U.S. patent application number 10/755087 was filed with the patent office on 2005-01-06 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..
Application Number | 20050003383 10/755087 |
Document ID | / |
Family ID | 26839458 |
Filed Date | 2005-01-06 |
United States Patent
Application |
20050003383 |
Kind Code |
A1 |
Rosson, Reinhardt A. ; et
al. |
January 6, 2005 |
Linoleate isomerase
Abstract
The present invention provides an isolated
(trans,cis)-10,12-linoleate isomerase and its nucleic acid and
amino acid sequences. The present invention also provides a method
for producing conjugated linoleic acid or conjugated linolenic acid
(CLA), or derivatives thereof, from an oil using an immobilized
cell and/or an isolated linoleate isomerase. The present invention
also provides an isolated lipase-like protein and its nucleic acid
and amino acid sequences. The present invention also provides an
isolated acetyltransferase-like enzyme and its nucleic acid and
amino acid sequences.
Inventors: |
Rosson, Reinhardt A.;
(Manitowoc, WI) ; Deng, Ming-De; (Manitowoc,
WI) ; Grund, Alan D.; (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: |
26839458 |
Appl. No.: |
10/755087 |
Filed: |
January 9, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10755087 |
Jan 9, 2004 |
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09561077 |
Apr 28, 2000 |
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6706501 |
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60141798 |
Jun 30, 1999 |
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Current U.S.
Class: |
435/134 ;
435/233; 435/252.3; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12N 9/90 20130101; C12P
7/6427 20130101; C12N 9/1029 20130101; C12N 15/52 20130101; C12P
7/6472 20130101; C12N 9/18 20130101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/134; 435/233; 435/252.3; 536/023.2 |
International
Class: |
C12Q 001/68; C07H
021/04; C12P 007/64; C12N 009/90 |
Claims
1. An isolated protein, comprising an amino acid sequence selected
from the group consisting of: a. an amino acid sequence selected
from the group consisting of SEQ ID NO:42 and SEQ ID NO:61; and, b.
a homologue of said amino acid sequence of (a), wherein said
homologue is at least about 35% identical to SEQ ID NO:61 over at
least about 170 contiguous amino acids of SEQ ID NO:61; wherein
said protein has 10,12-linoleate isomerase enzymatic activity.
2-10. (Cancelled)
11. The isolated protein of claim 1, wherein said protein comprises
an amino acid sequence selected from the group consisting of SEQ ID
NO:42 and SEQ ID NO:61.
12. The isolated protein of claim 1, wherein said protein comprises
an amino acid sequence SEQ ID NO:61.
13. The isolated protein of claim 1, wherein said protein is
selected from the group consisting of Propionibacterium acnes,
Propionibacterium acidipropionici and Propionibacterium
freudenreichii linoleate isomerases.
14. (Cancelled)
15. The isolated protein of claim 1, wherein said linoleate
isomerase converts linoleic acid and linolenic acid to (trans,
cis)-10,12-linoleic acid.
16. The isolated protein of claim 1, wherein said protein has a
specific linoleic acid isomerization activity of at least about 10
nmoles CLA mg.sup.-1 min.sup.-1.
17-18. (Cancelled)
19. The isolated protein of claim 1, wherein said protein is a
soluble enzyme.
20. The isolated protein of claim 1, wherein said protein comprises
a leader sequence which causes insertion of said protein into the
membrane of a cell which expresses said protein.
21. The isolated protein of claim 1, wherein said protein is bound
to a solid support.
22-23. (Cancelled)
24. An isolated antibody that selectively binds to the isolated
protein of claim 1.
25. An isolated protein comprising an amino acid sequence selected
from the group consisting of: a. an amino acid sequence selected
from the group consisting of SEQ ID NO:42 and SEQ ID NO:61; and, b.
a homologue of said amino acid sequence of (a), wherein said
homologue comprises an amino acid sequence that aligns with SEQ ID
NO:73 using Martinez/Needleman-Wunsch DNA alignment method with a
minimum match of 9, a gap penalty of 1.10 and a gap length penalty
of 0.33, wherein amino acid residues in said amino acid sequence
align with at least about 70% of non-Xaa residues in SEQ ID NO:73;
wherein said protein has 10,12-linoleate isomerase enzymatic
activity.
26. (Cancelled)
27. A method for producing CLA (conjugated linoleic acid or
conjugated linolenic acid) or derivatives thereof, comprising
contacting an oil, said oil comprising a fatty acid selected from
the group consisting of linoleic acid, linolenic acid, and a
derivative of linoleic or linolenic acid, with an isolated protein
having 10,12-linoleate isomerase enzymatic activity, to convert at
least a portion of said fatty acid to CLA or a derivative thereof,
said isolated protein comprising an amino acid sequence selected
from the group consisting of: a. an amino acid sequence selected
from the group consisting of SEQ ID NO:42 and SEQ ID NO:61; and, b.
a homologue of said amino acid sequence of (a), wherein said
homologue is at least about 35% identical to SEQ ID NO:61 over at
least about 170 contiguous amino acids of SEQ ID NO:61.
28-31. (Cancelled)
32. The method of claim 27, wherein said protein comprises an amino
acid sequence selected from the group consisting of SEQ ID NO:42
and SEQ ID NO:61.
33. The method of claim 27, wherein said protein comprises amino
acid sequence SEQ ID NO:61.
34. The method of claim 27, wherein said fatty acid is in the form
of a triglyceride and wherein said method further comprises
contacting said oil with a hydrolysis enzyme to convert at least a
portion of said triglyceride to free fatty acids.
35. The method of claim 34, wherein said hydrolysis enzyme is
selected from the group consisting of lipases, phospholipases and
esterases.
36. The method of claim 27, further comprising the step of
recovering said CLA or derivative thereof.
37. The method of claim 27, wherein said 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.
38. The method of claim 27, wherein said linoleate isomerase enzyme
is bound to a solid support.
39. The method of claim 38, wherein said solid support is selected
from the group consisting of organic supports, artificial
membranes, biopolymer supports and inorganic supports.
40-116. (Cancelled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C. .sctn.
119(e) to U.S. Provisional Application Ser. No. 60/141,798, filed
Jun. 30, 1999. The entire disclosure of U.S. Provisional
Application Ser. No. 60/141,798 is incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates to an isolated
(trans,cis)-10,12-linoleate isomerase enzyme, to a nucleic acid
molecule encoding a (trans,cis)-10,12-linoleate isomerase enzyme,
to immobilized cells containing a linoleate isomerase enzyme, to an
immobilized (trans,cis)-10,12-linoleate isomerase enzyme, and to a
method for converting linoleic acid or linolenic acid to CLA or
derivatives thereof 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. U.S. Pat. No. 6,015,833,
issued Jan. 18, 2000, to S.ae butted.b.o slashed. et al. describes
the chemical production of CLA compositions from seed oils with a
total CLA content of at least 50%, and with less than 1%
contaminating octadecadienoic acid isomers. 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. The above-described methods, however, do not typically
allow for the production of high purity, single isomer CLA, and if
single isomer production is achieved on a large scale level, such a
process is expected to be expensive.
[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 enzyme;
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] Another research group, Park and colleagues, published an
article in J. Food Science Nutrition (Vol. 1: 244-251, 1996),
describing the purification of a protein which Park et al. believed
to be the Butyrivibrio fibrisolvens linoleate isomerase. However,
based on the initial characterization of the enzyme's activity by
Kepler and Tove (see above) and the present inventors'
purification, sequencing and characterization of three demonstrated
linoleate isomerases, the present inventors believe that it is very
unlikely that the protein that was purified and described by Park
et al. is actually a linoleate isomerase. More particularly, it is
well established in the art that for successful purification of
particulate enzymes, such enzymes must first be converted into a
soluble form. Although Park et al. demonstrate that the
Butyrivibrio fibrisolvens linoleic acid isomerase is membrane
bound, Park et al. describe no such solubilization of the enzyme.
Instead, an isolated protein pellet was simply resuspended in
phosphate buffer, a procedure that will generally not solubilize
any membrane protein, and therefore raises significant doubts about
the described purification, particularly in view of previously
described purification attempts by Kepler and Tove (J. Biol. Chem.
242:5686-5692, 1967). Indeed, as discussed above, Kepler and Tove
had described their extensive but unsuccessful efforts using well
accepted solubilization methods (e.g., chelators, organic solvents,
high salt, detergents) to attempt to solubilize the isomerase.
Furthermore, in contrast to the 19 kD molecular weight of the
putative isomerase that was eventually reported by Park et al., the
main isomerase activity eluted quite early from the column during
purification, indicating an apparent molecular weight of several
hundred kD, and not 19 kD. When this initial material was applied
to a phenyl sepharose 4B column, multiple broad peaks of activity
were observed. This is not typical, and again indicates that the
isomerase preparation was heterogeneous, had not been solubilized
properly, and was undoubtedly associated with other membrane
proteins. One of these activity peaks was then applied to a
Superose 6 gel filtration column, yielding a single 19 kD band on
gel electrophoresis. Finally, this sample was assayed by Park et
al. for isomerase activity using HPLC, which is not appropriate for
detection of CLA, since it does not resolve the various positional
isomers. The retention time shown for the standard CLA was
significantly different than the retention time for the putative
CLA formed from linoleic acid using the 19 kD putative linoleate
isomerase, and should have lead Park et al. to the firm conclusion
that the peak was not CLA, but something else. Therefore, the
present inventors believe that the data presented by Park et al.
does not support the conclusion that a linoleate isomerase had been
purified.
[0010] 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
[0011] 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.
[0012] One embodiment of the invention relates to an isolated
10,12-linoleate isomerase. Included in the invention are linoleate
isomerases from Propionibacterium, and particularly, from
Propionibacterium acnes, Propionibacterium acidipropionici, and
Propionibacterium freudenreichii. Particularly preferred linoleate
isomerases include linoleate isomerases from Propionibacterium
acnes. In one embodiment, an isolated linoleate isomerase of the
present invention converts linoleic acid and linolenic acid to CLA,
including (trans, cis)-10,12-linoleic acid. In one embodiment, the
protein has a specific linoleic acid isomerization activity of at
least about 10 nmoles CLA mg.sup.-1 min.sup.-1.
[0013] One embodiment of the present invention relates to an
isolated protein, comprising an amino acid sequence selected from
the group of: (a) an amino acid sequence selected from the group of
SEQ ID NO:42 and SEQ ID NO:61; and, (b) a homologue of the amino
acid sequence of (a), wherein the homologue is at least about 35%
identical to SEQ ID NO:61 over at least about 170 contiguous amino
acids of SEQ ID NO:61. In this embodiment, the protein
10,12-linoleate isomerase enzymatic activity. In one embodiment,
the protein is encoded by a nucleic acid molecule comprising a
nucleic acid sequence that hybridizes under low, moderate, or high
stringency hybridization conditions to the complement of SEQ ID
NO:60. In another embodiment, the protein comprises an amino acid
sequence comprising at least 15 contiguous amino acids of SEQ ID
NO:61, and more preferably, at least 30 contiguous amino acids of
SEQ ID NO:61, and even more preferably, at least 45 contiguous
amino acids of SEQ ID NO:61. In one embodiment, the protein is
encoded by a nucleic acid molecule comprising a nucleic acid
sequence comprising at least 24 contiguous nucleotides of SEQ ID
NO:60. In a preferred embodiment, the protein is encoded by a
nucleic acid molecule comprising a nucleic acid sequence selected
from the group consisting of SEQ ID NO:59 and SEQ ID NO:60, with
SEQ ID NO:60 being most preferred. In another preferred embodiment,
the protein comprises an amino acid sequence selected from the
group consisting of SEQ ID NO:42 and SEQ ID NO:61, with SEQ ID
NO:61 being most preferred. In another embodiment, the protein
comprises an amino acid sequence that aligns with SEQ ID NO:73
using Martinez/Needleman-Wunsch DNA alignment method with a minimum
match of 9, a gap penalty of 1.10 and a gap length penalty of 0.33,
wherein amino acid residues in the amino acid sequence align with
at least about 70%, and in another embodiment, with at least about
90%, of non-Xaa residues in SEQ ID NO:73.
[0014] In one embodiment, the protein is a soluble enzyme. In
another embodiment, the protein comprises a leader sequence which
causes insertion of the protein into the membrane of a cell which
expresses the protein. In one embodiment, the linoleate isomerase
is bound to a solid support, which includes, but is not limited to
artificial membranes, organic supports, biopolymer supports and
inorganic supports.
[0015] Another embodiment of the present invention relates to an
isolated antibody that selectively binds to the isolated linoleate
isomerase of the present invention.
[0016] Yet another embodiment of the present invention relates to a
method for producing CLA or derivatives thereof, including
contacting an oil, which comprises a compound selected from the
group of linoleic acid, linolenic acid, and/or derivatives thereof,
with an isolated linoleate isomerase enzyme of the present
invention to convert at least a portion of the compound to CLA or
derivatives thereof (e.g., when the substrate is a derivative): 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 is
preferably (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.
[0017] Another embodiment of the present invention relates to an
isolated nucleic acid molecule comprising a nucleic acid sequence
selected from the group of: (a) a nucleic acid sequence encoding a
protein having 10,12-linoleate isomerase enzymatic activity,
wherein the protein comprises an amino acid sequence selected from
the group consisting of SEQ ID NO:42 and SEQ ID NO:61; (b) a
nucleic acid sequence encoding a homologue of a protein of (a),
wherein the homologue has 10,12-linoleate isomerase enzymatic
activity, and wherein the homologue is at least about 35% identical
to SEQ ID NO:61 over at least about 170 contiguous amino acids of
SEQ ID NO:61; and/or, (c) a nucleic acid sequence that is fully
complementary to any of the nucleic acid sequences of (a) or (b).
In one embodiment, the nucleic acid sequence of (b) hybridizes
under low, moderate, or high stringency hybridization conditions to
the complement of SEQ ID NO:60.
[0018] In another embodiment, the homologue comprises at least 15
contiguous amino acids of SEQ ID NO:61, and more preferably, at
least 30 contiguous amino acids of SEQ ID NO:61, and even more
preferably, at least 45 contiguous amino acids of SEQ ID NO:61. In
another embodiment, the nucleic acid sequence of (b) comprises at
least 24 contiguous nucleotides of SEQ ID NO:60. The nucleic acid
molecule preferably comprises a nucleic acid sequence selected from
the group of SEQ ID NO:59 and SEQ ID NO:60, with SEQ ID NO:60 being
most preferred. Preferably, the nucleic acid molecule comprises a
nucleic acid sequence encoding an amino acid sequence selected from
the group of SEQ ID NO:42 and SEQ ID NO:61, with SEQ ID NO:61 being
most preferred.
[0019] The isolate nucleic acid molecule of the present invention
includes linoleate isomerase nucleic acid molecules from
microorganisms including, but not limited to, Propionibacterium,
with Propionibacterium acnes, Propionibacterium acidipropionici,
and Propionibacterium freudenreichii being particularly preferred.
Most preferred linoleate isomerase nucleic acid molecules are from
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,
Propionibacterium acnes, Propionibacterium freudenreichii,
Propionibacterium acidipropionici, Escherichia coli, Bacillus
subtilis, or Bacillus licheniformis, 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 transfected with an isolated nucleic acid molecule
encoding linoleate isomerase.
[0022] Another embodiment of the present invention relates to a
method for producing CLA or derivatives thereof, including
contacting an oil which comprises a compound selected from the
group of linoleic acid, linolenic acid, and/or derivatives thereof,
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 and/or a derivative thereof.
[0023] Yet another embodiment of the present invention relates to
an immobilized cell having a genetic modification that increases
the action of linoleate isomerase. The cell can be any cell,
including immobilized bacterial, fungal (e.g., yeast), microalgal,
insect, plant or mammalian cells. In one embodiment, the cell is a
microorganism which includes, but is not limited to
Propionibacterium, Escherichia, Bacillus or yeast cells. In one
embodiment, the genetic modification results in overexpression of
linoleate isomerase by the 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 transfection 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.
[0024] In one embodiment, the recombinant nucleic acid molecule is
integrated into the genome of the cell. In another embodiment, the
recombinant nucleic acid molecule is a plasmid
transformed/transfected into a 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 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 or a derivative thereof, including
contacting an oil which includes a fatty acid selected from the
group of linoleic acid, linolenic acid, and derivatives thereof
with an immobilized cell having a linoleate isomerase, to convert
at least a portion of the compound to CLA or a derivative thereof.
Such cells are described above. The cell can be a naturally
occurring bacterial cell having a linoleate isomerase, or a
genetically modified cell, such as a genetically modified
microorganism, as described above. Preferably, a genetically
modified cell has increased linoleate isomerase action. The fatty
acid can include fatty acids 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.
[0027] Another embodiment of the present invention relates to an
isolated lipase-like protein. Such a protein comprises an amino
acid sequence selected from the group of: (a) SEQ ID NO:64; and,
(b) a homologue of SEQ ID NO:64, wherein the homologue is at least
about 35% identical to SEQ ID NO:64. In one embodiment, the protein
is encoded by a nucleic acid molecule comprising a nucleic acid
sequence that hybridizes under moderate or high stringency
conditions to the complement of SEQ ID NO:63. In another
embodiment, the protein is encoded by a nucleic acid sequence
comprising at least 24 contiguous nucleotides of SEQ ID NO:63, and
more preferably, the protein is encoded by a nucleic acid molecule
comprising a nucleic acid sequence represented by SEQ ID NO:63. In
one embodiment, the protein comprises amino acid sequence SEQ ID
NO:64. In another embodiment, the protein comprises an amino acid
sequence having an esterase/lipase/thioresterase active site
denoted by ProfileScan Profile No. PS50187. In yet another
embodiment, the protein comprises an amino acid sequence having a
carboxylesterase type-B active site denoted by ProfileScan Profile
No. GC0265. Preferably, the protein has lipase enzymatic activity.
Also included in the present invention is an isolated nucleic acid
molecule comprising a nucleic acid sequence encoding any of the
above-described lipase-like proteins.
[0028] Yet another embodiment of the present invention relates to
an isolated acetyltransferase-like protein. Such a protein
comprises an amino acid sequence selected from the group of: (a)
SEQ ID NO:69; and, (b) a homologue of SEQ ID NO:69, wherein the
homologue is at least about 40% identical to SEQ ID NO:69 over at
least about 60 contiguous amino acid residues of SEQ ID NO:69. In
one embodiment, such a protein is encoded by a nucleic acid
molecule comprising a nucleic acid sequence that hybridizes under
moderate or high stringency conditions to the complement of SEQ ID
NO:68. In another embodiment, the protein is encoded by a nucleic
acid molecule comprising a nucleic acid sequence represented by SEQ
ID NO:68. In one embodiment, the protein comprises amino acid
sequence SEQ ID NO:69. In another embodiment, the protein comprises
an amino acid sequence having an acetyltransferase (GNAT) family
profile denoted by ProScan Profile No. PF00583. Preferably, the
protein has acetyltransferase enzymatic activity. Also included in
the present invention is an isolated nucleic acid molecule
comprising a nucleic acid sequence encoding any of the
above-identified acetyltransferase proteins.
BRIEF DESCRIPTION OF THE FIGURES
[0029] FIG. 1A is a line graph showing whole cell biotransformation
of CLA from linoleic acid by Clostridium sporogenes ATCC 25762
under aerobic conditions.
[0030] FIG. 1B is a line graph showing whole cell biotransformation
of CLA from linoleic acid by Clostridium sporogenes ATCC 25762
under anaerobic conditions.
[0031] FIG. 2A is a line graph illustrating whole cell
biotransformation of CLA from linoleic acid by C. bifermentans ATCC
638 under aerobic conditions.
[0032] FIG. 2B is a line graph illustrating whole cell
biotransformation of CLA from linoleic acid by C. bifermentans ATCC
638 under anaerobic conditions.
[0033] FIG. 3A is a line graph showing whole cell biotransformation
of CLA from linoleic acid by Propionibacterium jensenii ATCC
14073.
[0034] FIG. 3B is a line graph showing whole cell biotransformation
of CLA from linoleic acid by P. acnes ATCC 6919.
[0035] FIG. 4 is a line graph demonstrating whole cell
biotransformation of CLA from linoleic acid by P. acidipropionici
ATCC 25562.
[0036] FIG. 5 is a line graph illustrating whole cell
biotransformation of CLA from linoleic acid by L. reuteri PYR8.
[0037] FIG. 6 is a line graph showing DEAE chromatography of
detergent solubilized isomerase by L. reuteri PYR8.
[0038] FIG. 7 is a line graph demonstrating hydroxyapatite
chromatography of isomerase activity by L. reuteri PYR8.
[0039] FIG. 8 is a line graph illustrating chromatofocusing of
linoleic acid isomerase activity by L. reuteri PYR8.
[0040] FIG. 9 is a schematic illustration of the linoleate
isomerase genes and flanking open reading frames in L. reuteri
PYR8.
[0041] FIG. 10 is a schematic illustration of the putative
transcription terminator in the linoleate isomerase gene.
[0042] FIG. 11 is an illustration of several constructs for
linoleate isomerase expression in E. coli.
[0043] FIG. 12 is an illustration of several constructs for
linoleate isomerase expression in Bacillus.
[0044] 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.
[0045] FIG. 14 is a line graph showing the formation of
trans10,cis12-CLA from linoleic acid using whole cells of P.
acnes.
[0046] FIG. 15 is a flow diagram showing the cell fractionation
protocol for P. acnes ATCC 6919.
[0047] FIG. 16 is a line graph showing the effect of pH on
linoleate isomerase activity in crude extracts of P. acnes ATCC
6919.
[0048] FIG. 17 is a line graph showing the time course of CLA
formation in crude extracts of P. acnes ATCC 6919.
[0049] 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.
[0050] 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.
[0051] FIG. 20 is a graph illustrating DEAE ion exchange
chromatography of total soluble protein from P. acnes ATCC
6919.
[0052] FIG. 21 is a graph illustrating hydrophobic interaction
chromatography of total soluble protein from P. acnes ATCC
6919.
[0053] FIG. 22 is a graph illustrating chromatofocusing of
isomerase activity from P. acnes ATCC 6919.
[0054] FIG. 23A is a graph showing a time course of CLA formation
by C. sporogenes ATCC 25762 under aerobic conditions at room
temperature.
[0055] FIG. 23B is a graph showing a time course of CLA formation
by C. sporogenes ATCC 25762 under anaerobic conditions at room
temperature.
[0056] FIG. 23C is a graph showing a time course of CLA formation
by C. sporogenes ATCC 25762 under aerobic conditions at 37.degree.
C.
[0057] FIG. 23D is a graph showing a time course of CLA formation
by C. sporogenes ATCC 25762 under anaerobic conditions at
37.degree. C.
[0058] FIG. 24 is a flow diagram showing an extraction protocol for
C. sporogenes ATCC 25762.
[0059] FIG. 25 is a line graph showing linoleate isomerase optimum
pH and temperature in C. sporogenes ATCC 25762.
[0060] FIG. 26 is a line graph showing optimum linoleic acid
concentration for C. sporogenes ATCC 25762 linoleate isomerase.
[0061] FIG. 27 is a graph showing the time course for CLA formation
by C. sporogenes ATCC 25762 linoleate isomerase.
[0062] FIG. 28 is a bar graph illustrating the stability of C.
sporogenes ATCC 25762 linoleate isomerase in Tris and phosphate
buffers.
[0063] FIG. 29 is an elution profile of fresh C. sporogenes ATCC
25762 linoleate isomerase extracts from DEAE-5PW.
[0064] FIG. 30 is a bar graph demonstrating the effect of culture
medium on C. sporogenes ATCC 25762 growth and linoleate isomerase
activity.
[0065] FIG. 31 is a bar graph showing the effect of CaCl.sub.2 on
C. sporogenes ATCC 25762 linoleate isomerase activity.
[0066] FIG. 32 is a bar graph showing the effect of chelating
agents on C. sporogenes ATCC 25762.
[0067] FIG. 33 is a bar graph showing the effect of chelating
agents on stability of linoleate isomerase.
[0068] FIG. 34 is a line graph illustrating the effect of pH on
extraction efficiency of linoleate isomerase in C. sporogenes ATCC
25762.
[0069] FIG. 35 is a line graph demonstrating the half lives of
linoleate isomerase in C. sporogenes ATCC 25762 versus pH.
[0070] FIG. 36 is a bar graph showing the effect of buffer system
on the activity of linoleate isomerase in C. sporogenes ATCC
25762.
[0071] 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.
[0072] FIG. 38 is a line graph showing the stability of detergent
solubilized linoleate isomerase in C. sporogenes ATCC 25762.
[0073] FIG. 39 is an elution profile of C. sporogenes ATCC 25762
linoleate isomerase on DEAE Mono Q.
[0074] FIG. 40 is an elution profile of C. sporogenes ATCC 25762
detergent solubilized linoleate isomerase on DEAE-5PW column.
[0075] FIG. 41 is an elution profile showing separation of
partially purified C. sporogenes ATCC 25762 linoleate isomerase by
chromatofocusing.
[0076] FIG. 42 is a digitized image of a Western blot analysis in
cell lysates prepared from different strains of linoleate isomerase
using rabbit antibodies specific for the cloned L. reuteri PYR8
isomerase.
[0077] FIG. 43 is an absorbence profile showing the time course of
isomerization of linoleic acid.
[0078] FIG. 44 is a line graph illustrating the effect of pH on
isomerization of linoleic acid to CLA by C. sporogenes ATCC 25762
linoleate isomerase.
[0079] FIG. 45 is a line graph showing the effect of substrate
concentration on the rate of linoleic acid isomerization.
[0080] FIG. 46 is a Lineweaver-Burke plot of reaction kinetics of
C. sporogenes ATCC 25762 linoleate isomerase.
[0081] FIG. 47 is a bar graph showing the effect of oleic acid on
isomerase activity.
[0082] FIG. 48 is a secondary plot of oleic acid inhibition.
[0083] FIG. 49 is a secondary plot of palmitoleic acid
inhibition.
[0084] FIG. 50 is a Lineweaver-Burke plot of linoleic acid
isomerization kinetics in the presence of absence of oleic
acid.
[0085] FIG. 51 is a Hanes-Woolf plot of oleic acid inhibition of
linoleic acid.
[0086] FIG. 52 is a schematic illustration of the linoleate
isomerase gene and flanking open reading frames in P. acnes.
[0087] FIG. 53 is a map of the expression vector pET-PAISOM
containing the complete P. acnes linoleate isomerase coding
sequence.
[0088] FIG. 54 is a digitized image of an SDS-PAGE showing IPTG
induction of the expression of the recombinant P. acnes linoleate
isomerase in E. coli.
[0089] FIG. 55 is a graph showing the ultraviolet absorbence
spectrum of the CLA produced by using the recombinant P. acnes
linoleate isomerase.
[0090] FIG. 56A is a graph showing the resolution of CLA isomers
using a 100-m SP-2380 column.
[0091] FIG. 56B is a graph showing the CLA produced by using the
recombinant P. acnes linoleate isomerase.
[0092] FIG. 57 is a graph of GC-MS spectrum of the DMOX derivative
of the CLA produced by the recombinant P. acnes linoleate
isomerase.
[0093] FIG. 58 is a sequence alignment showing a putative
NAD-binding domain shared by linoleate isomerases of the present
invention and other enzymes.
DETAILED DESCRIPTION OF THE INVENTION
[0094] One embodiment of the present invention is an isolated
linoleate isomerase enzyme. According to the present invention, the
isolated linoleate isomerase can be used to produce conjugated
double bonds in fatty acids, in derivatives of fatty acids, and/or
in related molecules. More particularly, the isolated linoleate
isomerase can be used to produce CLA from linoleic acid, linolenic
acid or their derivatives (e.g., (cis, cis, cis)-6, 9,
12-octadecatrienoic acid (18:3) (.gamma.-linolenic acid); (cis,
cis, cis, cis)-6, 9, 12, 15 octadecatetraenoic acid (18:4)
(stearidonic acid); (cis, cis)-11, 14 eicosadienoic acid (20:2);
and methyl esters and branched forms of CLA). 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-conjugated diene moiety which is then further
metabolized to other fatty acids containing a 9-11 monoene moiety.
For example, most linoleate isomerases convert
(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, produce a linoleate isomerase which converts
(cis,cis)-9,12-linoleic acid to (trans,cis)-10,12-linoleic acid.
According to the present invention, the term "CLA" is used herein
as a generic term to describe both conjugated linoleic acid and
conjugated linolenic acid, and the term "CLA-derivative" is used to
describe derivatives of CLA which are formed from derivatives of
linoleic acid or linolenic acid. Such derivatives of CLA include,
but are not limited to CLA-lipids, CLA-methyl-esters, and branched
forms of CLA. For example, using derivatives of linoleic acid or
linolenic acid as a substrate (e.g., (cis, cis, cis)-6, 9,
12-octadecatrienoic acid (18:3) (.gamma.-linolenic acid); (cis,
cis, cis, cis)-6, 9, 12, 15 octadecatetraenoic acid (18:4)
(stearidonic acid); (cis, cis)-11, 14 eicosadienoic acid (20:2)),
CLA-lipid derivatives can be formed.
[0095] 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. Isolated linoleate isomerases
are described in related U.S. patent application Ser. No.
09/221,014, filed Dec. 23, 1998, incorporated herein by reference
in its entirety.
[0096] 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 10 nmoles CLA
mg.sup.-1 min.sup.-1, and more preferably at least about 25 nmoles
CLA mg.sup.-1 min.sup.-1, and more preferably at least about 100
nmoles CLA mg.sup.-1 min.sup.-1, and morepreferably at least about
250 nmoles CLA mg.sup.-1 min.sup.-1, and more preferably at least
about 500 nmoles CLA mg.sup.-1 min.sup.-1, and more preferably at
least about 1000 nmoles CLA mg.sup.-1 min.sup.-1, and even more
preferably at least about 10,000 nmoles CLA mg.sup.-1
min.sup.-1.
[0097] 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. For example, an isolated linoleate
isomerase from Lactobacillus reuteri has a K.sub.m for linoleic
acid of at least about 8.1 .mu.M at a pH of about 7.5 and at a
temperature of about 20.degree. C. An isolated linoleate isomerase
from Clostridium sporogenes has a K.sub.m for linoleic acid of at
least about 11.3 .mu.M at a pH of about 7.5 and at a temperature of
about 20.degree. C. An isolated linoleate isomerase from
Propionibacterium acnes has a K.sub.m for linoleic acid of at least
about 17.2 .mu.M at a pH of about 7.5 and at a temperature of about
20.degree. C.
[0098] 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.
For example, an isolated (cis, trans)-9,11-linoleate isomerase of
the present invention has a K.sub.i of from about 20 .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, from about 50 .mu.M to about
100 .mu.M, and even more preferably, greater than 100 .mu.M, with
no inhibition being most preferred.
[0099] 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. For example,
the maximum initial velocity rate of an isolated 9,11-linoleate
isomerase, such as an isolated linoleate isomerase from
Lactobacillus reuteri or Clostridium sporogenes, at a pH of about
7.5 is at least about 100 nmoles/min/mg of protein, more preferably
at least about 1,000 mnoles/min/mg of protein, and most preferably
at least about 10,000 nmoles/min/mg of protein. The maximum initial
velocity rate of an isolated 10,12-linoleate isomerase, such as an
isolated linoleate isomerase from Propionibacterium acnes, at a pH
of about 7.3 is at least about 100 nmoles/min/mg of protein, more
preferably at least about 1,000 nmoles/min/mg of protein, and most
preferably at least about 10,000 nmoles/min/mg of protein.
[0100] 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. The pH optimum for a linoleate isomerase
from P. acnes is about 6.8 to about 7.3 and most preferably, about
7.3.
[0101] 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. As used
herein, reference to an isolated linoleate isomerase includes
full-length linoleate isomerase proteins, fusion proteins, or any
homologue of such a protein. According to the present invention, a
homologue of a linoleate isomerase protein 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 or fragment), 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 15
contiguous amino acid residues (i.e., contiguous amino acid
residues having 100% identity with), and preferably 30 contiguous
amino acid residues of SEQ ID NO:42 or SEQ ID NO:61. In a preferred
embodiment, a homologue of a linoleate isomerase amino acid
sequence includes amino acid sequences comprising at least 45, and
more preferably, at least 60, and more preferably at least 120, and
even more preferably, at least 240, contiguous amino acid residues
of SEQ ID NO:61. A linoleate isomerase protein homologue includes
proteins encoded by a nucleic acid sequence comprising at least 24,
and preferably at least 45, and more preferably at least 90, and
more preferably at least 180, and more preferably at least 360, and
even more preferably at least 720, contiguous nucleotides of SEQ ID
NO:60. In a preferred embodiment, a linoleate isomerase protein
homologue has measurable linoleate isomerase enzymatic activity
(i.e., has biological activity). Methods of detecting and measuring
linoleate isomerase biological activity are described in detail in
the Examples section. In another embodiment, a linoleate isomerase
homologue may or may not have measurable linoleate isomerase
enzymatic activity, but is used for the preparation of antibodies
or the development of oligonucleotides useful for identifying other
linoleate isomerases.
[0102] According to the present invention, the term "contiguous" or
"consecutive", with regard to nucleic acid or amino acid sequences
described herein, means to be connected in an unbroken sequence.
For example, for a first sequence to comprise 15 contiguous (or
consecutive) amino acids of a second sequence, means that the first
sequence includes an unbroken sequence of 15 amino acid residues
that is 100% identical to an unbroken sequence of 15 amino acid
residues in the second sequence. Similarly, 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.
[0103] In one embodiment, a linoleate isomerase protein homologue
comprises an amino acid sequence that is at least about 35%
identical to SEQ ID NO:61 over at least about 170 contiguous amino
acids of SEQ ID NO:61. Preferably, a linoleate isomerase protein
homologue comprises an amino acid sequence that is at least about
45%, and more preferably, at least about 55%, and more preferably,
at least about 65%, and more preferably at least about 75%, and
more preferably at least about 85%, and even more preferably at
least about 95% identical to SEQ ID NO:61 over at least about 170
amino acids of SEQ ID NO:61, and more preferably over at least
about 200 amino acids, and more preferably over at least about 250
amino acids, and more preferably over at least about 300 amino
acids, and more preferably over at least about 350 amino acids, and
even sore preferably over at least about 400 amino acids of SEQ ID
NO:61. As discussed above, such a linoleate isomerase protein
homologue preferably has linoleate isomerase enzymatic activity
(i.e., (trans,cis)-10,12-linol- eate isomerase enzymatic activity).
According to the present invention, the terms
"(trans,cis)-10,12-linoleate isomerase activity" and
"10,12-linoleate isomerase activity" can be used
interchangeably.
[0104] As used herein, unless otherwise specified, reference to a
percent (%) identity refers to an evaluation of homology which is
performed using: (1) a BLAST 2.0 Basic BLAST homology search
(http://www.ncbi.nlm.nih.gov/BLAST) using blastp for amino acid
searches and blastn for nucleic acid searches with standard default
parameters, wherein the query sequence is filtered for low
complexity regions by default (described in Altschul, S. F.,
Madden, T. L., Schffer, A. A., Zhang, J., Zhang, Z., Miller, W.
& Lipman, D. J. (1997) "Gapped BLAST and PSI-BLAST: a new
generation of protein database search programs." Nucleic Acids Res.
25:3389-3402, incorporated herein by reference in its entirety);
(2) a BLAST 2 alignment (using the parameters described below)
(http://www.ncbi.nlm.nih.gov/BLAST; or (3) both BLAST 2.0 and BLAST
2. It is noted that due to some differences in the standard
parameters between BLAST 2.0 Basic BLAST and BLAST 2, two specific
sequences might be recognized as having significant homology using
the BLAST 2 program, whereas a search performed in BLAST 2.0 Basic
BLAST using one of the sequences as the query sequence may not
identify the second sequence in the top matches. Therefore, it is
to be understood that percent identity can be determined by using
either one or both of these programs.
[0105] Two specific sequences can be aligned to one another using
BLAST 2 sequence as described in Tatusova and Madden, (1999),
"Blast 2 sequences--a new tool for comparing protein and nucleotide
sequences", FEMS Microbiol Lett. 174:247-250, incorporated herein
by reference in its entirety. BLAST 2 sequence alignment is
performed in blastp or blastn using the BLAST 2.0 algorithm to
perform a Gapped BLAST search (BLAST 2.0) between the two sequences
allowing for the introduction of gaps (deletions and insertions) in
the resulting alignment. For purposes of clarity herein, a BLAST 2
sequence alignment is performed using the standard default
parameters as follows.
[0106] For blastn, using 0 BLOSUM62 matrix:
[0107] Reward for match=1
[0108] Penalty for mismatch=-2
[0109] Open gap (5) and extension gap (2) penalties
[0110] gap x_dropoff (50) expect (10) word size (11) filter
(on)
[0111] For blastp, using 0 BLOSUM62 matrix:
[0112] Open gap (11) and extension gap (1) penalties
[0113] gap x_dropoff (50) expect (10) word size (3) filter (on)
[0114] In some embodiments, as indicated, to align and calculate
the percent identity between two amino acid sequences, the
Martinez/Needleman-Wunsch DNA alignment method is used. This method
is provided by the Lasergene MegAlign, a module within the DNASTAR
program (DNASTAR, Inc., Madison, Wis.), and the standard default
parameters are used as follows:
[0115] (1) Minimum match=9;
[0116] (2) Gap penalty=1.10;
[0117] (3) Gap length penalty=0.33.
[0118] In another embodiment, a linoleate isomerase, including a
linoleate isomerase homologue, includes 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 low, moderate or high
stringency conditions (described below) 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). Preferably, a homologue
of a linoleate isomerase protein is encoded by a nucleic acid
molecule comprising a nucleic acid sequence that hybridizes under
low, moderate, or high stringency conditions to the complement of a
nucleic acid sequence that encodes a protein comprising an amino
acid sequence represented by SEQ ID NO:42 or SEQ ID NO:61. Even
more preferably, a homologue of a linoleate isomerase protein is
encoded by a nucleic acid molecule comprising a nucleic acid
sequence that hybridizes under low, moderate, or high stringency
conditions to the complement of SEQ ID NO:60. Such hybridization
conditions are described in detail below. A nucleic acid sequence
complement of nucleic acid sequence encoding a 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 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:42 or SEQ
ID NO:61, and/or with the complement of the nucleic acid that
encodes amino acid sequence selected from the group of SEQ ID NO:42
or SEQ ID NO:61. 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.
[0119] 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:42 or SEQ
ID NO:61, 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.
[0120] 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.
[0121] 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. In particular,
an isolated linoleate isomerase of the present invention is capable
of converting linoleic acid and/or linolenic acid to (trans,
cis)-10,12-linoleic acid.
[0122] The minimum size of a protein and/or homologue of the
present invention is a size sufficient to have linoleate isomerase
biological activity or, when the protein is not required to have
such enzyme activity, sufficient to be useful for another purpose
associated with a linoleate isomerase of the present invention,
such as for the production of antibodies that bind to a naturally
occurring linoleate isomerase. As such, the minimum size of
linoleate isomerase protein or homologue of the present invention
is a size suitable to form at least one epitope that can be
recognized by an antibody, and is typically at least 8 amino acids
in length, and preferably 10, and more preferably 15, and more
preferably 20, and more preferably 25, and even more preferably 30
amino acids in length, with preferred sizes depending on whether
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. There is no limit, other than a
practical limit, on the maximum size of such a protein in that the
protein can include a portion of a linoleate isomerase (including
linoleate isomerase homologues) or a full-length linoleate
isomerase.
[0123] Similarly, the minimum size of a nucleic acid molecule of
the present invention is a size sufficient to encode a protein
having linoleate isomerase activity, sufficient to encode a protein
comprising at least one epitope which binds to an antibody, or
sufficient to form a probe or oligonucleotide primer that is
capable of forming a stable hybrid with the complementary sequence
of a nucleic acid molecule encoding a natural linoleate isomerase
(e.g., under low, moderate or high stringency conditions). As such,
the size of the nucleic acid molecule encoding such a protein can
be dependent on nucleic acid composition and percent homology or
identity 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 a nucleic acid molecule that is used as an
oligonucleotide primer or as a probe 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. There is no limit, other than a practical limit, on
the maximal size of a nucleic acid molecule of the present
invention, in that the nucleic acid molecule can include a portion
of a linoleate isomerase encoding sequence, a nucleic acid sequence
encoding a full-length linoleate isomerase (including a linoleate
isomerase gene), or multiple genes, or portions thereof.
[0124] Preferred linoleate isomerases of the present invention
include proteins which comprise an amino acid sequence having at
least about 35%, and preferably at least about 40%, and more
preferably at least about 50%, and more preferably at least about
60%, and more preferably 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:42 and/or SEQ
ID NO:61. Preferred linoleate isomerases of the present invention
also include proteins which comprise an amino acid sequence
selected from SEQ ID NO:42 and/or SEQ ID NO:61. Preferred linoleate
isomerases of the present invention also include proteins which
comprise a protein selected from PPAISOM.sub.35 (also known as
PCLA.sub.35) and/or PPAISOM.sub.424. It is noted that a protein of
the present invention can be identified as a protein by use of the
letter "P" at the beginning, by its apparent size (e.g., subscript
"35" is a 35 amino acid protein), by association with its function,
substrate or product (e.g., CLA or ISOM designates a linoleate
isomerase of the present invention), and in some instances, by its
source (e.g., PPAISOM.sub.424 is a linoleate isomerase protein from
Propionibacterium acnes which is about 424 amino acids in length).
As discussed above, as used herein, percent identity between two or
more amino acid sequences is determined using a BLAST 2.0 Basic
BLAST search or alignment, using the standard default
parameters.
[0125] In one embodiment of the present invention, an isolated
linoleate isomerase comprises a putative NAD/FAD binding domain.
Preferably, the NAD/FAD binding domain corresponds to ProfileScan
PROSITE Profile No. PS50205, from ProfileScan at www.expasy.ch.
Such an NAD/FAD binding domain has the signature sequence
Gly-Xaa-Gly-(Xaa).sub.2-Gly-(Xaa).sub.3- -Ala-(Xaa).sub.6-Gly
(positions 1 through 21 of SEQ ID NO:73, minus four additional Xaa
residues from positions 14-17 of SEQ ID NO:73). Such a sequence is
present in many different enzymes, as set forth in Example 13. To
align two or more sequences such as SEQ ID NO:73 and another
sequence, and to compare the homology/percent identity between such
sequences, for example, a module contained within DNASTAR (DNASTAR,
Inc., Madison, Wis.) is preferably used. In particular, to align
and calculate the percent identity between two amino acid
sequences, the Martinez/Needleman-Wunsch DNA alignment method is
used. This method is provided by the Lasergene MegAlign module
within the DNASTAR program, with the following parameters, also
referred to herein as the standard default parameters:
[0126] (1) Minimum match=9;
[0127] (2) Gap penalty=1.10;
[0128] (3) Gap length penalty=0.33.
[0129] Using the Martinez/Needleman-Wunsch method with these
parameters, for example, the alignment and calculation of percent
identity between the amino acid sequences shown in FIG. 58 were
performed. In a preferred embodiment, an isolated linoleate
isomerase of the present invention comprises an amino acid sequence
that aligns with SEQ ID NO:73 using the Martinez/Needleman-Wunsch
alignment program as defined above, wherein amino acid residues in
the amino acid sequence align with and are identical to at least
about 50% of the non-Xaa residues in SEQ ID NO:73. More preferably,
an isolated linoleate isomerase of the present invention comprises
an amino acid sequence that aligns with SEQ ID NO:73 using this
alignment program, wherein amino acid residues in the amino acid
sequence align with and are identical to at least about 60%, and
more preferably at least about 70%, and more preferably at least
about 80%, and even more preferably at least about 90% of the
non-Xaa residues in SEQ ID NO:73. Even more preferably, an isolated
linoleate isomerase of the present invention comprises an amino
acid sequence that is identified as having a match with a
ProfileScan PROSITE Profile No. PS50205, using the standard default
parameters for normalized match score (NScores) with sensitivity
set to include weak matches. The ProfileScan program can be
accessed publicly through www.expasy.ch, ExPASy (Expert Protein
Analysis System) proteomics server of the Swiss Institute of
Bioinformatics (SIB); Pattern and Profile Searches.
[0130] The present invention also includes a fusion protein that
includes a linoleate isomerase-containing domain (including a
homologue of a linoleate isomerase) 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 transfected 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.
[0131] 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.
Microorganisms which have (trans,cis)-10,12-linoleate isomerase
activity according to the present invention include bacteria of the
genus Propionibacterium. A particularly preferred linoleate
isomerase of the present invention is a Propionibacterium acnes
linoleate isomerase.
[0132] Further embodiments of the present invention include nucleic
acid molecules that encode linoleate isomerases. A nucleic acid
molecule of the present invention includes a nucleic acid molecule
comprising a nucleic acid sequence encoding any of the isolated
linoleate isomerase proteins, including a linoleate isomerase
homologue, described above. In one embodiment, such nucleic acid
molecules include isolated nucleic acid molecules that hybridize
under low stringency conditions, and more preferably under moderate
stringency conditions, and even more preferably under high
stringency conditions with the complement of a nucleic acid
sequence encoding a naturally occurring P. acnes linoleate
isomerase (i.e., including naturally occurring allelic variants
encoding a P. acnes linoleate isomerase). Preferably, an isolated
nucleic acid molecule comprises a nucleic acid sequence that
hybridizes under low, moderate, or high stringency conditions to
the complement of a nucleic acid sequence that encodes a protein
comprising an amino acid sequence represented by SEQ ID NO:42 or
SEQ ID NO:61. In one embodiment, an isolated nucleic acid molecule
comprises a nucleic acid sequence that hybridizes under low,
moderate, or high stringency conditions to the complement of a
nucleic acid sequence represented by SEQ ID NO:60. 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:42 or SEQ ID NO:61,
and an isolated nucleic acid molecule having a nucleic acid
sequence of SEQ ID NO:60.
[0133] As used herein, 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.
[0134] More particularly, low stringency hybridization and washing
conditions, as referred to herein, refer to conditions which permit
isolation of nucleic acid molecules having at least about 35%
nucleic acid sequence identity with the nucleic acid molecule being
used to probe in the hybridization reaction (i.e., conditions
permitting about 65% or less mismatch of nucleotides). Moderate
stringency hybridization and washing conditions, as referred to
herein, refer to conditions which permit isolation of nucleic acid
molecules having at least about 55% nucleic acid sequence identity
with the nucleic acid molecule being used to probe in the
hybridization reaction (i.e., conditions permitting about 45% or
less mismatch of nucleotides). High stringency hybridization and
washing conditions, as referred to herein, refer to conditions
which permit isolation of nucleic acid molecules having at least
about 75% nucleic acid sequence identity with the nucleic acid
molecule being used to probe in the hybridization reaction (i.e.,
conditions permitting about 25% or less mismatch of nucleotides).
As discussed above, one of skill in the art can use the formulae in
Meinkoth et al., ibid. to calculate the appropriate hybridization
and wash conditions to achieve these particular levels of
nucleotide mismatch. 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 60%. Alternatively, Tm can be
calculated empirically as set forth in Sambrook et al., supra,
pages 9.31 TO 9.62.
[0135] In one embodiment of the present invention, a nucleic acid
molecule encoding a linoleate isomerase of the present invention
comprises a nucleic acid sequence that encodes an amino acid
sequence that is at least about 35% identical to SEQ ID NO:61 over
at least about 170 contiguous amino acids of SEQ ID NO:61.
Preferably, a nucleic acid molecule encoding a linoleate isomerase
of the present invention comprises a nucleic acid sequence that
encodes an amino acid sequence that is at least about 45%, and more
preferably, at least about 55%, and more preferably, at least about
65%, and more preferably at least about 75%, and more preferably at
least about 85%, and even more preferably at least about 95%
identical to SEQ ID NO:61 over at least about 170 amino acids of
SEQ ID NO:61, and more preferably over at least about 200 amino
acids, and more preferably over at least about 250 amino acids, and
more preferably over at least about 300 amino acids, and more
preferably over at least about 350 amino acids, and even more
preferably over at least about 400 amino acids of SEQ ID NO:61.
Such a nucleic acid sequence can include a nucleic acid sequence
encoding a linoleate isomerase protein homologue, and can therefore
be referred to as a homologue of a nucleic acid sequence encoding a
naturally occurring linoleate isomerase (i.e., a nucleic acid
sequence homologue).
[0136] Preferred linoleate isomerase nucleic acid molecules of the
present invention include nucleic acid molecules which comprise a
nucleic acid sequence having at least about 35%, and more
preferably at least about 45%, and more preferably at least about
55%, and more preferably, at least about 65%, and more preferably,
at least about 75%, and even more preferably, at least about 85%,
and most preferably, at least about 95% identity with a nucleic
acid sequence that encodes a protein having an amino acid sequence
selected from SEQ ID NO:42 and SEQ ID NO:61. In another embodiment,
preferred linoleate isomerase nucleic acid molecules of the present
invention include nucleic acid molecules which comprise a nucleic
acid sequence having at least about 35%, and more preferably at
least about 45%, and more preferably at least about 55%, and more
preferably, at least about 65%, and more preferably, at least about
75%, and even more preferably, at least about 85%, and most
preferably, at least about 95% identity with a nucleic acid
sequence represented by SEQ ID NO:60. Preferred linoleate isomerase
nucleic acid molecules of the present invention also include
nucleic acid molecules which comprise a nucleic acid sequence
encoding a protein comprising an amino acid sequence represented by
SEQ ID NO:42 and/or SEQ ID NO:61, or a nucleic acid molecule
comprising a nucleic acid sequence represented by SEQ ID NO:60.
Preferred linoleate isomerase nucleic acid molecules of the present
invention also include nucleic acid molecules which comprise a
nucleic acid molecule selected from nPAISOM.sub.5275 and
nPAISOM.sub.1275. Percent identity is determined using BLAST 2.0
Basic BLAST default parameters, as described above.
[0137] 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,
linoleate isomerase genes, natural allelic variants of linoleate
isomerase genes, linoleate isomerase coding regions or portions
thereof, and linoleate isomerase coding and/or regulatory regions
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. 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. It is noted that an
isolated linoleate isomerase nucleic acid molecule of the present
invention is not required to encode a protein having linoleate
isomerase activity. A linoleate isomerase nucleic acid molecule can
encode a truncated, mutated or inactive protein, for example. Such
nucleic acid molecules and the proteins encoded by such nucleic
acid molecules are useful in diagnostic assays, for example, or for
other purposes such as antibody production, as is described in the
Examples below.
[0138] According to the present invention, reference to a linoleate
isomerase gene includes all nucleic acid sequences related to a
natural (i.e. wild-type) linoleate isomerase gene, such as
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 the coding region itself.
[0139] In another embodiment, an linoleate isomerase gene can be a
naturally occurring allelic variant that includes a similar but not
identical sequence to the nucleic acid sequence encoding a given
linoleate isomerase. Allelic variants have been previously
described above. The phrases "nucleic acid molecule" and "gene" can
be used interchangeably when the nucleic acid molecule comprises a
gene as described above.
[0140] A linoleate isomerase nucleic acid molecule homologue (i.e.,
encoding a linoleate isomerase protein 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 homologue of the
present invention includes a nucleic acid sequence comprising at
least 24 contiguous nucleotides of SEQ ID NO:60, and more
preferably, at least about 45, and more preferably, at least about
90, and even more preferably, at least about 135, and even more
preferably at least about 180, and even more preferably at least
about 360, and even more preferably at least about 720 contiguous
nucleotides of SEQ ID NO:60. Similarly, a nucleic acid molecule
homologue of the present invention encodes a protein comprising an
amino acid sequence including at least 15, and preferably 30
contiguous amino acid residues of SEQ ID NO:42 and/or SEQ ID NO:61.
In another embodiment, a preferred nucleic acid sequence homologue
encodes a protein comprising an amino acid sequence including at
least 45, and more preferably at least 60, and more preferably at
least 120, and even more preferably, at least 240, contiguous amino
acid residues of SEQ ID NO:61.
[0141] One embodiment of the present invention includes a
recombinant nucleic acid molecule, which includes at least one
isolated nucleic acid molecule of the present invention inserted
into any nucleic acid vector (e.g., a recombinant vector) which is
suitable for cloning, sequencing, and/or otherwise manipulating the
nucleic acid molecule, such as expressing and/or 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, although the
vector can also contain regulatory nucleic acid sequences (e.g.,
promoters, untranslated regions) which are naturally found adjacent
to nucleic acid molecules of the present invention (discussed in
detail below). The vector can be either RNA or DNA, either
prokaryotic or eukaryotic, and typically is a virus or a plasmid.
The vector can be maintained as an extrachromosomal element (e.g.,
a plasmid) or it can be integrated into the chromosome. The entire
vector can remain in place within a host cell, 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.
[0142] Typically, a recombinant molecule includes a nucleic acid
molecule of the present invention operatively linked to one or more
transcription control sequences. As used herein, the phrase
"recombinant molecule" or "recombinant nucleic acid 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", when such nucleic acid molecule is a recombinant
molecule as discussed herein. 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, or 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.
[0143] 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.
[0144] One type of recombinant molecule, referred to herein as a
recombinant virus, includes a recombinant nucleic acid 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.
[0145] 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 transfected 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 (trans,cis)-10,12-linoleate isomerase of the
present invention, including, but not limited to, bacterial cells
of the genera Propionibacterium, 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. Other particularly preferred host cells include
fungal cells suitable as industrial expression hosts including, but
not limited to, Saccharomyces sp., Hansenula sp., Pichia sp.,
Kluveromyces sp., and Phaffia sp., as well as other fungal
expression systems.
[0146] According to the present invention, the term "transfection"
is used to refer to any method by which an exogenous nucleic acid
molecule (i.e., a recombinant nucleic acid molecule) can be
inserted into the cell. The term "transformation" can be used
interchangeably with the term "transfection" when such term is used
to refer to the introduction of nucleic acid molecules into
microbial cells, such as bacteria and yeast. In microbial systems,
the term "transformation" is used to describe an inherited change
due to the acquisition of exogenous nucleic acids by the
microorganism and is essentially synonymous with the term
"transfection". However, in animal cells, transformation has
acquired a second meaning which can refer to changes in the growth
properties of cells in culture after they become cancerous, for
example. Therefore, to avoid confusion, with regard to the
introduction of exogenous nucleic acids into animal cells, the term
"transfection" is preferably used, and the term "transfection" will
be used herein to generally encompass both transfection of animal
cells and transformation of microbial cells, to the extent that the
terms pertain to the introduction of exogenous nucleic acids into a
cell. Transfection techniques include, but are not limited to,
transformation, electroporation, microinjection, lipofection,
adsorption, infection and protoplast fusion.
[0147] 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.
[0148] 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.
[0149] 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.
[0150] To produce significantly high yields of CLA by the methods
of the present invention, a microorganism can be 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 a bacterium, fungus, microalga, and particularly, 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.
[0151] 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.
[0152] 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.
[0153] 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 enzyme (e.g.,
specific activity or in vivo enzymatic activity), reduced
inhibition or degradation of the enzyme, and overexpression of the
enzyme. 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.
[0154] 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.
[0155] Therefore, it is an embodiment of the present invention to
provide a bacterium which is transformed with a recombinant nucleic
acid molecule comprising a nucleic acid sequence encoding a
linoleate isomerase. 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:42 or SEQ ID NO:61. Other preferred recombinant nucleic
acid molecules of the present invention include nucleic acid
molecules which comprise a nucleic acid sequence represented by SEQ
ID NO:60. The above identified nucleic acid molecules represent
nucleic acid molecules comprising wild-type (i.e., naturally
occurring or natural) 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 above.
[0156] 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 nucleic acid sequence encoding
the linoleate isomerase 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 molecules 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.
Another method for modifying a recombinant nucleic acid molecule
encoding a linoleate isomerase is gene shuffling (i.e.,
molecularbreeding) (See, for example, U.S. Pat. No. 5,605,793 to
Stemmer; Minshull and Stemmer; 1999, Curr. Opin. Chem. Biol.
3:284-290; Stemmer, 1994, P.N.A.S. USA 91:10747-10751, all of which
are incorporated herein by reference in their entirety). This
technique can be used to efficiently introduce multiple
simultaneous positive changes in the linoleate isomerase enzyme
action. 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 remains the same, or actually is decreased,
relative to a naturally occurring linoleate Is 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.
[0157] 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 have been described in
detail herein.
[0158] Another embodiment of the present invention is a method for
producing CLA or derivatives thereof 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 fatty acid selected from the group consisting of free
fatty acids, salts of free fatty acids (e.g., soaps), and mixtures
containing linoleic acid, linolenic acid, derivatives of linoleic
or linolenic acid, and mixtures thereof. As discussed previously
herein, derivatives of linoleic acid or linolenic acid include any
derivatives, including, but not limited to lipid derivatives,
methyl-ester derivatives and branched forms. Some lipid derivatives
of linoleic acid and linolenic acid which can be used as a
substrate for a linoleate isomerase of the present invention
include, but are not limited to: (cis, cis, cis)-6, 9,
12-octadecatrienoic acid (18:3) (.gamma.-linolenic acid); (cis,
cis, cis, cis)-6, 9, 12, 15 octadecatetraenoic acid (18:4)
(stearidonic acid); (cis, cis)-11, 14 eicosadienoic acid (20:2)).
Such derivatives are described in Example 19, Table 6.
[0159] Preferably, the oil comprises at least about 50% by weight
of the fatty acid, 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 fatty acid to
CLA. Preferably at least about 30% the oil is converted to CLA, and
more preferably, at least about 50% of the oil is converted to CLA,
and more preferably at least about 70%, and most preferably at
least about 95%.
[0160] 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.
[0161] When the fatty acid 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. The use of enzymes to hydrolyze a
triglyceride is well known to one skilled in the art.
[0162] 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.
[0163] 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 cells which
contain a linoleate isomerase enzyme (including immobilized
bacterial, fungal (e.g., yeast), microalgal, insect, plant or
mammalian cells), 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, insect cells or
mammalian cells 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 and other cells and in such an embodiment,
the cells can be lysed.
[0164] 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.
[0165] Briefly, a solid support refers to any solid organic
supports, artificial membranes, biopolymer supports, 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.
[0166] 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.
[0167] 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.
[0168] 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.).
[0169] 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.
[0170] CLA produced by a method of the present invention can be
recovered by conventional methods.
[0171] 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.
[0172] Yet another embodiment of the present invention relates to a
method for producing CLA which utilizes industrial expression
systems formed from the microorganisms (or insect or mammalian
cells), 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, insect cell
or mammalian cell as described herein (including recombinant
microorganisms, insect cells or mammalian cells), wherein the
microorganism or other cell 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.
[0173] Yet another embodiment of the present invention relates to a
nucleic acid molecule that encodes a lipase-like protein, and to
the lipase like protein encoded thereby. In one embodiment, a
nucleic acid molecule encoding a lipase-like protein of the present
invention is denoted nPALPL.sub.1073. The nucleic acid sequence of
nPALPL.sub.1073 spans from nucleotide positions 1 to 1073 on the
complement of SEQ ID NO:59 (with the positions recited with regard
to the sense strand), and is represented herein by SEQ ID NO:63.
SEQ ID NO:63 encodes a protein having an amino acid sequence of 358
amino acid residues with an incomplete C-terminus. This sequence is
referred to herein as PPALPL.sub.358 (SEQ ID NO:64). PPALPL.sub.358
shows some homology to lipases (see below) and is therefore
designated LPL (lipase-like).
[0174] A sequence of 22 contiguous nucleotides (positions 815-836
of SEQ ID NO:63) was determined to be identical to a segment of the
Bordetella pertussis RNA polymerase sigma 80 subunit gene (Sanger
520, B. pertussis Contig54). The BLAST 2.0 search with the sequence
PPALPL.sub.358 showed that the protein sequence shares a low but
significant homology to some lipases. For example, the region
spanning the positions 146-356 of SEQ ID NO:63 shares 26% identical
and 42% similar amino acid residues with lipC from Mycobacterium
tuberculosis. It is noted that PPALPL.sub.358 does not share
significant homology with a different lipase gene previously cloned
from P. acnes (GenBank X99255). However, the sequence GDSAG,
located at positions 244-249 of SEQ ID NO:63, is conserved in many
lipases and conforms to the active-site serine motif (GXSXG) which
is shared by various lipases, esterases and other hydrolytic
enzymes. To provide additional evidence that PPALPL.sub.358 is a
lipase, a ProfileScan (protein pattern and profile search) was
carried out with the protein sequence SEQ ID NO:64. An
esterase/lipase/thioresterase active site (PROSITE Profile No.
PS50187) was found in the region 167-261 of SEQ ID NO:64. In
addition, the region from the positions 213 to 268 of SEQ ID NO:64
contained a carboxylesterase type-B active site (GC0265). However,
the sequence PPALPL.sub.358 does not contain the exact lipase
prosites (PROSITE Profile Nos. PS01173 and PS01174) that are
present in the P. acnes lipase (GenBank X99255). Therefore, the
present inventors have concluded that the protein encoded by SEQ ID
NO:63 and represented by amino acid sequence SEQ ID NO:64 is a
novel lipase or lipase-like enzyme.
[0175] Therefore, one embodiment of the present invention relates
to an isolated lipase-like protein. Such a protein comprises an
amino acid sequence selected from the group of: (a) SEQ ID NO:64;
and, (b) a homologue of SEQ ID NO:64, wherein the homologue is at
least about 35% identical to SEQ ID NO:64. As discussed above,
identity of one amino acid sequence to another is determined using
BLAST 2.0. The general definition of a homologue of a protein has
been described in detail above with respect to a linoleate
isomerase of the present invention and applies to a lipase-like
protein of the present invention as well. Preferably, a lipase-like
protein of the present invention comprises an amino acid sequence
that is at least about 45%, and more preferably, at least about
55%, and more preferably, at least about 65%, and more preferably
at least about 75%, and more preferably at least about 85%, and
even more preferably at least about 95% identical to SEQ ID NO:64.
In a more preferred embodiment, a lipase-like protein of the
present invention is encoded by a nucleic acid molecule comprising
a nucleic acid sequence represented by SEQ ID NO:63. Most
preferably, a lipase-like protein of the present invention
comprises an amino acid sequence SEQ ID NO:64. 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 lipase-like
protein of the present invention.
[0176] In one embodiment, a protein homologue having an amino acid
sequence that is sufficiently similar to a natural lipase-like
protein of the present invention that a nucleic acid sequence
encoding the homologue is capable of hybridizing under low,
moderate or high stringency conditions (described above) to (i.e.,
with) a nucleic acid molecule encoding the natural lipase-like
protein (i.e., to the complement of the nucleic acid strand
encoding the natural lipase-like protein amino acid sequence).
Preferably, a homologue of a lipase-like protein is encoded by a
nucleic acid molecule comprising a nucleic acid sequence that
hybridizes under low, moderate, or high stringency conditions to
the complement of a nucleic acid sequence that encodes a protein
comprising an amino acid sequence represented by SEQ ID NO:64. Even
more preferably, a homologue of a lipase-like protein is encoded by
a nucleic acid molecule comprising a nucleic acid sequence that
hybridizes under low, moderate, or high stringency conditions to
the complement of SEQ ID NO:63. Such hybridization conditions are
described in detail above.
[0177] In another embodiment, a lipase-like protein homologue
includes proteins having an amino acid sequence comprising at least
15 contiguous amino acid residues (i.e., 15 contiguous amino acid
residues having 100% identity with), apd preferably at least 30,
and more preferably at least 45, and more preferably, at least 60,
and more preferably at least 120, and even more preferably, at
least 240, and even more preferably at least 300, contiguous amino
acid residues of SEQ ID NO:64. A lipase-like protein homologue
includes proteins encoded by a nucleic acid sequence comprising at
least 24, and preferably at least 45, and more preferably at least
90, and more preferably at least 180, and more preferably at least
360, and even more preferably at least 720, and even more
preferably at least 900, contiguous nucleotides of SEQ ID NO:63. In
a preferred embodiment, a lipase-like protein homologue has
measurable lipase enzymatic activity (i.e., has biological
activity). Methods of detecting and measuring lipase enzymatic
activity are described in Kurooka et al., 1977, "A novel and simple
colorimetric assay for human serum lipase", J. Biochem. (Tokyo)
81:361-369, incorporated herein by reference in its entirety. In
another embodiment, a lipase-like protein homologue may or may not
have measurable lipase enzymatic activity, but is used for the
preparation of antibodies (e.g., can be used to generate antibodies
that bind to a natural lipase-like protein of the present invention
such as a protein having an amino acid sequence comprising SEQ ID
NO:64), or for the development of oligonucleotides useful for
identifying other lipases.
[0178] In one embodiment, a lipase-like protein of the present
invention comprises an amino acid sequence having an
esterase/lipase/thioresterase active site denoted by ProfileScan
Profile No. PS50187. In another embodiment, a lipase-like protein
of the present invention comprises an amino acid sequence having a
carboxylesterase type-B active site denoted by ProfileScan Profile
No. GC0265.
[0179] One embodiment of the present invention relates to an
isolated nucleic acid molecule comprising a nucleic acid sequence
encoding any of the lipase-like proteins of the present invention
described above. Also included in the present invention are
recombinant nucleic acid molecules and cells comprising such an
isolated nucleic acid molecule, and methods of using such molecules
to produce a lipase-like protein of the present invention. In one
embodiment, isolated nucleic acid molecules comprising nucleic acid
sequence encoding a lipase-like protein can be used with isolated
nucleic acid molecules encoding linoleate isomerase proteins of the
present invention in any of the methods described above.
[0180] As described in Example 14 below, one embodiment of the
present invention is a nucleic acid molecule, denoted
nPAUNK.sub.783, which spans 1604 to 2386 of the sequence
nPAISOM.sub.5275 (SEQ ID NO:59) and is represented by SEQ ID NO:65,
located upstream of SEQ ID NO:60. SEQ ID NO:65 is an open reading
frame encoding a protein of 260 amino acids residues which is
designated PPAUNK.sub.260 (SEQ ID NO:66). A putative ribosome
binding site GAAGGAG (SEQ ID NO:67) is located up-stream from the
first ATG codon, with a 4-base spacing. Therefore, this ATG codon
is very likely the actual translation initiation codon of this open
reading frame. This open reading frame does not show a significant
homology with any sequences in GenBank or unfinished microbial
genomes Therefore, the function of PPAUNK.sub.260 is presently
unknown.
[0181] Another embodiment of the present invention relates to an
isolated nucleic acid molecule encoding an acetyltransferase-like
protein, and the acetyltransferase protein encoded thereby. A
nucleic acid molecule encoding an acetyltransferase-like enzyme is
referred to herein as nPAATL.sub.582 and has a nucleic acid
sequence represented by SEQ ID NO:68. SEQ ID NO:68 spans positions
4129 to 4710 of SEQ ID NO:59 (nPAISOM.sub.5275) and is located on
nPAISOM.sub.5275 downstream from SEQ ID NO:60 on the same nucleic
acid strand. This open reading frame encodes an
acetyltransferase-like enzyme (ATL) of 193 amino acid residues, the
amino acid sequence of which is represented by SEQ ID NO:69. A
protein having the amino acid sequence of SEQ ID NO:69 is referred
herein as PPAATL.sub.193.
[0182] Although a BLAST 2.0 search with the nucleotide sequence
nPAATL.sub.582 did not reveal any significant homology with other
sequences, a ProfileScan using the protein sequence PPAATL.sub.193
(SEQ ID NO:69) showed that PPAATL.sub.193 contains an
acetyltransferase (GNAT) family profile (ProfileScan PROSITE
Profile document PF00583). A BLAST 2.0 search also showed that
PPAATL.sub.193 has a low homology to three putative
acetyltransferase genes in the database (See Example 14).
Therefore, the present inventors believe that the protein encoded
by nucleic acid sequence SEQ ID NO:68 and represented by amino acid
sequence SEQ ID NO:69 is an acetyltransferase enzyme or an
acetyltransferase-like protein.
[0183] Therefore, one embodiment of the present invention relates
to an isolated acetyltransferase-like protein. Such a protein
comprises an amino acid sequence selected from the group of: (a)
SEQ ID NO:69; and, (b) a homologue of SEQ ID NO:69, wherein the
homologue is at least about 40% identical to SEQ ID NO:69 over at
least 60 contiguous amino acids of SEQ ID NO:69. As discussed
above, identity of one amino acid sequence to another is determined
using BLAST 2.0. The general definition of a homologue of a protein
has been described in detail above with respect to a linoleate
isomerase of the present invention and applies to an
acetyltransferase-like protein of the present invention as well.
Preferably, an acetyltransferase-like protein of the present
invention comprises an amino acid sequence that is at least about
50%, and more preferably, at least about 60%, and more preferably,
at least about 70%, and more preferably at least about 80%, and
more preferably at least about 90%, identical to SEQ ID NO:69 over
at least about 60 contiguous amino acids, and more preferably over
at least 100 contiguous amino acids, and more preferably over at
least 150 contiguous amino acids of SEQ ID NO:69. In a more
preferred embodiment, an acetyltransferase-like protein of the
present invention is encoded by a nucleic acid molecule comprising
a nucleic acid sequence represented by SEQ ID NO:68. Most
preferably, an acetyltransferase-like protein of the present
invention comprises an amino acid sequence SEQ ID NO:69. 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 an
acetyltransferase-like protein of the present invention.
[0184] In one embodiment, a protein homologue having an amino acid
sequence that is sufficiently similar to a natural
acetyltransferase-like protein of the present invention that a
nucleic acid sequence encoding the homologue is capable of
hybridizing under low, moderate or high stringency conditions
(described above) to (i.e., with) a nucleic acid molecule encoding
the natural acetyltransferase-like protein (i.e., to the complement
of the nucleic acid strand encoding the natural
acetyltransferase-like protein amino acid sequence). Preferably, a
homologue of an acetyltransferase-like protein is encoded by a
nucleic acid molecule comprising a nucleic acid sequence that
hybridizes under low, moderate, or high stringency conditions to
the complement of a nucleic acid sequence that encodes a protein
comprising an amino acid sequence represented by SEQ ID NO:69. Even
more preferably, a homologue of an acetyltransferase-like protein
is encoded by a nucleic acid molecule comprising a nucleic acid
sequence that hybridizes under low, moderate, or high stringency
conditions to the complement of SEQ ID NO:68. Such hybridization
conditions are described in detail above.
[0185] In another embodiment, an acetyltransferase-like protein
homologue includes proteins having an amino acid sequence
comprising at least 15 contiguous amino acid residues (i.e., 15
contiguous amino acid residues having 100% identity with), and
preferably at least 30, and more preferably at least 45, and more
preferably, at least 60, and more preferably at least 120, and even
more preferably, at least 150, contiguous amino acid residues of
SEQ ID NO:69. An acetyltransferase-like protein homologue includes
proteins encoded by a nucleic acid sequence comprising at least 24,
and preferably at least 45, and more preferably at least 90, and
more preferably at least 180, and more preferably at least 360, and
even more preferably at least 450, contiguous nucleotides of SEQ ID
NO:68. In a preferred embodiment, an acetyltransferase-like protein
homologue has measurable acetyltransferase enzymatic activity
(i.e., has biological activity). Methods of detecting and measuring
acetyltransferase enzymatic activity are described in Freeman et
al., 1983, "Acetyl CoA: alpha-glucosamidnide N-acetyl transferase:
partial purification from human liver", Biochem. Int. 6:663-671,
incorporated herein by reference in its entirety. In another
embodiment, an acetyltransferase-like protein homologue may or may
not have measurable acetyltransferase enzymatic activity, but is
used for the preparation of antibodies (e.g., can be used to
generate antibodies that bind to a natural acetyltransferase-like
protein of the present invention such as a protein having an amino
acid sequence comprising SEQ ID NO:69), or for the development of
oligonucleotides useful for identifying other
acetyltransferases.
[0186] In one embodiment, an acetyltransferase-like protein of the
present invention comprises an amino acid sequence having an
acetyltransferase (GNAT) family profile denoted by profile document
PF00583.
[0187] One embodiment of the present invention relates to an
isolated nucleic acid molecule comprising a nucleic acid sequence
encoding any of the acetyltransferase-like proteins of the present
invention described above. Also included in the present invention
are recombinant nucleic acid molecules and cells comprising such an
isolated nucleic acid molecule, and methods of using such molecules
to produce a acetyltransferase-like protein of the present
invention. In one embodiment, isolated nucleic acid molecules
comprising nucleic acid sequence encoding an acetyltransferase-like
protein can be used with isolated nucleic acid molecules encoding
linoleate isomerase proteins of the present invention in any of the
methods described above.
EXAMPLES
[0188] 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
[0189] 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.
[0190] 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.
[0191] Lactobacillus reuteriPYR8 (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.
[0192] 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.
[0193] Aerobic biotransformations were carried out in baffled 250
mL shake flasks agitated at 200 rpm on a shaker at room
temperature.
[0194] 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.
[0195] 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.
[0196] 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.
[0197] 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).
[0198] 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.
[0199] 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
[0200] This example describes a procedure for fatty acid analysis
to determine the amount of conversion of linoleic acid to CLA.
[0201] 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
[0202] This Example describes the purification of linoleate
isomerase from L. reuteri.
[0203] 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. 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.
[0204] Method A
[0205] 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).
[0206] Method B
[0207] 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.
[0208] 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.
[0209] Method C
[0210] 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.
[0211] The purification of the L. reuteri 9,11-linoleate isomerase
is summarized in Table 1 A.
1TABLE 1A Lactobacillus reuteri Step Protein Total Activity
Specific Activity Yield Crude extract 1125 2170 1.93 100 Detergent
soluble 147 836 5.68 38.5 DEAE 21.5 529 24.6 24.4 Chromatofocusing
1.60 148 92.5 6.8 Gel filtration 0.21 86 407 3.9 Protein in
milligrams. Enzyme activity units are nanomoles CLA formed per
minute. Specific activity is units per milligram protein.
Example 4
[0212] 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.
[0213] 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.
[0214] 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 absorbence 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.
[0215] 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
[0216] This Example shows the nucleic acid cloning and sequencing
of a Lactobacillus reuteri linoleate isomerase nucleic acid
molecule of the present invention.
[0217] 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.
[0218] 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.
[0219] The first oligonucleotide primer, designated CLAO1, had the
following sequence:
[0220] 5'-cgt gaa ttc ATG TA(T/C) TA(T/C) (T/A)(C/G)N AA(T/C) GGN
AA-3' (including an EcoRI site and 3 extra bases (shown as lower
case letters) at the 5' end) (SEQ ID NO:2).
[0221] The second oligonucleotide primer, designated CLAO2, had the
following sequence:
[0222] 5'-act gga tCC NAC (T/A/G)AT (A/G)AT NGC (A/G)TG (C/T)TT-3'
(including an Bam HI site and 3 extra bases (shown as lower case
letters) at the 5' end) (SEQ ID NO:3).
[0223] 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.
[0224] 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.
[0225] 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).
[0226] 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 SrfI site into the vector
pPCR-Script(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.
[0227] The sequences immediately flanking CLA03 and CLA04 are
identical to the sequence in nCLA.sub.87, confirming the identity
of the cloned PCR product.
[0228] 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 BaniHI, 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.
[0229] 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.
[0230] 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 CLA3 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.
[0231] 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.
[0232] 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.
[0233] 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 PCLA.sub.59
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.
[0234] 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 2.0 search parameters were the standard
default values as described above. No defined functions have been
previously described for either the Streptococcus pyogenes (U09352)
or the Staphylococcus aureus (L19300) sequences.
[0235] 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.
[0236] 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 encodesat 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 2.0) 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
[0237] The following example demonstrates the cloning of sequences
flanking the isomerase-gene in the L. reuteri PYR8 genome.
[0238] 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).
[0239] 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.
[0240] The identity of nSAL3684 was confirmed by the sequences
flanking the primers CLAo9 and CLAo10. The sequence nSAL3684
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.
[0241] 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.
[0242] 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 2.0) 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 2.0 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.
[0243] 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.
[0244] 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.
[0245] 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 2.0) 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.
[0246] 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.
[0247] 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.540 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.
[0248] 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.
[0249] 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
[0250] The following example demonstrates the expression of L.
reuteri linoleate isomerase in E. coli.
[0251] 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):
[0252] 5'-gcagtcgacGGAGTTAAGACTGAATTAG-3'
[0253] 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):
[0254] 5'-ctagtcgacGCAGTTTCTGTCATGAC-3'
[0255] The PCR product of 2.3 kb was ligated with blunt ends into
pPCR-Script(Amp)SK at the Srf1 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.
[0256] 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.
[0257] 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 (See Example 10). 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.
[0258] In additional experiments, E. coli transformed with and
expressing the PYR8 isom erase 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).
[0259] 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 (See Example
10).
[0260] 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
[0261] The following example describes expression of a linoleate
isomerase of the present invention in Bacillus.
[0262] 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):
[0263] 5'catATGTATTATTCAAACGGGAATTATGAAGC-3'.
[0264] 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):
[0265] 5'tgatcaTCTATACCAGCAGTTTCTGTCATG-3'.
[0266] 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.
[0267] 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.
[0268] 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.
[0269] 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.
[0270] 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.
[0271] 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 a-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 proteins 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
[0272] The following example demonstrates the expression of the L.
reuteri PYR8 (cis, trans)-9, 11-linoleate isomerase gene off of the
native PYR8 promoter in L. reuteri (type strain) ATCC 23272.
[0273] L. reuteri PYR8 isomerase gene (promoter-ORF-terminator) was
amplified from PYR8 genomic DNA using the primer pair CLAo7 (SEQ ID
NO:38, see Example 7) and CLAo8B (SEQ ID NO:39, see Example 7). The
PCR amplified isomerase gene product (2.3 kb) contained the
complete coding sequence plus 381 bp of immediately upstream
sequence containing the native promoter, as well as downstream
sequence containing the putative transcription terminator (up to
124 bp downstream from the stop codon). The PCR product was cloned
with blunted ends into pPCR-Script Amp SK (+). The isomerase gene
was isolated from the recombinant plasmid by Sal I digestion and
ligated with the Lactobacillus/E. coli shuttle vector pTRKH2, which
had been predigested with Sal I. The ligated DNA was transformed
into E. coli cells. Transformants were selected and checked by
restriction analysis. The recombinant plasmids with the isomerase
gene positioned either under control (downstream), or reverse to,
the lac promoter were transformed into cells of L. reuteri ATCC
23272 (type strain) by electroporation.
[0274] Cells of L. reuteri 23272 transformed with the isomerase
gene under control of or reverse to the lac promoter were grown at
37.degree. C. for 28 hours before harvesting. Total cellular
protein was analyzed by SDS-PAGE and Western Blot. A new protein
band of about 70 kD was detected with rabbit antibodies specific
for L. reuteri PYR8 isomerase in the cells of both types of
transformants. This indicates that the promoter sequence of L.
reuteri PYR8 linoleate isomerase gene functions in L. reuteri
23272.
[0275] Although a substantial amount of protein was produced, the
transformed L. reuteri 23272 cells did not show measurable enzyme
activity. Neither CLA nor the hydroxylated linoleic acid derivative
(formed in B. subtilis) was detected in the linoleic acid
biotransformation assay. It is still not clear if the protein was
produced as a membrane protein integrated into the membrane, or as
an inclusion body (similar to expression of the gene from the T7
promoter in E. coli). It is unlikely that the protein is produced
in soluble form. The present inventors are currently investigating
methods to improve the quality of the PYR8 isomerase gene
constructs, including confirmation of nucleic acid sequence, and
methods to induce the measurable enzyme activity in the transformed
cells.
Example 10
[0276] The following example describes the production and
characterization of a rabbit antibody made from the cloned L.
reuteri PYR8 (cis, trans)-9, 11-linoleate isomerase gene in
Escherichia coli.
[0277] An L. reuteri (cis, trans)-9, 11-linoleate isomerase-histag
fusion protein was synthesized in E. coli as inclusion bodies as
described in Example 7 and was further purified under denaturing
conditions using His-Bind Resin and Buffer kit (NOVAGEN, Catalogue
# 70239-3), following the vendors protocols. The purified isomerase
protein was used to immunize two rabbits. Serum collected from the
fourth bleeding was tested in Western Blot analysis (FIG. 42). FIG.
42 shows that the rabbit antibodies showed a strong specificity for
the isomerase produced in its native host L. reuteri PRY8 and for
isomerase expressed in heterologous hosts, although a low
background level of cross-reaction was observed when protein
samples were overloaded.
[0278] The antibodies showed a very strong signal with L. reuteri
isomerase-histag fusion protein expressed in E. coli (FIG. 42, lane
1). B. subtilis transformed with construct #1 (See FIG. 12:
isomerase coding sequence under the HpaII promoter control in the
plasmid vector pBH1) produced a protein of about 70 kD which could
be seen on Coomassie-Blue stained SDS-gel (data not shown). This
protein band was readily detected by the rabbit antiserum (FIG. 42,
lane 4). This signal was not detected in wild type cells of
Bacillus subtilis (FIG. 42, lane 3). The antibodies recognized a
peptide of about 70 kD in L. reuteri 23272 cells transformed with
constructs containing the L. reuteri PYR8 isomerase gene (FIG. 42;
lane 5: L. reuteri 23272 wild type; lane 6: L. reuteri 23272
transformed with the vector pTRKH2 containing the isomerase gene
under the control of both its native promoter and the lac promoter;
and lane 7: L. reuteri 23272 transformed with the vector pTRKH2
containing the isomerase gene under the control of its native
promoter; See Example 9).
[0279] The rabbit antibodies reacted specifically with the 70 kD
linoleate isomerase synthesized in the native L. reuteri PYR8 (FIG.
42, lane 2). The present inventors attempted to detect
cross-reaction of the antibodies with (cis, trans)-9, 11-linoleate
isomerase of C. sporogenes and (trans, cis)-10,12-linoleate
isomerase of P. acnes. A highly purified P. acnes isomerase showed
a single protein band of about 55 kD on SDS gel (data not shown).
The rabbit L. reuteri PYR8 antibody recognized the 55 kD P. acnes
(trans, cis)-10, 12-linoleate isomerase in a total cell lysate
(FIG. 42, lane 8). Similarly, the L. reuteri PYR8 antibody reacted
with the 45 kD (cis, trans)-9, 11-linoleate isomerase of C.
sporogenes (FIG. 42, lane 9). Significant nonspecific antibody
cross-reaction was observed with C. sporogenes lysates and to a
lesser extent with the P. acnes lysates.
Example 11
[0280] The following example describes the purification of
linoleate isomerase from Propionibacterium acnes.
[0281] P. acnes ATCC 6919 is the only microorganism known to
produce trans10,cis12-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
trans10,cis12-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 trans10,cis12-CLA. No
cis9,trans11-CLA was observed. With prolonged incubation, the level
of trans10,cis12-CLA changed only slightly, while nearly all
remaining linoleic acid disappeared. At present it is unclear how
linoleic acid is metabolized inthisorganism. In other experiments,
trans10,cis12-CLA rose in concentration, but later disappeared
completely, as did all of the linoleic acid (results not shown).
This suggests that trans10,cis12-CLA may be subject to further
metabolism, possibly by a reductase. Linoleic acid may also be a
substrate for enzymes other than the isomerase.
[0282] 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.
[0283] 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).
[0284] 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).
[0285] Formation of CLA was determined by measuring the absorbence
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.
[0286] 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.
[0287] 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.
[0288] 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.
[0289] 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).
[0290] 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.
[0291] Following HIC chromatography, a single fraction containing
high isomerase activity was applied to a Pharmacia MonoP
chromatofocusing column equilibrated with 20 mM bis-Tris (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 55 kD.
[0292] The purification of the P. acnes 10,12-linoleate isomerase
is summarized in Table 1B.
2TABLE 1B Propionibacterium acnes Total Step Protein Activity
Specific Activity Yield Crude extract 419 1365 3.26 100 DEAE 34.8
774 22.2 56.7 Hydrophobic 1.83 250 137 18.3 interaction
Chromatofocusing .107 51.1 478 3.75 Protein in milligrams. Enzyme
activity units are nanomoles CLA formed per minute. Specific
activity is units per milligram protein.
[0293] 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.
[0294] 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 50-55 kD in
size (data not shown). Other active fractions exhibited three to
four additional bands. These fractions will be applied to a gel
filtration column if further purification is required. N-terminal
sequencing of the P. acnes linoleate isomerase has been completed
(See Example 12).
Example 12
[0295] The following example demonstrates the sequencing of the
N-terminal amino acid sequence of the purified P. acnes soluble
(trans, cis)-10,12-linoleate isomerase.
[0296] Linoleate isomerase was purified from P. acnes ATCC 6919 to
apparent homogeneity as described in Example 11; only a single
peptide band of about 55 kD could be detected on SDS PAGE stained
by Coomassie Blue. The N-terminal peptide sequence (35 amino acid
residues) of the purified protein was determined as follows:
[0297] SISKD SRIAI IGAGP AGLAA GMYLW QAGFX DYTIL (SEQ ID NO:42)
[0298] A protein having the sequence of SEQ ID NO:42 is referred to
herein as PPAISOM.sub.35 (formerly called PCS-CLA.sub.35 in U.S.
Provisional Application Ser. No. 60/141,798, from which this
application claims priority). It should be noted that since amino
acid sequencing technology is not entirely error-free, SEQ ID NO:42
represents, at best, an apparent partial N-terminal amino acid
sequence.
[0299] No significant homology was detected when the PPAISOM.sub.35
amino acid sequence was initially compared to the linoleate
isomerase peptide deduced from the DNA sequence cloned from L.
reuteri PYR8 (SEQ ID NO:18) or of the directly determined amino
acid sequence of the PYR8 isomerase (SEQ ID NO:1). We note that the
L. reuteri PYR8 and P. acnes linoleate isomerases have different
mass; the isomerase from L. reuteri is about 70 kD while the
isomerase from P. acnes is about 55 kD. A comparison of the
complete isomerase sequences from L. reuteri and P. acnes (see
Example 13) will be more meaningful (See Example 13).
[0300] The N-terminal sequence of the P. acnes isomerase also
showed no homology with the N-terminal peptide sequence from the
putative Butyrivibrio fibrisolvens (cis, trans)-9, 11-linoleate
isomerase (Park et al., 1996), although, as discussed above
(Background section), the present inventors consider it to be
unlikely that the sequence described by Park et al. is actually a
linoleate isomerase. The N-terminal peptide sequence was also
analyzed against the sequences in the databases using Blastp
program with standard settings. The best-matched sequence is the
putative E. coli oxidoreductase, Fe--S subunit (gi887828) showing
71% identity in a region of 28 amino acid residues. However, no
homology could be detected with any sequences in the data base when
low complexity filtering is used in Blastp analysis. Low complexity
regions commonly give spuriously high scores that reflect
compositional bias rather than significant position-by-position
alignment. Filtering is designed to eliminate these potentially
confounding matches. Therefore, the true level of homology between
P. acnes linoleate isomerase and the putative E. coli
oxidoreductase Fe--S subunit remains to be determined when
full-length P. acnes isomerase gene sequence is determined.
[0301] Nucleic acid sequences encoding SEQ ID NO:42 can be deduced
from the amino acid sequence by those of ordinary skill in the art.
Isolated nucleic acid molecules comprising such nucleic acid
sequences are encompassed by the present invention.
Example 13
[0302] The following example describes the nucleic acid cloning and
sequencing of a Propionibacterium acnes linoleate isomerase nucleic
acid molecule of the present invention.
[0303] Peptide Sequences Determined for the Purified Isomerase.
[0304] The purified P. acnes linoleic acid isomerase, purified as
described in Examples 11 and 12, was subjected to digestion with
the enzyme endo LYS-C to generate peptide fragments. The resulting
peptides were separated by HPLC chromatography. Peptide fragments
from three different peaks were sequenced individually. None of
these peptide fragments are identical, entirely or partially, to
the N-terminal sequence. Therefore, these fragments represent
internal peptide fragments of the P. acnes linoleate isomerase.
[0305] A sequence of 14 amino acids was determined for the peptide
in HPLC peak number one and is represented herein as SEQ ID NO:44.
A peptide having the sequence of SEQ ID NO:44 is referred herein as
PPAISOM.sub.14. A sequence of 9 amino acid residues was determined
for the peptide in HPLC peak number two and is represented herein
as SEQ ID NO:45. A peptide having the sequence of SEQ ID NO:45 is
referred herein as PPAISOM.sub.9. A sequence of 15 amino acid was
determined for the peptide in HPLC peak number three and is
represented herein as SEQ ID NO:46. A peptide having the sequence
of SEQ ID NO:46 is referred herein as PPAISOM.sub.15. It should be
noted that the amino acid signals detected in this sequencing
experiment were very weak. There were, therefore, alternative
choices at certain positions of the sequence due to ambiguous
reads. The secondary choices at ambiguous amino acid positions in
SEQ ID NO:46 are presented as lowercase letters in parentheses.
[0306] Cloning the DNA Sequence Coding the N-Terminal Peptide
Residues.
[0307] Two sets of degenerated oligonucleotide primers were
synthesized according to SEQ ID NO:42. The first oligonucleotide
primer set corresponds to amino acid residues 8-14 of SEQ ID NO:42
and is designated PA05 (SEQ ID NO:47). The second oligonucleotide
primer set corresponds to amino acid residues 22-28 of SEQ ID NO:42
and is designated PA011 (SEQ ID NO:48). Two known genes in the
GenBank database have been previously cloned from Propionibacterium
acnes: hyaluronidase (GenBank U15927) and lipase (GenBank X99255).
The codon bias of these two genes was used to decrease the
degeneracy at the 5' end of the primer PA05 (SEQ ID NO:47). A PCR
reaction using P. acnes DNA as template generated large quantities
of non-specific products of different sizes and also a product of
the expected size (62 bp) in lesser amount. The PCR product of 62
bp was isolated, purified and cloned into pPCR-Script SK (+) Amp
(Stratagene). Putative recombinant plasmids were analyzed by
restriction digestions. Four clones were selected and sequenced.
Although different primer molecules were involved in the synthesis
of the PCR products, the deduced amino acid sequences of all four
clones matched exactly to residues 12 through 28 of the N-terminus
of the purified linoleate isomerase protein (SEQ ID NO:42), thus
confirming the identity of the PCR products. The sequence of the
cloned PCR product (SEQ ID NO:49) is denoted herein as
nPAISOM.sub.62. Differences in nucleotides found in the different
clones of SEQ ID NO:49 are indicated in the Sequence Listing as
alternate nucleotides. The deduced amino acid sequence (SEQ ID
NO:50) is referred herein as PPAISOM.sub.21.
[0308] Cloning of a Larger Portion of the Isomerase Gene by Inverse
PCR.
[0309] An inverse PCR amplification approach was used to clone the
DNA sequences flanking the DNA sequence nPAISOM.sub.62. Two
oligonucleotide primers, designated PA016 and PA017 (SEQ ID NO:51
and SEQ ID NO:52, respectively), were synthesized. The primer PA016
corresponded to nucleotides 7-23 of nPAISOM.sub.62, and PA017 to
nucleotides 30-47 of nPAISOM.sub.62.
[0310] Genomic DNA from P. acnes was digested with the following
restriction enzymes, or combinations of two enzymes that produce
compatible ends: BamHI, EcoRI, HindIII, PvuII, SalI, Sau3A,
BamHI/BglII, XbaI/NheI, XbaI/SpeI, XhoI/SalI. Each DNA digest was
purified using a PCR purification kit (Qiagen) and circularized
with T4 DNA ligase. PCR reactions were carried out with the primer
pair PA016/PA017 using aliquots of the circularized DNA digests as
template. A PCR product of about 570 bp was generated with the
circularized BamHI digest. The PCR product was purified and cloned
into the plasmid vector pCR2.1-TOPO (Invitrogen) and sequenced.
With reference to the BamHI site and the sequence nPAISOM.sub.62,
the cloned DNA sequence was edited to generate a sequence of 569
bp, referred herein as nPAISOM.sub.596 (SEQ ID NO:53). This
sequence contained an open reading frame (ORF) of 104 amino acid
residues, with its C-terminus still incomplete, and with the start
codon occurring at positions 259-261 of SEQ ID NO:53. The deduced
amino acid sequence of this incomplete ORF is denoted
PPAISOM.sub.104 (SEQ ID NO:54).
[0311] Southern Blot Analysis of P. acnes DNA with the Cloned
Partial Isomerase Sequence, nPAISOM.sub.569.
[0312] P. acnes DNA was digested with the following restriction
enzymes: BglI, EcoRI, FokI, HindIII, PvuII, XhoI. The digests were
analyzed on Southern Blots using the sequence nPAISOM.sub.569 (SEQ
ID NO:53) labeled with biotinylated nucleotides (NEBlot Phototope
kit, New England Biolabs). With the enzymes EcoRI, HindIII, PvuI
and XhoI (which do not cut the sequence nPAISOM.sub.569), only one
hybridization band was observed. This indicates that only one copy
of the linoleate isomerase gene is present in the genome of P.
acnes. As expected for DNA digested by FokI and SalI, which have
unique sites in the sequence nPAISOM.sub.569, two hybridization
bands were shown on Southern blot. Similarly, two bands were
observed hybridizing to nPAISOM.sub.569 in BglI digests although
additional bands of weak signal intensity and of higher molecular
weight were seen on the blot (which is likely caused by a problem
of incomplete DNA digestion).
[0313] Second Round of Inverse PCR to Clone the Entire Isomerase
Gene and Flanking Sequences.
[0314] The XhoI digests showed one band (about 3 kb) hybridizing to
nPAISOM.sub.569 while the BglI digests showed two major
hybridization bands of around 3 kb. Inverse PCR amplification and
cloning of both BglI fragments would cover a sequence of about 5.5
kb in total. Therefore, two pairs of oligonucleotide primers were
synthesized. The first pair of primers, designated PA021 (SEQ ID
NO:55) and PA022 (SEQ ID NO:56), respectively, were synthesized for
inverse PCR amplification of the upstream BglI fragment. The second
pair of primers, designated PA023 (SEQ ID NO:57) and PA024 (SEQ ID
NO:58), respectively, were synthesized for inverse PCR
amplification of the down-stream BglI fragment. As an alternative
approach, P. acnes genomic DNA was also digested with the enzyme
XhoI, circularized and used as template for inverse PCR reaction
with the primer pair PA021/PA024.
[0315] No PCR product of expected size could be generated with T4
DNA ligase treated Bgl I digest using the primer pair PA023/PA024.
One explanation for this observation is that the two ends of the
downstream BglI fragment were not compatible and therefore the
fragment could not be circularized. However, PCR amplification with
circularized BglI digest of P. acnes DNA generated a PCR product of
about 2.5 kb using the primer pair PA021/PA022. A PCR product of
about 2.5 kb was also amplified with circularized XhoI digest using
the primer pair PA021/PA024. Both PCR products were purified and
cloned into pPCR-Script Amp SK (+). Both of the cloned BglI
fragment and the cloned XhoI fragment were completely sequenced.
The complete sequence of the BglI fragment and the XhoI fragment
and the sequence nPAISOM.sub.569 were edited to generate a sequence
of about 5.3 kb (SEQ ID NO:59), which is referred herein as
nPAISOM.sub.5275.
[0316] Analysis of the Isomerase Gene and Flanking Sequences.
[0317] As schematically illustrated in FIG. 52, the sequence
nPAISOM.sub.5275 (SEQ ID NO:59) contains the entire P. acnes
linoleate isomerase open reading frame (ISOM) as well as two ORFs
(A and B) upstream and one ORF (C) downstream. The open reading
frames B, C and the isomerase gene (ISOM) are coded by the same DNA
strand while the ORF A is coded by the opposite DNA strand. No
obvious transcription terminator or structures similar to promoter
elements, common to other organisms, were found between the ORF B
and the ORF for the linoleate isomerase (ISOM). At this point, it
is not clear if the three open reading frames (B, C and ISOM) are
transcribed as a single transcript or as multiple transcripts. RNA
probing or primer extension experiments may be needed to determine
the answer.
[0318] Linoleate Isomerase Open Reading Frame.
[0319] The P. acnes linoleate isomerase open reading frame spans
from nucleotide positions 2735 to 4009 of the sequence
nPAISOM.sub.5275, and is represented by SEQ ID NO:60. A nucleic
acid molecule which has a nucleic acid sequence represented by SEQ
ID NO:60 is referred herein as nPAISOM.sub.1275. nPAISOM.sub.1275
encodes a linoleate isomerase protein of 424 amino acid residues
with an amino acid sequence represented herein as SEQ ID NO:61. A
protein having the amino acid sequence represented by SEQ ID NO:61
is referred herein as PPAISOM.sub.424.
[0320] The deduced molecular weight of the isomerase protein
PPAISOM.sub.424 (SEQ ID NO:61) is about 48 kDa, which is in
reasonably good agreement with the molecular weight of the purified
linoleate isomerase protein (i.e., about 50-55 kDa as previously
estimated by SDS-PAGE). The N-terminal peptide sequence of the
purified linoleate isomerase (SEQ ID NO:42) is identical to
positions 2-36 of the sequence PPAISOM.sub.424, with the exception
of two residues at positions 25 and 30, as discussed below. It
appears that the methionine residue coded by the start codon of the
ORF is removed after translation. The absence of additional
in-frame ATG codons in the up-stream DNA sequence indicates that
there is no signal peptide sequence associated with the isomerase
protein. Analysis of the amino acid sequence of PPAISOM.sub.424
(SEQ ID NO:61) shows that the unsolved residue at the position 30
of PPAISOM.sub.35 (SEQ ID NO:42) is a histidine (H), and that
residue 25 of PPAISOM.sub.35 is a glutamate (E) in stead of a
tryptophan (W). The sequences of the three internal peptide
fragments resulting from endo-LYS-C digestion of the purified
isomerase were also mapped within the deduced amino acid sequence
PPAISOM.sub.424. The peptide sequence of HPLC peak number one (SEQ
ID NO:44) matched residues 183 through 196 of PPAISOM424. The
peptide sequence of peak number three (SEQ ID NO:46) matched
residues 275 through 289 of PPAISOM.sub.424 with the only exception
at position 286. The peptide sequence of peak number two (SEQ ID
NO:45) matched the C-terminal residues (positions 416 through 424
of SEQ ID NO:61) of PPAISOM.sub.424.
[0321] A ribosome binding site-like structure (AAGGAAG), referred
to herein as SEQ ID NO:62, was found up-stream from the
translational initiation codon of the isomerase open reading frame
in nPAISOM.sub.1275. The spacing between this putative ribosome
binding site and the translation initiation codon is only 4 bases,
much shorter than the usual spacing of 6-9. Although rare, a 4-base
spacing was also found in other P. acnes genes such as the ORF B in
the sequence nPAUNKA.sub.783 (see below), and has been reported
previously, for instance, in the protoporphyrinogen oxidase gene
from P. freudenreichii (GenBank, D85417).
[0322] The (trans, cis)-10,12-linoleate isomerase open reading
frame does not show a significant homology with the (cis,
trans)-9,11-linoleate isomerase gene cloned from L. reuteri (SEQ ID
NO:17), with the N-terminal sequence of the (cis, trans)-9,
11-linoleate isomerase purified from C. sporogenes (SEQ ID NO:43),
or with the N-terminal peptide sequence of a protein purified from
Butyrivibrio that Park et al alleged to be a (cis, trans)-9,
11-linoleate isomerase (discussed in Background Section above).
[0323] A protein pattern and profile search (ProfileScan at
www.expasy.ch) was performed with the sequence PPAISOM.sub.424 (SEQ
ID NO:61) in order to detect putative functional domains in the
isomerase. The results suggested that the N-terminal domain of the
linoleate isomerase contained a putative NAD/FAD binding domain
(PROSITE Profile No. PS50205). Specifically, this domain is located
in the region spanning from amino acid residues 9-38 of SEQ ID
NO:61. The NAD/FAD binding domain with the signature sequence
Gly-Xaa-Gly-(Xaa).sub.2-Gly-(Xaa).sub.3-Ala-(Xaa).sub.- 6-Gly
(positions 1 through 21 of SEQ ID NO:73, minus four additional Xaa
residues from positions 14-17 of SEQ ID NO:73) is present in many
different enzymes, such as dihydropyrimidine dehydrogenase (FIG.
58; SEQ ID NO:74), tryptophane monoxygenase (FIG. 58; SEQ ID
NO:75), glutamate synthase (FIG. 58; SEQ ID NO:76),
6-hydroxy-L-nicotine oxidase (FIG. 58; SEQ ID NO:77), zeta-carotein
desaturase (FIG. 58; SEQ ID NO:78), phytoene dehydrogenase (FIG.
58; SEQ ID NO:79) and polyamine oxidase (FIG. 58; SEQ ID NO:80).
The consensus sequence is shown on the top of FIG. 58 (SEQ ID
NO:73), with the position of the first residue in different enzymes
shown on the left. The name of the enzymes along with their origin
and GenBank Accession Nos. are given in the lower part of the
figure. In addition to the characteristic signature residues, the
present inventors found that a lysine residue is also conserved in
this putative NAD-binding domain among the listed enzymes. The
homology of the 10,12 linoleate isomerase with other enzymes
extended slightly beyond the NAD-binding domain.
[0324] Interestingly, the (cis, trans)-9,11 linoleate isomerase
from Lactobacillus reuteri (SEQ ID NO:18) also contained a putative
FAD/NAD binding domain at positions 19 through 79 of SEQ ID NO:18.
The putative FAD/NAD binding domain in SEQ ID NO:18 aligns with the
consensus sequence of SEQ ID NO:73 as shown in FIG. 58, with a
spacer of four extra amino acid residues between the consensus
leucine at position 41 of SEQ ID NO:18 (position 13 of SEQ ID
NO:73) and the consensus glycine at position 49 of SEQ ID NO:18
(position 21 of SEQ ID NO:73). Therefore, two different linoleate
isomerases (i.e., a 9,11-linoleate isomerase and a 10,12-linoleate
isomerase) share 40% identical amino acid residues over 34 residues
(positions 11 through 44 of SEQ ID NO:61 and positions 27 through
41 and 46 through 63 of SEQ ID NO:18) if the 4-residue spacer at
positions 42-45 of SEQ ID NO:18 is excluded. Also, the putative
FAD/NAD binding domain is located near the N-terminus of both the
9,11- and the 10,12-linoleate isomerase protein sequence.
Therefore, the (cis, trans)-9, 11-linoleate isomerase from
Lactobacillus reuteri and the (trans, cis)-10,12 linoleate
isomerase from Propionibacterium acnes seem to share a putative
functional domain, despite a lack of overall sequence homology
between sequences.
[0325] The significance of the presence of a NAD/FAD binding site
in linoleate isomerase is unclear at the present. It could play an
important role in the conjugation of the double bonds even though
the isomerase does not require NAD, NADP, FAD or other cofactors
for its catalytic activity. The actual mechanism of the double bond
isomerization of linoleic acid is currently poorly understood. This
linoleate isomerase domain may be a good target for mutagenesis to
study the structure/function of the isomerase. It is important to
note that the (cis, trans)-9, 11 linoleate isomerase is a membrane
protein, with a major hydrophobic domain and some other regions of
relatively weak hydrophobicity. The major hydrophobic domain
located near the N-terminus seems to be the single putative
transmembrane domain, which is supposed to also function as an
uncleaved signal sequence for directing the protein into the
membrane. In addition, this putative transmembrane domain overlaps
the putative NAD-binding domain. On the other hand, the (trans,
cis)-10, 12 linoleate isomerase from P. acnes is a soluble protein
with a few domains of weak hydrophobicity and the putative
NAD-binding domain is located in the major hydrophobic domain at
the N-terminus. The association of the (cis, trans)-9, 11 linoleate
isomerase with membrane and the 4-residue spacer presented in the
putative NAD-binding domain may be related to a difference in
positional and geometric specificity with regard to the (trans,
cis)-10, 12 linoleate isomerase.
[0326] A BLAST 2.0 search was performed with the sequences
PPAISOM.sub.424 using the standard parameters as set forth above.
The linoleate isomerase was found to share a relatively low
homology with a variety of different enzymes. In most cases, the
homology was constrained to the short region of the putative NAD
binding site. The isomerase shares 31% identical residues and 51%
similar residues with polyamine oxidase precursor from Zea mays
(GenBank, O64411) within a region of 115 amino acid residues
(positions 8 through 122 of SEQ ID NO:61). It also shares 28%
identical residues and 42% similarresidues with tryptophan
2-monooxygenase from Agrobacterium vitis (GenBank, AAC77909.1) in
an overlapping region of 167 residues (positions 8-175 of SEQ ID
NO:61). The homology with the small subunit of the glutamate
synthase from Deinococcus radiodurans (GenBank, AE001880) was 27%
identical and 46% similar over an 88 amino acid sequence (positions
3-90 of SEQ ID NO:61). Another enzyme showing homology to linoleate
isomerase is phytoene dehydrogenase, such as the one from
Cercospora nicotianae (GenBank, P48537): 29% identical residues and
48% similar residues in an overlapping region of 163 amino acid
residues (positions 2-165-of SEQ ID NO:61). The Fe--S subunit of
the putative oxidoreductase from E. coli (GenBank, U28375) shares
11 contiguous identical amino acid residues with linoleate
isomerase residues 12 though 22. Residues 10 through 21 of the
isomerase were identical to the amino acid sequence deduced from a
segment of the incompletely sequenced Vibrio cholerae genome (The
Institute for Genomic Research, V. cholerae 6661752).
[0327] The nucleotide sequence of the isomerase coding region,
nPAISOM.sub.1275 (SEQ ID NO:60), does not show any significant
homology to the nucleotide sequence of the (cis,
trans)-9,11-linoleate isomerase cloned from L. reuteri (SEQ ID
NO:17). A BLAST 2.0 search was conducted with the nucleotide
sequence nPAISOM.sub.1275 (SEQ ID NO:60) using the standard
parameters as set forth in the detailed description above. The
isomerase gene showed no significant homology to other genes in the
database (i.e., GenBank). The closest sequence sharing some
homology with SEQ ID NO:60 was a phytoene dehydrogenase gene from
Cercospora nicotianae (GenBank Accession No. U03903), which had
only 30.1% similarity (i.e., not identity) with SEQ ID NO:60. The
isomerase has 22 contiguous nucleotides (positions 393 through 414
of SEQ ID NO:60) identical to Vicia faba
UDP-glucose:D-fructose-2-glucosyltransferase coding sequence
(GenBank, M97551). A different stretch of 21 contiguous nucleotides
of the isomerase (positions 633-653) was found in a segment of the
incomplete Sinorhizobium meliloti genome (Stanford 382, smelil
423017E10.x1).
Example 14
[0328] The following example describes the sequencing and
characterization of other open reading frames in the nucleic acid
molecule nPAISOM.sub.5275 of P. acnes.
[0329] The open reading frame A (ORFA) in the sequence
nPAISOM.sub.5275 (SEQ ID NO:59) spans from nucleotide positions 1
to 1073 (FIG. 52, A), and is represented by SEQ ID NO:63. A nucleic
acid molecule having SEQ ID NO:63 is referred to herein as
nPALPL.sub.1073. A protein sequence of 358 amino acid residues with
an incomplete C-terminus was deduced from nPALPL.sub.1073 using the
reverse complement of SEQ ID NO:63, with positions 1073-1071
(positions with regard to SEQ ID NO:63) forming the start codon.
This protein showed some homology to lipases (see below) and is
therefore designated LPL (lipase-like). This sequence is referred
herein as PPALPL.sub.358 (SEQ ID NO:64). The protein sequence
PPALPL.sub.358 is coded on the opposite DNA strand with respect to
the open reading frame B (ORFB) and the linoleate isomerase gene.
No obvious ribosome binding site could be identified with a
reasonable spacing upstream from the first ATG codon of the open
reading frame. Therefore, the actual translation start codon could
not be determined at the present.
[0330] By BLAST 2.0 search no significant homology was found with
respect to the nucleic acid sequence nPALPL.sub.1073. A stretch of
22 contiguous nucleotides (positions 815-836 of SEQ ID NO:63) was
identical to a segment of the Bordatella pertussis RNA polymerase
sigma 80 subunit gene (Sanger 520, B. pertussis Contig54). The
BLAST 2.0 search with the sequence PPALPL.sub.358 showed that the
protein sequence shares a low but significant homology to lipases.
For example, the region spanning the positions 146-356 of SEQ ID
NO:63 shares 26% identical and 42% similar amino acid residues with
lipC from Mycobacterium tuberculosis. However, PPALPL.sub.358 does
not share significant homology to the lipase gene previously cloned
from P. acnes (GenBank Accession No. X99255). The sequence GDSAG
(positions 244-249 of SEQ ID NO:63) was conserved in many lipases
and it fits the active-site serine motif (GXSXG) which is shared by
various lipases, esterases and other hydrolytic enzymes.
[0331] A ProfileScan (protein pattern and profile search) was
carried out with the protein sequence PPALPL.sub.358. An
esterase/lipase/thioresteras- e active site (PROSITE Profile No.
PS50187) was found in the region 167-261 of SEQ ID NO:64. In
addition, the region from the positions 213 to 268 of SEQ ID NO:64
contained a carboxylesterase type-B active site (GC0265). The
sequence PPALPL.sub.358 does not contain the exact lipase prosites
(PROSITE Profile Nos. PS01173 and PS01174) that are present in the
P. acnes lipase (GenBank Accession No. X99255). From these data, it
was concluded that the open reading frame A in the sequence
nPPALPL.sub.1075 is likely to encode a lipase-like enzyme.
[0332] The open reading frame B (ORFB) (FIG. 52; B) is located
up-stream to the P. acnes isomerase gene and is coded by the same
strand as the isomerase gene. It spans the positions 1604 to 2386
of the sequence nPAISOMS.sub.5275 and is represented by SEQ ID
NO:65. A nucleic acid molecule having SEQ ID NO:65 is referred
herein as nPAUNK.sub.783. This open reading frame codes an unknown
protein of 260 amino acids residues and is designated
PPAUNK.sub.260 (SEQ ID NO:66). A putative ribosome binding site
GAAGGAG (SEQ ID NO:67) is located up-stream from the first ATG
codon, with a 4-base spacing. Therefore, this ATG codon is very
likely the actual translation initiation codon of this open reading
frame.
[0333] This open reading frame does not show a significant homology
with any sequences in GenBank or unfinished microbial genomes. The
highest BLAST score of the search with the protein sequence
PPAUNK.sub.260 was only 32 and the BLAST score for with the
nucleotide sequence nPAUNK.sub.783 was 40.
[0334] The open reading frame C (ORFC) (FIG. 52; C) is located down
stream from the linoleate isomerase gene and is coded on the same
DNA strand as the isomerase gene. It spans positions 4129 to 4710
in the sequence nPAISOM.sub.5275 and is represented by SEQ ID
NO:68. A nucleic acid molecule having SEQ ID NO:68 is referred to
herein as nPAATL.sub.582. This open reading frame may encode an
acetyltransferase-like enzyme (ATL) of 193 amino acid residues (see
below) and is denoted SEQ ID NO:69. A protein having the amino acid
sequence of SEQ ID NO:69 is referred herein as PPAATL.sub.193.
[0335] The first ATG codon of the open reading frame SEQ ID NO:68
is located 7 bases down stream from a putative ribosome binding
site GGTAGGA (SEQ ID NO:70). In a BLAST 2.0 search with the
nucleotide sequence nPAATL.sub.582, no significant homology could
be found with other sequences in the databases. However, the open
reading frame shares 38% identical and 53% similar amino acid
residues with a hypothetical protein from Streptomyces coelicolor
(GenBank AL109732) in 56 amino acid residues overlap. It also shows
a limited homology to a putative phosphoglucomutase from the plant
Arabidopsis thaliana (GenBank AC008148) with 31% identical and 47%
similar residues in 78 amino acid residues overlap.
[0336] More importantly, the protein sequence PPAATL.sub.193 (SEQ
ID NO:69) contains an acetyltransferase (GNAT) family profile
(profile document PF00583). BLAST 2.0 search also shows that it has
a low homology to three putative acetyltransferase genes in the
database. It shares 29% identical and 35% similar amino acid
residues over a region spanning positions 24-151 of SEQ ID NO:69
with the gene from Deinococcus radiodurans (GenBank AE001875). It
shares 35% identical and 47% similar residues in 55 amino acid
residues overlap with the N-terminal acetyltransferase complex,
subunit ARD1 from Methanolbacterium thermoautotrophicum (GenBank
AE000872). It shares 30% identical and 53% similar residues in 48
amino acid residues overlap with the acetyltransferase-related
protein from Thermotoga maritima (GenBank AE001774). Therefore, the
ORF C may encode an acetyltransferase-like protein.
[0337] In summary, the genomic region containing the isomerase
gene, ORF B encodes an unknown protein. The other two ORFs encode
enzymes related to lipases (ORF A) and acetyltransferases (ORF C),
respectively. Therefore, this could be a genomic region rich in is
genes related to lipid/fatty acid modifications. Without being
bound by theory, the present inventors believe that some of these
genes could be involved in the rapid metabolism of (trans, cis)-10,
12-CLA, accounting for the lack of accumulation of CLA in P.
acnes.
Example 15
[0338] The following example describes the expression of the cloned
P. acnes linoleate isomerase in E. coli.
[0339] Two oligonucleotide primers were synthesized to amplify the
complete linoleate isomerase open reading frame. The primer,
designated PA041-NdeI (SEQ ID NO:71) corresponds to positions 1-20
of the sequence nPAISOM.sub.1275 (SEQ ID NO:60). This primer
contains a NdeI site and 4 extra bases at the 5' end. The primer
PA042-stop-XhoI (SEQ ID NO:72), is specific to the lower strand of
the sequence nPAISOM.sub.1275 and spans the position 1258-1275 of
SEQ ID NO:60. It contains a XhoI site and 4 extra bases at the 5'
end.
[0340] Genomic DNA prepared from P. acnes was used as template in a
PCR reaction with the primers PA041-NdeI and PA042-Stop-XhoI. A PCR
product of expected size of about 1.3 kb was generated,
gel-purified and ligated with the expression vector pET24a(+) which
had been digested with NdeI and XhoI. The ligation products were
transferred into the E. coli host, One Shot TOP10 (Novagen).
Potential clones carrying recombinant plasmid, designated
pET-PAISOM, were analyzed by restriction digestion with NdeI and
XhoI. Plasmid DNA showing the expected restriction patterns was
transferred into an expression host, E. coli BL21 (DE3).
[0341] In the recombinant plasmid pET-PAISOM (FIG. 53), the
complete isomerase open reading frame was positioned downstream
from the T7 promoter (for transcription control). The ribosome
binding site with optimal spacing to the ATG codon was supplied by
the vector. No extra codons or codon changes were introduced to the
isomerase coding sequence.
[0342] An aliquot of a fresh overnight culture of the E. coli BL21
(DE3) cells containing the plasmid pET-PAISOM was used at a 1/100
ratio to inoculate 50 ml of LB medium supplemented with kanamycin
at the concentration of 30 .mu.g/ml. After a growth period of 3 hrs
at 37.degree. C., IPTG was added to the culture at the final
concentration of 1 mM to induce the expression of the isomerase
protein. Cells were grown for an additional period of 2 hrs after
IPTG induction.
[0343] Cells were sampled at the time of IPTG induction (0) and 2
hrs after IPTG induction (2) and the total cellular protein was
analyze by SDS-PAGE. Four different clones (A-D) of plasmid
pET-PAISOM were compared along with E. coli BL21 (DE3) cells
without the isomerase gene as control. On SDS-PAGE, a peptide of
about 50 kDa was seen as a predominant protein in the extract
prepared from IPTG induced cells hosting pET-PAISOM (FIG. 54). This
is consistent with the prediction made from the amino acid sequence
and also with the size of the native linoleate isomerase purified
from P. acnes. Without IPTG induction, the expression of the
isomerase protein was very low. The 50-kDa protein band was not
seen in the cell lysate of BL21 (DE3) without the isomerase gene.
The data demonstrate a very high level expression in E. coli of the
cloned linoleate isomerase protein with expected size.
Example 16
[0344] The following example describes the functional confirmation
of the cloned linoleate isomerase.
[0345] To confirm the identity of the cloned isomerase gene, crude
cell extracts prepared from E. coli cells expressing the isomerase
protein were tested for isomerase enzyme activity by
biotransformation of linoleic acid. The IPTG-induced culture of E.
coli BL21 (DE3) cells hosting pET-PAISOM were harvested by
centrifugation at 10,000 g at 4.degree. C. The cell pellet was
suspended into 5 ml of a lysis buffer (100 mM Tris-HCl, pH5.8; 10
mM NaCl, 10% glycerol). Cells were broken by a single pass through
French Press (10,000 psi). The crude extract (cell lysate) was
immediately incubated with 350 ppm of linoleic acid at room
temperature with shaking at 150 rpm for one hour or overnight.
Aqueous samples taken from enzyme reactions were extracted with 1
ml hexane. The hexane layer was removed and the absorbence at 234
nm was measured using a HP 8452A diode array spectrophotometer.
Typically the entire spectrum in the 190-400 nm region was scanned
to ensure that an actual peak at 234 nm was present. FIG. 55 shows
the UV spectrum of such an extract. An absorbence peak at 234 nm
was observed, as is typical of conjugated linoleic acids.
[0346] In order to determine which of the CLA isomers was produced
by the recombinant isomerase, methyl esters were prepared and
analyzed by gas chromatography. Fatty acid methyl esters (FAMES)
were formed by treatment with 4% HCl in methanol for 30 min at room
temperature, followed by extraction with hexane. FAMES were
analyzed by gas chromatography using a HP 6890 model chromatograph
fitted with a flame ionization detector. The detector and injector
were held at 250.degree. C. After splitless injection, the column
(Supelco SP-2380, 100 m, 0.25 mm ID) was held at 155.degree. C. for
15 min, followed by an increase to 180.degree. C. at a rate of
1.degree. C./min. After a 30-min hold at 180.degree. C., the
temperature was increased to 220.degree. C. at 10.degree. C./min,
and held at 220.degree. C. for 5 min. The separation of a typical
commercial CLA mixture is shown in FIG. 56A. Chemically synthesized
CLA consists of a large number of structural and geometric isomers,
most of which can be resolved on a 100-m column. The peak at 62.414
min represents the (trans, cis)-10, 12-CLA isomer. FIG. 56B shows a
typical analysis of a reaction mixture using crude cell extract
containing the cloned isomerase. The only CLA peak observed elutes
at essentially the same retention time as the (trans, cis)-10,
12-CLA isomer.
[0347] To further elucidate the double bond positions of the CLA
molecules produced by the cloned isomerase, a DMOX
(2-alkenyl-4,4-dimethyloxazoline- ) derivative was made and
analyzed by gas chromatography-electron mass spectrometry. DMOX
derivatives were formed by refluxing fatty acids with 500 .mu.l
2-amino-2-methyl-1-propanol under nitrogen for 18 hrs at
160.degree. C. After cooling, 5 ml water and 1 ml hexane were
added, shaken, and the hexane layer removed for analysis. Samples
were analyzed using a HP 5890A gas chromatograph fitted with a HP
5970 MS quadropole mass spectrometer. The injector and detector
were held at 300.degree. C. Splitless injection was made onto a
Restec Rtx-5MS column (15 m, 0.25 mm ID). The oven temperature was
initially 150.degree. C., and was increased to 250.degree. C. at
10.degree. C./min with a final 10 min hold at this temperature.
FIG. 57 shows the mass spectrum observed from a DMOX derivative of
the putative (trans, cis)-10, 12-CLA isomer formed by using the
cloned isomerase. The fragmentation pattern clearly demonstrates
unsaturations at positions 10 and 12, consistent with the other
data already presented.
[0348] All of the biochemical data presented herein (i.e., the UV
spectra, retention time on GC, and GC-MS spectra of DMOX derivative
of the product of linoleic acid conversion) support the conclusion
that the cloned isomerase gene from P. acnes indeed encodes a
(trans, cis)-10, 12-linoleic acid isomerase.
Example 17
[0349] The following example describes the purification and
characterization of a linoleate isomerase from Clostridium
sporogenes.
[0350] 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 will be tested for
functionality. It will also be compared to the isomerase genes
cloned from L. reuteri and P. acnes.
[0351] 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 of
growth. The cell pellet was washed with 0.1 M Tris, pH 6.0,
containing 15 mM NaCl.
[0352] Biotransformation of linoleic acid to 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. (D & D). A rapid production of cis9,
trans11-CLA with a simultaneous decrease in linoleic acid was
observed within 30 minutes under all conditions tested. Similar
amounts of CLA were formed under aerobic and anaerobic conditions.
Upon further incubation, t9, trans11-CLA accumulated at the expense
of cis9, trans11-CLA. Upon extended incubation (15-20 hours), cis9,
trans11-CLA disappeared. Apparently the CLA was metabolized
further.
[0353] The cells were extracted as described in FIG. 24 to give
four principal fractions. Tables 2 and 3 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 Specific Activity (OD.sub.234/60 min/mg Fraction Protein
(mg/ml) Total Protein (mg) *OD.sub.234 protein) Crude Extract 6.8
408 0.26 .+-. 0.01 0.38 45K Supernatant 4.8 288 0.12 .+-. 0.01 0.25
45K OTGP Soluble 1.2 30 0.05 .+-. 0.00 0.41 10K OTGP Soluble 2.9 73
0.05 .+-. 0.01 0.17 *OD.sub.234 was determined in an assay using
0.1 ml of enzyme extract
[0354]
4TABLE 3 Linoleate Isomerase Activity - High Salt Extracts of Fresh
Cells Specific Activity (OD.sub.234/60 min/mg Fraction Protein
(mg/ml) Total Protein (mg) *OD.sub.234 protein) Crude Extract 6.0
390 0.66 .+-. 0.02 1.10 45K Supernatant 6.0 390 0.31 .+-. 0.02 0.51
45K OTGP Soluble 0.8 20 0.16 .+-. 0.01 20.0 10K OTGP Soluble 2.0 44
0.25 .+-. 0.01 12.5 *OD.sub.234 was determined in an assay using
0.1 ml of enzyme extract
[0355] Temperature sensitivity of the isomerase in crude extracts
was tested 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 containing 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).
[0356] The concentration of linoleic acid was tested from 0 to 100
ppm (FIG. 26). The optimum concentration for linoleic acid 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).
[0357] 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.
[0358] The detergent soluble fraction was further purified by
Method A, B or D, infra.
[0359] Method A
[0360] 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 1M
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 cis9, trans11-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).
[0361] A rapid spectrophotometric assay for CLA, measuring
absorbence at OD.sub.234 (detects conjugated double bonds in fatty
acids 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 enzyme activity.
[0362] 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.
[0363] 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, OTGP (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% OTGP. Less total protein was solubilized with a mixture of
0.1% Triton X-100 and 0.3% OTGP. The efficiency of solubilization
by 1 M NaCl alone was very low, indicating that the isomerase is an
integral membrane protein.
[0364] Method B
[0365] 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 the isomerase. None provided
measurable benefit.
[0366] 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).
[0367] 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).
[0368] 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.
[0369] 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).
[0370] 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).
[0371] 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 in crude extracts for one week. However, half-life of
detergent solubilized isomerase was only three and six days in Tris
and phosphate buffer, respectively.
[0372] 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. SDS-PAGE analysis of the
protein from this column showed a band corresponding to a molecular
weight of approximately 70 kD (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.
[0373] 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.
[0374] 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 Triton X-100
combined with a low salt concentration resulted in lower protein
extraction from the membrane pellet, but similar enzyme activity,
and eliminates the requirement for the desalting step.
[0375] 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 Activity Specific Activity Sample
(*OD.sub.234) Protein (mg/ml) (OD.sub.234/60 min/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 *OD.sub.234 was
determined in an assay using 0.1 ml of enzyme extract
[0376] 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.
[0377] Method C
[0378] 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 during further purification. The
membrane fraction was solubilized with 1.5% OG in 50 mM potassium
phosphate buffer, pH7.5. OG solubilized proteins was dialyzed
against 20 mM potassium phosphate buffer, pH 7.5, 10 mM NaCl, 2 mM
dithiothreitol and 0.3% OTGP. After 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.
[0379] 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 cis9, trans11-CLA, as determined by GC analysis of
methyl ester products. Peak II was chosen for further
purification.
[0380] 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 the pooled chromatofocusing fractions
with high isomerase-activity revealed two major protein 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
[0381] Method D.
[0382] The pooled chromotofocusing fractions with high isomerase
activity were further purified using gel filtration. This method
separates the proteins by size. For isomerase purification, the gel
filtration was carried out using a 1.6.times.55 cm Superdex-200
column. The elution buffer was composed of 100 mM K-phosphate, pH
7.5, containing 0.1-0.3 M NaCl, 013% OTGP, and 10% glycerol. Assay
of isomerase activity in different fractions revealed a single peak
of isomerase activity. SDS-PAGE analysis showed a single protein
band after staining with Coomassie Blue of about 45 kD.
[0383] The purification of the C. sporogenes 9,11-linoleate
isomerase is summarized in Table 1C.
7TABLE 1C Clostridium sporogenes Step Protein Total Activity
Specific Activity Yield Crude extract 570 653 1.1 100.0 OG extract
157 470 3.0 72.0 DEAE-5PW 12 132 11.0 20.2 Chromatofocusing 0.677
64 94.6 9.8 Gel-Filtration 0.030 10.5 350.0 1.6 Protein in
milligrams. Enzyme activity units are nanomoles CLA formed per
minute. Specific activity is units per milligram protein.
Example 18
[0384] The following example describes the sequencing of the
N-terminal amino acid sequence of the purified C. sporogenes
integral membrane (cis, trans)-9, 11-linoleate isomerase.
[0385] Clostridium isomerase was purified using multi-steps of the
chromatography method (Example 17). This purified protein showed a
single band of an approximate mass of 45 kD on SDS gel and was used
for N-terminal sequencing. The first attempt to obtain N-terminal
amino acid sequence suggested the protein was N-terminally blocked.
It has been estimated that 40-70% of all proteins are N-terminally
blocked either in the native form in the cell or as a result of
artificial events during protein extraction and purification. To
limit the possibility of an artificial N-terminal block, a protein
sample that did not go through the final step of gel filtration was
used. This protein sample had a very high isomerase activity and
contained predominantly a 45 kD protein by SDS PAGE analysis. After
blotting onto a PVDF (polyvinylidene difluoride) membrane, the 45
kD band was excised and used for sequencing. A sequence of 21 amino
acid residues was determined as follows:
[0386] MFNLK NRNFL TLMDF TPXEI Q (SEQ ID NO:43)
[0387] A protein having the sequence of SEQ ID NO:43 is referred to
herein as PCLA.sub.21. It should be noted that since amino acid
sequencing technology is not entirely error-free, SEQ ID NO:43
represents, at best, an apparent partial N-terminal amino acid
sequence.
[0388] The sequence shows no significant homology to the linoleate
isomerase cloned from L. reuteri PYR8 (SEQ ID NO:18), nor to the
N-terminal sequences of 55 kD isomerase purified from P. acnes ATCC
6919 (SEQ ID NO:42) nor the putative 19 kD isomerase peptide
purified from Butyrivibrio fibrisolvens (Park et al., 1996; see
previous comments regarding Park et al. in Background section). The
C. sporogenes isomerase N-terminal sequence was analyzed against
sequences in the databases using Blastp program with standard
settings. The sequence was found to share 57 to 75% identical amino
acid sequences with the ornithine carbamoyltransferase isolated
from 6 different organisms, including C. perfringens (gi 1321787).
However, the Clostridium omithine carbamoyltransferase is a protein
of 37-kD--much smaller than the C. sporogenes, P. acnes, or the L.
reuteri linoleate isomerases. The Clostridium omithine
carbamoyltransferase shares no significant sequence homology with
the linoleate isomerase peptide deduced from DNA sequence cloned
from L. reuteri PYR8 or of the directly determined amino acid
sequence of the PYR8 isomerase or to the N-terminal sequence of C.
acnes isomerase. Therefore, the significance of the homology
between the Clostridium linoleate isomerase and the enzyme involved
in arginine metabolism is not clear. As discussed above with the P.
acnes isomerase, comparison with the complete isomerase sequence
will be necessary.
[0389] The entire C. sporogenes linoleate isomerase nucleic acid
and amino acid sequence are currently being derived using standard
methods in the art and as described for the P. acnes linoleate
isomerase described in Example 13. Nucleic acid sequences encoding
SEQ ID NO:43 can be deduced from the amino acid sequence by those
of ordinary skill in the art. Isolated nucleic acid molecules
comprising such nucleic acid sequences are encompassed by the
present invention.
Example 19
[0390] The following example demonstrates the enzyme activity of
the C. sporogenes linoleate isomerase.
[0391] Similar kinetic data have been developed for L. reuteri
(Example 4). In this experiment, all kinetic experiments were
performed in a quartz cuvette (1-cm light-path with a magnetic
stirrer) at room temperature using a HP 8452A diode array
spectrophotometer. The cuvette was filled with 1 ml of incubation
buffer containing 50 .mu.g protein, 0.1 M potassium phosphate,
pH7.5, 10 mM NaCl and 10% 1,2-propane diol. The isomerization was
initiated by adding a small aliquot (1-2 .mu.l) of freshly diluted
substrate-stock (.about.20 .mu.M linoleic acid) in 1,2-propane diol
at 50 seconds. The increase in absorbence at 234 nm was monitored
and recorded. FIG. 43 shows a typical progress curve for the
enzymatic catalyzed conversion of linoleic acid to CLA versus time.
The amount of product formed was calculated based on a molar
extinction coefficient of 24,000.
[0392] Using partially purified enzyme (a mixture of DEAE active
fractions), the following C. sporogenes linoleate isomerase kinetic
parameters were characterized: substrate specificity, inhibitors of
the enzyme activity, pH, cofactors, and the effect of various fatty
acids and their derivatives on catalysis.
[0393] pH Dependence of the Isomerization
[0394] The effect of pH on the isomerase activity was determined
using a series of potassium phosphate buffers (0.1M) at different
pH values. A plot of isomerization versus pH for C. sporogenes
linoleate isomerase is shown in FIG. 44. The optimum pH for
isomerization is about pH 7.5, which was also the optimum for the
L. reuteri PYR8 isomerase. Therefore, pH 7.5 buffer was used for
all kinetic analyses.
8TABLE 6 Substrate specificity of the linoleate isomerase from
Clostridium sporogenes.dagger-dbl. RELATIVE SUBSTRATE ACTIVITY %
(cis, cis)-9, 12-octadecadienoic acid (18:2) (linoleic acid) 100
(cis, cis, cis)-9, 12, 15 octadecatrienoic acid (18:3) 84
(linolenic acid) (cis, cis, cis)-6, 9, 12-octadecatrienoic acid
(18:3) 77 (.gamma.-linolenic acid) (cis, cis, cis, cis)-6, 9, 12,
15 octadecatetraenoic acid 60 (18:4) (stearidonic acid) (cis,
cis)-11, 14 eicosadienoic acid (20:2) 20 (cis, cis, cis)-8, 11, 14
eicosatrienoic acid (20:3) 0 (cis, cis)-13, 16 docosadienoic acid
(22:2) 0 (cis, cis)-9, 12-octadecadien-1-ol (18:2) (linoleyl
alcohol) 0 (cis, cis)-linoleic acid methyl ester(18:2) 0 (methyl
linoleate) (cis, cis)-11, 14 eicosadienoic acid methyl ester (20:2)
0 (trans, trans)-9, 12-octadecadienoic acid (18:2) 0 (linolelaidic
acid) (cis)-9:10-epoxyoctadecanoic (18:0) (epoxystearic 0 acid;
oleic acid oxide) (cis)-13-docosaenoic acid (18:1) (erucic acid) 0
(cis)-9 octadecenoic acid (18:1) (oleic acid) 0 (cis)-9
hexadecenoic acid (16:1) (palmitoleic acid) 0 .dagger-dbl.Isomerase
activity was determined with the individual substrate
concentrations fixed at 10 PPM. The activity determined with
linoleic acid (LA) alone is set as 100%. The data determined with
other substrates are presented as relative activity (percent of the
isomerase activity determined with linoleic acid as substrate).
[0395] Substrate Specificity
[0396] The substrate specificity of C. sporogenes linoleate
isomerase was studied using a number of unsaturated fatty acids and
their esterified or alcoholized derivatives as substrate. Substrate
specificity trends are summarized in Table 6. The isomerase shows a
definite preference towards substrates containing "Z" double bonds
at the 9, 12 position of C18 fatty acids. Compounds that possess
additional double bonds are also good substrates, but the turnover
rate decreased with increasing number of double bonds. Among the
other dienoic acids tested (C18-C22), only (cis, cis)-11,
14-eicosadienoic acid was isomerised. This suggests that the
isomerase uses C18 and C20 unsaturated fatty acids having nine
carbon atom after the first double-bond position.
[0397] The isomerase was also incubated with linoleyl alcohol and
methyl linoleate. As shown in Table 6, alcoholized linoleic acid
and esterified linoleic acid do not serve as substrates. It is
clear that the isomerase only uses compounds that contain a free
carboxyl group. This is in agreement with the results obtained by
Kepler and his co-workers with Butyrivibrio fibrisolvens isomerase
and in our studies with the L. reuteri PLR8 and P. acnes 6919
isomerases.
[0398] Affinity to Substrates
[0399] Since the C. sporogenes linoleate isomerase showed a high
specificity for "Z" double bond compounds with a chain length of 18
carbons, the affinity of the enzyme for similar compounds (linoleic
acid, linolenic acid, r-limolenic acid and (cis, cis, cis,
cis)-6,9,12,15 octadecatetraenoic acid) was investigated.
[0400] Kinetic analysis of the substrate concentration dependency
of isomerization (FIG. 45) provided evidence that:
[0401] 1. The reaction velocity is strongly dependent on substrate
concentration; and,
[0402] 2. The substrate has an inhibitory effect at concentrations
above 20, 40, 15 and 120 .mu.M for linoleic acid, linolenic acid,
r-linolenic acid and (cis, cis, cis, cis)-6, 9, 12, 15
octadecatetraenoic acid, respectively.
[0403] FIG. 46 shows a Lineweaver-Burke plot of 1/v versus 1/[S].
Calculation from this plot yielded a Km of 11.3 .mu.M and a maximal
velocity (Vmax) of 350 .mu.mol CLA/min/mg protein for linoleic
acid. The values of Km and Vmax determinated using other C18
unsaturated fatty acids are shown in Table 7A. All of the
substrates tested showed a normal Michaelis-Menten behavior over
the range of concentrations tested. The Km value increased with
increasing number of double bonds in substrates. The enzyme had
similar Km values for the isomers with three double bonds:
linolenic acid and r-linolenic acid. From a comparison of Km,
linoleic acid is clearly the best substrate for the C. sporogenes
linoleate isomerase.
[0404] Table 7A compares key kinetic parameters of linoleate
isomerases from L. reuteri PYR8, and C. sporogenes 23272, all
isolated by the present inventors, and the B. fibrisolvens A-38
linoleate isomerase (Kepler and Tove, 1967, J. Biochem.
242:5686-5692).
9TABLE 7A Comparison of kinetic constants for linoleate isomerases
from different organisms Vmax (nmole/min/mg protein) Km (.mu.M)
Organism LA* LnA.sup..dagger-dbl. .gamma.-LnA.sup.#
.DELTA.-LA.sup.f LA* LnA.sup..dagger-dbl. .gamma.-LnA.sup.#
.DELTA.-LA.sup.f C. sporogenes 27232 350 811 236 204 11.3 22.5 23.8
18.9 L. reuteri PYR8 880 ND ND ND 8.1 ND ND ND B. fibrisolvens
A-38.sup..dagger. 55 130 ND ND 12.0 23.0 ND ND *(cis, cis)-9,
12-octadecadienoic acid (18:2) (linoleic acid) .dagger-dbl. (cis,
cis, cis)-9, 12, 15 octadecatrienoic acid (18:3) (linolenic acid) #
(cis, cis, cis)-6, 9, 12-octadecatrienoic acid (18:3)
(.gamma.-linolenic acid) f (cis, cis, cis, cis)-6, 9, 12, 15
octadecatetraenoic acid (18:4) (stearidonic acid) .dagger. Kepler
and Tove (1967) J. Biol. Chem. 242: 5686-5692 U .mu.mole CLA/min ND
Not determined
[0405] As seen in Table 7A, the kinetic data for the C. sporogenes
linoleate isomerase is qualitatively similar to other linoleate
isomerases. However, the rates of isomerization are much higher
than those reported by Kepler for unpurified Butyrivibrio
fibrisolvens isomerase (See Kepler and Tove, 1967, ibid.).
[0406] Table 7B is a summary of the kinetic data and
characterization of linoleate isomerases from P. acnes (crude
extract prior to DEAE purification as in Example 11, except that
the Vmax was determined using enzyme purified through the
chromatofocusing step as in Example 11), L. reuteri (enzyme
purified through the gel filtration step as in Example 3), and C.
sporogenes (enzyme purified through the gel filtration step as in
Example 17), all isolated by the present inventors, and the B.
fibrisolvens A-38 linoleate isomerase (crude extract; Kepler and
Tove, 1967, J. Biochem. 242:5686-5692). The substrate in these
experiments was linoleic acid.
10TABLE 7B Linoleate Isomerase Characterization Vmax Enzyme
Organism CLA Isomer.sup.1 Optimal pH Km (.mu.M) (nmol/min/mg)
Solubility.sup.2 P. acnes 10,12 7.3 17.2.sup.4 478.sup.5 S L.
reuteri 9,11 7.5 8.1.sup.5 880.sup.5 M C. sporogenes 9,11 7.5
11.3.sup.5 350.sup.5 M B. fibrisolvens.sup.3 9,11 7.2 12.4.sup.4
55.sup.4 M .sup.1CLA isomer produced. 10,12 = t10, c12-CLA; 9,11 =
c9, t11-CLA .sup.2Enzyme solubility. S = soluble; M = integral
membrane protein .sup.3Kepler & Tove 1969 = Kepler, Carol R.,
and S. B. Tove. 1969. Linoleate .DELTA..sup.12-cis,
.DELTA..sup.11trans-isomerase. Methods Enzymol. 14: 105-110.
.sup.4Crude extract .sup.5Purified enzyme
[0407] Effect of Cofactors on Isomerase Activity
[0408] Two substrates, linoleic acid (LA) and (cis, cis, cis,
cis)-6, 9, 12, -15 octadecatetraenoic acid (tetra LA), were used to
test the effect of cofactors. The reaction mixtures contained 10
PPM (LA) or 20 PPM (tetra LA) substrate, and one or more of the
following cofactors or additions: 1 mM DTT, 50 .mu.M ATP, ADP, NAD,
NADH, NADPH and CoA. As seen in Table 8 and Table 9, none of the
cofactors or additions has a strong effect on the isomerase
activity, suggesting the isomerization does not require the
addition of external cofactors or energy.
11TABLE 8 Effect of cofactors on the isomerization of linoleic acid
by the linoleate isomerase from C. sporogenes 23272 Activity
Cofactor (nmol/min) (%) None 10.6 .+-. 2.3 100 ATP 11.1 .+-. 1.4
105 ADP 11.1 .+-. 1.7 105 NAD 10.8 .+-. 1.3 102 NADH 10.5 .+-. 1.0
99 NADPH 11.1 .+-. 1.5 105 DTT 12.8 .+-. 3.8 121
[0409]
12TABLE 9 Effect of cofactors on the isomerization of (cis, cis,
cis, cis)-6, 9, 12, 15 octadecatetraenoic acid Activity Cofactor
(nmol/min) (%) None 6.2 100 CoA 6.5 105 ATP 6.1 98 ADP 5.7 92 NAD
5.6 90 NADH 6.4 103 NADPH 6.3 102 DTT 5.9 95 DTT + ATP 6.2 100
[0410] Effect of Fatty Acids and Their Derivatives on Linoleic Acid
Isomerization
[0411] The effect of fatty acids and their derivatives was
investigated. The concentration of fatty acids was fixed at 35
.mu.M. Table 10 shows the summary of the data. The results
demonstrated that:
[0412] 1. The saturated fatty acids tested apparently do not affect
activity of the C. sporogenes linoleate isomerase;
[0413] 2. Isomerase activity is strongly inhibited by all of the
unsaturated fatty acids studied; and,
[0414] 3. Ester derivatives of linoleic acid carboxyl group, methyl
linoleate and linoleyl alcohol are also inhibitors of the C.
sporogenes linoleate isomerase.
13TABLE 10 Effect of fatty acids and derivatives on the
isomerization of linoleic acid.sup.a catalyzed by C. sporogenes
linoleate isomerase RELATIVE ACTIVITY.sup.c ADDITION.sup.b (%) none
100 octadecanoic acid (18:0) (stearic acid) 107 hexadecanoic acid
(16:0) (palmitic acid) 93 (cis, cis)-9, 12-octadecadien-1-ol (18:2)
(linoleyl alcohol) 81 (cis, cis)-linoleic acid methyl ester (18:2)
(methyl linoleate) 69 (cis)-9-hexadecenoic acid (16:1) (palmitoleic
acid) 27 (cis)-9-octadecenoic acid (18:1) (oleic acid) 25 (trans,
trans)-9, 12-octadecadienoic acid (18:2) 19 (linolelaidic acid)
(cis, cis)-11, 14 ecosadienoic acid (20:2) 0 .sup.athe reaction
mixture contained 35 .mu.M substrate (LA), 50 mg partially purified
protein, 100 mM potassium phosphate buffer, pH 7.5, 10%, 1,2
propane diol and 10 mM NaCl .sup.bcompounds were added to the
reaction mixture at a concentration of 35 .mu.M the activity
determined with linoleic acid (LA) alone is set as 100%. The effect
of addition of .sup.cspecific compounds on isomerase activity is
presented as relative activity (percent of the isomerase activity
determined with linoleic acid as substrate).
[0415] Inhibitory Effect of Oleic Acid and Palmitoleic Acid
[0416] The inhibitory effect of oleic acid and palmitoleic acid was
further characterized:
[0417] 1. Both C18 and C16 unsaturated fatty acids containing a cis
double bound at the 9 carbon (counting from carboxyl end) inhibited
isomerization of linoleic acid;
[0418] 2. The isomerase activity was dramatically reduced with
increasing concentration of oleic acid (FIG. 47); in the presence
of 70 .mu.M oleic acid (20 PPM), the isomerase activity was almost
completely lost; and,
[0419] 3. The inhibition constants (Ki) for oleic acid and
palmitoleic acid, calculated from secondary plots (1/v Vs [S]), are
23.8 and 33.1 .mu.M, respectively (FIGS. 48 and 49).
[0420] Kepler and Tove and co-workers reported that oleic acid
competitively inhibited isomerization of linoleic acid to CLA
(Kepler and Tove, 1967, ibid.). To determine whether oleic acid is
a competitive inhibitor of the C. sporogenes linoleate isomerase,
the inhibitory effect of oleic acid was further investigated using
Lineweaver-Burke (1/V versus 1/[S]) and Hanes-Woolf ([S]N versus
[S]) plots. The kinetic analyses were performed in the presence of
0, 24 and 48 .mu.M oleic acid with varied concentration of linoleic
acid. Parallel lines in a Lineweaver-Burke plot (FIG. 50) and a
common intercept in the Hanes-Woolf plot (FIG. 51) were obtained.
These data are consistent with oleic acid being an uncompetitive
inhibitor of the C. sporogenes linoleate isomerase. Similar results
were obtained with the L. reuteri PYR8 (cis, trans)-9, 11-linoleate
isomerase. These results contrast with the competitive inhibition
by oleic acid reported for the B. fibrisolvens (cis, trans)-9,
11-linoleate isomerase.
Example 20
[0421] The following example describes the optimization of growth
conditions for L. reuteri PYR8.
[0422] 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.
[0423] 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.
[0424] 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 beefextract, 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 11.
14TABLE 11 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
[0425] The vitamin mixture contained riboflavin, pantothenic acid,
pyridoxal, nicotinic acid, folic acid, choline chloride, biotin and
thiamine.
[0426] 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/l 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.
[0427] 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
12.
15TABLE 12 Nitrogen Sources Evaluated as Medium Components Nitrogen
Source Name Type Manufacturer 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
Lactalbumin Quest 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 Protein Red Star Amberferm 4002 Acid
Hydrolyzed Vegetable Protein Red Star Blend Amberferm Enzyme
Hydrolyzed Soy Protein Red Star 4015G 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
[0428] 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.
[0429] 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.
[0430] 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.
[0431] The medium with 30 g/l yeast extract, 10 g/l Hy-Soy and 30
g/l glucose was chosen for further optimizations steps.
[0432] 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.
[0433] 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.
[0434] 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.
[0435] 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 21
[0436] The following example describes the determination of
conditions to improve enzyme stability and performance and on
testing the limitations of the biotransformation process. Whole
cells of L. reuteri PYR8 were used in all biotransformation
experiments described below.
[0437] 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.
[0438] 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.
[0439] 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.
[0440] 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.
[0441] 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.
[0442] 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.
[0443] 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 coinhibitors of the 9,11 isomerase than the product
itself.
[0444] 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).
[0445] 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.
[0446] 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.
[0447] 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.
[0448] 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 22
[0449] The following example describes a preferred
biotransformation protocol.
[0450] 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 23
[0451] The following example describes the biotransformation of
linoleic acid to 10,12 CLA with P. acnes whole cells.
[0452] 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.
[0453] 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.
[0454] 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).
[0455] 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.
[0456] 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
80 1 35 PRT Lactobacillus reuteri UNSURE (30) Xaa = any amino acid
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 unsure
(1)..(29) n = a, c, g, or t 2 cgtgaattca tgtaytayws naayggnaa 29 3
27 DNA Lactobacillus reuteri unsure (1)..(27) n = a, c, g, or t 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 6
ggtcgagcaa aggcttc 17 7 17 DNA Lactobacillus reuteri 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) Xaa = any amino acid 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 12
ccaattccag gacgatc 17 13 19 DNA Lactobacillus reuteri 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 (1)..(726) n = a,
c, g, or t 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 UNSURE (1)..(242) 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 23 aatctagtga tggttctc 18
24 18 DNA Lactobacillus reuteri 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 (1)..(7113) n = a, c,
g, or t 26 gtcgactgga gttctggttt gctatgatta aggttgttac gattattgca
atgattattc 60 ttggtttact ggttatcgtt cttgggttag gtaataactg
gcacccagtt gggatttcta 120 atttgtggtc tcatggcgga ttctttaccg
gtggctttat gggctttatg ttctcgctat 180 ctgtgattgc tggttcttat
cagggaattg agttattggg aatcactgct ggtgaagctg 240 aatcaccacg
tcatgcgatt gtgaaatcag ttaagtccgt tatctggcgg atcttaatct 300
tctatattgg tgcaattttc gtcattgttt ctatttaccc atggaacgaa ttgaagtccg
360 ttggctcacc attcgttgaa accttcacga aggttggaat tactggagca
gccggaatca 420 ttaactttgt tgttttgacg gcagctcttt ctggagctaa
ctctggaatt tacagtgcta 480 gtcggatgtt gttcaagctt tctgttgatg
gggaagtacc aaagttcttt agtaagcttt 540 ccaagcgcgt tgttcctaat
gttgcaatcc tcacgatttc ttcctggatc ttccttggct 600 ttgtaattaa
tgaattaatg tcgattttta gttctgctgc tcaaaatatt ttcgtcattg 660
tatatagttc cagtgttctt ccagggatgg taccatggtt tatcattctc ttgtcagaac
720 ttcacttcag aaaagaacac cctgaacagc ttaaagatca tccattcaag
atgccgcttt 780 acccggctta taactacttt agtttgattg ccttgactgt
gatcttgatc ttcatgttct 840 ttaacccaga tactcgagtt tcagtatcag
ttggtgttat cttcttgatt atcatgagta 900 ttatttatcg 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 (1)..(744) n = a, c, g, or t 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
UNSURE (1)..(247) 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 38 gcagtcgacg gagttaagac tgaattag 28 39 26
DNA Lactobacillus reuteri 39 ctagtcgacg cagtttctgt catgac 26 40 32
DNA Lactobacillus reuteri 40 catatgtatt attcaaacgg gaattatgaa gc 32
41 30 DNA Lactobacillus reuteri 41 tgatcatcta taccagcagt ttctgtcatg
30 42 35 PRT Propionibacterium acnes UNSURE (30) Xaa = any amino
acid 42 Ser Ile Ser Lys Asp Ser Arg Ile Ala Ile Ile Gly Ala Gly Pro
Ala 1 5 10 15 Gly Leu Ala Ala Gly Met Tyr Leu Trp Gln Ala Gly Phe
Xaa Asp Tyr 20 25 30 Thr Ile Leu 35 43 21 PRT Clostridium
sporogenes UNSURE (18) Xaa = any amino acid 43 Met Phe Asn Leu Lys
Asn Arg Asn Phe Leu Thr Leu Met Asp Phe Thr 1 5 10 15 Pro Xaa Glu
Ile Gln 20 44 14 PRT Propionibacterium acnes 44 Lys Tyr Leu Asp Phe
Val Thr Met Met Ser Phe Ala Lys Gly 1 5 10 45 9 PRT
Propionibacterium acnes 45 Lys Asp Leu Val Thr Arg Phe Phe Val 1 5
46 15 PRT Propionibacterium acnes UNSURE (2) Xaa = Ile or Ser 46
Lys Xaa Ile Xaa Gln Xaa Tyr Met Val Xaa Ala Xaa Leu Val Lys 1 5 10
15 47 20 DNA Artificial Sequence Description of Artificial
Sequenceprimer 47 atcgcgatna tnggngcngg 20 48 20 DNA Artificial
Sequence Description of Artificial Sequenceprimer 48 ccngcytgcc
anarrtacat 20 49 62 DNA Propionibacterium acnes 49 atcgagatva
trggggctgg cccggccggg ctggctgccg gaatgtacct ctggcargcs 60 gg 62 50
21 PRT Propionibacterium acnes UNSURE (2) xaa = ala or glu 50 Ile
Xaa Xaa Xaa Gly Ala Gly Pro Ala Gly Leu Ala Ala Gly Met Tyr 1 5 10
15 Leu Trp Gln Ala Gly 20 51 17 DNA Artificial Sequence Description
of Artificial Sequenceprimer 51 gggccagccc cyatnat 17 52 18 DNA
Artificial Sequence Description of Artificial Sequenceprimer 52
gctggctgcc ggaatgta 18 53 569 DNA Propionibacterium acnes 53
ggatcccaac tggccgcctg ccccggggga ggagtaccac gccgacatcg aaggcaacaa
60 tgcccgtaac gggtggaccg aggacacccc ggccgtcaat gatgcccagg
ccgagcggcg 120 ggccaaggag ctggcagcac atctcgatga gatggcacgt
ggtcggcgaa ctgcccgctg 180 agatgtttcg cgacctatac cattaccgac
cccattcatc gccgaactta ttcaccacta 240 catcgacaag gaagaacgat
gtccatctcg aaggattcac gtatcgccat catcggggct 300 ggcccggccg
ggctggctgc cggaatgtac ctcgaacagg ccggatttca cgactacacg 360
atcctggaac gcaccgacca cgtcggaggc aagtgccact caccgaacta ccacggccgt
420 cgttatgaga tgggggccat catgggcgtc cccagttacg acaccatcca
ggagatcatg 480 gatcgcactg gcgacaaggt cgacgggccg aaactgcgtc
gcgagttcct gcacgaggac 540 ggcgagatct acgtcccgga aaaggatcc 569 54
104 PRT Propionibacterium acnes 54 Met Ser Ile Ser Lys Asp Ser Arg
Ile Ala Ile Ile Gly Ala Gly Pro 1 5 10 15 Ala Gly Leu Ala Ala Gly
Met Tyr Leu Glu Gln Ala Gly Phe His Asp 20 25 30 Tyr Thr Ile Leu
Glu Arg Thr Asp His Val Gly Gly Lys Cys His Ser 35 40 45 Pro Asn
Tyr His Gly Arg Arg Tyr Glu Met Gly Ala Ile Met Gly Val 50 55 60
Pro Ser Tyr Asp Thr Ile Gln Glu Ile Met Asp Arg Thr Gly Asp Lys 65
70 75 80 Val Asp Gly Pro Lys Leu Arg Arg Glu Phe Leu His Glu Asp
Gly Glu 85 90 95 Ile Tyr Val Pro Glu Lys Asp Pro 100 55 17 DNA
Artificial Sequence Description of Artificial Sequenceprimer 55
cgatgtcggc gtggtac 17 56 18 DNA Artificial Sequence Description of
Artificial Sequenceprimer 56 tcacgtatcg ccatcatc 18 57 17 DNA
Artificial Sequence Description of Artificial Sequenceprimer 57
aatccggcct gttcgag 17 58 17 DNA Artificial Sequence Description of
Artificial Sequenceprimer 58 aggacggcga gatctac 17 59 5275 DNA
Propionibacterium acnes 59 gccgggcggg cacgattgac gaatttccgc
acactggatg gcggaacaaa ggtgtcgtga 60 tttccctgga tcaccattgt
tggtggtgcc tgaggagtga tccaggtgga actgttgaca 120 gcgcgataac
ggtcggggaa ttgcttgggg gtgccaccga tatacatttt ggcggcattg 180
cccgcgctca gtgtggtgac cgactcgacg gtaccgacat ccaccgttgg atagagggcg
240 aggactgact tcggggcccg tattgagccg caggaactct tcaactttcc
actggcggcg 300 ccgtaggcga gattaatggc cattccacca ccagcggaat
cacccatgat cgatacctgt 360 gaagggtcgc caccgagttc tttcacgtgg
gacaggctcc aggcccaggc acatgcgacc 420 tgttttgggg cggtattcca
ggtggggtgg ccctgggtgg ccagggtgta cgaggggcga 480 atgactaacc
agccatgatc ggaaaaccat ctcaacgtgg cgggcatggt ggcgtcggtg 540
ctccatcctt caccatgaat gtcgacaagt accggggcat tgtggttatg ggcacggtag
600 atctgggccg tctcgtcagg gccggatcca taccggaccg tttcgtcagg
gtggtcggac 660 atcgacgaca ccgcagctgc cgagacgacg ttgatacgtc
caccggggcg gtccgtgatc 720 cacgccgtcg tcgccgttgc cgccactggc
acgatgaggg ccatcaccga gaagacaacg 780 gccaccactc gcagaccacc
tcgtcccaaa agagcgagga cgaaggcgat gacggcgatg 840 accagagccg
gtacagccaa cgatcccacc agaacggagg agatgaaggt gagggcattg 900
tgtgagggga ggatcgcggc cactgaccac gccagtaccg gcagggtcag gatcagcccg
960 acgagaccgg aagtgatgcg tagccaggaa tgacgggagg ttttcgtgtc
agccacgcgt 1020 ccaccgtact cacgggacat ggtcgatagg atcttcgcgc
aggagggacc catggctatc 1080 aggatcagac aggttgctac cgaagacagg
ccggccgtcg cgcggatgtc ccacggcgag 1140 tatgtttggc tggctgtcga
caccgagtca gtcgttgatg gccttctcta ggtcttcgga 1200 ttcgcctggg
ccggggtgca ccttgacgaa cctactccgc tggaactgga aagtctctac 1260
gtccgttctc gtctgtacgg caccgggctc ggccaggccc tcatgaatac cgtcatcggc
1320 gattcccggg cctatgtcat ggtctatccc gacaacaccc aggccaaggc
attctaccgc 1380 cgtaacggat tctctcccga tggtcatctc gacgattacc
gcgacgagga tccggcctac 1440 gtcctggagt gctggattcg ctgaatcccc
ttggttcttg ctcgcgacaa gctaggataa 1500 attaaattta tttatttctt
gtgtcgatgc gccacgacga cgtagtggga ccggctcagg 1560 ggatgacgac
ccggtcccgg gccgtgagtc acgaaggagt gccatgtcca taacaccacg 1620
aaagtgcaag gctgccgccc ttgccacagc gccggtggcc gctgccctcg gtgcttacgg
1680 atttcttaaa ggggcgacga agttctattc cagccaggtt aacggaactc
ccgagcagta 1740 caagatgacc cttcctggtg acgacctcgt cccggaaggt
tcgccgcgct tcaagcgcct 1800 cacccatgtg gaggatctcg acgccccctg
cgacgaggtc tggaagcacg tctaccagct 1860 caacaccacg accgccggct
tctactcctt caccttcttc gagaagatgt tcggactgtc 1920 ggtcgacaac
accttcatgg tggaacaggc ttggcaggcc ccggactact acaagcccgg 1980
tgacatgttc tgttggagtt acgccggttt cggtgccgag gtcgccgaca tggtccccgg
2040 caagtatctg gtgtggttcg ctgacacccg tgacggcacc aggacaccgg
gcgcaagttt 2100 cctgctaccg cctggaatgc cgtggaaccg ctggagttgg
gtcatcgccc tggaacccct 2160 cgacagtggc aaccggacgc gcatctactc
ccggtggaac atctcggcct ccgaggagtc 2220 cagtccgatc tcggtcttcc
tcatggatct ggtcatgatg gacggcggcg gcatggtgaa 2280 ccgtcggatg
ttccaagggc tggagaaggc tgccgtcgga actgctcgca agaacatcgt 2340
tcctgcgcgc ctatcagcgg ttcatgggca agtcctacgg cactgacgac gacctgcagt
2400 accgcgttcc gtacccggag atccgctggt cccgcgactt ccctcgagtg
gccagcgaac 2460 gggcctcctt caccgaggat cccaactggc cgcctgcccc
gggggaggag taccacgccg 2520 acatcgaagg caacaatgcc cgtaacgggt
ggaccgagga caccccggcc gtcaatgatg 2580 cccaggccga gcggcgggcc
aaggagctgg cagcacatct cgatgagatg gcacgtggtc 2640 ggcgaactgc
ccgctgagat gtttcgcgac ctataccatt accgacccca ttcatcgccg 2700
aacttattca ccactacatc gacaaggaag aacgatgtcc atctcgaagg attcacgtat
2760 cgccatcatc ggggctggcc cggccgggct ggctgccgga atgtacctcg
aacaggccgg 2820 atttcacgac tacacgatcc tggaacgcac cgaccacgtc
ggaggcaagt gccactcacc 2880 gaactaccac ggccgtcgtt atgagatggg
ggccatcatg ggcgtcccca gttacgacac 2940 catccaggag atcatggatc
gcactggcga caaggtcgac gggccgaaac tgcgtcgcga 3000 gttcctgcac
gaggacggcg agatctacgt cccggaaaag gatccagtgc gtggtccgca 3060
ggtcatggca gcagtgcaga agctgggcca gttgctcgcg acgaagtacc agggatatga
3120 cgccaacggc cactacaaca aggttcacga ggacctcatg ctgcccttcg
acgagttcct 3180 cgccctcaac gggtgcgagg ccgcccgaga cctgtggatc
aaccccttca cggccttcgg 3240 ctacgggcac ttcgacaacg tcccggccgc
ctacgtgctg aagtacctcg acttcgtcac 3300 catgatgtcc tttgccaagg
gagatctgtg gacgtgggcc gacggcaccc aggcgatgtt 3360 cgagcacctc
aacgccaccc tggagcaccc ggccgaacgc aacgttgaca tcactcgcat 3420
cacccgcgag gacggcaagg tccacattca caccacggac tgggatcgcg agtccgacgt
3480 cctcgtcctc accgtcccgc tggaaaagtt cctcgactac tccgacgcgg
acgatgacga 3540 gcgggagtac ttctcgaaga tcatccacca gcagtacatg
gtggatgcct gcctggtgaa 3600 ggagtacccg accatctccg ggtacgtccc
cgacaacatg aggcccgaac gtctcgggca 3660 cgtcatggtt tactaccacc
gctgggctga tgatccgcac cagatcatca cgacctacct 3720 gctacgtaac
catccggact acgcggacaa gactcaggag gagtgccgcc agatggtcct 3780
cgacgacatg gagaccttcg gtcatccggt cgagaagatc atcgaggagc agacctggta
3840 ctacttcccg cacgttagct cggaggacta caaggccggg tggtacgaga
aggtcgaggg 3900 aatgcagggt cgtcgcaaca ccttctacgc cggagaaatt
atgagtttcg gtaatttcga 3960 cgaggtgtgc cactactcga aggacctggt
gacgcggttc ttcgtgtgag gtgtattccc 4020 gcattgctgc ggggatgaga
atggggggtg gtaccgggtt cggtaccacc ccccatcgac 4080 cgtcgcgaac
cgggcctctg tgaggcttcg ggccggtagg atcaggttat ggatacttca 4140
gtcaatgtcg acacgtcgtc aagaccggcg cacgaaccgg ccaccgctcc cggtcgtttc
4200 gtcgtcagag atgcctgtca cgaggacctg cctgaagccg cggctgttca
ggccgtgtgc 4260 gtccgagaga tcggccaggg ggtgatccct aatgacgtcc
ttaccgaggt cactggcccc 4320 ggtatcgtcc acaccaccat tgagcagtgg
aaccacttta tggatgatgg tgcgatcttc 4380 aagatccttg ttgatcgcct
cgatatgagg actgtcgggg ttgccatggc ccgggtctct 4440 acaagttctg
atgctcccac accgtgggag atcgcgaccc tccatgtact gccagaggcg 4500
cgaaactgcg gagcgtcaga caacctcctc gatgcttgta tcgggaaccg gtcggcctat
4560 gtgtgggtct ttgccgataa tgctcgcgcc atttcgttct accaacgcca
tgggttccac 4620 gtcgacgcgg ccgacggtgc cgttgacgat tccctcggcg
gggtagagct gcagcggctg 4680 atccgcgagg acatcatcga gtcgcagtga
tgatggatgg gtagctcccg tggctcgtcg 4740 gcatgccagc acataggtct
agcgctgcct cagccgacga tggtcctcac acatgggacg 4800 agagcttggt
ggtgtcatcc tgaatatgca gggcgacttg cttgagcttg tcttcgtggg 4860
ctcgggcatg gtgcgcgcag aaaaggagct cgccaccgtt gcgcagcgtg atgcgcacat
4920 aggcttgtgc gccgcaacga tcacaacggt ccgcagtggt aagggcttgg
tgttcgatca 4980 tcgtggtgct catgacaacc tcctccatct gaatcatcgg
atcacctact agacaaccta 5040 cgctatcgtc ggaatgttct catacgtatc
gaaagatgga tggctggggg cgaacacggt 5100 gccgggattc cgtgtcgtcg
gctgtcgata agctgccacc gtgaccatgg acaacatctc 5160 gacctcatca
gccaacagct cggaaacgcc acgtggtaag ggcgataccg tgcgcacggc 5220
gtcgactagc cgggagtacg gcgccaagaa tttattggtg ttggaggggc tcgag 5275
60 1275 DNA Propionibacterium acnes CDS (1)..(1272) 60 atg tcc atc
tcg aag gat tca cgt atc gcc atc atc ggg gct ggc ccg 48 Met Ser Ile
Ser Lys Asp Ser Arg Ile Ala Ile Ile Gly Ala Gly Pro 1 5 10 15 gcc
ggg ctg gct gcc gga atg tac ctc gaa cag gcc gga ttt cac gac 96 Ala
Gly Leu Ala Ala Gly Met Tyr Leu Glu Gln Ala Gly Phe His Asp 20 25
30 tac acg atc ctg gaa cgc acc gac cac gtc gga ggc aag tgc cac tca
144 Tyr Thr Ile Leu Glu Arg Thr Asp His Val Gly Gly Lys Cys His Ser
35 40 45 ccg aac tac cac ggc cgt cgt tat gag atg ggg gcc atc atg
ggc gtc 192 Pro Asn Tyr His Gly Arg Arg Tyr Glu Met Gly Ala Ile Met
Gly Val 50 55 60 ccc agt tac gac acc atc cag gag atc atg gat cgc
act ggc gac aag 240 Pro Ser Tyr Asp Thr Ile Gln Glu Ile Met Asp Arg
Thr Gly Asp Lys 65 70 75 80 gtc gac ggg ccg aaa ctg cgt cgc gag ttc
ctg cac gag gac ggc gag 288 Val Asp Gly Pro Lys Leu Arg Arg Glu Phe
Leu His Glu Asp Gly Glu 85 90 95 atc tac gtc ccg gaa aag gat cca
gtg cgt ggt ccg cag gtc atg gca 336 Ile Tyr Val Pro Glu Lys Asp Pro
Val Arg Gly Pro Gln Val Met Ala 100 105 110 gca gtg cag aag ctg ggc
cag ttg ctc gcg acg aag tac cag gga tat 384 Ala Val Gln Lys Leu Gly
Gln Leu Leu Ala Thr Lys Tyr Gln Gly Tyr 115 120 125 gac gcc aac ggc
cac tac aac aag gtt cac gag gac ctc atg ctg ccc 432 Asp Ala Asn Gly
His Tyr Asn Lys Val His Glu Asp Leu Met Leu Pro 130 135 140 ttc gac
gag ttc ctc gcc ctc aac ggg tgc gag gcc gcc cga gac ctg 480 Phe Asp
Glu Phe Leu Ala Leu Asn Gly Cys Glu Ala Ala Arg Asp Leu 145 150 155
160 tgg atc aac ccc ttc acg gcc ttc ggc tac ggg cac ttc gac aac gtc
528 Trp Ile Asn Pro Phe Thr Ala Phe Gly Tyr Gly His Phe Asp Asn Val
165 170 175 ccg gcc gcc tac gtg ctg aag tac ctc gac ttc gtc acc atg
atg tcc 576 Pro Ala Ala Tyr Val Leu Lys Tyr Leu Asp Phe Val Thr Met
Met Ser 180 185 190 ttt gcc aag gga gat ctg tgg acg tgg gcc gac ggc
acc cag gcg atg 624 Phe Ala Lys Gly Asp Leu Trp Thr Trp Ala Asp Gly
Thr Gln Ala Met 195 200 205 ttc gag cac ctc aac gcc acc ctg gag cac
ccg gcc gaa cgc aac gtt 672 Phe Glu His Leu Asn Ala Thr Leu Glu His
Pro Ala Glu Arg Asn Val 210 215 220 gac atc act cgc atc acc cgc gag
gac ggc aag gtc cac att cac acc 720 Asp Ile Thr Arg Ile Thr Arg Glu
Asp Gly Lys Val His Ile His Thr 225 230 235 240 acg gac tgg gat cgc
gag tcc gac gtc ctc gtc ctc acc gtc ccg ctg 768 Thr Asp Trp Asp Arg
Glu Ser Asp Val Leu Val Leu Thr Val Pro Leu 245 250 255 gaa aag ttc
ctc gac tac tcc gac gcg gac gat gac gag cgg gag tac 816 Glu Lys Phe
Leu Asp Tyr Ser Asp Ala Asp Asp Asp Glu Arg Glu Tyr 260 265 270 ttc
tcg aag atc atc cac cag cag tac atg gtg gat gcc tgc ctg gtg 864 Phe
Ser Lys Ile Ile His Gln Gln Tyr Met Val Asp Ala Cys Leu Val 275 280
285 aag gag tac ccg acc atc tcc ggg tac gtc ccc gac aac atg agg ccc
912 Lys Glu Tyr Pro Thr Ile Ser Gly Tyr Val Pro Asp Asn Met Arg Pro
290 295 300 gaa cgt ctc ggg cac gtc atg gtt tac tac cac cgc tgg gct
gat gat 960 Glu Arg Leu Gly His Val Met Val Tyr Tyr His Arg Trp Ala
Asp Asp 305 310 315 320 ccg cac cag atc atc acg acc tac ctg cta cgt
aac cat ccg gac tac 1008 Pro His Gln Ile Ile Thr Thr Tyr Leu Leu
Arg Asn His Pro Asp Tyr 325 330 335 gcg gac aag act cag gag gag tgc
cgc cag atg gtc ctc gac gac atg 1056 Ala Asp Lys Thr Gln Glu Glu
Cys Arg Gln Met Val Leu Asp Asp Met 340 345 350 gag acc ttc ggt cat
ccg gtc gag aag atc atc gag gag cag acc tgg 1104 Glu Thr Phe Gly
His Pro Val Glu Lys Ile Ile Glu Glu Gln Thr Trp 355 360 365 tac tac
ttc ccg cac gtt agc tcg gag gac tac aag gcc ggg tgg tac 1152 Tyr
Tyr Phe Pro His Val Ser Ser Glu Asp Tyr Lys Ala Gly Trp Tyr 370 375
380 gag aag gtc gag gga atg cag ggt cgt cgc aac acc ttc tac gcc gga
1200 Glu Lys Val Glu Gly Met Gln Gly Arg Arg Asn Thr Phe Tyr Ala
Gly 385 390 395 400 gaa att atg agt ttc ggt aat ttc gac gag gtg tgc
cac tac tcg aag 1248 Glu Ile Met Ser Phe Gly Asn Phe Asp Glu Val
Cys His Tyr Ser Lys 405 410 415 gac ctg gtg acg cgg ttc ttc gtg tga
1275 Asp Leu Val Thr Arg Phe Phe Val 420 61 424 PRT
Propionibacterium acnes 61 Met Ser Ile Ser Lys Asp Ser Arg Ile Ala
Ile Ile Gly Ala Gly Pro 1 5 10 15 Ala Gly Leu Ala Ala Gly Met Tyr
Leu Glu Gln Ala Gly Phe His Asp 20 25 30 Tyr Thr Ile Leu Glu Arg
Thr Asp His Val Gly Gly Lys Cys His Ser 35 40 45 Pro Asn Tyr His
Gly Arg Arg Tyr Glu Met Gly Ala Ile Met Gly Val 50 55 60 Pro Ser
Tyr Asp Thr Ile Gln Glu Ile Met Asp Arg Thr Gly Asp Lys 65 70 75 80
Val Asp Gly Pro Lys Leu Arg Arg Glu Phe Leu His Glu Asp Gly Glu 85
90 95 Ile Tyr Val Pro Glu Lys Asp Pro Val Arg Gly Pro Gln Val Met
Ala 100 105 110 Ala Val Gln Lys Leu Gly Gln Leu Leu Ala Thr Lys Tyr
Gln Gly Tyr 115 120 125 Asp Ala Asn Gly His Tyr Asn Lys Val His Glu
Asp Leu Met Leu Pro 130 135 140 Phe Asp Glu Phe Leu Ala Leu Asn Gly
Cys Glu Ala Ala Arg Asp Leu 145 150 155 160 Trp Ile Asn Pro Phe Thr
Ala Phe Gly Tyr Gly His Phe Asp Asn Val 165 170 175 Pro Ala Ala Tyr
Val Leu Lys Tyr Leu Asp Phe Val Thr Met Met Ser 180 185 190 Phe Ala
Lys Gly Asp Leu Trp Thr Trp Ala Asp Gly Thr Gln Ala Met 195 200 205
Phe Glu His Leu Asn Ala Thr Leu Glu His Pro Ala Glu Arg Asn Val 210
215 220 Asp Ile Thr Arg Ile Thr Arg Glu Asp Gly Lys Val His Ile His
Thr 225 230 235 240 Thr Asp Trp Asp Arg Glu Ser Asp Val Leu Val Leu
Thr Val Pro Leu 245 250 255 Glu Lys Phe Leu Asp Tyr Ser Asp Ala Asp
Asp Asp Glu Arg Glu Tyr 260 265 270 Phe Ser Lys Ile Ile His Gln Gln
Tyr Met Val Asp Ala Cys Leu Val 275 280 285 Lys Glu Tyr Pro Thr Ile
Ser Gly Tyr Val Pro Asp Asn Met Arg Pro 290 295 300 Glu Arg Leu Gly
His Val Met Val Tyr Tyr His Arg Trp Ala Asp Asp 305 310 315 320 Pro
His Gln Ile Ile Thr Thr Tyr Leu Leu Arg Asn His Pro Asp Tyr 325 330
335 Ala Asp Lys Thr Gln Glu Glu Cys Arg Gln Met Val Leu Asp Asp Met
340 345 350 Glu Thr Phe Gly His Pro Val Glu Lys Ile Ile Glu Glu Gln
Thr Trp 355 360 365 Tyr Tyr Phe Pro His Val Ser Ser Glu Asp Tyr Lys
Ala Gly Trp Tyr 370 375 380 Glu Lys Val Glu Gly Met Gln Gly Arg Arg
Asn Thr Phe Tyr Ala Gly 385 390 395 400 Glu Ile Met Ser Phe Gly Asn
Phe Asp Glu Val Cys His Tyr Ser Lys 405 410 415 Asp Leu Val Thr Arg
Phe Phe Val 420 62 7 DNA Propionibacterium acnes RBS (1)..(7) 62
aaggaag 7 63 1073 DNA Propionibacterium acnes 63 gccgggcggg
cacgattgac gaatttccgc acactggatg gcggaacaaa ggtgtcgtga 60
tttccctgga tcaccattgt tggtggtgcc tgaggagtga tccaggtgga actgttgaca
120 gcgcgataac ggtcggggaa ttgcttgggg gtgccaccga tatacatttt
ggcggcattg 180 cccgcgctca gtgtggtgac cgactcgacg gtaccgacat
ccaccgttgg atagagggcg 240 aggactgact tcggggcccg tattgagccg
caggaactct tcaactttcc actggcggcg 300 ccgtaggcga gattaatggc
cattccacca ccagcggaat cacccatgat cgatacctgt 360 gaagggtcgc
caccgagttc tttcacgtgg gacaggctcc aggcccaggc acatgcgacc 420
tgttttgggg cggtattcca ggtggggtgg ccctgggtgg ccagggtgta cgaggggcga
480 atgactaacc agccatgatc ggaaaaccat ctcaacgtgg cgggcatggt
ggcgtcggtg 540 ctccatcctt caccatgaat gtcgacaagt accggggcat
tgtggttatg ggcacggtag 600 atctgggccg tctcgtcagg gccggatcca
taccggaccg tttcgtcagg gtggtcggac 660 atcgacgaca ccgcagctgc
cgagacgacg ttgatacgtc caccggggcg gtccgtgatc 720 cacgccgtcg
tcgccgttgc cgccactggc acgatgaggg ccatcaccga gaagacaacg 780
gccaccactc gcagaccacc tcgtcccaaa agagcgagga cgaaggcgat gacggcgatg
840 accagagccg gtacagccaa cgatcccacc agaacggagg agatgaaggt
gagggcattg 900 tgtgagggga ggatcgcggc cactgaccac gccagtaccg
gcagggtcag gatcagcccg 960 acgagaccgg aagtgatgcg tagccaggaa
tgacgggagg ttttcgtgtc agccacgcgt 1020 ccaccgtact cacgggacat
ggtcgatagg atcttcgcgc aggagggacc cat 1073 64 358 PRT
Propionibacterium acnes 64 Met Gly Pro Ser Cys Ala Lys Ile Leu Ser
Thr Met Ser Arg Glu Tyr 1 5 10 15 Gly Gly Arg Val Ala Asp Thr Lys
Thr Ser Arg His Ser Trp Leu Arg 20 25 30 Ile Thr Ser Gly Leu Val
Gly Leu Ile Leu Thr Leu Pro Val Leu Ala 35 40 45 Trp Ser Val Ala
Ala Ile Leu Pro Ser His Asn Ala Leu Thr Phe Ile 50 55 60 Ser Ser
Val Leu Val Gly Ser Leu Ala Val Pro Ala Leu Val Ile Ala 65 70 75 80
Val Ile Ala Phe Val Leu Ala Leu Leu Gly Arg Gly Gly Leu Arg Val 85
90 95 Val Ala Val Val Phe Ser Val Met Ala Leu Ile Val Pro Val Ala
Ala 100 105 110 Thr Ala Thr Thr Ala Trp Ile Thr Asp Arg Pro Gly Gly
Arg Ile Asn 115 120 125 Val Val Ser Ala Ala Ala Val Ser Ser Met Ser
Asp His Pro Asp Glu 130 135 140 Thr Val Arg Tyr Gly Ser Gly Pro Asp
Glu Thr Ala Gln Ile Tyr Arg 145 150 155 160 Ala His Asn His Asn Ala
Pro Val Leu Val Asp Ile His Gly Glu Gly 165 170 175 Trp Ser Thr Asp
Ala Thr Met Pro Ala Thr Leu Arg Trp Phe Ser Asp 180 185 190 His Gly
Trp Leu Val Ile Arg Pro Ser Tyr Thr Leu Ala Thr Gln Gly 195 200 205
His Pro Thr Trp Asn Thr Ala Pro Lys Gln Val Ala Cys Ala Trp Ala 210
215 220 Trp Ser Leu Ser His Val Lys Glu Leu Gly Gly Asp Pro Ser Gln
Val 225 230 235 240 Ser Ile Met Gly Asp Ser Ala Gly Gly Gly Met Ala
Ile Asn Leu Ala 245 250 255 Tyr Gly Ala Ala Ser Gly Lys Leu Lys Ser
Ser Cys Gly Ser Ile Arg 260 265 270 Ala Pro Lys Ser Val Leu Ala Leu
Tyr Pro Thr Val Asp Val Gly Thr 275 280 285 Val Glu Ser Val Thr Thr
Leu Ser Ala Gly Asn Ala Ala Lys Met Tyr 290 295 300 Ile Gly Gly Thr
Pro Lys Gln Phe Pro Asp Arg Tyr Arg Ala Val Asn 305 310 315 320 Ser
Ser Thr Trp Ile Thr Pro Gln Ala Pro Pro Thr Met Val Ile Gln 325 330
335 Gly Asn His Asp Thr Phe Val Pro Pro Ser Ser Val Arg Lys Phe Val
340 345 350 Asn Arg Ala Arg Pro Ala 355 65 783 DNA
Propionibacterium acnes CDS (1)..(783) 65 atg tcc ata aca cca cga
aag tgc aag gct gcc gcc ctt gcc aca gcg 48 Met Ser Ile Thr Pro Arg
Lys Cys Lys Ala Ala Ala Leu Ala Thr Ala 1 5 10 15 ccg gtg gcc gct
gcc ctc ggt gct tac gga ttt ctt aaa ggg gcg acg 96 Pro Val Ala Ala
Ala Leu Gly Ala Tyr Gly Phe Leu Lys Gly Ala Thr 20 25 30 aag ttc
tat tcc agc cag gtt aac gga act ccc gag cag tac aag atg 144 Lys Phe
Tyr Ser Ser Gln Val Asn Gly Thr Pro Glu Gln Tyr Lys Met 35 40 45
acc ctt cct ggt gac gac ctc gtc ccg gaa ggt tcg ccg cgc ttc aag 192
Thr Leu Pro Gly Asp Asp Leu Val Pro Glu Gly Ser Pro Arg Phe Lys 50
55 60 cgc ctc acc cat gtg gag gat ctc gac gcc ccc tgc gac gag gtc
tgg 240 Arg Leu Thr His Val Glu Asp Leu Asp Ala Pro Cys Asp Glu Val
Trp 65 70 75 80 aag cac gtc tac cag ctc aac acc acg acc gcc ggc ttc
tac tcc ttc 288 Lys His Val Tyr Gln Leu Asn Thr Thr Thr Ala Gly Phe
Tyr Ser Phe 85 90 95 acc ttc ttc gag aag atg ttc gga ctg tcg gtc
gac aac acc ttc atg 336 Thr Phe Phe Glu Lys Met Phe Gly Leu Ser Val
Asp Asn Thr Phe Met 100 105 110 gtg gaa cag gct tgg cag gcc ccg gac
tac tac aag ccc ggt gac atg 384 Val Glu Gln Ala Trp Gln Ala Pro Asp
Tyr Tyr Lys Pro Gly Asp Met 115 120 125 ttc tgt tgg agt tac gcc ggt
ttc ggt gcc gag gtc gcc gac atg gtc 432 Phe Cys Trp Ser Tyr Ala Gly
Phe Gly Ala Glu Val Ala Asp Met Val 130 135 140 ccc ggc aag tat ctg
gtg tgg ttc gct gac acc cgt gac ggc acc agg 480 Pro Gly Lys Tyr Leu
Val Trp Phe Ala Asp Thr Arg Asp Gly Thr Arg 145 150 155 160 aca ccg
ggc gca agt ttc ctg cta ccg cct gga atg ccg tgg aac cgc 528 Thr Pro
Gly Ala Ser Phe Leu Leu Pro Pro Gly Met Pro Trp Asn Arg 165 170
175 tgg agt tgg gtc atc gcc ctg gaa ccc ctc gac agt ggc aac cgg acg
576 Trp Ser Trp Val Ile Ala Leu Glu Pro Leu Asp Ser Gly Asn Arg Thr
180 185 190 cgc atc tac tcc cgg tgg aac atc tcg gcc tcc gag gag tcc
agt ccg 624 Arg Ile Tyr Ser Arg Trp Asn Ile Ser Ala Ser Glu Glu Ser
Ser Pro 195 200 205 atc tcg gtc ttc ctc atg gat ctg gtc atg atg gac
ggc ggc ggc atg 672 Ile Ser Val Phe Leu Met Asp Leu Val Met Met Asp
Gly Gly Gly Met 210 215 220 gtg aac cgt cgg atg ttc caa ggg ctg gag
aag gct gcc gtc gga act 720 Val Asn Arg Arg Met Phe Gln Gly Leu Glu
Lys Ala Ala Val Gly Thr 225 230 235 240 gct cgc aag aac atc gtt cct
gcg cgc cta tca gcg gtt cat ggg caa 768 Ala Arg Lys Asn Ile Val Pro
Ala Arg Leu Ser Ala Val His Gly Gln 245 250 255 gtc cta cgg cac tga
783 Val Leu Arg His 260 66 260 PRT Propionibacterium acnes 66 Met
Ser Ile Thr Pro Arg Lys Cys Lys Ala Ala Ala Leu Ala Thr Ala 1 5 10
15 Pro Val Ala Ala Ala Leu Gly Ala Tyr Gly Phe Leu Lys Gly Ala Thr
20 25 30 Lys Phe Tyr Ser Ser Gln Val Asn Gly Thr Pro Glu Gln Tyr
Lys Met 35 40 45 Thr Leu Pro Gly Asp Asp Leu Val Pro Glu Gly Ser
Pro Arg Phe Lys 50 55 60 Arg Leu Thr His Val Glu Asp Leu Asp Ala
Pro Cys Asp Glu Val Trp 65 70 75 80 Lys His Val Tyr Gln Leu Asn Thr
Thr Thr Ala Gly Phe Tyr Ser Phe 85 90 95 Thr Phe Phe Glu Lys Met
Phe Gly Leu Ser Val Asp Asn Thr Phe Met 100 105 110 Val Glu Gln Ala
Trp Gln Ala Pro Asp Tyr Tyr Lys Pro Gly Asp Met 115 120 125 Phe Cys
Trp Ser Tyr Ala Gly Phe Gly Ala Glu Val Ala Asp Met Val 130 135 140
Pro Gly Lys Tyr Leu Val Trp Phe Ala Asp Thr Arg Asp Gly Thr Arg 145
150 155 160 Thr Pro Gly Ala Ser Phe Leu Leu Pro Pro Gly Met Pro Trp
Asn Arg 165 170 175 Trp Ser Trp Val Ile Ala Leu Glu Pro Leu Asp Ser
Gly Asn Arg Thr 180 185 190 Arg Ile Tyr Ser Arg Trp Asn Ile Ser Ala
Ser Glu Glu Ser Ser Pro 195 200 205 Ile Ser Val Phe Leu Met Asp Leu
Val Met Met Asp Gly Gly Gly Met 210 215 220 Val Asn Arg Arg Met Phe
Gln Gly Leu Glu Lys Ala Ala Val Gly Thr 225 230 235 240 Ala Arg Lys
Asn Ile Val Pro Ala Arg Leu Ser Ala Val His Gly Gln 245 250 255 Val
Leu Arg His 260 67 7 DNA Propionibacterium acnes RBS (1)..(7) 67
gaaggag 7 68 582 DNA Propionibacterium acnes CDS (1)..(582) 68 atg
gat act tca gtc aat gtc gac acg tcg tca aga ccg gcg cac gaa 48 Met
Asp Thr Ser Val Asn Val Asp Thr Ser Ser Arg Pro Ala His Glu 1 5 10
15 ccg gcc acc gct ccc ggt cgt ttc gtc gtc aga gat gcc tgt cac gag
96 Pro Ala Thr Ala Pro Gly Arg Phe Val Val Arg Asp Ala Cys His Glu
20 25 30 gac ctg cct gaa gcc gcg gct gtt cag gcc gtg tgc gtc cga
gag atc 144 Asp Leu Pro Glu Ala Ala Ala Val Gln Ala Val Cys Val Arg
Glu Ile 35 40 45 ggc cag ggg gtg atc cct aat gac gtc ctt acc gag
gtc act ggc ccc 192 Gly Gln Gly Val Ile Pro Asn Asp Val Leu Thr Glu
Val Thr Gly Pro 50 55 60 ggt atc gtc cac acc acc att gag cag tgg
aac cac ttt atg gat gat 240 Gly Ile Val His Thr Thr Ile Glu Gln Trp
Asn His Phe Met Asp Asp 65 70 75 80 ggt gcg atc ttc aag atc ctt gtt
gat cgc ctc gat atg agg act gtc 288 Gly Ala Ile Phe Lys Ile Leu Val
Asp Arg Leu Asp Met Arg Thr Val 85 90 95 ggg gtt gcc atg gcc cgg
gtc tct aca agt tct gat gct ccc aca ccg 336 Gly Val Ala Met Ala Arg
Val Ser Thr Ser Ser Asp Ala Pro Thr Pro 100 105 110 tgg gag atc gcg
acc ctc cat gta ctg cca gag gcg cga aac tgc gga 384 Trp Glu Ile Ala
Thr Leu His Val Leu Pro Glu Ala Arg Asn Cys Gly 115 120 125 gcg tca
gac aac ctc ctc gat gct tgt atc ggg aac cgg tcg gcc tat 432 Ala Ser
Asp Asn Leu Leu Asp Ala Cys Ile Gly Asn Arg Ser Ala Tyr 130 135 140
gtg tgg gtc ttt gcc gat aat gct cgc gcc att tcg ttc tac caa cgc 480
Val Trp Val Phe Ala Asp Asn Ala Arg Ala Ile Ser Phe Tyr Gln Arg 145
150 155 160 cat ggg ttc cac gtc gac gcg gcc gac ggt gcc gtt gac gat
tcc ctc 528 His Gly Phe His Val Asp Ala Ala Asp Gly Ala Val Asp Asp
Ser Leu 165 170 175 ggc ggg gta gag ctg cag cgg ctg atc cgc gag gac
atc atc gag tcg 576 Gly Gly Val Glu Leu Gln Arg Leu Ile Arg Glu Asp
Ile Ile Glu Ser 180 185 190 cag tga 582 Gln 69 193 PRT
Propionibacterium acnes 69 Met Asp Thr Ser Val Asn Val Asp Thr Ser
Ser Arg Pro Ala His Glu 1 5 10 15 Pro Ala Thr Ala Pro Gly Arg Phe
Val Val Arg Asp Ala Cys His Glu 20 25 30 Asp Leu Pro Glu Ala Ala
Ala Val Gln Ala Val Cys Val Arg Glu Ile 35 40 45 Gly Gln Gly Val
Ile Pro Asn Asp Val Leu Thr Glu Val Thr Gly Pro 50 55 60 Gly Ile
Val His Thr Thr Ile Glu Gln Trp Asn His Phe Met Asp Asp 65 70 75 80
Gly Ala Ile Phe Lys Ile Leu Val Asp Arg Leu Asp Met Arg Thr Val 85
90 95 Gly Val Ala Met Ala Arg Val Ser Thr Ser Ser Asp Ala Pro Thr
Pro 100 105 110 Trp Glu Ile Ala Thr Leu His Val Leu Pro Glu Ala Arg
Asn Cys Gly 115 120 125 Ala Ser Asp Asn Leu Leu Asp Ala Cys Ile Gly
Asn Arg Ser Ala Tyr 130 135 140 Val Trp Val Phe Ala Asp Asn Ala Arg
Ala Ile Ser Phe Tyr Gln Arg 145 150 155 160 His Gly Phe His Val Asp
Ala Ala Asp Gly Ala Val Asp Asp Ser Leu 165 170 175 Gly Gly Val Glu
Leu Gln Arg Leu Ile Arg Glu Asp Ile Ile Glu Ser 180 185 190 Gln 70
7 DNA Propionibacterium acnes RBS (1)..(7) 70 ggtagga 7 71 27 DNA
Artificial Sequence Description of Artificial Sequenceprimer 71
cagacatatg tccatctcga aggattc 27 72 29 DNA Artificial Sequence
Description of Artificial Sequenceprimer 72 ctatctcgag tcacacgaag
aaccgcgtc 29 73 53 PRT Artificial Sequence Description of
Artificial Sequenceconsensus 73 Gly Xaa Gly Xaa Xaa Gly Xaa Xaa Xaa
Ala Xaa Xaa Leu Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Gly Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Glu Xaa Xaa Xaa 20 25 30 Xaa Xaa Gly Gly Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Xaa Xaa 35 40 45 Xaa Xaa Xaa
Xaa Gly 50 74 43 PRT Homo sapiens 74 Ser Glu Ala Tyr Ser Ala Lys
Ile Ala Leu Phe Gly Ala Gly Pro Ala 1 5 10 15 Ser Ile Ser Cys Ala
Ser Phe Leu Ala Arg Leu Gly Tyr Ser Asp Ile 20 25 30 Thr Ile Phe
Glu Lys Gln Glu Tyr Val Gly Gly 35 40 75 41 PRT Agrobacterium vitis
75 Lys Val Ala Ile Val Gly Ala Gly Leu Ser Gly Leu Val Val Ala Ser
1 5 10 15 Glu Leu Leu His Ala Gly Ile Asp Asp Val Thr Leu Tyr Glu
Ala Ser 20 25 30 Asp Arg Ile Gly Gly Lys Leu Trp Ser 35 40 76 45
PRT Deinococcus radiodurans 76 Val Lys Thr Gly Lys Lys Val Ala Val
Val Gly Ser Gly Pro Ala Gly 1 5 10 15 Leu Ala Ala Ala Gln Gln Leu
Ala Arg Ala Gly His Asp Val Thr Val 20 25 30 Phe Glu Lys Asn Asp
Arg Val Gly Gly Arg Ile Glu Gln 35 40 45 77 37 PRT Arthrobacter
nicotinovorans 77 Val Val Gly Gly Gly Phe Ser Gly Leu Lys Ala Ala
Arg Asp Leu Thr 1 5 10 15 Asn Ala Gly Lys Lys Val Leu Leu Leu Glu
Gly Gly Glu Arg Leu Gly 20 25 30 Gly Arg Ala Tyr Ser 35 78 52 PRT
Synechocystis sp. 78 Arg Ile Ala Ile Ile Gly Ala Gly Leu Ala Gly
Met Ala Thr Ala Val 1 5 10 15 Glu Leu Val Asp Ala Gly His Glu Val
Glu Leu Tyr Glu Ala Arg Ser 20 25 30 Phe Ile Gly Gly Lys Val Gly
Ser Trp Val Asp Gly Asp Gly Asn His 35 40 45 Ile Glu Met Gly 50 79
57 PRT Cercospora nicotianae 79 Ser Thr Ser Lys Arg Pro Thr Ala Ile
Val Ile Gly Ser Gly Val Gly 1 5 10 15 Gly Val Ser Thr Ala Ala Arg
Leu Ala Arg Ala Gly Phe His Val Thr 20 25 30 Val Leu Glu Lys Asn
Asn Phe Thr Gly Gly Arg Cys Ser Leu Ile His 35 40 45 His Glu Gly
Tyr Arg Phe Asp Gln Gly 50 55 80 52 PRT Zea mays 80 Arg Val Ile Val
Val Gly Ala Gly Met Ser Gly Ile Ser Ala Ala Lys 1 5 10 15 Arg Leu
Ser Glu Ala Gly Ile Thr Asp Leu Leu Ile Leu Glu Ala Thr 20 25 30
Asp His Ile Gly Gly Arg Met His Lys Thr Asn Phe Ala Gly Ile Asn 35
40 45 Val Glu Leu Gly 50
* * * * *
References