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THIS IS VOLUME 1 OF 2
NOTE: For additional vohxmes please contact the Canadian Patent Oi~ice.
CA 02549710 2006-06-14
WO 2005/056801 PCT/NZ2003/000278
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;
1
CA 02549710 2006-06-14
WO 2005/056801 PCT/NZ2003/000278
Yasui et al., Antonie van Leeuwenhoek 76:383-389, 1999; Hirayama and Rafter,
Antonie van
Leeuwenhoek 76:391-394, 1999; Ouwehand, 1998. In: Lactic Acid Bacteria,
Salminen S and
von Wright A (eds)., Marcel Dekker Inc, New York, Basel, Hong Kong, pp. 139-
159; Isolauri
et al., S 1998. In: Lactic Acid Bacteria, Salminen S and von Wright A (eds).,
Marcel Dekker
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.
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WO 2005/056801 PCT/NZ2003/000278
Immune system modulation and treatment of autoimmune disorders and allergies -
due
to enhancement of non-specific and antigen-specific defence against infection
and tumors;,
enhanced mucosal immunity; adjuvant effect in antigen-specific immune
responses; and
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.
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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
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CA 02549710 2006-06-14
WO 2005/056801 PCT/NZ2003/000278
starter cultures that grow up during the manufacture of the cheese, or
adventitious and adjunct
non-starter lactic acid bacteria that grow in the cheese as it ripens (Law
Haandrikman, Int.
Dairy J. 7:1-11, 1997).
Many other enzymes can also influence dairy product flavor, and functional and
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
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CA 02549710 2006-06-14
WO 2005/056801 PCT/NZ2003/000278
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
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CA 02549710 2006-06-14
WO 2005/056801 PCT/NZ2003/000278
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
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WO 2005/056801 PCT/NZ2003/000278
frames or full-length genes, that then can be used as expressible or otherwise
functional DNA
in transgenic organisms. Similarly, RNA sequences, reverse sequences,
complementary
sequences, antisense sequences and the like, corresponding to the
polynucleotides of the
present invention, may be routinely ascertained and obtained using the
polynucleotides
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
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WO 2005/056801 PCT/NZ2003/000278
techniques well known in the art, such as polymerase chain reaction (PCR) and
DNA
hybridization, as detailed below.
The inventive polynucleotides and polypeptides may also be employed in methods
for
the selection and production of more effective probiotic bacteria; as
"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
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WO 2005/056801 PCT/NZ2003/000278
identifier into an organism, the polynucleotide comprising at least a portion
of a
polynucleotide of the present invention.
The polynucleotides of the present invention may also be used as promoters,
gene
regulators, origins of DNA replication, secretion signals, cell wall or
membrane anchors for
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.
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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.
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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.
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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-
145
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.
13
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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-
14
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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
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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
16
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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.
17
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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
18
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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
19
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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
CA 02549710 2006-06-14
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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.
21
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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
.
22
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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
23
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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.
24
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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.
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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
26
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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
27
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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
28
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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
.
29
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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
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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
31
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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
32
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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).
33
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.
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
34
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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
CA 02549710 2006-06-14
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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
36
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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.
37
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SEQ SEQ ID
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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
38
CA 02549710 2006-06-14
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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
39
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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
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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.
41
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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
42
CA 02549710 2006-06-14
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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
43
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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
44
CA 02549710 2006-06-14
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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
CA 02549710 2006-06-14
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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
46
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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
47
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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
48
CA 02549710 2006-06-14
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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
49
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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
CA 02549710 2006-06-14
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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).
51
CA 02549710 2006-06-14
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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
52
CA 02549710 2006-06-14
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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
53
CA 02549710 2006-06-14
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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.
54
CA 02549710 2006-06-14
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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:
CA 02549710 2006-06-14
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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
56
CA 02549710 2006-06-14
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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
57
CA 02549710 2006-06-14
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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,
58
CA 02549710 2006-06-14
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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
59
CA 02549710 2006-06-14
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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.
CA 02549710 2006-06-14
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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.
61
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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
62
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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).
63
CA 02549710 2006-06-14
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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
64
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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
CA 02549710 2006-06-14
WO 2005/056801 PCT/NZ2003/000278
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).
66
CA 02549710 2006-06-14
WO 2005/056801 PCT/NZ2003/000278
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
67
CA 02549710 2006-06-14
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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
68
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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
69
CA 02549710 2006-06-14
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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
CA 02549710 2006-06-14
WO 2005/056801 PCT/NZ2003/000278
fragment of the corresponding polynucleotide, and the definition of
"polynucleotide" therefore
includes all operable antisense fragments. Antisense polynucleotides and
techniques involving
antisense polynucleotides are well known in the art and are described, for
example, in
Robinson-Benion, et al., "Antisense techniques," Methods in Enzymol. 254(23):
363-375,
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
71
CA 02549710 2006-06-14
WO 2005/056801 PCT/NZ2003/000278
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.
72
CA 02549710 2006-06-14
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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
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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
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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.
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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;
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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
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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
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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,
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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
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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,
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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
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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
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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
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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
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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
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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.
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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.
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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
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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
LACTOBACILLUS RHAMNOSUS STRAIN HNOO1
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.
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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-
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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.
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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,
101
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
180-182
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
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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.
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TABLE 2
Polynucleotide Polypeptide
SEQ ID NO: ORF SEQ ID NO:
1 5831-7288 81
2 4395-5630 82
3 1445-2791 83
4 316-1413 84
1392-2444 _85
6 1-1083 86
7 2881-4071 87
8 1859-3295 88
9 265-1023 89
1160-2005 90
11 2324-3604 91
11 548-1696 92
12 1102-2358 93
12 188-1020 94
13 140-1138 95
14 5612-6413 96
84-2276 97
16 1130-2275 98
17 1644-2645 99
18 500-2404 100
19 110-1153 101
167-718 102
21 1-3669 103
22 2327-4951 104
23 522-1694 105
24 973-2928 106
133-1296 107
26 1938-3497 108
27 69-716 109
28 125-1054 110
29 84-3377 111
555-887 112
30 226-558 113
31 77-862 114
32 3135-4673 115
32 6384-7877 116
33 412-828 117
33 863-1663 118
34 1642-2682 119
814-2037 120
35 3510-5084 121
36 101-1222 122
37 113-760 123
38 1-477 124
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Polynucleotide Polypeptide
SEQ ID NO: ORF SEQ ID NO:
39 12447-13400 125
40 1186-2439 126
41 7973-8812 127
41 6950-7924 128
41 1925-2773 129
41 3916-4956 130
41 1023-1895 ~ 131
41 8822-10489 132
42 102-860 133
43 3759-4343 134
44 622-1113 135
45 1129-1722 136
46 1216-2439 137
46 2345-3835 138
47 6155-8361 139
48 550-1176 140
49 455-901 141
51 31-1179 143
52 766-2142 144
53 2336-3817 145
54 7567-9057 146
55 3713-4447 147
56 426-1625 148
57 138-851 149
58 3066-4769 150
59 31-1188 151
60 132-1328 152
61 94-534 153
62 652-1998 154
63 1033-1905 155
63 1902-2789 156
64 951-1646 157
64 1824-3227 158
65 179-1030 159
66 1244-2425 160
67 6885-7589 161
68 333-1121 162
69 2638-4251 163
69 1312-2622 164
70 1587-2441 165
71 4642-6489 166
71 7554-8516 16?
71 6625-7536 168
71 8518-9469 169
72 __23_44-3732170
72~ 3755-5674 171
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Polynucleotide Polypeptide
SEQ ID NO: ORIi' SEQ ID NO:
73 102-1874 172
74 2993-4429 173
75 1008-1499 174
76 3565-5967 175
77 759-1708 176
77 1488-2837 177
77 3295-4074 178
?7 4071-5096 179
77 2692-3291 180
78 165-842 181
79 66-2291 182
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
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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
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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
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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.
100
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