Tryptophan is modification, important for utagenesis amplificationavor development. Amino of acid enetic material or egradation products greatly for other enetic or protein ontribute to flavor or manipulations, to off flavors texed survival characteristics:(IZijnen et al., Appl.
Environ.

urvival of industrialicrobioL 65:4873-4880, processes, 1999).

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.

77 177 tered amino acid omologue of trpB, encoding metabolism.

emoval of undesirabletophan synthase beta flavor chain' (EC

haracteristics. .2.1.20). TrpB catalyzes the roduction of desirableondensation of indole flavors. c to a serine-odified flavor, aromaerived aminoacrylate and/or moiety bound exture attributes. o pyridoxal phosphate.
Seven onstruction of genetictructural genes are required vectors for or controlled expressiontophan biosynthesis:
of RNA

dlor protein, fusionABCDEFG. TrpB encodes protein the roduction, genetic tophan synthase beta modification, chain (EC

utagenesis amplification.2.1.20). Tryptophan of is important for enetic material or avor development. Amino for other acid enetic or protein egradation products greatly manipulations.

tered survival characteristics:ontribute to flavor or to off flavors urvival of industrialRijnen et al., Appl.
processes, ( Environ.

owth or stora a in icrobiol. 65:4873-4880, roduct 1999).

SEQ SEQ ID

NO: NO: Utility Description DNA Pol a tide ormats, persistence in gut nvironment.

tered metabolic properties.

tered probiotic attributes.

odified health properties (including immunoregulatory, ticancer, gut health).

odified antibiotic resistance.

77 178 tered amino acid metabolism.omologue of trpC, encoding the emoval of undesirableifunctional enzyme flavor haracteristics. hosphoribosylanthranilate isomerase roduction of desirable(EC 5.3.1.24) -indoleglycerol flavors.

odified flavor, aromahosphate synthetase (EC
and/or 4.1.1.48).

exture attributes. rpC catalyzes the ring closure of 1-Construction of genetic(2-carboxyphenylamino)-1-vectors or controlled expressioneoxyribulose 5'-phosphate of RNA to d/or protein, fusion ' doleglycerol phosphate, protein the fifth roduction, genetic tep in the pathway of modification, tryptophan utagenesis amplificationiosynthesis from chorismate.
of Seven enetic material or tructural genes are required for other for _ tophan biosynthesis:
enetic or protein manipulations.

tered survival characteristics:ABCDEFG. Tryptophan is urvival of industrialportant for flavor development.
processes, owth or storage in ' o acid degradation product products ormats, persistence eatly contribute to flavor in gut or to off nvironment. avors (Rijnen et al., Appl. Environ.

tered metabolic properties.icrobiol. 65:48?3-4880, 1999).

tered probiotic attributes.

odified health properties (including immunoregulatory, ticancer, gut health).

odified antibiotic resistance.

77 179 tered amino acid metabolism.omologue of trpD, encoding emoval of undesirablehosphoribosyl anthranilate flavor haracteristics. ansferase (EC 2.4.2.18),.
TrpD

roduction of desirableatalyzes the reaction flavors. N-(5-phospho-odified flavor, aroma-ribosyl)-anthranilate andlor + diphosphate exture attributes. anthranilate + 5-phospho-a-D-Construction of genetic'bose 1-diphosphate.
vectors Seven or controlled expressiontructural genes are required of RNA for d/or protein, fusion tophan biosynthesis:
protein roduction, genetic ABCDEFG. Tryptophan is modification, utagenesis amplificationportant for flavor development.
of enetic material or ' o acid degradation for other products enetic or rotein manieatl contribute to flavor ulations. or to off SEQ SEQ ID

NO : NO: Utility Description DNA Pol a e tid tered survival characteristics:avors (Rijnen et al., Appl. Environ.

survival of industrialicrobiol. 65:4873-4880, processes, 1999).

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.

77 180 tered amino acid metabolism.omologue oftrpF, encoding emoval of undesirablehosphoribosylanthranilate flavor isomerase characteristics. C 5.3.1.24). TrpF catalyzes the roduction of desirableconversion of N-(5'-flavors.

odified flavor, aroma'phosphoribosyl)anthranilate and/or to t-exture attributes. (o-carboxyphenylamino)-1-Construction of geneticeoxyribulose 5 phosphate.
vectors Seven or controlled expressiontructural genes are required of RNA for d/or protein, fusion tophan biosynthesis:
protein roduction, genetic ABCDEFG. Tryptophan is modification, utagenesis amplificationportant for flavor development.
of enetic material or ' o acid degradation products for other enetic or protein eatly contribute to flavor manipulations. or to off ltered survival characteristics:avors (Rijnen et al., Appl. Environ.

survival of industrialicrobiol. 65:4873-4880, processes, 1999).

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.

78 181 C onstruction of geneticomologue of purine nucleoside vectors or controlled expressionhosphorylase (PNP) (EC
of RNA 2.4.2.1).

a nd/or protein, fusionNP catalyzes the reversible protein roduction, genetic hosphorolysis of (2'-deoxy)purine modification, utagenesis amplificationbonucleosides to free of ' base and (2'-enetic material or eoxy)ribose-1-phosphate for other and has a enetic or protein etabolic role in purine manipulations. salvage.

roduction of desirabletracellular phosphate flavors. levels odified flavor, aromauence survival of bacteria and/or in e xture attributes. vironmental stress conditions, n and SEQ SEQ ID

NO : NO: ~ Utility Description DNA Pol a tide tered survival characteristics:a 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.

tered metabolic properties-or egulation of metabolic athways.

tered probiotic attributes.

79 182 Construction of geneticomologue to relA. ReIA
vectors plays a role or controlled expression' synthesis and degradation of RNA of the d/or protein, fusion'ghly phosphorylated protein guanosine roduction, genetic ucleotides (p)ppGp. Intracellular modification, utagenesis amplificationhosphate levels influence of survival o enetic material or acteria in environmental for other stress enetic or protein onditions, and are involved manipulations. in the roduction of desirable' gent response.
flavors.

odified flavor, aroma and/or exture attributes.

tered survival characteristics:

urvival of industrial processes, owth or storage in product ormats, persistence in gut nvironment.

tered viability in response to tress conditions. .

tered metabolic properties or egulation of metabolic athways.

tered probiotic attributes.

80 183 tered cell wall or omologue of lysostaphin, cell surface an haracteristics, structurestimicrobial immunity or factor of ctions. taphylococcus simulans biovar proved antimicrobialtaphylolyticus active against roperties taphylococcus aureas.
Lysostaphin odifled adhesion s currently being investigated to human or ' for use al cells or cell gainst mastitis in dairy lines. cattle, caused roduction of desirabley Staphylococcus aureus flavors. (Kerr et odified flavor, aromal., Nat. Biotechnol.
andlor 19:66-70, 2001).

exture attributes. a gene contains the cohserved Construction of geneticotif GPHLHF, which is vectors also present or controlled expressionn several secreted peptidases.
of RNA ' d/or rotein, fusion sosta hin has utility rotein as an SEQ SEQ ID
NO : NO: Utility Description DNA Pol a tide roduction, genetic timicrobial for human modification, and utagenesis amplificationeterinary use.
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 atkributes.

odified health properties (including immunoregulatory, ticancer, gut health)..

odified antibiotic resistance.

proved fermentation properties o r other industrially useful rocesses.

Isolated polynucleotides of the present invention include the polynucleotides identified herein as SEQ ID NOS: 1-80; isolated polynucleotides comprising a polynucleotide sequence 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 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 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 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, 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' to 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, 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.
The polynucleotides identified as SEQ )Z7 NOS: 1-80 contain open reading frames ("ORFs"), or partial open reading frames, encoding polypeptides. Additionally, polynucleotides identified as SEQ )D 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 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, 3o Wellcome Trust Genome Campus, Hinxton, Cambridge CB 10 1 SA, 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 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 reading frames encoding polypeptides may be identified using the polynucleotides of the presentinvention.
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, l0 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 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 the polynucleotides identified as SEQ ID NOS: 1-80. The value ofx 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 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 3o purified products, or may be produced parhially or wholly using recombinant techniques. Such polypeptides may be glycosylated with bacterial, fungal, mammalian or other eukaryotic carbohydrates or may be non-glycosylated.

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 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, to 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 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 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 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 Mernfield solid-phase synthesis method, where amino acids are sequentially added to a 3o growing amino acid chain (See Mernfield, 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 using standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis (Kunkel, Proc. Natl. Acad. Sci. USA 82: 488-492, 1985). Sections of DNA
sequences may also be removed using standard techniques to permit preparation of truncated polypeptides.
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 to 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%, 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.
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
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 (NCB)], National Library of Medicine, 3o Building 38A, Room 8N805, Bethesda, MD 20894, USA. The BLASTN algorithm Version 2Ø4 [Feb-24-1998], Version 2Ø6 [Sept-16-1998] and Version 2Ø11 (Tan-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 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.
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.Ou4 [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.
l0 T'he 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 -a 10 -GO -EO
-r 1 -v 30 -b 30 -i queryseq -o results; the parameters are: -p Program Name [String]; -d Database [String]; -a 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 2o InJ; 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 ~-a 10 -G 0 -E
0 -v 30 -b 30 -i queryseq -o results; the parameters are: -p Program Name [String]; -d Database [String]; -a 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 OutJ Optional. The "hits" to one or more database sequences by a queried sequence produced by BLASTN, FASTA, BLASTP or a similar 3o 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.

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 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 to 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 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 2o 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 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 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;

and then multiplying by 100 to detemline 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 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 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 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 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 65°C, 6X SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in 1X SSC, 0.1 % SDS at 65° C and two washes of 30 minutes each in 0.2X SSC, 0.1 %
SDS at 65°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 3o the present invention. Thus, polynucleotides comprising sequences that differ from the polynucleotide sequences recited in SEQ 117 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 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 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 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 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 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 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 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 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 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.
to 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 polynucleotides comprising a 20-mer, a 40-mer, a 60-mer, an 80-mer, a 100-mer, a 120-mer, a 1 SO-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 ID 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
2o 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 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 3o 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, more usually less than about 500 mM and 'preferably less than about 200 mM.
Hybridization temperatures can be as low as 5°C, but are generally greater than about 22°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 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.
1o 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.
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 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, 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 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
D7 NOS: 1-80.
so 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 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, to 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.
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 2o 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 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 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 1 mm2.
Preferably each discrete area is less than about 10,000 mm2, more preferably less than about 100 mmz.
In this manner, 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 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 to 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 32P, 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 probe arrays is available from Af~ymetrix, 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 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 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.
In this manner, oligonucleotide probe kits of the present invention may be employed to examine the presencelabsence (or relative amounts in case of mixtures) of polynucleotides in different samples or products containing different materials rapidly and in a cost-effective manner. Examples of microbial species which may be examined using the present invention, include lactic acid bacteria, such as Lactobacillus rhamnosus, and other miczobial 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 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 to 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 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 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 m NOS: 1-80, and variants of these polynucleotides.
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 3o 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 termination sequences are functional 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 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 to 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, 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 2o 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, 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 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 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:
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
Lactobacillus coryniformis, 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 hetero, Lactobacillus hilgardii, Lactobacillus homohiochii, Lactobacillus japonicus, Lactobacillus johnsonii, Lactobacillus kefiri, Lactobacillus lactis, Lactobacillus leichmannii, Lactobacillus lindneri, Lactobacillus mall, Lactobacillus maltaromicus, Lactobacillus manihotivorans, Lactobacillus mucosae, Lactobacillus murinus, Lactobacillus oris, Lactobacillus panis, Lactobacillus paracasei, Lactobacillus paracasei subsp.
2o 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 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 micxobe, 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.
3o 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 Forest Res. Papers, Vol. 595, 199; 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 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 l0 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, 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 2o 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 of plants, (Current Plant Science and Biotechnology in Agriculture), 20(12):471-540, 1995. Specific protocols for the 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.
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 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 tags) in a sample of material.
Detection of the tags) may be accomplished using a variety of conventional techniques, and 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 to 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 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 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 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 3o 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.

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 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
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 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 2o 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.
Tm_m__unogenlc 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 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 viral expression systems. . Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter and temvnator 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.

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 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 Garner will vary depending on the mode of administration. For parenteral administration, such 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 also be employed as Garners 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 2o 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, N~.
Other suitable adjuvants include alum, biodegradable nlicrospheres, 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 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 ~,g per kg of 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.
Example 1 ISOLATION AND CHARACTERIZATION OF DNA SEQUENCES FROM

1o 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 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 37°C with shaking (220 2o 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 NaCI, 30 mM
Tris pH
8.0, 0.5 n1M 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 ~.g/ml mutanolysin) and incubated at 37 °C for 45 min.
Equal volumes of EDTA (0.25 M) 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 mirl. 50 ~tl 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 3o (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.

After resuspension in 500 ~.1 TE buffer, DNase-free RNase was added to a final concentraion of 100 ~.g/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 chloroform:isoamylalcohol (24:1) extraction and dissolved in 250 ~,l TE
buffer.
DNA was digested with Sau3AI at a concentration of 0.004 U/~g in a total volume of 1480 ~,1, with 996 ~.1 DNA, 138.75 ~,1 lOX REACT 4 buffer and 252.75 ~1 H20.
Following incubation for 1 hour at 37 °C, DNA was divided into two tubes. 31 ~l 0.5 M EDTA was added to stop the digestion and 17 ~1 samples were taken for agarose gel analysis. Samples to 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 (1M NaCI, 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 loaded onto the gradient, with 3 ml of DNA solution on top. The gradients were centrifuged at 26K for 18 hours at 4°C in a Centricon T-2060 centrifuge using a Kontron TST 28-38 rotor.
After deceleration without braking (approx. 1 hour), the gradients were removed and fractions collected using an auto Densi-Flow (Haake-Buckler Instruments). Agarose gel was used to analyze the fractions. The best two pairs of fractions were pooled and diluted to contain less 2o 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 -20°C.
. DNA pellets were resuspended in 300 ~tl 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 pelleted again, dried and resuspended in 10 p,l TE buffer.
DNA was ligated into dephosphorylated BamHI-digested pBluescript SK lI+ and dephosphorylated BamHI-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.
3o 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-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
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 (Bobbins Scientific, Sunnyvale, CA) for liquid handling. DNA amplification was done in a 9700 PCB machine (Perkin Eliner/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 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.
BIASTNPolynucleotideAnal 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 2Ø11 [Jan-20-2000], set to the following running parameters: Unix running command: blastall -p blastn -d embldb -a 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 3o the computer algorithm BLASTN, as described above. The cDNA sequence of SEQ
ID N0:
33 was determined to have less than 90% identity, determined as described above, to sequences in the EMBL database using BLASTN, as described above.

BLASTP Amino Acid Analysis 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 databases (using BLASTP) were made as of August 12, 2002 using BLASTN
algorithm Version 2Ø11 [Jan-20-2000], and the following Unix running command: blastall -p blastp -d swissprottrembledb -a 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, 103, 108, 111, 114, 116, 119-122, 124, 125, 130, 134-136, 140, 146, 147, 152, 156, 159, 162, 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 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 2o 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.
BLASTX Polynucleotide 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 BLASTS were made as of August 12, 2002 using BLAST algorithm Version 2Ø11 [Jan-20-2000], and the following Unix running command: blastall p blastx -d swissprottrembleldb -a 10 -GO -EO -v 30 -b 30 -i queryseq -o.
The cDNA sequences of SEQ 117 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 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.
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, l0 respectively.
Several of the sequences provided in SEQ ID NO: 1-80 were found to be full-lengkh 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.

Polynucleotide Polypeptide SEQ ID NO: ORF SEQ ID NO:

1392-2444 _85 Polynucleotide Polypeptide SEQ ID NO: ORF SEQ ID NO:

41 1023-1895 ~ 131 71 7554-8516 16?

72 __23_44-3732170 72~ 3755-5674 171 Polynucleotide Polypeptide SEQ ID NO: ORIi' SEQ ID NO:

?7 4071-5096 179 80 269-1006 183 j Example 2 ISOLATION AND CHARACTERIZATION OF PURINE NUCLEOSIDE PHOSPHORYLASE
$ FROM L. RHAMNOSUS STRAIN HNOO 1 The full-length polynucleotide sequence of the deoD purine nucleoside phosphorylase gene AQI from L. rhamnosus HN001 is given in SEQ ID NO: ?8 and shown in Fig. 1 (with ATG initiation and translation stop codons boxed). The polypeptide sequence ofAQl is given 1o in SEQ U~ NO: 181 and shown in Fig. 2.
A 634 by internal AQI 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 is phagemid pBlueScript (pBS-SK+) (Stratagene, La Jolla CA, USA). The ampicillin resistance gene in pBS-SK+ was removed by digestion with RcaI (Roche, Auckland, New Zealand), and the 1,953 by fragment containing the ColEl 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 2o pVA891 (Macrina et al., Gene 25:145-50, 1983) with CIaI and HindIII and treatment with Klenow to give blunt ends. The 1.6 kb Em fragment was ligated to the 1,953 by pBS-SK+
fragment, transformed into E. coli TGl (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 g.glml Em. Maintenance of a-complementation for blue/white color selection of recombinant pBEryl clones was confirmed by growing E coli colonies on agar plates containing IPTG and X-gal.
The resulting pBEryl construct encoding the HN001 deoD purine nucleoside phosphorylase AQI gene was transformed into competent HN001 cells and grown anaerobically for 48 hrs at 37 °C on MRS lactobacilli agar (Difco, Detroit MI) containing 2.5 p.g/ml Em. Em-resistant HN001 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 to endogenous HN001 deoD purine nucleoside phosphorylase AQl gene were assessed for increased resistance to UV irradiation. Briefly, single colonies of wild-type ofAQl -knockout HN001 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 OD6oo of approximately 0.3) at 37 °C, and then 5 ml of culture taken and cells collected by centrifugation. Cells were resuspended in 1 ml normal saline (0.9% NaCI), 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, 2o 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 forAQl' HN001 strain (~) and wild-type HN001 (~).
Results indicate that the AQI - HN001 mutant strain showed enhanced survival to exposure to ITV light compared to wild-type HN001. UV light exposure of 20 seconds appeared to have no effect onAQl- viability while the viability of wild-type HN001 cells had dropped to.34.7%.
After 30 seconds LTV light exposure, 86.9% of AQI' cells survived compared to only 27.9%
for wild type. Therefore, removal of AQI gene expression led to enhanced survival of LTV
light exposure, indicating that AQI encodes the HN001 deoD purine nucleoside phosphorylase.
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 W
light exposure, as well as high salt, pH and temperature (Duwat et al, Int J Food Microbiol.
55:83-6, 2000).
Applications for HN001 purine nucleoside phosphorylase AQ1 include:
~ methods of enhanced survival of industrial processes;
~ improved colonization of human intestinal environment; and ~ improved survival of multiple stress conditions.
Example 3 ISOLATION AND CHARACTERIZATION OF GTP PYROPHOSPHOKINASE
FROM L. RHAMNOSUS HNOO 1 The full-length polynucleotide sequence of the relA GTP pyrophosphokinase gene AMl 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 AM1 is given in SEQ ID N0: 182 and shown in Fig. 5.
A 798 by internal AMl 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 AMI gene was transformed into competent HN001 cells and grown 2o anaerobically for 48 hrs at 37 °C on MRS lactobacilli agar (Difco, Detroit MIJ containing 2.5 pglml 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 AM1 internal fragment-specific primers.
Colonies giving PCR patterns consistent with the insertional inactivation of the endogenous HN001 relA GTP pyrophosphokinase AMl gene, were assessed for increased resistance to LTV irradiation as described in Example 2.
Fig. 6 shows the results of a LTV light exposure assay measuring relative viability in response to increasing doses of W light in AMI' HN001 ( ~ ) and wild-type HN001 (~) strains. Results indicate that the AMI' HN001 mutant strain showed enhanced survival to exposure to UV light compared to wild-type HN001. LTV light exposure of 20 seconds appeared to have little effect on AMI' cell viability (91.7%) while the viability of wild-type HN001 cells had dropped to 34.7%. After 30 seconds IJV light exposure, 61.1%
ofAMl' cells survived compared to only 27.9% for wild type. Therefore, removal of AMI gene expression led to enhanced survival of UV light exposure, indicating that AMI 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 nutrient starvation and other environmental stresses (reviewed in Chatterji and Ojha, Curr Opin Microbiol. 4:160-5, 2001). Studies have indicated that sunnression 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 ,I. Food Microbiol. 55:83-6, 2000).
Applications for to 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.
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.
While in the foregoing specification this invention has been described in relation to certain prefer-ed 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.

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Claims (28)

We claim:

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 100-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.

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.

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.

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.