U.S. patent application number 10/832946 was filed with the patent office on 2005-08-25 for linoleate isomerase.
This patent application is currently assigned to Arkion Life Sciences LLC d/b/a Bio-Technical Resources Division, Arkion Life Sciences LLC d/b/a Bio-Technical Resources Division. Invention is credited to Deng, Ming-De, Grund, Alan D., Rosson, Reinhardt A., Sanchez-Riera, Fernando.
Application Number | 20050186583 10/832946 |
Document ID | / |
Family ID | 32329722 |
Filed Date | 2005-08-25 |
United States Patent
Application |
20050186583 |
Kind Code |
A1 |
Rosson, Reinhardt A. ; et
al. |
August 25, 2005 |
Linoleate isomerase
Abstract
The present invention provides an isolated linoleate isomerase
and its nucleic acid and amino acid sequence. The present invention
also provides a method for producing CLA from an oil using an
immobilized bacterial cell or an isolated linoleate isomerase.
Inventors: |
Rosson, Reinhardt A.;
(Manitowoc, WI) ; Grund, Alan D.; (Manitowoc,
WI) ; Deng, Ming-De; (Manitowoc, WI) ;
Sanchez-Riera, Fernando; (Manitowoc, WI) |
Correspondence
Address: |
SHERIDAN ROSS PC
1560 BROADWAY
SUITE 1200
DENVER
CO
80202
|
Assignee: |
Arkion Life Sciences LLC d/b/a
Bio-Technical Resources Division
|
Family ID: |
32329722 |
Appl. No.: |
10/832946 |
Filed: |
April 26, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10832946 |
Apr 26, 2004 |
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09221014 |
Dec 23, 1998 |
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6743609 |
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60068617 |
Dec 23, 1997 |
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60089560 |
Jun 17, 1998 |
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Current U.S.
Class: |
435/134 ;
435/252.3; 530/388.4 |
Current CPC
Class: |
C12P 7/6472 20130101;
C12N 11/00 20130101; C12N 11/02 20130101; C12N 9/90 20130101; C12Y
502/01005 20130101; C12P 7/6427 20130101 |
Class at
Publication: |
435/006 ;
435/134; 435/252.3; 530/388.4 |
International
Class: |
C12Q 001/68; C07F
009/02; C12P 007/64; C07K 016/12; C12N 001/21 |
Claims
1-27. (canceled)
28. An isolated antibody that selectively binds to an isolated
linoleate isomerase of selected from the group consisting of
Lactobacillus, Clostridium, Propionibacterium, Butyrivibrio, and
Eubacterium linoleate isomerases.
29-44. (canceled)
45. An immobilized bacterial cell selected from the group
consisting of immobilized Lactobacillus, Clostridium,
Propionibacterium, Butyrivibrio, Escherichia, Bacillus and
Eubacterium cells, wherein said cell comprises a genetic
modification that increases the action of linoleate isomerase.
46-49. (canceled)
50. The immobilized bacterial cell of claim 47, wherein said
recombinant nucleic acid molecule comprises a nucleic acid sequence
that hybridizes under stringent hybridization conditions to a
nucleic acid molecule comprising the complement of a sequence
selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ
ED NO:10, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17
and SEQ ID NO:26.
51. The immobilized bacterial cell of claim 47, wherein said
recombinant nucleic acid molecule comprises a nucleic acid sequence
selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ
ID NO:10, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17
and SEQ ID NO:26.
52. The immobilized bacterial cell of claim 47, wherein said
recombinant nucleic acid molecule comprises nucleic acid sequence
SEQ ID NO:17.
53. The immobilized bacterial cell of claim 47, wherein said
recombinant nucleic acid molecule encodes a protein comprising an
amino acid sequence with at least about 70% identity with the amino
acid sequence selected from the group consisting of SEQ ID NO:1,
SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:11 and SEQ ID NO:18.
54. The immobilized bacterial cell of claim 47, wherein said
recombinant nucleic acid molecule encodes a protein comprising
amino acid sequence SEQ ID NO:18.
55. (canceled)
56. The immobilized bacterial cell of claim 47, wherein said
recombinant nucleic acid molecule encoding linoleate isomerase
comprises a genetic modification which increases the action of said
linoleate isomerase.
57-60. (canceled)
61. The immobilized bacterial cell of claim 45, wherein said cell
has been immobilized by crosslinking with a bifunctional or
multifunctional crosslinking agent.
62. (canceled)
63. Amended) A method for producing CLA, comprising contacting an
oil, said oil comprising a compound selected from the group
consisting of linoleic acid and linolenic acid, with the
immobilized bacterial cell of claim 45.
64-78. (canceled)
79. An isolated nucleic acid molecule selected from the group
consisting of: (a) a nucleic acid molecule comprising a nucleic
acid sequence that encodes a protein having an amino acid sequence
selected from the group consisting of SEQ ID NO:1, SEQ ID NO:5, SEQ
ID NO:9, SEQ ID NO:11, and SEQ ID NO:18; (b) a nucleic acid
molecule encoding a homologue of any of said amino acid sequences
of (a), wherein said homologue comprises at least 8 contiguous
amino acids having 100% identity with amino acids in said amino
acid sequence selected from the group consisting of SEQ ID NO:1,
SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:11, and SEQ ID NO:18; (c) a
nucleic acid molecule comprising a naturally occurring allelic
variant of a nucleic acid molecule encoding any of said amino acid
sequences of (a); and, (d) a nucleic acid molecule that is
complementary to any of said nucleic acid molecules of (a), (b) or
(c).
80-81. (canceled)
82. The nucleic acid molecule of claim 79, wherein said nucleic
acid molecule comprises a nucleic acid sequence with at least about
70% identity with a nucleic acid sequence selected from the group
consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:14,
SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:26.
83. The nucleic acid molecule of claim 79, wherein said nucleic
acid molecule comprises a nucleic acid sequence having at least 24
contiguous nucleotides having 100% identity with nucleic acid
sequence SEQ ID NO:17.
84. (canceled)
85. The isolated nucleic acid molecule of claim 79, wherein said
nucleic acid molecule encodes a protein comprising amino acid
sequence SEQ ID NO:18.
86. The isolated nucleic acid molecule of claim 79, wherein said
nucleic acid molecule is selected from the group consisting of a
nucleic acid molecule having a sequence selected from the group
consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:14,
SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:26.
87. The isolated nucleic acid molecule of claim 79, wherein said
nucleic acid molecule comprises nucleic acid sequence SEQ ID
NO:17.
88-90. (canceled)
91. A recombinant molecule comprising an isolated nucleic acid
molecule as set forth in claim 79 operatively linked to a
transcription control sequence.
92. A recombinant virus comprising an isolated nucleic acid
molecule as set forth in claim 79.
93. A recombinant cell comprising an isolated nucleic acid molecule
as set forth in claim 79, wherein said cell expresses said nucleic
acid molecule.
94-96. (canceled)
97. Amended) A method to produce linoleate isomerase, comprising
culturing a recombinant cell of claim 93.
98-99. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. Provisional
Application No. 60/068,617, filed Dec. 23, 1997, and from U.S.
Provisional Application No. 60/089,560, filed Jun. 17, 1998, both
of which are incorporated herein by reference in their
entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to an isolated linoleate
isomerase enzyme, an nucleic acid molecule encoding a linoleate
isomerase enzyme, to immobilized cells containing a linoleate
isomerase enzyme, and to a method for converting linoleic acid or
linolenic acid to CLA using the isolated linoleate isomerase
enzyme, nucleic acid molecule and/or immobilized cells.
BACKGROUND OF THE INVENTION
[0003] The term "CLA" is used herein as a generic term to describe
both conjugated linoleic acid and conjugated linolenic acid. The
CLA compounds (cis,trans)-9,11-linoleic acid and
(trans,cis)-10,12-linoleic acid are recognized nutritional
supplements and effective inhibitors of epidermal carcinogenesis
and forestomach neoplasia in mice, and of carcinogen-induced rat
mammary tumors. CLA has also been shown to prevent adverse effects
caused by immune stimulation in chicks, mice and rats, and has been
shown to decrease the ratio of low density lipoprotein cholesterol
to high density lipoprotein cholesterol in rabbits fed an
atherogenic diet. CLA also reduces body fat in mouse, rat, chick
and pig models. CLA has also been shown to be effective in treating
skin lesions when included in the diet.
[0004] CLA occurs naturally in various amounts in virtually all
foods. The principle natural sources of CLA are dairy products,
beef and foods derived from ruminant animals. In the U.S., beef,
beef tallow, veal, lamb (3-4 mg CLA/g fat; 84% cis-9, trans-11) and
dairy products (3-7 mg CLA/g fat; 80-90% cis-9, trans-11) have the
highest concentration of CLA. CLA concentrations 2-3 times higher
are found in Australian dairy products and pasture-fed beef and
lamb. Very low concentrations of CLA (0.1-0.7 mg CLA/g fat; ca. 40%
each cis-9, trans-11 and trans-10, cis-12) are found in commercial
vegetable oils.
[0005] CLA is a normal intermediate of linoleic acid metabolism. In
cows, (cis,trans)-9,11-CLA produced by natural bacterial flora that
is not further metabolized is incorporated into lipids and then
into host tissues and milk. Animals take up and incorporate CLA
into normal tissue and milk from dietary sources such as milk, milk
products or meat containing CLA, or from CLA dietary
supplements.
[0006] CLA can be synthetically obtained from alkaline
isomerization of linoleic or linolenic acid, or of vegetable oils
which contain linoleic acid, linolenic acid or their derivatives.
Heating vegetable oil at about 180.degree. C. under alkaline
conditions catalyzes two reactions: (1) fatty acid ester bonds from
the triglyceride lipid backbone are hydrolyzed, producing free
fatty acids; and (2) unconjugated unsaturated fatty acids with two
or more appropriate double bonds are conjugated. Commercial CLA
oils available at the present time, typically made from sunflower
oil, are sold without further purification. They contain a mixture
of CLA isomers as well as other saturated and unsaturated fatty
acids. Generally, chemical synthesis produces about 20-35%
(cis,trans)-9,11-CLA and about 20-35% (trans,cis)-10,12-CLA, and
the balance as a variety of other isomers. The presence of the
non-active, non-natural isomers introduces the need to purify
(cis,trans)-9,11-CLA and/or (trans,cis)-10,12-CLA, or to
demonstrate the safety and seek regulatory approval of these
non-beneficial, non-natural isomers for human use. It is not
feasible economically, however, to isolate single isomers of CLA
from the CLA made by alkaline isomerization. Using a fractional
crystallization procedure, it is possible to enrich 9,11-CLA
relative to 10,12-CLA and vice versa. Another approach, described
in WO 97/18320 to Loders Croklaan B. V. uses lipases to selectively
esterify 10,12-CLA and thus enrich the 9,11-CLA fraction. None of
the above-described methods, however, allow for the production of
high purity, single isomer CLA.
[0007] One method of overcoming the shortcomings of chemical
transformation is a whole cell transformation or an enzymatic
transformation of linoleic acid, linolenic acid or their
derivatives to CLA. It is well known that a biological system can
be an effective alternative to chemical synthesis in producing a
desired chemical compound where such a biological system is
available. The existence of linoleate isomerase enzyme to convert
linoleic acid to CLA has been known for over thirty years, however,
no one has yet successfully isolated the enzyme. And because it has
not yet been isolated, the linoleate isomerase enzyme has not been
sequenced.
[0008] In many microorganisms, the linoleate isomerase enzyme
converts linoleic acid to CLA as an intermediate in the
biohydrogenation step. Kepler and Tove have identified this enzyme
in Butyrivibrio fibrisolvens. Kepler and Tove, J. Biol. Chem.,
1966, 241, 1350. However, they could not solubilize the activity,
i.e., they were unable to isolate the enzyme in any significantly
pure form. Kepler and Tove, J. Biol. Chem., 1967, 242, 5686. In
addition, earlier studies have indicated that only compounds which
possess a free carboxyl group and a cis-9, cis-12 double bond
moieties are isomerized by linoleate isomerase. See Kepler and
Tove, Methods in Enzymology, 1969, 14, 105-109, and Kepler et al.,
J. Biol. Chem., 1970, 245, 3612.
[0009] Therefore, there remains a need for purifying and
identifying a linoleate isomerase enzyme and/or producing one by
recombinant techniques. There also remains a need for finding and
identifying an linoleate isomerase enzyme which does not require
presence of a free carboxylic acid group in the fatty acid for
isomerization. In addition, there remains a need for a method for
producing CLA utilizing whole cells or isolated linoleate isomerase
enzyme.
SUMMARY OF THE INVENTION
[0010] The present invention generally relates to isolated
linoleate isomerase nucleic acid molecules, isolated linoleate
isomerase proteins, immobilized bacterial cells having a genetic
modification that increases the action of linoleate isomerase, and
methods of using such nucleic acid molecules, proteins and cells to
produce CLA.
[0011] One embodiment of the invention relates to an isolated
linoleate isomerase. Included in the invention are linoleate
isomerases from Lactobacillus, Clostridium, Propionibacterium,
Butyrivibrio and Eubacterium, and particularly, from Lactobacillus
reuteri, Clostridium sporogenes, Propionibacterium acnes,
Butyrivibrio fibrisolvens, Propionibacterium acidipropionici,
Propionibacterium freudenreichii and Eubacterium lentum.
Particularly preferred linoleate isomerases include linoleate
isomerases from Lactobacillus reuteri, Clostridium sporogenes, and
Propionibacterium acnes.
[0012] In one embodiment, an isolated linoleate isomerase of the
present invention converts linoleic acid and linolenic acid to CLA,
including (cis, trans)-9,11-linoleic acid and/or (trans,
cis)-10,12-linoleic acid. A linoleate isomerase of the present
invention includes linoleate isomerases having one or more of the
following biochemical characteristics: a size of about 50 kDa or
about 67 kDa; an optimum pH of about 6.8 or about 7.5; a specific
linoleic acid isomerization activity of at least about 1000 nmoles
mg.sup.-1 min.sup.-1; a Km of about 8.1 .mu.M for linoleic acid, a
pH optimum of about 7.5, and a Ki of about 80 .mu.M for oleic acid;
and/or an initial velocity that decreases at about 60 .mu.M
linoleic acid. A linoleate isomerase of the present invention can
be either a membrane bound or a soluble enzyme. The linoleate
isomerase of the present invention can include lipid material.
[0013] In another embodiment, an isolated linoleate isomerase of
the present invention includes an amino acid sequence encoded by a
nucleic acid molecule that hybridizes under stringent hybridization
conditions to a nucleic acid molecule comprising the complement of
a sequence selected from the group of SEQ ID NO:4, SEQ ID NO:8, SEQ
ID NO:10, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17
and SEQ ID NO:26. In another embodiment, the isolated linoleate
isomerase is encoded by a nucleic acid molecule which includes a
nucleic acid sequence having at least 24 contiguous nucleotides
having 100% identity with nucleic acid sequence SEQ ID NO:17. In
yet another embodiment, an isolated linoleate isomerase of the
present invention includes an amino acid sequence with at least
about 70% identity with an amino acid sequence selected from the
group of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:11 and
SEQ ID NO:18.
[0014] Preferably, an isolated linoleate isomerase is encoded by a
nucleic acid molecule comprising a nucleic acid sequence selected
from the group of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, SEQ ID
NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:26,
with SEQ ID NO:17 being the most preferred. Isolated linoleate
isomerases of the present invention include proteins having an
amino acid sequence selected from the group of SEQ ID NO:1, SEQ ID
NO:5, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:18, with SEQ ID NO:18
being most preferred. Also included in the present invention are
homologues of linoleate isomerase proteins, including proteins
having an amino acid sequence having at least 8 contiguous amino
acids with 100% identity to SEQ ID NO:18, and proteins encoded by
naturally occurring allelic variants of linoleate isomerase nucleic
acid molecules.
[0015] In one embodiment, the linoleate isomerase is bound to a
solid support, which includes, but is not limited to organic
supports, biopolymer supports and inorganic supports.
[0016] Another embodiment of the present invention relates to an
isolated antibody that selectively binds to the isolated linoleate
isomerase of the present invention.
[0017] Yet another embodiment of the present invention relates to a
method for producing CLA, including contacting an oil; which
comprises a compound selected from the group of linoleic acid and
linolenic acid, with an isolated linoleate isomerase enzyme of the
present invention to convert at least a portion of the compound to
CLA. In one embodiment, the compound is in the form of a
triglyceride and the method further includes contacting the oil
with a hydrolysis enzyme to convert at least a portion of the
triglyceride to free fatty acids. Such a hydrolysis enzyme can
include lipases, phospholipases and esterases. The method of the
present invention can also include a step of recovering the CLA.
The CLA can included (cis, trans)-9,11-linoleic acid and/or (trans,
cis)-10,12-linoleic acid. The oil can include, but is not limited
to, sunflower oil, safflower oil, corn oil, linseed oil, palm oil,
rapeseed oil, sardine oil, herring oil, mustard seed oil, peanut
oil, sesame oil, perilla oil, cottonseed oil, soybean oil,
dehydrated castor oil and walnut oil. In one embodiment of the
method, the linoleate isomerase enzyme is bound to a solid support,
which can include organic supports, biopolymer supports and
inorganic supports.
[0018] Another aspect of the present invention relates to an
isolated nucleic acid molecule selected from the group of: (a) a
nucleic acid molecule comprising a nucleic acid sequence that
encodes a protein having an amino acid sequence selected from the
group of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:11, and
SEQ ID NO:18; (b) a nucleic acid molecule encoding a homologue of
any of such amino acid sequences of (a), wherein the homologue
comprises at least 8 contiguous amino acids having 100% identity
with amino acids in such amino acid sequences; (c) a nucleic acid
molecule comprising a naturally occurring allelic variant of a
nucleic acid molecule encoding any of such amino acid sequences of
(a); and, (d) a nucleic acid molecule that is complementary to any
of the nucleic acid molecules of (a), (b) or (c). Preferably, an
isolated nucleic acid molecule of the present invention encodes a
linoleate isomerase, including a linoleate isomerase homologue. In
one embodiment, such an isolated nucleic acid molecule hybridizes
under stringent conditions to a nucleic acid molecule having a
nucleic acid sequence selected from the group of SEQ ID NO:4, SEQ
ID NO:8, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16,
SEQ ID NO:17, SEQ ID NO:26, and/or the complement of any of such
nucleic acid sequences. In another embodiment, an isolated nucleic
acid molecule of the present invention includes a nucleic acid
sequence having at least about 70% identity with a nucleic acid
sequence selected from the group of SEQ ID NO:4, SEQ ID NO:8, SEQ
ID NO:10, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17
and SEQ ID NO:26. In yet another embodiment, an isolated nucleic
acid molecule of the present invention includes a nucleic acid
sequence having at least 24 contiguous nucleotides having 100%
identity with nucleic acid sequence SEQ ID NO:17. Preferred nucleic
acid molecules of the present invention include molecules that
hybridize under stringent hybridization conditions with a nucleic
acid molecule selected from the group of nCLA.sub.87, nCLA.sub.596,
nCLA.sub.1709, nCLA.sub.3551, nCLA.sub.1776 and nCLA.sub.7113. More
preferably, an isolated nucleic acid molecule of the present
invention has a sequence selected from the group of SEQ ID NO:4,
SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:15, SEQ ID
NO:16, SEQ ID NO:17 and SEQ ID NO:26, with SEQ ID NO:17 being more
preferred. An isolated nucleic acid molecule of the present
invention preferably encodes a linoleate isomerase protein of the
present invention as described above.
[0019] The isolate nucleic acid molecule of the present invention
includes linoleate isomerase nucleic acid molecules from
microorganisms including, but not limited to, Lactobacillus,
Clostridium, Propionibacterium, Butyrivibrio, and Eubacterium, with
Lactobacillus reuteri, Clostridium sporogenes, Propionibacterium
acnes, Butyrivibrio fibrisolvens, Propionibacterium
acidipropionici, Propionibacterium freudenreichii and Eubacterium
lentum being particularly preferred. Most preferred linoleate
isomerase nucleic acid molecules are from Lactobacillus reuteri,
Clostridium sporogenes, or Propionibacterium acnes.
[0020] Also included in the present invention are recombinant
molecules, recombinant viruses and recombinant cells which include
an isolated nucleic acid molecule of the present invention. In one
embodiment, as recombinant cell of the present invention is from a
microorganism which includes, but is not limited to, Lactobacillus
reuteri, Clostridium sporogenes, Propionibacterium acnes,
Propionibacterium freudenreichii, Propionibacterium
acidipropionici, Escherichia coli, Bacillus subtilis, Bacillus
licheniformis, Butyrivibrio fibrisolvens, or Eubacterium lentum,
with Escherichia coli, Bacillus subtilis and Bacillus licheniformis
being most preferred.
[0021] Yet another embodiment of the present invention relates to a
method to produce linoleate isomerase, comprising culturing a
recombinant cell transformed with an isolated nucleic acid molecule
encoding linoleate isomerase.
[0022] Another embodiment of the present invention relates to a
method for producing CLA, including contacting an oil which
comprises a compound selected from the group of linoleic acid and
linolenic acid, with an isolated linoleate isomerase enzyme encoded
by the isolated nucleic acid molecule of the present invention to
convert at least a portion of the compound to CLA.
[0023] Yet another embodiment of the present invention relates to
an immobilized bacterial cell having a genetic modification that
increases the action of linoleate isomerase. The cell can be a
microorganism which includes, but is not limited to Lactobacillus,
Clostridium, Propionibacterium, Butyrivibrio, Escherichia, Bacillus
or Eubacterium cells. In one embodiment, the genetic modification
results in overexpression of linoleate isomerase by the bacterial
cell. The genetic modification can result in at least one amino
acid modification selected from the group consisting of deletion,
insertion, inversion, substitution and derivatization of at least
one amino acid residue of the linoleate isomerase, wherein such
modification results in increased linoleate isomerase action,
reduced substrate inhibition, and/or reduced product inhibition. In
another embodiment, the genetic modification includes
transformation of the cell with a recombinant nucleic acid molecule
encoding a linoleate isomerase of the present invention, wherein
the recombinant nucleic acid molecule is operatively linked to a
transcription control sequence. The recombinant nucleic acid
molecule can include any of the isolated nucleic acid molecules
described above, including a nucleic acid sequence encoding a
homologue of linoleate isomerase. In one embodiment, such a
homologue has an amino acid sequence having at least 8 contiguous
amino acids with 100% identity to amino acid sequence SEQ ID
NO:18.
[0024] In one embodiment, the recombinant nucleic acid molecule is
integrated into the genome of the bacterial cell. In another
embodiment, the recombinant nucleic acid molecule encoding
linoleate isomerase comprises a genetic modification which
increases the action of the linoleate isomerase and in another
embodiment, the genetic modification reduces substrate and/or
product inhibition of the linoleate isomerase.
[0025] In another embodiment, an immobilized bacterial cell of the
present invention can be lysed. The cell can be immobilized by
crosslinking with a bifunctional or multifunctional crosslinking
agent, including, but not limited to glutaraldehyde.
[0026] Yet another embodiment of the present invention relates to a
method for producing CLA, including contacting an oil which
includes a compound selected from the group of linoleic acid and
linolenic acid, with an immobilized bacterial cell having a
linoleate isomerase, to convert at least a portion of the compound
to CLA. The bacterial cell can be from a microorganism including
Lactobacillus, Clostridium, Propionibacterium, Butyrivibrio,
Escherichia, Bacillus and Eubacterium cells, preferably
Lactobacillus reuteri, Clostridium sporogenes, Propionibacterium
acnes, Propionibacterium freudenreichii, Propionibacterium
acidipropionici, Escherichia coli, Bacillus subtilis, Bacillus
licheniformis, Butyrivibrio fibrisolvens, and Eubacterium lentum,
and most preferably, Escherichia coli, Bacillus subtilis or
Bacillus licheniformis. The cell can be a naturally occurring
bacterial cell having a linoleate isomerase, or a genetically
modified microorganism as described above. Preferably, a
genetically modified microorganism has increased linoleate
isomerase action. The compound can include compounds in the form of
a triglyceride such that at least a portion of the triglycerides
are converted to free fatty acids. Other features of the method are
as described above in the method to produce CLA.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1A is a line graph showing whole cell biotransformation
of CLA from linoleic acid by Clostridium sporogenes ATCC 25762
under aerobic conditions.
[0028] FIG. 1B is a line graph showing whole cell biotransformation
of CLA from linoleic acid by Clostridium sporogenes ATCC 25762
under anaerobic conditions.
[0029] FIG. 2A is a line graph illustrating whole cell
biotransformation of CLA from linoleic acid by C. bifermentans ATCC
638 under aerobic conditions.
[0030] FIG. 2B is a line graph illustrating whole cell
biotransformation of CLA from linoleic acid by C. bifermentans ATCC
638 under anaerobic conditions.
[0031] FIG. 3A is a line graph showing whole cell biotransformation
of CLA from linoleic acid by Propionibacterium jensenii ATCC
14073.
[0032] FIG. 3B is a line graph showing whole cell biotransformation
of CLA from linoleic acid by P. acnes ATCC 6919.
[0033] FIG. 4 is a line graph demonstrating whole cell
biotransformation of CLA from linoleic acid by P. acidipropionici
ATCC 25562.
[0034] FIG. 5 is a line graph illustrating whole cell
biotransformation of CLA from linoleic acid by L. reuteri PYR8.
[0035] FIG. 6 is a line graph showing DEAE chromatography of
detergent solubilized isomerase by L. reuteri PYR8.
[0036] FIG. 7 is a line graph demonstrating hydroxyapatite
chromatography of isomerase activity by L. reuteri PYR8.
[0037] FIG. 8 is a line graph illustrating chromatofocusing of
linoleic acid isomerase activity by L. reuteri PYR8.
[0038] FIG. 9 is a schematic illustration of the linoleate
isomerase genes and flanking open reading frames in L. reuteri
PYR8.
[0039] FIG. 10 is a schematic illustration of the putative
transcription terminator in the linoleate isomerase gene.
[0040] FIG. 11 is an illustration of several constructs for
linoleate isomerase expression in E. coli.
[0041] FIG. 12 is an illustration of several constructs for
linoleate isomerase expression in Bacillus.
[0042] FIG. 13 is a flow diagram of the experimental protocol for
the preparation of different protein fractions of E. coli which
have expressed recombinant linoleate isomerase.
[0043] FIG. 14 is a line graph showing the formation of t10,c12-CLA
from linoleic acid using whole cells of P. acnes.
[0044] FIG. 15 is a flow diagram showing the cell fractionation
protocol for P. acnes ATCC 6919.
[0045] FIG. 16 is a line graph showing the effect of pH on
linoleate isomerase activity in crude extracts of P. acnes ATCC
6919.
[0046] FIG. 17 is a line graph showing the time course of CLA
formation in crude extracts of P. acnes ATCC 6919.
[0047] FIG. 18 is a line graph showing the time course for the
formation of CLA in crude extracts of P. acnes ATCC 6919 at
different levels of linoleic acid.
[0048] FIG. 19. is a line graph showing end point for formation of
CLA in crude extracts of P. acnes ATCC 6919 at different levels of
linoleic acid.
[0049] FIG. 20 is a graph illustrating DEAE ion exchange
chromatography of total soluble protein from P. acnes ATCC
6919.
[0050] FIG. 21 is a graph illustrating hydrophobic interaction
chromatography of total soluble protein from P. acnes ATCC
6919.
[0051] FIG. 22 is a graph illustrating chromatofocusing of
isomerase activity from P. acnes ATCC 6919.
[0052] FIG. 23A is a graph showing a time course of CLA formation
by C. sporogenes ATCC 25762 under aerobic conditions at room
temperature.
[0053] FIG. 23B is a graph showing a time course of CLA formation
by C. sporogenes ATCC 25762 under anaerobic conditions at room
temperature.
[0054] FIG. 23C is a graph showing a time course of CLA formation
by C. sporogenes ATCC 25762 under aerobic conditions at 37.degree.
C.
[0055] FIG. 23D is a graph showing a time course of CLA formation
by C. sporogenes ATCC 25762 under anaerobic conditions at
37.degree. C.
[0056] FIG. 24 is a flow diagram showing an extraction protocol for
C. sporogenes ATCC 25762.
[0057] FIG. 25 is a line graph showing linoleate isomerase optimum
pH and temperature in C. sporogenes ATCC 25762.
[0058] FIG. 26 is a line graph showing optimum linoleic acid
concentration for C. sporogenes ATCC 25762 linoleate isomerase.
[0059] FIG. 27 is a graph showing the time course for CLA formation
by C. sporogenes ATCC 25762 linoleate isomerase.
[0060] FIG. 28 is a bar graph illustrating the stability of C.
sporogenes ATCC 25762 linoleate isomerase in Tris and phosphate
buffers.
[0061] FIG. 29 is an elution profile of fresh C. sporogenes ATCC
25762 linoleate isomerase extracts from DEAE-5PW.
[0062] FIG. 30 is a bar graph demonstrating the effect of culture
medium on C. sporogenes ATCC 25762 growth and linoleate isomerase
activity.
[0063] FIG. 31 is a bar graph showing the effect of CaCl.sub.2 on
C. sporogenes ATCC 25762 linoleate isomerase activity.
[0064] FIG. 32 is a bar graph showing the effect of chelating
agents on C. sporogenes ATCC 25762.
[0065] FIG. 33 is a bar graph showing the effect of chelating
agents on stability of linoleate isomerase.
[0066] FIG. 34 is a line graph illustrating the effect of pH on
extraction efficiency of linoleate isomerase in C. sporogenes ATCC
25762.
[0067] FIG. 35 is a line graph demonstrating the half lives of
linoleate isomerase in C. sporogenes ATCC 25762 versus pH.
[0068] FIG. 36 is a bar graph showing the effect of buffer system
on the activity of linoleate isomerase in C. sporogenes ATCC
25762.
[0069] FIG. 37 is a line graph illustrating the effect of glycerol
and salt concentration on the stability of crude extracts of
linoleate isomerase in C. sporogenes ATCC 25762.
[0070] FIG. 38 is a line graph showing the stability of detergent
solubilized linoleate isomerase in C. sporogenes ATCC 25-762.
[0071] FIG. 39 is an elution profile of C. sporogenes ATCC 25762
linoleate isomerase on DEAE Mono Q.
[0072] FIG. 40 is an elution profile of C. sporogenes ATCC 25762
detergent solubilized linoleate isomerase on DEAE-5PW column.
[0073] FIG. 41 is an elution profile showing separation of
partially purified C. sporogenes ATCC 25762 linoleate isomerase by
chromatofocusing.
DETAILED DESCRIPTION OF THE INVENTION
[0074] One embodiment of the present invention is an isolated
linoleate isomerase enzyme. The isolated linoleate isomerase can be
used to produce CLA from linoleic acid, linolenic acid or their
derivatives. More specifically, isolated linoleate isomerase can
convert linoleic acid to conjugated linoleic acid and/or linolenic
acid to conjugated linolenic acid. The term "conjugated" refers to
a molecule which has two or more double bonds which alternate with
single bonds in an unsaturated compound. Linoleate isomerase is a
part of a biohydrogenation pathway in microorganisms which convert
linoleic acid and other unsaturated fatty acids containing a
9,12-diene moiety into a 9,11-conjugate diene moiety which is then
further metabolized to other fatty acids containing a 9-11 monoene
moiety. For example, most linoleate isomerase converts
(cis,cis)-9,12-linoleic acid to (cis,trans)-9,11-linoleic acid as
an intermediate in the biohydrogenation pathway. In many cases, the
formation of CLA is followed by metabolism to other CLA isomers as
well as metabolism to non-CLA compounds, such as a monoene fatty
acid. Lactobacillus reuteri, however, produces and accumulates CLA
as an end product. Other microorganisms such as Propionibacterium
acnes convert (cis,cis)-9,12-linoleic acid to
(trans,cis)-10,12-linoleic acid.
[0075] The term "isolated linoleate isomerase" refers to a
linoleate isomerase outside of its natural environment in a pure
enough form to achieve a significant increase in activity over
crude extracts having linoleate isomerase activity. Such a
linoleate isomerase can include, but is not limited to, purified
linoleate isomerase, recombinantly produced linoleate isomerase,
membrane bound linoleate isomerase, linoleate isomerase complexed
with lipids, linoleate isomerase having an artificial membrane,
soluble linoleate isomerase and isolated linoleate isomerase
containing other proteins. An "artificial membrane" refers to any
membrane-like structure that is not part of the natural membrane
which contain linoleate isomerase.
[0076] An isolated linoleate isomerase of the present invention can
be characterized by its specific activity. A "specific activity"
refers to the rate of conversion of linoleic acid to CLA by the
enzyme. More specifically, it refers to the number of molecules of
linoleic acid converted to CLA per mg of the enzyme per time unit.
Preferably, the isolated linoleate isomerase of the present
invention has a specific activity of at least about 1000 nmoles
mg.sup.-1 min.sup.-1, more preferably at least about 10,000 nmoles
mg.sup.-1 min.sup.-1, and most preferably at least about 100,000
nmoles mg.sup.-1 min.sup.-1.
[0077] Another way to characterize the isolated linoleate isomerase
is by its Michaelis-Menten constant (K.sub.m). K.sub.m is a kinetic
(i.e., rate) constant of the enzyme-linoleic acid complex under
conditions of the steady state. Preferably, the isolated linoleate
isomerase of the present invention has K.sub.m of at least about
8.1 .mu.M at a pH of about 7.5 and at a temperature of about
20.degree. C.
[0078] Yet another way to characterize the linoleate isomerase is
by oleic acid inhibition rate constant (K.sub.i). Specifically,
K.sub.i is a dissociation rate of the oleic acid-enzyme complex.
Preferably, the isolated linoleate isomerase of the present
invention has K.sub.i of from about 50 .mu.M to about 100 .mu.M at
a pH of about 7.5 and at a temperature of about 20.degree. C., and
more preferably, greater than 100 .mu.M, with no inhibition being
most preferred.
[0079] Still another way to characterize the isolated linoleate
isomerase is by its initial velocity (v.sub.0), i.e., initial rate
of product formation. The initial velocity (v.sub.0) refers to the
initial conversion rate of linoleic acid to CLA by the enzyme.
Specifically, it refers to the number of molecules of linoleic acid
converted to CLA per mg of the enzyme per time unit. Preferably,
the maximum initial velocity rate of the isolated linoleate
isomerase at a pH of about 7.5 is least about 100 nmoles/(sec-mg of
protein), more preferably at least about 1,000 nmoles/(sec-mg of
protein), and most preferably at least about 10,000 nmoles/(sec-mg
of protein).
[0080] The isolated linoleate isomerase can be further
characterized by its optimum pH. The optimum pH refers to the pH at
which the linoleate isomerase has a maximum initial velocity.
Preferably the optimum pH is between about 5 and about 10, more
preferably between about 6 and about 8, and most preferably from
about 6.8 to about 7.5.
[0081] Further embodiments of the isolated linoleate isomerase of
the present invention include proteins which are encoded by any of
the nucleic acid molecules which are described below.
[0082] Further embodiments of the present invention include nucleic
acid molecules that encode linoleate isomerases. In one embodiment,
such nucleic acid molecules include isolated nucleic acid molecules
that hybridize under stringent hybridization conditions with: the
complement of a gene encoding a naturally occurring linoleate
isomerase, a nucleic acid molecule comprising the complement of a
nucleic acid molecule encoding an amino acid sequence selected from
the group consisting of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9, SEQ
ID NO:11 and/or SEQ ID NO:18, or a nucleic acid molecule comprising
the complement of a nucleic acid molecule having a nucleic acid
sequence SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:14, SEQ
ID NO:15, SEQ ID NO:16, SEQ ID NO:17 or SEQ ID NO:26. In other
embodiments, the present invention includes an isolated nucleic
acid molecule that encodes a protein comprising amino acid sequence
selected from the group consisting of SEQ ID NO:1, SEQ ID NO:5, SEQ
ID NO:9, SEQ ID NO:11 and/or SEQ ID NO:18 and an isolated nucleic
acid molecule having a nucleic acid sequence of SEQ ID NO:4, SEQ ID
NO:8, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ
ID NO:17 or SEQ ID NO:26.
[0083] As used herein, a linoleate isomerase gene (i.e., nucleic
acid molecule which encodes a linoleate isomerase) of the present
invention can include an isolated natural linoleate isomerase gene
or a homologue thereof, the latter of which is described in more
detail below. A nucleic acid molecule of the present invention can
include one or more regulatory regions that control production of
the linoleate isomerase protein encoded by that gene (such as, but
not limited to, transcription, translation or post-translation
control regions) as well as a full-length or partial coding region
itself. It is to be noted that an isolated linoleate isomerase
nucleic acid molecule of the present invention need not encode a
protein having linoleate isomerase activity. A linoleate isomerase
nucleic acid molecule can encode a truncated, mutated or inactive
protein, for example. Such genes and the proteins encoded by such
genes are useful in diagnostic assays, for example, or for other
purposes such as antibody production, as is described in the
Examples below.
[0084] As used herein, stringent hybridization conditions refer to
standard hybridization conditions under which nucleic acid
molecules are used to identify similar nucleic acid molecules. Such
standard conditions are disclosed, for example, in Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs
Press, 1989. Sambrook et al., ibid., is incorporated by reference
herein in its entirety (see specifically, pages 9.31-9.62). In
addition, formulae to calculate the appropriate hybridization and
wash conditions to achieve hybridization permitting varying degrees
of mismatch of nucleotides are disclosed, for example, in Meinkoth
et al., 1984, Anal. Biochem. 138, 267-284; Meinkoth et al., ibid.,
is incorporated by reference herein in its entirety.
[0085] More particularly, stringent hybridization and washing
conditions, as referred to herein, refer to conditions which permit
isolation of nucleic acid molecules having at least about 70%
nucleic acid sequence identity with the nucleic acid molecule being
used to probe in the hybridization reaction, more particularly at
least about 75%, and most particularly at least about 80%. Such
conditions will vary, depending on whether DNA:RNA or DNA:DNA
hybrids are being formed. Calculated melting temperatures for
DNA:DNA hybrids are 10.degree. C. less than for DNA:RNA hybrids. In
particular embodiments, stringent hybridization conditions for
DNA:DNA hybrids include hybridization at an ionic strength of
6.times.SSC (0.9 M Na.sup.+) at a temperature of between about
20.degree. C. and about 35.degree. C., more preferably, between
about 28.degree. C. and about 40.degree. C., and even more
preferably, between about 35.degree. C. and about 45.degree. C. In
particular embodiments, stringent hybridization conditions for
DNA:RNA hybrids include hybridization at an ionic strength of
6.times.SSC (0.9 M Na.sup.+) at a temperature of between about
30.degree. C. and about 45.degree. C., more preferably, between
about 38.degree. C. and about 50.degree. C., and even more
preferably, between about 45.degree. C. and about 55.degree. C.
These values are based on calculations of a melting temperature for
molecules larger than about 100 nucleotides, 0% formamide and a G+C
content of about 40%. Alternatively, T.sub.m can be calculated
empirically as set forth in Sambrook et al., supra, pages 9.31 TO
9.62.
[0086] Preferred linoleate isomerase nucleic acid molecules of the
present invention include nucleic acid molecules which comprise a
nucleic acid sequence having at least about 70%, more preferably,
at least about 80% and most preferably, at least about 90% identity
with a nucleic acid sequence selected from SEQ ID NO:4, SEQ ID
NO:8, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ
ID NO:17 and/or SEQ ID NO:26. Preferred linoleate isomerase nucleic
acid molecules of the present invention also include nucleic acid
molecules which comprise a nucleic acid sequence selected from SEQ
ID NO:4, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:15, SEQ
ID NO:16, SEQ ID NO:17 and/or SEQ ID NO:26. Preferred linoleate
isomerase nucleic acid molecules of the present invention also
include nucleic acid molecules which comprise a nucleic acid
molecule selected from nCLA.sub.87, nCLA.sub.596, nCLA.sub.1709,
nCLA.sub.1.1, nCLA.sub.2.3, nCLA.sub.3551, nCLA.sub.1776 and/or
nCLA.sub.7113. As used herein, reference to a percent (%) identity
refers to a BLAST homology search with the default parameters
identified in Table 1.
1TABLE 1 BLAST Search Parameters HISTOGRAM Display a histogram of
scores for each search; default is yes. (See parameter H in the
BLAST Manual). DESCRIPTIONS Restricts the number of short
descriptions of matching sequences reported to the number
specified; default limit is 100 descriptions. (See parameter V in
the manual page). See also EXPECT and CUTOFF. ALIGNMENTS Restricts
database sequences to the number specified for which high-scoring
segment pairs (HSPs) are reported; the default limit is 50. If more
database sequences than this happen to satisfy the statistical
significance threshold for reporting (see EXPECT and CUTOFF below),
only the matches ascribed the greatest statistical significance are
reported. (See parameter B in the BLAST Manual). EXPECT The
statistical significance threshold for reporting matches against
database sequences; the default value is 10, such that 10 matches
are expected to be found merely by chance, according to the
stochastic model of Karlin and Altschul (1990). If the statistical
significance ascribed to a match is greater than the EXPECT
threshold, the match will not be reported. Lower EXPECT thresholds
are more stringent, leading to fewer chance matches being reported.
Fractional values are acceptable. (See parameter E in the BLAST
Manual). CUTOFF Cutoff score for reporting high-scoring segment
pairs. The default value is calculated from the EXPECT value (see
above). HSPs are reported for a database sequence only if the
statistical significance ascribed to them is at least as high as
would be ascribed to a lone HSP having a score equal to the CUTOFF
value. Higher CUTOFF values are more stringent, leading to fewer
chance matches being reported. (See parameter S in the BLAST
Manual). Typically, significance thresholds can be more intuitively
managed using EXPECT. MATRIX Specify an alternate scoring matrix
for BLASTP, BLASTX, TBLASTN and TBLASTX. The default matrix is
BLOSUM62 (Henikoff & Henikoff, 1992). The valid alternative
choices include: PAM40, PAM120, PAM250 and IDENTITY. No alternate
scoring matrices are available for BLASTN; specifying the MATRIX
directive in BLASTN requests returns an error response. STRAND
Restrict a TBLASTN search to just the top or bottom strand of the
database sequences; or restrict BLASTN, BLASTX or TBLASTX search to
just reading frames on the top or bottom strand of the query
sequence. FILTER Mask off segments of the query sequence that have
low compositional complexity, as determined by the SEG program of
Wootton & Federhen (Computers and Chemistry, 1993), or segments
consisting of short-periodicity internal repeats, as determined by
the SNU program of Claverie & States (Computers and Chemistry,
1993), or, for BLASTN, by the DUST program of Tatusov and Lipman
(in preparation). Filtering can eliminate statistically significant
but biologically uninteresting reports from the blast output (e.g.,
hits against common acidic-, basic- or proline-rich regions),
leaving the more biologically interesting regions of the query
sequence available for specific matching against database
sequences. Low complexity sequence found by a filter program is
substituted using the letter "N" in nucleotide sequence (e.g.,
"NNNNNNNNNNNNN") and the letter "X" in protein sequences (e.g.,
"XXXXXXXXX"). Users may turn off filtering by using the "Filter"
option on the "Advanced options for the BLAST server" page.
Filtering is only applied to the query sequence (or its translation
products), not to database sequences. Default filtering is DUST for
BLASTN, SEG for other programs. It is not unusual for nothing at
all to be masked by SEG, SNU, or both, when applied to sequences in
SWISS-PROT, so filtering should not be expected to always yield an
effect. Furthermore, in some cases, sequences are masked in their
entirety, indicating that the statistical significance of any
matches reported against the unfiltered query sequence should be
suspect. NCBI-gi Causes NCBI gi identifiers to be shown in the
output, in addition to the accession and/or locus name.
[0087] In accordance with the present invention, an isolated
nucleic acid molecule is a nucleic acid molecule that has been
removed from its natural milieu (i.e., that has been subject to
human manipulation) and can include DNA, RNA, or derivatives of
either DNA or RNA. As such, "isolated" does not reflect the extent
to which the nucleic acid molecule has been purified. An isolated
linoleate isomerase nucleic acid molecule of the present invention
can be isolated from its natural source or produced using
recombinant DNA technology (e.g., polymerase chain reaction (PCR)
amplification, cloning) or chemical synthesis. Isolated linoleate
isomerase nucleic acid molecules can include, for example, natural
allelic variants and nucleic acid molecules modified by nucleotide
insertions, deletions, substitutions, and/or inversions in a manner
such that the modifications do not substantially interfere with the
nucleic acid molecule's ability to encode a linoleate isomerase of
the present invention or to form stable hybrids under stringent
conditions with natural gene isolates (i.e., a linoleate isomerase
nucleic acid homologue). An isolated linoleate isomerase nucleic
acid molecule can include degeneracies. As used herein, nucleotide
degeneracies refers to the phenomenon that one amino acid can be
encoded by different nucleotide codons. Thus, the nucleic acid
sequence of a nucleic acid molecule that encodes a linoleate
isomerase of the present invention can vary due to
degeneracies.
[0088] A linoleate isomerase nucleic acid molecule homologue can be
produced using a number of methods known to those skilled in the
art (see, for example, Sambrook et al.). For example, nucleic acid
molecules can be modified using a variety of techniques including,
but not limited to, by classic mutagenesis and recombinant DNA
techniques (e.g., site-directed mutagenesis, chemical treatment,
restriction enzyme cleavage, ligation of nucleic acid fragments
and/or PCR amplification), or synthesis of oligonucleotide mixtures
and ligation of mixture groups to "build" a mixture of nucleic acid
molecules and combinations thereof. Nucleic acid molecule
homologues can be selected by hybridization with a linoleate
isomerase gene or by screening the function of a protein encoded by
a nucleic acid molecule (e.g., ability to convert linoleic acid to
CLA). Additionally, a nucleic acid molecule homologues of the
present invention can include nucleic acid sequences comprising at
least 24 contiguous nucleotides having 100% identity with nucleic
acid sequence SEQ ID NO:17, and more preferably, at least about 30,
and even more preferably, at least about 42 contiguous mucleotides
having 100% identity with nucleic acid sequence SEQ ID NO:17.
Similarly, nucleic acid molecule homologues encode proteins having
an amino acid sequence comprising at least 8, and preferably 10,
and even more preferably 14 contiguous amino acid residues having
100% identity with amino acid sequence SEQ ID NO:18. According to
the present invention, the term "contiguous" means to be connected
in an unbroken sequence. For a first sequence to have "100%
identity" with a second sequence means that the first sequence
exactly matches the second sequence with no gaps between
nucleotides or amino acids.
[0089] One embodiment of the present invention includes a
recombinant vector, which includes at least one isolated nucleic
acid molecule of the present invention, inserted into any vector
capable of delivering the nucleic acid molecule into a host cell to
form a recombinant cell. Such a vector contains heterologous
nucleic acid sequences, that is nucleic acid sequences that are not
naturally found adjacent to nucleic acid molecules of the present
invention. The vector can be either RNA or DNA, either prokaryotic
or eukaryotic, and typically is a virus or a plasmid. Recombinant
vectors can be used in the cloning, sequencing, and/or otherwise
manipulating of linoleate isomerase nucleic acid molecules of the
present invention. The vector can be expressed as an
extrachromosomal element (e.g., a plasmid) or it can be integrated
into the chromosome. The entire vector can remain in place, or
under certain conditions, the plasmid DNA can be deleted leaving
behind the nucleic acid molecule of the present invention. The
integrated nucleic acid molecule can be under chromosomal promoter
control, under native or plasmid promoter control, or under a
combination of several promoter controls. Single or multiple copies
of the nucleic acid molecule can be integrated into the
chromosome.
[0090] One type of recombinant vector, referred to herein as a
recombinant molecule, includes a nucleic acid molecule of the
present invention operatively linked to one or more transcription
control sequences to form a recombinant molecule. As used herein,
the phrase "recombinant molecule" primarily refers to a nucleic
acid molecule or nucleic acid sequence operatively linked to a
transcription control sequence, but can be used interchangeably
with the phrase "nucleic acid molecule". According to the present
invention, the phrase "operatively linked" refers to linking a
nucleic acid molecule to a transcription control sequence in a
manner such that the molecule is able to be expressed when
transfected (i.e., transformed, transduced, transfected, conjugated
or conduced) into a host cell. Transcription control sequences are
sequences which control the initiation, elongation, and termination
of transcription. Particularly important transcription control
sequences are those which control transcription initiation, such as
promoter, enhancer, operator and repressor sequences. Suitable
transcription control sequences include any transcription control
sequence that can function in at least one of the recombinant cells
useful for expressing a linoleate isomerase of the present
invention. A variety of such transcription control sequences are
known to those skilled in the art. Preferred transcription control
sequences include those which function in bacterial, fungal (e.g.,
yeast), insect, plant or animal cells.
[0091] Recombinant molecules of the present invention, which can be
either DNA or RNA, can also contain additional regulatory
sequences, such as translation regulatory sequences, origins of
replication, and other regulatory sequences that are compatible
with the recombinant cell. In one embodiment, a recombinant
molecule of the present invention, including those which are
integrated into the host cell chromosome, also contains secretory
signals (i.e., signal segment nucleic acid sequences) to enable an
expressed linoleate isomerase to be secreted from the cell that
produces the protein. Suitable signal segments include a signal
segment that is naturally associated with a linoleate isomerase of
the present invention or any heterologous signal segment capable of
directing the secretion of a linoleate isomerase according to the
present invention. In another embodiment, a recombinant molecule of
the present invention comprises a leader sequence to enable an
expressed linoleate isomerase to be delivered to and inserted into
the membrane of a host cell. Suitable leader sequences include a
leader sequence that is naturally associated with a linoleate
isomerase of the present invention, or any heterologous leader
sequence capable of directing the delivery and insertion of a
linoleate isomerase to the membrane of a cell.
[0092] One or more recombinant molecules of the present invention
can be used to produce an encoded product (i.e., a linoleate
isomerase protein) of the present invention. In one embodiment, an
encoded product is produced by expressing a nucleic acid molecule
as described herein under conditions effective to produce the
protein. A preferred method to produce an encoded protein is by
transfecting a host cell with one or more recombinant molecules to
form a recombinant cell. Suitable host cells to transfect include
any bacterial, fungal (e.g., yeast), insect, plant or animal cell
that can be transfected. Host cells can be either untransfected
cells or cells that are already transformed with at least one
nucleic acid molecule. Preferred host cells for use in the present
invention include any microorganism cell which is suitable for
expression of a linoleate isomerase of the present invention,
including, but not limited to, bacterial cells of the genera
Lactobacillus, Clostridium, Propionibacterium, Butyrivibrio,
Eubacterium, Escherichia and Bacillus. Particularly preferred host
cells include bacterial cells suitable as industrial expression
hosts including, but not limited to Escherichia coli and Bacillus
species, and particularly including, but not limited to Escherichia
coli, Bacillus subtilis and Bacillus licheniformis.
[0093] In one embodiment, an isolated linoleate isomerase protein
of the present invention is produced by culturing a cell that
expresses the protein under conditions effective to produce the
protein, and recovering the protein. A preferred cell to culture is
a recombinant cell of the present invention. Effective culture
conditions include, but are not limited to, effective media,
bioreactor, temperature, pH and oxygen conditions that permit
protein production. An effective, medium refers to any medium in
which a cell is cultured to produce a linoleate isomerase protein
of the present invention. Such medium typically comprises an
aqueous medium having assimilable carbon, nitrogen and phosphate
sources, and appropriate salts, minerals, metals and other
nutrients, such as vitamins. Examples of suitable media and culture
conditions are discussed in detail in the Examples section. Cells
of the present invention can be cultured in conventional
fermentation bioreactors, shake flasks, test tubes, microtiter
dishes, and petri plates. Culturing can be carried out at a
temperature, pH and oxygen content appropriate for a recombinant
cell. Such culturing conditions are within the expertise of one of
ordinary skill in the art.
[0094] Depending on the vector and host system used for production,
resultant proteins of the present invention may either remain
within the recombinant cell; be secreted into the fermentation
medium; be secreted into a space between two cellular membranes,
such as the periplasmic space in E. coli; or be retained on the
outer surface of a cell or viral membrane.
[0095] The phrase "recovering the protein" refers to collecting the
whole fermentation medium containing the protein and need not imply
additional steps of separation or purification. Proteins of the
present invention can be purified using a variety of standard
protein purification techniques, such as, but not limited to,
affinity chromatography, ion exchange chromatography, filtration,
electrophoresis, hydrophobic interaction chromatography, gel
filtration chromatography, reverse phase chromatography,
concanavalin A chromatography, chromatofocusing and differential
solubilization. Proteins of the present invention are preferably
retrieved in "substantially pure" form. As used herein,
"substantially pure" refers to a purity that allows for the
effective use of the protein as a biocatalyst or other reagent.
[0096] Another type of recombinant vector, referred to herein as a
recombinant virus, includes a recombinant molecule of the present
invention that is packaged in a viral coat and that can be
expressed in a cell after delivery of the virus to the cell. A
number of recombinant virus particles can be used, including, but
not limited to, those based on alphaviruses, baculoviruses,
poxviruses, adenoviruses, herpesviruses, and retroviruses.
[0097] As used herein, descriptions of nucleic acid molecules in
relation to linoleate isomerase proteins (e.g., nucleic acid
molecules which encode a linoleate isomerase) refer to isolated
linoleate isomerases which can be full-length linoleate isomerase
proteins, truncated linoleate isomerase proteins, fusion proteins,
or any homologue of such a protein. According to the present
invention, a linoleate isomerase protein homologue includes
linoleate isomerase proteins in which at least one or a few, but
not limited to one or a few, amino acids have been deleted (e.g., a
truncated version of the protein, such as a peptide), inserted,
inverted, substituted and/or derivatized (e.g., by glycosylation,
phosphorylation, acetylation, myristoylation, prenylation,
palmitation, amidation and/or addition of glycosylphosphatidyl
inositol). A linoleate isomerase protein homologue includes
proteins having an amino acid sequence comprising at least 8, and
preferably at least 10, and more preferably, at least 14,
contiguous amino acid residues having 100% identity to any of the
linoleate isomerase amino acid sequences disclosed herein, and
particularly, to amino acid sequence SEQ ID NO:18. Similarly, a
linoleate isomerase protein homologue includes proteins encoded by
a nucleic acid sequence comprising at least 24, and preferably at
least 30, and more preferably at least 42, contiguous nucleotides
having 100% identity to any of the linoleate isomerase nucleic acid
molecules disclosed herein, and particularly, to SEQ ID NO:17. A
linoleate isomerase protein homologue can also be identified as a
protein having at least one epitope which elicits an immune
response against a protein having an amino acid sequence selected
from the group of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9, SEQ ID
NO:11 and/or SEQ ID NO:18. In some embodiments, a linoleate
isomerase protein homologue has measurable linoleate isomerase
enzymatic activity. In another embodiment, a homologue of a
linoleate isomerase is a protein having an amino acid sequence that
is sufficiently similar to a natural linoleate isomerase amino acid
sequence that a nucleic acid sequence encoding the homologue is
capable of hybridizing under stringent conditions to (i.e., with) a
nucleic acid molecule encoding the natural linoleate isomerase
(i.e., to the complement of the nucleic acid strand encoding the
natural linoleate isomerase amino acid sequence). A nucleic acid
sequence complement of nucleic acid sequence encoding linoleate
isomerase of the present invention refers to the nucleic acid
sequence of the nucleic acid strand that is complementary to (i.e.,
can form a complete double helix with) the strand for which the
nucleic acid sequence encodes linoleate isomerase. It will be
appreciated that a double stranded DNA which encodes a given amino
acid sequence comprises a single strand DNA and its complementary
strand having a sequence that is a complement to the single strand
DNA. As such, nucleic acid molecules which encode the linoleate
isomerase of the present invention can be either double-stranded or
single-stranded, and include those nucleic acid molecules that form
stable hybrids under stringent hybridization conditions with a
nucleic acid sequence that encodes the amino acid sequence selected
from the group consisting of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9,
SEQ ID NO:11 and/or SEQ ID NO:18, and/or with the complement of the
nucleic acid that encodes amino acid sequence selected from the
group consisting of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9, SEQ ID
NO:11 and/or SEQ ID NO:18. Methods to deduce a complementary
sequence are known to those skilled in the art. It should be noted
that since amino acid sequencing and nucleic acid sequencing
technologies are not entirely error-free, the sequences presented
herein, at best, represent apparent sequences of linoleate
isomerase of the present invention.
[0098] Linoleate isomerase homologues can be the result of natural
allelic variation or natural mutation. Linoleate isomerase
homologues of the present invention can also be produced using
techniques known in the art including, but not limited to, direct
modifications to the protein or modifications to the gene encoding
the protein using, for example, classic or recombinant DNA
techniques to effect random or targeted mutagenesis. A naturally
occurring allelic variant of a nucleic acid encoding linoleate
isomerase is a gene that occurs at essentially the same locus (or
loci) in the genome as the gene which encodes an amino acid
sequence selected from the group consisting of SEQ ID NO:1, SEQ ID
NO:5, SEQ ID NO:9, SEQ ID NO:11 and/or SEQ ID NO:18, but which, due
to natural variations caused by, for example, mutation or
recombination, has a similar but not identical sequence. Natural
allelic variants typically encode proteins having similar activity
to that of the protein encoded by the gene to which they are being
compared. One class of allelic variants can encode the same protein
but have different nucleic acid sequences due to the degeneracy of
the genetic code. Allelic variants can also comprise alterations in
the 5' or 3' untranslated regions of the gene (e.g., in regulatory
control regions). Allelic variants are well known to those skilled
in the art and would be expected to be found within a given
bacterial species since the genome is haploid and/or among a group
of two or more bacterial species.
[0099] Linoleate isomerase proteins also include expression
products of gene fusions (for example, used to overexpress soluble,
active forms of the recombinant enzyme), of mutagenized genes (such
as genes having codon modifications to enhance gene transcription
and translation), and of truncated genes (such as genes having
membrane binding domains removed to generate soluble forms of a
membrane enzyme, or genes having signal sequences removed which are
poorly tolerated in a particular recombinant host). It is noted
that linoleate isomerase proteins and protein homologues of the
present invention include proteins which do not have linoleate
isomerase enzymatic activity. Such proteins are useful, for
example, for the production of antibodies and for diagnostic
assays.
[0100] An isolated linoleate isomerase of the present invention,
including full-length proteins, truncated proteins, fusion proteins
and homologues, can be identified in a straight-forward manner by:
the proteins' ability to convert linoleic acid and/or linolenic
acid to CLA, such as is illustrated in the Examples; the
biochemical properties of the protein as described in the Examples;
by selective binding to an antibody against a linoleate isomerase;
and/or by homology with other linoleate isomerase amino acid and
nucleic acid sequences as disclosed in the Examples.
[0101] The minimal size of a protein and/or homologue of the
present invention is a size sufficient to be encoded by a nucleic
acid molecule capable of forming a stable hybrid with the
complementary sequence of a nucleic acid molecule encoding the
corresponding natural protein. As such, the size of the nucleic
acid molecule encoding such a protein is dependent on nucleic acid
composition and percent homology between the nucleic acid molecule
and complementary sequence as well as upon hybridization conditions
per se (e.g., temperature, salt concentration, and formamide
concentration). The minimal size of such nucleic acid molecules is
typically at least about 12 to about 15 nucleotides in length if
the nucleic acid molecules are GC-rich and at least about 15 to
about 18 bases in length if they are AT-rich. As such, the minimal
size of a nucleic acid molecule used to encode linoleate isomerase
protein or homologue of the present invention is from about 12 to
about 18 nucleotides in length. There is no limit, other than a
practical limit, on the maximal size of such a nucleic acid
molecule in that the nucleic acid molecule can include a portion of
a gene, an entire gene, or multiple genes, or portions thereof.
Similarly, the minimal size of linoleate isomerase protein or
homologue of the present invention is from about 4 to about 6 amino
acids in length, with preferred sizes depending on whether a
full-length, multivalent (i.e., fusion protein having more than one
domain each of which has a function), or functional portions of
such proteins are desired.
[0102] Preferred linoleate isomerases of the present invention
include proteins which comprise an amino acid sequence having at
least about 70%, more preferably, at least about 80% and most
preferably, at least about 90% identity with an amino acid sequence
selected from SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:11
and/or SEQ ID NO:18. Preferred linoleate isomerases of the present
invention also include proteins which comprise an amino acid
sequence selected from SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9, SEQ
ID NO:11 and/or SEQ ID NO:18. Preferred linoleate isomerases of the
present invention also include proteins which comprise a protein
selected from PCLA.sub.35, PCLA.sub.28, PCLA.sub.158, PCLA.sub.324
and/or PCLA.sub.591.
[0103] The present invention also includes a fusion protein that
includes a linoleate isomerase-containing domain attached to one or
more fusion segments. Suitable fusion segments for use with the
present invention include, but are not limited to, segments that
can: enhance a protein's stability; provide other enzymatic
activity (e.g., lipase, phospholipase, or esterase to hydrolyze
esters of 9,12-diene fatty acids to 9,12-fatty acids); and/or
assist purification of a linoleate isomerase (e.g., by affinity
chromatography). A suitable fusion segment can be a domain of any
size that has the desired function (e.g., imparts increased
stability, solubility, action or activity; provides other enzymatic
activity such as hydrolysis of esters; and/or simplifies
purification of a protein). Fusion segments can be joined to amino
and/or carboxyl termini of the linoleate isomerase-containing
domain of the protein and can be susceptible to cleavage in order
to enable straight-forward recovery of a linoleate isomerase.
Fusion proteins are preferably produced by culturing a recombinant
cell transformed with a fusion nucleic acid molecule that encodes a
protein including the fusion segment attached to either the
carboxyl and/or amino terminal end of a linoleate
isomerase-containing domain.
[0104] Linoleate isomerases can be isolated from a various
microorganisms including bacteria and fungi. For example, bacterial
genera such as Lactobacillus, Clostridium, Propionibacterium,
Butyrivibrio, and Eubacterium have linoleate isomerase activity. In
particular, bacterial species such as Lactobacillus reuteri,
Clostridium sporogenes, Propionibacterium acnes, Butyrivibrio
fibrisolvens, Propionibacterium acidipropionici, Propionibacterium
freudenreichii and Eubacterium lentum contain linoleate isomerase.
A particularly preferred linoleate isomerase is Lactobacillus
reuteri linoleate isomerase. Other preferred linoleate isomerases
are linoleate isomerases from Propionibacterium acnes and
Clostridium sporogenes.
[0105] To produce significantly high yields of CLA by the methods
of the present invention, a microorganism is genetically modified
to increase the action of linoleate isomerase, and preferably, to
enhance production of linoleate isomerase, and thereby, CLA. As
used herein, a genetically modified microorganism, such as any of
the preferred genera of bacteria described herein, has a genome
which is modified (i.e., mutated or changed) from its normal (i.e.,
wild-type or naturally occurring) form such that the desired result
is achieved (i.e., increase the action of linoleate isomerase).
Genetic modification of a microorganism can be accomplished using
classical strain development and/or molecular genetic techniques.
Such techniques are generally disclosed, for example, in Sambrook
et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Labs Press. The reference Sambrook et al., ibid., is
incorporated by reference herein in its entirety. Additionally,
techniques for genetic modification of a microorganism through
recombinant technology are described in detail in the Examples
section.
[0106] A genetically modified microorganism can include a
microorganism in which nucleic acid molecules have been inserted,
deleted or modified (i.e., mutated; e.g., by insertion, deletion,
substitution, and/or inversion of nucleotides), in such a manner
that such modifications provide the desired effect within the
microorganism.
[0107] According to the present invention, a genetically modified
microorganism includes a microorganism that has been modified using
recombinant technology. As used herein, genetic modifications which
result in a decrease in gene expression, in the function of the
gene, or in the function of the gene product (i.e., the protein
encoded by the gene) can be referred to as inactivation (complete
or partial), deletion, interruption, blockage or down-regulation of
a gene. For example, a genetic modification in a gene which results
in a decrease in the function of the protein encoded by such gene,
can be the result of a complete deletion of the gene (i.e., the
gene does not exist, and therefore the protein does not exist), a
mutation in the gene which results in incomplete or no translation
of the protein (e.g., the protein is not expressed), or a mutation
in the gene which decreases or abolishes the natural function of
the protein (e.g., a protein is expressed which has decreased or no
enzymatic activity or action). Genetic modifications which result
in an increase in gene expression or function can be referred to as
amplification, overproduction, overexpression, activation,
enhancement, addition, or up-regulation of a gene.
[0108] In one embodiment of the present invention, a genetic
modification of a microorganism increases or decreases the action
of a linoleate isomerase. Such a genetic modification includes any
type of modification and specifically includes modifications made
by recombinant technology and by classical mutagenesis. It should
be noted that reference to increasing the action (or activity) of
linoleate isomerase refers to any genetic modification in the
microorganism in question which results in increased functionality
of the enzyme and includes higher activity of the enzymes (e.g.,
specific activity or in vivo enzymatic activity), reduced
inhibition or degradation of the enzymes and overexpression of the
enzymes. For example, gene copy number can be increased, expression
levels can be increased by use of a promoter that gives higher
levels of expression than that of the native promoter, or a gene
can be altered by genetic engineering or classical mutagenesis to
increase the action of an enzyme. Similarly, reference to
decreasing the action of an enzyme refers to any genetic
modification in the microorganism in question which results in
decreased functionality of the enzymes and includes decreased
activity of the enzymes (e.g., specific activity), increased
inhibition or degradation of the enzymes and a reduction or
elimination of expression of the enzymes. For example, the action
of an enzyme of the present invention can be decreased by blocking
or reducing the production of the enzyme, "knocking out" the gene
encoding the enzyme, reducing enzyme activity, or inhibiting the
activity of the enzyme. Blocking or reducing the production of an
enzyme can include placing the gene encoding the enzyme under the
control of a promoter that requires the presence of an inducing
compound in the growth medium. By establishing conditions such that
the inducer becomes depleted from the medium, the expression of the
gene encoding the enzyme (and therefore, of enzyme synthesis) could
be turned off. Blocking or reducing the activity of an enzyme could
also include using an excision technology approach similar to that
described in U.S. Pat. No. 4,743,546, incorporated herein by
reference. To use this approach, the gene encoding the enzyme of
interest is cloned between specific genetic sequences that allow
specific, controlled excision of the gene from the genome. Excision
could be prompted by, for example, a shift in the cultivation
temperature of the culture, as in U.S. Pat. No. 4,743,546, or by
some other physical or nutritional signal.
[0109] In one embodiment of the present invention, a genetically
modified microorganism includes a microorganism which has an
enhanced ability to synthesize CLA. According to the present
invention, "an enhanced ability to synthesize" a product refers to
any enhancement, or up-regulation, in a pathway related to the
synthesis of the product such that the microorganism produces an
increased amount of the product compared to the wild-type
microorganism cultured under the same conditions. In one embodiment
of the present invention, enhancement of the ability of a
microorganism to synthesize CLA is accomplished by amplification of
the expression of the linoleate isomerase gene. Amplification of
the expression of Linoleate isomerase can be accomplished in a
bacterial cell, for example, by introduction of a recombinant
nucleic acid molecule encoding the linoleate isomerase gene, or by
modifying regulatory control over a native linoleate isomerase
gene.
[0110] Therefore, it is an embodiment of the present invention to
provide a bacteria which is transformed with a recombinant nucleic
acid molecule comprising a nucleic acid sequence encoding a
linoleate isomerase gene. Preferred recombinant nucleic acid
molecules comprising such a nucleic acid sequence include
recombinant nucleic acid molecules comprising a nucleic acid
sequence which encodes a linoleate isomerase comprising an amino
acid sequence selected from SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9,
SEQ ID NO:11 or SEQ ID NO:18. Other preferred recombinant nucleic
acid molecules of the present invention include nucleic acid
molecules which comprise a nucleic acid sequence selected from the
group of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:14, SEQ
ID NO:15, SEQ ID NO:16, SEQ ID NO:17 or SEQ ID NO:26. The above
identified nucleic acid molecules represent nucleic acid molecules
comprising wild-type (i.e., naturally occurring) nucleic acid
sequences encoding linoleate isomerases. Genetically modified
nucleic acid molecules which include nucleic acid
sequences-encoding homologues of (i.e., modified and/or mutated)
linoleate isomerases are also encompassed by the present invention
and are described in detail herein.
[0111] Therefore, it is yet another embodiment of the present
invention to provide a microorganism having a linoleate isomerase
with reduced substrate inhibition and/or reduced product
inhibition. A linoleate isomerase with reduced substrate and/or
product inhibition can be a mutated (i.e.; genetically modified)
linoleate isomerase gene, for example, and can be produced by any
suitable method of genetic modification. For example, a recombinant
nucleic acid molecule encoding linoleate isomerase can be modified
by any method for inserting, deleting, and/or substituting
nucleotides, such as by error-prone PCR. In this method, the gene
is amplified under conditions that lead to a high frequency of
misincorporation errors by the DNA polymerase used for the
amplification. As a result, a high frequency of mutations are
obtained in the PCR products. The resulting linoleate isomerase
gene mutants can then be screened for reduced substrate and/or
product inhibition by testing the mutant genes for the ability to
confer increased CLA production onto a test microorganism, as
compared to a microorganism carrying the non-mutated recombinant
linoleate isomerase nucleic acid molecule. It should be noted that
decreased substrate and/or product inhibition of linoleate
isomerase will typically result in a linoleate isomerase with
increased action, even when the specific activity of the enzyme is
remains the same, or actually is decreased, relative to a naturally
occurring linoleate isomerase enzyme. Therefore, it is an
embodiment of the present invention to produce a genetically
modified linoleate isomerase with increased action and increased in
vivo enzymatic activity, which has unmodified or even decreased
specific activity as compared to a naturally occurring linoleate
isomerase. Also encompassed by the present invention are
genetically modified linoleate isomerases with increased specific
activity.
[0112] Therefore, it is an embodiment of the present invention to
provide a microorganism which is transformed with a genetically
modified recombinant nucleic acid molecule comprising a nucleic
acid sequence encoding a mutant, or homologue, linoleate isomerase.
Such linoleate isomerases can be referred to herein as linoleate
isomerase homologues. Protein homologues are described in detail
herein. Preferred recombinant nucleic acid molecules comprising
such a nucleic acid sequence include recombinant nucleic acid
molecules comprising a nucleic acid sequence which encodes a
linoleate isomerase comprising an amino acid sequence selected from
the group of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:11 or
SEQ ID NO:18. Other preferred recombinant nucleic acid molecules
comprise a nucleic acid sequence selected from the group of SEQ ID
NO:4, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO.: 14, SEQ ID NO:15, SEQ
ID NO:16, SEQ ID NO:17 or SEQ ID NO:26. Particularly preferred
genetically modified recombinant nucleic acid molecules useful in
the present invention include nucleic acid molecules comprising
nucleic acid molecules selected from the group of nCLA.sub.87 ,
nCLA.sub.596, nCLA.sub.1709, nCLA.sub.1.1, nCLA.sub.2.3,
nCLA.sub.3551 , nCLA.sub.1776 and/or nCLA.sub.7113.
[0113] Another embodiment of the present invention is a method for
producing CLA from an oil using an isolated linoleate isomerase
enzyme. The method can be operated in batch or continuous mode
using a stirred tank, a plug-flow column reactor or other apparatus
known to those skilled in the art. The oil comprises a compound
selected from the group consisting of free fatty acids, salts of
free fatty acids (e.g., soaps), and mixtures containing linoleic
acid, linolenic acid and mixtures thereof. Preferably the oil
comprises at least about 50% by weight of the compound, more
preferably at least about 60% by weight, and most preferably at
least about 80% by weight. The method of the present invention
converts at least a portion of the compound to CLA. Preferably at
least about 50% of the oil is converted to CIA, more preferably at
least about 70%, and most preferably at least about 95%.
[0114] A variety of animal and plant sources are available which
contain oil that is useful for the foregoing method of the present
invention. Preferably, the oil is selected from the group
consisting of sunflower oil, safflower oil, corn oil, linseed oil,
palm oil, rapeseed oil, sardine oil, herring oil, mustard seed oil,
peanut oil, sesame oil, perilla oil, cottonseed oil, soybean oil,
dehydrated castor oil and walnut oil.
[0115] When the compound is in the form of a triglyceride, the
method includes contacting the oil with a hydrolysis enzyme to
convert at least a portion of the triglyceride to free fatty acids.
Hydrolysis enzymes include any enzyme which can cleave an ester
bond of a triglyceride to provide a free fatty acid. Preferably,
hydrolysis enzyme is selected from the group consisting of lipases,
phospholipases, and esterases. Use of enzymes to hydrolyze a
triglyceride is well known to one skilled in the art.
[0116] Alternatively, the oil comprising a triglyceride of a fatty
acid can be chemically hydrolyzed to convert at least a portion of
the triglyceride to free fatty acids. Chemical conversion of
triglyceride to free fatty acids is well known to one skilled in
the art. For example, a triglyceride can be hydrolyzed to provide a
free fatty acid under a basic condition using a base such as
hydroxides, carbonates and bicarbonates. Exemplary bases include
sodium hydroxide, calcium hydroxide, potassium hydroxide, sodium
carbonate, lithium hydroxide, magnesium hydroxide, calcium
carbonate, sodium bicarbonate, lithium carbonate, and lithium
bicarbonate. Alternatively, triglycerides can be hydrolyzed to
provide a free fatty acid under an acidic condition using an acid.
Exemplary acids include, hydrochloric acid, sulfuric acid,
phosphoric acid, and carboxylic acids such as acetic acid and
formic acid.
[0117] In a preferred method of the present invention, the
linoleate isomerase is bound to a solid support, i.e., an
immobilized enzyme. As used herein, a linoleate isomerase bound to
a solid support (i.e., an immobilized linoleate isomerase) includes
immobilized isolated linoleate isomerases, immobilized bacterial
cells which contain a linoleate isomerase enzyme, stabilized intact
cells and stabilized cell/membrane homogenates. Stabilized intact
cells and stabilized cell/membrane homogenates include cells and
homogenates from naturally occurring microorganisms expressing
linoleate isomerase or from genetically modified microorganisms as
disclosed elsewhere herein. Thus, although methods for immobilizing
linoleate isomerase are discussed below, it will be appreciated
that such methods are equally applicable to immobilizing bacterial
cells and in such an embodiment, the cells can be lysed.
[0118] A variety of methods for immobilizing an enzyme are
disclosed in Industrial Enzymology 2nd Ed., Godfrey, T. and West,
S. Eds., Stockton Press, New York, N.Y., 1996, pp. 267-272;
Immobilized Enzymes, Chibata, I. Ed., Halsted Press, New York,
N.Y., 1978; Enzymes and Immobilized Cells in Biotechnology, Laskin,
A. Ed., Benjamin/Cummings Publishing Co., Inc., Menlo Park, Calif.,
1985; and Applied Biochemistry and Bioengineering, Vol. 4, Chibata,
I. and Wingard, Jr., L. Eds, Academic Press, New York, N.Y., 1983,
which are incorporated herein in their entirety.
[0119] Briefly, a solid support refers to any solid organic,
biopolymer or inorganic supports that can form a bond with
linoleate isomerase without significantly effecting the activity of
isolated linoleate isomerase enzyme. Exemplary organic solid
supports include polymers such as polystyrene, nylon,
phenol-formaldehyde resins, acrylic copolymers (e.g.,
polyacrylamide), stabilized intact whole cells, and stabilized
crude whole cell/membrane homogenates. Exemplary biopolymer
supports include cellulose, polydextrans (e.g., Sephadex.RTM.),
agarose, collagen and chitin. Exemplary inorganic supports include
glass beads (porous and nonporous), stainless steel, metal oxides
(e.g., porous ceramics such as ZrO.sub.2, TiO.sub.2,
Al.sub.2O.sub.3, and NiO) and sand. Preferably, the solid support
is selected from the group consisting of stabilized intact cells
and/or crude cell homogenates. Preparation of such supports
requires a minimum of handling and cost. Additionally, such
supports provide excellent stability of the enzyme.
[0120] Stabilized intact cells and/or cell/membrane homogenates can
be produced, for example, by using bifunctional crosslinkers (e.g.,
glutaraldehyde) to stabilize cells and cell homogenates. In both
the intact cells and the cell membranes, the cell wall and
membranes act as immobilizing supports. In such a system, integral
membrane proteins are in the "best" lipid membrane environment.
Whether starting with intact cells or homogenates, in this system
the cells are either no longer "alive" or "metabolizing", or
alternatively, are "resting" (i.e., the cells maintain metabolic
potential and active linoleate isomerase, but under the culture
conditions are not growing); in either case, the immobilized cells
or membranes serve as biocatalysts.
[0121] Linoleate isomerase can be bound to a solid support by a
variety of methods including adsorption, cross-linking (including
covalent bonding), and entrapment. Adsorption can be through van
del Waal's forces, hydrogen bonding, ionic bonding, or hydrophobic
binding. Exemplary solid supports for adsorption immobilization
include polymeric adsorbents and ion-exchange resins. Solid
supports in a bead form are particularly well-suited. The particle
size of an adsorption solid support can be selected such that the
immobilized enzyme is retained in the reactor by a mesh filter
while the substrate (e.g., the oil) is allowed to flow through the
reactor at a desired rate. With porous particulate supports it is
possible to control the adsorption process to allow linoleate
isomerases or bacterial cells to be embedded within the cavity of
the particle, thus providing protection without an unacceptable
loss of activity.
[0122] Cross-linking of a linoleate isomerase to a solid support
involves forming a chemical bond between a solid support and a
linoleate isomerase. It will be appreciated that although
cross-linking generally involves linking a linoleate isomerase to a
solid support using an intermediary compound, it is also possible
to achieve a covalent bonding between the enzyme and the solid
support directly without the use of an intermediary compound.
Cross-linking commonly uses a bifunctional or multifunctional
reagent to activate and attach a carboxyl group, amino group,
sulfur group, hydroxy group or other functional group of the enzyme
to the solid support. The term "activate" refers to a chemical
transformation of a functional group which allows a formation of a
bond at the functional group. Exemplary amino group activating
reagents include water-soluble carbodiimides, glutaraldehyde,
cyanogen bromide, N-hydroxysuccinimide esters, triazines, cyanuric
chloride, and carbonyl diimidazole. Exemplary carboxyl group
activating reagents include water-soluble carbodiimides, and
N-ethyl-5-phenylisoxazolium-3-sulfonate. Exemplary tyrosyl group
activating reagents include diazonium compounds. And exemplary
sulfhydryl group activating reagents include
dithiobis-5,5'-(2-nitrobenzoic acid), and glutathione-2-pyridyl
disulfide. Systems for covalently linking an enzyme directly to a
solid support include Eupergit.RTM., a polymethacrylate bead
support available from Rohm Pharma (Darmstadt, Germany), kieselguhl
(Macrosorbs), available from Sterling Organics, kaolinite available
from English China Clay as "Biofix" supports, silica gels which can
be activated by silanization, available from W.R. Grace, and
high-density alumina, available from UOP (Des Plains, Ill.).
[0123] Entrapment can also be used to immobilize linoleate
isomerase. Entrapment of linoleate isomerase involves formation of,
inter alia, gels (using organic or biological polymers), vesicles
(including microencapsulation), semipermeable membranes or other
matrices. Exemplary materials used for entrapment of an enzyme
include collagen, gelatin, agar, cellulose triacetate, alginate,
polyacrylamide, polystyrene, polyurethane, epoxy resins,
carrageenan, and egg albumin. Some of the polymers, in particular
cellulose triacetate, can be used to entrap the enzyme as they are
spun into a fiber. Other materials such as polyacrylamide gels can
be polymerized in solution to entrap the enzyme. Still other
materials such as polyglycol oligomers that are functionalized with
polymerizable vinyl end groups can entrap enzymes by forming a
cross-linked polymer with UV light illumination in the presence of
a photosensitizer.
[0124] CLA produced by a method of the present invention can be
recovered by conventional methods.
[0125] CLA can be produced in a two-phase aqueous-oil system with
emulsified oil (e.g., emulsified with lecithin), in a co-solvent
system, or most preferably, in a two-phase aqueous oil system
comprising an oil stream containing very little water (i.e, only
the minimum water required for enzyme activity). A further
characteristic of linoleate isomerases of the present invention is
that they are not inhibited by higher log P solvents. In fact, it
has been surprisingly found that in some cases linoleate isomerases
of the present invention provide higher conversion of linoleic acid
to CLA when immiscible solvents are used. CLA can be produced using
a variety of solvent systems. For example, CLA can be produced
using an aqueous system or a combination of an aqueous and an
organic system. Preferably, a solvent system for CLA production
using a linoleate isomerase comprises a solvent selected from the
group consisting of water, hexane decane, hexadecane, and propylene
glycol.
[0126] Yet another embodiment of the present invention relates to a
method for producing CLA which utilizes industrial expression
systems formed from the microorganisms, nucleic acid molecules, and
proteins of the present invention which have been disclosed herein.
In this method, immobilized intact whole cells or cell/membrane
homogenates formed from naturally occurring microorganisms
expressing linoleate isomerase or from a genetically modified
microorganism as described herein (including recombinant
microorganisms), wherein the microorganism stably expresses a
linoleate isomerase of the present invention, will be grown in a
suitable culture system (e.g., fermentors). The stabilized cells or
homogenates will serve as a biocatalyst in a biotransformation
process to convert linoleic acid and/or linolenic acid to CLA,
according to the parameters specified elsewhere herein. In one
embodiment, the biocatalyst will be reused (i.e., recycled) several
times. In a preferred embodiment, the linoleic and/or linolenic
acid-containing oil stream is added to the biocatalyst in the
presence of a minimum amount of water.
[0127] Yet another embodiment of the present invention relates to a
nucleic acid molecule denoted nCSN.sub.726, which is located on the
strand of nCLA.sub.3551 that is complementary to the nucleic acid
sequence SEQ ID NO:16, and which comprises an open reading frame
having nucleic acid sequence SEQ ID NO:21. SEQ ID NO:21 is
positioned on the strand that is complementary to nucleotide
positions 1 through 726 of SEQ ID NO:16, with a start codon
approximately 275 nucleotides up-stream from the putative start
codon of a nucleic acid molecule of the present invention which
encodes a linoleate isomerase. The deduced amino acid sequence of
SEQ ID NO:21 is represented by SEQ ID NO:22. A protein comprising
SEQ ID NO:22 is referred to herein as PCSN.sub.242. The C-terminal
portion of the protein comprising SEQ ID NO:22 is not included in
SEQ ID NO:22. The present inventors have shown (See Example 5) that
the nucleic acid sequence of SEQ ID NO:21 is 66% identical to a
competence-specific nuclease (DNA entry nuclease) from
Streptococcus pneumoniae (Q03158), with the amino acid sequence SEQ
ID NO:22 being 51-72% identical to the amino acid sequence for this
competence-specific nuclease. Therefore, it is believed to be
possible that PCSN.sub.242 represents at least a portion of a
competence-specific nuclease. PCSN.sub.242 is contained within a
protein denoted PCSN.sub.247, which has an amino acid sequence
represented herein by SEQ ID NO:34. SEQ ID NO:34 is encoded by a
nucleic acid molecule denoted nCSN.sub.744, which is represented
herein by SEQ ID NO:33. The amino acid sequence SEQ ID NO:34 is
about 57% identical and about 71% similar to the amino acid
sequence for the above-mentioned competence-specific nuclease
(BLAST, standard parameters).
[0128] Yet another embodiment of the present invention relates to a
nucleic acid molecule denoted nBSP.sub.941, which comprises an open
reading frame located upstream from an open reading frame encoding
a linoleate isomerase of the present invention, and having nucleic
acid sequence SEQ ID NO:27. The deduced amino acid sequence of SEQ
ID NO:27 is represented by SEQ ID NO:28. A protein comprising SEQ
ID NO:28 is referred to herein as PBSP.sub.312. The present
inventors have shown (See Example 5) that the amino acid sequence
of SEQ ID NO:28 is about 56% identical and about 74% similar (using
standard BLAST parameters) to a permease from Bacillus subtilis
(p54425). Therefore, it is believed to be possible that
PBSP.sub.312 represents at least a portion of a permease.
[0129] As described in Example 5 below, one embodiment of the
present invention is a nucleic acid molecule, denoted
nUNK1.sub.656, which comprises an open reading frame located about
122 nucleotides downstream from an open reading frame encoding a
linoleate isomerase of the present invention. The nucleic acid
molecule nUNK1.sub.656 comprises a nucleic acid sequence
represented by SEQ ID NO:19, the deduced amino acid sequence of
which is represented by SEQ ID NO:20. A protein having SEQ ID NO:20
is referred to herein as PUNK1.sub.218. PUNK1.sub.218 is contained
within a larger protein, denoted PUNK1.sub.513 having a deduced
amino acid sequence represented by SEQ ID NO:36. SEQ ID NO:36 is
encoded by a nucleic acid molecule denoted nUNK1.sub.1540, the
nucleic acid sequence of which is represented by SEQ ID NO:35. The
C-terminal sequence of PUNK1.sub.513 is incomplete.
[0130] Yet another embodiment of the present invention is a nucleic
acid molecule, denoted nUNK2.sub.600, which comprises an open
reading frame located upstream from an open reading frame encoding
a linoleate isomerase of the present invention. The nucleic acid
molecule nUNK2.sub.600 comprises a nucleic acid sequence
represented by SEQ ID NO:29, the deduced amino acid sequence of
which is represented by SEQ ID NO:30. A protein having SEQ ID NO:30
is referred to herein as PUNK2.sub.199.
[0131] Yet another embodiment of the present invention is a nucleic
acid molecule, denoted nUNK3.sub.849, which comprises an open
reading frame located upstream from an open reading frame encoding
a linoleate isomerase of the present invention. The nucleic acid
molecule nUNK3.sub.849 comprises a nucleic acid sequence
represented by SEQ ID NO:31, the deduced amino acid sequence of
which is represented by SEQ ID NO:32. A protein having SEQ ID NO:32
is referred to herein as PUNK3.sub.282.
EXAMPLES
[0132] It is to be noted that the Examples include a number of
molecular biology, microbiology, immunology and biochemistry
techniques considered to be known to those skilled in the art.
Disclosure of such techniques can be found, for example, in
Sambrook et al., id. and related references. Unless otherwise
noted, all column chromatography was performed at 4.degree. C.
Example 1
[0133] This example illustrates CLA production from linoleic acid
using whole cell biotransformations with a variety of
microorganisms. The term "whole cell biotransformation" refers to a
conversion of a suitable substrate to CLA by a microorganism.
[0134] A variety of other microorganisms were purchased from ATCC
(American Type Culture Collection) and grown on Brain Heart
Infusion Broth (Difco) supplemented with 0.5% yeast extract,
0.0005% hemin, 0.001% vitamin K.sub.1, 0.05% cysteine, and 0.001%
resazurin. Cultures were grown in closed containers with limited
head space for about 12 to about 16 hours at 37.degree. C.,
harvested and washed with fresh medium. Culture stocks were
maintained in 10% glycerol at about -80.degree. C.
[0135] Lactobacillus reuteri PYR8 (ATCC Accession No. 55739,
deposited on Feb. 15, 1996 with the American Type Culture
Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110,
USA, in connection with U.S. Pat. No. 5,674,901 to Cook et al.,
issued Oct. 7, 1997, incorporated herein by reference in its
entirety) was obtained from Dr. Michael Pariza of the Food Research
Institute at University of Wisconsin at Madison. The organism was
grown on MRS Lactobacillus Broth (BBL) in closed containers with
limited head space. Large scale cultures were grown (1-2% inoculum)
in 2-L bottles without agitation at 37.degree. C. for about 36 to
about 40 hours, harvested by centrifugation, washed once with 0.1 M
Bis-Tris, 0.9% NaCl pH 6.0 buffer, and was used immediately or
stored at about -80.degree. C.
[0136] Cultures were grown and harvested as described above. Washed
cells were resuspended in either fresh growth medium or 0.1 M Tris
pH 8.0 buffer containing linoleic acid at various
concentrations.
[0137] Aerobic biotransformations were carried out in baffled 250
mL shake flasks agitated at 200 rpm on a shaker at room
temperature.
[0138] Anaerobic biotransformations were carried out in sealed 150
mL serum bottles under a 95% nitrogen/5% carbon dioxide head space
at 37.degree. C. Media were prepared anaerobically by boiling under
a N.sub.2/CO.sub.2 atmosphere for 15 minutes, sealed with a crimped
septum and autoclaved. MRS broth (BBL) was used with L. reuteri.
Supplemented Brain Heart Infusion broth was used in anaerobic
biotransformations with other microorganisms.
[0139] Samples were taken at appropriate intervals and analyzed for
CLA as described in Example 2. In some experiments, various
detergents were added to 0.1-0.5% final concentration. In
experiments using organic solvents, linoleic acid was provided as a
5% (v/v) stock dissolved in hexane (log P=3.6), decane (log P=5.6)
or hexadecane (log P=8.6). About 5 mL solvent was added to about 20
mL aqueous cell suspension in a 125 mL baffled shake flask
incubated at room temperature.
[0140] FIGS. 1A and 1B show the results of whole cell
biotransformation by Clostridium sporogenes ATCC 25762 under
aerobic (FIG. 1A) and anaerobic (FIG. 1B) conditions. As FIG. 1A
shows, under aerobic conditions, C. sporogenes ATCC 25762 rapidly
converts linoleic acid to (cis,trans)-9,11-CLA. However, prolonged
whole cell biotransformation results in a, decrease in
(cis,trans)-9,11-CLA and an increase in (trans,trans)-9,11-CLA and
(trans,trans)-10,12-CLA. C. sporogenes ATCC 25762 also produces
(cis,trans)-9,11-CLA from linoleic acid under anaerobic conditions
(FIG. 1B); however, no (trans,trans)-CLA is observed under
anaerobic conditions.
[0141] FIGS. 2A and 2B show the results of whole cell
biotransformation by C. bifermentans ATCC 638 under aerobic (FIG.
2A) and anaerobic (FIG. 2B) conditions. Linoleic acid is more
rapidly converted to (cis,trans)-9,11-CLA by C. bifermentans ATCC
638 under aerobic conditions (FIG. 2A) than under anaerobic
conditions (FIG. 2B). The highest (cis,trans)-9,11-CLA
concentration is observed at about 1 to about 5 hours under aerobic
conditions. C. sordellii ATCC 9714 also converts linoleic acid to
(cis,trans)-9,11-CLA under both aerobic and anaerobic conditions
(data not shown).
[0142] FIGS. 3A, 3B and 4 show the results of whole cell
biotransformation by Propionibacterium jensenii ATCC 14073 (FIG.
3A), P. acnes ATCC 6919 (FIG. 3B), and P. acidipropionici ATCC
25562 (FIG. 4), respectively. Interestingly, it has been found that
while P. acidipropionici converts linoleic acid to
(cis,trans)-9,11-CLA, P. acnes converts linoleic acid to
(trans,cis)-10,12-CLA under aerobic conditions.
[0143] FIG. 5 shows the results of whole cell biotransformation by
Lactobacillus reuteri. Unlike other microorganisms, the
concentration of (cis,trans)-9,11-CLA formed by L. reuteri from
linoleic acid does not decrease significantly with time. Addition
of various nonionic detergents, such as Tween-80 or Triton X-100,
does not significantly increase (cis,trans)-9,11-CLA formation.
Example 2
[0144] This example describes a procedure for fatty acid analysis
to determine the amount of conversion of linoleic acid to CLA.
[0145] Fatty acids were extracted from about 1 mL to about 2.5 mL
aqueous samples with 0.5 mL of 5 M NaCl added. The samples were
shaken with 5 mL of 2:1 mixture of chloroform/methanol in a glass
screw cap tube with Teflon lined cap. The two phases were separated
and about 1 to 2 mL of the chloroform layer was removed. The
organic layer was dried with Na.sub.2SO.sub.4 and concentrated. The
concentrated fatty acids were converted to methyl esters by a
modification of the procedure of Chin et al., J. Food Composition,
1992, 5:185-192. About 6 mL of 4% HCl in methanol preheated to
60.degree. C. was added to the glass tube containing the fatty acid
sample. The tubes were sealed with a Teflon lined cap and incubated
in a tube heater at 60.degree. C. for 20 minutes, then cooled to
room temperature, and 2 mL of water and 3 mL of hexane are added.
After shaking, the organic layer was separated, dried with
Na.sub.2SO.sub.4, and analyzed by gas chromatography. The order of
four CLA peaks was (1) (cis,trans)-9,11-CLA, (2)
(trans,cis)-10,12-CLA, (3) (cis,cis)-9-11-CLA and
(cis,cis)-10,12-CLA, and (4) (trans,trans)-9,11-CLA and
(trans,trans)-10,12-CLA.
Example 3
[0146] This Example describes the purification of linoleate
isomerase from L. reuteri.
[0147] Detergent soluble protein fractions were prepared as
follows. Frozen cells were thawed and suspended in breakage buffer
on ice. The standard breakage buffer for L. reuteri comprised 0.1 M
Bis-Tris (Calbiochem Ultrol grade) pH 5.8 (4.degree. C.), 10 mM
NaCl, 10% glycerol, 2 mM dithiothreitol. For other organisms, Tris
buffer at pH 7.5 was used in place of Bis-Tris buffer. The cell
suspensions were broken at 18,000 psi using a SLM Aminco French
press. The extract was centrifuged at 12,000.times. g for 30
minutes. The supernatant was further fractionated by centrifugation
at 100,000.times. g for 90 minutes to yield a soluble fraction and
a membrane pellet.
[0148] The membrane pellets were resuspended (approximately 5
mg/mL) and extracted with detergent buffer (0.1 M Bis-Tris pH 5.8,
0.25 M NaCl, 10% -glycerol, 2 mM dithiothreitol, 0.3%
octylthioglucopyranoside (OTGP, Calbiochem Ultrol grade)) at.
4.degree. C. for 4-18 hours with gentle stirring using a magnetic
flea. After centrifugation at 100,000.times. g for 90 minutes, the
supernatants (i.e., the detergent soluble protein fraction) were
further purified by Method A, B or C, infra.
[0149] Method A
[0150] Detergent soluble protein fractions were dialyzed overnight
against low salt buffer (0.1 M Bis-Tris pH 5.8, 10 mM NaCl, 2 mM
dithiothreitol, 10% glycerol, 0.3% OTGP), and. applied to a
2.1.times.15 cm DEAE-5PW column (TosoHaas) previously equilibrated
with low salt buffer. The column was washed (4 mL/min) with the
same buffer containing 1 M NaCl (high salt buffer). The results of
this step are shown in FIG. 6. Protein concentration was monitored
continuously at 280 nm. About 4 mL fractions were collected and
assayed for isomerase activity. Isomerase activity in the extracts
was measured at 20 ppm linoleic acid. Fractions with significant
isomerase activity were combined and concentrated using an Amicon
ultrafiltration cell. Concentrated fractions were then applied to a
1.6.times.55 cm Superdex-200 (Pharmacia) gel filtration column. The
column was developed with a buffer comprising 0.1 M Bis-Tris pH
5.8, 0.2 M NaCl, 10% glycerol, 2 mM dithiothreitol, 0.3% OTGP at
0.5 mL/min. Fractions of 2.0 mL were collected and assayed for
isomerase activity. Active fractions were collected, concentrated
and applied to a hydroxylapatite column (Bio-Rad 5 mL CHT-II
cartridge) equilibrated with 0.1 M Bis-Tris pH 5.8, 10 mM
KH.sub.2PO.sub.4, 10% glycerol, 2 mM dithiothreitol, 0.3% OTGP, 0.2
M NaCl. The column was washed (1 mL/min) with increasing phosphate
using the same buffer containing 400 mM KH.sub.2PO.sub.4 with the
results shown in FIG. 7. Active fractions were subjected to SDS
PAGE using the Pharmacia Phast System on 12.5% acrylamide gels. The
isomerase activity correlated with four bands on the gel, ranging
from 45 to 70 kilodaltons (kD).
[0151] Method B
[0152] Detergent soluble protein fractions were applied to an
affinity column. The affinity column was then sequentially washed
(1 mL/min) with low salt buffer (75 mL), high salt buffer (50 mL)
and linoleic acid buffer (100 mL) comprising 0.1 M Bis-Tris pH 5.8,
1 M NaCl, 0.3% OTGP, 2 mM dithiothreitol, 10% glycerol, 20%
1,2-propane diol.
[0153] The affinity column was prepared as follows. Pharmacia EAH
Sepharose 4B was washed and suspended as a slurry in deionized
water. A five-fold excess of ligand (linoleic acid or oleic acid)
was added in an equal volume of 1,2-propane diol. Solid
N-ethyl-N'-(3-dimethylaminopropyl- )carbodiimide hydrochloride
(EDC) was added to 0.1 M, pH adjusted to around 5.0, and the slurry
was mixed by slow inversion overnight at room temperature. The gel
was collected on a glass fritted funnel and extensively washed
successively with 50% 1,2-propane diol, 0.1 M potassium acetate pH
4.0, 0.5 M NaCl, and 0.1 M tris pH 8.2. The resin was then washed,
suspended in low salt buffer and used to prepare a 1.6.times.20 cm
affinity column.
[0154] Method C
[0155] Detergent soluble protein fractions were purified by a
chromatography using DEAE-5PW column as described in Method A. The
fractions containing isomerase activity were combined,
concentrated, and desalted by ultrafiltration. The resulting sample
was applied to a Mono PHR 5/20 column (Pharmacia, 0.5.times.20 cm)
which has been previously equilibrated with a buffer comprising 25
mM triethanolamine, 1 mM dithiothreitol, 0.3% OTGP at pH 8.3. The
column was then eluted with a buffer comprising 10% Polybuffer 96
(Pharmacia), 0.3% OTGP, 1 mM dithiothreitol at pH 6.5 and 1 mL
fractions were collected. As shown in FIG. 8, some of the proteins
were present in early fractions (fractions 5-15) and fractions
containing isomerase activity were eluted typically between
fractions 27 and 47. The fractions containing isomerase activity
were combined and further purified by a chromatography using
Superdex-200 gel filtration column as described in Method A. The
fraction containing isomerase activity was eluted as a single band
with a mass of about 160 kD. This same band was run on a denaturing
SDS-PAGE gel and resulted in a single band of about 70 kD. This 70
kD band was excised and subjected to N-terminal amino acid
sequencing using techniques known to those skilled in the art. A
partial N-terminal amino acid sequence of about 35 amino acids was
deduced and is represented herein as SEQ ID NO:1. A protein having
the sequence of SEQ ID NO:1 is referred to herein as PCLA.sub.35.
It should be noted that since amino acid sequencing technology is
not entirely error-free, SEQ ID NO:1 represents, at best, an
apparent partial N-terminal amino acid sequence.
Example 4
[0156] This example describes the procedure for determining
presence of isomerase activity of a fraction or a protein. This
example also describes a method for conducting a kinetic assay.
[0157] Linoleic isomerase activity was assayed either via CLA
quantification by gas chromatography as described in Example 2 or
by spectrophotometry. The enzyme assay was carried out in 0.1 M
Tris buffer pH 7.5, 10 mM NaCl, 1 mM dithiothreitol, with linoleic
acid at 20 parts per million (ppm), unless otherwise noted.
[0158] About 50 to about 250 .mu.L of enzyme sample was added to
1.5 mL of enzyme assay buffer for reaction. About 1 to about 2 mL
of aqueous phase was separated from the enzyme reaction and was
extracted with about 3 mL of hexane. In some experiments, and with
chromatography fractions containing detergent, 0.5 mL of methanol
and 0.5 mL of 5 M NaCl solution were first added to enhance phase
separation. The organic layer was separated and the absorbance at
234 nm was measured using a HP 8452A diode array spectrophotometer.
Depending on the level of activity, assay mixtures of
chromatography fractions were incubated at room temperature from
about 1 to about 24 hours before extraction with hexane.
[0159] Kinetic assays were performed directly in a 0.5 mL quartz
cuvette at room temperature and were continuously monitored at 234
nm. Reactions were initiated by addition of linoleic acid from a
concentrated stock prepared in 1,2-propane diol. Reaction buffer
was the same as above except it contained 10% 1,2-propane diol.
Example 5
[0160] This Example shows the nucleic acid cloning and sequencing
of a Lactobacillus reuteri linoleate isomerase nucleic acid
molecule of the present invention.
[0161] It should be noted that since amino acid sequencing and
nucleic acid sequencing technologies are not entirely error-free,
the sequences presented in this example and those below, at best,
represent apparent sequences of a linoleate isomerase of the
present invention.
[0162] Two sets of fully degenerated oligonucleotide primers: were
synthesized, corresponding the sequences of the amino acid residues
1-7 and 23-29 of SEQ ID NO:1.
[0163] The first oligonucleotide primer, designated CLAO1, had the
following sequence:
[0164] 5'-cgt gaa ttc ATG TA(T/C) TA(T/C) (T/A) (C/G)N AA(T/C) GGN
AA-3'
[0165] (including an EcoRI site and 3 extra bases (shown as lower
case letters) at the 5' end) (SEQ ID NO:2).
[0166] The second oligonucleotide primer, designated CLAO2, had the
following sequence:
[0167] 5'-act gga tCC NAC (T/A/G)AT (A/G)AT NGC (A/G)TG
(C/T)TT-3'
[0168] (including an Bam HI site and 3 extra bases (shown as lower
case letters) at the 5' end) (SEQ ID NO:3).
[0169] PCR products were amplified from L. reuteri genomic DNA
under optimized PCR conditions and gel purified. A single band of
PCR product with the expected size (about 100 bp) was detected on
3% agarose gel. The PCR product was purified and cloned at the Srf
I site into the vector pPCR-Script(Amp)SK(+) (Stratagen). Potential
recombinant plasmids were analyzed by restriction digestion and
sequenced.
[0170] Four clones which were sequenced contain inserts of about 87
nucleotides with the same sequence (SEQ ID NO:4) denoted herein as
nCLA.sub.87. The deduced amino acid sequence (SEQ ID NO:5) matches
the N-terminal sequence of the linoleate isomerase identified in
Example 3. A protein having the sequence of SEQ ID NO:5 is referred
to herein as PCLA.sub.28.
[0171] An approach of inverse PCR amplification was used to clone
the DNA fragments flanking the N-terminus coding sequence,
nCLA.sub.87. Two oligonucleotide primers, designated CLA03 and
CLA04 (SEQ ID NO:6 and SEQ ID NO:7, respectively) were designed for
inverse PCR. CLA03 corresponded to nucleotides 25-41 of nCLA.sub.87
(SEQ ID NO:4), and CLA04 nucleotides 46-67 of nCLA.sub.87 (SEQ ID
NO:4).
[0172] Genomic DNA from Lactobacillus reuteri PYR8 was digested
with the restriction enzyme Bam HI, treated with T4 DNA ligase to
circularize the molecules, and the resulting molecules were used as
a template in PCR reactions. A PCR product of 592 nucleotides was
purified and cloned at the Srf I site into the vector pPCR-Scrip
t(Amp)SK(+) (Stratagen) and sequenced. A 596 bp edited version of
this molecule is denoted herein as nCLA.sub.596 (SEQ ID NO:8).
nCLA.sub.596 contains both the 5' upstream and 3' downstream
sequences of a linoleate isomerase gene. The site of Bam HI in the
sequence would indicate the junction point. However, no Bam HI site
was detected in the sequence. Therefore, the sequence in
nCLA.sub.596 was tentatively edited with reference to its ORF and
the sequence nCLA.sub.87. This tentatively edited sequence contains
an ORF of 475 nucleotides. The deduced amino acid sequence of this
ORF is denoted PCLA.sub.158 (SEQ ID NO:9). A protein having the
sequence of SEQ ID NO:9 is referred to herein as PCLA.sub.158.
[0173] The sequences immediately flanking CLA03 and CLA04 are
identical to the sequence in nCLA.sub.87, confirming the identity
of the cloned PCR product.
[0174] nCLA.sub.596 was labeled with .sup.32P and hybridized to a
Southern blot of Lactobacillus reuteri PYR8 genomic DNA digested
with different restriction enzymes. The partial linoleate isomerase
sequence of nCLA.sub.596 contains one AgeI site and one Eco 58 I
site. As expected, two hybridization bands were observed on the
Southern blot when the genomic DNA were digested with these two
enzymes individually. Only one hybridization band was detected in
the digests prepared with enzymes which do not cut the partial
isomerase sequence such as BamHI, HindIII, PvuI, SalI, and XhoI
while a more diffused hybridization signal in the high molecular
mass region (>10 kb) was observed with EcoRI, SacI and SphI
digests. These data indicate that the linoleate isomerase gene is
present as a single copy in the genome of Lactobacillus reuteri
PYR8.
[0175] In order to clone the entire linoleate isomerase gene, an
approach of inverse PCR was followed. As set forth above, a
restriction enzyme AgeI site is present in the middle of SEQ ID
NO:8 (nCLA.sub.596) at nucleotide position 295. Southern
hybridization showed two bands in AgeI digests of L. reuteri PYR8
genomic DNA: about 1.1 and about 2.3 kb, respectively. Genomic DNA
from L. reuteri PYR8 was digested with AgeI, religated with T4 DNA
ligase and used as template in a PCR reaction. Under optimized
conditions, a PCR product of about 1.1 kb was generated using the
primer set of CLA03 and CLA04 as well as a product of 2.3 kb using
the primer set CLA05 and CLA06 (SEQ ID NO:12 and SEQ ID NO:13,
respectively). CLA05 corresponds to nucleotides 326-342 of
nCLA.sub.596 and CLA06 corresponds to nucleotides 396-414 of
nCLA.sub.596. These results are consistent with the Southern blot
data.
[0176] Both of the PCR products were cloned into
pPCR-Script(Amp)SK(+) (Stratagen). The clone containing the 1.1 kb
fragment is denoted nCLA.sub.1.1 and the clone with the 2.3 kb
fragment is denoted nCLA.sub.2.3. Initially about 700 nucleotides
of sequencing data upstream from CLA03 in nCLA.sub.1.1 and about
700 nucleotides of sequencing data down-stream of CLA06 in
nCLA.sub.2.3 were obtained. The sequences of nCLA.sub.596 partial
nCLA.sub.1.1, and partial nCLA.sub.2.3 were edited to generate a
composite sequence denoted herein as nCLA.sub.1709 (SEQ ID NO:10).
The deduced amino acid sequence of SEQ ID NO:10 is represented
herein as SEQ ID NO:11. A protein having the sequence of SEQ ID
NO:11 is referred to herein as PCLA.sub.324.
[0177] nCLA.sub.1709 contains part of the isomerase coding sequence
as well as 5' upstream sequence. The 737 nucleotide sequence
upstream from the ATG codon corresponding to the first amino acid
of the purified polypeptide was compared against known sequences by
using Blastx (open reading frames) and Blastn (nucleotides)
searches of the BLAST network. No significant homology has been
found with any entry, with the score being below 176 for Blastn and
153 for Blastx. The coding sequence downstream from the ATG start
codon showed a homology with 67 kD myosin-crossreactive
streptococcal antigen from Streptococcus pyogenes (U09352) : 69%
identity at the amino acid level and 66% at the nucleotide level.
The longest stretch of identical nucleotides is of 23 nucleotides.
The isomerase coding sequence shows also a homology to an ORF from
Staphylococcus aureus (L19300): 62% identity at both the amino acid
level and the nucleotide level.
[0178] Subsequent to the initial sequencing of nCLA.sub.1.1 and
nCLA.sub.2.3, both clones were completely sequenced (SEQ ID NO:14
and SEQ ID NO:15, respectively) and these sequences, along with the
sequence of nCLA.sub.596 (SEQ ID NO: 8), were assembled to generate
a nucleic acid sequence of 3551 nucleotides, which is denoted
nCLA.sub.3551 and which is represented herein by SEQ ID NO:16. SEQ
ID NO:14 spans from nucleotide 1 to 1173 of SEQ ID NO:16, and SEQ
ID NO:15 spans from nucleotide 1174 to 3551 of SEQ ID NO:16.
[0179] nCLA.sub.3551 contains three open reading frames (FIG. 9;
ISOM, D and E), with the first ORF (FIG. 9, ISOM) being an about
1.8 kb nucleic acid molecule spanning from nucleotide positions
1000 to 2775 of SEQ ID NO:16, and represented herein as SEQ ID
NO:17. SEQ ID NO:17 encodes a linoleate isomerase of the present
invention. A nucleic acid molecule having a nucleic acid sequence
represented by SEQ ID NO:17 is referred to herein as nCLA.sub.1776,
and encodes an approximately 67 kD (deduced) protein of 591 amino
acid residues having an amino acid sequence represented by SEQ ID
NO:18. A protein having amino acid sequence SEQ ID NO:18 is
referred to herein as PCLA.sub.591. The deduced size of PCLA591 is
consistent with the size of the purified isomerase protein
determined on an SDS gel. Seven nucleotides upstream from the
initiation codon of this first ORF (SEQ ID NO:17) is a sequence
similar to the consensus ribosome-binding-site which has been
reported in Lactobacillus. Also, upstream from this first ORF are
sequences similar to -10 and -35 promoter sequences. These sequence
characteristics are consistent with a conclusion that the start
codon at position 1000 of nCLA.sub.3551 is the translation start
codon. Alternatively, the sequence of nCLA.sub.3551 has, upstream
from the first ORF, at 36 nucleotides upstream from the start codon
at position 1000, in frame, two ATG start codons in tandem. If one
of these codons is a translation start codon, then a leader peptide
of about 12 amino acids may be produced which is subsequently
cleaved to form a mature isomerase.
[0180] The complete coding sequence for the linoleate isomerase
gene determined as described above (SEQ ID NO:17) was compared
against known sequences by using Blastx (open reading frames) and
Blastn (nucleotides) searches of the BLAST network. The linoleate
isomerase encoded by SEQ ID NO:17 showed 67% identity at the
nucleic acid level and 70% identity at the amino acid level with
the previously-mentioned Staphylococcus pyogenes (U09352) 67 kD
myosin-crossreactive streptococcal antigen. The Staphylococcus
pyogenes (U09352) protein has 590 amino acid residues. The homology
between the linoleate isomerase encoded by SEQ ID NO:17 and the
above-described Streptococcus aureus (L19300) gene is slightly
lower: about 60% at the nucleic acid level and about 62% at the
amino acid level. The BLAST Search Parameters with default values
for these searches are shown in Table 1 (see above). No defined
functions have been previously described for either the
Streptococcus pyogenes (U09352) or the Staphylococcus aureus
(L19300) sequences.
[0181] The second open reading frame of nCLA.sub.3551 (FIG. 9, E)
is from nucleotide positions 2896 to 3551 of SEQ ID NO:16, and is
represented by SEQ ID NO:19. A nucleic acid molecule having SEQ ID
NO:19 is referred to herein as nUNK1.sub.656 which encodes a
protein of about 218 amino acid residues having an amino acid
sequence of SEQ ID NO:20. A protein having SEQ ID NO:20 is referred
to herein as PUNK1.sub.218. The function of PUNK1.sub.218 is
unknown. The sequence of nUNK1.sub.656 was compared with known
sequences for homology and no significant homology was identified.
This second reading frame is located 122 nucleotides downstream
from the first open reading frame (SEQ ID NO:17) encoding the
linoleate isomerase.
[0182] The third open reading frame of nCLA.sub.3551 (FIG. 9, D) is
located on the strand of nCLA.sub.3551 that is complementary to SEQ
ID NO:16, and is represented herein as SEQ ID NO:21. SEQ ID NO:21
is positioned on the strand that is complementary to nucleotide
positions 1 through 726 of SEQ ID NO:16, with start codon 275
nucleotides up-stream from position 1000 of the putative start
codon of SEQ ID NO:17. A nucleic acid molecule having SEQ ID NO:21
is referred to herein as nCSN.sub.726 which encodes at least a
portion of a protein having an amino acid sequence of SEQ ID NO:22.
The C-terminal portion of the protein comprising SEQ ID NO:22 was
not present in the isolated clones. A 242 amino acid residue
protein having SEQ ID NO:22 is referred to herein as PCSN.sub.242.
A database search (BLAST) showed that the nucleic acid sequence of
this third ORF (SEQ ID NO:21) is about 66% identical to a
competence-specific nuclease (DNA entry nuclease) from
Streptococcus pneumoniae (Q03158), with the amino acid sequence SEQ
ID NO:22 being about 51-72% identical to the amino acid sequence
for this competence-specific nuclease. Therefore, it is believed to
be possible that the third ORF identified on the complementary
strand of SEQ ID NO:16 encodes a competence-specific nuclease.
Example 6
[0183] The following example demonstrates the cloning of sequences
flanking the isomerase gene in the L. reuteri PYR8 genome.
[0184] A third round of inverse PCR was carried out on the
circularized genomic DNA from Lactobacillus reuteri PYR8 as
described in Example 5. This third round was designed to clone more
sequences flanking the isomerase gene. Two oligonucleotide primers,
designated CLAo9 and CLAo10 (SEQ ID NO:23 and SEQ ID NO:24,
respectively) were designed for this round of PCR. CLAo9 (SEQ ID
NO:23) was designed close to the 5' end of the sequence of
nCLA.sub.3551 (nucleotides 63-40 of SEQ ID NO:16). CLAo10 was
designed to correspond to the 3' end of the nCLA.sub.3551 sequence
(nucleotides 3505-3522 of SEQ ID NO:16).
[0185] More particularly, L. reuteri PYR8 genomic DNA was digested
with SalI, religated and amplified with oligonucleotide primers
CLAo9 and CLAo10. A PCR product of about 3.5-4.0 kb was cloned into
pPCR-Script Amp SK(+) and sequenced. This nucleic acid molecule was
denoted nSAL.sub.3684 and is represented herein by SEQ ID
NO:25.
[0186] The identity of nSAL.sub.3684 was confirmed by the sequences
flanking the primers CLAo9 and CLAo10. The sequence nSAL.sub.3684
contains a unique SalI site, which indicates the junction point of
the inverse PCR product. Therefore, the sequence nSAL.sub.3648 was
spliced at the SalI site and added to the 3' and 5' ends of the
sequence of nCLA.sub.3551 (SEQ ID NO:16). This approximately 7 kb
nucleic acid molecule is denoted nCLA.sub.7113 and is represented
herein by SEQ ID NO:26.
[0187] The approximately 7 kb L. reuteri PYR8 genomic DNA (SEQ ID
NO:26) contains 6 open reading frames, schematically illustrated in
FIG. 9. There are four ORF's (A, B, C and D) located 5' upstream of
the isomerase gene (ISOM) and one ORF located 3' downstream of the
isomerase gene.
[0188] The first open reading frame of nCLA.sub.7113 (FIG. 9, A)
spans from nucleotide positions 1 to 941 of SEQ ID NO:26, and is
represented by SEQ ID NO:27. A nucleic acid molecule having SEQ ID
NO:27 is referred to herein as nBSP.sub.941 which encodes a protein
of about 312 amino acid residues having an amino acid sequence of
SEQ ID NO:28. A protein having SEQ ID NO:28 is referred to herein
as PBSP.sub.312. A database search (BLAST) showed that the amino
acid sequence (SEQ ID NO:28) of the protein encoded by this first
ORF A (SEQ ID NO:27) is about 56% identical and 74% similar (using
standard BLAST parameters) to a permease from Bacillus subtilis
(p54425). Therefore, it is believed to be possible that the first
ORF A of SEQ ID NO:26 encodes a permease.
[0189] The second open reading frame of nCLA.sub.7113 (FIG. 9, B)
spans from nucleotide positions 1146 to 1745 of SEQ ID NO:26, and
is represented by SEQ ID NO:29. A nucleic acid molecule having SEQ
ID NO:29 is referred to herein as nUNK2.sub.600 which encodes a
protein of about 199 amino acid residues having an amino acid
sequence of SEQ ID NO:30. A protein having SEQ ID NO:30 is referred
to herein as PUNK2.sub.199. The function of PUNK2.sub.199 is
unknown. The sequence of nUNK2.sub.600 was compared with known
sequences for homology and no significant homology was identified.
The highest Blastp score using standard defaults was 51.
[0190] The third open reading frame of nCLA.sub.7113 (FIG. 9, C)
spans from nucleotide positions 1742 to 2590 of SEQ ID NO:26, and
is represented by SEQ ID NO:31. A nucleic acid molecule having SEQ
ID NO:31 is referred to herein as nUNK3.sub.849 which encodes a
protein of about 282 amino acid residues having an amino acid
sequence of SEQ ID NO:32. A protein having SEQ ID NO:32 is referred
to herein as PUNK3.sub.282. The function of PUNK3.sub.282 is
unknown. The sequence of nUNK3.sub.849 was compared with known
sequences for homology and no significant homology was identified.
The highest Blastp score using standard defaults was 68.
[0191] The fourth open reading frame of nCLA.sub.7113 (FIG. 9, D)
spans from nucleotide positions 2662 to 3405 of SEQ ID NO:26, and
is represented by SEQ ID NO:33. A nucleic acid molecule having SEQ
ID NO:33 is referred to herein as nCSN.sub.744 which encodes a
protein having an amino acid sequence of SEQ ID NO:34. A 247 amino
acid residue protein having SEQ ID NO:34 is referred to herein as
PCSN.sub.247. PCSN.sub.242 (SEQ ID NO:22), described above in
Example 5 (the third ORF identified in nCLA.sub.3551) is included
in PCSN.sub.247 spanning from amino acid position 1 to 242 of SEQ
ID NO:34. Similarly, the nucleic acid sequence of nCSN.sub.726 (SEQ
ID NO:21) spans from nucleotides 1 to 726 of SEQ ID NO:33. A
database search (BLAST) showed that the amino acid sequence SEQ ID
NO:34 is about 57% identical and about 71% similar (using standard
parameters) to the amino acid sequence for the above-mentioned
Streptococcus pneumoniae competence-specific nuclease.
[0192] The fifth open reading frame of nCLA.sub.7113 (FIG. 9, ISOM)
is a nucleic acid molecule (nCLA.sub.1776, SEQ ID NO:17) encoding
the linoleate isomerase (PCLA.sub.591, SEQ ID NO:18) of the present
invention, as described above in Example 5.
[0193] The sixth open reading frame of nCLA.sub.7113 (FIG. 9, E)
spans from nucleotide positions 5574-7113 of SEQ ID NO:26, and is
represented by SEQ ID NO:35. A nucleic acid molecule having SEQ ID
NO:35 is referred to herein as nUNK1.sub.1540 which encodes a
protein having an amino acid sequence of SEQ ID NO:36. A 513 amino
acid residue protein having SEQ ID NO:36 is referred to herein as
PUNK1.sub.513. PUNK1.sub.218 (SEQ ID NO:20), described above in
Example 5 (the second ORF identified in nCLA.sub.3551) is included
in PUNK1.sub.513, spanning from amino acid position 1 to 218 of SEQ
ID NO:36. Similarly, the nucleic acid sequence of nUNK1.sub.656
(SEQ ID NO:19) spans from nucleotides 1 to 656 of SEQ ID NO:35. The
sequence of nUNK1.sub.1540 was compared with known sequences for
homology and no significant homology was identified. The highest
Blastp score for PUNK1.sub.513 using standard defaults was 51. The
C-terminal sequence of PUNK1.sub.513 is incomplete.
[0194] The isomerase gene is very likely transcribed as a
monocistron. This conclusion is based on two observations. First,
the ORF that is located immediately upstream from the isomerase
gene (FIG. 9, D) is coded on the opposite strand. Secondly, a
reverse-repeat DNA sequence was observed in the region downstream
from the stop codon of the isomerase gene (FIG. 10). This 28
nucleotide structure (SEQ ID NO:37), starting at the base 6 after
the stop codon, has only one unmatched base. This structure could
function as a rho-dependent stem-loop transcription terminator of
the isomerase gene. Therefore, it is concluded that the isomerase
gene is most likely transcribed as a monocistron and that the open
reading frame downstream from the isomerase gene seems to be in a
separate transcription unit.
[0195] Linoleate isomerase from L. reuteri is a membrane protein
since its activity is detected mostly in membrane fraction of
cellular protein extracts and detergent is needed to solubilize the
enzyme. Consistent with this data, the hydrophilicity plot of the
isomerase ORF shows a major hydrophobic domain close to the
N-terminal sequence, from amino acid residue 27 through 42. This
hydrophobic domain may function as a transmembrane segment as well
as part of an uncleaved signal peptide, which plays an important
role in directing the protein into the membrane. Also, it is
interesting to notice that the peptide contains 4 cysteine residues
at amino acid positions 89, 124, 336 and 430, suggesting the native
protein may have one or two internal disulfide bonds.
Example 7
[0196] The following example demonstrates the expression of L.
reuteri linoleate isomerase in E. coli.
[0197] Two oligonucleotides were synthesized to amplify the
isomerase gene (Promoter-ORF-Terminator) from L. reuteri PYR8
genomic DNA (described in Example 5). Nucleotide CLAo7 (SEQ ID
NO:38), the forward primer, corresponds to the positions 3296
through 3314 of the sequence nCLA.sub.7113 (SEQ ID NO:26) and it
includes a SalI site and 3 extra bases at the 5' end (lower
case):
[0198] 51-gcagtcgacGGAGTTAAGACTGAATTAG-3'
[0199] The nucleotide CLAo8B (SEQ ID NO:39), the reverse primer,
corresponds to the positions 5577 through 5593 of the sequence
nCLA.sub.7113 (SEQ ID NO:26) and it includes a SalI site and 3
extra bases at the 5' end (lower case):
[0200] 51-ctagtcgacGCAGTTTCTGTCATGAC-3'
[0201] The PCR product of 2.3 kb was ligated with blunt ends into
pPCR-Script(Amp)SK at the SrfI site. Ligated DNA was transformed
into E. coli cells. Clones with inserts in both orientations were
selected and tested for expression of the isomerase gene. In the
construct #1 (FIG. 11), the isomerase gene was placed downstream
from the lac promoter. In the construct #2 (FIG. 11), the isomerase
gene was placed reverse to the lac promoter.
[0202] To detect isomerase activity, E. coli cells transformed with
the different isomerase constructs were grown to mid log phase,
induced with or without IPTG for 1 to 3 hours and harvested for
testing in an isomerase activity assay. Linoleic acid was incubated
with E. coli cells (biotransformation) or with a crude cell lysate.
Fatty acids were extracted by hexane and analyzed on gas
chromatography. With both plasmid construct #1 and plasmid
construct #2 in E. coli, no isomerase activity was detected by
biotransformation or by crude cell lysate. SDS-gel analysis,
however, showed that IPTG induced expression of a 67 kD protein in
cells transformed with construct #1. The size of the expressed
isomerase protein is that predicted from the isomerase gene
sequence analysis and is in good agreement with the size of the
native isomerase purified from L. reuteri PYR8. The lack of
catalytic activity may be a result of incorrect folding and/or
membrane insertion of the isomerase in the heterologous system.
[0203] pET vectors were used to develop isomerase gene constructs
where the isomerase coding sequence is fused to a His tag at the
C-terminus. Using a commercial antibody specific to His tag, it
would be possible to monitor the levels of isomerase-His tag fusion
protein synthesized in E. coli, Lactobacillus, Bacillus, or any
other appropriate expression host by Western blot analysis, even if
the enzyme was inactive. Since the, constructs would be made with
E. coli plasmids, E. coli systems could be used to test the method.
The isomerase-His tag protein was expressed in E. coli to produce
large amounts of isomerase protein. This protein can be further
purified under denaturing conditions with nickel columns and used
in the production of antibodies specific to the L. reuteri PYR8
linoleate isomerase. Isomerase expression in the native host and
recombinant systems can be monitored with these antibodies.
Additionally, the antibodies can be used in immunoscreening to
identify new microorganism strains that produce linoleate
isomerases, and eventually to aid in the cloning of additional
linoleate isomerase genes.
[0204] In additional experiments, E. coli transformed with and
expressing the PYR8 isomerase gene with a His tag (construct #3,
FIG. 11) were grown under standard conditions to study expression
of the isomerase protein. On Coomassie Blue stained SDS gel, a band
between 60 and 70 kD was predominant in the cell lysate. This band
was present at a high level even before induction. The addition of
IPTG, however, induced a very strong overproduction of the protein
(data not shown). The highest expression level was achieved two
hours after IPTG induction. This protein band was strongly
recognized by anti-His tag antibody on Western blot, confirming
that this protein corresponds to the correct linoleate isomerase
fusion protein (data not shown).
[0205] The cells expressing the linoleate isomerase gene were
harvested four hours after IPTG induction and analyzed to determine
the location of the isomerase-His fusion protein. FIG. 13 outlines
the experimental protocol for the preparation of different protein
fractions. Briefly, E. coli cells expressing the isomerase-His tag
fusion protein were lysed in a non-denaturing buffer with lysozyme
and broken by sonication. The total cell lysate was centrifuged at
low speed to pellet the inclusion bodies. The crude inclusion
bodies were washed twice with 0.25% Tween 20 and 0.1 mM EGTA. The
proteins retained in the washed pellets were highly insoluble
aggregates of improperly folded peptides (inclusion bodies). The
supernatant generated by low speed centrifugation of the total cell
lysate was subjected to an ultra centrifugation step to separate
membrane (pellet) from soluble proteins. Detergent was used to
solubilize membrane proteins, which were then separated from other
insoluble membrane components by ultra-centrifugation. The total
cell lysate and different protein fractions were analyzed on SDS
gel and by Western blot. In the total cell lysate of E. coli cells
expressing the isomerase gene, only the protein band between 60 and
70 kD can be seen after Coomassie staining. This protein band was
recovered in the inclusion body fraction and was confirmed to be
the isomerase-His tag fusion protein by Western blot. Under the
conditions used in these experiments, the antibody did not
cross-react with other proteins in the cell lysate of E. coli that
did not contain the isomerase gene construct. The amount of fusion
protein in the soluble and membrane fractions was under the
detectable limit. The fusion protein in the inclusion body fraction
was extensively washed with EGTA and Tween 20 to remove other
contaminant proteins. The purified peptide will be used to produce
antibodies specific for the PYR8 linoleate isomerase.
[0206] Additional strategies for expressing a linoleate isomerase
of the present invention include, but are not limited to: (1)
deleting the single hydrophobic domain of the sequence to try to
convert the isomerase into a functional soluble protein for use in
determination of fusion protein synthesis, solubility and isomerase
activity; (2) developing constructs for production of the isomerase
in L. reuteri using both the native promoter and non-native
inducible or constitutive promoters, including an isomerase-His tag
fusion gene under the control of the isomerase native promoter; (3)
cloning the promoter from the erythromycin resistance gene for
control of isomerase gene expression in L. reuteri ATCC 23272; and
(4) knocking out the wild-type linoleate isomerase gene in the
native L. reuteri PYR8 strain and recovering the activity by
transforming the strain with the cloned isomerase gene. In this
fourth strategy, a plasmid has been generated to knock out the
wild-type gene which contains a non-functional isomerase gene
interrupted by an erythromycin resistance gene as a selectable
marker.
Example 8
[0207] The following example describes expression of a linoleate
isomerase of the present invention in Bacillus.
[0208] To express the L. reuteri PYR8 linoleate isomerase gene
described in Example 5 in Bacillus subtilis and Bacillus
licheniformis, two oligonucleotides were synthesized to amplify
isomerase coding sequence from L. reuteri genomic DNA. The forward
primer (SEQ ID NO:40) corresponds to nucleotide positions 3678
through 3706 of nCLA.sub.7113 (SEQ ID NO:26), with a NdeI site
containing the ATG start codon at the 5' end (lower case):
2 5'catATGTATTATTCAAACGGGAATTATGAAGC-3'.
[0209] The reverse primer (SEQ ID NO:41) corresponds to nucleotide
position 5579 through 5602 of the sequence nCLA.sub.7113 (SEQ ID
NO:26) with a BclI site at the 5' end (lower case):
[0210] 5'tgatcaTCTATACCAGCAGTTTCTGTCATG-3'
[0211] The PCR product of 1.9 kb was cloned as blunt ends at the
SrfI site into pPCR-Script Amp SK and transformed into cells of E.
coli strain NovaBlue. Since dam methylation in this host prevents
BclI digestion, the recombinant plasmid was transformed into cells
of E. coli strain GM2163, which is a dam minus strain. Recombinant
plasmid DNA was digested with the restriction enzymes NdeI and BclI
and ligated to the vector pBHAI which had been digested with NdeI
and BamHI. Recombinant plasmid DNA was digested with SacI to remove
the E. coli portion of the vector, recircularized, and transformed
into B. subtilis 23856.
[0212] In this construct, the isomerase coding sequence was placed
under the control of the HpaII promoter (FIG. 12, #1 construct) and
its native ribosome-binding site was replaced by the counterpart in
the vector. Clones of transformants were grown to mid-log phase and
then harvested for biotransformation of linoleic acid. No CLA was
detected by GC analysis in the hexane extract of fatty acids.
However, after incubation for 1 hour, 2 hours, and 3 hours, the
level of linoleic acid decreased drastically, being about 40% after
a 3 hour incubation. The same results were observed with all
sixteen B. subtilis clones tested. The use of linoleic acid was
dependent on the presence of the cloned isomerase gene since the
level of linoleic acid was constant during the incubation of B.
subtilis wild type cell without the plasmid and the cells
transformed with the empty vector. The same results were observed
when the isomerase construct was transformed into B. licheniformis
T399.
[0213] Experiments were carried out to investigate why CLA did not
accumulate while linoleic acid was used up. One possibility was
that linoleic acid might be converted to CLA, which was rapidly
metabolized or degraded. That implied that B. subtilis and B.
licheniformis cells have the ability to metabolize CLA. To test
this hypothesis, Bacillus wild type cells and cell transformed with
the isomerase gene construct were incubated with single 9,11 isomer
produced in a biotransformation using L. reuteri PYR8 cells and
with chemically synthesized CLA, which contains 9,11 and other CLA
isomers. Bacillus cells could not metabolize the CLA. The same
conclusion was also drawn with crude cell extracts.
[0214] Furthermore, a peak of unknown product (retention time=20
minutes) on GC spectra of the biotransformation with cells
containing the isomerase gene was observed. Also, the conversion of
linoleic acid and formation of the unknown product seemed to be at
a 1:1 ratio. Preliminary GC-MS analysis indicated that this unknown
product has a molecular weight consistent with that of a
hydroxylated linoleic acid derivative. Further structural analysis
by different methods may help to determine the identity of the
product.
[0215] Without being bound by theory, the present inventors believe
that this unknown product may be an intermediate of linoleic acid
conjugation. When this product was incubated with L. reuteri PYR8
cells or crude enzyme extracts, however, it could not be converted
to CLA. It is possible that the intermediate has to be bound with
the enzyme or membrane during the conjugation, and once it is
released the conjugation could not be completed.
[0216] Further experiments include developing a series of
constructs based on the vector pLAT10 to explore the advantage of
including the His tag (FIG. 12). pLAT10 is a plasmid that can be
used to directly transform B. subtilis and B. licheniformis. It has
the promoter, coding sequence and the terminator of the LAT gene
encoding .alpha.-amylase. Also present is a signal peptide sequence
for mobilizing proteins into or across the Bacillus membrane. In
construct #2, the isomerase coding sequence was placed under
amylase promoter control as a fusion to its signal peptide.
Normally, the LAT signal sequence directs the protein into or
across the membrane. Soluble proteins typically are secreted into
the culture broth and in the process, the signal peptide is removed
by specific proteases. Membrane protein would migrate to and
integrate into the membrane. Since the hydrophobic domain of the
isomerase peptide may function both as an uncleaved signal sequence
and transmembrane segment in L. reuteri, it is not known if such a
domain of the protein would interfere with the proper function of
the secretion machinery in Bacillus and the LAT signal-isomerase
may not fold into proper conformation. In construct #3 (FIG. 12),
the entire coding sequence of the isomerase gene is fused to His
tag at the C-terminus while in construct #4, the isomerase sequence
without the hydrophobic domain is fused to His tag. In constructs
#5 and #6, the secretion signal peptide is removed. With these new
constructs, it can be determined whether the isomerase protein is
synthesized in Bacillus cells and in which cellular fractions the
protein is located.
Example 9
[0217] The following example describes the purification of
linoleate isomerase from Propionibacterium acnes.
[0218] P. acnes ATCC 6919 is the only microorganism known to
produce t10,c12-CLA directly from linoleic acid. Experiments
described in Example 1 using whole cells confirmed the presence of
a 10,12-linoleate isomerase in this organism. Enzyme extracts were
prepared by French Press. FIG. 14 shows the formation of
t10,c12-CLA from linoleic acid using whole cells of P. acnes.
Cultures were grown anaerobically to stationary phase in a complex
brain heart infusion medium, harvested and resuspended in the same
medium containing 500 ppm linoleic acid. Cells were incubated
aerobically with shaking at ambient temperature. The level of
linoleic acid decreased about 50% in 24 hours. About half of this
missing linoleic acid could be detected as t10,c12-CLA. No
c9,t11-CLA was observed. With prolonged incubation, the level of
t10,c12-CLA changed only slightly, while nearly all remaining
linoleic acid disappeared. At present it is unclear how linoleic
acid is metabolized in this organism. In other experiments,
t10,c12-CLA rose in concentration, but later disappeared
completely, as did all of the linoleic acid (results not shown).
This suggests that t10,c12-CLA may be subject to further
metabolism, possibly by a reductase. Linoleic acid may also be a
substrate for enzymes other than the isomerase.
[0219] Enzyme extracts were prepared by French Press and the
extract fractionated as outlined in FIG. 15. Taking the total
isomerase activity in fraction A as 100%, over 93% of the activity
was detected in the soluble protein fraction (B). Less than 1% of
the isomerase activity was found in the washed pellet, or membrane
fraction (C). Approximately 2% of the activity was located in the
buffer fraction (D), after the pellet washing and centrifugation
steps. Thus, the P. acnes isomerase clearly is not a membrane
protein, unlike the isomerase activities in L. reuteri PYR8 and
other strains examined to date.
[0220] Isomerase activity, using a crude soluble enzyme
preparation, was not significantly affected by overnight dialysis.
A number of possible cofactors were tested for their effect on
isomerase activity, including NAD, NADH, NADP, NADPH, FAD, FMN,
ADP, ATP and glutathione. No significant effect was observed in 60
minute assays with any of these compounds. Calcium and magnesium
also had no effect. Isomerase activity was not inhibited by the
chelators EDTA (5 mM) or 1,10-phenanthroline (1 mM), or the
sulfhydryl reagents p-chloromercuribenzoate (5 .mu.M) or
N-ethylmaleimide (100 .mu.M).
[0221] The effect of pH on enzyme activity in crude extracts was
examined. The isomerase activity exhibits a pH optimum centered
around 6.8 (FIG. 16).
[0222] Formation of CLA was determined by measuring the absorbance
at 234 nm. FIG. 17 shows a typical time course experiment using the
crude isomerase extract as enzyme source. Generally, the isomerase
was assayed using an endpoint assay after 30 to 60 minutes
incubation at room temperature. FIG. 18 (time course assay at
different linoleic acid levels) and FIG. 19 (end point assay at
different linoleic acid levels) show the effect of increasing
substrate concentration on formation of linoleic acid. These data
suggest that the enzyme in P. acnes is not subject to the same type
of substrate inhibition observed in the linoleic acid isomerases of
C. sporogenes, L. reuteri and B. fibrisolvens.
[0223] The effect of temperature on isomerase activity has been
examined to a limited extent. The enzyme works very slowly at
4.degree. C., demonstrating much better activity at room
temperature. CLA formation was virtually the same at 37.degree. C.
as at room temperature (data not shown). These results again differ
significantly from those observed with the particulate L. reuteri
isomerase.
[0224] Purification of the linoleate isomerase in P. acnes was
initiated. Following preparation of a centrifuged crude extract,
samples were applied to several columns to determine applicability
and suitable conditions. Typical chromatograms for some of these
pilot experiments are shown in FIG. 20 and FIG. 21 for DEAE and
hydrophobic interaction chromatography (HIC), respectively. The
initial purification trial consisted of DEAE followed by HIC and
gel filtration chromatography. After these three columns, however,
multiple bands were seen on SDS PAGE.
[0225] This initial attempt at purification clearly highlighted the
need to optimize separation conditions. The DEAE step was optimized
further by altering the salt gradient program. Following a linear
gradient to 0.175 M NaCl, the salt level was held at this level for
70 ml. The isomerase eluted at this point, after which time the
gradient was continued to elute other proteins.
[0226] The isomerase binds very tightly to the phenyl HIC column,
and is only released with ethylene glycol. A large number of other
proteins were also released, however, with stepwise exposure to 20%
ethylene glycol. The HIC chromatography step was altered by use of
an ethylene glycol gradient from 5 to 30%. This resulted in a
somewhat sharper elution profile for the isomerase than previously
obtained (results not shown).
[0227] Following DEAE and HIC chromatography, chromatofocusing was
employed. This method separates molecules on the basis of
isoelectric point. Protein was applied to a weak anion exchange
column at high pH, and eluted as the pH decreased by application of
a lower pH "Polybuffer". Preliminary experiments showed that a pH
gradient from 6.5 to 4.0 resulted in elution of the isomerase at a
pH around 4.4. Clearly the isomerase is a fairly acidic
protein.
[0228] Following HIC chromatography, a single fraction containing
high isomerase activity was applied to a Pharmacia MonoP
chromatofocusing column equilibrated with 20 mM bis-Tris is (pH
6.5). The pH gradient was formed using 10% Polybuffer 74 (pH 4.0).
Results are shown in FIG. 22. The isomerase activity eluted in a
sharp peak around pH 4.5. The three fractions containing activity
were examined for purity by SDS PAGE. Fraction 32 appeared on 12.5%
and 20% gels as a single protein band with a mass of 50 kDa.
[0229] This material was submitted for amino acid sequencing. After
running the sample on a SDS PAGE gel, the single band was
transferred and N-terminal sequencing performed at the UW Medical
College of Wisconsin. Surprisingly, several signals were obtained,
indicating the presence of multiple peptides or that the N-terminal
portion of the peptide was highly degraded (unlikely) in this
apparent single band. Subsequent analysis of isomerase purified
further (described below) determined that the N-terminus of the
protein was blocked.
[0230] To further modify the purification scheme to obtain pure
isomerase, the protocols previously used were revised and improved
to enhance the purification. As before, soluble crude extract was
prepared by cell disruption. This material was fractionated using
DEAE chromatography. Fractions from several runs containing
significant isomerase activity were pooled, dialyzed, and reapplied
to the same column. The active fractions were pooled, made 1 molar
in (NH.sub.4).sub.2SO.sub.4, and applied to a phenyl hydrophobic
column in several runs. Active fractions were concentrated, if
necessary, and analyzed by SDS PAGE chromatography. Fractions
having high isomerase activity exhibited a large number of protein
bands at this stage. Selected fractions from the HIC column were
pooled, concentrated, dialyzed, and applied to a chromatofocusing
column. Protein elution was accomplished with a shallower pH
gradient than was previously used, from 5.5 to 4.0. The isomerase
activity eluted as a sharp peak at about pH 4.2. Active fractions
were examined for purity by SDS PAGE. At this point, several
fractions appear to contain a single band approximately 50 kDa in
size (data not shown). This material is currently being carefully
checked for homogeneity. Other active fractions exhibited three to
four additional bands. These fractions will be applied to a gel
filtration column if further purification is required.
[0231] N-terminal sequencing of the P. acnes linoleate isomerase
has been completed. The N-terminal amino acid is blocked, and
therefore, N-terminal sequence cannot be determined directly. After
fragmentation with protease, internal fragments will be
chromatographically separated and the amino acid sequence will be
determined. Upon obtaining the sequence, the entire linoleate
isomerase nucleic acid and amino acid sequence will be derived
using methods described for the L. reuteri linoleate isomerase in
Example 5, and as described below.
[0232] In preparation for cloning the 10,12 linoleate isomerase
gene from Proprionibacterium acnes, a genomic DNA library is being
created. Using a modified protocol for DNA isolation from
Gram-positive bacteria, genomic DNA of good quality was isolated
from P. acnes. After small-scale tests, genomic DNA was partially
digested with Sau3AI, partially filled with dGTP and dATP, size
selected by electrophoresis through agarose gel, and purified from
agarose gel by electroelution. This purified DNA digest will be
ligated to XhoI half-site arm of the BlueStar phage vector,
packaged and plated. The quality of the library (titer and
percentage of recombinant phage) will be tested. Oligonucleotide
probes will be created based on the determination of the internal
fragment sequences of the P. acnes linoleate isomerase and will be
used to screen the library and isolate one or more clones for
sequence analysis.
Example 10
[0233] The following example describes the purification and
characterization of a linoleate isomerase from Clostridium
sporogenes.
[0234] Previous work by the present inventors and by others has
shown that C. sporogenes is capable of converting significant
amounts of linoleic acid to CLA. The linoleate isomerase from C.
sporogenes appears to have activity levels and characteristics most
similar to that of L. reuteri PYR8. The following experiments
describe the purification and characterization of the linoleate
isomerase from this microorganism, with the goal to clone this
isomerase gene, as has been described for L. reuteri in Example 5.
The cloned C. sporogenes isomerase gene activity and functionality
will then be compared to the recombinant L. reuteri activity.
[0235] C. sporogenes ATCC 25762 was grown in a Brain Heart Infusion
Broth (BHI) medium under anaerobic conditions. Bacterial growth was
measured with a spectrophotometer at 600 nm. When cells were grown
at 37.degree. C., pH 7.5, stationary phase was reached after 6
hours incubation. Further incubation resulted in rapid lysis of the
culture. Cultures were harvested, therefore, after about 6 hours
growth. The cell pellet was washed with 0. 1 M Tris, pH 6.0,
containing 15 mM NaCl.
[0236] Biological transformation of CLA was performed by
resuspending harvested cells in fresh growth medium containing 200
ppm linoleic acid. After incubation, fatty acids were extracted
with hexane and analyzed by gas chromatography using an isothermal
program at 215.degree. C. for 14 minutes. FIG. 23A-D shows a time
course of biotransformation of linoleic acid by C. sporogenes.
Resuspended cells were grown under aerobic (FIGS. 23A & C) or
anaerobic (B & D) conditions at room temperature (A & B) or
at 37.degree. C. (C & D). A rapid production of c9, t11-CLA
with a simultaneous decrease in linoleic acid was observed within
30 minutes under all conditions tested. Upon further incubation,
t9, t11-CLA accumulated at the expense of c9, t11-CLA. Upon
extended incubation (15-20 hours), c9, t11-CLA disappeared.
Apparently the CLA was metabolized further. Similar amounts of CLA
were formed under aerobic and anaerobic conditions.
[0237] The cells were extracted as described in FIG. 24 to give
four principal fractions. Tables 1 and 2 show the distribution of
isomerase activity and protein concentration in fractions which
were prepared with low salt (10 mm NaCl) from frozen cells and with
high salt (500 mM NaCl) from fresh cells, respectively. Enzyme
activity was detected in all fractions. The highest activity was
found in the 45 k/0.3% OTGP soluble fraction. It has been reported
that detergents require high concentration of salt for effective
solubilization of membrane proteins. Addition of NaCl in extract
buffer resulted in increasing specific activity (Table 2),
indicating the effectiveness of high salt. The specific activity
was at least 50-fold higher in high salt detergent soluble
fractions (Table 3) than in low salt detergent soluble fractions
(Table 2). Conditions under which the active cultures are stored
could also affect activity. These results suggested that the C.
sporogenes linoleate isomerase has characteristics similar to the
L. reuteri PYR8 membrane-associated enzyme.
3TABLE 2 Linoleate Isomerase Activity - Low Salt Extracts of Frozen
Cells Total Specific Activity Protein Protein (OD.sub.234/60
Fraction (mg/ml) (mg) OD.sub.234 min/mg protein) Crude Extract 6.8
408 0.26 .+-. 0.01 0.38 45K Superna- 4.8 288 0.12 .+-. 0.01 0.25
tant 45K OTGP 1.2 30 0.05 .+-. 0.00 0.41 Soluble 10K OTGP 2.9 73
0.05 .+-. 0.01 0.17 Soluble
[0238]
4TABLE 3 Linoleate Isomerase Activity - High Salt Extracts of Fresh
Cells Total Specific Activity Protein Protein (OD.sub.234/60
Fraction (mg/ml) (mg) OD.sub.234 min/mg protein) Crude Extract 6.0
390 0.66 .+-. 0.02 1.10 45K Superna- 6.0 390 0.31 .+-. 0.02 0.51
tant 45K OTGP 0.8 20 0.16 .+-. 0.01 20.0 Soluble 10K OTGP 2.0 44
0.25 .+-. 0.01 12.5 Soluble
[0239] Temperature optimization with crude extracts was carried out
at 4.degree. C., room temperature (.about.25.degree. C.) and
37.degree. C. The best temperature for the isomerase activity was
room temperature (FIG. 25). The isomerase activity decreased to 73%
of optimum at 4.degree. C. and 63% of optimum at 37.degree. C. The
optimum pH was monitored by adjusting the pH from 5.0 to 9.0 using
the 0.1M Tris buffer with 10 mM NaCl, 1 mM DTT and 40 ppm linoleic
acid. The optimum pH was found to be 7.5, 8.0 and 9.0 for
incubating at 4.degree. C., room temperature and 37.degree. C.,
respectively (FIG. 25).
[0240] The concentration of linoleic acid was tested from 0 to 100
ppm (FIG. 26). The optimum concentration for linoleic add was
determined to be 40 ppm. A time course study indicated that the
activity responded linearly within 20 minutes and showed a slight
decrease upon 60 minutes incubation at optimum pH, temperature and
substrate concentration (FIG. 27).
[0241] The C. sporogenes isomerase was alternatively extracted by
sonication in 0.1 M Tris, pH7.5, 10 mM NaCl, 2 mM DTT and 10%
glycerol. This extraction was of higher efficiency (about 20%) than
that by French press. This is different from the isomerase isolated
from L. reuteri, wherein it was observed that sonication resulted
in a total loss of activity. Isomerase activity was higher in
phosphate buffer, pH 7.5, than in Tris buffer, pH 7.5 (FIG. 28).
The enzyme was most stable in phosphate buffer.
[0242] The detergent soluble fraction was further purified by
Method A, B or C, infra.
[0243] Method A
[0244] Experimental conditions for purification of the isomerase by
DEAE-5PW chromatography have been established. Under these
conditions, 75% of the isomerase was recovered. FIG. 29 gives an
overview of the purification of the isomerase from OTGP solubilized
protein. Three experiments were performed with similar results: the
C. sporogenes isomerase eluted from the column by 0.5 M NaCl. The
peak fractions (#48 to #51) contained 60% of the isomerase loaded
on the column, resulting in a 6-fold purification to an average
specific activity of 32. The column was eluted further with 1 M
NaCl, and putative enzyme activity was detected by UV analysis
(linoleic acid (LA) was apparently converted into products with
spectra identical to CLA). However, analysis of these fractions by
GC showed that the major product of the conversion had a retention
time of 13 minutes, while the retention time of c9, t11-CLA is
about 10 minutes. Although this peak was minor as compared to the
peak eluted by 0.5 M NaCl, this data suggested that C. sporogenes
cells may have the ability to produce other isomers of CLA. It was
observed that a freshly prepared extract should be used to achieve
a high recovery of isomerase activity by ion-exchange
chromatography, because the detergent solubilized protein tended to
lose activity or it precipitated after storage at 4.degree. C. for
more than 3 days (data not shown).
[0245] A rapid spectrophotometric assay for CLA, measuring
absorbance at OD.sub.234 (detects conjugated fatty acid bonds as
well as other UV absorbing compounds), was used to estimate CLA
concentration in column eluate fractions. It was important to
confirm that the OD.sub.234 absorbing material was CLA before
attempting further purification. Gas chromatography was used for
this purpose. Comparison of the OD.sub.234 data with the result
obtained by gas chromatography showed good correlation, indicating
that the "active" fractions collected contained the desired
isomerase activity.
[0246] Isomerase was partially purified on DEAE with acceptable
minimal loss of activity as described above. However, the activity
of pooled enzyme fractions decreased by 50% after overnight storage
at 4.degree. C. In some experiments, no activity was detected after
DEAE purification. Therefore, it would be important to maintain the
enzyme activity to continue purification.
[0247] It was demonstrated that the C. sporogenes linoleate
isomerase is a membrane protein. The detergent,
octyl-thioglucopyranoside (OTGP), has been used successfully to
solubilize isomerase. Unfortunately, OPTG (and the solubilized
enzyme) precipitated slowly during purification at 4.degree. C. A
nondenaturing detergent, Triton X-100 with a high concentration of
salt, is commonly used to distinguish between peripheral and
integral membrane proteins. No significant difference was found in
the efficiency of the solubilization between 1% Triton X-100. and
0.3% OPTG. Less total protein was solubilized with a mixture of 0.1
% Triton X-100 and 0.3% OPTG. The efficiency of solubilization by 1
M NaCl alone was very low, indicating that the isomerase is an
integral membrane protein.
[0248] Method B
[0249] It was clearly necessary to improve the activity and
stability of the C. sporogenes isomerase. Two different media, BHI
and MRS, were tested. Results are shown in FIG. 30. At pH 7.0, less
isomerase was produced in MRS medium, although a higher total
protein was obtained. There is no difference in stability of
isomerase produced in either medium. Protease inhibitors, PMSF
(0.1-1.0 mM), iodoacetamide (1 mM) and pepstatin A were tested for
their effects on the stability of isomerase. None provided
measurable benefit.
[0250] It has been reported that solubilization in the presence of
lysophosphatidylcholine (LPC) allows higher detergent
concentrations to be used, thus allowing more complete membrane
protein solubilization. CaCl.sub.2 can activate enzymes, such as
some nucleases. Added CaCl.sub.2 plus LPC has been demonstrated to
stabilize detergent solubilized sodium channel membrane proteins.
None of these positive effects was observed on the linoleate
isomerase. Moreover, CaCl.sub.2 decreased the enzyme activity in
both Tris and phosphate buffer systems. At temperatures higher than
37.degree. C., CaCl.sub.2 had no effect on the activity, but the
isomerase activity was reduced to 50% at the temperatures of
42.degree. C. and 60.degree. C. (FIG. 31).
[0251] FIG. 32 shows the effect of the iron-chelating agents,
phenanthroline and EDTA, on the enzyme activity and stability. It
seems that the enzyme in crude extracts was protected by
phenanthroline. This protection was more effective when 1 mM of
phenanthroline was combined with 1 mM EDTA, although addition of 1
mM EDTA had a negative effect. In contrast, the addition of
iron-chelating compounds to detergent buffer resulted in a loss of
activity during the enzyme preparation, but a slight increase in
stability (FIG. 33).
[0252] Prior to efforts to further purify the 9,11-linoleic acid
isomerase from C. sporogenes, endeavors were made to increase the
stability of the enzyme in crude extracts and in the detergent
soluble fraction to be able to complete the procedures necessary
for purification.
[0253] The effect of pH and type of buffer used during enzyme
extraction, on extraction efficiency and on enzyme stability were
examined. Crude extracts were prepared with 0.1 M Tris buffer at pH
5.0-9.0. The pH during extraction had a strong impact on both
extraction efficiency and enzyme stability. The optimum pH for the
extraction of the isomerase was 7.5 (FIG. 34), and at this pH, the
half-life of isomerase was extended from one day at pH 8.0 to one
week (FIG. 35).
[0254] The effect of the type of buffer was also significant. Tris
buffer, potassium phosphate buffer, and Hepes buffer were compared,
and the results are shown in FIG. 36. Phosphate buffer was the most
effective in extraction and solubilization of the isomerase. This
buffer produced a distinct increase in the activity obtained. In
crude extracts, activity was about double that obtained with Tris,
and in detergent soluble fractions a four- to seven-fold increase
was measured. Further improvements (FIG. 37) were obtained by
increasing the NaCl (20% increase in activity) and glycerol
concentrations (30% increase).
[0255] The enzyme stability was compared at pH 7.5. In general, the
isomerase was more stable in crude extracts than in detergent
solubilized fractions (FIG. 38). A half-life of 10, 11 and 13 days
was measured in Tris, phosphate and Hepes crude extracts,
respectively. Increasing glycerol and salt concentration provided
major improvements on stability, resulting in near full retention
of activity for one week. However, half-life of detergent
solubilized isomerase was only three and six days in Tris and
phosphate buffer, respectively.
[0256] A small-scale purification was performed using a Pharmacia
DEAE Mono Q column with enzyme solubilized with 0.3%
octyl-thioglucopyranoside (OTGP), as described above, and with
phosphate buffer replacing the previously used Tris. A single peak
of activity, eluting at approximately 250 mM NaCl, was obtained
(FIG. 39). The specific activity after this step increased 2.5
fold. This result was reproducible and repeatable. SDS-PAGE
analysis of the protein from this column showed a band
corresponding to a molecular weight of approximately 70 kDa (data
not shown). The molecular mass is similar to that of the 9,11
isomerase of L. reuteri, suggesting that the two isomerases may
have similar characteristics.
[0257] OTGP has been used successfully to solubilize the isomerase.
However, the detergent (and the solubilized enzyme) slowly
precipitates at 4.degree. C. This precipitation results in more
than 50% loss of activity after desalting of the enzyme solution by
dialysis, but more importantly, it clogs the ion exchange columns,
rendering them unusable.
[0258] Therefore, detergents that could efficiently solubilize the
isomerase while avoiding the precipitation problem were sought.
Triton X-100 has a good performance as solubilizing agent for the
isomerase, and the amount of protein solubilized increased with
increasing Triton X-100 concentrations. Isomerase extraction was
also enhanced at high salt concentration (500 mM NaCl). However, it
was determined that enzyme activity was completely lost when the
solution was dialyzed before ion exchange. The use of low salt
concentration resulted in lower protein extraction from the
membrane pellet, but similar enzyme activity and eliminates the
requirement for the desalting step.
[0259] Extraction efficiency similar to that obtained with OTGP has
been achieved using 2% Triton X-100 in 50 mM phosphate buffer. A
comparison of soluble protein and specific activity in the two
detergent systems is shown in Table 4. The enzyme stability is
reduced in Triton with respect to OTGP, which is one remaining
disadvantage of this new detergent system. However, the conditions
would still give a workable time frame to purify the enzyme by
multiple steps of chromatography. The continued purification scheme
for the isomerase is DEAE chromatography, followed by
chromatofocusing, as has been done for the isomerases described in
Examples 5 and 9.
5TABLE 4 Preparation of C. sporogenes 9, 11 Isomerase Extracts with
OTGP and Triton X-100 Enzyme Specific Activity Protein Activity
Sample (A234) (mg/ml) (A234/mg) Crude Extract 0.84 7.6 1.10 45K
Soluble 0.12 6.6 0.18 0.3% OTGP Soluble 0.40 3.3 1.22 2% Triton -
50 mM NaCl 0.42 2.8 1.50
[0260] A third nonionic detergent, octyl glucoside (OG) was tested
for its ability to solubilize the C. sporogenes linoleate
isomerase. OG effectively solubilized the isomerase and the
activity of the solubilized enzyme was stable. OG at 1.5% (2.times.
critical micelle concentration) produced an isomerase specific
activity about 20% higher than that of OTGP solubilized isomerase.
No precipitation was observed in the solubilized membrane protein
sample after dialysis.
[0261] Method C
[0262] While OG can be used to solubilize linoleate isomerase, this
detergent is too expensive to use in large-scale isomerase
purification. The protocol to solubilize linoleate isomerase was
modified to initially solubilize isomerase with OG, and then keep
the enzyme solubilized with OTGP. the membrane fraction was
solubilized with 1.5% OG in 50 mM potassium phosphate buffer at
15.degree. C. OG solubilized proteins were dialyzed against 20 mM
potassium phosphate buffer, pH 7.5, 10 mM NaCl, 2 mM dithiothreitol
and 0.3% OTGP. Afte4r centrifugation at 45,000 g for 30 minutes,
the solubilized proteins were applied to a DEAE-5PW column,
equilibrated with low salt buffer (20 mM bis-Tris, pH7.5, 10 mM
NaCl, 0.3% OTGP, 1 mM dithiothreitol, 1 mM EDTA and 1 .mu.M
pepstatin A) and eluted with a linear gradient of NaCl from 0 to
0.5 M at 16.degree. C.
[0263] The DEAE-5PW column chromatography achieved a 4-fold
purification. Two distinct peaks of isomerase activity were
revealed (FIG. 40). Peak II, which eluted at higher ionic strength,
was observed in all previous DEAE chromatography experiments. Peak
I, which was eluted at lower ionic strength (0.18M NaCl), was
observed for the first time. Both peaks catalyzed isomerization of
linoleic acid to c9, t11-CLA, as determined by GC analysis of
methyl ester products. Peak II was chosen for further
purification.
[0264] Active fractions (fraction 43-47) from DEAE peak II were
pooled, concentrated and dialyzed against 25 mM bis-Tris, pH 7.1
containing 0.3% OTGP, and then loaded on a Mono-p chromatofocusing
gel column. Elution was carried out with 100 ml of 10%
polybuffer74, lowering the final pH to 3.5. The isomerase activity
was retained on the column. Following completion of the
polybuffer74 gradient, the isomerase activity was eluted with 1 M
NaCl in 50 mM bis-Tris, pH 7.1 (FIG. 41). This chromatofocusing
step achieved another 2 to 6 fold purification (Table 5).
Examinations by SDS PAGE of chromatofocusing fractions possessing
enzymatic activity revealed two major bands.
6TABLE 5 Summary of Chromatofocusing Fraction A234 [P] (mg/ml)
Specific Activity Fold A/S* 1.76 0.97 9 1 F73 1.51 0.35 22 2 F74
2.11 0.66 16 2 F75 2.01 0.26 39 4 F76 1.90 0.18 53 6 F77 1.72 0.26
33 4 F78 1.69 0.21 40 4 F79 1.11 0.29 19 2 F80 0.72 0.18 20 2 F82
0.37 0.07 26 2 Total loading: A234 = 88 Fractions: A235 = 75
Recovering = 85% *A/S: Applied Sample
[0265] This protocol can be used to purify sufficient C. sporogenes
linoleate isomerase protein to determine the N-terminal sequence
for the isomerase, and to subsequently clone and sequence the
entire enzyme, as described for the L. reuteri linoleate isomerase
described above.
Example 1
[0266] The following example describes the optimization of growth
conditions for L. reuteri PYR8.
[0267] Fermentation work was concentrated on the optimization of
growth conditions for L. reuteri PYR8. A fermentation medium that
could consistently support cell growth well and isomerase
production, thus eliminating the variability previously observed
was pursued.
[0268] Working with MRS medium, it was determined that the
linoleate isomerase activity was variable, mainly due to the medium
composition and sterilization procedures that had some effect on
cell growth. The number of inoculum stages and the inoculum size
did not affect final cell concentration. Mixed versus static
growth, suspected to affect the gas balance in the medium, did not
appear to be a significant variable. Given the medium richness,
toxic concentrations of some compounds were suspected as a possible
reason for the variability. However, it was determined that
different dilutions of MRS resulted in proportional lower cell
densities (data not shown) indicating a nutritional limitation in
the medium. Additionally, high variability was observed when using
two batches of the same medium.
[0269] Experiments performed in one and ten-liter fermentors
indicated that a different medium (AV) with composition similar to
MRS, but with higher yeast extract, peptone and acetate
concentration, and without beef extract, gave consistently better
results in fermentors than MRS with respect to both cell growth and
isomerase activity. We adopted this medium as our base medium for
further work. Its composition is shown in Table 6.
7TABLE 6 Composition of AV Medium Component Concentration Yeast
Extract 10 g/l Proteose Peptone #3 (Difco) 20 g/l Sodium Acetate 10
g/l Glucose 20 g/l Tween 80 1 ml/l MgSO4 0.028 g/l MnSO4.2H2O 0.012
g/l FeSO4.7H2O 0.0034 g/l Vitamin Mixture 10 ml/l
[0270] The vitamin mixture contained riboflavin, pantothenic acid,
pyridoxal, nicotinic acid, folic acid, choline chloride, biotin and
thiamine.
[0271] A full factorial experiment was run in bottles, dividing
this medium into seven categories (yeast extract, peptone, acetate,
glucose, Tween 80, salts and vitamins), and studying the impact of
two concentrations of the components in each category as follows:
2.5 and 10 g/l yeast extract, 10 and 20 g/11 peptone, 10 and 20 g/l
glucose, 5 and 10 g/l acetate, 0.5 and 1 ml/l Tween 80, 0.5.times.
and 1.times. salts concentration and no addition vs. addition of
vitamins. This study demonstrated clearly that yeast extract
concentration had the most significant impact on growth, followed
by glucose and the combined effect of glucose and yeast extract.
Peptone effect was marginal and the other components did not affect
growth. The concentration of Tween 80 seemed to affect isomerase
activity, as measured by conversion of linoleic acid to CLA.
[0272] Difco yeast extract was successfully replaced by KAT yeast
extract, and several industrial type nitrogen sources were tested
as replacements for Peptone #3. These are summarized in Table
7.
8TABLE 7 Nitrogen Sources Evaluated as Medium Components Nitrogen
Source Manufac- Name Type turer N-Z-Amine A Enzyme Hydrolysate of
Casein Quest N-Z-Amine YT Enzyme Hydrolysate of Casein Quest
Pepticase Enzyme Hydrolysate of Casein Quest Amicase Acid
Hydrolysate of Casein Quest Edamin K Enzyme Hydrolysate of Lact-
Quest albumin Amisoy Acid Hydrolysate of Soy Quest Hy-soy Enzyme
Hydrolysate of Soy Quest Primatone RL Enzyme Hydrolysate of Meat
Quest Primatone HS Enzyme Hydrolysate of Meat Quest Primagen Enzyme
Hydrolysate of Animal Quest Tissue Pancase Pancreatic Digest of
Casein Red Star Amberferm 2000 Proteolyzed Dairy Protein Red Star
Amberferm 2234 Proteolyzed Dairy Protein Red Star Amberferm 4000
Acid Hydrolyzed Vegetable Red Star Protein Amberferm 4002 Acid
Hydrolyzed Vegetable Red Star Protein Blend Amberferm 4015G Enzyme
Hydrolyzed Soy Protein Red Star Amberferm 4016 Enzyme Hydrolyzed
Soy Red Star Whey Protein Corn Steep Liquor Roquette Concentrate
Nutrisoy Soy Hydrolyze in the Lab with Neutrase ADM Flour Nutrisoy
Soy Hydrolyze in the Lab with Neutrase ADM Flour with Added Oils
Pharmamedia Cottonseed Flour Traders
[0273] Most of these nitrogen sources supported growth of L.
reuteri PYR8, but isomerase activity was not always detected. The
most promising ones, N-Z-Amine A, Amberferm 2234, Amberferm 4015,
Amisoy and Hy-Soy were further tested in fermentors. Hy-soy was
determined not just to be a good replacement for peptone, but to
actually improve growth over peptone.
[0274] Lactose, fructose and galactose were compared to glucose as
possible carbon sources. The organism did not grow on fructose and
lactose, and galactose did not offer any advantage over
glucose.
[0275] Fermentations performed with peptone and increasing levels
of yeast extract up to 20 g/l indicated that yeast extract
concentrations above 10 g/l were still beneficial. Further
optimization proceeded with a full factorial experiment in
fermentors where the effects two levels of yeast extract (20 and 30
g/l), Hy-Soy (10 and 20 g/l) and glucose (20 and 30 g/l) were
compared. pH control at 4.8 was adopted to avoid low pH inhibition
due to the higher acid production from the higher glucose
concentrations. The growth results from these fermentations showed
that even though there was not a statistically significant
difference between conditions, higher yeast extract fermentors
resulted in slightly higher optical density (data not shown). The
culture in the medium with high level of the three components grew
faster and reached a higher cell density.
[0276] The medium with 30 g/l yeast extract, 10 g/l Hy-Soy and 30
g/l glucose was chosen for further optimizations steps.
[0277] The effects of growth temperature and Tween 80 concentration
were studied. Fermentations were performed at 11 and 1.5 ml/l Tween
80 and 37.degree. C., 40.degree. C. and 43.degree. C. Medium with
Hy-Soy at 20 g/l was also compared at 37.degree. C. and 43.degree.
C. The growth and conversion results indicated clearly that higher
temperatures were beneficial for growth and isomerase activity
(data not shown). The increase in Tween 80 concentration did not
seem to impact linoleic acid conversion significantly, although a
higher conversion rate was observed at 43.degree. C. with higher
Tween 80 concentration.
[0278] A temperature of 40.degree. C. was adopted as the preferred
growth temperature and the medium containing 30 g/l yeast extract,
10 g/l Hy-Soy, 30 g/l glucose and 1.5 ml/l Tween, as the new base
medium. In another set of fermentations, the reproducibility of the
process was tested in triplicate fermentors. Higher concentrations
of yeast extract, Hy-Soy and glucose were also compared at
40.degree. C. The results showed that good reproducibility can be
obtained with this medium and growing conditions, with respect to
final cell density and isomerase activity (data not shown). Further
increases in the concentrations of the main components favored cell
growth. An optical density above 10 units was obtained with 40 g/l
yeast extract, 20 g/l Hy-Soy and 40 g/l glucose. The specific
enzyme activity and activity per cell was similar under all these
different conditions. Therefore, an increase in cell density
resulted in increased isomerase activity.
[0279] The medium with 30 g/l yeast extract, 10 g/l Hy-Soy and 30
g/l glucose plus the additional components of the AV medium
described above, resulted in cell densities (measured by OD and
DCW) twice as high as those obtained with MRS, and a much more
reliable performance. With these conditions, the fermentation
performed consistently better in fermentors than in static bottles.
The cultures were harvested at 24 and 30 hours to determine
isomerase activity as a function of culture age. No difference was
found in the rate of conversion of linoleic acid to CLA between
cells of different age for any of the media tested. The shorter
fermentation time is due to the faster growth in this medium and at
the higher temperature. At 24 hours, the culture had already
reached stationary phase.
[0280] Several substances were tested as possible inducers of the
9,11 linoleate isomerase. The materials tested included: lauric
acid, myristic acid, palmitic acid, palmitoleic acid, oleic acid,
linoleic acid, linolenic acid, oleic acid stearyl ester, linoleyl
alcohol, linoleic acid methyl ester, linoleic acid ethyl ester,
stearic acid and linoleic acid methyl ester. They were added to the
growth medium at a 100 mg/l level. No positive effect was found
with any of the compounds, and some of them were detrimental to the
expression and/or activity of the isomerase. The determination of
the existence of a positive effect may be obscured by the required
presence of Tween 80, which may be an inducer in itself, but which
cannot be eliminated because it is required for growth.
Example 12
[0281] The following example describes the determination of
conditions to improve enzyme stability and performance and on
testing the limitations of tile biotransformation process. Whole
cells of L. reuteri PYR8 were used in all biotransformation
experiments described below.
[0282] One aspect of the preservation of the enzyme activity is the
handling of the cells immediately after harvesting and the
determination of suitable storage conditions. The preservation of
activity in cells maintained in different buffers was investigated,
and it was determined that reduced buffers such as TKM/EDTA/NaCl
(50 mM Tris.HCl, 25 mM KCl, 5 mM MgCl.sub.2, 1.25 mM EDTA, 0.1 mM
NaCl, pH 7.5) with 20 mM cysteine or 20 mM DTT preserved isomerase
activity much better than other buffers or culture medium. Cells
maintained in 100 mM Bis-Tris pH 5.8 with 10 mM NaCl, 10% glycerol
and 2 mM DTT (breakage buffer) did not lose any activity in 48
hours. It was also determined that the biotransformation rate
measured in this buffer was very similar to that measured in the
culture medium (MRS) which had been used as the preferred medium to
perform the reaction.
[0283] Isomerase activity could also be preserved by freezing the
cell paste. The cell paste was frozen immediately after harvesting
with and without washing with either MRS or breakage buffer. No
differences were observed. Some interesting results were also
obtained when the cells were directly preserved in the culture
broth with or without harvesting. A comparison was made between
activity in cells immediately after harvest, cells that were
harvested and maintained as a cell paste (no washing) at 4.degree.
C. for 24 hours, cells that were kept without harvesting in the
culture broth at 4.degree. C. for 24 hours, and cells from culture
broth that were kept at room temperature for 24 hours. The
conversion of linoleic acid to CLA was very similar in every case,
with only a slight decrease observed in those cells that had been
maintained at room temperature for a day.
[0284] In another experiment, the isomerase activity was followed
in cells that were handled in different ways after harvesting.
Cells were resuspended in either MRS, breakage buffer or culture
supernatant (pH adjusted to 5.8). Isomerase activity, compared as
conversion of 1000 ppm linoleic acid, was measured in the cells
immediately after harvesting, after being held for 24 hours at
4.degree. C. and after a four hour period at 22.degree. C. followed
by 20 hours at 4.degree. C. The results from these different
experiments indicated that the enzyme activity is better preserved
when the cells are maintained under strictly non-growing conditions
(data not shown). In several repeat experiments, cells resuspended
in MRS gave more variable results than cells resuspended in
breakage buffer. When cells in MRS were kept at room temperature,
the deterioration was even more marked. Breakage buffer was
selected as the medium of choice to perform the biotransformation
because of the better enzyme stability. Cells can also be preserved
prior to harvesting in the culture broth at the low pH reached at
the end of the fermentation.
[0285] Another aspect of the biotransformation investigated was the
possible presence of mass transfer limitations between the oil, the
water phase and the membrane bound enzyme. Experiments were done
using different methods of addition of the linoleic acid and
performing the isomerization reaction in stirred jars at different
agitation rates.
[0286] Linoleic acid was added as 99% LA, dissolved in
propyleneglycol (100 mg/ml solution) or emulsified with 0.5, 5 or
30% lecithin. The emulsion was prepared by blending the linoleic
acid and the lecithin with the reaction medium before the addition
of the cells. Linoleic acid was added at 1000 and 2000 ppm. The
results indicated that there was no significant difference between
adding the pure acid or the propyleneglycol solution, and that the
reaction was slightly faster with both than when the acid was
emulsified with lecithin (data not shown). High levels of lecithin
seemed to negatively affect the final conversion.
[0287] Two biotransformation reactions were performed in stirred
jars with 300 ml of reaction medium. Cells were concentrated
10-fold with respect to the original culture density. The reaction
was run between 6.degree. C. and 8.degree. C., in MRS, and linoleic
acid was added dissolved in propyleneglycol. 1000 ppm were added at
time 0 and another 1000 ppm at two hours. Agitation was kept at 200
rpm in one reactor and 1000 rpm in the other. Results showed that
no significant difference was found between the two conditions.
These experiments indicated that mass transfer limitations are not
a major problem when working with this enzyme.
[0288] The effect of substrate and product on the enzyme
performance was also investigated, as well as the possibility of
recycling the cells. The effect of CLA on the reaction was studied
by adding different concentrations of either a mixture of isomers
(Sigma material, approximately 41% 9,11 isomer and 48% 10,12 CLA),
or just 9,11 CLA (Matreya material, approximately 77% 9,11 CLA).
Concentrations from 500 to 3000 ppm were tested. Some experiments
were also performed recycling the broth from a previous
biotransformation reaction with L. reuteri PYR8, resulting in an
initial CLA concentration around 700 ppm. In every case, 1000 ppm
linoleic acid were added. With both the Sigma and the Matreya CLA,
the reaction was completely inhibited even at the lowest
concentration tested, and linoleic acid and CLA remained constant
over the four-hour period that the reaction was followed. In the
same period, almost complete conversion of the linoleic acid was
obtained in the control without exogenous CLA. However, the effect
was not detected when the CLA present came from recycled reaction
broth. In this case, there was no difference in conversion rate
between no CLA presence and 700 ppm. The results may indicate that
some of the impurities present in the chemically produced CLA may
be stronger inhibitors of the 9,11 isomerase than the product
itself.
[0289] Three separate experiments were performed where the cells
were recycled after a first biotransformation step. In the first
experiment, a biotransformation step with 1000 ppm linoleic acid
was completed in three hours in both MRS and breakage buffer.
98-99% of the linoleic acid was isomerized to 9,11 CLA. Cells were
recovered by centrifugation, resuspended in the same medium, 1000
ppm linoleic acid were added, and the reaction proceeded for
another three hours. Very good conversions were obtained in every
case (data not shown).
[0290] In the second experiment, cell recycle was studied with
cells that have performed the biotransformation at different levels
of linoleic acid. Cells were harvested, resuspended in breakage
buffer at a 10-fold concentration, and linoleic acid was added at
1000, 1500, 2000, 2500 and 3000 ppm level. Given the higher
linoleic acid concentration, the reaction was allowed to proceed
for six hours. At that time, the cells were recovered by
centrifugation, washed with buffer to remove (at least partially)
non reacted linoleic acid and CLA, and to place all the cells under
comparable conditions. 1000 ppm linoleic acid were added and the
reaction was followed for four hours. The conversions and CLA
concentrations obtained during the first stage indicated that with
cells with good activity, no substrate inhibition was detected up
to 3000 ppm linoleic acid. The reactions at higher linoleic acid
did not reach completion in seven hours, but the rate of formation
of CLA was very similar at the different substrate concentrations.
However, when the cells were recycled and supplied with linoleic
acid in a second stage, the reaction did not take place and
isomerase activity was not detected in any of the cells, regardless
of the linoleic acid level to which they had been exposed. Cells
from the same lot that were not exposed to linoleic acid maintained
full activity after 24 hours.
[0291] These results prompted the need to investigate the length of
exposure to the reaction mix in relation to the loss of activity
observed. Since it was clear that the activity was not lost in
cells which had not performed the reaction, either the substrate,
or more likely the product, might interact with the enzyme and
affect its activity.
[0292] In the third recycle experiment, cells were concentrated as
usual and the reaction was started at 2000 ppm. Aliquots were taken
while the reaction proceeded, every two hours up to eight hours
with one final sample at 25 hours. The cells from each aliquot were
recovered by centrifugation and resuspended in buffer. 1000 ppm
linoleic acid was added. The reaction was then followed for three
hours. The results clearly indicated that the activity was slowly
being lost in the cells. The reaction slowed down over time in the
first stage and the activity was not recovered when the cells were
placed in fresh reaction medium. While cells recycled after two
hours had very good activity and could quickly transform the 1000
ppm linoleic acid to CLA, cells recycled after eight hours had no
activity. Once again, full activity was preserved in the control
(no reaction) after 25 hours.
[0293] This experiment provided a clear demonstration that the
enzyme is either inactivated or becomes inaccessible to the
substrate during the reaction. The reason is not clear, but an
interaction with the product is suggested, as the activity seems to
be lost as the product accumulates. It is not clear at this time
the nature of this interaction or if it is directly related to the
enzyme or the physical conditions of the cells. Studies will be
required with immobilized enzyme to better understand this
effect.
Example 13
[0294] The following example describes a preferred
biotransformation protocol.
[0295] Cells of Lactobacillus reuteri (or another organism carrying
the linoleate isomerase gene) are grown in modified AV medium with
40 g/l yeast extract, 20 g/l Hy-soy and 40 g/l glucose (or other
appropriate medium for other organisms) to a cell density of about
3-4 g/l dry cell weight. When the cells reach stationary phase,
they are harvested and resuspended in breakage buffer at a
concentration between 5 and 20 g dry cell weight per liter. The
biotransformation reaction should be preferably carried out at a
temperature between 4.degree. C. and 8.degree. C. to maintain the
enzyme activity. The linoleic acid can be added as a 99% oil, as a
component of another oil, as an oil phase, or dissolved in a
cosolvent such as propylene glycol. It can be added at
concentrations between 0.5 and 4 g/l. The addition should
preferably be done in several steps of smaller amounts. To obtain
higher CLA concentrations, it is also possible to add the cells in
successive steps while the reaction proceeds. Under these
conditions, and at these linoleic acid concentrations, conversion
of linoleic acid to CLA between 80% and 100% is expected within 2
to 8 hours.
Example 14
[0296] The following example describes the biotransformation of
linoleic acid to 10,12 CLA with P. acnes whole cells.
[0297] The objective of these studies was to begin the
characterization of the behavior of the 10,12 linoleic acid
isomerase and to determine the conditions to enhance its
performance.
[0298] P. acnes is a strict anaerobe for which growth in the medium
currently used is very poor. Some previous experiments suggested
that P. acnes was able to further metabolize 10,12 CLA. The new
experiments also indicated this to be the case, but it seems to be
a slow process which may depend on the conditions of the reaction.
It must be noted that with the current cell concentration achieved
in the culture, and the same bioconversion protocol used in the
production of the 9,11 CLA isomer (10-fold-concentration of the
cells, resuspension in buffer, addition of linoleic acid dissolved
in propyleneglycol), the reaction proceeds at a much lower rate
than that of the 9,11 isomerase, and much lower conversions are
achieved.
[0299] The reaction was compared in culture medium vs. breakage
buffer, at different temperatures and in the presence and absence
of air. Temperature had a strong effect on the reaction rate. The
reaction proceed very slowly at 4.degree. C. and the rate increased
with temperature, from room temperature to 37.degree. C. No
significant difference in conversion was found between the use of
growth medium or a buffer, or the presence or absence of oxygen
during the time frame allowed for the reaction to proceed (30
hours) (data not shown).
[0300] Further experiments with different cell and substrate
concentrations had shown an apparent decrease in the CLA
concentration when the reaction proceeded more than 48 hours, while
the formation of CLA from linoleic acid seemed to stop at that
time.
[0301] Those skilled in the art will appreciate that numerous
changes and modifications may be made to the preferred embodiments
of the invention and that such changes and modifications may be
made without departing from the spirit of the invention. It is
therefore intended that the appended claims cover all such
equivalent variations as fall within the true spirit and scope of
the invention.
Sequence CWU 1
1
41 1 35 PRT Lactobacillus reuteri UNSURE (30) 1 Met Tyr Tyr Ser Asn
Gly Asn Tyr Glu Ala Phe Ala Arg Pro Lys Lys 1 5 10 15 Pro Ala Gly
Val Asp Lys Lys His Ala Tyr Ile Val Gly Xaa Gly Leu 20 25 30 Ala
Ser Leu 35 2 29 DNA Lactobacillus reuteri misc_feature (1)..(29)
PRIMER 2 cgtgaattca tgtaytayws naayggnaa 29 3 27 DNA Lactobacillus
reuteri misc_feature (1)..(27) PRIMER 3 actggatccn acdatratng
crtgytt 27 4 87 DNA Lactobacillus reuteri CDS (1)..(87) 4 atg tat
tat tcg aac gga aat tat gaa gcc ttt gct cga cca aag aag 48 Met Tyr
Tyr Ser Asn Gly Asn Tyr Glu Ala Phe Ala Arg Pro Lys Lys 1 5 10 15
cct gct ggc gtt gat aag aaa cac gcc tac ata gtc gga 87 Pro Ala Gly
Val Asp Lys Lys His Ala Tyr Ile Val Gly 20 25 5 29 PRT
Lactobacillus reuteri 5 Met Tyr Tyr Ser Asn Gly Asn Tyr Glu Ala Phe
Ala Arg Pro Lys Lys 1 5 10 15 Pro Ala Gly Val Asp Lys Lys His Ala
Tyr Ile Val Gly 20 25 6 17 DNA Lactobacillus reuteri misc_feature
(1)..(17) PRIMER 6 ggtcgagcaa aggcttc 17 7 17 DNA Lactobacillus
reuteri misc_feature (1)..(17) PRIMER 7 aagcctgctg gcgttga 17 8 596
DNA Lactobacillus reuteri CDS (122)..(595) 8 aaaaattatt tagaattaat
ttataagttc attgtgttta ataaaattga cactttcaac 60 cgctttcact
aaaattaagg tagttatgat gcacttgttt actgagaagg gagtcgtcaa 120 a atg
tat tat tca aac ggg aat tat gaa gcc ttt gct cga cca aag aag 169 Met
Tyr Tyr Ser Asn Gly Asn Tyr Glu Ala Phe Ala Arg Pro Lys Lys 1 5 10
15 cct gct ggc gtt gat aag aaa cat gcc tac att gtc ggt ggt ggt tta
217 Pro Ala Gly Val Asp Lys Lys His Ala Tyr Ile Val Gly Gly Gly Leu
20 25 30 gct ggt tta tcg gcc gcc gtg ttt tta att cgt gat gcc caa
atg ccg 265 Ala Gly Leu Ser Ala Ala Val Phe Leu Ile Arg Asp Ala Gln
Met Pro 35 40 45 ggt gag aat atc cat att tta gag gaa tta ccg gtt
gcc ggt ggt tct 313 Gly Glu Asn Ile His Ile Leu Glu Glu Leu Pro Val
Ala Gly Gly Ser 50 55 60 ctt gat ggt gaa gat cgt cct gga att ggt
ttt gtt act cgt gga ggc 361 Leu Asp Gly Glu Asp Arg Pro Gly Ile Gly
Phe Val Thr Arg Gly Gly 65 70 75 80 cgg gaa atg gag aac cat ttc gag
tgt atg tgg gac atg tat cgt tca 409 Arg Glu Met Glu Asn His Phe Glu
Cys Met Trp Asp Met Tyr Arg Ser 85 90 95 att cca tca ctt gaa atc
cca ggt gct tcc tac ctt gat gaa tac tac 457 Ile Pro Ser Leu Glu Ile
Pro Gly Ala Ser Tyr Leu Asp Glu Tyr Tyr 100 105 110 tgg tta gat aag
gaa gat cca aac agt tct aat tgt cgt tta acc tat 505 Trp Leu Asp Lys
Glu Asp Pro Asn Ser Ser Asn Cys Arg Leu Thr Tyr 115 120 125 aag cgg
gga aat gaa gtt cca tcg gac ggt aaa tat ggt tta agt aaa 553 Lys Arg
Gly Asn Glu Val Pro Ser Asp Gly Lys Tyr Gly Leu Ser Lys 130 135 140
aag gca atc aaa gag ctg act aag cta att atg acc cct aaa g 596 Lys
Ala Ile Lys Glu Leu Thr Lys Leu Ile Met Thr Pro Lys 145 150 155 9
158 PRT Lactobacillus reuteri 9 Met Tyr Tyr Ser Asn Gly Asn Tyr Glu
Ala Phe Ala Arg Pro Lys Lys 1 5 10 15 Pro Ala Gly Val Asp Lys Lys
His Ala Tyr Ile Val Gly Gly Gly Leu 20 25 30 Ala Gly Leu Ser Ala
Ala Val Phe Leu Ile Arg Asp Ala Gln Met Pro 35 40 45 Gly Glu Asn
Ile His Ile Leu Glu Glu Leu Pro Val Ala Gly Gly Ser 50 55 60 Leu
Asp Gly Glu Asp Arg Pro Gly Ile Gly Phe Val Thr Arg Gly Gly 65 70
75 80 Arg Glu Met Glu Asn His Phe Glu Cys Met Trp Asp Met Tyr Arg
Ser 85 90 95 Ile Pro Ser Leu Glu Ile Pro Gly Ala Ser Tyr Leu Asp
Glu Tyr Tyr 100 105 110 Trp Leu Asp Lys Glu Asp Pro Asn Ser Ser Asn
Cys Arg Leu Thr Tyr 115 120 125 Lys Arg Gly Asn Glu Val Pro Ser Asp
Gly Lys Tyr Gly Leu Ser Lys 130 135 140 Lys Ala Ile Lys Glu Leu Thr
Lys Leu Ile Met Thr Pro Lys 145 150 155 10 1709 DNA Lactobacillus
reuteri 10 cggaaggcat caaaatccca atgaattccc accaaactta gtgcataggg
caagaagggt 60 gtcccgcgat tggtatgcat ggattggaac ccgcctttaa
gattaatgcg cctgaaggaa 120 gccagctggt cgccaatccg tagcaccatt
ccctgggcaa ttcggctttt atattgaccg 180 agttgtcctg tttaaccagg
catcaccttg ccacgccctt ccttgacggt caagatgatt 240 tacagcatag
ggtgcacttg caatcttagc gttaagattt gtttggttat tattgataat 300
aaacgcaccg gctttgttcc aggtaattga aatgccaagt tgttggcgaa cagccggagt
360 taagactgaa ttagcctgtt cctgagttgg cggtaatgtt tttttgatcg
ttgtgactgg 420 ttttcttcca ataagcaatt ttactaatat ggtttaacga
agcatttgtt agctgaggtt 480 gctggataac tccagtaact actaataaac
cagcaagagc aaataaaagg tgatagaggc 540 gtttcttaag tttcataaat
tcactccatt tctaataatt ccaaagtcta ttttactagt 600 ttgaacatac
gtttggaata attatttaga attaatttat aagttcattg tgtttaataa 660
aattgacact ttcaaccgct ttcactaaaa ttaaggtagt tatgatgcac ttgtttactg
720 agaagggagt cgtcaaaatg tattattcaa acgggaatta tgaagccttt
gctcgaccaa 780 agaagcctgc tggcgttgat aagaaacatg cctacattgt
cggtggtggt ttagctggtt 840 tatcggccgc cgtgttttta attcgtgatg
cccaaatgcc gggtgagaat atccatattt 900 tagaggaatt accggttgcc
ggtggttctc ttgatggtga agatcgtcct ggaattggtt 960 ttgttactcg
tggaggccgg gaaatggaga accatttcga gtgtatgtgg gacatgtatc 1020
gttcaattcc atcacttgaa atcccaggtg cttcctacct tgatgaatac tactggttag
1080 ataaggaaga tccaaacagt tctaattgtc gtttaaccta taagcgggga
aatgaagttc 1140 catcggacgg taaatatggt ttaagtaaaa aggcaatcaa
agagctgact aagctaatta 1200 tgacccctga agaaaaattg ggaagggaga
ctattggtga atacttctct gatgatttct 1260 ttgaaagcaa tttctggatt
tattggtcaa caatgtttgc gtttgaacgg tggcactctc 1320 tagctgaaat
gcgtcgttat atgatgcggt ttattcacca tattgatggt ttaccggatt 1380
tcactgcact gaagtttaat aagtataacc aatatgaatc aatgaccaag ccgctattgg
1440 cctacctgaa agatcatcat gtcaagattg agtacgatac ccaggtaaag
aatgttattg 1500 ttgatactca tgggcggcaa aagcacgcta agcgaatctt
attaactcaa gccggtaaag 1560 ataaagttgt tgagttaacg gacaatgacc
ttgtctttgt cacaaacggt tcaattacag 1620 aaagttctac ttacggcagt
caccatcaag ccagctcgac caacgcagca cttggtgggt 1680 agttggaaac
tgtgggaaaa ccttgctcc 1709 11 324 PRT Lactobacillus reuteri UNSURE
(315) 11 Met Tyr Tyr Ser Asn Gly Asn Tyr Glu Ala Phe Ala Arg Pro
Lys Lys 1 5 10 15 Pro Ala Gly Val Asp Lys Lys His Ala Tyr Ile Val
Gly Gly Gly Leu 20 25 30 Ala Gly Leu Ser Ala Ala Val Phe Leu Ile
Arg Asp Ala Gln Met Pro 35 40 45 Gly Glu Asn Ile His Ile Leu Glu
Glu Leu Pro Val Ala Gly Gly Ser 50 55 60 Leu Asp Gly Glu Asp Arg
Pro Gly Ile Gly Phe Val Thr Arg Gly Gly 65 70 75 80 Arg Glu Met Glu
Asn His Phe Glu Cys Met Trp Asp Met Tyr Arg Ser 85 90 95 Ile Pro
Ser Leu Glu Ile Pro Gly Ala Ser Tyr Leu Asp Glu Tyr Tyr 100 105 110
Trp Leu Asp Lys Glu Asp Pro Asn Ser Ser Asn Cys Arg Leu Thr Tyr 115
120 125 Lys Arg Gly Asn Glu Val Pro Ser Asp Gly Lys Tyr Gly Leu Ser
Lys 130 135 140 Lys Ala Ile Lys Glu Leu Thr Lys Leu Ile Met Thr Pro
Glu Glu Lys 145 150 155 160 Leu Gly Arg Glu Thr Ile Gly Glu Tyr Phe
Ser Asp Asp Phe Phe Glu 165 170 175 Ser Asn Phe Trp Ile Tyr Trp Ser
Thr Met Phe Ala Phe Glu Arg Trp 180 185 190 His Ser Leu Ala Glu Met
Arg Arg Tyr Met Met Arg Phe Ile His His 195 200 205 Ile Asp Gly Leu
Pro Asp Phe Thr Ala Leu Lys Phe Asn Lys Tyr Asn 210 215 220 Gln Tyr
Glu Ser Met Thr Lys Pro Leu Leu Ala Tyr Leu Lys Asp His 225 230 235
240 His Val Lys Ile Glu Tyr Asp Thr Gln Val Lys Asn Val Ile Val Asp
245 250 255 Thr His Gly Arg Gln Lys His Ala Lys Arg Ile Leu Leu Thr
Gln Ala 260 265 270 Gly Lys Asp Lys Val Val Glu Leu Thr Asp Asn Asp
Leu Val Phe Val 275 280 285 Thr Asn Gly Ser Ile Thr Glu Ser Ser Thr
Tyr Gly Ser His His Gln 290 295 300 Ala Ser Ser Thr Asn Ala Ala Leu
Gly Gly Xaa Leu Glu Thr Val Gly 305 310 315 320 Lys Pro Cys Ser 12
17 DNA Lactobacillus reuteri misc_feature (1)..(17) PRIMER 12
ccaattccag gacgatc 17 13 19 DNA Lactobacillus reuteri misc_feature
(1)..(19) PRIMER 13 acatgtatcg ttcaattcc 19 14 1165 DNA
Lactobacillus reuteri 14 aagcctgctg gcgttgataa gaaacatgcc
tacattgtcg gtggtggttt agctggttta 60 tcggccgccg tgtttttaat
tcgtgatgcc caaatgccgg gtgagaatat ccatatttta 120 gaggaattac
cggttgaata attaatggta atgtttcttt ggacattcgg aacaaagaca 180
ttgtattcta gagaaccatc actagattta gcttcgatat gagcacctgc cggaacgata
240 ttattaccgt cataaatatt ggtaactcgg tagcgaactt gcttattctg
atctaatgct 300 tttctcacca gaccttcgta gtaattttgc cctgttgagt
tcttacttcg tgcttcattt 360 gcccaggcag tttgcgtggc aatattagat
ggatttgatt cggatgcatc aaatccatga 420 atacccacca actagtgcat
agggcaagaa ggtgtccgcg atcgtatgca tgattgtacc 480 cgcctttaag
attatgcgcc tgaaaggaag ccagctggtc gccaatccgt agcaccattc 540
cctgggcaat tcggctttta tattgaccga gttgtcctgt ttaaccaggc atcaccttgc
600 cacgcccttc cttgacggtc aagatgattt acagcatagg gtgcacttgc
aatcttagcg 660 ttaagatttg tttggttatt attgataata aacgcaccgg
ctttgttcca ggtaattgaa 720 atgccaagtt gttggcgaac agccggagtt
aagactgaat tagcctgttc ctgagttggc 780 ggtaatgttt ttttgatcgt
tgtgactggt tttcttccaa taagcaattt tactaatatg 840 gtttaacgaa
gcatttgtta gctgaggttg ctggataact ccagtaacta ctaataaacc 900
agcaagagca aataaaaggt gatagaggcg tttcttaagt ttcataaatt cactccattt
960 ctaataattc caaagtctat tttactagtt tgaacatacg tttggaataa
ttatttagaa 1020 ttaatttata agttcattgt gtttaataaa attgacactt
tcaaccgctt tcactaaaat 1080 taaggtagtt atgatgcact tgtttactga
gaagggagtc gtcaaaatgt attattcaaa 1140 cgggaattat gaagcctttg ctcga
1165 15 2319 DNA Lactobacillus reuteri 15 ccaattccag gacgatcttc
accatcaaga gaaccaccgg caaccggtcc cttaccgcta 60 tcctgatctt
tctttccttc ctcaacttgc ttttgagctg cctttactag gttcatagta 120
aagaagggct tcaatactgg cttaaaatcc tttttaaagt ggtcagtaag gttttggtat
180 aagcggacat cattgtcaaa taccaatact tcttcaaatt gatttcggtg
agcatcaaat 240 gaagcttcgt ctaaatctac actcccaaga atcacacggg
aatcatgagt agttgaacta 300 cttaacaagt aaaacttaga atggataacc
tgagtaggcg cgattgatac gcgaaaaaga 360 ttatttaaga cgttcgtttg
gttgtcactg gttaacgctg agaagagttt agcagcttct 420 ttatttgctg
aactaaggag agcattggta agtgcaacct ttgtcaccat ctcatcagca 480
cttaattcac tagttgattg ggagcttaac gctacattaa tactgataaa attactaagg
540 tatttattaa tgaagtcagc agtaattttc ccagttactg cgattaactg
atcgtatttt 600 tgtgaatcaa ataattgatg gatctttaat ggtggtgttt
cttgaccatc aaaaacaata 660 tgaatttttc ttataccagc agtttctgtc
atgaccataa tcctttacta tcaataaata 720 tattagtttt attttcgact
atttaatccc tttttgcaag tggttccccg ataagctata 780 taaaaaaaga
agccggaaat ttccagcttc tttcatcttt atagtaagtg ctgttgctcc 840
attaattcac caatccacgt tccttggagt ttctttaata atggcttttc aacaatcttt
900 ggaattggca agtccatgtc ttttaacggc ttcttatcat tcatgtaata
cattgcccgc 960 attaactctc gaagatcata aatagagtta aagacttctg
gaactccccg atcaacatct 1020 aatagagtgt agacggcttc cattgcggtc
cgtactgaat attccgtggt aaatacggta 1080 tctcgacttg gagattcagc
aaagttacca ataaatgcca agttagcgga tccttctgga 1140 acaacgtctg
gacggtcgcc cttaactcgt ggcataaagt agctagtgat aaatggcata 1200
tatactggaa cagtattaat tgaactctcc ttagccaaat cgtcaattaa cggcttctgg
1260 aacccccaga tggatagcca ttctttagta atctcttcac cagtacaatc
aacgatccgt 1320 ttcttaatat agtttccctt tgtattagag tacagaccgt
aaatccaaac aatggtttca 1380 tttttctttt gtttcttgaa gtgcggttga
cggtgaattg tccaggaaag catccaatta 1440 gagtcagtga ccgtaatgat
tccaccagta ttaactttgc catcatggag atctcgcttg 1500 gttaagcgtt
caatgtatgg ttcaacttgc gggttcttaa cggttgcagt agcggaaatg 1560
aaccagcttc tccctggaag attcttgcaa aagacatcag gatgaccaaa atcagctgac
1620 tgccgagcaa ggttttccca cagtttccaa ctaccaccaa gtgctgcgtt
ggtcgagctg 1680 cttgatggtg actgccgtaa gtagaacttt ctgtaattga
accgtttgtg acaaagacaa 1740 ggtcattgtc cgttaactca acaactttat
ctttaccggc ttgagttaat aagattcgct 1800 tagcgtgctt ttgccgccca
tgagtatcaa caataacatt ctttacctgg gtatcgtact 1860 caatcttgac
atgatgatct ttcaggtagg ccaatagcgg cttggtcatt gattcatatt 1920
ggttatactt attaaacttc agtgcagtga aatccggtaa accatcaata tggtgaataa
1980 accgcatcat ataacgacgc atttcagcta gagagtgcca ccgttcaaac
gcaaacattg 2040 ttgaccaata aatccagaaa ttgctttcaa agaaatcatc
agagaagtat tcaccaatag 2100 tctcccttcc caatttttct tcaggggtca
taattagctt agtcagctct ttgattgcct 2160 ttttacttaa accatattta
ccgtccgatg gaacttcatt tccccgctta taggttaaac 2220 gacaattaga
actgtttgga tcttccttat ctaaccagta gtattcatca aggtaggaag 2280
cacctgggat ttcaagtgat ggaattgaac gatacatgt 2319 16 3551 DNA
Lactobacillus reuteri 16 accggttgaa taattaatgg taatgtttct
ttggacattc ggaacaaaga cattgtattc 60 tagagaacca tcactagatt
tagcttcgat atgagcacct gccggaacga tattattacc 120 gtcataaata
ttggtaactc ggtagcgaac ttgcttattc tgatctaatg cttttctcac 180
cagaccttcg tagtaatttt gccctgttga gttcttactt cgtgcttcat ttgcccaggc
240 agtttgcgtg gcaatattag atggatttga ttcggatgca tcaaatccat
gaataccacc 300 aactagtgca taggcaagaa ggtgtccgcg atcgtatgca
tgattgtacc cgcctttaag 360 attatgcgcc tgaaaggaag ccagctggtc
gccaatccgt agcaccattc cctgtggcaa 420 atttcggctt ttatattgac
cgagttgtcc tgtttaacca ggcatcacct tgccacgccc 480 ttccttgacg
gtcaagatga tttacagcat agggtgcact tgcaatctta gcgttaagat 540
ttgtttggtt attattgata ataaacgcac cggctttgtt ccaggtaatt gaaatgccaa
600 gttgttggcg aacagccgga gttaagactg aattagcctg ttcctgagtt
ggcggtaatg 660 tttttttgat cgttgtgact ggttttcttc caataagcaa
ttttactaat atggtttaac 720 gaagcatttg ttagctgagg ttgctggata
actccagtaa ctactaataa accagcaaga 780 gcaaataaaa ggtgatagag
gcgtttctta agtttcataa attcactcca tttctaataa 840 ttccaaagtc
tattttacta gtttgaacat acgtttggaa taattattta gaattaattt 900
ataagttcat tgtgtttaat aaaattgaca ctttcaaccg ctttcactaa aattaaggta
960 gttatgatgc acttgtttac tgagaaggga gtcgtcaaaa tgtattattc
aaacgggaat 1020 tatgaagcct ttgctcgacc aaagaagcct gctggcgttg
ataagaaaca tgcctacatt 1080 gtcggtggtg gtttagctgg tttatcggcc
gccgtgtttt taattcgtga tgcccaaatg 1140 ccgggtgaga atatccatat
tttagaggaa ttaccggttg ccggtggttc tcttgatggt 1200 gaagatcgtc
ctggaattgg ttttgttact cgtggaggcc gggaaatgga gaaccatttc 1260
gagtgtatgt gggacatgta tcgttcaatt ccatcacttg aaatcccagg tgcttcctac
1320 cttgatgaat actactggtt agataaggaa gatccaaaca gttctaattg
tcgtttaacc 1380 tataagcggg gaaatgaagt tccatcggac ggtaaatatg
gtttaagtaa aaaggcaatc 1440 aaagagctga ctaagctaat tatgacccct
gaagaaaaat tgggaaggga gactattggt 1500 gaatacttct ctgatgattt
ctttgaaagc aatttctgga tttattggtc aacaatgttt 1560 gcgtttgaac
ggtggcactc tctagctgaa atgcgtcgtt atatgatgcg gtttattcac 1620
catattgatg gtttaccgga tttcactgca ctgaagttta ataagtataa ccaatatgaa
1680 tcaatgacca agccgctatt ggcctacctg aaagatcatc atgtcaagat
tgagtacgat 1740 acccaggtaa agaatgttat tgttgatact catgggcggc
aaaagcacgc taagcgaatc 1800 ttattaactc aagccggtaa agataaagtt
gttgagttaa cggacaatga ccttgtcttt 1860 gtcacaaacg gttcaattac
agaaagttct acttacggca gtcaccatca agcagctcga 1920 ccaacgcaag
cacttggtgg tagttggaaa ctgtgggaaa accttgctcg gcagtcagct 1980
gattttggtc atcctgatgt cttttgcaag aatcttccag ggagaagctg gttcatttcc
2040 gctactgcaa ccgttaagaa cccgcaagtt gaaccataca ttgaacgctt
aaccaagcga 2100 gatctccatg atggcaaagt taatactggt ggaatcatta
cggtcactga ctctaattgg 2160 atgctttcct ggacaattca ccgtcaaccg
cacttcaaga aacaaaagaa aaatgaaacc 2220 attgtttgga tttacggtct
gtactctaat acaaagggaa actatattaa gaaacggatc 2280 gttgattgta
ctggtgaaga gattactaaa gaatggctat ccatctgggg gttccagaag 2340
ccgttaattg acgatttggc taaggagagt tcaattaata ctgttccagt atatatgcca
2400 tttatcacta gctactttat gccacgagtt aagggcgacc gtccagacgt
tgttccagaa 2460 ggatccgcta acttggcatt tattggtaac tttgctgaat
ctccaagtcg agataccgta 2520 tttaccacgg aatattcagt acggaccgca
atggaagccg tctacactct attagatgtt 2580 gatcggggag ttccagaagt
ctttaactct atttatgatc ttcgagagtt aatgcgggca 2640 atgtattaca
tgaatgataa gaagccgtta aaagacatgg acttgccaat tccaaagatt 2700
gttgaaaagc cattattaaa gaaactccaa ggaacgtgga ttggtgaatt aatggagcaa
2760 cagcacttac tataaagatg aaagaagctg gaaatttccg gcttcttttt
ttatatagct 2820 tatcggggaa ccacttgcaa aaagggatta aatagtcgaa
aataaaacta atatatttat 2880 tgatagtaaa ggattatggt catgacagaa
actgctggta taagaaaaat tcatattgtt 2940 tttgatggtc aagaaacacc
accattaaag atccatcaat tatttgattc acaaaaatac 3000 gatcagttaa
tcgcagtaac tgggaaaatt actgctgact tcattaataa ataccttagt 3060
aattttatca gtattaatgt agcgttaagc tcccaatcaa ctagtgaatt aagtgctgat
3120 gagatggtga caaaggttgc acttaccaat gctctcctta gttcagcaaa
taaagaagct 3180 gctaaactct tctcagcgtt aaccagtgac aaccaaacga
acgtcttaaa taatcttttt 3240 cgcgtatcaa tcgcgcctac tcaggttatc
cattctaagt tttacttgtt aagtagttca 3300 actactcatg attcccgtgt
gattcttggg agtgtagatt tagacgaagc ttcatttgat 3360 gctcaccgaa
atcaatttga agaagtattg gtatttgaca atgatgtccg cttataccaa
3420 aaccttactg accactttaa aaaggatttt aagccagtat tgaagccctt
ctttactatg 3480 aacctagtaa aggcagctca aaagcaagtt gaggaaggaa
agaaagatca ggatagcggt 3540 aagggaccgg t 3551 17 1776 DNA
Lactobacillus reuteri CDS (1)..(1776) 17 atg tat tat tca aac ggg
aat tat gaa gcc ttt gct cga cca aag aag 48 Met Tyr Tyr Ser Asn Gly
Asn Tyr Glu Ala Phe Ala Arg Pro Lys Lys 1 5 10 15 cct gct ggc gtt
gat aag aaa cat gcc tac att gtc ggt ggt ggt tta 96 Pro Ala Gly Val
Asp Lys Lys His Ala Tyr Ile Val Gly Gly Gly Leu 20 25 30 gct ggt
tta tcg gcc gcc gtg ttt tta att cgt gat gcc caa atg ccg 144 Ala Gly
Leu Ser Ala Ala Val Phe Leu Ile Arg Asp Ala Gln Met Pro 35 40 45
ggt gag aat atc cat att tta gag gaa tta ccg gtt gcc ggt ggt tct 192
Gly Glu Asn Ile His Ile Leu Glu Glu Leu Pro Val Ala Gly Gly Ser 50
55 60 ctt gat ggt gaa gat cgt cct gga att ggt ttt gtt act cgt gga
ggc 240 Leu Asp Gly Glu Asp Arg Pro Gly Ile Gly Phe Val Thr Arg Gly
Gly 65 70 75 80 cgg gaa atg gag aac cat ttc gag tgt atg tgg gac atg
tat cgt tca 288 Arg Glu Met Glu Asn His Phe Glu Cys Met Trp Asp Met
Tyr Arg Ser 85 90 95 att cca tca ctt gaa atc cca ggt gct tcc tac
ctt gat gaa tac tac 336 Ile Pro Ser Leu Glu Ile Pro Gly Ala Ser Tyr
Leu Asp Glu Tyr Tyr 100 105 110 tgg tta gat aag gaa gat cca aac agt
tct aat tgt cgt tta acc tat 384 Trp Leu Asp Lys Glu Asp Pro Asn Ser
Ser Asn Cys Arg Leu Thr Tyr 115 120 125 aag cgg gga aat gaa gtt cca
tcg gac ggt aaa tat ggt tta agt aaa 432 Lys Arg Gly Asn Glu Val Pro
Ser Asp Gly Lys Tyr Gly Leu Ser Lys 130 135 140 aag gca atc aaa gag
ctg act aag cta att atg acc cct gaa gaa aaa 480 Lys Ala Ile Lys Glu
Leu Thr Lys Leu Ile Met Thr Pro Glu Glu Lys 145 150 155 160 ttg gga
agg gag act att ggt gaa tac ttc tct gat gat ttc ttt gaa 528 Leu Gly
Arg Glu Thr Ile Gly Glu Tyr Phe Ser Asp Asp Phe Phe Glu 165 170 175
agc aat ttc tgg att tat tgg tca aca atg ttt gcg ttt gaa cgg tgg 576
Ser Asn Phe Trp Ile Tyr Trp Ser Thr Met Phe Ala Phe Glu Arg Trp 180
185 190 cac tct cta gct gaa atg cgt cgt tat atg atg cgg ttt att cac
cat 624 His Ser Leu Ala Glu Met Arg Arg Tyr Met Met Arg Phe Ile His
His 195 200 205 att gat ggt tta ccg gat ttc act gca ctg aag ttt aat
aag tat aac 672 Ile Asp Gly Leu Pro Asp Phe Thr Ala Leu Lys Phe Asn
Lys Tyr Asn 210 215 220 caa tat gaa tca atg acc aag ccg cta ttg gcc
tac ctg aaa gat cat 720 Gln Tyr Glu Ser Met Thr Lys Pro Leu Leu Ala
Tyr Leu Lys Asp His 225 230 235 240 cat gtc aag att gag tac gat acc
cag gta aag aat gtt att gtt gat 768 His Val Lys Ile Glu Tyr Asp Thr
Gln Val Lys Asn Val Ile Val Asp 245 250 255 act cat ggg cgg caa aag
cac gct aag cga atc tta tta act caa gcc 816 Thr His Gly Arg Gln Lys
His Ala Lys Arg Ile Leu Leu Thr Gln Ala 260 265 270 ggt aaa gat aaa
gtt gtt gag tta acg gac aat gac ctt gtc ttt gtc 864 Gly Lys Asp Lys
Val Val Glu Leu Thr Asp Asn Asp Leu Val Phe Val 275 280 285 aca aac
ggt tca att aca gaa agt tct act tac ggc agt cac cat caa 912 Thr Asn
Gly Ser Ile Thr Glu Ser Ser Thr Tyr Gly Ser His His Gln 290 295 300
gca gct cga cca acg caa gca ctt ggt ggt agt tgg aaa ctg tgg gaa 960
Ala Ala Arg Pro Thr Gln Ala Leu Gly Gly Ser Trp Lys Leu Trp Glu 305
310 315 320 aac ctt gct cgg cag tca gct gat ttt ggt cat cct gat gtc
ttt tgc 1008 Asn Leu Ala Arg Gln Ser Ala Asp Phe Gly His Pro Asp
Val Phe Cys 325 330 335 aag aat ctt cca ggg aga agc tgg ttc att tcc
gct act gca acc gtt 1056 Lys Asn Leu Pro Gly Arg Ser Trp Phe Ile
Ser Ala Thr Ala Thr Val 340 345 350 aag aac ccg caa gtt gaa cca tac
att gaa cgc tta acc aag cga gat 1104 Lys Asn Pro Gln Val Glu Pro
Tyr Ile Glu Arg Leu Thr Lys Arg Asp 355 360 365 ctc cat gat ggc aaa
gtt aat act ggt gga atc att acg gtc act gac 1152 Leu His Asp Gly
Lys Val Asn Thr Gly Gly Ile Ile Thr Val Thr Asp 370 375 380 tct aat
tgg atg ctt tcc tgg aca att cac cgt caa ccg cac ttc aag 1200 Ser
Asn Trp Met Leu Ser Trp Thr Ile His Arg Gln Pro His Phe Lys 385 390
395 400 aaa caa aag aaa aat gaa acc att gtt tgg att tac ggt ctg tac
tct 1248 Lys Gln Lys Lys Asn Glu Thr Ile Val Trp Ile Tyr Gly Leu
Tyr Ser 405 410 415 aat aca aag gga aac tat att aag aaa cgg atc gtt
gat tgt act ggt 1296 Asn Thr Lys Gly Asn Tyr Ile Lys Lys Arg Ile
Val Asp Cys Thr Gly 420 425 430 gaa gag att act aaa gaa tgg cta tcc
atc tgg ggg ttc cag aag ccg 1344 Glu Glu Ile Thr Lys Glu Trp Leu
Ser Ile Trp Gly Phe Gln Lys Pro 435 440 445 tta att gac gat ttg gct
aag gag agt tca att aat act gtt cca gta 1392 Leu Ile Asp Asp Leu
Ala Lys Glu Ser Ser Ile Asn Thr Val Pro Val 450 455 460 tat atg cca
ttt atc act agc tac ttt atg cca cga gtt aag ggc gac 1440 Tyr Met
Pro Phe Ile Thr Ser Tyr Phe Met Pro Arg Val Lys Gly Asp 465 470 475
480 cgt cca gac gtt gtt cca gaa gga tcc gct aac ttg gca ttt att ggt
1488 Arg Pro Asp Val Val Pro Glu Gly Ser Ala Asn Leu Ala Phe Ile
Gly 485 490 495 aac ttt gct gaa tct cca agt cga gat acc gta ttt acc
acg gaa tat 1536 Asn Phe Ala Glu Ser Pro Ser Arg Asp Thr Val Phe
Thr Thr Glu Tyr 500 505 510 tca gta cgg acc gca atg gaa gcc gtc tac
act cta tta gat gtt gat 1584 Ser Val Arg Thr Ala Met Glu Ala Val
Tyr Thr Leu Leu Asp Val Asp 515 520 525 cgg gga gtt cca gaa gtc ttt
aac tct att tat gat ctt cga gag tta 1632 Arg Gly Val Pro Glu Val
Phe Asn Ser Ile Tyr Asp Leu Arg Glu Leu 530 535 540 atg cgg gca atg
tat tac atg aat gat aag aag ccg tta aaa gac atg 1680 Met Arg Ala
Met Tyr Tyr Met Asn Asp Lys Lys Pro Leu Lys Asp Met 545 550 555 560
gac ttg cca att cca aag att gtt gaa aag cca tta tta aag aaa ctc
1728 Asp Leu Pro Ile Pro Lys Ile Val Glu Lys Pro Leu Leu Lys Lys
Leu 565 570 575 caa gga acg tgg att ggt gaa tta atg gag caa cag cac
tta cta taa 1776 Gln Gly Thr Trp Ile Gly Glu Leu Met Glu Gln Gln
His Leu Leu 580 585 590 18 591 PRT Lactobacillus reuteri 18 Met Tyr
Tyr Ser Asn Gly Asn Tyr Glu Ala Phe Ala Arg Pro Lys Lys 1 5 10 15
Pro Ala Gly Val Asp Lys Lys His Ala Tyr Ile Val Gly Gly Gly Leu 20
25 30 Ala Gly Leu Ser Ala Ala Val Phe Leu Ile Arg Asp Ala Gln Met
Pro 35 40 45 Gly Glu Asn Ile His Ile Leu Glu Glu Leu Pro Val Ala
Gly Gly Ser 50 55 60 Leu Asp Gly Glu Asp Arg Pro Gly Ile Gly Phe
Val Thr Arg Gly Gly 65 70 75 80 Arg Glu Met Glu Asn His Phe Glu Cys
Met Trp Asp Met Tyr Arg Ser 85 90 95 Ile Pro Ser Leu Glu Ile Pro
Gly Ala Ser Tyr Leu Asp Glu Tyr Tyr 100 105 110 Trp Leu Asp Lys Glu
Asp Pro Asn Ser Ser Asn Cys Arg Leu Thr Tyr 115 120 125 Lys Arg Gly
Asn Glu Val Pro Ser Asp Gly Lys Tyr Gly Leu Ser Lys 130 135 140 Lys
Ala Ile Lys Glu Leu Thr Lys Leu Ile Met Thr Pro Glu Glu Lys 145 150
155 160 Leu Gly Arg Glu Thr Ile Gly Glu Tyr Phe Ser Asp Asp Phe Phe
Glu 165 170 175 Ser Asn Phe Trp Ile Tyr Trp Ser Thr Met Phe Ala Phe
Glu Arg Trp 180 185 190 His Ser Leu Ala Glu Met Arg Arg Tyr Met Met
Arg Phe Ile His His 195 200 205 Ile Asp Gly Leu Pro Asp Phe Thr Ala
Leu Lys Phe Asn Lys Tyr Asn 210 215 220 Gln Tyr Glu Ser Met Thr Lys
Pro Leu Leu Ala Tyr Leu Lys Asp His 225 230 235 240 His Val Lys Ile
Glu Tyr Asp Thr Gln Val Lys Asn Val Ile Val Asp 245 250 255 Thr His
Gly Arg Gln Lys His Ala Lys Arg Ile Leu Leu Thr Gln Ala 260 265 270
Gly Lys Asp Lys Val Val Glu Leu Thr Asp Asn Asp Leu Val Phe Val 275
280 285 Thr Asn Gly Ser Ile Thr Glu Ser Ser Thr Tyr Gly Ser His His
Gln 290 295 300 Ala Ala Arg Pro Thr Gln Ala Leu Gly Gly Ser Trp Lys
Leu Trp Glu 305 310 315 320 Asn Leu Ala Arg Gln Ser Ala Asp Phe Gly
His Pro Asp Val Phe Cys 325 330 335 Lys Asn Leu Pro Gly Arg Ser Trp
Phe Ile Ser Ala Thr Ala Thr Val 340 345 350 Lys Asn Pro Gln Val Glu
Pro Tyr Ile Glu Arg Leu Thr Lys Arg Asp 355 360 365 Leu His Asp Gly
Lys Val Asn Thr Gly Gly Ile Ile Thr Val Thr Asp 370 375 380 Ser Asn
Trp Met Leu Ser Trp Thr Ile His Arg Gln Pro His Phe Lys 385 390 395
400 Lys Gln Lys Lys Asn Glu Thr Ile Val Trp Ile Tyr Gly Leu Tyr Ser
405 410 415 Asn Thr Lys Gly Asn Tyr Ile Lys Lys Arg Ile Val Asp Cys
Thr Gly 420 425 430 Glu Glu Ile Thr Lys Glu Trp Leu Ser Ile Trp Gly
Phe Gln Lys Pro 435 440 445 Leu Ile Asp Asp Leu Ala Lys Glu Ser Ser
Ile Asn Thr Val Pro Val 450 455 460 Tyr Met Pro Phe Ile Thr Ser Tyr
Phe Met Pro Arg Val Lys Gly Asp 465 470 475 480 Arg Pro Asp Val Val
Pro Glu Gly Ser Ala Asn Leu Ala Phe Ile Gly 485 490 495 Asn Phe Ala
Glu Ser Pro Ser Arg Asp Thr Val Phe Thr Thr Glu Tyr 500 505 510 Ser
Val Arg Thr Ala Met Glu Ala Val Tyr Thr Leu Leu Asp Val Asp 515 520
525 Arg Gly Val Pro Glu Val Phe Asn Ser Ile Tyr Asp Leu Arg Glu Leu
530 535 540 Met Arg Ala Met Tyr Tyr Met Asn Asp Lys Lys Pro Leu Lys
Asp Met 545 550 555 560 Asp Leu Pro Ile Pro Lys Ile Val Glu Lys Pro
Leu Leu Lys Lys Leu 565 570 575 Gln Gly Thr Trp Ile Gly Glu Leu Met
Glu Gln Gln His Leu Leu 580 585 590 19 656 DNA Lactobacillus
reuteri CDS (1)..(654) 19 atg gtc atg aca gaa act gct ggt ata aga
aaa att cat att gtt ttt 48 Met Val Met Thr Glu Thr Ala Gly Ile Arg
Lys Ile His Ile Val Phe 1 5 10 15 gat ggt caa gaa aca cca cca tta
aag atc cat caa tta ttt gat tca 96 Asp Gly Gln Glu Thr Pro Pro Leu
Lys Ile His Gln Leu Phe Asp Ser 20 25 30 caa aaa tac gat cag tta
atc gca gta act ggg aaa att act gct gac 144 Gln Lys Tyr Asp Gln Leu
Ile Ala Val Thr Gly Lys Ile Thr Ala Asp 35 40 45 ttc att aat aaa
tac ctt agt aat ttt atc agt att aat gta gcg tta 192 Phe Ile Asn Lys
Tyr Leu Ser Asn Phe Ile Ser Ile Asn Val Ala Leu 50 55 60 agc tcc
caa tca act agt gaa tta agt gct gat gag atg gtg aca aag 240 Ser Ser
Gln Ser Thr Ser Glu Leu Ser Ala Asp Glu Met Val Thr Lys 65 70 75 80
gtt gca ctt acc aat gct ctc ctt agt tca gca aat aaa gaa gct gct 288
Val Ala Leu Thr Asn Ala Leu Leu Ser Ser Ala Asn Lys Glu Ala Ala 85
90 95 aaa ctc ttc tca gcg tta acc agt gac aac caa acg aac gtc tta
aat 336 Lys Leu Phe Ser Ala Leu Thr Ser Asp Asn Gln Thr Asn Val Leu
Asn 100 105 110 aat ctt ttt cgc gta tca atc gcg cct act cag gtt atc
cat tct aag 384 Asn Leu Phe Arg Val Ser Ile Ala Pro Thr Gln Val Ile
His Ser Lys 115 120 125 ttt tac ttg tta agt agt tca act act cat gat
tcc cgt gtg att ctt 432 Phe Tyr Leu Leu Ser Ser Ser Thr Thr His Asp
Ser Arg Val Ile Leu 130 135 140 ggg agt gta gat tta gac gaa gct tca
ttt gat gct cac cga aat caa 480 Gly Ser Val Asp Leu Asp Glu Ala Ser
Phe Asp Ala His Arg Asn Gln 145 150 155 160 ttt gaa gaa gta ttg gta
ttt gac aat gat gtc cgc tta tac caa aac 528 Phe Glu Glu Val Leu Val
Phe Asp Asn Asp Val Arg Leu Tyr Gln Asn 165 170 175 ctt act gac cac
ttt aaa aag gat ttt aag cca gta ttg aag ccc ttc 576 Leu Thr Asp His
Phe Lys Lys Asp Phe Lys Pro Val Leu Lys Pro Phe 180 185 190 ttt act
atg aac cta gta aag gca gct caa aag caa gtt gag gaa gga 624 Phe Thr
Met Asn Leu Val Lys Ala Ala Gln Lys Gln Val Glu Glu Gly 195 200 205
aag aaa gat cag gat agc ggt aag gga ccg gt 656 Lys Lys Asp Gln Asp
Ser Gly Lys Gly Pro 210 215 20 218 PRT Lactobacillus reuteri 20 Met
Val Met Thr Glu Thr Ala Gly Ile Arg Lys Ile His Ile Val Phe 1 5 10
15 Asp Gly Gln Glu Thr Pro Pro Leu Lys Ile His Gln Leu Phe Asp Ser
20 25 30 Gln Lys Tyr Asp Gln Leu Ile Ala Val Thr Gly Lys Ile Thr
Ala Asp 35 40 45 Phe Ile Asn Lys Tyr Leu Ser Asn Phe Ile Ser Ile
Asn Val Ala Leu 50 55 60 Ser Ser Gln Ser Thr Ser Glu Leu Ser Ala
Asp Glu Met Val Thr Lys 65 70 75 80 Val Ala Leu Thr Asn Ala Leu Leu
Ser Ser Ala Asn Lys Glu Ala Ala 85 90 95 Lys Leu Phe Ser Ala Leu
Thr Ser Asp Asn Gln Thr Asn Val Leu Asn 100 105 110 Asn Leu Phe Arg
Val Ser Ile Ala Pro Thr Gln Val Ile His Ser Lys 115 120 125 Phe Tyr
Leu Leu Ser Ser Ser Thr Thr His Asp Ser Arg Val Ile Leu 130 135 140
Gly Ser Val Asp Leu Asp Glu Ala Ser Phe Asp Ala His Arg Asn Gln 145
150 155 160 Phe Glu Glu Val Leu Val Phe Asp Asn Asp Val Arg Leu Tyr
Gln Asn 165 170 175 Leu Thr Asp His Phe Lys Lys Asp Phe Lys Pro Val
Leu Lys Pro Phe 180 185 190 Phe Thr Met Asn Leu Val Lys Ala Ala Gln
Lys Gln Val Glu Glu Gly 195 200 205 Lys Lys Asp Gln Asp Ser Gly Lys
Gly Pro 210 215 21 726 DNA Lactobacillus reuteri CDS (1)..(726)
unsure (12) unsure (297) misc_feature (10)..(12) Xaa = Tyr or stop
21 atg ctt cgt tan acc ata tta gta aaa ttg ctt att gga aga aaa cca
48 Met Leu Arg Xaa Thr Ile Leu Val Lys Leu Leu Ile Gly Arg Lys Pro
1 5 10 15 gtc aca acg atc aaa aaa aca tta ccg cca act cag gaa cag
gct aat 96 Val Thr Thr Ile Lys Lys Thr Leu Pro Pro Thr Gln Glu Gln
Ala Asn 20 25 30 tca gtc tta act ccg gct gtt cgc caa caa ctt ggc
att tca att acc 144 Ser Val Leu Thr Pro Ala Val Arg Gln Gln Leu Gly
Ile Ser Ile Thr 35 40 45 tgg aac aaa gcc ggt gcg ttt att atc aat
aat aac caa aca aat ctt 192 Trp Asn Lys Ala Gly Ala Phe Ile Ile Asn
Asn Asn Gln Thr Asn Leu 50 55 60 aac gct aag att gca agt gca ccc
tat gct gta aat cat ctt gac cgt 240 Asn Ala Lys Ile Ala Ser Ala Pro
Tyr Ala Val Asn His Leu Asp Arg 65 70 75 80 caa gga agg gcg tgg caa
ggt gat gcc tgg tta aac agg aca act cgg 288 Gln Gly Arg Ala Trp Gln
Gly Asp Ala Trp Leu Asn Arg Thr Thr Arg 85 90 95 tca ata tan aag
ccg aaa ttt gcc aca ggg aat ggt gct acg gat tgg 336 Ser Ile Xaa Lys
Pro Lys Phe Ala Thr Gly Asn Gly Ala Thr Asp Trp 100 105 110 cga cca
gct ggc ttc ctt cag gcg cat aat ctt aaa ggc ggg tac aat 384 Arg Pro
Ala Gly Phe Leu Gln Ala His Asn Leu Lys Gly Gly Tyr Asn 115 120 125
cat gca tac gat cgc gga cac ctt ctt gcc tat gca cta gtt ggt ggt 432
His Ala Tyr Asp Arg Gly His Leu Leu Ala Tyr Ala Leu Val Gly Gly 130
135 140 att cat gga ttt gat gca tcc gaa tca aat cca tct aat att gcc
acg 480 Ile His Gly Phe Asp Ala Ser Glu Ser Asn Pro Ser Asn Ile Ala
Thr 145 150 155 160 caa act gcc tgg gca aat gaa gca cga agt aag aac
tca aca ggg
caa 528 Gln Thr Ala Trp Ala Asn Glu Ala Arg Ser Lys Asn Ser Thr Gly
Gln 165 170 175 aat tac tac gaa ggt ctg gtg aga aaa gca tta gat cag
aat aag caa 576 Asn Tyr Tyr Glu Gly Leu Val Arg Lys Ala Leu Asp Gln
Asn Lys Gln 180 185 190 gtt cgc tac cga gtt acc aat att tat gac ggt
aat aat atc gtt ccg 624 Val Arg Tyr Arg Val Thr Asn Ile Tyr Asp Gly
Asn Asn Ile Val Pro 195 200 205 gca ggt gct cat atc gaa gct aaa tct
agt gat ggt tct cta gaa tac 672 Ala Gly Ala His Ile Glu Ala Lys Ser
Ser Asp Gly Ser Leu Glu Tyr 210 215 220 aat gtc ttt gtt ccg aat gtc
caa aga aac att acc att aat tat tca 720 Asn Val Phe Val Pro Asn Val
Gln Arg Asn Ile Thr Ile Asn Tyr Ser 225 230 235 240 acc ggt 726 Thr
Gly 22 242 PRT Lactobacillus reuteri misc_feature 4 Xaa = Tyr or
stop 22 Met Leu Arg Xaa Thr Ile Leu Val Lys Leu Leu Ile Gly Arg Lys
Pro 1 5 10 15 Val Thr Thr Ile Lys Lys Thr Leu Pro Pro Thr Gln Glu
Gln Ala Asn 20 25 30 Ser Val Leu Thr Pro Ala Val Arg Gln Gln Leu
Gly Ile Ser Ile Thr 35 40 45 Trp Asn Lys Ala Gly Ala Phe Ile Ile
Asn Asn Asn Gln Thr Asn Leu 50 55 60 Asn Ala Lys Ile Ala Ser Ala
Pro Tyr Ala Val Asn His Leu Asp Arg 65 70 75 80 Gln Gly Arg Ala Trp
Gln Gly Asp Ala Trp Leu Asn Arg Thr Thr Arg 85 90 95 Ser Ile Xaa
Lys Pro Lys Phe Ala Thr Gly Asn Gly Ala Thr Asp Trp 100 105 110 Arg
Pro Ala Gly Phe Leu Gln Ala His Asn Leu Lys Gly Gly Tyr Asn 115 120
125 His Ala Tyr Asp Arg Gly His Leu Leu Ala Tyr Ala Leu Val Gly Gly
130 135 140 Ile His Gly Phe Asp Ala Ser Glu Ser Asn Pro Ser Asn Ile
Ala Thr 145 150 155 160 Gln Thr Ala Trp Ala Asn Glu Ala Arg Ser Lys
Asn Ser Thr Gly Gln 165 170 175 Asn Tyr Tyr Glu Gly Leu Val Arg Lys
Ala Leu Asp Gln Asn Lys Gln 180 185 190 Val Arg Tyr Arg Val Thr Asn
Ile Tyr Asp Gly Asn Asn Ile Val Pro 195 200 205 Ala Gly Ala His Ile
Glu Ala Lys Ser Ser Asp Gly Ser Leu Glu Tyr 210 215 220 Asn Val Phe
Val Pro Asn Val Gln Arg Asn Ile Thr Ile Asn Tyr Ser 225 230 235 240
Thr Gly 23 18 DNA Lactobacillus reuteri misc_feature (1)..(18)
PRIMER 23 aatctagtga tggttctc 18 24 18 DNA Lactobacillus reuteri
misc_feature (1)..(18) PRIMER 24 caagttgagg aaggaaag 18 25 3684 DNA
Lactobacillus reuteri 25 caagttgagg aaggaaagaa agatcaggat
agcggtaagg gaccggttat ccttgataat 60 gaaacaacag ataagatcgc
tgaaacagac atggtggatc tgttgaagca tgaccttcag 120 catgatattg
accataatct tgttcctgaa atgatcacaa agtcaatgcg tgatattacc 180
ataaatcgtt ctcaagcaaa ggagaaaatt gctaagcagg ttaagcaaca tgatacgatt
240 tatactttgc aaaaagaagc ggtctctcct cgggcagcta agccaaaact
aaagactcga 300 gaaaaaatta ccaagcaggt tcaggatgct ttgatcagtg
gaatgtcacc acagcaacgg 360 gatgctgaga aaaagtacac gacttttctg
tacgatcggc caatggaacg aaacattgcg 420 aataacaata gtggcctata
cgttcctaat gatacgggaa ctcacccaat cccatttggt 480 aaaattgcaa
ctatttctga aattcgtgac ggtttaaaga gcattgatgc tgttatgaag 540
ggctatcagc agtttgtcgt tgattatgat gctgactacg ggaagcggtt ctttgaagca
600 attttgtata gttttactgc accgttttta tgggaaattc gttctaaagc
tagcctgaac 660 cctgaagatg ggaatgatgt tcctaatttc ctaatcctag
gggcaacggc tggttccgga 720 aagtctaccc ttcttcggat tattaatcag
ctcacgtgga acactgatcg ctcgttgatt 780 gactttggaa cgatctaccc
gtcgcaaact cctcaaaaga aggcaaagac tgttgaggcg 840 atggaacatt
atatgaaact tggtagttca tacccggttt tgttagatga aattgaaccg 900
tacttcttcc agcaagatca atatagtcga ctggagttct ggtttgctat gattaaggtt
960 gttacgatta ttgcaatgat tattcttggt ttactggtta tcgttcttgg
gttaggtaat 1020 aactggcacc cagttgggat ttctaatttg tggtctcatg
gcggattctt taccggtggc 1080 tttatgggct ttatgttctc gctatctgtg
attgctggtt cttatcaggg aattgagtta 1140 ttgggaatca ctgctggtga
agctgaatca ccacgtcatg cgattgtgaa atcagttaag 1200 tccgttatct
ggcggatctt aatcttctat attggtgcaa ttttcgtcat tgtttctatt 1260
tacccatgga acgaattgaa gtccgttggc tcaccattcg ttgaaacctt cacgaaggtt
1320 ggaattactg gagcagccgg aatcattaac tttgttgttt tgacggcagc
tctttctgga 1380 gctaactctg gaatttacag tgctagtcgg atgttgttca
agctttctgt tgatggggaa 1440 gtaccaaagt tctttagtaa gctttccaag
cgcgttgttc ctaatgttgc aatcctcacg 1500 atttcttcct ggatcttcct
tggctttgta attaatgaat taatgtcgat ttttagttct 1560 gctgctcaaa
atattttcgt cattgtatat agttccagtg ttcttccagg gatggtacca 1620
tggtttatca ttctcttgtc agaacttcac ttcagaaaag aacaccctga acagcttaaa
1680 gatcatccat tcaagatgcc gctttacccg gcttataact actttagttt
gattgccttg 1740 actgtgatct tgatcttcat gttctttaac ccagatactc
gagtttcagt atcagttggt 1800 gttatcttct tgattatcat gagtattatt
tatcgtgttc gtgttcatga aggaaaagaa 1860 aagtaaatat atagctaaag
cagctttgta aatcctgcgt acaatacccc ttagggttga 1920 cactttaaat
aataaaagtg tgaatcctag ggggtgtttt gcattgtaag ttattcaact 1980
attgaaaagc ttaaattact tcatgattat cagaaatcgg attatggttt aacggtgtac
2040 tccgattacc atggtgtccg accagcaaac atgagtaagt ggattaagca
attcctactc 2100 gctggattgg cgggattaat tagacctaag cataatcaga
agtactcatt agagactaag 2160 ttaactgctg taaaagctta tctttctggc
aagtatacta atcaagcaat tctccagcag 2220 tatcaaatta gaaatatttc
tcaactacat caatgggtta tcagttacaa taatgacaaa 2280 ctccgagtta
atcagacaac gagaaagcga gtcagaaaaa tgggacgaaa agtaaccttt 2340
gatgaaaaga ggcagattgt ccgatggaca attgaacata acaataacta taaagcggct
2400 gcagagaagt atgatattag ttaccaacga gtttattctt gggtacggaa
gtaccgagta 2460 aatagcgact gggaagtact aaaagataac cgtgggcgta
ataaaggaaa agagcccact 2520 aatgaactag aaaaactaag gaaacgagtt
cgtgagctag aagatcgtga ccgtgaacgg 2580 gagctgcaaa tcgctttcgc
aaaaaaatta gtcgaaatac gcaatcggga ggtgaaacga 2640 ccggacgata
tcaagcgatt caagaaatga acaatgaagg ttattccatt agtgaattgg 2700
ccaaggtcgc tggaattact agacaggctt actacaaatg gttgaaacat gaaccgacta
2760 aatatgagat tgaagaatcg gagattctcc aattgattaa acagttagaa
aatgaacata 2820 agcaaagcgt tggttatgac aaaatgacta ggttaatcaa
gttaagtcag cagatctctt 2880 ataccgttaa taagaaacga gtcattcgta
ttatgaaagg ccatagtatc aaggccgact 2940 atcgtcagcc aaccgacaaa
cgtattcaag cccagcaaac ttatgaagct gaaaatattc 3000 ttaaccgaca
atttgaccaa actgcagcta accaagtttg ggttacggat acgacggaac 3060
tgaattacgg aatctggctt aataaagttc gtctacatat agtattagat ttatatggtc
3120 aatacccagt aagctggtta attacaccta cagaaaccgc tgaaggagta
gttcaagtgt 3180 tcgagcaagc acggatgaaa gaaggagcac tagctccgtt
aattcatact gatcgtggtg 3240 cggcgtatac ttccaaagca tttaatcagt
atttagtagt taatggtgcc caacacagtt 3300 attcagcacc agggacaccg
gctgacaatg ccgtaataga acattggtgg gcagatttta 3360 aggctatttg
gatcgcacat ctacctaaag cacaaacatt attagaacta gaagaacaag 3420
ttagagaagg aattacctat ttcactgaaa aatttatctc agcgaagaga aatgacctta
3480 ccgcagcgga ataccgcttt ggcaaggcca actaattttt attatttaat
gtgtaaactt 3540 gacagggcac agtaccctgt ttgaggggac tcacaaagct
gcttttttag ttttgtttta 3600 ctgcaccggt tgaataatta atggtaatgt
ttctttggac attcggaaca aagacattgt 3660 attctagaga accatcacta gatt
3684 26 7113 DNA Lactobacillus reuteri unsure (3109) unsure (3394)
26 gtcgactgga gttctggttt gctatgatta aggttgttac gattattgca
atgattattc 60 ttggtttact ggttatcgtt cttgggttag gtaataactg
gcacccagtt gggatttcta 120 tttgtggtc tcatggcgga ttctttaccg
gtggctttat gggctttatg ttctcgctat 180 tgtgattgc tggttcttat
cagggaattg agttattggg aatcactgct ggtgaagctg 240 atcaccacg
tcatgcgatt gtgaaatcag ttaagtccgt tatctggcgg atcttaatct 300
ctatattgg tgcaattttc gtcattgttt ctatttaccc atggaacgaa ttgaagtccg
360 tggctcacc attcgttgaa accttcacga aggttggaat tactggagca
gccggaatca 420 taactttgt tgttttgacg gcagctcttt ctggagctaa
ctctggaatt tacagtgcta 480 tcggatgtt gttcaagctt tctgttgatg
gggaagtacc aaagttcttt agtaagcttt 540 caagcgcgt tgttcctaat
gttgcaatcc tcacgatttc ttcctggatc ttccttggct 600 tgtaattaa
tgaattaatg tcgattttta gttctgctgc tcaaaatatt ttcgtcattg 660
atatagttc cagtgttctt ccagggatgg taccatggtt tatcattctc ttgtcagaac
720 tcacttcag aaaagaacac cctgaacagc ttaaagatca tccattcaag
atgccgcttt 780 cccggctta taactacttt agtttgattg ccttgactgt
gatcttgatc ttcatgttct 840 taacccaga tactcgagtt tcagtatcag
ttggtgttat cttcttgatt atcatgagta 900 tatttatcg tgttcgtgtt
catgaaggaa aagaaaagta aatatatagc taaagcagct 960 ttgtaaatcc
tgcgtacaat accccttagg gttgacactt taaataataa aagtgtgaat 1020
cctagggggt gttttgcatt gtaagttatt caactattga aaagcttaaa ttacttcatg
1080 attatcagaa atcggattat ggtttaacgg tgtactccga ttaccatggt
gtccgaccag 1140 caaacatgag taagtggatt aagcaattcc tactcgctgg
attggcggga ttaattagac 1200 ctaagcataa tcagaagtac tcattagaga
ctaagttaac tgctgtaaaa gcttatcttt 1260 ctggcaagta tactaatcaa
gcaattctcc agcagtatca aattagaaat atttctcaac 1320 tacatcaatg
ggttatcagt tacaataatg acaaactccg agttaatcag acaacgagaa 1380
agcgagtcag aaaaatggga cgaaaagtaa cctttgatga aaagaggcag attgtccgat
1440 ggacaattga acataacaat aactataaag cggctgcaga gaagtatgat
attagttacc 1500 aacgagttta ttcttgggta cggaagtacc gagtaaatag
cgactgggaa gtactaaaag 1560 ataaccgtgg gcgtaataaa ggaaaagagc
ccactaatga actagaaaaa ctaaggaaac 1620 gagttcgtga gctagaagat
cgtgaccgtg aacgggagct gcaaatcgct ttcgcaaaaa 1680 aattagtcga
aatacgcaat cgggaggtga aacgaccgga cgatatcaag cgattcaaga 1740
aatgaacaat gaaggttatt ccattagtga attggccaag gtcgctggaa ttactagaca
1800 ggcttactac aaatggttga aacatgaacc gactaaatat gagattgaag
aatcggagat 1860 tctccaattg attaaacagt tagaaaatga acataagcaa
agcgttggtt atgacaaaat 1920 gactaggtta atcaagttaa gtcagcagat
ctcttatacc gttaataaga aacgagtcat 1980 tcgtattatg aaaggccata
gtatcaaggc cgactatcgt cagccaaccg acaaacgtat 2040 tcaagcccag
caaacttatg aagctgaaaa tattcttaac cgacaatttg accaaactgc 2100
agctaaccaa gtttgggtta cggatacgac ggaactgaat tacggaatct ggcttaataa
2160 agttcgtcta catatagtat tagatttata tggtcaatac ccagtaagct
ggttaattac 2220 acctacagaa accgctgaag gagtagttca agtgttcgag
caagcacgga tgaaagaagg 2280 agcactagct ccgttaattc atactgatcg
tggtgcggcg tatacttcca aagcatttaa 2340 tcagtattta gtagttaatg
gtgcccaaca cagttattca gcaccaggga caccggctga 2400 caatgccgta
atagaacatt ggtgggcaga ttttaaggct atttggatcg cacatctacc 2460
taaagcacaa acattattag aactagaaga acaagttaga gaaggaatta cctatttcac
2520 tgaaaaattt atctcagcga agagaaatga ccttaccgca gcggaatacc
gctttggcaa 2580 ggccaactaa tttttattat ttaatgtgta aacttgacag
ggcacagtac cctgtttgag 2640 gggactcaca aagctgcttt tttagttttg
ttttactgca ccggttgaat aattaatggt 2700 aatgtttctt tggacattcg
gaacaaagac attgtattct agagaaccat cactagattt 2760 agcttcgata
tgagcacctg ccggaacgat attattaccg tcataaatat tggtaactcg 2820
gtagcgaact tgcttattct gatctaatgc ttttctcacc agaccttcgt agtaattttg
2880 ccctgttgag ttcttacttc gtgcttcatt tgcccaggca gtttgcgtgg
caatattaga 2940 tggatttgat tcggatgcat caaatccatg aataccacca
actagtgcat aggcaagaag 3000 gtgtccgcga tcgtatgcat gattgtaccc
gcctttaaga ttatgcgcct gaaggaagcc 3060 agctggtcgc caatccgtag
caccattccc tgtggcaaat ttcggcttnt atattgaccg 3120 agttgtcctg
tttaaccagg catcaccttg ccacgccctt ccttgacggt caagatgatt 3180
tacagcatag ggtgcacttg caatcttagc gttaagattt gtttggttat tattgataat
3240 aaacgcaccg gctttgttcc aggtaattga aatgccaagt tgttggcgaa
cagccggagt 3300 taagactgaa ttagcctgtt cctgagttgg cggtaatgtt
tttttgatcg ttgtgactgg 3360 ttttcttcca ataagcaatt ttactaatat
ggtntaacga agcatttgtt agctgaggtt 3420 gctggataac tccagtaact
actaataaac cagcaagagc aaataaaagg tgatagaggc 3480 gtttcttaag
tttcataaat tcactccatt tctaataatt ccaaagtcta ttttactagt 3540
ttgaacatac gtttggaata attatttaga attaatttat aagttcattg tgtttaataa
3600 aattgacact ttcaaccgct ttcactaaaa ttaaggtagt tatgatgcac
ttgtttactg 3660 agaagggagt cgtcaaaatg tattattcaa acgggaatta
tgaagccttt gctcgaccaa 3720 agaagcctgc tggcgttgat aagaaacatg
cctacattgt cggtggtggt ttagctggtt 3780 tatcggccgc cgtgttttta
attcgtgatg cccaaatgcc gggtgagaat atccatattt 3840 tagaggaatt
accggttgcc ggtggttctc ttgatggtga agatcgtcct ggaattggtt 3900
ttgttactcg tggaggccgg gaaatggaga accatttcga gtgtatgtgg gacatgtatc
3960 gttcaattcc atcacttgaa atcccaggtg cttcctacct tgatgaatac
tactggttag 4020 ataaggaaga tccaaacagt tctaattgtc gtttaaccta
taagcgggga aatgaagttc 4080 catcggacgg taaatatggt ttaagtaaaa
aggcaatcaa agagctgact aagctaatta 4140 tgacccctga agaaaaattg
ggaagggaga ctattggtga atacttctct gatgatttct 4200 ttgaaagcaa
tttctggatt tattggtcaa caatgtttgc gtttgaacgg tggcactctc 4260
tagctgaaat gcgtcgttat atgatgcggt ttattcacca tattgatggt ttaccggatt
4320 tcactgcact gaagtttaat aagtataacc aatatgaatc aatgaccaag
ccgctattgg 4380 cctacctgaa agatcatcat gtcaagattg agtacgatac
ccaggtaaag aatgttattg 4440 ttgatactca tgggcggcaa aagcacgcta
agcgaatctt attaactcaa gccggtaaag 4500 ataaagttgt tgagttaacg
gacaatgacc ttgtctttgt cacaaacggt tcaattacag 4560 aaagttctac
ttacggcagt caccatcaag cagctcgacc aacgcaagca cttggtggta 4620
gttggaaact gtgggaaaac cttgctcggc agtcagctga ttttggtcat cctgatgtct
4680 tttgcaagaa tcttccaggg agaagctggt tcatttccgc tactgcaacc
gttaagaacc 4740 cgcaagttga accatacatt gaacgcttaa ccaagcgaga
tctccatgat ggcaaagtta 4800 atactggtgg aatcattacg gtcactgact
ctaattggat gctttcctgg acaattcacc 4860 gtcaaccgca cttcaagaaa
caaaagaaaa atgaaaccat tgtttggatt tacggtctgt 4920 actctaatac
aaagggaaac tatattaaga aacggatcgt tgattgtact ggtgaagaga 4980
ttactaaaga atggctatcc atctgggggt tccagaagcc gttaattgac gatttggcta
5040 aggagagttc aattaatact gttccagtat atatgccatt tatcactagc
tactttatgc 5100 cacgagttaa gggcgaccgt ccagacgttg ttccagaagg
atccgctaac ttggcattta 5160 ttggtaactt tgctgaatct ccaagtcgag
ataccgtatt taccacggaa tattcagtac 5220 ggaccgcaat ggaagccgtc
tacactctat tagatgttga tcggggagtt ccagaagtct 5280 ttaactctat
ttatgatctt cgagagttaa tgcgggcaat gtattacatg aatgataaga 5340
agccgttaaa agacatggac ttgccaattc caaagattgt tgaaaagcca ttattaaaga
5400 aactccaagg aacgtggatt ggtgaattaa tggagcaaca gcacttacta
taaagatgaa 5460 agaagctgga aatttccggc ttcttttttt atatagctta
tcggggaacc acttgcaaaa 5520 agggattaaa tagtcgaaaa taaaactaat
atatttattg atagtaaagg attatggtca 5580 tgacagaaac tgctggtata
agaaaaattc atattgtttt tgatggtcaa gaaacaccac 5640 cattaaagat
ccatcaatta tttgattcac aaaaatacga tcagttaatc gcagtaactg 5700
ggaaaattac tgctgacttc attaataaat accttagtaa ttttatcagt attaatgtag
5760 cgttaagctc ccaatcaact agtgaattaa gtgctgatga gatggtgaca
aaggttgcac 5820 ttaccaatgc tctccttagt tcagcaaata aagaagctgc
taaactcttc tcagcgttaa 5880 ccagtgacaa ccaaacgaac gtcttaaata
atctttttcg cgtatcaatc gcgcctactc 5940 aggttatcca ttctaagttt
tacttgttaa gtagttcaac tactcatgat tcccgtgtga 6000 ttcttgggag
tgtagattta gacgaagctt catttgatgc tcaccgaaat caatttgaag 6060
aagtattggt atttgacaat gatgtccgct tataccaaaa ccttactgac cactttaaaa
6120 aggattttaa gccagtattg aagcccttct ttactatgaa cctagtaaag
gcagctcaaa 6180 agcaagttga ggaaggaaag aaagatcagg atagcggtaa
gggaccggtt atccttgata 6240 atgaaacaac agataagatc gctgaaacag
acatggtgga tctgttgaag catgaccttc 6300 agcatgatat tgaccataat
cttgttcctg aaatgatcac aaagtcaatg cgtgatatta 6360 ccataaatcg
ttctcaagca aaggagaaaa ttgctaagca ggttaagcaa catgatacga 6420
tttatacttt gcaaaaagaa gcggtctctc ctcgggcagc taagccaaaa ctaaagactc
6480 gagaaaaaat taccaagcag gttcaggatg ctttgatcag tggaatgtca
ccacagcaac 6540 gggatgctga gaaaaagtac acgacttttc tgtacgatcg
gccaatggaa cgaaacattg 6600 cgaataacaa tagtggccta tacgttccta
atgatacggg aactcaccca atcccatttg 6660 gtaaaattgc aactatttct
gaaattcgtg acggtttaaa gagcattgat gctgttatga 6720 agggctatca
gcagtttgtc gttgattatg atgctgacta cgggaagcgg ttctttgaag 6780
caattttgta tagttttact gcaccgtttt tatgggaaat tcgttctaaa gctagcctga
6840 accctgaaga tgggaatgat gttcctaatt tcctaatcct aggggcaacg
gctggttccg 6900 gaaagtctac ccttcttcgg attattaatc agctcacgtg
gaacactgat cgctcgttga 6960 ttgactttgg aacgatctac ccgtcgcaaa
ctcctcaaaa gaaggcaaag actgttgagg 7020 cgatggaaca ttatatgaaa
cttggtagtt catacccggt tttgttagat gaaattgaac 7080 cgtacttctt
ccagcaagat caatatagtc gac 7113 27 941 DNA Lactobacillus reuteri CDS
(3)..(941) 27 gt cga ctg gag ttc tgg ttt gct atg att aag gtt gtt
acg att att 47 Arg Leu Glu Phe Trp Phe Ala Met Ile Lys Val Val Thr
Ile Ile 1 5 10 15 gca atg att att ctt ggt tta ctg gtt atc gtt ctt
ggg tta ggt aat 95 Ala Met Ile Ile Leu Gly Leu Leu Val Ile Val Leu
Gly Leu Gly Asn 20 25 30 aac tgg cac cca gtt ggg att tct aat ttg
tgg tct cat ggc gga ttc 143 Asn Trp His Pro Val Gly Ile Ser Asn Leu
Trp Ser His Gly Gly Phe 35 40 45 ttt acc ggt ggc ttt atg ggc ttt
atg ttc tcg cta tct gtg att gct 191 Phe Thr Gly Gly Phe Met Gly Phe
Met Phe Ser Leu Ser Val Ile Ala 50 55 60 ggt tct tat cag gga att
gag tta ttg gga atc act gct ggt gaa gct 239 Gly Ser Tyr Gln Gly Ile
Glu Leu Leu Gly Ile Thr Ala Gly Glu Ala 65 70 75 gaa tca cca cgt
cat gcg att gtg aaa tca gtt aag tcc gtt atc tgg 287 Glu Ser Pro Arg
His Ala Ile Val Lys Ser Val Lys Ser Val Ile Trp 80 85 90 95 cgg atc
tta atc ttc tat att ggt gca att ttc gtc att gtt tct att 335 Arg Ile
Leu Ile Phe Tyr Ile Gly Ala Ile Phe Val Ile Val Ser Ile 100 105 110
tac cca tgg aac gaa ttg aag tcc gtt ggc tca cca ttc gtt gaa acc 383
Tyr Pro Trp Asn Glu Leu Lys Ser Val Gly Ser Pro Phe Val Glu Thr 115
120 125 ttc acg aag gtt gga att act gga gca gcc gga atc att aac ttt
gtt 431 Phe Thr Lys Val Gly Ile Thr Gly Ala Ala Gly Ile Ile Asn Phe
Val 130 135 140 gtt ttg acg gca gct ctt tct gga gct aac tct gga att
tac agt gct
479 Val Leu Thr Ala Ala Leu Ser Gly Ala Asn Ser Gly Ile Tyr Ser Ala
145 150 155 agt cgg atg ttg ttc aag ctt tct gtt gat ggg gaa gta cca
aag ttc 527 Ser Arg Met Leu Phe Lys Leu Ser Val Asp Gly Glu Val Pro
Lys Phe 160 165 170 175 ttt agt aag ctt tcc aag cgc gtt gtt cct aat
gtt gca atc ctc acg 575 Phe Ser Lys Leu Ser Lys Arg Val Val Pro Asn
Val Ala Ile Leu Thr 180 185 190 att tct tcc tgg atc ttc ctt ggc ttt
gta att aat gaa tta atg tcg 623 Ile Ser Ser Trp Ile Phe Leu Gly Phe
Val Ile Asn Glu Leu Met Ser 195 200 205 att ttt agt tct gct gct caa
aat att ttc gtc att gta tat agt tcc 671 Ile Phe Ser Ser Ala Ala Gln
Asn Ile Phe Val Ile Val Tyr Ser Ser 210 215 220 agt gtt ctt cca ggg
atg gta cca tgg ttt atc att ctc ttg tca gaa 719 Ser Val Leu Pro Gly
Met Val Pro Trp Phe Ile Ile Leu Leu Ser Glu 225 230 235 ctt cac ttc
aga aaa gaa cac cct gaa cag ctt aaa gat cat cca ttc 767 Leu His Phe
Arg Lys Glu His Pro Glu Gln Leu Lys Asp His Pro Phe 240 245 250 255
aag atg ccg ctt tac ccg gct tat aac tac ttt agt ttg att gcc ttg 815
Lys Met Pro Leu Tyr Pro Ala Tyr Asn Tyr Phe Ser Leu Ile Ala Leu 260
265 270 act gtg atc ttg atc ttc atg ttc ttt aac cca gat act cga gtt
tca 863 Thr Val Ile Leu Ile Phe Met Phe Phe Asn Pro Asp Thr Arg Val
Ser 275 280 285 gta tca gtt ggt gtt atc ttc ttg att atc atg agt att
att tat cgt 911 Val Ser Val Gly Val Ile Phe Leu Ile Ile Met Ser Ile
Ile Tyr Arg 290 295 300 gtt cgt gtt cat gaa gga aaa gaa aag taa 941
Val Arg Val His Glu Gly Lys Glu Lys 305 310 28 312 PRT
Lactobacillus reuteri 28 Arg Leu Glu Phe Trp Phe Ala Met Ile Lys
Val Val Thr Ile Ile Ala 1 5 10 15 Met Ile Ile Leu Gly Leu Leu Val
Ile Val Leu Gly Leu Gly Asn Asn 20 25 30 Trp His Pro Val Gly Ile
Ser Asn Leu Trp Ser His Gly Gly Phe Phe 35 40 45 Thr Gly Gly Phe
Met Gly Phe Met Phe Ser Leu Ser Val Ile Ala Gly 50 55 60 Ser Tyr
Gln Gly Ile Glu Leu Leu Gly Ile Thr Ala Gly Glu Ala Glu 65 70 75 80
Ser Pro Arg His Ala Ile Val Lys Ser Val Lys Ser Val Ile Trp Arg 85
90 95 Ile Leu Ile Phe Tyr Ile Gly Ala Ile Phe Val Ile Val Ser Ile
Tyr 100 105 110 Pro Trp Asn Glu Leu Lys Ser Val Gly Ser Pro Phe Val
Glu Thr Phe 115 120 125 Thr Lys Val Gly Ile Thr Gly Ala Ala Gly Ile
Ile Asn Phe Val Val 130 135 140 Leu Thr Ala Ala Leu Ser Gly Ala Asn
Ser Gly Ile Tyr Ser Ala Ser 145 150 155 160 Arg Met Leu Phe Lys Leu
Ser Val Asp Gly Glu Val Pro Lys Phe Phe 165 170 175 Ser Lys Leu Ser
Lys Arg Val Val Pro Asn Val Ala Ile Leu Thr Ile 180 185 190 Ser Ser
Trp Ile Phe Leu Gly Phe Val Ile Asn Glu Leu Met Ser Ile 195 200 205
Phe Ser Ser Ala Ala Gln Asn Ile Phe Val Ile Val Tyr Ser Ser Ser 210
215 220 Val Leu Pro Gly Met Val Pro Trp Phe Ile Ile Leu Leu Ser Glu
Leu 225 230 235 240 His Phe Arg Lys Glu His Pro Glu Gln Leu Lys Asp
His Pro Phe Lys 245 250 255 Met Pro Leu Tyr Pro Ala Tyr Asn Tyr Phe
Ser Leu Ile Ala Leu Thr 260 265 270 Val Ile Leu Ile Phe Met Phe Phe
Asn Pro Asp Thr Arg Val Ser Val 275 280 285 Ser Val Gly Val Ile Phe
Leu Ile Ile Met Ser Ile Ile Tyr Arg Val 290 295 300 Arg Val His Glu
Gly Lys Glu Lys 305 310 29 600 DNA Lactobacillus reuteri CDS
(1)..(597) 29 atg agt aag tgg att aag caa ttc cta ctc gct gga ttg
gcg gga tta 48 Met Ser Lys Trp Ile Lys Gln Phe Leu Leu Ala Gly Leu
Ala Gly Leu 1 5 10 15 att aga cct aag cat aat cag aag tac tca tta
gag act aag tta act 96 Ile Arg Pro Lys His Asn Gln Lys Tyr Ser Leu
Glu Thr Lys Leu Thr 20 25 30 gct gta aaa gct tat ctt tct ggc aag
tat act aat caa gca att ctc 144 Ala Val Lys Ala Tyr Leu Ser Gly Lys
Tyr Thr Asn Gln Ala Ile Leu 35 40 45 cag cag tat caa att aga aat
att tct caa cta cat caa tgg gtt atc 192 Gln Gln Tyr Gln Ile Arg Asn
Ile Ser Gln Leu His Gln Trp Val Ile 50 55 60 agt tac aat aat gac
aaa ctc cga gtt aat cag aca acg aga aag cga 240 Ser Tyr Asn Asn Asp
Lys Leu Arg Val Asn Gln Thr Thr Arg Lys Arg 65 70 75 80 gtc aga aaa
atg gga cga aaa gta acc ttt gat gaa aag agg cag att 288 Val Arg Lys
Met Gly Arg Lys Val Thr Phe Asp Glu Lys Arg Gln Ile 85 90 95 gtc
cga tgg aca att gaa cat aac aat aac tat aaa gcg gct gca gag 336 Val
Arg Trp Thr Ile Glu His Asn Asn Asn Tyr Lys Ala Ala Ala Glu 100 105
110 aag tat gat att agt tac caa cga gtt tat tct tgg gta cgg aag tac
384 Lys Tyr Asp Ile Ser Tyr Gln Arg Val Tyr Ser Trp Val Arg Lys Tyr
115 120 125 cga gta aat agc gac tgg gaa gta cta aaa gat aac cgt ggg
cgt aat 432 Arg Val Asn Ser Asp Trp Glu Val Leu Lys Asp Asn Arg Gly
Arg Asn 130 135 140 aaa gga aaa gag ccc act aat gaa cta gaa aaa cta
agg aaa cga gtt 480 Lys Gly Lys Glu Pro Thr Asn Glu Leu Glu Lys Leu
Arg Lys Arg Val 145 150 155 160 cgt gag cta gaa gat cgt gac cgt gaa
cgg gag ctg caa atc gct ttc 528 Arg Glu Leu Glu Asp Arg Asp Arg Glu
Arg Glu Leu Gln Ile Ala Phe 165 170 175 gca aaa aaa tta gtc gaa ata
cgc aat cgg gag gtg aaa cga ccg gac 576 Ala Lys Lys Leu Val Glu Ile
Arg Asn Arg Glu Val Lys Arg Pro Asp 180 185 190 gat atc aag cga ttc
aag aaa tga 600 Asp Ile Lys Arg Phe Lys Lys 195 30 199 PRT
Lactobacillus reuteri 30 Met Ser Lys Trp Ile Lys Gln Phe Leu Leu
Ala Gly Leu Ala Gly Leu 1 5 10 15 Ile Arg Pro Lys His Asn Gln Lys
Tyr Ser Leu Glu Thr Lys Leu Thr 20 25 30 Ala Val Lys Ala Tyr Leu
Ser Gly Lys Tyr Thr Asn Gln Ala Ile Leu 35 40 45 Gln Gln Tyr Gln
Ile Arg Asn Ile Ser Gln Leu His Gln Trp Val Ile 50 55 60 Ser Tyr
Asn Asn Asp Lys Leu Arg Val Asn Gln Thr Thr Arg Lys Arg 65 70 75 80
Val Arg Lys Met Gly Arg Lys Val Thr Phe Asp Glu Lys Arg Gln Ile 85
90 95 Val Arg Trp Thr Ile Glu His Asn Asn Asn Tyr Lys Ala Ala Ala
Glu 100 105 110 Lys Tyr Asp Ile Ser Tyr Gln Arg Val Tyr Ser Trp Val
Arg Lys Tyr 115 120 125 Arg Val Asn Ser Asp Trp Glu Val Leu Lys Asp
Asn Arg Gly Arg Asn 130 135 140 Lys Gly Lys Glu Pro Thr Asn Glu Leu
Glu Lys Leu Arg Lys Arg Val 145 150 155 160 Arg Glu Leu Glu Asp Arg
Asp Arg Glu Arg Glu Leu Gln Ile Ala Phe 165 170 175 Ala Lys Lys Leu
Val Glu Ile Arg Asn Arg Glu Val Lys Arg Pro Asp 180 185 190 Asp Ile
Lys Arg Phe Lys Lys 195 31 849 DNA Lactobacillus reuteri CDS
(1)..(849) 31 atg aac aat gaa ggt tat tcc att agt gaa ttg gcc aag
gtc gct gga 48 Met Asn Asn Glu Gly Tyr Ser Ile Ser Glu Leu Ala Lys
Val Ala Gly 1 5 10 15 att act aga cag gct tac tac aaa tgg ttg aaa
cat gaa ccg act aaa 96 Ile Thr Arg Gln Ala Tyr Tyr Lys Trp Leu Lys
His Glu Pro Thr Lys 20 25 30 tat gag att gaa gaa tcg gag att ctc
caa ttg att aaa cag tta gaa 144 Tyr Glu Ile Glu Glu Ser Glu Ile Leu
Gln Leu Ile Lys Gln Leu Glu 35 40 45 aat gaa cat aag caa agc gtt
ggt tat gac aaa atg act agg tta atc 192 Asn Glu His Lys Gln Ser Val
Gly Tyr Asp Lys Met Thr Arg Leu Ile 50 55 60 aag tta agt cag cag
atc tct tat acc gtt aat aag aaa cga gtc att 240 Lys Leu Ser Gln Gln
Ile Ser Tyr Thr Val Asn Lys Lys Arg Val Ile 65 70 75 80 cgt att atg
aaa ggc cat agt atc aag gcc gac tat cgt cag cca acc 288 Arg Ile Met
Lys Gly His Ser Ile Lys Ala Asp Tyr Arg Gln Pro Thr 85 90 95 gac
aaa cgt att caa gcc cag caa act tat gaa gct gaa aat att ctt 336 Asp
Lys Arg Ile Gln Ala Gln Gln Thr Tyr Glu Ala Glu Asn Ile Leu 100 105
110 aac cga caa ttt gac caa act gca gct aac caa gtt tgg gtt acg gat
384 Asn Arg Gln Phe Asp Gln Thr Ala Ala Asn Gln Val Trp Val Thr Asp
115 120 125 acg acg gaa ctg aat tac gga atc tgg ctt aat aaa gtt cgt
cta cat 432 Thr Thr Glu Leu Asn Tyr Gly Ile Trp Leu Asn Lys Val Arg
Leu His 130 135 140 ata gta tta gat tta tat ggt caa tac cca gta agc
tgg tta att aca 480 Ile Val Leu Asp Leu Tyr Gly Gln Tyr Pro Val Ser
Trp Leu Ile Thr 145 150 155 160 cct aca gaa acc gct gaa gga gta gtt
caa gtg ttc gag caa gca cgg 528 Pro Thr Glu Thr Ala Glu Gly Val Val
Gln Val Phe Glu Gln Ala Arg 165 170 175 atg aaa gaa gga gca cta gct
ccg tta att cat act gat cgt ggt gcg 576 Met Lys Glu Gly Ala Leu Ala
Pro Leu Ile His Thr Asp Arg Gly Ala 180 185 190 gcg tat act tcc aaa
gca ttt aat cag tat tta gta gtt aat ggt gcc 624 Ala Tyr Thr Ser Lys
Ala Phe Asn Gln Tyr Leu Val Val Asn Gly Ala 195 200 205 caa cac agt
tat tca gca cca ggg aca ccg gct gac aat gcc gta ata 672 Gln His Ser
Tyr Ser Ala Pro Gly Thr Pro Ala Asp Asn Ala Val Ile 210 215 220 gaa
cat tgg tgg gca gat ttt aag gct att tgg atc gca cat cta cct 720 Glu
His Trp Trp Ala Asp Phe Lys Ala Ile Trp Ile Ala His Leu Pro 225 230
235 240 aaa gca caa aca tta tta gaa cta gaa gaa caa gtt aga gaa gga
att 768 Lys Ala Gln Thr Leu Leu Glu Leu Glu Glu Gln Val Arg Glu Gly
Ile 245 250 255 acc tat ttc act gaa aaa ttt atc tca gcg aag aga aat
gac ctt acc 816 Thr Tyr Phe Thr Glu Lys Phe Ile Ser Ala Lys Arg Asn
Asp Leu Thr 260 265 270 gca gcg gaa tac cgc ttt ggc aag gcc aac taa
849 Ala Ala Glu Tyr Arg Phe Gly Lys Ala Asn 275 280 32 282 PRT
Lactobacillus reuteri 32 Met Asn Asn Glu Gly Tyr Ser Ile Ser Glu
Leu Ala Lys Val Ala Gly 1 5 10 15 Ile Thr Arg Gln Ala Tyr Tyr Lys
Trp Leu Lys His Glu Pro Thr Lys 20 25 30 Tyr Glu Ile Glu Glu Ser
Glu Ile Leu Gln Leu Ile Lys Gln Leu Glu 35 40 45 Asn Glu His Lys
Gln Ser Val Gly Tyr Asp Lys Met Thr Arg Leu Ile 50 55 60 Lys Leu
Ser Gln Gln Ile Ser Tyr Thr Val Asn Lys Lys Arg Val Ile 65 70 75 80
Arg Ile Met Lys Gly His Ser Ile Lys Ala Asp Tyr Arg Gln Pro Thr 85
90 95 Asp Lys Arg Ile Gln Ala Gln Gln Thr Tyr Glu Ala Glu Asn Ile
Leu 100 105 110 Asn Arg Gln Phe Asp Gln Thr Ala Ala Asn Gln Val Trp
Val Thr Asp 115 120 125 Thr Thr Glu Leu Asn Tyr Gly Ile Trp Leu Asn
Lys Val Arg Leu His 130 135 140 Ile Val Leu Asp Leu Tyr Gly Gln Tyr
Pro Val Ser Trp Leu Ile Thr 145 150 155 160 Pro Thr Glu Thr Ala Glu
Gly Val Val Gln Val Phe Glu Gln Ala Arg 165 170 175 Met Lys Glu Gly
Ala Leu Ala Pro Leu Ile His Thr Asp Arg Gly Ala 180 185 190 Ala Tyr
Thr Ser Lys Ala Phe Asn Gln Tyr Leu Val Val Asn Gly Ala 195 200 205
Gln His Ser Tyr Ser Ala Pro Gly Thr Pro Ala Asp Asn Ala Val Ile 210
215 220 Glu His Trp Trp Ala Asp Phe Lys Ala Ile Trp Ile Ala His Leu
Pro 225 230 235 240 Lys Ala Gln Thr Leu Leu Glu Leu Glu Glu Gln Val
Arg Glu Gly Ile 245 250 255 Thr Tyr Phe Thr Glu Lys Phe Ile Ser Ala
Lys Arg Asn Asp Leu Thr 260 265 270 Ala Ala Glu Tyr Arg Phe Gly Lys
Ala Asn 275 280 33 744 DNA Lactobacillus reuteri CDS (1)..(744)
unsure (12) unsure (297) misc_feature (10)..(12) Xaa = Tyr or stop
33 atg ctt cgt tan acc ata tta gta aaa ttg ctt att gga aga aaa cca
48 Met Leu Arg Xaa Thr Ile Leu Val Lys Leu Leu Ile Gly Arg Lys Pro
1 5 10 15 gtc aca acg atc aaa aaa aca tta ccg cca act cag gaa cag
gct aat 96 Val Thr Thr Ile Lys Lys Thr Leu Pro Pro Thr Gln Glu Gln
Ala Asn 20 25 30 tca gtc tta act ccg gct gtt cgc caa caa ctt ggc
att tca att acc 144 Ser Val Leu Thr Pro Ala Val Arg Gln Gln Leu Gly
Ile Ser Ile Thr 35 40 45 tgg aac aaa gcc ggt gcg ttt att atc aat
aat aac caa aca aat ctt 192 Trp Asn Lys Ala Gly Ala Phe Ile Ile Asn
Asn Asn Gln Thr Asn Leu 50 55 60 aac gct aag att gca agt gca ccc
tat gct gta aat cat ctt gac cgt 240 Asn Ala Lys Ile Ala Ser Ala Pro
Tyr Ala Val Asn His Leu Asp Arg 65 70 75 80 caa gga agg gcg tgg caa
ggt gat gcc tgg tta aac agg aca act cgg 288 Gln Gly Arg Ala Trp Gln
Gly Asp Ala Trp Leu Asn Arg Thr Thr Arg 85 90 95 tca ata tan aag
ccg aaa ttt gcc aca ggg aat ggt gct acg gat tgg 336 Ser Ile Xaa Lys
Pro Lys Phe Ala Thr Gly Asn Gly Ala Thr Asp Trp 100 105 110 cga cca
gct ggc ttc ctt cag gcg cat aat ctt aaa ggc ggg tac aat 384 Arg Pro
Ala Gly Phe Leu Gln Ala His Asn Leu Lys Gly Gly Tyr Asn 115 120 125
cat gca tac gat cgc gga cac ctt ctt gcc tat gca cta gtt ggt ggt 432
His Ala Tyr Asp Arg Gly His Leu Leu Ala Tyr Ala Leu Val Gly Gly 130
135 140 att cat gga ttt gat gca tcc gaa tca aat cca tct aat att gcc
acg 480 Ile His Gly Phe Asp Ala Ser Glu Ser Asn Pro Ser Asn Ile Ala
Thr 145 150 155 160 caa act gcc tgg gca aat gaa gca cga agt aag aac
tca aca ggg caa 528 Gln Thr Ala Trp Ala Asn Glu Ala Arg Ser Lys Asn
Ser Thr Gly Gln 165 170 175 aat tac tac gaa ggt ctg gtg aga aaa gca
tta gat cag aat aag caa 576 Asn Tyr Tyr Glu Gly Leu Val Arg Lys Ala
Leu Asp Gln Asn Lys Gln 180 185 190 gtt cgc tac cga gtt acc aat att
tat gac ggt aat aat atc gtt ccg 624 Val Arg Tyr Arg Val Thr Asn Ile
Tyr Asp Gly Asn Asn Ile Val Pro 195 200 205 gca ggt gct cat atc gaa
gct aaa tct agt gat ggt tct cta gaa tac 672 Ala Gly Ala His Ile Glu
Ala Lys Ser Ser Asp Gly Ser Leu Glu Tyr 210 215 220 aat gtc ttt gtt
ccg aat gtc caa aga aac att acc att aat tat tca 720 Asn Val Phe Val
Pro Asn Val Gln Arg Asn Ile Thr Ile Asn Tyr Ser 225 230 235 240 acc
ggt gca gta aaa caa aac taa 744 Thr Gly Ala Val Lys Gln Asn 245 34
247 PRT Lactobacillus reuteri misc_feature 4 Xaa = Tyr or stop 34
Met Leu Arg Xaa Thr Ile Leu Val Lys Leu Leu Ile Gly Arg Lys Pro 1 5
10 15 Val Thr Thr Ile Lys Lys Thr Leu Pro Pro Thr Gln Glu Gln Ala
Asn 20 25 30 Ser Val Leu Thr Pro Ala Val Arg Gln Gln Leu Gly Ile
Ser Ile Thr 35 40 45 Trp Asn Lys Ala Gly Ala Phe Ile Ile Asn Asn
Asn Gln Thr Asn Leu 50 55 60 Asn Ala Lys Ile Ala Ser Ala Pro Tyr
Ala Val Asn His Leu Asp Arg 65 70 75 80 Gln Gly Arg Ala Trp Gln Gly
Asp Ala Trp Leu Asn Arg Thr Thr Arg 85 90 95 Ser Ile Xaa Lys Pro
Lys Phe Ala Thr Gly Asn Gly Ala Thr Asp Trp 100 105 110 Arg Pro Ala
Gly Phe Leu Gln Ala His Asn Leu Lys Gly Gly Tyr Asn 115 120 125 His
Ala Tyr Asp Arg Gly His Leu Leu Ala Tyr Ala Leu Val Gly Gly 130 135
140 Ile His Gly Phe Asp Ala Ser Glu Ser Asn Pro Ser Asn Ile Ala Thr
145 150
155 160 Gln Thr Ala Trp Ala Asn Glu Ala Arg Ser Lys Asn Ser Thr Gly
Gln 165 170 175 Asn Tyr Tyr Glu Gly Leu Val Arg Lys Ala Leu Asp Gln
Asn Lys Gln 180 185 190 Val Arg Tyr Arg Val Thr Asn Ile Tyr Asp Gly
Asn Asn Ile Val Pro 195 200 205 Ala Gly Ala His Ile Glu Ala Lys Ser
Ser Asp Gly Ser Leu Glu Tyr 210 215 220 Asn Val Phe Val Pro Asn Val
Gln Arg Asn Ile Thr Ile Asn Tyr Ser 225 230 235 240 Thr Gly Ala Val
Lys Gln Asn 245 35 1540 DNA Lactobacillus reuteri CDS (1)..(1539)
35 atg gtc atg aca gaa act gct ggt ata aga aaa att cat att gtt ttt
48 Met Val Met Thr Glu Thr Ala Gly Ile Arg Lys Ile His Ile Val Phe
1 5 10 15 gat ggt caa gaa aca cca cca tta aag atc cat caa tta ttt
gat tca 96 Asp Gly Gln Glu Thr Pro Pro Leu Lys Ile His Gln Leu Phe
Asp Ser 20 25 30 caa aaa tac gat cag tta atc gca gta act ggg aaa
att act gct gac 144 Gln Lys Tyr Asp Gln Leu Ile Ala Val Thr Gly Lys
Ile Thr Ala Asp 35 40 45 ttc att aat aaa tac ctt agt aat ttt atc
agt att aat gta gcg tta 192 Phe Ile Asn Lys Tyr Leu Ser Asn Phe Ile
Ser Ile Asn Val Ala Leu 50 55 60 agc tcc caa tca act agt gaa tta
agt gct gat gag atg gtg aca aag 240 Ser Ser Gln Ser Thr Ser Glu Leu
Ser Ala Asp Glu Met Val Thr Lys 65 70 75 80 gtt gca ctt acc aat gct
ctc ctt agt tca gca aat aaa gaa gct gct 288 Val Ala Leu Thr Asn Ala
Leu Leu Ser Ser Ala Asn Lys Glu Ala Ala 85 90 95 aaa ctc ttc tca
gcg tta acc agt gac aac caa acg aac gtc tta aat 336 Lys Leu Phe Ser
Ala Leu Thr Ser Asp Asn Gln Thr Asn Val Leu Asn 100 105 110 aat ctt
ttt cgc gta tca atc gcg cct act cag gtt atc cat tct aag 384 Asn Leu
Phe Arg Val Ser Ile Ala Pro Thr Gln Val Ile His Ser Lys 115 120 125
ttt tac ttg tta agt agt tca act act cat gat tcc cgt gtg att ctt 432
Phe Tyr Leu Leu Ser Ser Ser Thr Thr His Asp Ser Arg Val Ile Leu 130
135 140 ggg agt gta gat tta gac gaa gct tca ttt gat gct cac cga aat
caa 480 Gly Ser Val Asp Leu Asp Glu Ala Ser Phe Asp Ala His Arg Asn
Gln 145 150 155 160 ttt gaa gaa gta ttg gta ttt gac aat gat gtc cgc
tta tac caa aac 528 Phe Glu Glu Val Leu Val Phe Asp Asn Asp Val Arg
Leu Tyr Gln Asn 165 170 175 ctt act gac cac ttt aaa aag gat ttt aag
cca gta ttg aag ccc ttc 576 Leu Thr Asp His Phe Lys Lys Asp Phe Lys
Pro Val Leu Lys Pro Phe 180 185 190 ttt act atg aac cta gta aag gca
gct caa aag caa gtt gag gaa gga 624 Phe Thr Met Asn Leu Val Lys Ala
Ala Gln Lys Gln Val Glu Glu Gly 195 200 205 aag aaa gat cag gat agc
ggt aag gga ccg gtt atc ctt gat aat gaa 672 Lys Lys Asp Gln Asp Ser
Gly Lys Gly Pro Val Ile Leu Asp Asn Glu 210 215 220 aca aca gat aag
atc gct gaa aca gac atg gtg gat ctg ttg aag cat 720 Thr Thr Asp Lys
Ile Ala Glu Thr Asp Met Val Asp Leu Leu Lys His 225 230 235 240 gac
ctt cag cat gat att gac cat aat ctt gtt cct gaa atg atc aca 768 Asp
Leu Gln His Asp Ile Asp His Asn Leu Val Pro Glu Met Ile Thr 245 250
255 aag tca atg cgt gat att acc ata aat cgt tct caa gca aag gag aaa
816 Lys Ser Met Arg Asp Ile Thr Ile Asn Arg Ser Gln Ala Lys Glu Lys
260 265 270 att gct aag cag gtt aag caa cat gat acg att tat act ttg
caa aaa 864 Ile Ala Lys Gln Val Lys Gln His Asp Thr Ile Tyr Thr Leu
Gln Lys 275 280 285 gaa gcg gtc tct cct cgg gca gct aag cca aaa cta
aag act cga gaa 912 Glu Ala Val Ser Pro Arg Ala Ala Lys Pro Lys Leu
Lys Thr Arg Glu 290 295 300 aaa att acc aag cag gtt cag gat gct ttg
atc agt gga atg tca cca 960 Lys Ile Thr Lys Gln Val Gln Asp Ala Leu
Ile Ser Gly Met Ser Pro 305 310 315 320 cag caa cgg gat gct gag aaa
aag tac acg act ttt ctg tac gat cgg 1008 Gln Gln Arg Asp Ala Glu
Lys Lys Tyr Thr Thr Phe Leu Tyr Asp Arg 325 330 335 cca atg gaa cga
aac att gcg aat aac aat agt ggc cta tac gtt cct 1056 Pro Met Glu
Arg Asn Ile Ala Asn Asn Asn Ser Gly Leu Tyr Val Pro 340 345 350 aat
gat acg gga act cac cca atc cca ttt ggt aaa att gca act att 1104
Asn Asp Thr Gly Thr His Pro Ile Pro Phe Gly Lys Ile Ala Thr Ile 355
360 365 tct gaa att cgt gac ggt tta aag agc att gat gct gtt atg aag
ggc 1152 Ser Glu Ile Arg Asp Gly Leu Lys Ser Ile Asp Ala Val Met
Lys Gly 370 375 380 tat cag cag ttt gtc gtt gat tat gat gct gac tac
ggg aag cgg ttc 1200 Tyr Gln Gln Phe Val Val Asp Tyr Asp Ala Asp
Tyr Gly Lys Arg Phe 385 390 395 400 ttt gaa gca att ttg tat agt ttt
act gca ccg ttt tta tgg gaa att 1248 Phe Glu Ala Ile Leu Tyr Ser
Phe Thr Ala Pro Phe Leu Trp Glu Ile 405 410 415 cgt tct aaa gct agc
ctg aac cct gaa gat ggg aat gat gtt cct aat 1296 Arg Ser Lys Ala
Ser Leu Asn Pro Glu Asp Gly Asn Asp Val Pro Asn 420 425 430 ttc cta
atc cta ggg gca acg gct ggt tcc gga aag tct acc ctt ctt 1344 Phe
Leu Ile Leu Gly Ala Thr Ala Gly Ser Gly Lys Ser Thr Leu Leu 435 440
445 cgg att att aat cag ctc acg tgg aac act gat cgc tcg ttg att gac
1392 Arg Ile Ile Asn Gln Leu Thr Trp Asn Thr Asp Arg Ser Leu Ile
Asp 450 455 460 ttt gga acg atc tac ccg tcg caa act cct caa aag aag
gca aag act 1440 Phe Gly Thr Ile Tyr Pro Ser Gln Thr Pro Gln Lys
Lys Ala Lys Thr 465 470 475 480 gtt gag gcg atg gaa cat tat atg aaa
ctt ggt agt tca tac ccg gtt 1488 Val Glu Ala Met Glu His Tyr Met
Lys Leu Gly Ser Ser Tyr Pro Val 485 490 495 ttg tta gat gaa att gaa
ccg tac ttc ttc cag caa gat caa tat agt 1536 Leu Leu Asp Glu Ile
Glu Pro Tyr Phe Phe Gln Gln Asp Gln Tyr Ser 500 505 510 cga c 1540
Arg 36 513 PRT Lactobacillus reuteri 36 Met Val Met Thr Glu Thr Ala
Gly Ile Arg Lys Ile His Ile Val Phe 1 5 10 15 Asp Gly Gln Glu Thr
Pro Pro Leu Lys Ile His Gln Leu Phe Asp Ser 20 25 30 Gln Lys Tyr
Asp Gln Leu Ile Ala Val Thr Gly Lys Ile Thr Ala Asp 35 40 45 Phe
Ile Asn Lys Tyr Leu Ser Asn Phe Ile Ser Ile Asn Val Ala Leu 50 55
60 Ser Ser Gln Ser Thr Ser Glu Leu Ser Ala Asp Glu Met Val Thr Lys
65 70 75 80 Val Ala Leu Thr Asn Ala Leu Leu Ser Ser Ala Asn Lys Glu
Ala Ala 85 90 95 Lys Leu Phe Ser Ala Leu Thr Ser Asp Asn Gln Thr
Asn Val Leu Asn 100 105 110 Asn Leu Phe Arg Val Ser Ile Ala Pro Thr
Gln Val Ile His Ser Lys 115 120 125 Phe Tyr Leu Leu Ser Ser Ser Thr
Thr His Asp Ser Arg Val Ile Leu 130 135 140 Gly Ser Val Asp Leu Asp
Glu Ala Ser Phe Asp Ala His Arg Asn Gln 145 150 155 160 Phe Glu Glu
Val Leu Val Phe Asp Asn Asp Val Arg Leu Tyr Gln Asn 165 170 175 Leu
Thr Asp His Phe Lys Lys Asp Phe Lys Pro Val Leu Lys Pro Phe 180 185
190 Phe Thr Met Asn Leu Val Lys Ala Ala Gln Lys Gln Val Glu Glu Gly
195 200 205 Lys Lys Asp Gln Asp Ser Gly Lys Gly Pro Val Ile Leu Asp
Asn Glu 210 215 220 Thr Thr Asp Lys Ile Ala Glu Thr Asp Met Val Asp
Leu Leu Lys His 225 230 235 240 Asp Leu Gln His Asp Ile Asp His Asn
Leu Val Pro Glu Met Ile Thr 245 250 255 Lys Ser Met Arg Asp Ile Thr
Ile Asn Arg Ser Gln Ala Lys Glu Lys 260 265 270 Ile Ala Lys Gln Val
Lys Gln His Asp Thr Ile Tyr Thr Leu Gln Lys 275 280 285 Glu Ala Val
Ser Pro Arg Ala Ala Lys Pro Lys Leu Lys Thr Arg Glu 290 295 300 Lys
Ile Thr Lys Gln Val Gln Asp Ala Leu Ile Ser Gly Met Ser Pro 305 310
315 320 Gln Gln Arg Asp Ala Glu Lys Lys Tyr Thr Thr Phe Leu Tyr Asp
Arg 325 330 335 Pro Met Glu Arg Asn Ile Ala Asn Asn Asn Ser Gly Leu
Tyr Val Pro 340 345 350 Asn Asp Thr Gly Thr His Pro Ile Pro Phe Gly
Lys Ile Ala Thr Ile 355 360 365 Ser Glu Ile Arg Asp Gly Leu Lys Ser
Ile Asp Ala Val Met Lys Gly 370 375 380 Tyr Gln Gln Phe Val Val Asp
Tyr Asp Ala Asp Tyr Gly Lys Arg Phe 385 390 395 400 Phe Glu Ala Ile
Leu Tyr Ser Phe Thr Ala Pro Phe Leu Trp Glu Ile 405 410 415 Arg Ser
Lys Ala Ser Leu Asn Pro Glu Asp Gly Asn Asp Val Pro Asn 420 425 430
Phe Leu Ile Leu Gly Ala Thr Ala Gly Ser Gly Lys Ser Thr Leu Leu 435
440 445 Arg Ile Ile Asn Gln Leu Thr Trp Asn Thr Asp Arg Ser Leu Ile
Asp 450 455 460 Phe Gly Thr Ile Tyr Pro Ser Gln Thr Pro Gln Lys Lys
Ala Lys Thr 465 470 475 480 Val Glu Ala Met Glu His Tyr Met Lys Leu
Gly Ser Ser Tyr Pro Val 485 490 495 Leu Leu Asp Glu Ile Glu Pro Tyr
Phe Phe Gln Gln Asp Gln Tyr Ser 500 505 510 Arg 37 26 DNA
Lactobacillus reuteri terminator (1)..(26) 37 aaagaagctg aaatttcggc
ttcttt 26 38 28 DNA Lactobacillus reuteri misc_feature (1)..(28)
PRIMER 38 gcagtcgacg gagttaagac tgaattag 28 39 26 DNA Lactobacillus
reuteri misc_feature (1)..(26) PRIMER 39 ctagtcgacg cagtttctgt
catgac 26 40 32 DNA Lactobacillus reuteri misc_feature (1)..(32)
PRIMER 40 catatgtatt attcaaacgg gaattatgaa gc 32 41 30 DNA
Lactobacillus reuteri misc_feature (1)..(30) PRIMER 41 tgatcatcta
taccagcagt ttctgtcatg 30
* * * * *