U.S. patent application number 10/911160 was filed with the patent office on 2005-09-08 for proteins.
Invention is credited to de Kreij, Arno, Mikkelson, Jorn Dalgaard, Soe, Jorn Borch.
Application Number | 20050196766 10/911160 |
Document ID | / |
Family ID | 34913646 |
Filed Date | 2005-09-08 |
United States Patent
Application |
20050196766 |
Kind Code |
A1 |
Soe, Jorn Borch ; et
al. |
September 8, 2005 |
Proteins
Abstract
The present invention relates to a method of producing a variant
lipid acyltransferase enzyme comprising: (a) selecting a parent
enzyme which is a lipid acyltransferase enzyme characterised in
that the enzyme comprises the amino acid sequence motif GDSX,
wherein X is one or more of the following amino acid residues L, A,
V, I, F, Y, H, Q, T N, M or S; (b) modifying one or more amino
acids to produce a variant lipid acyltransferase; (c) testing the
variant lipid acyltransferase for activity on a galactolipid
substrate, and optionally a phospholipid substrate and/or
optionally a triglyceride substrate; (d) selecting a variant enzyme
with an enhanced activity towards galactolipids compared with the
parent enzyme; and optionally (e) preparing a quantity of the
variant enzyme. The present invention further relates to variant
lipid acyltransferase enzyme characterised in that the enzyme
comprises the amino acid sequence motif GDSX, wherein X is one or
more of the following amino acid residues L, A, V, I, F, Y, H, Q, T
N, M or S, and wherein the variant enzyme comprises one or more
amino acid modifications compared with a parent sequence at any one
or more of the following amino acid residues when aligned to SEQ ID
No. 2: Ser3, Leu17, Ala114, Trp111, Tyr117, Leu118, Pro156, Gly159,
Gln160, Asn161, Pro162, Ser163, Ala164, Arg165, Ser166, Gln167,
Lys168, Val169, Val170, Glu171, Ala172, Tyr179, Gln182, Lys187,
His180, Asn181, Met209, Leu210, Arg211, Asn215, Lys284, Met285,
Gln289, Val290, Ala309, Ser310, Lys22, Met23, Gly40, Asn80, Pro81,
Lys82, Val112, Asn87, Asn88, -318.
Inventors: |
Soe, Jorn Borch; (Tilst,
DK) ; Mikkelson, Jorn Dalgaard; (Hvidovre, DK)
; de Kreij, Arno; (Papendrecht, NL) |
Correspondence
Address: |
FROMMER LAWRENCE & HAUG
745 FIFTH AVENUE- 10TH FL.
NEW YORK
NY
10151
US
|
Family ID: |
34913646 |
Appl. No.: |
10/911160 |
Filed: |
August 2, 2004 |
Current U.S.
Class: |
435/6.11 ;
435/193; 435/320.1; 435/325; 435/6.18; 435/69.1 |
Current CPC
Class: |
C12P 7/62 20130101; C11B
3/003 20130101; A23L 33/195 20160801; C12P 7/6454 20130101; A21D
8/042 20130101; A23L 15/25 20160801; A23L 33/10 20160801; C12P
7/6481 20130101; C12P 7/6436 20130101; C12N 9/1029 20130101 |
Class at
Publication: |
435/006 ;
435/325; 435/320.1; 435/193; 435/069.1 |
International
Class: |
C12Q 001/68; C12N
009/10; C12N 015/09 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 24, 2003 |
GB |
0330016.7 |
Jul 16, 2004 |
GB |
0415999.2 |
Jan 15, 2004 |
WO |
PCT/IB04/00655 |
Claims
1. A method of producing a variant lipid acyltransferase enzyme
comprising: (a) selecting a parent enzyme which is a lipid
acyltransferase enzyme characterised in that the enzyme comprises
the amino acid sequence motif GDSX, wherein X is one or more of the
following amino acid residues L, A, V, I, F, Y, H, Q, T N, M or S;
(b) modifying one or more amino acids to produce a variant lipid
acyltransferase; (c) testing the variant lipid acyltransferase for
activity on a galactolipid substrate, and optionally a phospholipid
substrate and/or optionally a triglyceride substrate; (d) selecting
a variant enzyme with an enhanced activity towards galactolipids
compared with the parent enzyme; and optionally (e) preparing a
quantity of the variant enzyme.
2. The method according to claim 1 wherein one or more of the one
or more of the following amino acid residues identified by
alignment with SEQ ID No. 2 is modified compared with a parent
sequence SEQ ID No. 2: Ser3, Leu17, Ala114, Trp111, Tyr117, Leu118,
Pro156, Gly159, Gln160, Asn161, Pro162, Ser163, Ala164, Arg165,
Ser166, Gln167, Lys168, Val169, Val170, Glu171, Ala172, Tyr179,
Gln182, Lys187, His180, Asn181, Met209, Arg211, Asn215, Lys284,
Met285, Gln289, Val290, Ala309, Ser310, Lys22, Met23, Gly40, Asn80,
Pro81, Lys82, Val112, Asn87, Asn88, -318.
3. The method according to claim 1 wherein the parent enzyme
comprises an amino acid sequence as shown as SEQ ID No. 2, SEQ ID
No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ
ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20, SEQ ID No.
22, SEQ ID No. 24, SEQ ID No. 26, SEQ ID No. 28, SEQ ID No. 30, SEQ
ID No. 33, SEQ ID No. 34, SEQ ID No. 36, SEQ ID No. 37, SEQ ID No.
39, SEQ ID No. 41, SEQ ID No. 43 or SEQ ID No. 45, or an amino acid
sequence which has at least 70% identity therewith.
4. The method according to claim 1 wherein amino acid residue 18 of
the parent sequence identified by alignment with SEQ ID No. 2 is
substituted by one of the following amino acids A, L, M, F, W, K,
Q, E, P, I, C, Y, H, R, N, D, T.
5. The method according to claim 1 wherein amino acid residue 30 of
the parent sequence identified by alignment with SEQ ID No. 2 is by
one of the following amino acids A, G, L, M, W, K, Q, S, E, P, V,
I, C, H, R, N, D, T.
6. The method according to claim 1 wherein amino acid residue 20 of
the parent sequence identified by alignment with SEQ ID No. 2 is by
one of the following amino acids A, G, L, M, W, K, Q, S, E, P, V,
I, C, H, R, N, D, T.
7. The method according to claim 1 wherein the parent enzyme is an
enzyme which comprises the amino acid sequence shown as SEQ ID No.
2 and/or SEQ ID No. 28.
8. The method according to claim 1 wherein the X of the GDSX motif
is L.
9. The method according to claim 1 wherein the method further
comprises one or more of the following steps: structural homology
mapping or sequence homology alignment.
10. The method according to claim 9 wherein the structural homology
mapping comprises one or more of the following steps: a) aligning a
parent sequence with a structural model (1IVN.PDB) shown in FIG.
52; b) selecting one or more amino acid residue within a 10 .ANG.
sphere centred on the central carbon atom of the glycerol molecule
in the active site (see FIG. 53); and c) modifying one or more
amino acids selected in accordance with step (b) in said parent
sequence.
11. The method according to claim 9 wherein the structural homology
mapping comprises one or more of the following steps: a) aligning a
parent sequence with a structural model (1IVN.PDB) shown in FIG.
52; b) selecting one or more amino acids within a 10 .ANG. sphere
centred on the central carbon atom of the glycerol molecule in the
active site (see FIG. 53); c) determining if one or more amino acid
residues selected in accordance with step (b) are highly conserved
(particularly are active site residues and/or part of the GDSx
motif and/or part of the GANDY motif, SEQ ID NO: 54); and d)
modifying one or more amino acids selected in accordance with step
(b), excluding conserved regions identified in accordance with step
(c) in said parent sequence.
12. The method according to claim 9 wherein the sequence homology
alignment comprises one or more of the following steps: a)
selecting a first parent lipid acyltransferase; b) identifying a
second related lipid acyltransferase having a desirable activity;
c) aligning said first parent lipid acyltransferase and the second
related lipid acyltransferase; d) identifying amino acid residues
that differ between the two sequences; and e) modifying one or more
of the amino acid residues identified in accordance with step (iv)
in said parent lipid acyltransferase.
13. The method according to claim 9 wherein the sequence homology
alignment may comprise one or more of the following steps: i.
selecting a first parent lipid acyltransferase; ii. identifying a
second related lipid acyltransferase having a desirable activity;
iii. aligning said first parent lipid acyltransferase and the
second related lipid acyltransferase; iv. identifying amino acid
residues that differ between the two sequences; v. determining if
one or more amino acid residues selected in accordance with step
(iv) are highly conserved (particularly are active site residues
and/or part of the GDSx motif and/or part of the GANDY motif; SEQ
ID NO: 54); and vi. modifying one or more of the amino acid
residues identified in accordance with step (iv) excluding
conserved regions identified in accordance with step (v) in said
parent sequence.
14. The method according to claim 1 wherein one or more of the
following modifications is made to the parent enzyme: S3A, C, D, E,
F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; D157A, C, E, F,
G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; Q182A, C, D, E, F,
G, H, I, K, L, M, N, Q, P, R, S, T, V, W, or Y; A309A, C, D, E, F,
G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; Y230A, C, D, E, G,
H, I, K, L, M, N, P, Q, R, S, T, V or W; a C-terminal addition
(-318) of at least one amino acid.
15. The method according to claim 14 wherein one or more of the
following modifications is made to the parent enzyme S3T, S3N, S3Q,
S3K, S3R, S3P, S3M; D157 is substituted with a polar uncharged
amino acid; Q182 is substituted with an aliphatic amino acid
residue; A309 is substituted with an aliphatic residue; Y230 is
substituted with an aliphatic amino acid or one of the following
amino acid residues G, D, T, V, R or M; a C-terminal addition
comprising one or more of I, L or V.
16. The method according to claim 1 wherein one or more of the
following modifications is made to the parent enzyme K187D, E309A,
Y230T, Y230G, S3Q.
17. The method according to claim 1 wherein one or more of the
following modifications is made to the parent enzyme K187D, K187D,
Y230G, Y230T, Y230R, Y230M, Y230V, D157C, E309A, G2181.
18. The method according to claim 1 wherein one or more of the
following modifications is made to the parent enzyme S3K, S3R, S3Q,
S3N, S3P, S3M.
19. The method according to claim 1 wherein one or more of the
following modifications is made to the parent enzyme Y230T, K187D,
Y230G, E309A
20. A variant lipid acyltransferase enzyme characterised in that
the enzyme comprises the amino acid sequence motif GDSX, wherein X
is one or more of the following amino acid residues L, A, V, I, F,
Y, H, Q, T N, M or S, and wherein the variant enzyme comprises one
or more amino acid modifications compared with a parent sequence at
any one or more of the following amino acid residues when aligned
to SEQ ID No. 2: Ser3, Leu17, Ala114, Trp111, Tyr117, Leu118,
Pro156, Gly159, Gln160, Asn161, Pro162, Ser163, Ala164, Arg165,
Ser166, Gln167, Lys168, Val169, Val170, Glu171, Ala172, Tyr179,
Gln182, Lys187, His180, Asn181, Met209, Leu210, Arg211, Asn215,
Lys284, Met285, Gln289, Val290, Ala309, Ser310, Lys22, Met23,
Gly40, Asn80, Pro81, Lys82, Val112, Asn87, Asn88, -318.
21. The variant lipid acyltransferase enzyme according to claim 20
wherein the variant enzyme comprises an amino acid sequence, which
amino acid sequence is shown as SEQ ID No. 2, SEQ ID No. 3, SEQ ID
No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 14,
SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20, SEQ ID No. 22, SEQ ID
No. 24, SEQ ID No. 26, SEQ ID No. 28, SEQ ID No. 30, SEQ ID No. 33,
SEQ ID No. 34, SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 39, SEQ ID
No. 41, SEQ ID No. 43 or SEQ ID No. 45 except for one or more amino
acid modifications at any one or more of the following amino acid
residues identified by sequence alignment with SEQ ID No. 2: Ser3,
Leu17, Ala114, Trp111, Tyr117, Leu118, Pro156, Gly159, Gln160,
Asn161, Pro162, Ser163, Ala164, Arg165, Ser166, Gln167, Lys168,
Val169, Val170, Glu171, Ala172, Tyr179, Gln182, Lys187, His180,
Asn181, Met209, Leu210, Arg211, Asn215, Lys284, Met285, Gln289,
Val290, Ala309, Ser310, Lys22, Met23, Gly40, Asn80, Pro81, Lys82,
Val112, Asn87, Asn88, -318.
22. The variant lipid acyltransferase enzyme according to claim 20
wherein the enzyme comprises one or more of the following amino
acid modifications S18A, L, M, F, W, K, Q, E, P, I, C, Y, H, R, N,
D, T; Y30A, G, L, M, W, K, Q, S, E, P, V, I, C, H, R, N, D, T;
Y230A, G, L, M, W, K, Q, S, E, P, V, I, C, H, R, N, D, T.
23. The variant lipid acyltransferase enzyme according to claim 20
wherein the enzyme comprises one or more of the following amino
acid modifications: S3A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,
S, T, V, W, or Y; D157A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S,
T, V, W or Y; Q182A, C, D, E, F, G, H, I, K, L, M, N, Q, P, R, S,
T, V, W, or Y; A309A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,
T, V, W or Y; Y230A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V
or W; a C-terminal addition (-318) of at least one amino acid.
24. The variant lipid acyltransferase enzyme according to claim 20
wherein the enzyme comprises one or more of the following amino
acid modifications: S3T, S3N, S3Q, S3K, S3R, S3P, S3M; D157 is
substituted with a polar uncharged amino acid; Q182 is substituted
with an aliphatic amino acid residue; A309 is substituted with an
aliphatic residue; Y230 is substituted with an aliphatic amino acid
or one of the following amino acid residues G, D, T, V, R or M; a
C-terminal addition comprising one or more of I, L or V.
25. The variant lipid acyltransferase enzyme according to claim 20
wherein the enzyme comprises one or more of the following amino
acid modifications: K187D, E309A, Y230T, Y230G, S3Q.
26. The variant lipid acyltransferase enzyme according to claim 20
wherein the enzyme comprises one or more of the following amino
acid modifications: K187D, K187D, Y230G, Y230T, Y230R, Y230M,
Y230V, D157C, E309A, G218I.
27. The variant lipid acyltransferase enzyme according to claim 20
wherein the enzyme comprises one or more of the following amino
acid modifications: S3K, S3R, S3Q, S3N, S3P, S3M.
28. The variant lipid acyltransferase enzyme according to claim 20
wherein the enzyme comprises one or more of the following amino
acid modifications: Y230T, K187D, Y230G, E309A.
29. The variant lipid acyltransferase enzyme according to claim 20
wherein the variant enzyme has an enhanced ratio of activity on
galactolipids to either phospholipids and/or triglycerides when
compared with the parent enzyme.
30. The variant lipid acyltransferase enzyme according to claim 20
wherein the variant enzyme is an enzyme which comprises an amino
acid sequence, which amino acid sequence is shown as SEQ ID No. 2
or SEQ ID No. 28 except for one or more amino acid modifications at
any one or more of the following amino acid residues: Ser3, Leu17,
Ala114, Trp111, Tyr117, Leu118, Pro156, Gly159, Gln160, Asn161,
Pro162, Ser163, Ala164, Arg165, Ser166, Gln167, Lys168, Val169,
Val170, Glu171, Ala172, Tyr179, Gln182, Lys187, His180, Asn181,
Met209, Leu210, Arg211, Asn215, Lys284, Met285, Gln289, Val290,
Ala309, Ser310, Lys22, Met23, Gly40, Asn80, Pro81, Lys82, Val112,
Asn87, Asn88.
31. A method of using a variant lipolytic enzyme according to claim
20 in a substrate for preparing a lyso-glycolipid, for example
digalactosyl monoglyceride (DGMG) or monogalactosyl monoglyceride
(MGMG) by treatment of a glycolipid (e.g. digalactosyl diglyceride
(DGDG) or monogalactosyl diglyceride (MGDG)) with the variant
lipolytic enzyme according to the present invention or obtained by
a method according to the present invention to produce the partial
hydrolysis product, i.e. the lyso-glycolipid.
32. A method of using a variant lipolytic enzyme obtained by the
method according to claim 1 in a substrate for preparing a
lyso-glycolipid, for example digalactosyl monoglyceride (DGMG) or
monogalactosyl monoglyceride (MGMG) by treatment of a glycolipid
(e.g. digalactosyl diglyceride (DGDG) or monogalactosyl diglyceride
(MGDG)) with the variant lipolytic enzyme according to the present
invention or obtained by a method according to the present
invention to produce the partial hydrolysis product, i.e. the
lyso-glycolipid.
33. The method according to claim 31 wherein the substrate is a
foodstuff.
34. The method according to claim 32 wherein the substrate is a
foodstuff.
35. A method of preparing a foodstuff the method comprising adding
a variant lipolytic enzyme according to claim 20 to one or more
ingredients of the foodstuff.
36. A method of preparing a foodstuff the method comprising adding
a variant lipolytic enzyme obtained by the method according to
claim 1 to one or more ingredients of the foodstuff.
37. A method of preparing a baked product from a dough, the method
comprising adding a variant lipolytic enzyme according to claim 20
to the dough.
38. A method of preparing a baked product from a dough, the method
comprising adding a variant lipolytic enzyme obtained by the method
according to claim 1 to the dough.
39. A method of using a variant lipolytic enzyme according to claim
20 in a process of treating egg or egg-based products to produce
lysophospholipids.
40. A method of using a variant lipolytic enzyme obtained by the
method according to claim 1 in a process of treating egg or
egg-based products to produce lysophospholipids.
41. A process of enzymatic degumming of vegetable or edible oils,
comprising treating the edible or vegetable oil with a variant
lipolytic enzyme according to claim 20 so as to hydrolyse a major
part of the polar lipids (e.g. phospholipid and/or glycolipid).
42. A process of enzymatic degumming of vegetable or edible oils,
comprising treating the edible or vegetable oil with a variant
lipolytic enzyme obtained by the method according to claim 1 so as
to hydrolyse a major part of the polar lipids (e.g. phospholipid
and/or glycolipid).
43. A method of using a variant lipolytic enzyme according to claim
20 in a process for reducing the content of a phospholipid in an
edible oil, comprising treating the oil with said variant lipolytic
enzyme so as to hydrolyse a major part of the phospholipid, and
separating an aqueous phase containing the hydrolysed phospholipid
from the oil.
44. A method of using a variant lipolytic enzyme obtained by the
method according to claim 1 in a process for reducing the content
of a phospholipid in an edible oil, comprising treating the oil
with said variant lipolytic enzyme so as to hydrolyse a major part
of the phospholipid, and separating an aqueous phase containing the
hydrolysed phospholipid from the oil.
45. A method of using a variant lipolytic enzyme according to claim
20 in the bioconversion of polar lipids (preferably glycolipids) to
make high value products, such as carbohydrate esters and/or
protein esters and/or protein subunit esters and/or a hydroxy acid
ester.
46. A method of using a variant lipolytic enzyme obtained by the
method according to claim 1 in the bioconversion of polar lipids
(preferably glycolipids) to make high value products, such as
carbohydrate esters and/or protein esters and/or protein subunit
esters and/or a hydroxy acid ester.
47. An immobilised variant lipolytic enzyme according to claim
20.
48. An immobilised variant lipolytic enzyme obtained by the method
according to claim 1.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from United Kingdom
Application Number GB 0330016.7 filed on 24 Dec. 2003,
International Patent Application Number PCT/IB2004/000655 filed on
15 Jan. 2004 and United Kingdom Application Number GB 0415999.2
filed on 16 Jul. 2004.
[0002] Reference is also made to the following related
applications: U.S. application Ser. No. 09/750,990 filed on 20 Jul.
1999; U.S. application Ser. No. 10/409,391 and U.S. Application
Ser. No. 60/489,441 filed on 23 Jul. 2003.
[0003] Each of these applications and each of the documents cited
in each of these applications ("application cited documents"), and
each document referenced or cited in the application cited
documents, either in the text or during the prosecution of those
applications, as well as all arguments in support of patentability
advanced during such prosecution, are hereby incorporated herein by
reference. Various documents are also cited in this text ("herein
cited documents"). Each of the herein cited documents, and each
document cited or referenced in the herein cited documents, is
hereby incorporated herein by reference.
FIELD OF INVENTION
[0004] The present invention relates to methods of producing
variant enzymes. The present invention further relates to novel
variant enzymes and to the use of these novel variant enzymes.
TECHNICAL BACKGROUND
[0005] Lipid:cholesterol acyltransferase enzymes have been known
for some time (see for example Buckley--Biochemistry 1983, 22,
5490-5493). In particular, glycerophospholipid:cholesterol acyl
transferases (GCATs) have been found, which like the plant and/or
mammalian lecithin:cholesterol acyltransferases (LCATs), will
catalyse fatty acid transfer between phosphatidylcholine and
cholesterol.
[0006] Upton and Buckley (TIBS 20, May 1995, p 178-179) and Brumlik
and Buckley (J. of Bacteriology Apr. 1996, p 2060-2064) teach a
lipase/acyltransferase from Aeromonas hydrophila which has the
ability to carry out acyl transfer to alcohol receptors in aqueous
media.
[0007] A putative substrate binding domain and active site of the
A. hydrophila acyltransferase have been identified (see for example
Thornton et al 1988 Biochem. et Biophys. Acta. 959, 153-159 and
Hilton & Buckley 1991 J. Biol. Chem. 266, 997-1000) for this
enzyme.
[0008] Buckley et al (J. Bacteriol 1996, 178(7) 2060-4) taught that
Ser16, Asp116 and His291 are essential amino acids which must be
retained for enzyme activity to be maintained.
[0009] Robertson et al (J. Biol. Chem. 1994, 269, 2146-50) taught
some specific mutations, namely Y226F, Y230F, Y30F, F13S, S18G,
S18V, of the A. hydrophila acyltransferase, none of which are
encompassed by the present invention.
SUMMARY ASPECTS OF THE PRESENT INVENTION
[0010] The present invention is predicated upon the finding of
specific variants of a GDSx containing lipid acyltransferase
enzyme, which variants have an increased hydrolytic activity and/or
transferase activity compared with a parent enzyme. In particular,
the variants according to the present invention have an enhanced
hydrolytic activity towards galactolipids and/or an enhanced
transferase activity using galactolipid as an acyl donor as
compared with a parent enzyme. The variants according to the
present invention may additionally have an enhanced ratio of
activity towards galactolipids to phospholipids and/or towards
galactolipids to triacylglyerides compared with a parent
enzyme.
[0011] According to a first aspect the present invention provides a
method of producing a variant lipid acyltransferase enzyme
comprising: (a) selecting a parent enzyme which is a lipid
acyltransferase enzyme characterised in that the enzyme comprises
the amino acid sequence motif GDSX, wherein X is one or more of the
following amino acid residues L, A, V, I, F, Y, H, Q, T N, M or S;
(b) modifying one or more amino acids to produce a variant lipid
acyltransferase; (c) testing the variant lipid acyltransferase for
activity on a galactolipid substrate, and optionally a phospholipid
substrate and/or optionally a triglyceride substrate; (d) selecting
a variant enzyme with an enhanced activity towards galactolipids
compared with the parent enzyme; and optionally (e) preparing a
quantity of the variant enzyme.
[0012] In another aspect the present invention provides a variant
lipid acyltransferase enzyme characterised in that the enzyme
comprises the amino acid sequence motif GDSX, wherein X is one or
more of the following amino acid residues L, A, V, I, F, Y, H, Q, T
N, M or S, and wherein the variant enzyme comprises one or more
amino acid modifications compared with a parent sequence at any one
or more of the following amino acid residues when aligned to SEQ ID
No. 2: Ser3, Leu17, Ala114, Trp111, Tyr117, Leu118, Pro156, Gly159,
Gln160, Asn161, Pro162, Ser163, Ala164, Arg165, Ser166, Gln167,
Lys168, Val169, Val170, Glu171, Ala172, Tyr179, Gln182, Lys187,
His180, Asn181, Met209, Leu210, Arg211, Asn215, Lys284, Met285,
Gln289, Val290, Ala309, Ser310, Lys22, Met23, Gly40, Asn80, Pro81,
Lys82, Val112, Asn87, Asn88, -318.
[0013] In a further aspect the present invention provides a variant
lipid acyltransferase enzyme characterised in that the enzyme
comprises the amino acid sequence motif GDSX, wherein X is one or
more of the following amino acid residues L, A, V, I, F, Y, H, Q, T
N, M or S, and wherein the variant enzyme comprises one or more
amino acid modifications compared with a parent sequence at any one
or more of the following amino acid residues identified by said
parent sequence being structurally aligned with the structural
model of P10480 defined herein, which is preferably obtained by
structural alignment of P10480 crystal structure coordinates with
1IVN.PDB and/or 1DEO.PDB as taught herein: Ser3, Leu17, Ala114,
Trp111, Tyr117, Leu118, Pro156, Gly159, Gln160, Asn161, Pro162,
Ser163, Ala164, Arg165, Ser166, Gln167, Lys168, Val169, Val170,
Glu171, Ala172, Tyr179, Gln182, Lys187, His180, Asn181, Met209,
Leu210, Arg211, Asn215, Lys284, Met285, Gln289, Val290, Ala309,
Ser310, Lys22, Met23, Gly40, Asn80, Pro81, Lys82, Val112, Asn87,
Asn88, -318.
[0014] The present invention yet further provides a variant lipid
acyltransferase enzyme characterised in that the enzyme comprises
the amino acid sequence motif GDSX, wherein X is one or more of the
following amino acid residues L, A, V, I, F, Y, H, Q, T N, M or S,
and wherein the variant enzyme comprises one or more amino acid
modifications compared with a parent sequence at any one or more of
the following amino acid residues identified when said parent
sequence is aligned to the pfam consensus sequence (SEQ ID No. 1)
and modified according to a structural model of P10480 to ensure
best fit overlap (see FIG. 55) as taught herein: Ala114, Trp111,
Tyr117, Pro156, Tyr179, Gln182, Lys187, His180, Asn181, Met209,
Leu210, Arg211, Asn215, Met285, Gln289, Val290, Asn80, Pro81,
Lys82.
[0015] According to a further aspect the present invention provides
a variant lipid acyltransferase enzyme wherein the variant enzyme
comprises an amino acid sequence, which amino acid sequence is
shown as SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5,
SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID
No. 18, SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 26,
SEQ ID No. 28, SEQ ID No. 30, SEQ ID No. 33, SEQ ID No. 34, SEQ ID
No. 36, SEQ ID No. 37, SEQ ID No. 39, SEQ ID No. 41, SEQ ID No. 43
or SEQ ID No. 45 except for one or more amino acid modifications at
any one or more of the following amino acid residues identified by
sequence alignment with SEQ ID No. 2: Ser3, Leu17, Ala114, Trp111,
Tyr117, Leu118, Pro156, Gly159, Gln160, Asn161, Pro162, Ser163,
Ala164, Arg165, Ser166, Gln167, Lys168, Val169, Val170, Glu171,
Ala172, Tyr179, Gln182, Lys187, His180, Asn181, Met209, Leu210,
Arg211, Asn215, Lys284, Met285, Gln289, Val290, Ala309, Ser310,
Lys22, Met23, Gly40, Asn80, Pro81, Lys82, Val112, Asn87, Asn88,
-318.
[0016] In a further aspect the present invention provides a variant
lipid acyltransferase enzyme wherein the variant enzyme comprises
an amino acid sequence, which amino acid sequence is shown as SEQ
ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6,
SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID
No. 20, SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 26, SEQ ID No. 28,
SEQ ID No. 30, SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 36, SEQ ID
No. 37, SEQ ID No. 39, SEQ ID No. 41, SEQ ID No. 43 or SEQ ID No.
45 except for one or more amino acid modifications at any one or
more of the following amino acid residues identified by said parent
sequence being structurally aligned with the structural model of
P10480 defined herein, which is preferably obtained by structural
alignment of P10480 crystal structure coordinates with 1IVN.PDB
and/or 1DEO.PDB as taught herein: Ser3, Leu17, Ala114, Trp111,
Tyr117, Leu118, Pro156, Gly159, Gln160, Asn161, Pro162, Ser163,
Ala164, Arg165, Ser166, Gln167, Lys168, Val169, Val170, Glu171,
Ala172, Tyr179, Gln182, Lys187, His180, Asn181, Met209, Leu210,
Arg211, Asn215, Lys284, Met285, Gln289, Val290, Ala309, Ser310,
Lys22, Met23, Gly40, Asn80, Pro81, Lys82, Val112, Asn87, Asn88,
-318.
[0017] According to a further aspect the present invention provides
a variant lipid acyltransferase enzyme wherein the variant enzyme
comprises an amino acid sequence, which amino acid sequence is
shown as SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5,
SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID
No. 18, SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 26,
SEQ ID No. 28, SEQ ID No. 30, SEQ ID No. 33, SEQ ID No. 34, SEQ ID
No. 36, SEQ ID No. 37, SEQ ID No. 39, SEQ ID No. 41, SEQ ID No. 43
or SEQ ID No. 45 except for one or more amino acid modifications at
any one or more of the following amino acid residues identified
when said parent sequence is aligned to the pfam consensus sequence
(SEQ ID No. 1) and modified according to a structural model of
P10480 to ensure best fit overlap (see FIG. 55) as taught herein:
Ala114, Trp111, Tyr117, Pro156, Tyr179, Gln182, Lys187, His180,
Asn181, Met209, Leu210, Arg211, Asn215, Met285, Gln289, Val290,
Asn80, Pro81, Lys82.
[0018] The present invention yet further provides the use of a
variant lipolytic enzyme according to the present invention or
obtained by a method according to the present invention in a
substrate (preferably a foodstuff) for preparing a lyso-glycolipid,
for example digalactosyl monoglyceride (DGMG) or monogalactosyl
monoglyceride (MGMG) by treatment of a glycolipid (e.g.
digalactosyl diglyceride (DGDG) or monogalactosyl diglyceride
(MGDG)) with the variant lipolytic enzyme according to the present
invention or obtained by a method according to the present
invention to produce the partial hydrolysis product, i.e. the
lyso-glycolipid.
[0019] In a further aspect, the present invention provides the use
of a variant lipolytic enzyme according to the present invention or
obtained by a method according to the present invention in a
substrate (preferably a foodstuff) for preparing a
lyso-phospholipid, for example lysolecithin, by treatment of a
phospholipid (e.g. lecithin) with the variant lipolytic enzyme
according to the present invention or obtained by a method
according to the present invention to produce a partial hydrolysis
product, i.e a lyso-phospholipid.
[0020] In one aspect the present invention relates to a method of
preparing a foodstuff the method comprising adding a variant
lipolytic enzyme according to the present invention or obtained by
a method according to the present invention to one or more
ingredients of the foodstuff.
[0021] Another aspect of the present invention relates to a method
of preparing a baked product from a dough, the method comprising
adding a variant lipolytic enzyme according to the present
invention or obtained by a method according to the present
invention to the dough.
[0022] In another aspect of the present invention there is provided
the use of a variant lipolytic enzyme according to the present
invention or obtained by a method according to the present
invention in a process of treating egg or egg-based products to
produce lysophospholipids.
[0023] A further aspect of the present invention provides a process
of enzymatic degumming of vegetable or edible oils, comprising
treating the edible or vegetable oil with a variant lipolytic
enzyme according to the present invention or obtained by a method
according to the present invention so as to hydrolyse a major part
of the polar lipids (e.g. phospholipid and/or glycolipid).
[0024] In another aspect the present invention provides the use of
a variant lipolytic enzyme according to the present invention or
obtained by a method according to the present invention in a
process comprising treatment of a phospholipid so as to hydrolyse
fatty acyl groups.
[0025] In another aspect the present invention provides the use of
a variant lipolytic enzyme according to the present invention or
obtained by a method according to the present invention in a
process for reducing the content of a phospholipid in an edible
oil, comprising treating the oil with said variant lipolytic enzyme
so as to hydrolyse a major part of the phospholipid, and separating
an aqueous phase containing the hydrolysed phospholipid from the
oil.
[0026] There is also provided a method of preparing a variant
lipolytic enzyme according to the present invention or obtained by
a method according to the present invention, the method comprising
transforming a host cell with a recombinant nucleic acid comprising
a nucleotide sequence coding for said variant lipolytic enzyme, the
host cell being capable of expressing the nucleotide sequence
coding for the polypeptide of the lipolytic enzyme, cultivating the
transformed host cell under conditions where the nucleic acid is
expressed and harvesting the variant lipolytic enzyme.
[0027] In a further aspect the present invention relates to the use
of a variant lipolytic enzyme according to the present invention or
obtained by a method according to the present invention in the
bioconversion of polar lipids (preferably glycolipids) to make high
value products, such as carbohydrate esters and/or protein esters
and/or protein subunit esters and/or a hydroxy acid ester.
[0028] The present invention yet further relates to an immobilised
variant lipolytic enzyme according to the present invention or
obtained by a method according to the present invention.
[0029] Aspects of the present invention are presented in the claims
and in the following commentary.
[0030] Other aspects concerning the nucleotide sequences which can
be used in the present invention include: a construct comprising
the sequences of the present invention; a vector comprising the
sequences for use in the present invention; a plasmid comprising
the sequences for use in the present invention; a transformed cell
comprising the sequences for use in the present invention; a
transformed tissue comprising the sequences for use in the present
invention; a transformed organ comprising the sequences for use in
the present invention; a transformed host comprising the sequences
for use in the present invention; a transformed organism comprising
the sequences for use in the present invention. The present
invention also encompasses methods of expressing the nucleotide
sequence for use in the present invention using the same, such as
expression in a host cell; including methods for transferring same.
The present invention further encompasses methods of isolating the
nucleotide sequence, such as isolating from a host cell.
[0031] Other aspects concerning the amino acid sequence for use in
the present invention include: a construct encoding the amino acid
sequences for use in the present invention; a vector encoding the
amino acid sequences for use in the present invention; a plasmid
encoding the amino acid sequences for use in the present invention;
a transformed cell expressing the amino acid sequences for use in
the present invention; a transformed tissue expressing the amino
acid sequences for use in the present invention; a transformed
organ expressing the amino acid sequences for use in the present
invention; a transformed host expressing the amino acid sequences
for use in the present invention; a transformed organism expressing
the amino acid sequences for use in the present invention. The
present invention also encompasses methods of purifying the amino
acid sequence for use in the present invention using the same, such
as expression in a host cell; including methods of transferring
same, and then purifying said sequence.
[0032] For the ease of reference, these and further aspects of the
present invention are now discussed under appropriate section
headings. However, the teachings under each section are not
necessarily limited to each particular section.
DETAILED ASPECTS OF THE PRESENT INVENTION
[0033] The variant lipid acyltransferase enzyme according to the
present invention may in addition (or alternatively) to the
modifications taught above, may comprise one of the following amino
acid modifications at Ser18: S18A, L, M, F, W, K, Q, E, P, I, C, Y,
H, R, N, D, T.
[0034] The variant lipid acyltransferase enzyme according to the
present invention may in addition (or alternatively) to the
modifications taught above, may comprise one of the following amino
acid modifications at Y30: Y30A, G, L, M, W, K, Q, S, E, P, V, I,
C, H, R, N, D, T.
[0035] The variant lipid acyltransferase enzyme according to the
present invention may in addition (or alternatively) to the
modifications taught above, may comprise one of the following amino
acid modifications at Y230: Y230A, G, L, M, W, K, Q, S, E, P, V, I,
C, H, R, N, D, T.
[0036] Preferably, the parent lipid acyltransferase enzyme
comprises any one of the following amino acid sequences: SEQ ID No.
2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID
No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20,
SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 26, SEQ ID No. 28, SEQ ID
No. 30, SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 36, SEQ ID No. 37,
SEQ ID No. 39, SEQ ID No. 41, SEQ ID No. 43 or SEQ ID No. 45 or an
amino acid sequence which has 75% or more identity with any one of
the sequences shown as SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4,
SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 14, SEQ ID
No. 16, SEQ ID No. 18, SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 24,
SEQ ID No. 26, SEQ ID No. 28, SEQ ID No. 30, SEQ ID No. 33, SEQ ID
No. 34, SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 39, SEQ ID No. 41,
SEQ ID No. 43 or SEQ ID No. 45.
[0037] Suitably, the parent lipid acyltransferase enzyme according
to the present invention comprises an amino acid sequence which has
at least 80%, preferably at least 85%, more preferably at least
90%, more preferably at least 95%, more at least 98% homology with
any one of the sequences shown as SEQ ID No. 2, SEQ ID No. 3, SEQ
ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 14,
SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20, SEQ ID No. 22, SEQ ID
No. 24, SEQ ID No. 26, SEQ ID No. 28, SEQ ID No. 30, SEQ ID No. 33,
SEQ ID No. 34, SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 39, SEQ ID
No. 41, SEQ ID No. 43 or SEQ ID No. 45.
[0038] Suitably, the parent lipid acyltransferase enzyme may be
encoded by any one of the following nucleotide sequences: SEQ ID
No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11,
SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, SEQ ID
No. 21, SEQ ID No. 23, SEQ ID No. 25, SEQ ID No.27, SEQ ID No. 29,
SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 35, SEQ ID No. 38, SEQ ID
No. 40, SEQ ID No. 42, SEQ ID No. 44 or SEQ ID No. 46 or a
nucleotide sequence which has at least 75% or more identity with
any one of the sequences shown as SEQ ID No. 7, SEQ ID No. 8, SEQ
ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No.
15, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21, SEQ ID No. 23, SEQ
ID No. 25, SEQ ID No.27, SEQ ID No. 29, SEQ ID No. 31, SEQ ID No.
32, SEQ ID No. 35, SEQ ID No. 38, SEQ ID No. 40, SEQ ID No. 42, SEQ
ID No. 44 or SEQ ID No. 46.
[0039] Suitably, the nucleotide sequence may have 80% or more,
preferably 90% or more, more preferably 95% or more, even more
preferably 98% or more identity with any one of the sequences shown
as SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID
No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19,
SEQ ID No. 21, SEQ ID No. 23, SEQ ID No. 25, SEQ ID No.27, SEQ ID
No. 29, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 35, SEQ ID No. 38,
SEQ ID No. 40, SEQ ID No. 42, SEQ ID No. 44 or SEQ ID No. 46.
[0040] Preferably, the parent enzyme is modified at one or more of
the following amino acid residues Ser3, Leu17, Ala114, Trp111,
Tyr117, Leu118, Pro156, Gly159, Gln160, Asn161, Pro162, Ser163,
Ala164, Arg165, Ser166, Gln167, Lys168, Val169, Val170, Glu171,
Ala172, Tyr179, Gln182, Lys187, His180, Asn181, Met209, Leu210,
Arg211, Asn215, Lys284, Met285, Gln289, Val290, Ala309, Ser310,
Lys22, Met23, Gly40, Asn80, Pro81, Lys82, Val112, Asn87, Asn88 when
aligned to the reference sequence (SEQ ID No. 2) or structurally
aligned to the structural model of P10480, or aligned to the pfam
consensus sequence and modified according to the structural model
of P10480.
[0041] Suitably the variant enzyme may have an enhanced ratio of
activity on galactolipids to either phospholipids and/or
triglycerides when compared with the parent enzyme.
[0042] The term "enhanced activity towards galactolipids" means the
enzyme has an enhanced (i.e. higher) hydrolytic activity towards
galactolipids and/or an enhanced (i.e. higher) transferase activity
wherein the lipid acyl donor is a galactolipid.
[0043] The term "modifying" as used herein means adding,
substituting and/or deleting. Preferably the term "modifying" means
"substituting".
[0044] For the avoidance of doubt, when an amino acid is
substituted in the parent enzyme it is preferably substituted with
an amino acid which is different from that originally found at that
position in the parent enzyme. In other words, the term
"substitution" is not intended to cover the replacement of an amino
acid with the same amino acid.
[0045] Preferably, the parent enzyme is an enzyme which comprises
the amino acid sequence shown as SEQ ID No. 2 and/or SEQ ID No.
28.
[0046] Preferably, the variant enzyme is an enzyme which comprises
an amino acid sequence, which amino acid sequence is shown as SEQ
ID No. 2 except for one or more amino acid modifications at any one
or more of the following amino acid residues: Ser3, Leu17, Ala114,
Trp111, Tyr117, Leu118, Pro156, Gly159, Gln160, Asn161, Pro162,
Ser163, Ala164, Arg165, Ser166, Gln167, Lys168, Val169, Val170,
Glu171, Ala172, Tyr179, Gln182, Lys187, His180, Asn181, Met209,
Leu210, Arg211, Asn215, Lys284, Met285, Gln289, Val290, Ala309,
Ser310, Lys22, Met23, Gly40, Asn80, Pro81, Lys82, Val112, Asn87,
Asn88.
[0047] Preferably, X of the GDSX motif is L. Thus, preferably the
parent enzyme comprises the amino acid motif GDSL.
[0048] Preferably the method of producing a variant lipid
acyltransferase enzyme further comprises one or more of the
following steps:
[0049] 1) structural homology mapping or
[0050] 2) sequence homology alignment.
[0051] Suitably, the structural homology mapping may comprise one
or more of the following steps:
[0052] i) aligning a parent sequence with a structural model
(1IVN.PDB) shown in FIG. 52;
[0053] ii) selecting one or more amino acid residue within a 10
.ANG. sphere centred on the central carbon atom of the glycerol
molecule in the active site (see FIG. 53); and
[0054] iii) modifying one or more amino acids selected in
accordance with step (ii) in said parent sequence.
[0055] In one embodiment preferably the amino acid residue selected
in within an 9, preferably within a 8, 7, 6, 5, 4, or 3 .ANG.
sphere centred on the central carbon atom of the glycerol molecule
in the active site (see FIG. 53).
[0056] Suitably, the structural homology mapping may comprise one
or more of the following steps:
[0057] i) aligning a parent sequence with a structural model
(1IVN.PDB) shown in FIG. 52;
[0058] ii) selecting one or more amino acids within a 10 .ANG.
sphere centred on the central carbon atom of the glycerol molecule
in the active site (see FIG. 53);
[0059] iii) determining if one or more amino acid residues selected
in accordance with step (ii) are highly conserved (particularly are
active site residues and/or part of the GDSx motif and/or part of
the GANDY motif); and
[0060] iv) modifying one or more amino acids selected in accordance
with step (ii), excluding conserved regions identified in
accordance with step (iii) in said parent sequence.
[0061] In one embodiment preferably the amino acid residue selected
in within an 9, preferably within a 8, 7, 6, 5, 4, or 3 .ANG.
sphere centred on the central carbon atom of the glycerol molecule
in the active site (see FIG. 53).
[0062] Suitably, the sequence homology alignment may comprise one
or more of the following steps:
[0063] i) selecting a first parent lipid acyltransferase;
[0064] ii) identifying a second related lipid acyltransferase
having a desirable activity;
[0065] iii) aligning said first parent lipid acyltransferase and
the second related lipid acyltransferase;
[0066] iv) identifying amino acid residues that differ between the
two sequences; and
[0067] v) modifying one or more of the amino acid residues
identified in accordance with step (iv) in said parent lipid
acyltransferase.
[0068] Suitably, the sequence homology alignment may comprise one
or more of the following steps:
[0069] i) selecting a first parent lipid acyltransferase;
[0070] ii) identifying a second related lipid acyltransferase
having a desirable activity;
[0071] iii) aligning said first parent lipid acyltransferase and
the second related lipid acyltransferase;
[0072] iv) identifying amino acid residues that differ between the
two sequences;
[0073] v) determining if one or more amino acid residues selected
in accordance with step (iv) are highly conserved (particularly are
active site residues and/or part of the GDSx motif and/or part of
the GANDY motif); and
[0074] vi) modifying one or more of the amino acid residues
identified in accordance with step (iv) excluding conserved regions
identified in accordance with step (v) in said parent sequence.
[0075] Suitably, said first parent lipid acyltransferase may
comprise any one of the following amino acid sequences: SEQ ID No.
2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID
No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20,
SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 26, SEQ ID No. 28, SEQ ID
No. 30, SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 36, SEQ ID No. 37,
SEQ ID No. 39, SEQ ID No. 41, SEQ ID No. 43 or SEQ ID No. 45.
[0076] Suitably, said second related lipid acyltransferase may
comprise any one of the following amino acid sequences: SEQ ID No.
2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID
No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20,
SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 26, SEQ ID No. 28, SEQ ID
No. 30, SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 36, SEQ ID No. 37,
SEQ ID No. 39, SEQ ID No. 41, SEQ ID No. 43 or SEQ ID No. 45.
[0077] Suitably the variant enzyme may comprise at least one amino
acid modification. In some embodiments, the variant enzyme may
comprise at least 2, preferably at least 3, preferably at least 4,
preferably at least 5, preferably at least 6, preferably at least
7, preferably at least 8, preferably at least 9, preferably at
least 10 amino acid modifications compared with the parent
enzyme.
[0078] In order to align a GDSx polypeptide sequence (parent
sequence) with SEQ ID No. 2 (P01480), sequence alignment such as
pairwise alignment can be used
(http://www.ebi.ac.uk/emboss/align/index.html). Thereby, the
equivalent amino acids in alternative parental GDSx polypeptides,
which correspond to one or more of the following amino acids Ser3,
Leu17, Ala114, Trp111, Tyr117, Leu118, Pro156, Gly159, Gln160,
Asn161, Pro162, Ser163, Ala164, Arg165, Ser166, Gln167, Lys168,
Val169, Val170, Glu171, Ala172, Tyr179, Gln182, Lys187, His180,
Asn181, Met209, Leu210, Arg211, Asn215, Lys284, Met285, Gln289,
Val290, Ala309, Ser310, Lys22, Met23, Gly40, Asn80, Pro81, Lys82,
Val112, Asn87, Asn88 of SEQ ID No. 2 can be determined and
modified. As the skilled person will readily appreciate, when using
the emboss pairwise alignment, standard settings usually suffice.
Corresponding residues can be identified using "needle" in order to
make an alignment that covers the whole length of both sequences.
However, it is also possible to find the best region of similarity
between two sequences, using "water".
[0079] Alternatively, particularly in instances where parent GDSx
polypeptides share low homology with SEQ ID No. 2, the
corresponding amino acids in alternative parental GDSx polypeptides
which correspond to one or more of the following amino acids Ser3,
Leu17, Ala114, Trp111, Tyr117, Leu118, Pro156, Gly159, Gln160,
Asn161, Pro162, Ser163, Ala164, Arg165, Ser166, Gln167, Lys168,
Val169, Val170, Glu171, Ala172, Tyr179, Gln182, Lys187, His180,
Asn181, Met209, Leu210, Arg211, Asn215, Lys284, Met285, Gln289,
Val290, Ala309, Ser310, Lys22, Met23, Gly40, Asn80, Pro81, Lys82,
Val112, Asn87, Asn88 of SEQ ID No. 2 can be determined by
structural alignment to the structural model of P10480, obtained by
the structural alignment of P10480 crystal structure coordinates of
1IVN.PDB and 1DEO.PDB using the `Deep View Swiss-PDB viewer`
(obtained from www.expasy.org/spdbv/) (FIG. 53 and Example 1).
Equivalent residues are identified as those overlapping or in
closest proximity to the residues in the obtained structural model
of P010480.
[0080] Alternatively, particularly in instances where a parent GDSx
polypeptide shares a low homology with SEQ ID No. 2, the equivalent
amino acids in alternative parental GDSx polypeptides, which
correspond to one or more of the following amino acids Ser3, Leu17,
Ala114, Trp111, Tyr117, Leu118, Pro156, Gly159, Gln160, Asn161,
Pro162, Ser163, Ala164, Arg165, Ser166, Gln167, Lys168, Val169,
Val170, Glu171, Ala172, Tyr179, Gln182, Lys187, His180, Asn181,
Met209, Leu210, Arg211, Asn215, Lys284, Met285, Gln289, Val290,
Ala309, Ser310, Lys22, Met23, Gly40, Asn80, Pro81, Lys82, Val112,
Asn87, Asn88 of SEQ ID No. 2 can be determined from an alignment
obtained from the PFAM database (PFAM consensus) modified based on
the structural alignment as shown in Alignment 1 (FIG. 55). The
modification based on the structural models may be necessary to
slightly shift the alignment in order to ensure a best fit overlap.
Alignment 1 (FIG. 55) provides guidance in this regard.
[0081] Suitably the variant enzyme may be prepared using site
directed mutagenesis.
[0082] Alternatively, one can introduce mutations randomly for
instance using a commercial kit such as the GeneMorph PCR
mutagenesis kit from Stratagene, or the Diversify PCR random
mutagenesis kit from Clontech. EP 0 583 265 refers to methods of
optimising PCR based mutagenesis, which can also be combined with
the use of mutagenic DNA analogues such as those described in EP 0
866 796. Error prone PCR technologies are suitable for the
production of variants of lipid acyl transferases with preferred
characteristics. WO0206457 refers to molecular evolution of
lipases.
[0083] A third method to obtain novel sequences is to fragment
non-identical nucleotide sequences, either by using any number of
restriction enzymes or an enzyme such as Dnase I, and reassembling
full nucleotide sequences coding for functional proteins
(hereinafter referred to as "shuffling"). Alternatively one can use
one or multiple non-identical nucleotide sequences and introduce
mutations during the reassembly of the full nucleotide sequence.
DNA shuffling and family shuffling technologies are suitable for
the production of variants of lipid acyl transferases with
preferred characteristics. Suitable methods for performing
`shuffling` can be found in EP0 752 008, EP1 138 763, EP1 103 606.
Shuffling can also be combined with other forms of DNA mutagenesis
as described in U.S. Pat. No. 6,180,406 and WO 01/34835.
[0084] Thus, it is possible to produce numerous site directed or
random mutations into a nucleotide sequence, either in vivo or in
vitro, and to subsequently screen for improved functionality of the
encoded variant polypeptide by various means.
[0085] As a non-limiting example, In addition, mutations or natural
variants of a polynucleotide sequence can be recombined with either
the wild type or other mutations or natural variants to produce new
variants. Such new variants can also be screened for improved
functionality of the encoded polypeptide.
[0086] Suitably, the variant lipid acyltransferase according to the
present invention retains at least 70%, preferably at least 80%,
preferably at least 90%, preferably at least 95%, preferably at
least 97%, preferably at least 99% homology with the parent
enzyme.
[0087] Suitable parent enzymes may include any enzyme with esterase
or lipase activity.
[0088] Preferably, the parent enzyme aligns to the pfam00657
consensus sequence.
[0089] In a preferable embodiment a variant lipid acyltransferase
enzyme retains or incorporates at least one or more of the
pfam00657 consensus sequence amino acid residues found in the GDSx,
GANDY and HPT blocks.
[0090] Enzymes, such as lipases with no or low lipid
acyltransferase activity in an aqueous environment may be mutated
using molecular evolution tools to introduce or enhance the
transferase activity, thereby producing a variant lipid
acyltransferase enzyme with significant transferase activity
suitable for use in the compositions and methods of the present
invention.
[0091] Suitably, the lipid acyltransferase for use in the invention
may be a variant with enhanced enzyme activity on polar lipids,
preferably glycolipids, when compared to the parent enzyme.
Preferably, such variants also have low or no activity on lyso
polar lipids. The enhanced activity on polar lipids, preferably
glycolipids may be the result of hydrolysis and/or transferase
activity or a combination of both.
[0092] Variant lipid acyltransferases for use in the invention may
have decreased activity on triglycerides, and/or monoglycerides
and/or diglycerides compared with the parent enzyme.
[0093] Suitably the variant enzyme may have no activity on
triglycerides and/or monoglycerides and/or diglycerides.
[0094] When referring to specific amino acid residues herein the
numbering is that obtained from alignment of the variant sequence
with the reference sequence shown as SEQ ID No. 2.
[0095] In one aspect preferably the variant enzyme comprises one or
more of the following amino acid substitutions:
[0096] S3A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,
or Y; and/or
[0097] L17A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,
or Y; and/or
[0098] S18A, C, D, E, F, H, I, K, L, M, N, P, Q, R, T, W, or Y;
and/or
[0099] K22A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or
Y; and/or
[0100] M23A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,
or Y; and/or
[0101] G40A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or
Y; and/or
[0102] N80A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or
Y; and/or
[0103] N87A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or
Y; and/or
[0104] N88A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or
Y; and/or
[0105] P81A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or
Y; and/or
[0106] L82A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or
Y; and/or
[0107] V112A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or
Y; and/or
[0108] A114C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or
Y; and/or
[0109] W111A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or
Y; and/or
[0110] Y117A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or
W; and/or
[0111] L118A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or
Y; and/or
[0112] P156A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or
Y; and/or
[0113] G159A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or
Y; and/or
[0114] Q160A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or
Y; and/or
[0115] N161A, C, D, E, F, G, H, I, K, L, M P, Q, R, S, T, V, W, or
Y; and/or
[0116] P162A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or
Y; and/or
[0117] S163A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or
Y; and/or
[0118] A164C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or
Y; and/or
[0119] R165A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or
Y; and/or
[0120] S166A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or
Y; and/or
[0121] Q167A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or
Y; and/or
[0122] K168A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or
Y; and/or
[0123] V169A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or
Y; and/or
[0124] V170A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or
Y; and/or
[0125] E171A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or
Y; and/or
[0126] A172C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or
Y; and/or
[0127] Y179A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or
W; and/or
[0128] H180A, C, D, E, F, G, I, K, L, M, P, Q, R, S, T, V, W, or Y;
and/or
[0129] N181A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or
Y; and/or
[0130] Q182A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or
Y; and/or
[0131] M209A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or
Y; and/or
[0132] L210A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or
Y; and/or
[0133] R211A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or
Y; and/or
[0134] N215A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or
Y; and/or
[0135] Y230A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V or W;
and/or
[0136] K284A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or
Y; and/or
[0137] M285A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or
Y; and/or
[0138] Q289A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or
Y; and/or
[0139] V290A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or
Y; and/or
[0140] A309C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or
Y; and/or
[0141] S310A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or
Y; and/or
[0142] In addition or alternatively thereto there may be one or
more C-terminal extensions. Preferably the additional C-terminal
extension is comprised of one or more aliphatic amino acids,
preferably a non-polar amino acid, more preferably of I, L, V or G.
Thus, the present invention further provides for a variant enzyme
comprising one or more of the following C-terminal extensions:
318I, 318L, 318V, 318G.
[0143] When it is the case that the residues in the parent backbone
differ from those in P10480 (SEQ ID No. 2), as determined by
homology alignment and/or structural alignment to P10480 and/or
1IVN, it may be desirable to replace the residues which align to
any one or more of the following amino acid residues in P10480 (SEQ
ID No. 2): Ser3, Leu17, Ala114, Trp111, Tyr117, Leu118, Pro156,
Gly159, Gln160, Asn161, Pro162, Ser163, Ala164, Arg165, Ser166,
Gln167, Lys168, Val169, Val170, Glu171, Ala172, Tyr179, Gln182,
Lys187, His180, Asn181, Met209, Leu210, Arg211, Asn215, Lys284,
Met285, Gln289, Val290, Ala309, Ser310, Lys22, Met23, Gly40, Asn80,
Pro81, Lys82, Val112, Ser18, Asn87, Asn88, -318, Tyr230, Tyr30,
with the residue found in P10480.
[0144] Preferably, the His amino acid at residue 180 is substituted
for one of the following A, D, E, F, G, I, K, L, P, R, V, W, or
Y.
[0145] Preferably, the Gln amino acid at residue 182 is substituted
for a polar amino acid, most preferably K, R, D, or E.
[0146] Preferably, the Tyr amino acid at residue 230 is substituted
for one of the following amino acids A, C, D, E, G, I, K, L, M, N,
P, Q, R, S, T, V, or Y
[0147] In one aspect preferably the variant enzyme comprises one or
more of the following amino acid substitutions: S3T, Q182K, E309A,
S310E.
[0148] In a further aspect, preferably the variant enzyme comprises
a C-terminal addition, namely -318G.
[0149] Suitably, the variant enzyme may comprise one or more of the
following modifications: S3T, Q182K, E309A, S310E, -318G.
[0150] Variant enzymes which have an increased hydrolytic activity
against a polar lipid may also have an increased transferase
activity from a polar lipid.
[0151] Variant enzymes which have an increased hydrolytic activity
against a phospholipid, such as phosphatidylcholine (PC) may also
have an increased transferase activity from a phospholipid.
[0152] Variant enzymes which have an increased hydrolytic activity
against a galactolipid, such as DGDG, may also have an increased
transferase activity from a galactolipid.
[0153] Variants enzymes which have an increased transferase
activity from a phospholipid, such as phosphatidylcholine (PC), may
also have an increased hydrolytic activity against a
phospholipid.
[0154] Variants enzymes which have an increased transferase
activity from a galactolipid, such as DGDG, may also have an
increased hydrolytic activity against a galactolipid.
[0155] Variants enzymes which have an increased transferase
activity from a polar lipid may also have an increased hydrolytic
activity against a polar lipid.
[0156] Suitably, one or more of the following sites may be involved
in substrate binding: Leu17; Ala114; Tyr179; His180; Asn181;
Met209, Leu210; Arg211; Asn215; Lys284; Met285; Gln289; Val290.
[0157] The variant enzyme in accordance with the present invention
may have one or more of the following functionalities compared with
the parent enzyme:
[0158] i) improved activity towards a phospholipid, such as
phosphatidylcholine;
[0159] ii) improved activity towards a galactolipid, such as
DGDG;
[0160] iii) improved specificity towards a galactolipid, in
particular DGDG;
[0161] iv) improved galactolipid:phospholipid ratio);
[0162] v) improved transferase activity with a phospholipid, such
as phosphatidylcholine, as the lipid acyl donor;
[0163] vi) improved transferase activity with a galactolipid, such
as DGDG, as the lipid acyl donor
[0164] The following modifications may result in variants having an
improved activity towards a polar lipid substrate (phospholipids
and/or galactolipids):
[0165] S3A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,
or Y, preferably S3 is substituted with an aliphatic amino acid or
one of the following residues S3T, S3N, S3Q, S3K, S3R, S3P, S3M;
and/or
[0166] D157A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or
Y, preferably D157 is substituted with a polar uncharged amino
acid, preferably with C, S, T or M, more preferably C; and/or
[0167] Q182A, C, D, E, F, G, H, I, K, L, M, N, Q, P, R, S, T, V, W,
or Y, preferably Q182 is substituted with an aliphatic amino acid
residue, preferably a polar amino acid, more preferably a polar
charged amino acid, more preferably D or E, most preferably D;
and/or
[0168] A309A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,
or Y, preferably A309 is substituted with an aliphatic residue,
preferably a non-polar residue, preferably G, A, or P, more
preferably A.
[0169] The following modifications may result in variants having an
improved activity towards a galactolipid, such as DGDG:
[0170] S3A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,
or Y, preferably S3 is substituted with an aliphatic amino acid or
one of the following amino acid residues S3G, S3A, S3T, S3N, S3Q,
S3K, S3R, S3P, S3M, or a polar charged amino acid, preferably C, S,
T, M, N or Q, more preferably N or Q; and/or
[0171] Y230A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W,
preferably Y230 is substituted with an aliphatic amino acid or one
of the following amino acid residues G, D, T, V, R or M, more
preferably G, D, T, V, R or M, more preferably G or T; and/or
[0172] Q182A, C, D, E, F, G, H, I, K, L, M, N, Q, P, R, S, T, V, W,
or Y, preferably Q182 is substituted with an aliphatic amino acid,
preferably a polar amino acid, preferably a polar charged amino
acid, more preferably D or E, most preferably D; and/or
[0173] A309A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,
or Y, preferably A309 is substituted with an aliphatic amino acid,
preferably a non-polar amino acid, preferably G, A, or P, more
preferably A; and/or
[0174] A C-terminal addition (-318) of at least one amino acid,
preferably one amino acid, wherein the additional amino acid is
preferably an aliphatic amino acid, preferably a non-polar amino
acid, more preferably I, L or V. I
[0175] The following modifications may result in variants having an
improved specificity towards a galactolipid, in particular
DGDG:
[0176] Y230A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W,
preferably Y230 is substituted with an aliphatic amino acid or one
of the following amino acid residues G, D, T, V, R or M, more
preferably G, D, T, V, R or M;
[0177] The following modifications may result in variants having an
improved galactolipid:phospholipid ratio:
[0178] Y230A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W,
preferably Y230 is substituted with an aliphatic amino acid or one
of the following amino acid residues G, D, T, V, R or M, more
preferably G, D, T, V, R or M.
[0179] The following modifications may result in variants having an
improved activity with a phospholipid, such as phosphatidylcholine,
as the lipid acyl donor:
[0180] A309A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,
or Y, preferably A309 is substituted with an aliphatic amino acid,
preferably a non-polar amino acid, preferably G, A, or P, more
preferably A; and/or
[0181] S3A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,
or Y, preferably SA is substituted with a polar uncharged and/or
polar charged amino acid, preferably one of the following amino
acids residues S3T, S3N, S3Q, S3K, S3R, S3P, S3M, more preferably
S3Q, S3K, or S3R.
[0182] The following modifications may result in variants having an
improved transferase activity with a phospholipid, such as
phosphatidylcholine, as the lipid acyl donor:
[0183] S3A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,
or Y, preferably SA is substituted with a polar uncharged and/or
polar charged amino acid pore preferably one of the following amino
acids residues S3T, S3N, S3Q, S3D, S3K, S3R, S3P, S3M; and/or
[0184] Q182A, C, D, E, F, G, H, I, K, L, M, N, Q, P, R, S, T, V, W,
or Y, preferably Q182 is substituted with an aliphatic amino acid
residue, preferably a polar amino acid, preferably a polar charged
amino acid, more preferably D or E, most preferably D; and/or
[0185] A309A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,
or Y, preferably A309 is substituted with an aliphatic residue,
preferably a non-polar residue, preferable G, A, or P, more
preferably A.
[0186] The following modifications may result in variants having an
improved transferase activity using a galactolipid acyl, such as
DGDG, as the lipid acyl donor:
[0187] Q182A, C, D, E, F, G, H, I, K, L, M, N, Q, P, R, S, T, V, W,
or Y, preferably Q182 is substituted by an aliphatic amino acid
residue, preferably a polar amino acid, preferably a polar charged
amino acid, more preferably D or E, most preferably D; and/or
[0188] Y230A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W,
preferably Y230 is substituted with an aliphatic amino acid or one
of the following amino acid residues G, D, T, V, R or M, more
preferably G, D, T, V, R or M, more preferably G or T; and/or
[0189] A309A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,
or Y, preferably A309 is substituted with an aliphatic residue,
preferably a non-polar residue, preferable G, A, or P, more
preferably A.
[0190] The following modifications may result in variants having an
improved transferase activity with a polar lipid, such as a
galactolipid (e.g. DGDG) and/or a phospholipid (e.g.
phosphatidylcholine) as the lipid acyl donor:
[0191] S3A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,
or Y, preferably S3 is substituted with a polar uncharged and/or
polar charged amino acid, more preferably one of the following
amino acids residues S3T, S3N, S3Q, S3D, S3K, S3R, S3P, S3M;
and/or
[0192] Y230A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W,
preferably Y230 is substituted with an aliphatic amino acid or one
of the following amino acid residues G, D, T, V, R or M, more
preferably G, D, T, V, R or M, more preferably G or T; and/or
[0193] Q182A, C, D, E, F, G, H, I, K, L, M, N, Q, P, R, S, T, V, W,
or Y, preferably an aliphatic amino acid residue, preferably a
polar amino acid, preferably a polar charged amino acid, more
preferably D or E, most preferably D; and/or
[0194] S3A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,
or Y, preferably S3 is substituted with a polar uncharged and/or
polar charged amino acid, more preferably one of the following
amino acids residues S3T, S3N, S3Q, S3D, S3K, S3R, S3P, S3M;
and/or
[0195] A309A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,
or Y, preferably an aliphatic residue, preferably a non-polar
residue, preferable G, A, or P, more preferably A.
[0196] The following modifications result in variants having
improved activity towards PC:
[0197] S3N, Q, K, R, P, and/or M
[0198] The following modifications result in variants having
improved activity towards DGDG:
[0199] K187D, E309A, Y230T, Y230G, S3Q
[0200] The following modifications result in variants having
improved specificity towards DGDG:
[0201] K187D, K187D, Y230G, Y230T, Y230R, Y230M, Y230V, D157C,
E309A, G218I
[0202] The following modifications result in variants having
improved transferase activity with PC as the acyl donor:
[0203] S3K, S3R, S3Q, S3N, S3P, S3M
[0204] The following modifications result in variants having
improved transferase activity with DGDG as the acyl donor:
[0205] Y230T, K187D, Y230G, E309A
[0206] As noted above, when referring to specific amino acid
residues herein the numbering is that obtained from alignment of
the variant sequence with the reference sequence shown as SEQ ID
No. 2.
[0207] For the avoidance of doubt, when a particular amino acid is
taught at a specific site, for instance K187 for instance, this
refers to the specific amino acid at residue number 187 in SEQ ID
No. 2. However, the amino acid residue at site 187 in a different
parent enzyme may be different from lysine.
[0208] Thus, when taught to substitute an amino acid at residue
187, although reference may be made to K187 it would be readily
understood by the skilled person that when the parent enzyme is
other than that shown in SEQ ID No. 2, the amino acid being
substituted may not be lysine. It is, therefore, possible that when
substituting an amino acid sequence in a parent enzyme which is not
the enzyme having the amino acid sequence shown as SEQ ID No. 2,
the new (substituting) amino acid may be the same as that taught in
SEQ ID No. 2. This may be the case, for instance, where the amino
acid at say residue 187 is not lysine and is, therefore different
from the amino acid at residue 187 in SEQ ID No. 2. In other words,
at residue 187 for example, if the parent enzyme has at that
position an amino acid other than lysine, this amino acid may be
substituted with lysine in accordance with the present
invention.
[0209] The term "lipid acyltransferase" as used herein means an
enzyme which has acyltransferase activity (generally classified as
E.C. 2.3.1.x in accordance with the Enzyme Nomenclature
Recommendations (1992) of the Nomenclature Committee of the
International Union of Biochemistry and Molecular Biology), whereby
the enzyme is capable of transferring an acyl group from a lipid to
one or more acceptor substrates, such as one or more of the
following: a sterol; a stanol; a carbohydrate; a protein; a protein
subunit; glycerol.
[0210] Preferably, the lipid acyltransferase variant according to
the present invention and/or for use in the methods and/or uses of
the present invention is capable of transferring an acyl group from
a lipid (as defined herein) to one or more of the following acyl
acceptor substrates: a sterol, a stanol, a carbohydrate, a protein
or subunits thereof, or a glycerol.
[0211] For some aspects the "acyl acceptor" according to the
present invention may be any compound comprising a hydroxy group
(--OH), such as for example, polyvalent alcohols, including
glycerol; sterol; stanols; carbohydrates; hydroxy acids including
fruit acids, citric acid, tartaric acid, lactic acid and ascorbic
acid; proteins or a sub-unit thereof, such as amino acids, protein
hydrolysates and peptides (partly hydrolysed protein) for example;
and mixtures and derivatives thereof. Preferably, the "acyl
acceptor" according to the present invention is not water.
[0212] In one embodiment, the acyl acceptor is preferably not a
monoglyceride and/or a diglyceride.
[0213] In one aspect, preferably the variant enzyme is capable of
transferring an acyl group from a lipid to a sterol and/or a
stanol.
[0214] In one aspect, preferably the variant enzyme is capable of
transferring an acyl group from a lipid to a carbohydrate.
[0215] In one aspect, preferably the variant enzyme is capable of
transferring an acyl group from a lipid to a protein or a subunit
thereof. Suitably the protein subunit may be one or more of the
following: an amino acid, a protein hydrolysate, a peptide, a
dipeptide, an oligopeptide, a polypeptide.
[0216] Suitably in the protein or protein subunit the acyl acceptor
may be one or more of the following constituents of the protein or
protein subunit: a serine, a threonine, a tyrosine, or a
cysteine.
[0217] When the protein subunit is an amino acid, suitably the
amino acid may be any suitable amino acid. Suitably the amino acid
may be one or more of a serine, a threonine, a tyrosine, or a
cysteine for example.
[0218] In one aspect, preferably the variant enzyme is capable of
transferring an acyl group from a lipid to glycerol.
[0219] In one aspect, preferably the variant enzyme is capable of
transferring an acyl group from a lipid to a hydroxy acid.
[0220] In one aspect, preferably the variant enzyme is capable of
transferring an acyl group from a lipid to a polyvalent
alcohol.
[0221] In one aspect, the variant lipid acyltransferase may, as
well as being able to transfer an acyl group from a lipid to a
sterol and/or a stanol, additionally be able to transfer the acyl
group from a lipid to one or more of the following: a carbohydrate,
a protein, a protein subunit, glycerol.
[0222] Preferably, the lipid substrate upon which the variant lipid
acyltransferase according to the present invention acts is one or
more of the following lipids: a phospholipid, such as a lecithin,
e.g. phosphatidylcholine, a triacylglyceride, a cardiolipin, a
diglyceride, or a glycolipid, such as digalactosyldiglyceride
(DGDG) for example. This lipid substrate may be referred to herein
as the "lipid acyl donor". The term lecithin as used herein
encompasses phosphatidylcholine, phosphatidylethanolamine,
phosphatidylinositol, phosphatidylserine and
phosphatidylglycerol.
[0223] For some aspects, preferably the lipid substrate upon which
the variant lipid acyltransferase acts is a phospholipid, such as
lecithin, for example phosphatidylcholine.
[0224] For some aspects, preferably the lipid substrate is a
glycolipid, such as DGDG for example.
[0225] Preferably the lipid substrate is a food lipid, that is to
say a lipid component of a foodstuff.
[0226] For some aspects, preferably the variant lipid
acyltransferase according to the present invention is incapable, or
substantially incapable, of acting on a triglyceride and/or a
1-monoglyceride and/or 2-monoglyceride.
[0227] Suitably, the lipid substrate or lipid acyl donor may be one
or more lipids present in one or more of the following substrates:
fats, including lard, tallow and butter fat; oils including oils
extracted from or derived from palm oil, sunflower oil, soya bean
oil, safflower oil, cotton seed oil, ground nut oil, corn oil,
olive oil, peanut oil, coconut oil, and rape seed oil. Lecithin
from soya, rape seed or egg yolk is also a suitable lipid
substrate. The lipid substrate may be an oat lipid or other plant
based material containing galactolipids.
[0228] In one aspect the lipid acyl donor is preferably lecithin
(such as phosphatidylcholine) in egg yolk.
[0229] For some aspects of the present invention, the lipid may be
selected from lipids having a fatty acid chain length of from 8 to
22 carbons.
[0230] For some aspects of the present invention, the lipid may be
selected from lipids having a fatty acid chain length of from 16 to
22 carbons, more preferably of from 16 to 20 carbons.
[0231] For some aspects of the present invention, the lipid may be
selected from lipids having a fatty acid chain length of no greater
than 14 carbons, suitably from lipids having a fatty acid chain
length of from 4 to 14 carbons, suitably 4 to 10 carbons, suitably
4 to 8 carbons.
[0232] Suitably, the variant lipid acyltransferase according to the
present invention may exhibit one or more of the following lipase
activities: glycolipase activity (E.C. 3.1.1.26), triacylglycerol
lipase activity (E.C. 3.1.1.3), phospholipase A2 activity (E.C.
3.1.1.4) or phospholipase A1 activity (E.C. 3.1.1.32). The term
"glycolipase activity" as used herein encompasses "galactolipase
activity".
[0233] Suitably, the variant lipid acyltransferase according to the
present invention may have at least one or more of the following
activities: glycolipase activity (E.C. 3.1.1.26) and/or
phospholipase A1 activity (E.C. 3.1.1.32) and/or phospholipase A2
activity (E.C. 3.1.1.4).
[0234] For some aspects, the variant lipid acyltransferase
according to the present invention may have at least glycolipase
activity (E.C. 3.1.1.26).
[0235] Suitably, for some aspects the variant lipid acyltransferase
according to the present invention may be capable of transferring
an acyl group from a glycolipid and/or a phospholipid to one or
more of the following acceptor substrates: a sterol, a stanol, a
carbohydrate, a protein, glycerol.
[0236] For some aspects, preferably the variant lipid
acyltransferase according to the present invention is capable of
transferring an acyl group from a glycolipid and/or a phospholipid
to a sterol and/or a stanol to form at least a sterol ester and/or
a stanol ester.
[0237] For some aspects, preferably the variant lipid
acyltransferase according to the present invention is capable of
transferring an acyl group from a glycolipid and/or a phospholipid
to a carbohydrate to form at least a carbohydrate ester.
[0238] For some aspects, preferably the variant lipid
acyltransferase according to the present invention is capable of
transferring an acyl group from a glycolipid and/or a phospholipid
to a protein to form at least protein ester (or a protein fatty
acid condensate).
[0239] For some aspects, preferably the variant lipid
acyltransferase according to the present invention is capable of
transferring an acyl group from a glycolipid and/or a phospholipid
to glycerol to form at least a diglyceride and/or a
monoglyceride.
[0240] For some aspects, preferably the variant lipid
acyltransferase according to the present invention does not exhibit
triacylglycerol lipase activity (E.C. 3.1.1.3).
[0241] In some aspects, the variant lipid acyltransferase may be
capable of transferring an acyl group from a lipid to a sterol
and/or a stanol. Thus, in one embodiment the "acyl acceptor"
according to the present invention may be either a sterol or a
stanol or a combination of both a sterol and a stanol.
[0242] In one embodiment suitably the sterol and/or stanol may
comprise one or more of the following structural features:
[0243] i) a 3-beta hydroxy group or a 3-alpha hydroxy group;
and/or
[0244] ii) A:B rings in the cis position or A:B rings in the trans
position or C.sub.5-C.sub.6 is unsaturated.
[0245] Suitable sterol acyl acceptors include cholesterol and
phytosterols, for example alpha-sitosterol, beta-sitosterol,
stigmasterol, ergosterol, campesterol, 5,6-dihydrosterol,
brassicasterol, alpha-spinasterol, beta-spinasterol,
gamma-spinasterol, deltaspinasterol, fucosterol, dimosterol,
ascosterol, serebisterol, episterol, anasterol, hyposterol,
chondrillasterol, desmosterol, chalinosterol, poriferasterol,
clionasterol, sterol glycosides, and other natural or synthetic
isomeric forms and derivatives.
[0246] In one aspect of the present invention suitably more than
one sterol and/or stanol may act as the acyl acceptor, suitably
more than two sterols and/or stanols may act as the acyl acceptor.
In other words, in one aspect of the present invention, suitably
more than one sterol ester and/or stanol ester may be produced.
Suitably, when cholesterol is the acyl acceptor one or more further
sterols or one or more stanols may also act as the acyl acceptor.
Thus, in one aspect, the present invention provides a method for
the in situ production of both a cholesterol ester and at least one
sterol or stanol ester in combination. In other words, the lipid
acyltransferase for some aspects of the present invention may
transfer an acyl group from a lipid to both cholesterol and at
least one further sterol and/or at least one stanol.
[0247] In one aspect, preferably the sterol acyl acceptor is one or
more of the following: alpha-sitosterol, beta-sitosterol,
stigmasterol, ergosterol and campesterol.
[0248] In one aspect, preferably the sterol acyl acceptor is
cholesterol. When it is the case that cholesterol is the acyl
acceptor for the variant lipid acyltransferase, the amount of free
cholesterol in the foodstuff is reduced as compared with the
foodstuff prior to exposure to the variant lipid acyltransferase
and/or as compared with an equivalent foodstuff which has not been
treated with the variant lipid acyltransferase.
[0249] Suitable stanol acyl acceptors include phytostanols, for
example beta-sitostanol or ss-sitostanol.
[0250] In one aspect, preferably the sterol and/or stanol acyl
acceptor is a sterol and/or a stanol other than cholesterol.
[0251] In some aspects, the foodstuff prepared in accordance with
the present invention may be used to reduce blood serum cholesterol
and/or to reduce low density lipoprotein. Blood serum cholesterol
and low density lipoproteins have both been associated with certain
diseases in humans, such as atherosclerosis and/or heart disease
for example. Thus, it is envisaged that the foodstuffs prepared in
accordance with the present invention may be used to reduce the
risk of such diseases.
[0252] Thus, in one aspect the present invention provides the use
of a foodstuff according to the present invention for use in the
treatment and/or prevention of atherosclerosis and/or heart
disease.
[0253] In a further aspect, the present invention provides a
medicament comprising a foodstuff according to the present
invention.
[0254] In a further aspect, the present invention provides a method
of treating and/or preventing a disease in a human or animal
patient which method comprising administering to the patient an
effective amount of a foodstuff according to the present
invention.
[0255] Suitably, the sterol and/or the stanol "acyl acceptor" may
be found naturally within the foodstuff. Alternatively, the sterol
and/or the stanol may be added to the foodstuff. When it is the
case that a sterol and/or a stanol is added to the foodstuff, the
sterol and/or stanol may be added before, simultaneously with,
and/or after the addition of the lipid acyltransferase according to
the present invention. Suitably, the present invention may
encompass the addition of exogenous sterols/stanols, particularly
phytosterols/phytostanols, to the foodstuff prior to or
simultaneously with the addition of the variant enzyme according to
the present invention.
[0256] For some aspects, one or more sterols present in the
foodstuff may be converted to one or more stanols prior to or at
the same time as the variant lipid acyltransferase is added
according to the present invention. Any suitable method for
converting sterols to stanols may be employed. For example, the
conversion may be carried out by chemical hydrogenation for
example. The conversion may be conducted prior to the addition of
the variant lipid acyltransferase in accordance with the present
invention or simultaneously with the addition of the variant lipid
acyltransferase in accordance with the present invention. Suitably
enzymes for the conversion of sterol to stanols are taught in
WO00/061771.
[0257] Suitably the present invention may be employed to produce
phytostanol esters in situ in a foodstuff. Phytostanol esters have
increased solubility through lipid membranes, bioavailability and
enhanced health benefits (see for example WO92/99640).
[0258] In some embodiments of the present invention the stanol
ester and/or the sterol ester may be a flavouring and/or a
texturiser. In which instances, the present invention encompasses
the in situ production of flavourings and/or texturisers.
[0259] For some aspects of the present invention, the variant lipid
acyltransferase according to the present invention may utilise a
carbohydrate as the acyl acceptor. The carbohydrate acyl acceptor
may be one or more of the following: a monosaccharide, a
disaccharide, an oligosaccharide or a polysaccharide. Preferably,
the carbohydrate is one or more of the following: glucose,
fructose, anhydrofructose, maltose, lactose, sucrose, galactose,
xylose, xylooligosacharides, arabinose, maltooligosaccharides,
tagatose, microthecin, ascopyrone P, ascopyrone T,
cortalcerone.
[0260] Suitably, the carbohydrate "acyl acceptor" may be found
naturally within the foodstuff. Alternatively, the carbohydrate may
be added to the foodstuff. When it is the case that the
carbohydrate is added to the foodstuff, the carbohydrate may be
added before, simultaneously with, and/or after the addition of the
variant lipid acyltransferase according to the present
invention.
[0261] Carbohydrate esters can function as valuable emulsifiers in
foodstuffs. Thus, when it is the case that the enzyme functions to
transfer the acyl group to a sugar, the invention encompasses the
production of a second in situ emulsifier in the foodstuff.
[0262] In some embodiments, the variant lipid acyltransferase may
utilise both a sterol and/or stanol and a carbohydrate as an acyl
acceptor.
[0263] The utilisation of a variant lipid acyltransferase which can
transfer the acyl group to a carbohydrate as well as to a sterol
and/or a stanol is particularly advantageous for foodstuffs
comprising eggs. In particular, the presence of sugars, in
particular glucose, in eggs and egg products is often seen as
disadvantageous. Egg yolk may comprise up to 1% glucose. Typically,
egg or egg based products may be treated with glucose oxidase to
remove some or all of this glucose. However, in accordance with the
present invention this unwanted sugar can be readily removed by
"esterifying" the sugar to form a sugar ester.
[0264] For some aspects of the present invention, the variant lipid
acyltransferase according to the present invention may utilise a
protein as the acyl acceptor. Suitably, the protein may be one or
more of the proteins found in a food product, for example in a
dairy product and/or a meat product. By way of example only,
suitable proteins may be those found in curd or whey, such as
lactoglobulin. Other suitable proteins include ovalbumin from egg,
gliadin, glutenin, puroindoline, lipid transfer proteins from
grains, and myosin from meat.
[0265] Preferably, the parent lipid acyltransferase enzyme
according to the present invention may be characterised using the
following criteria:
[0266] (i) the enzyme possesses acyl transferase activity which may
be defined as ester transfer activity whereby the acyl part of an
original ester bond of a lipid acyl donor is transferred to an acyl
acceptor to form a new ester; and
[0267] (ii) the enzyme comprises the amino acid sequence motif
GDSX, wherein X is one or more of the following amino acid residues
L, A, V, I, F, Y, H, Q, T, N, M or S.
[0268] Preferably, X of the GDSX motif is L. Thus, preferably the
enzyme according to the present invention comprises the amino acid
sequence motif GSDL.
[0269] The GDSX motif is comprised of four conserved amino acids.
Preferably, the serine within the motif is a catalytic serine of
the lipid acyltransferase enzyme. Suitably, the serine of the GDSX
motif may be in a position corresponding to Ser-16 in Aeromonas
hydrophila lipolytic enzyme taught in Brumlik & Buckley
(Journal of Bacteriology April 1996, Vol. 178, No. 7, p
2060-2064).
[0270] To determine if a protein has the GDSX motif according to
the present invention, the sequence is preferably compared with the
hidden markov model profiles (HMM profiles) of the pfam
database.
[0271] Pfam is a database of protein domain families. Pfam contains
curated multiple sequence alignments for each family as well as
profile hidden Markov models (profile HMMs) for identifying these
domains in new sequences. An introduction to Pfam can be found in
Bateman A et al. (2002) Nucleic Acids Res. 30; 276-280. Hidden
Markov models are used in a number of databases that aim at
classifying proteins, for review see Bateman A and Haft D H (2002)
Brief Bioinform 3; 236-245.
[0272]
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMe-
d&list uids=122 30032&dopt=Abstract
[0273]
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMe-
d&list uids=117 52314&dopt=Abstract
[0274] For a detailed explanation of hidden Markov models and how
they are applied in the Pfam database see Durbin R, Eddy S, and
Krogh A (1998) Biological sequence analysis; probabilistic models
of proteins and nucleic acids. Cambridge University Press, ISBN
0-521-62041-4. The Hammer software package can be obtained from
Washington University, St Louis, USA.
[0275] Alternatively, the GDSX motif can be identified using the
Hammer software package, the instructions are provided in Durbin R,
Eddy S, and Krogh A (1998) Biological sequence analysis;
probabilistic models of proteins and nucleic acids. Cambridge
University Press, ISBN 0-521-62041-4 and the references therein,
and the HMMER2 profile provided within this specification.
[0276] The PFAM database can be accessed, for example, through
several servers which are currently located at the following
websites.
[0277] http://www.sanger.ac.uk/Software/Pfam/index.shtml
[0278] http://pfam.wustl.edu/
[0279] http://pfam.jouy.inra.fr/
[0280] http://pfam.cgb.ki.se/
[0281] The database offers a search facility where one can enter a
protein sequence. Using the default parameters of the database the
protein sequence will then be analysed for the presence of Pfam
domains. The GDSX domain is an established domain in the database
and as such its presence in any query sequence will be recognised.
The database will return the alignment of the Pfam00657 consensus
sequence to the query sequence.
[0282] A multiple alignment, including Aeromonas salmonicida or
Aeromonas hydrophila can be obtained by:
[0283] a) manual
[0284] obtain an alignment of the protein of interest with the
Pfam00657 consensus sequence and obtain an alignment of P10480 with
the Pfam00657 consensus sequence following the procedure described
above;
[0285] or
[0286] b) through the database
[0287] After identification of the Pfam00657 consensus sequence the
database offers the option to show an alignment of the query
sequence to the seed alignment of the Pfam00657 consensus sequence.
P10480 is part of this seed alignment and is indicated by
GCAT_AERHY. Both the query sequence and P10480 will be displayed in
the same window.
[0288] The Aeromonas hydrophila reference sequence:
[0289] The residues of Aeromonas hydrophila GDSX lipase are
numbered in the NCBI file P10480, the numbers in this text refer to
the numbers given in that file which in the present invention is
used to determine specific amino acids residues which, in a
preferred embodiment are present in the lipid acyltransferase
enzymes of the invention.
[0290] The Pfam alignment was performed (FIG. 33 and FIG. 34):
[0291] The following conserved residues can be recognised and in a
preferable embodiment may be present in the variant enzymes for use
in the compositions and methods of the invention;
1 Block 1 - GDSX block hid hid hid hid Gly Asp Ser hid 28 29 30 31
32 33 34 35 Block 2 - GANDY block hid Gly hid Asn Asp hid 130 131
132 133 134 135 Block 3 - HPT block His 309
[0292] Where `hid` means a hydrophobic residue selected from Met,
Ile, Leu, Val, Ala, Gly, Cys, His, Lys, Trp, Tyr, Phe.
[0293] Preferably the parent and/or variant lipid acyltransferase
enzyme for use in the compositions/methods of the invention can be
aligned using the Pfam00657 consensus sequence.
[0294] Preferably, a positive match with the hidden markov model
profile (HMM profile) of the pfam00657 domain family indicates the
presence of the GDSL or GDSX domain according to the present
invention.
[0295] Preferably when aligned with the Pfam00657 consensus
sequence the parent and/or variant lipid acyltransferase for use in
the compositions/methods of the invention have at least one,
preferably more than one, preferably more than two, of the
following, a GDSx block, a GANDY block, a HPT block. Suitably, the
parent and/or variant lipid acyltransferase may have a GDSx block
and a GANDY block. Alternatively, the parent and/or variant enzyme
may have a GDSx block and a HPT block. Preferably the parent and/or
variant enzyme comprises at least a GDSx block.
[0296] Preferably, when aligned with the Pfam00657 consensus
sequence the parent and/or variant enzyme for use in the
compositions/methods of the invention have at least one, preferably
more than one, preferably more than two, preferably more than
three, preferably more than four, preferably more than five,
preferably more than six, preferably more than seven, preferably
more than eight, preferably more than nine, preferably more than
ten, preferably more than eleven, preferably more than twelve,
preferably more than thirteen, preferably more than fourteen, of
the following amino acid residues when compared to the reference A.
hydrophilia polypeptide sequence, namely SEQ ID No. 26: 28hid,
29hid, 30hid, 31hid, 32gly, 33Asp, 34Ser, 35hid, 130hid, 131 Gly,
132Hid, 133Asn, 134Asp, 135hid, 309His
[0297] The pfam00657 GDSX domain is a unique identifier which
distinguishes proteins possessing this domain from other
enzymes.
[0298] The pfam00657 consensus sequence is presented in FIG. 1 as
SEQ ID No. 1. This is derived from the identification of the pfam
family 00657, database version 6, which may also be referred to as
pfam00657.6 herein.
[0299] The consensus sequence may be updated by using further
releases of the pfam database. For example, FIGS. 33 and 34 show
the pfam alignment of family 00657, from database version 11, which
may also be referred to as pfam00657.11 herein.
[0300] The presence of the GDSx, GANDY and HPT blocks are found in
the pfam family 00657 from both releases of the database. Future
releases of the pfam database can be used to identify the pfam
family 00657.
[0301] Preferably, the parent lipid acyltransferase enzyme
according to the present invention may be characterised using the
following criteria:
[0302] (i) the enzyme possesses acyl transferase activity which may
be defined as ester transfer activity whereby the acyl part of an
original ester bond of a lipid acyl donor is transferred to acyl
acceptor to form a new ester;
[0303] (ii) the enzyme comprises the amino acid sequence motif
GDSX, wherein X is one or more of the following amino acid residues
L, A, V, I, F, Y, H, Q, T, N, M or S.;
[0304] (iii) the enzyme comprises His-309 or comprises a histidine
residue at a position corresponding to His-309 in the Aeromonas
hydrophila lipolytic enzyme shown in FIG. 2 (SEQ ID No. 2 or SEQ ID
No. 26).
[0305] Preferably, the amino acid residue of the GDSX motif is
L.
[0306] In SEQ ID No. 26 the first 18 amino acid residues form a
signal sequence. His-309 of the full length sequence, that is the
protein including the signal sequence, equates to His-291 of the
mature part of the protein, i.e. the sequence without the signal
sequence.
[0307] Preferably, the parent lipid acyltransferase enzyme
according to the present invention comprises the following
catalytic triad: Ser-16, Asp-116 and His-291 or comprises a serine
residue, an aspartic acid residue and a histidine residue,
respectively, at positions corresponding to Ser-16, Asp-116 and
His-291 in the Aeromonas hydrophila lipolytic enzyme shown in FIG.
2 (SEQ ID No. 2) or at positions corresponding to Ser-34, Asp-134
and His-309 of the full length sequence shown in FIG. 28 (SEQ ID
No. 26). As stated above, in the sequence shown in SEQ ID No. 26
the first 18 amino acid residues form a signal sequence. Ser-34,
Asp-134 and His-309 of the full length sequence, that is the
protein including the signal sequence, equate to Ser-16, Asp-116
and His-291 of the mature part of the protein, i.e. the sequence
without the signal sequence. In the pfam00657 consensus sequence,
as given in FIG. 1 (SEQ ID No. 1) the active site residues
correspond to Ser-7, Asp-157 and His-348.
[0308] Preferably, the parent lipid acyltransferase enzyme
according to the present invention may be characterised using the
following criteria:
[0309] (i) the enzyme possesses acyl transferase activity which may
be defined as ester transfer activity whereby the acyl part of an
original ester bond of a first lipid acyl donor is transferred to
an acyl acceptor to form a new ester; and
[0310] (ii) the enzyme comprises at least Gly-14, Asp-15, Ser-16,
Asp-116 and His-191 at positions corresponding to Aeromonas
hydrophila enzyme in FIG. 2 (SEQ ID No. 2) which is equivalent to
positions Gly-32, Asp-33, Ser-34, Asp-134 and His-309,
respectively, in FIG. 28 (SEQ ID No. 26).
[0311] Suitably, the parent lipid acyltransferase enzyme according
to the present invention may be obtainable, preferably obtained,
from organisms from one or more of the following genera: Aeromonas,
Corynebacterium, Novosphingobium, Termobifida, Streptomyces,
Saccharomyces, Lactococcus, Mycobacterium, Streptococcus,
Lactobacillus, Desulfitobacterium, Bacillus, Campylobacter,
Vibrionaceae, Xylella, Sulfolobus, Aspergillus,
Schizosaccharomyces, Listeria, Neisseria, Mesorhizobium, Ralstonia,
Xanthomonas and Candida.
[0312] Suitably, the parent lipid acyltransferase enzyme according
to the present invention may be obtainable, preferably obtained,
from one or more of the following organisms: Aeromonas hydrophila,
Aeromonas salmonicida, Streptomyces coelicolor, Streptomyces
rimosus, Mycobacterium, Streptococcus pyogenes, Lactococcus lactis,
Streptococcus pyogenes, Streptococcus thermophilus, Lactobacillus
helveticus, Desulfitobacterium dehalogenans, Bacillus sp,
Campylobacter jejuni, Vibrionaceae, Xylella fastidiosa, Sulfolobus
solfataricus, Saccharomyces cerevisiae, Aspergillus terreus,
Schizosaccharomyces pombe, Listeria innocua, Listeria
monocytogenes, Neisseria meningitidis, Mesorhizobium loti,
Ralstonia solanacearum, Xanthomonas campestris, Xanthomonas
axonopodis, Corynebacterium efficens, Novosphingobium
aromaticivorans, Termobifida fusca and Candida parapsilosis.
[0313] In one aspect, preferably the parent lipid acyltransferase
enzyme according to the present invention is obtainable, preferably
obtained, from one or more of Aeromonas hydrophila or Aeromonas
salmonicida.
[0314] In one aspect, the parent lipid acyltransferase according to
the present invention may be a lecithin:cholesterol
acyltransferases (LCAT) or variant thereof (for example a variant
made by molecular evolution)
[0315] Suitable LCATs are known in the art and may be obtainable
from one or more of the following organisms for example: mammals,
rat, mice, chickens, Drosophila melanogaster, plants, including
Arabidopsis and Oryza sativa, nematodes, fungi and yeast.
[0316] Preferably, when carrying out a method according to the
present invention the product (i.e. foodstuff) is produced without
increasing or substantially increasing the free fatty acids in the
foodstuff.
[0317] The term "transferase" as used herein is interchangeable
with the term "lipid acyltransferase".
[0318] Suitably, the lipid acyltransferase as defined herein
catalyses one or more of the following reactions:
interesterification, transesterification, alcoholysis,
hydrolysis.
[0319] The term "interesterification" refers to the enzymatic
catalysed transfer of acyl groups between a lipid donor and lipid
acceptor, wherein the lipid donor is not a free acyl group.
[0320] The term "transesterification" as used herein means the
enzymatic catalysed transfer of an acyl group from a lipid donor
(other than a free fatty acid) to an acyl acceptor (other than
water).
[0321] As used herein, the term "alcoholysis" refers to the
enzymatic cleavage of a covalent bond of an acid derivative by
reaction with an alcohol ROH so that one of the products combines
with the H of the alcohol and the other product combines with the
OR group of the alcohol.
[0322] As used herein, the term "alcohol" refers to an alkyl
compound containing a hydroxyl group.
[0323] As used herein, the term "hydrolysis" refers to the
enzymatic catalysed transfer of an acyl group from a lipid to the
OH group of a water molecule. Acyl transfer which results from
hydrolysis requires the separation of the water molecule.
[0324] The term "without increasing or without substantially
increasing the free fatty acids" as used herein means that
preferably the lipid acyl transferase according to the present
invention has 100% transferase activity (i.e. transfers 100% of the
acyl groups from an acyl donor onto the acyl acceptor, with no
hydrolytic activity); however, the enzyme may transfer less than
100% of the acyl groups present in the lipid acyl donor to the acyl
acceptor. In which case, preferably the acyltransferase activity
accounts for at least 5%, more preferably at least 10%, more
preferably at least 20%, more preferably at least 30%, more
preferably at least 40%, more preferably 50%, more preferably at
least 60%, more preferably at least 70%, more preferably at least
80%, more preferably at least 90% and more preferably at least 98%
of the total enzyme activity. The % transferase activity (i.e. the
transferase activity as a percentage of the total enzymatic
activity) may be determined by the following protocol:
[0325] Protocol for the Determination of % Acyltransferase
Activity:
[0326] A foodstuff to which a lipid acyltransferase according to
the present invention has been added may be extracted following the
enzymatic reaction with CHCl.sub.3:CH.sub.3OH 2:1 and the organic
phase containing the lipid material is isolated and analysed by GLC
according to the procedure detailed hereinbelow. From the GLC
analysis (and if necessary HPLC analysis) the amount of free fatty
acids and one or more of sterol/stanol esters; carbohydrate esters,
protein esters; diglycerides; or monoglycerides are determined. A
control foodstuff to which no enzyme according to the present
invention has been added, is analysed in the same way.
[0327] Calculation:
[0328] From the results of the GLC (and optionally HPLC analyses)
the increase in free fatty acids and sterol/stanol esters and/or
carbohydrate esters and/or protein esters and/or diglycerides
and/or monoglycerides can be calculated:
.DELTA. % fatty acid=% Fatty acid(enzyme)-% fatty acid(control); Mv
fatty acid=average molecular weight of the fatty acids;
A=.DELTA. % sterol ester/Mv sterol ester (where .DELTA. % sterol
ester=% sterol/stanol ester(enzyme)-% sterol/stanol ester(control)
and Mv sterol ester=average molecular weight of the sterol/stanol
esters)-applicable where the acyl acceptor is a sterol and/or
stanol;
B=.DELTA. % carbohydrate ester/Mv carbohydrate ester (where .DELTA.
% carbohydrate ester=% carbohydrate ester(enzyme)-% carbohydrate
ester(control) and Mv carbohydrate ester=average molecular weight
of the carbohydrate ester)-applicable where the acyl acceptor is a
carbohydrate;
C=.DELTA. % protein ester/Mv protein ester (where .DELTA. % protein
ester=% protein ester(enzyme)-% protein ester(control) and Mv
protein ester=average molecular weight of the protein
ester)-applicable where the acyl acceptor is a protein; and
D=absolute value of diglyceride and/or monoglyceride/Mv
di/monoglyceride (where .DELTA. % diglyceride and/or
monoglyceride=% diglyceride and/or monoglyceride (enzyme)-%
diglyceride and/or monoglyceride (control) and Mv
di/monoglyceride=average molecular weight of the diglyceride and/or
monoglyceride)-applicable where the acyl acceptor is glycerol.
[0329] The transferase activity is calculated as a percentage of
the total enzymatic activity: 1 % transferase activity = A * + B *
+ C * + D * .times. 100 A * + B * + C * + D * + % fatty acid / ( Mv
fatty acid ) . * - delete as appropriate .
[0330] The amino acids which fall within the terms "non-polar",
"polar--uncharged", "polar--charged" are given in the table below,
as are the amino acids falling within the terms "aliphatic" and
"aromatic". The term "polar" refers to both "polar--uncharged" and
"polar--charged" amino acids.
2 ALIPHATIC Non-polar G A P I L V Polar - uncharged C S T M N Q
Polar - charged D E K R AROMATIC H F W Y
[0331] GLC Analysis
[0332] Perkin Elmer Autosystem 9000 Capillary Gas Chromatograph
equipped with WCOT fused silica column 12.5 m.times.0.25 mm
ID.times.0.1.mu. film thickness 5% phenyl-methyl-silicone (CP Sil 8
CB from Chrompack).
[0333] Carrier gas: Helium.
[0334] Injector. PSSI cold split injection (initial temp 50.degree.
C. heated to 385.degree. C.), volume 1.0 .mu.l
[0335] Detector FID: 395.degree. C.
3 Oven program: 1 2 3 Oven temperature, 0.degree. C. 90 280 350
Isothermal, time, min. 1 0 10 Temperature rate, .degree. C./min. 15
4
[0336] Sample preparation: 30 mg of sample was dissolved in 9 ml
Heptane:Pyridin, 2:1 containing internal standard heptadecane, 0.5
mg/ml. 300 .mu.l sample solution was transferred to a crimp vial,
300 .mu.l MSTFA (N-Methyl-N-trimethylsilyl-trifluoraceamid) was
added and reacted for 20 minutes at 60.degree. C.
[0337] Calculation: Response factors for mono-di-triglycerides and
free fatty acid were determined from Standard 2
(mono-di-triglyceride), for Cholesterol, Cholesteryl palmitate and
Cholesteryl stearate the response factors were determined from pure
reference material (weighing for pure material 10 mg).
[0338] Advantages
[0339] Variants transferases of the present invention have one or
more of the following advantageous properties compared with the
parent enzyme:
[0340] i) an increased activity on polar lipids and/or an increased
activity on polar lipids compared to triglycerides.
[0341] ii) an increased activity on galactolipids (glycolipids),
such as one or more of digalactosyl diglyceride (DGDG) and/or
monogalactosyl diglyceride (MGDG).
[0342] iii) an increased ratio of activity on galactolipids
(glycolipids) compared to either phospholipids and/or
triglycerides
[0343] Preferably variants transferases of the invention have
increased activity on digalactosyl diglyceride (DGDG) and/or
monogalactosyl diglyceride (MGDG).
[0344] The variants transferases of the invention may also have an
increased activity on triglycerides.
[0345] The variants transferases of the invention may also have an
increased activity on phospholipids, such as lecithin, including
phosphatidyl choline.
[0346] Variants transferases of the present invention may have
decreased activity on triglycerides, and/or monoglycerides and/or
diglycerides.
[0347] The term polar lipid refers to the polar lipids usually
found in a dough, preferably galactolipids and phospholipids.
[0348] When used in preparation of a dough or baked product the
variant transferase of the invention may result in one or more of
the following unexpected technical effects in dough and/or baked
products: an improved specific volume of either the dough or the
baked products (for example of bread and/or of cake); an improved
dough stability; an improved crust score (for example a thinner
and/or crispier bread crust), an improved crumb score (for example
a more homogenous crumb distribution and/or a finer crumb structure
and/or a softer crumb); an improved appearance (for example a
smooth surface without blisters or holes or substantially without
blisters or holes); a reduced staling; an enhanced softness; an
improved odour; an improved taste.
[0349] Isolated
[0350] In one aspect, preferably the polypeptide or protein for use
in the present invention is in an isolated form. The term
"isolated" means that the sequence is at least substantially free
from at least one other component with which the sequence is
naturally associated in nature and as found in nature.
[0351] Purified
[0352] In one aspect, preferably the polypeptide or protein for use
in the present invention is in a purified form. The term "purified"
means that the sequence is in a relatively pure state--e.g. at
least about 51% pure, or at least about 75%, or at least about 80%,
or at least about 90% pure, or at least about 95% pure or at least
about 98% pure.
CLONING A NUCLEOTIDE SEQUENCE ENCODING A POLYPEPTIDE ACCORDING TO
THE PRESENT INVENTION
[0353] A nucleotide sequence encoding either a polypeptide which
has the specific properties as defined herein or a polypeptide
which is suitable for modification may be isolated from any cell or
organism producing said polypeptide. Various methods are well known
within the art for the isolation of nucleotide sequences.
[0354] For example, a genomic DNA and/or cDNA library may be
constructed using chromosomal DNA or messenger RNA from the
organism producing the polypeptide. If the amino acid sequence of
the polypeptide is known, labelled oligonucleotide probes may be
synthesised and used to identify polypeptide-encoding clones from
the genomic library prepared from the organism. Alternatively, a
labelled oligonucleotide probe containing sequences homologous to
another known polypeptide gene could be used to identify
polypeptide-encoding clones. In the latter case, hybridisation and
washing conditions of lower stringency are used.
[0355] Alternatively, polypeptide-encoding clones could be
identified by inserting fragments of genomic DNA into an expression
vector, such as a plasmid, transforming enzyme-negative bacteria
with the resulting genomic DNA library, and then plating the
transformed bacteria onto agar containing an enzyme inhibited by
the polypeptide, thereby allowing clones expressing the polypeptide
to be identified.
[0356] In a yet further alternative, the nucleotide sequence
encoding the polypeptide may be prepared synthetically by
established standard methods, e.g. the phosphoroamidite method
described by Beucage S. L. et al (1981) Tetrahedron Letters 22, p
1859-1869, or the method described by Matthes et al (1984) EMBO J.
3, p 801-805. In the phosphoroamidite method, oligonucleotides are
synthesised, e.g. in an automatic DNA synthesiser, purified,
annealed, ligated and cloned in appropriate vectors.
[0357] The nucleotide sequence may be of mixed genomic and
synthetic origin, mixed synthetic and cDNA origin, or mixed genomic
and cDNA origin, prepared by ligating fragments of synthetic,
genomic or cDNA origin (as appropriate) in accordance with standard
techniques. Each ligated fragment corresponds to various parts of
the entire nucleotide sequence. The DNA sequence may also be
prepared by polymerase chain reaction (PCR) using specific primers,
for instance as described in U.S. Pat. No. 4,683,202 or in Saiki R
K et al (Science (1988) 239, pp 487-491).
[0358] Nucleotide Sequences
[0359] The present invention also encompasses nucleotide sequences
encoding polypeptides having the specific properties as defined
herein. The term "nucleotide sequence" as used herein refers to an
oligonucleotide sequence or polynucleotide sequence, and variant,
homologues, fragments and derivatives thereof (such as portions
thereof). The nucleotide sequence may be of genomic or synthetic or
recombinant origin, which may be double-stranded or single-stranded
whether representing the sense or antisense strand.
[0360] The term "nucleotide sequence" in relation to the present
invention includes genomic DNA, cDNA, synthetic DNA, and RNA.
Preferably it means DNA, more preferably cDNA for the coding
sequence.
[0361] In a preferred embodiment, the nucleotide sequence per se
encoding a polypeptide having the specific properties as defined
herein does not cover the native nucleotide sequence in its natural
environment when it is linked to its naturally associated
sequence(s) that is/are also in its/their natural environment. For
ease of reference, we shall call this preferred embodiment the
"non-native nucleotide sequence". In this regard, the term "native
nucleotide sequence" means an entire nucleotide sequence that is in
its native environment and when operatively linked to an entire
promoter with which it is naturally associated, which promoter is
also in its native environment. Thus, the polypeptide of the
present invention can be expressed by a nucleotide sequence in its
native organism but wherein the nucleotide sequence is not under
the control of the promoter with which it is naturally associated
within that organism.
[0362] Preferably the polypeptide is not a native polypeptide. In
this regard, the term "native polypeptide" means an entire
polypeptide that is in its native environment and when it has been
expressed by its native nucleotide sequence.
[0363] Typically, the nucleotide sequence encoding polypeptides
having the specific properties as defined herein is prepared using
recombinant DNA techniques (i.e. recombinant DNA). However, in an
alternative embodiment of the invention, the nucleotide sequence
could be synthesised, in whole or in part, using chemical methods
well known in the art (see Caruthers M H et al (1980) Nuc Acids Res
Symp Ser 215-23 and Horn T et al (1980) Nuc Acids Res Symp Ser
225-232).
[0364] Molecular Evolution
[0365] Once an enzyme-encoding nucleotide sequence has been
isolated, or a putative enzyme-encoding nucleotide sequence has
been identified, it may be desirable to modify the selected
nucleotide sequence, for example it may be desirable to mutate the
sequence in order to prepare an enzyme in accordance with the
present invention.
[0366] Mutations may be introduced using synthetic
oligonucleotides. These oligonucleotides contain nucleotide
sequences flanking the desired mutation sites.
[0367] A suitable method is disclosed in Morinaga et al
(Biotechnology (1984) 2, p 646-649). Another method of introducing
mutations into enzyme-encoding nucleotide sequences is described in
Nelson and Long (Analytical Biochemistry (1989), 180, p
147-151).
[0368] Instead of site directed mutagenesis, such as described
above, one can introduce mutations randomly for instance using a
commercial kit such as the GeneMorph PCR mutagenesis kit from
Stratagene, or the Diversify PCR random mutagenesis kit from
Clontech. EP 0 583 265 refers to methods of optimising PCR based
mutagenesis, which can also be combined with the use of mutagenic
DNA analogues such as those described in EP 0 866 796. Error prone
PCR technologies are suitable for the production of variants of
lipid acyl transferases with preferred characterisitics. WO0206457
refers to molecular evolution of lipases.
[0369] A third method to obtain novel sequences is to fragment
non-identical nucleotide sequences, either by using any number of
restriction enzymes or an enzyme such as Dnase I, and reassembling
full nucleotide sequences coding for functional proteins.
Alternatively one can use one or multiple non-identical nucleotide
sequences and introduce mutations during the reassembly of the full
nucleotide sequence. DNA shuffling and family shuffling
technologies are suitable for the production of variants of lipid
acyl transferases with preferred characteristics. Suitable methods
for performing `shuffling` can be found in EP0 752 008, EP1 138
763, EP1 103 606. Shuffling can also be combined with other forms
of DNA mutagenesis as described in U.S. Pat. No. 6,180,406 and WO
01/34835.
[0370] Thus, it is possible to produce numerous site directed or
random mutations into a nucleotide sequence, either in vivo or in
vitro, and to subsequently screen for improved functionality of the
encoded polypeptide by various means. Using in silico and exo
mediated recombination methods (see WO 00/58517, U.S. Pat. No.
6,344,328, U.S. Pat. No. 6,361,974), for example, molecular
evolution can be performed where the variant produced retains very
low homology to known enzymes or proteins. Such variants thereby
obtained may have significant structural analogy to known
transferase enzymes, but have very low amino acid sequence
homology.
[0371] As a non-limiting example, In addition, mutations or natural
variants of a polynucleotide sequence can be recombined with either
the wild type or other mutations or natural variants to produce new
variants. Such new variants can also be screened for improved
functionality of the encoded polypeptide.
[0372] The application of the above-mentioned and similar molecular
evolution methods allows the identification and selection of
variants of the enzymes of the present invention which have
preferred characteristics without any prior knowledge of protein
structure or function, and allows the production of non-predictable
but beneficial mutations or variants. There are numerous examples
of the application of molecular evolution in the art for the
optimisation or alteration of enzyme activity, such examples
include, but are not limited to one or more of the following:
optimised expression and/or activity in a host cell or in vitro,
increased enzymatic activity, altered substrate and/or product
specificity, increased or decreased enzymatic or structural
stability, altered enzymatic activity/specificity in preferred
environmental conditions, e.g. temperature, pH, substrate
[0373] As will be apparent to a person skilled in the art, using
molecular evolution tools an enzyme may be altered to improve the
functionality of the enzyme.
[0374] Suitably, the lipid acyltransferase used in the invention
may be a variant, i.e. may contain at least one amino acid
substitution, deletion or addition, when compared to a parental
enzyme. Variant enzymes retain at least 25%, 30%, 40%, 50 %, 60%,
70%, 80%, 90%, 95%, 97%, 99% homology with the parent enzyme.
Suitable parent enzymes may include any enzyme with esterase or
lipase activity. Preferably, the parent enzyme aligns to the
pfam00657 consensus sequence.
[0375] In a preferable embodiment a variant lipid acyltransferase
enzyme retains or incorporates at least one or more of the
pfam00657 consensus sequence amino acid residues found in the GDSx,
GANDY and HPT blocks.
[0376] Enzymes, such as lipases with no or low lipid
acyltransferase activity in an aqueous environment may be mutated
using molecular evolution tools to introduce or enhance the
transferase activity, thereby producing a lipid acyltransferase
enzyme with significant transferase activity suitable for use in
the compositions and methods of the present invention.
[0377] Suitably, the lipid acyltransferase for use in the invention
may be a variant with enhanced enzyme activity on polar lipids,
preferably phospholipids and/or glycolipids when compared to the
parent enzyme. Preferably, such variants also have low or no
activity on lyso polar lipids. The enhanced activity on polar
lipids, phospholipids and/or glycolipids may be the result of
hydrolysis and/or transferase activity or a combination of
both.
[0378] Variant lipid acyltransferases for use in the invention may
have decreased activity on triglycerides, and/or monoglycerides
and/or diglycerides compared with the parent enzyme.
[0379] Suitably the variant enzyme may have no activity on
triglycerides and/or monoglycerides and/or diglycerides.
[0380] Alternatively, the variant enzyme for use in the invention
may have increased activity on triglycerides, and/or may also have
increased activity on one or more of the following, polar lipids,
phospholipids, lecithin, phosphatidylcholine, glycolipids,
digalactosyl monoglyceride, monogalactosyl monoglyceride.
[0381] Variants of lipid acyltransferases are known, and one or
more of such variants may be suitable for use in the methods and
uses according to the present invention and/or in the enzyme
compositions according to the present invention. By way of example
only, variants of lipid acyltransferases are described in the
following references may be used in accordance with the present
invention: Hilton & Buckley J Biol. Chem. Jan. 15, 1991: 266
(2): 997-1000; Robertson et al J. Biol. Chem. Jan. 21, 1994;
269(3):2146-50; Brumlik et al J. Bacteriol 1996 April; 178 (7):
2060-4; Peelman et al Protein Sci. 1998 March; 7(3):587-99.
[0382] Amino Acid Sequences
[0383] The present invention also encompasses amino acid sequences
of polypeptides having the specific properties as defined
herein.
[0384] As used herein, the term "amino acid sequence" is synonymous
with the term "polypeptide" and/or the term "protein". In some
instances, the term "amino acid sequence" is synonymous with the
term "peptide".
[0385] The amino acid sequence may be prepared/isolated from a
suitable source, or it may be made synthetically or it may be
prepared by use of recombinant DNA techniques.
[0386] Suitably, the amino acid sequences may be obtained from the
isolated polypeptides taught herein by standard techniques.
[0387] One suitable method for determining amino acid sequences
from isolated polypeptides is as follows:
[0388] Purified polypeptide may be freeze-dried and 100 .mu.g of
the freeze-dried material may be dissolved in 50 .mu.l of a mixture
of 8 M urea and 0.4 M ammonium hydrogen carbonate, pH 8.4. The
dissolved protein may be denatured and reduced for 15 minutes at
50.degree. C. following overlay with nitrogen and addition of 5
.mu.l of 45 mM dithiothreitol. After cooling to room temperature, 5
.mu.l of 100 mM iodoacetamide may be added for the cysteine
residues to be derivatized for 15 minutes at room temperature in
the dark under nitrogen.
[0389] 135 .mu.l of water and 5 .mu.g of endoproteinase Lys-C in 5
.mu.l of water may be added to the above reaction mixture and the
digestion may be carried out at 37.degree. C. under nitrogen for 24
hours.
[0390] The resulting peptides may be separated by reverse phase
HPLC on a VYDAC C18 column (0.46.times.15 cm; 10 .mu.m; The
Separation Group, California, USA) using solvent A: 0.1% TFA in
water and solvent B: 0.1% TFA in acetonitrile. Selected peptides
may be re-chromatographed on a Develosil C18 column using the same
solvent system, prior to N-terminal sequencing. Sequencing may be
done using an Applied Biosystems 476A sequencer using pulsed liquid
fast cycles according to the manufacturer's instructions (Applied
Biosystems, California, USA).
[0391] Sequence Identity or Sequence Homology
[0392] The present invention also encompasses the use of sequences
having a degree of sequence identity or sequence homology with
amino acid sequence(s) of a polypeptide having the specific
properties defined herein or of any nucleotide sequence encoding
such a polypeptide (hereinafter referred to as a "homologous
sequence(s)"). Here, the term "homologue" means an entity having a
certain homology with the subject amino acid sequences and the
subject nucleotide sequences. Here, the term "homology" can be
equated with "identity".
[0393] The homologous amino acid sequence and/or nucleotide
sequence should provide and/or encode a polypeptide which retains
the functional activity and/or enhances the activity of the
enzyme.
[0394] In the present context, a homologous sequence is taken to
include an amino acid sequence which may be at least 75, 85 or 90%
identical, preferably at least 95 or 98% identical to the subject
sequence. Typically, the homologues will comprise the same active
sites etc. as the subject amino acid sequence. Although homology
can also be considered in terms of similarity (i.e. amino acid
residues having similar chemical properties/functions), in the
context of the present invention it is preferred to express
homology in terms of sequence identity.
[0395] In the present context, a homologous sequence is taken to
include a nucleotide sequence which may be at least 75, 85 or 90%
identical, preferably at least 95 or 98% identical to a nucleotide
sequence encoding a polypeptide of the present invention (the
subject sequence). Typically, the homologues will comprise the same
sequences that code for the active sites etc. as the subject
sequence. Although homology can also be considered in terms of
similarity (i.e. amino acid residues having similar chemical
properties/functions), in the context of the present invention it
is preferred to express homology in terms of sequence identity.
[0396] Homology comparisons can be conducted by eye, or more
usually, with the aid of readily available sequence comparison
programs. These commercially available computer programs can
calculate % homology between two or more sequences.
[0397] % homology may be calculated over contiguous sequences, i.e.
one sequence is aligned with the other sequence and each amino acid
in one sequence is directly compared with the corresponding amino
acid in the other sequence, one residue at a time. This is called
an "ungapped" alignment. Typically, such ungapped alignments are
performed only over a relatively short number of residues.
[0398] Although this is a very simple and consistent method, it
fails to take into consideration that, for example, in an otherwise
identical pair of sequences, one insertion or deletion will cause
the following amino acid residues to be put out of alignment, thus
potentially resulting in a large reduction in % homology when a
global alignment is performed. Consequently, most sequence
comparison methods are designed to produce optimal alignments that
take into consideration possible insertions and deletions without
penalising unduly the overall homology score. This is achieved by
inserting "gaps" in the sequence alignment to try to maximise local
homology.
[0399] However, these more complex methods assign "gap penalties"
to each gap that occurs in the alignment so that, for the same
number of identical amino acids, a sequence alignment with as few
gaps as possible--reflecting higher relatedness between the two
compared sequences--will achieve a higher score than one with many
gaps. "Affine gap costs" are typically used that charge a
relatively high cost for the existence of a gap and a smaller
penalty for each subsequent residue in the gap. This is the most
commonly used gap scoring system. High gap penalties will of course
produce optimised alignments with fewer gaps. Most alignment
programs allow the gap penalties to be modified. However, it is
preferred to use the default values when using such software for
sequence comparisons. For example when using the GCG Wisconsin
Bestfit package the default gap penalty for amino acid sequences is
-12 for a gap and -4 for each extension.
[0400] Calculation of maximum % homology therefore firstly requires
the production of an optimal alignment, taking into consideration
gap penalties. A suitable computer program for carrying out such an
alignment is the GCG Wisconsin Bestfit package (Devereux et al 1984
Nuc. Acids Research 12 p 387). Examples of other software that can
perform sequence comparisons include, but are not limited to, the
BLAST package (see Ausubel et al 1999 Short Protocols in Molecular
Biology, 4.sup.th Ed--Chapter 18), FASTA (Altschul et al 1990 J.
Mol. Biol. 403-410) and the GENEWORKS suite of comparison tools.
Both BLAST and FASTA are available for offline and online searching
(see Ausubel et al 1999, pages 7-58 to 7-60). However, for some
applications, it is preferred to use the GCG Bestfit program. A new
tool, called BLAST 2 Sequences is also available for comparing
protein and nucleotide sequence (see FEMS Microbiol Lett 1999
174(2): 247-50; FEMS Microbiol Lett 1999 177(1): 187-8 and
tatiana@ncbi.nlm.nih.gov).
[0401] Although the final % homology can be measured in terms of
identity, the alignment process itself is typically not based on an
all-or-nothing pair comparison. Instead, a scaled similarity score
matrix is generally used that assigns scores to each pairwise
comparison based on chemical similarity or evolutionary distance.
An example of such a matrix commonly used is the BLOSUM62
matrix--the default matrix for the BLAST suite of programs. GCG
Wisconsin programs generally use either the public default values
or a custom symbol comparison table if supplied (see user manual
for further details). For some applications, it is preferred to use
the public default values for the GCG package, or in the case of
other software, the default matrix, such as BLOSUM62.
[0402] Alternatively, percentage homologies may be calculated using
the multiple alignment feature in DNASIS.TM. (Hitachi Software),
based on an algorithm, analogous to CLUSTAL (Higgins D G &
Sharp P M (1988), Gene 73(1), 237-244).
[0403] Once the software has produced an optimal alignment, it is
possible to calculate % homology, preferably % sequence identity.
The software typically does this as part of the sequence comparison
and generates a numerical result.
[0404] The sequences may also have deletions, insertions or
substitutions of amino acid residues which produce a silent change
and result in a functionally equivalent substance. Deliberate amino
acid substitutions may be made on the basis of similarity in
polarity, charge, solubility, hydrophobicity, hydrophilicity,
and/or the amphipathic nature of the residues as long as the
secondary binding activity of the substance is retained. For
example, negatively charged amino acids include aspartic acid and
glutamic acid; positively charged amino acids include lysine and
arginine; and amino acids with uncharged polar head groups having
similar hydrophilicity values include leucine, isoleucine, valine,
glycine, alanine, asparagine, glutamine, serine, threonine,
phenylalanine, and tyrosine.
[0405] Conservative substitutions may be made, for example
according to the Table below. Amino acids in the same block in the
second column and preferably in the same line in the third column
may be substituted for each other:
4 ALIPHATIC Non-polar G A P I L V Polar - uncharged C S T M N Q
Polar - charged D E K R AROMATIC H F W Y
[0406] The present invention also encompasses homologous
substitution (substitution and replacement are both used herein to
mean the interchange of an existing amino acid residue, with an
alternative residue) that may occur i.e. like-for-like substitution
such as basic for basic, acidic for acidic, polar for polar etc.
Non-homologous substitution may also occur i.e. from one class of
residue to another or alternatively involving the inclusion of
unnatural amino acids such as ornithine (hereinafter referred to as
Z), diaminobutyric acid ornithine (hereinafter referred to as B),
norleucine ornithine (hereinafter referred to as O), pyriylalanine,
thienylalanine, naphthylalanine and phenylglycine.
[0407] Replacements may also be made by unnatural amino acids.
[0408] Variant amino acid sequences may include suitable spacer
groups that may be inserted between any two amino acid residues of
the sequence including alkyl groups such as methyl, ethyl or propyl
groups in addition to amino acid spacers such as glycine or
.beta.-alanine residues. A further form of variation, involves the
presence of one or more amino acid residues in peptoid form, will
be well understood by those skilled in the art. For the avoidance
of doubt, "the peptoid form" is used to refer to variant amino acid
residues wherein the .alpha.-carbon substituent group is on the
residue's nitrogen atom rather than the .alpha.-carbon. Processes
for preparing peptides in the peptoid form are known in the art,
for example Simon R J et al., PNAS (1992) 89(20), 9367-9371 and
Horwell D C, Trends Biotechnol. (1995) 13(4), 132-134.
[0409] Nucleotide sequences for use in the present invention or
encoding a polypeptide having the specific properties defined
herein may include within them synthetic or modified nucleotides. A
number of different types of modification to oligonucleotides are
known in the art. These include methylphosphonate and
phosphorothioate backbones and/or the addition of acridine or
polylysine chains at the 3' and/or 5' ends of the molecule. For the
purposes of the present invention, it is to be understood that the
nucleotide sequences described herein may be modified by any method
available in the art. Such modifications may be carried out in
order to enhance the in vivo activity or life span of nucleotide
sequences.
[0410] The present invention also encompasses the use of nucleotide
sequences that are complementary to the sequences discussed herein,
or any derivative, fragment or derivative thereof. If the sequence
is complementary to a fragment thereof then that sequence can be
used as a probe to identify similar coding sequences in other
organisms etc.
[0411] Polynucleotides which are not 100% homologous to the
sequences of the present invention but fall within the scope of the
invention can be obtained in a number of ways. Other variants of
the sequences described herein may be obtained for example by
probing DNA libraries made from a range of individuals, for example
individuals from different populations. In addition, other
viral/bacterial, or cellular homologues particularly cellular
homologues found in mammalian cells (e.g. rat, mouse, bovine and
primate cells), may be obtained and such homologues and fragments
thereof in general will be capable of selectively hybridising to
the sequences shown in the sequence listing herein. Such sequences
may be obtained by probing cDNA libraries made from or genomic DNA
libraries from other animal species, and probing such libraries
with probes comprising all or part of any one of the sequences in
the attached sequence listings under conditions of medium to high
stringency. Similar considerations apply to obtaining species
homologues and allelic variants of the polypeptide or nucleotide
sequences of the invention.
[0412] Variants and strain/species homologues may also be obtained
using degenerate PCR which will use primers designed to target
sequences within the variants and homologues encoding conserved
amino acid sequences within the sequences of the present invention.
Conserved sequences can be predicted, for example, by aligning the
amino acid sequences from several variants/homologues. Sequence
alignments can be performed using computer software known in the
art. For example the GCG Wisconsin PileUp program is widely
used.
[0413] The primers used in degenerate PCR will contain one or more
degenerate positions and will be used at stringency conditions
lower than those used for cloning sequences with single sequence
primers against known sequences.
[0414] Alternatively, such polynucleotides may be obtained by site
directed mutagenesis of characterised sequences. This may be useful
where for example silent codon sequence changes are required to
optimise codon preferences for a particular host cell in which the
polynucleotide sequences are being expressed. Other sequence
changes may be desired in order to introduce restriction
polypeptide recognition sites, or to alter the property or function
of the polypeptides encoded by the polynucleotides.
[0415] Polynucleotides (nucleotide sequences) of the invention may
be used to produce a primer, e.g. a PCR primer, a primer for an
alternative amplification reaction, a probe e.g. labelled with a
revealing label by conventional means using radioactive or
non-radioactive labels, or the polynucleotides may be cloned into
vectors. Such primers, probes and other fragments will be at least
15, preferably at least 20, for example at least 25, 30 or 40
nucleotides in length, and are also encompassed by the term
polynucleotides of the invention as used herein.
[0416] Polynucleotides such as DNA polynucleotides and probes
according to the invention may be produced recombinantly,
synthetically, or by any means available to those of skill in the
art. They may also be cloned by standard techniques.
[0417] In general, primers will be produced by synthetic means,
involving a stepwise manufacture of the desired nucleic acid
sequence one nucleotide at a time. Techniques for accomplishing
this using automated techniques are readily available in the
art.
[0418] Longer polynucleotides will generally be produced using
recombinant means, for example using a PCR (polymerase chain
reaction) cloning techniques. This will involve making a pair of
primers (e.g. of about 15 to 30 nucleotides) flanking a region of
the lipid targeting sequence which it is desired to clone, bringing
the primers into contact with mRNA or cDNA obtained from an animal
or human cell, performing a polymerase chain reaction under
conditions which bring about amplification of the desired region,
isolating the amplified fragment (e.g. by purifying the reaction
mixture on an agarose gel) and recovering the amplified DNA. The
primers may be designed to contain suitable restriction enzyme
recognition sites so that the amplified DNA can be cloned into a
suitable cloning vector.
[0419] Hybridisation
[0420] The present invention also encompasses sequences that are
complementary to the sequences of the present invention or
sequences that are capable of hybridising either to the sequences
of the present invention or to sequences that are complementary
thereto.
[0421] The term "hybridisation" as used herein shall include "the
process by which a strand of nucleic acid joins with a
complementary strand through base pairing" as well as the process
of amplification as carried out in polymerase chain reaction (PCR)
technologies.
[0422] The present invention also encompasses the use of nucleotide
sequences that are capable of hybridising to the sequences that are
complementary to the subject sequences discussed herein, or any
derivative, fragment or derivative thereof.
[0423] The present invention also encompasses sequences that are
complementary to sequences that are capable of hybridising to the
nucleotide sequences discussed herein.
[0424] Hybridisation conditions are based on the melting
temperature (Tm) of the nucleotide binding complex, as taught in
Berger and Kimmel (1987, Guide to Molecular Cloning Techniques,
Methods in Enzymology, Vol. 152, Academic Press, San Diego Calif.),
and confer a defined "stringency" as explained below.
[0425] Maximum stringency typically occurs at about Tm-5.degree. C.
(5.degree. C. below the Tm of the probe); high stringency at about
5.degree. C. to 10.degree. C. below Tm; intermediate stringency at
about 10.degree. C. to 20.degree. C. below Tm; and low stringency
at about 20.degree. C. to 25.degree. C. below Tm. As will be
understood by those of skill in the art, a maximum stringency
hybridisation can be used to identify or detect identical
nucleotide sequences while an intermediate (or low) stringency
hybridisation can be used to identify or detect similar or related
polynucleotide sequences.
[0426] Preferably, the present invention encompasses sequences that
are complementary to sequences that are capable of hybridising
under high stringency conditions or intermediate stringency
conditions to nucleotide sequences encoding polypeptides having the
specific properties as defined herein.
[0427] More preferably, the present invention encompasses sequences
that are complementary to sequences that are capable of hybridising
under high stringent conditions (e.g. 65.degree. C. and
0.1.times.SSC {1.times.SSC=0.15 M NaCl, 0.015 M Na-citrate pH 7.0})
to nucleotide sequences encoding polypeptides having the specific
properties as defined herein.
[0428] The present invention also relates to nucleotide sequences
that can hybridise to the nucleotide sequences discussed herein
(including complementary sequences of those discussed herein).
[0429] The present invention also relates to nucleotide sequences
that are complementary to sequences that can hybridise to the
nucleotide sequences discussed herein (including complementary
sequences of those discussed herein).
[0430] Also included within the scope of the present invention are
polynucleotide sequences that are capable of hybridising to the
nucleotide sequences discussed herein under conditions of
intermediate to maximal stringency.
[0431] In a preferred aspect, the present invention covers
nucleotide sequences that can hybridise to the nucleotide sequences
discussed herein, or the complement thereof, under stringent
conditions (e.g. 50.degree. C. and 0.2.times.SSC).
[0432] In a more preferred aspect, the present invention covers
nucleotide sequences that can hybridise to the nucleotide sequences
discussed herein, or the complement thereof, under high stringent
conditions (e.g. 65.degree. C. and 0.1.times.SSC).
[0433] Expression of Polypeptides
[0434] A nucleotide sequence for use in the present invention or
for encoding a polypeptide having the specific properties as
defined herein can be incorporated into a recombinant replicable
vector. The vector may be used to replicate and express the
nucleotide sequence, in polypeptide form, in and/or from a
compatible host cell. Expression may be controlled using control
sequences which include promoters/enhancers and other expression
regulation signals. Prokaryotic promoters and promoters functional
in eukaryotic cells may be used. Tissue specific or stimuli
specific promoters may be used. Chimeric promoters may also be used
comprising sequence elements from two or more different promoters
described above.
[0435] The polypeptide produced by a host recombinant cell by
expression of the nucleotide sequence may be secreted or may be
contained intracellularly depending on the sequence and/or the
vector used. The coding sequences can be designed with signal
sequences which direct secretion of the substance coding sequences
through a particular prokaryotic or eukaryotic cell membrane.
[0436] Expression Vector
[0437] The term "expression vector" means a construct capable of in
vivo or in vitro expression.
[0438] Preferably, the expression vector is incorporated in the
genome of the organism. The term "incorporated" preferably covers
stable incorporation into the genome.
[0439] The nucleotide sequence of the present invention or coding
for a polypeptide having the specific properties as defined herein
may be present in a vector, in which the nucleotide sequence is
operably linked to regulatory sequences such that the regulatory
sequences are capable of providing the expression of the nucleotide
sequence by a suitable host organism, i.e. the vector is an
expression vector.
[0440] The vectors of the present invention may be transformed into
a suitable host cell as described below to provide for expression
of a polypeptide having the specific properties as defined
herein.
[0441] The choice of vector, e.g. plasmid, cosmid, virus or phage
vector, will often depend on the host cell into which it is to be
introduced.
[0442] The vectors may contain one or more selectable marker
genes--such as a gene which confers antibiotic resistance e.g.
ampicillin, kanamycin, chloramphenicol or tetracyclin resistance.
Alternatively, the selection may be accomplished by
co-transformation (as described in WO91/17243).
[0443] Vectors may be used in vitro, for example for the production
of RNA or used to transfect or transform a host cell.
[0444] Thus, in a further embodiment, the invention provides a
method of making nucleotide sequences of the present invention or
nucleotide sequences encoding polypeptides having the specific
properties as defined herein by introducing a nucleotide sequence
into a replicable vector, introducing the vector into a compatible
host cell, and growing the host cell under conditions which bring
about replication of the vector.
[0445] The vector may further comprise a nucleotide sequence
enabling the vector to replicate in the host cell in question.
Examples of such sequences are the origins of replication of
plasmids pUC19, pACYC177, pUB110, pE194, pAMB1 and pIJ702.
[0446] Regulatory Sequences
[0447] In some applications, a nucleotide sequence for use in the
present invention or a nucleotide sequence encoding a polypeptide
having the specific properties as defined herein may be operably
linked to a regulatory sequence which is capable of providing for
the expression of the nucleotide sequence, such as by the chosen
host cell. By way of example, the present invention covers a vector
comprising the nucleotide sequence of the present invention
operably linked to such a regulatory sequence, i.e. the vector is
an expression vector.
[0448] The term "operably linked" refers to a juxtaposition wherein
the components described are in a relationship permitting them to
function in their intended manner. A regulatory sequence "operably
linked" to a coding sequence is ligated in such a way that
expression of the coding sequence is achieved under conditions
compatible with the control sequences.
[0449] The term "regulatory sequences" includes promoters and
enhancers and other expression regulation signals.
[0450] The term "promoter" is used in the normal sense of the art,
e.g. an RNA polymerase binding site.
[0451] Enhanced expression of the nucleotide sequence encoding the
enzyme having the specific properties as defined herein may also be
achieved by the selection of heterologous regulatory regions, e.g.
promoter, secretion leader and terminator regions.
[0452] Preferably, the nucleotide sequence of the present invention
may be operably linked to at least a promoter.
[0453] Examples of suitable promoters for directing the
transcription of the nucleotide sequence in a bacterial, fungal or
yeast host are well known in the art.
[0454] Constructs
[0455] The term "construct"--which is synonymous with terms such as
"conjugate", "cassette" and "hybrid"--includes a nucleotide
sequence encoding a polypeptide having the specific properties as
defined herein for use according to the present invention directly
or indirectly attached to a promoter. An example of an indirect
attachment is the provision of a suitable spacer group such as an
intron sequence, such as the Sh1-intron or the ADH intron,
intermediate the promoter and the nucleotide sequence of the
present invention. The same is true for the term "fused" in
relation to the present invention which includes direct or indirect
attachment. In some cases, the terms do not cover the natural
combination of the nucleotide sequence coding for the protein
ordinarily associated with the wild type gene promoter and when
they are both in their natural environment.
[0456] The construct may even contain or express a marker which
allows for the selection of the genetic construct.
[0457] For some applications, preferably the construct comprises at
least a nucleotide sequence of the present invention or a
nucleotide sequence encoding a polypeptide having the specific
properties as defined herein operably linked to a promoter.
[0458] Host Cells
[0459] The term "host cell"--in relation to the present invention
includes any cell that comprises either a nucleotide sequence
encoding a polypeptide having the specific properties as defined
herein or an expression vector as described above and which is used
in the recombinant production of a polypeptide having the specific
properties as defined herein.
[0460] Thus, a further embodiment of the present invention provides
host cells transformed or transfected with a nucleotide sequence of
the present invention or a nucleotide sequence that expresses a
polypeptide having the specific properties as defined herein. The
cells will be chosen to be compatible with the said vector and may
for example be prokaryotic (for example bacterial), fungal, yeast
or plant cells. Preferably, the host cells are not human cells.
[0461] Examples of suitable bacterial host organisms are gram
negative bacterium or gram positive bacteria.
[0462] Depending on the nature of the nucleotide sequence encoding
a polypeptide having the specific properties as defined herein,
and/or the desirability for further processing of the expressed
protein, eukaryotic hosts such as yeasts or other fungi may be
preferred. In general, yeast cells are preferred over fungal cells
because they are easier to manipulate. However, some proteins are
either poorly secreted from the yeast cell, or in some cases are
not processed properly (e.g. hyperglycosylation in yeast). In these
instances, a different fungal host organism should be selected.
[0463] The use of suitable host cells, such as yeast, fungal and
plant host cells--may provide for post-translational modifications
(e.g. myristoylation, glycosylation, truncation, lapidation and
tyrosine, serine or threonine phosphorylation) as may be needed to
confer optimal biological activity on recombinant expression
products of the present invention.
[0464] The host cell may be a protease deficient or protease minus
strain.
[0465] Organism
[0466] The term "organism" in relation to the present invention
includes any organism that could comprise a nucleotide sequence
according to the present invention or a nucleotide sequence
encoding for a polypeptide having the specific properties as
defined herein and/or products obtained therefrom.
[0467] Suitable organisms may include a prokaryote, fungus, yeast
or a plant.
[0468] The term "transgenic organism" in relation to the present
invention includes any organism that comprises a nucleotide
sequence coding for a polypeptide having the specific properties as
defined herein and/or the products obtained therefrom, and/or
wherein a promoter can allow expression of the nucleotide sequence
coding for a polypeptide having the specific properties as defined
herein within the organism. Preferably the nucleotide sequence is
incorporated in the genome of the organism.
[0469] The term "transgenic organism" does not cover native
nucleotide coding sequences in their natural environment when they
are under the control of their native promoter which is also in its
natural environment.
[0470] Therefore, the transgenic organism of the present invention
includes an organism comprising any one of, or combinations of, a
nucleotide sequence coding for a polypeptide having the specific
properties as defined herein, constructs as defined herein, vectors
as defined herein, plasmids as defined herein, cells as defined
herein, or the products thereof. For example the transgenic
organism can also comprise a nucleotide sequence coding for a
polypeptide having the specific properties as defined herein under
the control of a heterologous promoter.
[0471] Transformation of Host Cells/Organism
[0472] As indicated earlier, the host organism can be a prokaryotic
or a eukaryotic organism. Examples of suitable prokaryotic hosts
include E. coli and Bacillus subtilis.
[0473] Teachings on the transformation of prokaryotic hosts is well
documented in the art, for example see Sambrook et al (Molecular
Cloning: A Laboratory Manual, 2nd edition, 1989, Cold Spring Harbor
Laboratory Press). If a prokaryotic host is used then the
nucleotide sequence may need to be suitably modified before
transformation--such as by removal of introns.
[0474] In another embodiment the transgenic organism can be a
yeast.
[0475] Filamentous fungi cells may be transformed using various
methods known in the art--such as a process involving protoplast
formation and transformation of the protoplasts followed by
regeneration of the cell wall in a manner known. The use of
Aspergillus as a host microorganism is described in EP 0 238
023.
[0476] Another host organism can be a plant. A review of the
general techniques used for transforming plants may be found in
articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991]
42:205-225) and Christou (Agro-Food-Industry Hi-Tech March/April
1994 17-27). Further teachings on plant transformation may be found
in EP-A-0449375.
[0477] General teachings on the transformation of fungi, yeasts and
plants are presented in following sections.
[0478] Transformed Fungus
[0479] A host organism may be a fungus--such as a filamentous
fungus. Examples of suitable such hosts include any member
belonging to the genera Thermomyces, Acremonium, Aspergillus,
Penicillium, Mucor, Neurospora, Trichoderma and the like.
[0480] Teachings on transforming filamentous fungi are reviewed in
U.S. Pat. No. 5,741,665 which states that standard techniques for
transformation of filamentous fungi and culturing the fungi are
well known in the art. An extensive review of techniques as applied
to N. crassa is found, for example in Davis and de Serres, Methods
Enzymol (1971) 17A: 79-143.
[0481] Further teachings on transforming filamentous fungi are
reviewed in U.S. Pat. No. 5,674,707.
[0482] In one aspect, the host organism can be of the genus
Aspergillus, such as Aspergillus niger.
[0483] A transgenic Aspergillus according to the present invention
can also be prepared by following, for example, the teachings of
Turner G. 1994 (Vectors for genetic manipulation. In: Martinelli S.
D., Kinghorn J. R.( Editors) Aspergillus: 50 years on. Progress in
industrial microbiology vol 29. Elsevier Amsterdam 1994. pp.
641-666).
[0484] Gene expression in filamentous fungi has been reviewed in
Punt et al. (2002) Trends Biotechnol 2002 May;20(5):200-6, Archer
& Peberdy Crit Rev Biotechnol (1997) 17(4):273-306.
[0485] Transformed Yeast
[0486] In another embodiment, the transgenic organism can be a
yeast.
[0487] A review of the principles of heterologous gene expression
in yeast are provided in, for example, Methods Mol Biol (1995),
49:341-54, and Curr Opin Biotechnol (1997) October;8(5):554-60
[0488] In this regard, yeast--such as the species Saccharomyces
cerevisi or Pichia pastoris (see FEMS Microbiol Rev (2000
24(1):45-66), may be used as a vehicle for heterologous gene
expression.
[0489] A review of the principles of heterologous gene expression
in Saccharomyces cerevisiae and secretion of gene products is given
by E Hinchcliffe E Kenny (1993, "Yeast as a vehicle for the
expression of heterologous genes", Yeasts, Vol 5, Anthony H Rose
and J Stuart Harrison, eds, 2nd edition, Academic Press Ltd.).
[0490] For the transformation of yeast, several transformation
protocols have been developed. For example, a transgenic
Saccharomyces according to the present invention can be prepared by
following the teachings of Hinnen et al., (1978, Proceedings of the
National Academy of Sciences of the USA 75, 1929); Beggs, J D
(1978, Nature, London, 275, 104); and Ito, H et al (1983, J
Bacteriology 153, 163-168).
[0491] The transformed yeast cells may be selected using various
selective markers--such as auxotrophic markers dominant antibiotic
resistance markers.
[0492] A suitable yeast host organism can be selected from the
biotechnologically relevant yeasts species such as, but not limited
to, yeast species selected from Pichia spp., Hansenula spp.,
Kluyveromyces, Yarrowinia spp., Saccharomyces spp., including S.
cerevisiae, or Schizosaccharomyce spp. including Schizosaccharomyce
pombe.
[0493] A strain of the methylotrophic yeast species Pichia pastoris
may be used as the host organism.
[0494] In one embodiment, the host organism may be a Hansenula
species, such as H. polymorpha (as described in WO01/39544).
[0495] Transformed Plants/Plant Cells
[0496] A host organism suitable for the present invention may be a
plant. A review of the general techniques may be found in articles
by Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991]
42:205-225) and Christou (Agro-Food-Industry Hi-Tech March/April
1994 17-27), or in WO01/16308. The transgenic plant may produce
enhanced levels of phytosterol esters and phytostanol esters, for
example.
[0497] Therefore the present invention also relates to a method for
the production of a transgenic plant with enhanced levels of
phytosterol esters and phytostanol esters, comprising the steps of
transforming a plant cell with a lipid acyltransferase as defined
herein (in particular with an expression vector or construct
comprising a lipid acyltransferase as defined herein), and growing
a plant from the transformed plant cell.
[0498] Secretion
[0499] Often, it is desirable for the polypeptide to be secreted
from the expression host into the culture medium from where the
enzyme may be more easily recovered. According to the present
invention, the secretion leader sequence may be selected on the
basis of the desired expression host. Hybrid signal sequences may
also be used with the context of the present invention.
[0500] Typical examples of heterologous secretion leader sequences
are those originating from the fungal amyloglucosidase (AG) gene
(glaA--both 18 and 24 amino acid versions e.g. from Aspergillus),
the a-factor gene (yeasts e.g. Saccharomyces, Kluyveromyces and
Hansenula) or the .alpha.-amylase gene (Bacillus).
[0501] Detection
[0502] A variety of protocols for detecting and measuring the
expression of the amino acid sequence are known in the art.
Examples include enzyme-linked immunosorbent assay (ELISA),
radioimmunoassay (RIA) and fluorescent activated cell sorting
(FACS).
[0503] A wide variety of labels and conjugation techniques are
known by those skilled in the art and can be used in various
nucleic and amino acid assays.
[0504] A number of companies such as Pharmacia Biotech (Piscataway,
N.J.), Promega (Madison, Wis.), and US Biochemical Corp (Cleveland,
Ohio) supply commercial kits and protocols for these
procedures.
[0505] Suitable reporter molecules or labels include those
radionuclides, enzymes, fluorescent, chemiluminescent, or
chromogenic agents as well as substrates, cofactors, inhibitors,
magnetic particles and the like. Patents teaching the use of such
labels include U.S. Pat. No. 3,817,837; U.S. Pat. No. 3,850,752;
U.S. Pat. No. 3,939,350; U.S. Pat. No. 3,996,345; U.S. Pat. No.
4,277,437; U.S. Pat. No 4,275,149 and U.S. Pat. No. 4,366,241.
[0506] Also, recombinant immunoglobulins may be produced as shown
in U.S. Pat. No. 4,816,567.
[0507] Fusion Proteins
[0508] A polypeptide having the specific properties as defined
herein may be produced as a fusion protein, for example to aid in
extraction and purification thereof. Examples of fusion protein
partners include glutathione-S-transferase (GST), 6.times.His, GAL4
(DNA binding and/or transcriptional activation domains) and
.beta.-galactosidase. It may also be convenient to include a
proteolytic cleavage site between the fusion protein partner and
the protein sequence of interest to allow removal of fusion protein
sequences. Preferably the fusion protein will not hinder the
activity of the protein sequence.
[0509] Gene fusion expression systems in E. coli have been reviewed
in Curr. Opin. Biotechnol. (1995) 6(5):501-6.
[0510] In another embodiment of the invention, the amino acid
sequence of a polypeptide having the specific properties as defined
herein may be ligated to a heterologous sequence to encode a fusion
protein. For example, for screening of peptide libraries for agents
capable of affecting the substance activity, it may be useful to
encode a chimeric substance expressing a heterologous epitope that
is recognised by a commercially available antibody.
[0511] The invention will now be described, by way of example only,
with reference to the following Figures and Examples.
[0512] FIG. 1 shows a pfam00657 consensus sequence from database
version 6 (SEQ ID No. 1);
[0513] FIG. 2 shows an amino acid sequence (SEQ ID No. 2) obtained
from the organism Aeromonas hydrophila (P10480; GI:121051). This
amino acid sequence is a reference enzyme, which may be a parent
enzyme in accordance with the present invention;
[0514] FIG. 3 shows an amino acid sequence (SEQ ID No. 3) obtained
from the organism Aeromonas salmonicida (AAG098404;
GI:9964017);
[0515] FIG. 4 shows an amino acid sequence (SEQ ID No. 4) obtained
from the organism Streptomyces coelicolor A3(2) (Genbank accession
number NP.sub.--631558);
[0516] FIG. 5 shows an amino acid sequence (SEQ ID No. 5) obtained
from the organism Streptomyces coelicolor A3(2) (Genbank accession
number: CAC42140);
[0517] FIG. 6 shows an amino acid sequence (SEQ ID No. 6) obtained
from the organism Saccharomyces cerevisiae (Genbank accession
number P41734);
[0518] FIG. 7 shows an alignment of selected sequences to pfam00657
consensus sequence;
[0519] FIG. 8 shows a pairwise alignment of SEQ ID No. 3 with SEQ
ID No. 2 showing 93% amino acid sequence identity. The signal
sequence is underlined. + denotes differences. The GDSX motif
containing the active site serine 16, and the active sites aspartic
acid 116 and histidine 291 are highlighted (see shaded regions).
Numbers after the amino acid is minus the signal sequence;
[0520] FIG. 9 shows a nucleotide sequence (SEQ ID No. 7) encoding a
lipid acyl transferase according to the present invention obtained
from the organism Aeromonas hydrophila;
[0521] FIG. 10 shows a nucleotide sequence (SEQ ID No. 8) encoding
a lipid acyl transferase according to the present invention
obtained from the organism Aeromonas salmonicida;
[0522] FIG. 11 shows a nucleotide sequence (SEQ ID No. 9) encoding
a lipid acyl transferase according to the present invention
obtained from the organism Streptomyces coelicolor A3(2) (Genbank
accession number NC.sub.--003888.1:8327480 . . . 8328367);
[0523] FIG. 12 shows a nucleotide sequence (SEQ ID No. 10) encoding
a lipid acyl transferase according to the present invention
obtained from the organism Streptomyces coelicolor A3(2) (Genbank
accession number AL939131.1:265480 . . . 266367);
[0524] FIG. 13 shows a nucleotide sequence (SEQ ID No. 11) encoding
a lipid acyl transferase according to the present invention
obtained from the organism Saccharomyces cerevisiae (Genbank
accession number Z75034);
[0525] FIG. 14 shows an amino acid sequence (SEQ ID No. 12)
obtained from the organism Ralstonia (Genbank accession number:
AL646052);
[0526] FIG. 15 shows a nucleotide sequence (SEQ ID No. 13) encoding
a lipid acyl transferase according to the present invention
obtained from the organism Ralstonia;
[0527] FIG. 16 shows SEQ ID No. 14. Scoe1 NCBI protein accession
code CAB39707.1 GI:4539178 conserved hypothetical protein
[Streptomyces coelicolor A3(2)];
[0528] FIG. 17 shows a nucleotide sequence shown as SEQ ID No. 15
encoding NCBI protein accession code CAB39707.1 GI:4539178
conserved hypothetical protein [Streptomyces coelicolor A3(2)];
[0529] FIG. 18 shows an amino acid shown as SEQ ID No. 16. Scoe2
NCBI protein accession code CAC01477.1 GI:9716139 conserved
hypothetical protein [Streptomyces coelicolor A3(2)];
[0530] FIG. 19 shows a nucleotide sequence shown as SEQ ID No. 17
encoding Scoe2 NCBI protein accession code CAC01477.1 GI:9716139
conserved hypothetical protein [Streptomyces coelicolor A3(2)];
[0531] FIG. 20 shows an amino acid sequence (SEQ ID No. 18) Scoe3
NCBI protein accession code CAB88833.1 GI:7635996 putative secreted
protein. [Streptomyces coelicolor A3(2)];
[0532] FIG. 21 shows a nucleotide sequence shown as SEQ ID No. 19
encoding Scoe3 NCBI protein accession code CAB88833.1 GI:7635996
putative secreted protein. [Streptomyces coelicolor A3(2)];
[0533] FIG. 22 shows an amino acid sequence (SEQ ID No. 20) Scoe4
NCBI protein accession code CAB89450.1 GI:7672261 putative secreted
protein. [Streptomyces coelicolor A3(2)];
[0534] FIG. 23 shows an nucleotide sequence shown as SEQ ID No. 21
encoding Scoe4 NCBI protein accession code CAB89450.1 GI:7672261
putative secreted protein. [Streptomyces coelicolor A3(2)];
[0535] FIG. 24 shows an amino acid sequence (SEQ ID No. 22) Scoe5
NCBI protein accession code CAB62724.1 GI:6562793 putative
lipoprotein [Streptomyces coelicolor A3(2)];
[0536] FIG. 25 shows a nucleotide sequence shown as SEQ ID No. 23,
encoding Scoe5 NCBI protein accession code CAB62724.1 GI:6562793
putative lipoprotein [Streptomyces coelicolor A3(2)];
[0537] FIG. 26 shows an amino acid sequence (SEQ ID No. 24) Srim1
NCBI protein accession code AAK84028.1 GI:15082088 GDSL-lipase
[Streptomyces rimosus];
[0538] FIG. 27 shows a nucleotide sequence shown as SEQ ID No. 25
encoding Srim1 NCBI protein accession code AAK84028.1 GI:15082088
GDSL-lipase [Streptomyces rimosus];
[0539] FIG. 28 shows an amino acid sequence (SEQ ID No. 26)--a
lipid acyl transferase from Aeromonas hydrophila (ATCC #7965);
[0540] FIG. 29 shows a nucleotide sequence (SEQ ID No. 27) encoding
a lipid acyltransferase from Aeromonas hydrophila (ATCC #7965);
[0541] FIG. 30 shows an amino acid sequence (SEQ ID No. 28) of a
lipid acyltransferase from Aeromonas salmonicida subsp. Salmonicida
(ATCC#14174);
[0542] FIG. 31 shows a nucleotide sequence (SEQ ID No. 29) encoding
a lipid acyltransferase from Aeromonas salmonicida subsp.
Salmonicida (ATCC#14174);
[0543] FIG. 32 shows that homologues of the Aeromonas genes can be
identified using the basic local alignment search tool service at
the National Center for Biotechnology Information, NIH, MD, USA and
the completed genome databases. The GDSX motif was used in the
database search and a number of sequences/genes potentially
encoding enzymes with lipolytic activity were identified. Genes
were identified from the genus Streptomyces, Xanthomonas and
Ralstonia. As an example below, the Ralstonia solanacearum was
aligned to the Aeromonas salmonicida (satA) gene. Pairwise
alignment showed 23% identity. The active site serine is present at
the amino terminus and the catalytic residues histidine and
aspartic acid can be identified;
[0544] FIG. 33 shows the Pfam00657.11 [family 00657, database
version 11] consensus sequence (hereafter called Pfam consensus)
and the alignment of various sequences to the Pfam consensus
sequence. The arrows indicate the active site residues, the
underlined boxes indicate three of the homology boxes indicated by
[Upton C and Buckley J T (1995) Trends Biochem Sci 20; 179-179].
Capital letters in the Pfam consensus indicate conserved residues
in many family members. The - symbol indicates a position where the
hidden Markov model of the Pfam consensus expected to find a
residue but did not, so a gap is inserted. The . symbol indicates a
residue without a corresponding residue in the Pfam consensus. The
sequences are the amino acid sequences listed in FIGS. 16, 18, 20,
22, 24, 26, 28 and 30.
[0545] FIG. 34 shows the Pfam00657.11 [family 00657, database
version 11] consensus sequence (hereafter called Pfam consensus)
and the alignment of various sequences to the Pfam consensus
sequence. The arrows indicate the active site residues, the
underlined boxes indicate three of the homology boxes indicated by
[Upton C and Buckley J T (1995) Trends Biochem Sci 20; 179-179].
Capital letters in the Pfam consensus indicate conserved residues
in many family members. The - symbol indicates a position where the
hidden Markov model of the Pfam consensus expected to find a
residue but did not, so a gap is inserted. The . symbol indicates a
residue without a corresponding residue in the Pfam consensus. The
sequences are the amino acid sequences listed in FIGS. 2, 16, 18,
20, 26, 28 and 30. All these proteins were found to be active
against lipid substrates.
[0546] FIG. 35 shows an amino acid sequence (SEQ ID No. 30) of the
fusion construct used for mutagenesis of the Aeromonas hydrophila
lipid acyltransferase gene in Example 7. The underlined amino acids
is a xylanase signal peptide;
[0547] FIG. 36 shows a nucleotide sequence (SEQ ID No. 31) encoding
a lipid acyltransferase enzyme from Aeromonas hydrophila including
a xylanase signal peptide;
[0548] FIG. 37 shows a nucleotide sequence encoding a lipid
acyltransferase enzyme from Streptomyces (SEQ ID No. 32);
[0549] FIG. 38 shows a polypeptide sequence of a lipid
acyltransferase enzyme from Streptomyces (SEQ ID No. 33);
[0550] FIG. 39 shows a polypeptide sequence of a lipid
acyltransferase enzyme from Termobifida (SEQ ID No. 34);
[0551] FIG. 40 shows a nucleotide sequence encoding a lipid
acyltransferase enzyme from Termobifida_(SEQ ID No. 35);
[0552] FIG. 41 shows a polypeptide sequence of a lipid
acyltransferase enzyme from Termobifida (SEQ ID No. 36);
[0553] FIG. 42 shows a polypeptide of a lipid acyltransferase
enzyme from Corynebacterium.backslash.effciens.backslash.GDSx 300
aa_(SEQ ID No. 37);
[0554] FIG. 43 shows a nucleotide sequence encoding a lipid
acyltransferase enzyme from
Corynebacterium.backslash.effciens.backslash.- GDSx 300 aa_(SEQ ID
No. 38);
[0555] FIG. 44 shows a polypeptide of a lipid acyltransferase
enzyme from
Novosphingobium.backslash.aromaticivorans.backslash.GDSx 284
aa_(SEQ ID No. 39);
[0556] FIG. 45 shows a nucleotide sequence encoding a lipid
acyltransferase enzyme from
Novosphingobium.backslash.aromaticivorans.bac- kslash.GDSx 284 aa
(SEQ ID No. 40);
[0557] FIG. 46 shows a polypeptide of a lipid acyltransferase
enzyme from Streptomyces coelicolor.backslash.GDSx 268 aa (SEQ ID
No. 41);
[0558] FIG. 47 shows a nucleotide sequence encoding a lipid
acyltransferase enzyme from Streptomyces coelicolor.backslash.GDSx
268 aa (SEQ ID No. 42);
[0559] FIG. 48 shows a polypeptide of a lipid acyltransferase
enzyme from Streptomyces averitilis.backslash.GDSx 269 aa (SEQ ID
No. 43);
[0560] FIG. 49 shows a nucleotide sequence encoding a lipid
acyltransferase enzyme from Streptomyces avermitilis.backslash.GDSx
269 aa (SEQ ID No. 44);
[0561] FIG. 50 shows a polypeptide of a lipid acyltransferase
enzyme from Streptomyces (SEQ ID No. 45);
[0562] FIG. 51 shows a nucleotide sequence encoding a lipid
acyltransferase enzyme from Streptomyces (SEQ ID No. 46);
[0563] FIG. 52 shows a ribbon representation of the 1IVN.PDB
crystal structure which has glycerol in the active site. The Figure
was made using the Deep View Swiss-PDB viewer;
[0564] FIG. 53 shows 1IVN.PDB Crystal Structure--Side View using
Deep View Swiss-PDB viewer, with glycerol in active site--residues
within 10 .ANG. of active site glycerol are coloured black;
[0565] FIG. 54 shows 1IVN.PDB Crystal Structure--Top View using
Deep View Swiss-PDB viewer, with glycerol in active site--residues
within 10 .ANG. of active site glycerol are coloured black;
[0566] FIG. 55 shows alignment 1;
[0567] FIG. 56 shows alignment 2;
[0568] FIGS. 57 and 58 show a alignments of 1IVN to P10480 (P10480
is the database sequence for A. hydrophila enzyme), this alignment
was obtained from the PFAM database and used in the model building
process;
[0569] FIG. 59 shows an alignment where P10480 is the database
sequence for Aeromonas hydrophila. This sequence is used for the
model construction and the site selection. Note that the full
protein is depicted, the mature protein (equivalent to SEQ ID No.
2) starts at residue 19. A. sal is Aeromonas salmonicida (SEQ ID
No. 28) GDSX lipase, A. hyd is Aeromonas hydrophila (SEQ ID No. 26)
GDSX lipase. The consensus sequence contains a * at the position of
a difference between the listed sequences;
[0570] FIG. 60 shows a typical set of 384 clones, the wild type
control lies at the intersection of 0.9PC, 0.8DGDG; and
[0571] FIG. 61 shows three areas of interest. Section 1 contains
mutants with an increased ratio R but lower activity towards DGDG.
Region 2 contains mutants with an increased ratio R and an
increased DGDG activity. Region 3 contains clones with an increased
PC or DGDG activity, but no increase in the ratio R.
EXAMPLE 1
[0572] Modelling of Aeromonas hydrophila GDSx Lipase on 1IVN
[0573] The alignment of the Aeromonas hydrophila GDSX lipase amino
acid sequence (P10480) to the Escherichia coli Tioesterase amino
acid sequence (1IVN) and the Aspergillus aculeatus
rhamnogalacturonan acetylesterase amino acid sequence (1DEO) was
obtained from the PFAM database in FASTA format. The alignment of
P10480 and 1IVN was fed into an automated 3D structure modeller
(SWISS-MODELLER server at www.expasy.org) together with the
1IVN.PDB crystal structure coordinates file FIG. 52). The obtained
model for P10480 was structurally aligned to the crystal structures
coordinates of 1IVN.PDB and 1DEO.PDB using the `Deep View Swiss-PDB
viewer` (obtained from www.expasy.org/spdbv/) (FIG. 53). The amino
acid alignment obtained from the PFAM database (alignment 1--(FIG.
55)) was modified based on the structural alignment of 1DEO.PDB and
1IVN.PDB. This alternative amino acid alignment is called alignment
2 (FIG. 56).
[0574] The 1IVN.PDB structure contains a glycerol molecule. This
molecule is considered to be in the active site it is in the
vicinity of the catalytic residues. Therefore, a selection can be
made of residues that are close to the active site which, due to
their vicinity, are likely to have an influence on substrate
binding, product release, and/or catalysis. In the 1IVN.PDB
structure, all amino acids within a 10 .ANG. sphere centered on the
central carbon atom of the glycerol molecule in the active site
were selected (amino acid set 1) (See FIG. 53 and FIG. 54).
[0575] The following amino acids were selected from the P10480
sequence; (1) all amino acids in P10480 corresponding to the amino
acid set 1 in alignment 1; (2) all amino acids in P10480
corresponding to the amino acid set 1 in alignment 2; (3) from the
overlay of the P10480 model and 1IVN all amino acids in the P10480
model within 12 .ANG. from the glycerol molecule in 1IVN. All three
groups combined give amino acid set 2.
[0576] Sequence P10480 was aligned to "AAG09804.1 GI:9964017
glycerophospholipid-cholesterol acyltransferase [Aeromonas
salmonicida]" and the residues in AAG09804 corresponding to amino
acid set 2 were selected in amino acid set 3.
[0577] Set 1, 2, and 3
[0578] Amino Acid Set 1 (Note That These are Amino Acids in
1IVN--FIG. 57 and FIG. 58.)
[0579] Gly8, Asp9, Ser10, Leu11, Ser12, Tyr15, Gly44, Asp45, Thr46,
Glu69, Leu70, Gly71, Gly72, Asn73, Asp74, Gly75, Leu76, Gln106,
Ile107, Arg108, Leu109, Pro110, Tyr113, Phe121, Phe139, Phe140,
Met141, Tyr145, Met151, Asp154, Gly155, Ile156, His157, Pro158
[0580] The highly conserved motifs, such as GDSx and catalytic
residues, were deselected from set 1 (residues underlined).
[0581] Amino Acid Set 2 (Note That the Numbering of the Amino Acids
Refers to the Amino Acids in the P10480 Mature Sequence)
[0582] Leu17, Lys22, Met23, Gly40, Asn80, Pro81, Lys82, Asn87,
Asn88, Trp111, Val112, Ala114, Tyr117, Leu118, Pro156, Gly159,
Gln160, Asn161, Pro162, Ser163, Ala164, Arg165, Ser166, Gln167,
Lys168, Val169, Val170, Glu171, Ala172, Tyr179, His180, Asn181,
Met209, Leu210, Arg211, Asn215, Lys284, Met285, Gln289, Val290
[0583] Amino acid set 3 is identical to set 2 but refers to the
Aeromonas salmonicida (SEQ ID No. 28) mature sequence, i.e. the
amino acid residue numbers are 18 higher in set 3 as this reflects
the difference between the amino acid numbering in the mature
protein (SEQ ID No. 2) compared with the protein including a signal
sequence (SEQ ID No. 28).
[0584] The mature proteins of Aeromonas salmonicida GDSX (SEQ ID
No. 28) and Aeromonas hydrophila GDSX (SEQ ID No. 26) differ in
five amino acids. These are Thr3Ser, Lys182Gln Glu309Ala,
Thr310Asn, Gly318-, where the salmonicida residue is listed first
and the hydrophila residue last (FIG. 59). The hydrophila protein
is only 317 amino acids long and lacks a residue on position 318.
The Aeromonas salmonicidae GDSX has considerably high activity on
polar lipids such as galactolipid substrates than the Aeromonas
hydrophila protein.
[0585] Amino acid set four=Thr3Ser, Lys182Gln Glu309Ala, Thr310Asn,
-318Gly
[0586] The Alignments 1 and 2 used to obtain the sets
[0587] From the crystal structure one can obtain the secondary
structure classification. That means, one can classify each amino
acid as being part of an alpha-helix or a beta-sheet. FIG. 57 shows
the PFAM alignment of 1DEO, 1IVN, and P10480 (the database
Aeromonas hydrophila). Added below each line of sequence is the
structural classification.
[0588] The PFAM database contains alignments of proteins with low
sequence identity. Therefore, these alignments are not very good.
Although the alignment algorithms (HAMMER profiles) are well suited
for recognizing conserved motifs the algorithm is not very good on
a detailed level. Therefore it is not surprising to find a
disparity between the PFAM alignment and a structural alignment. As
a skilled person would be readily aware, one can modify the PFAM
alignment based on the structural data. Meaning that one can align
those structural elements that overlap.
[0589] FIG. 55 shows the original PFAM alignment of 1DEO, 1IVN and
P10480. Added to the alignment is the secondary structure
information from the crystal structures of 1DEO and 1IVN. Alignment
2 in FIG. 56 shows a manually modified alignment where the match
between the secondary structure elements is improved. Based on
conserved residues between either 1DEO and P10480 or between 1IVN
and P10480 the alignment was modified for P10480 as well. To easily
distinguish the sequence blocks the sequence identifiers in
alignment 2 have an extra m (1DEOm, 1IVNm, P10480m).
[0590] Alignment 3 is a mix of 1 and 2, it gives the alignment per
block
EXAMPLE 2
Construction of Site Scan Libraries
[0591] The Quick Change Multi Site-Directed Mutagenesis Kit from
Stratagene was used according to the manufacturers instruction. For
each library a degenerate primer with one NNK or NNS (nucleotide
abbreviations) codon was designed. Primer design was performed
using the tools available on the Stratagene web site. Primer
quality control was further confirmed using standard analysis tools
which analyze the primer for the potential of forming hairpins or
of forming primer-dimers.
[0592] The main concepts of the method are as follows; using a
non-strand displacing high-fidelity DNA polymerase such as
Pfu-Turbo and a single primer one will linearly amplify the DNA
template. This is in contrast to the normal exponential
amplification process of a PCR reaction. This linear amplification
process ensures a low error frequency. The product is single
stranded non-methylated DNA and double stranded hemi-methylated
DNA. If the template is obtained from a suitable host organism,
then the template is double stranded methylated DNA. This means
that the template DNA can be digested with Dpn I endonuclease
without digesting the product DNA. Therefore upon transformation of
the DNA into a suitable host only a very low frequency of the
transformants with non-mutagenized plasmid.
EXAMPLE 3
Selection of Winners from a Site Scan Library
[0593] Two alternative approaches are described; library sequencing
followed by analysis of unique amino acids, or library analysis
followed by sequencing of the winners.
[0594] Selection of winners method 1; library sequencing followed
by analysis of unique amino acids.
[0595] Site scan libraries were constructed using a degenerate
oligo containing one NNK codon, where K stands for G or T and N
stands for A, C, G, or T. This means that a set of clones
constructed from an amplification reaction using an NNK primer
(also known as `a site scan library`) contains in principle 32
unique codons (4.times.4.times.2=32 combination options). Assuming
no bias due, the number of clones that one needs to pick to have a
95% chance of picking every one of the 32 codons at least once is
95. This can be calculated using the following formula
n={log(1-c)}/{log(1-f)} Formula 1;
[0596] Where n is the number of clones, c is the fraction value of
the confidence interval, for example the 95% confidence interval
has a value of 0.95 and the 99% confidence interval has a fraction
value of 0.99, and f is the frequency with which each individual
codon occurs, which for an NNK primer is {fraction (1/32)} or
0.03125. Solving the formula for n gives 94.36 or 95 clones. If a
95% confidence interval is deemed to be too low, or if one is
unable to avoid bias in one or more steps of the library
construction process, one can decide to assay or sequence more
clones. For example, in formula 1, if n is set to 384, f to
{fraction (1/32)} or 0.03125 then the confidence interval c is much
larger than 99%. Even if 60% of the clones contain the same
mutation or the wild type codon, then 363 clones will give a 99%
confidence of obtaining all 32 codons. From this one can conclude
that, 384 clones will have a 99% confidence of containing each of
the 32 codons at least once.
[0597] A colony PCR was performed (a PCR reaction on a bacterial
colony or on a bacterial liquid culture to amplify a fragment from
a plasmid inside a bacterium, and subsequently sequencing that part
of the fragment which has been mutagenised is an established
procedure. Colony PCR can be routinely performed for sets of 96 due
to the availability of prefabricated material (also known as kits)
for colony PCR, sequencing, and sequence purification. This entire
procedure is offered as a service by several commercial companies
such as AGOWA GmbH, Glienicker weg 185, D-12489 Berlin,
Germany.
[0598] After analysing the 96 sequence reactions, the individual
clones were selected representing one for each codon that is
available in the set of 96 sequences. Subsequently, the individual
clones were grown and the recombinant protein expressed. The unit
activity per quantity of protein in the assays described in Example
4 was performed.
[0599] Selection of winners method 2; library screening followed by
sequencing of the winners
[0600] Although one could choose to sequence 384 clones, one may
also assay them and select improved variants before sequencing.
[0601] A number of issues should be considered when such a number
of samples are screened. Without being exhaustive, although it is
possible to select variants with altered activity on one substrate,
the difference in expression level between 384 cultures can be
substantial even if one uses a 384 well microtiter plate, resulting
in a high background. Therefore, measuring two activities and
selecting winners based on a change in ratio is a preferred method.
To illustrate, if two activities have a certain ratio R then
regardless of the absolute amount of enzyme present, the ratio
between the two activities will always be R. A change in the R
value indicates a mutation that changed one activity relative to
the second activity.
[0602] FIG. 60 shows a data set obtained from the site scan
library. The clones are all tested for activity towards
phosphatidyl choline (PC) and digalactosyl diglyceride (DGDG). All
clones, which can be mutated or not, that exhibit no change in the
R value will lie on a straight line with a certain margin of error.
Disregarding these clones three groups of interest appear in FIG.
61.
[0603] Section 1 in FIG. 61 contains all the clones that have a
significantly higher R than the wild-type (not mutated) but lower
overall DGDG activity. Section 2 contains those clones that have
both a higher R value and a higher DGDG activity than the wild
type. Section 3 contains clones that do not have a higher R value,
but that do have a significantly higher DGDG or PC activity.
[0604] If one is interested in variants with an increased activity
towards DGDG then section 2 contains the most interesting variants
and section 3 contains variants of interest as well. The variants
in Section 3 which show a large increase in hydrolytic activity may
be accompanied by a decrease in transferase activity.
[0605] One thing is worth noticing, if a specificity determining
residue is hit, most of the 20 possible amino acids could yield a
very different R value. However, if the library contains a large
bias towards a single amino acid (for example 60% is Tyrosine) then
all those variants will still lie on a straight line.
EXAMPLE 4
Assays for PC and DGDG Activity in a 384 Well Microtiter Plate
[0606] Start Material
[0607] EM media
[0608] Plate with transformants
[0609] Plate with wild type
[0610] 384 plates
[0611] colony picker
[0612] Waco NEFA-C kit
[0613] PC and DGDG solutions in a 384 plate
[0614] Part 1--Picking Colonies
[0615] Pick colonies into a 384 plate filled with EM medium
[0616] Skip 4 wells and inoculate those with colonies containing
the non-mutated backbone
[0617] Grow o/n at 30.degree. C., 200 rpm shaking speed
[0618] Part 2--Incubation on Substrate
[0619] Centrifuge the o/n grown plates; 2500 rpm, 20 min
[0620] Transfer 10 .mu.l supernatant from each well to 2 empty 384
plates
[0621] Add 5 .mu.l 12.5 mM DGDG to one of the plates, add 5 .mu.l
12.5 mM PC to the other plate
[0622] Incubate both plates 2 hrs at 37.degree. C., shake at start
to mix then stop the shaking
[0623] Continue with the NEFA C procedure
[0624] Part 3--NEFA-C Procedure
[0625] Add 10 .mu.l A solution
[0626] Incubate 10 min 37.degree. C., 300 rpm
[0627] Add 20 .mu.l B solution
[0628] Incubate 10 min 37.degree. C., 300 rpm
[0629] Read the plate at 550 nm
[0630] Substrate Composition--in mM
[0631] 25 mM PC eller DGDG
[0632] 10 mM CaCl.sub.2
[0633] 60 mM Triton X 100
[0634] 15 mM NaN.sub.3
[0635] 20 mM Briton Robinson pH 5.0
EXAMPLE 5
Selected Variants
[0636] Determination of Enzyme Activity
[0637] To determine the enzymatic activity towards various
substrates 4 .mu.l enzyme solution was incubated with 11 .mu.l
substrate for 60 minutes at 37.degree. C. Subsequently the amount
of free fatty acids was determined using the WACO NEFA-C kit. To
the 15 .mu.l enzyme+substrate mix 75 .mu.l NEFA solution A was
added and incubated for 15 minutes at 37.degree. C. Subsequently
150 .mu.l NEFA solution B was added and incubated for 15 minutes.
Subsequently the optical density (OD) of the sample was measured at
550 nm.
[0638] As a control, from each variant 4 .mu.l enzyme solution was
incubated with 11 .mu.l HEPES buffer for 60 min at 37.degree. C.
Subsequently the amount of free fatty acids was determined as
described above. The OD values of this control sample was deducted
from the observed OD on each substrate to obtain a corrected
activity.
[0639] Four different substrates were used, the composition was in
general 30 mg lipid, 4.75 ml 50 mM HEPES buffer pH 7, 42.5 .mu.l
0.6 M CaCl2, 200 .mu.l 10% Triton X-100 H2O2-free. The 30 mg lipid
was either phosphatidyl choline (PC), PC with cholsterol in a 9 to
1 ratio, digalactosyl diglyceride (DGDG), or DGDG with cholesterol
in a 9 to 1 ratio.
[0640] Selection of Improved Variants
[0641] Variants with Improved Activity Towards PC
[0642] Those variants that showed an increase in the OD relative to
the wild type enzyme when incubated on PC were selected as variants
with improved phospholipase activity.
[0643] Variants with Improved Activity Towards DGDG
[0644] Those variants that showed an increase in the OD relative to
the wild type enzyme when incubated on DGDG were selected as
variants with improved activity towards DGDG.
[0645] Variants with Improved Specificity Towards DGDG
[0646] The specificity towards DGDG is the ratio between the
activity towards DGDG and the activity towards phosphatidylcholine
(PC). Those variants that showed a higher ratio between DGDG and PC
than the wild type were selected as variants with improved
specificity towards DGDG.
[0647] Variants with Improved Transferase Activity with PC as the
Acyl Donor
[0648] The difference in the amount of free fatty acids formed when
one incubates an enzyme on PC and on PC with cholesterol is an
indication of the amount of transferase activity relative to the
amount of hydrolytic activity. Transferase activity will not cause
the formation of free fatty acids. The transferase preference is
the ratio between the free fatty acids formed when PC is used as a
substrate and the free fatty acids formed when PC with cholesterol
is used as a substrate. Those variants that show an increase in the
transferase preference and show a higher than wild type activity
towards PC were selected as having improved transferase
activity.
[0649] Variants with Improved Transferase Activity with DGDG as the
Acyl Donor
[0650] The difference in the amount of free fatty acids formed when
one incubates an enzyme on DGDG and on DGDG with cholesterol is an
indication of the amount of transferase activity relative to the
amount of hydrolytic activity. Transferase activity will not cause
the formation of free fatty acids. The transferase preference is
the ratio between the free fatty acids formed when DGDG is used as
a substrate and the free fatty acids formed when DGDG with
cholesterol is used as a substrate. Those variants that show an
increase in the transferase preference and show a higher than wild
type activity towards DGDG were selected as having improved
transferase activity.
[0651] Selected Variants
[0652] For each of the four selection criteria above a number of
variants were selected.
[0653] The "wild type" enzyme in this example is A. salmonicida
(SEQ ID No. 28).
[0654] Variants with Improved Activity Towards PC:
5 PC Thr3Asn 158.0 Thr3Gln 151.5 Thr3Lys 141.5 Thr3Arg 133.0
Glu309Ala 106.0 Thr3Pro 101.5 Thr3Met 96.0 wild-type 86.5
[0655] Variants with Improved Activity Towards DGDG:
6 DGDG Lys182Asp 66.5 Glu309Ala 60 Tyr230Thr 59 Tyr230Gly 57.5
Tyr230Gly 51 Thr3Gln 44.5 wild-type 43.5
[0656] Variants with Improved Specificity Towards DGDG:
7 R .sub.DGDG/PC PC DGDG Lys182Asp 1.02 65.5 66.5 Tyr230Gly 0.79
72.5 57.5 Tyr230Gly 0.78 65.0 51.0 Tyr230Thr 0.75 78.5 59.0
Tyr230Val 0.71 58.0 41.0 Asp157Cys 0.69 48.0 33.0 Glu309Pro 0.58
73.5 42.5 Glu309Ala 0.57 106.0 60.0 Gly318Ile 0.53 69.5 36.5
Tyr230Arg 0.50 63.5 32.0 Tyr230Met 0.50 64.5 32.5 wild-type 0.50
86.5 43.5
[0657] Variants with Improved Transferase Activity with PC as the
Acyl Donor:
8 R.sub.PC+Cho/PC PC PC + Cho Thr3Lys 0.54 142 76 Thr3Arg 0.55 133
73 Thr3Gln 0.63 152 96 Thr3Asn 0.64 158 101 Thr3Pro 0.67 102 68
Thr3Met 0.78 96 75 wild-type 0.83 87 72
[0658] Variants with Improved Transferase Activity with DGDG as the
Acyl Donor:
9 R.sub.DGDG+Cho/DG .sub.DG DGDG Tyr230Thr 1.10 59 Lys182Asp 1.39
67 Tyr230Gly 1.55 58 Glu309Ala 1.78 60 wild-type 1.78 44
EXAMPLE 6
Transferase Assay Phospholipid:Cholesterol
[0659] Phospholipid can be replaced by DGDG to provide a
transferase assay from a galacolipid. Other acceptors for example,
glycerol, glucose, hydroxy acids, proteins or maltose can also be
used in the same assay.
[0660] 300 mg Phosphatidylcholine (Avanti
#441601):Cholesterol(Sigma C8503) 9:1 is scaled in a Wheaton glass.
10 ml 50 mM HEPES buffer pH 7.0 is added and stirring at 40.degree.
C. disperses the substrate
[0661] 0.5 ml substrate is transferred to a 4 ml vial and placed in
a heating block at 40.degree. C. 0.050 ml transferase solution is
added, also a control with 0.050 ml water is analysed in the same
way. The reaction mixture is agitated for 4 hours at 40.degree. C.
The sample is then frozen and lyophilised and analysed by GLC.
[0662] Calculation:
[0663] From the GLC analysis the content of free fatty acids and
cholesterol ester is calculated.
[0664] The enzymatic activity is calculated as: 2 % Transferase
activity = % cholesterol ester / ( Mv sterol ester ) .times. 100 %
cholesterol ester / ( Mv cholesterol ester ) + % fatty acid / ( Mv
fatty acid ) . % Hydrolyse activity = % fatty acid / ( Mv fatty
acid ) .times. 100 % cholesterol ester / ( Mv cholesterol ester ) +
% fatty acid / ( Mv fatty acid ) . Ratio Transferase/Hydrolyse=%
transferase activity/% Hydrolyse activity
Where:
.DELTA. % cholesterol ester=% cholesterol ester(sample)-%
cholesterol ester(control).
.DELTA. % fatty acid=% fatty acid(sample)-% fatty
acid(control).
[0665] Transferase Assay Galactolipid:Cholesterol.
[0666] 300 mg Digalactosyldiglyceride (>95%, from Wheat
lipid):Cholesterol(Sigma) 9:1 is scaled in a Wheaton glass. 10 ml
50 mM HEPES buffer pH 7.0 is added and stirring at 40.degree. C.
disperses the substrate.
[0667] 0.5 ml substrate is transferred to a 4 ml vial and placed in
a heating block at 40.degree. C. 0.050 ml transferase solution is
added, also a control with 0.050 ml water is analysed in the same
way. The reaction mixture is agitated for 4 hours at 40.degree. C.
The sample is then frozen and lyophilised and analysed by GLC.
[0668] Calculation:
[0669] From the GLC analysis the content of free fatty acids and
cholesterol ester is calculated.
[0670] The enzymatic activity is calculated as: 3 % Transferase
activity = % cholesterol ester / ( Mv sterol ester ) .times. 100 %
cholesterol ester / ( Mv cholesterol ester ) + % fatty acid / ( Mv
fatty acid ) . % Hydrolyse activity = % fatty acid / ( Mv fatty
acid ) .times. 100 % cholesterol ester / ( Mv cholesterol ester ) +
% fatty acid / ( Mv fatty acid ) . Ratio Transferase/Hydrolyse=%
transferase activity/% Hydrolyse activity
Where:
.DELTA. % cholesterol ester=% cholesterol ester(sample)-%
cholesterol ester(control).
.DELTA. % fatty acid=% fatty acid(sample)-% fatty acid(control)
EXAMPLE 7
Variants of a Lipid Acyltransferase for Aeromonas hydrophila (SEQ
ID No. 26)
[0671] Mutations were introduced using the QuikChange.TM.
Multi-Site Directed Mutagenesis kit from Stratagene, La Jolla,
Calif. 92037, USA following the instructions provided by
Stratagene.
[0672] Variants at Tyr256 showed an increased activity towards
phospholipids.
[0673] Variants at Tyr256 and Tyr260 showed an increased activity
towards galactolipids.
[0674] Variants at Tyr265 showed an increased transferase activity
with galactolipids as the acyl donor.
[0675] The numbers indicate positions on the following sequence: An
enzyme from Aeromonas hydrophila the amino acid sequence of which
is shown as SEQ ID No. 26. The nucleotide sequence is as shown as
SEQ ID No. 27.
[0676] The invention will now be further described by the following
numbered paragraphs:
[0677] 1. A method of producing a variant lipid acyltransferase
enzyme comprising: (a) selecting a parent enzyme which is a lipid
acyltransferase enzyme characterised in that the enzyme comprises
the amino acid sequence motif GDSX, wherein X is one or more of the
following amino acid residues L, A, V, I, F, Y, H, Q, T N, M or S;
(b) modifying one or more amino acids to produce a variant lipid
acyltransferase; (c) testing the variant lipid acyltransferase for
activity on a galactolipid substrate, and optionally a phospholipid
substrate and/or optionally a triglyceride substrate; (d) selecting
a variant enzyme with an enhanced activity towards galactolipids
compared with the parent enzyme; and optionally (e) preparing a
quantity of the variant enzyme.
[0678] 2. A method according to paragraph 1 wherein one or more of
the one or more of the following amino acid residues identified by
alignment with SEQ ID No. 2 is modified compared with a parent
sequence SEQ ID No. 2: Ser3, Leu17, Ala114, Trp111, Tyr117, Leu118,
Pro156, Gly159, Gln160, Asn161, Pro162, Ser163, Ala164, Arg165,
Ser166, Gln167, Lys168, Val169, Val170, Glu171, Ala172, Tyr179,
Gln182, Lys187, His180, Asn181, Met209, Leu210, Arg211, Asn215,
Lys284, Met285, Gln289, Val290, Ala309, Ser310, Lys22, Met23,
Gly40, Asn80, Pro81, Lys82, Val112, Asn87, Asn88, -318.
[0679] 3. A method according to paragraph 1 or paragraph 2 wherein
the parent enzyme comprises an amino acid sequence as shown as SEQ
ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6,
SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID
No. 20, SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 26, SEQ ID No. 28,
SEQ ID No. 30, SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 36, SEQ ID
No. 37, SEQ ID No. 39, SEQ ID No. 41, SEQ ID No. 43 or SEQ ID No.
45, or an amino acid sequence which has at least 70% identity
therewith.
[0680] 4. A method according to any one of paragraphs 1-3 wherein
amino acid residue 18 of the parent sequence identified by
alignment with SEQ ID No. 2 is substituted by one of the following
amino acids A, L, M, F, W, K, Q, E, P, I, C, Y, H, R, N, D, T.
[0681] 5. A method according to any one of the preceding paragraphs
wherein amino acid residue 30 of the parent sequence identified by
alignment with SEQ ID No. 2 is by one of the following amino acids
A, G, L, M, W, K, Q, S, E, P, V, I, C, H, R, N, D, T.
[0682] 6. A method according to any one of the preceding paragraphs
wherein amino acid residue 20 of the parent sequence identified by
alignment with SEQ ID No. 2 is by one of the following amino acids
A, G, L, M, W, K, Q, S, E, P, V, I, C, H, R, N, D, T.
[0683] 7. A method according to any one of the preceding paragraphs
wherein the parent enzyme is an enzyme which comprises the amino
acid sequence shown as SEQ ID No. 2 and/or SEQ ID No. 28.
[0684] 8. A method according to any one of the preceding paragraphs
wherein Preferably, the X of the GDSX motif is L.
[0685] 9. A method according to any one of the preceding paragraphs
wherein the method further comprises one or more of the following
steps: structural homology mapping or sequence homology
alignment.
[0686] 10. A method according to paragraph 9 wherein the structural
homology mapping comprises one or more of the following steps:
[0687] a) aligning a parent sequence with a structural model
(1IVN.PDB) shown in FIG. 52;
[0688] b) selecting one or more amino acid residue within a 10
.ANG. sphere centred on the central carbon atom of the glycerol
molecule in the active site (see FIG. 53); and
[0689] c) modifying one or more amino acids selected in accordance
with step (b) in said parent sequence.
[0690] 11. A method according to paragraph 9 wherein the structural
homology mapping comprises one or more of the following steps:
[0691] a) aligning a parent sequence with a structural model
(1IVN.PDB) shown in FIG. 52;
[0692] b) selecting one or more amino acids within a 10 .ANG.
sphere centred on the central carbon atom of the glycerol molecule
in the active site (see FIG. 53);
[0693] c) determining if one or more amino acid residues selected
in accordance with step (b) are highly conserved (particularly are
active site residues and/or part of the GDSx motif and/or part of
the GANDY motif); and
[0694] d) modifying one or more amino acids selected in accordance
with step (b), excluding conserved regions identified in accordance
with step (c) in said parent sequence.
[0695] 12. A method according to paragraph 9 wherein the sequence
homology alignment comprises one or more of the following
steps:
[0696] i) selecting a first parent lipid acyltransferase;
[0697] identifying a second related lipid acyltransferase having a
desirable activity;
[0698] aligning said first parent lipid acyltransferase and the
second related lipid acyltransferase;
[0699] identifying amino acid residues that differ between the two
sequences; and
[0700] modifying one or more of the amino acid residues identified
in accordance with step (iv) in said parent lipid
acyltransferase.
[0701] 13. A method according to paragraph 9 wherein the sequence
homology alignment may comprise one or more of the following
steps:
[0702] i) selecting a first parent lipid acyltransferase;
[0703] ii) identifying a second related lipid acyltransferase
having a desirable activity;
[0704] iii) aligning said first parent lipid acyltransferase and
the second related lipid acyltransferase;
[0705] iv) identifying amino acid residues that differ between the
two sequences;
[0706] v) determining if one or more amino acid residues selected
in accordance with step (iv) are highly conserved (particularly are
active site residues and/or part of the GDSx motif and/or part of
the GANDY motif); and
[0707] vi) modifying one or more of the amino acid residues
identified in accordance with step (iv) excluding conserved regions
identified in accordance with step (v) in said parent sequence.
[0708] 14. A method according to any one of the preceding
paragraphs wherein one or more of the following modifications is
made to the parent enzyme: S3A, C, D, E, F, G, H, I, K, L, M, N, P,
Q, R, S, T, V, W, or Y; D157A, C, E, F, G, H, I, K, L, M, N, P, Q,
R, S, T, V, W or Y; Q182A, C, D, E, F, G, H, I, K, L, M, N, Q, P,
R, S, T, V, W, or Y; A309A, C, D, E, F, G, H, I, K, L, M, N, P, Q,
R, S, T, V, W or Y; Y230A, C, D, E, G, H, I, K, L, M, N, P, Q, R,
S, T, V or W; a C-terminal addition (-318) of at least one amino
acid.
[0709] 15. A method according to paragraph 14 wherein one or more
of the following modifications is made to the parent enzyme S3T,
S3N, S3Q, S3K, S3R, S3P, S3M; D157 is substituted with a polar
uncharged amino acid, preferably with C, S, T or M, more preferably
C; Q182 is substituted with an aliphatic amino acid residue,
preferably a polar amino acid, more preferably a polar charged
amino acid, more preferably D or E, most preferably D; A309 is
substituted with an aliphatic residue, preferably a non-polar
residue, preferably G, A, or P, more preferably A; Y230 is
substituted with an aliphatic amino acid or one of the following
amino acid residues G, D, T, V, R or M, more preferably G, D, T, V,
R or M, more preferably G or T; a C-terminal addition comprising
one or more of I, L or V.
[0710] 16. A method according to any one of the preceding
paragraphs one or more of the following modifications is made to
the parent enzyme K187D, E309A, Y230T, Y230G, S3Q.
[0711] 17. A method according to any one of the preceding
paragraphs wherein one or more of the following modifications is
made to the parent enzyme K187D, K187D, Y230G, Y230T, Y230R, Y230M,
Y230V, D157C, E309A, G2181.
[0712] 18. A method according to any one of the preceding
paragraphs wherein one or more of the following modifications is
made to the parent enzyme S3K, S3R, S3Q, S3N, S3P, S3M.
[0713] 19. A method according to any one of the preceding
paragraphs wherein one or more of the following modifications is
made to the parent enzyme Y230T, K187D, Y230G, E309A
[0714] 20. A variant lipid acyltransferase enzyme characterised in
that the enzyme comprises the amino acid sequence motif GDSX,
wherein X is one or more of the following amino acid residues L, A,
V, I, F, Y, H, Q, T N, M or S, and wherein the variant enzyme
comprises one or more amino acid modifications compared with a
parent sequence at any one or more of the following amino acid
residues when aligned to SEQ ID No. 2: Ser3, Leu17, Ala114, Trp111,
Tyr117, Leu118, Pro156, Gly159, Gln160, Asn161, Pro162, Ser163,
Ala164, Arg165, Ser166, Gln167, Lys168, Val169, Val170, Glu171,
Ala172, Tyr179, Gln182, Lys187, His180, Asn181, Met209, Leu210,
Arg211, Asn215, Lys284, Met285, Gln289, Val290, Ala309, Ser310,
Lys22, Met23, Gly40, Asn80, Pro81, Lys82, Val112, Asn87, Asn88,
-318.
[0715] 21. A variant lipid acyltransferase enzyme according to
paragraph 20 wherein the variant enzyme comprises an amino acid
sequence, which amino acid sequence is shown as SEQ ID No. 2, SEQ
ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12,
SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20, SEQ ID
No. 22, SEQ ID No. 24, SEQ ID No. 26, SEQ ID No. 28, SEQ ID No. 30,
SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 36, SEQ ID No. 37, SEQ ID
No. 39, SEQ ID No. 41, SEQ ID No. 43 or SEQ ID No. 45 except for
one or more amino acid modifications at any one or more of the
following amino acid residues identified by sequence alignment with
SEQ ID No. 2: Ser3, Leu17, Ala114, Trp111, Tyr117, Leu118, Pro156,
Gly159, Gln160, Asn161, Pro162, Ser163, Ala164, Arg165, Ser166,
Gln167, Lys168, Val169, Val170, Glu171, Ala172, Tyr179, Gln182,
Lys187, His180, Asn181, Met209, Leu210, Arg211, Asn215, Lys284,
Met285, Gln289, Val290, Ala309, Ser310, Lys22, Met23, Gly40, Asn80,
Pro81, Lys82, Val112, Asn87, Asn88, -318.
[0716] 22. A variant lipid acyltransferase enzyme according to
paragraph 20 or paragraph 21 wherein the enzyme comprises one or
more of the following amino acid modifications S18A, L, M, F, W, K,
Q, E, P, I, C, Y, H, R, N, D, T; Y30A, G, L, M, W, K, Q, S, E, P,
V, I, C, H, R, N, D, T; Y230A, G, L, M, W, K, Q, S, E, P, V, I, C,
H, R, N, D, T.
[0717] 23. A variant lipid acyltransferase enzyme according to any
one of paragraphs 20-22 wherein the enzyme comprises one or more of
the following amino acid modifications: S3A, C, D, E, F, G, H, I,
K, L, M, N, P, Q, R, S, T, V, W, or Y; D157A, C, E, F, G, H, I, K,
L, M, N, P, Q, R, S, T, V, W or Y; Q182A, C, D, E, F, G, H, I, K,
L, M, N, Q, P, R, S, T, V, W, or Y; A309A, C, D, E, F, G, H, I, K,
L, M, N, P, Q, R, S, T, V, W or Y; Y230A, C, D, E, G, H, I, K, L,
M, N, P, Q, R, S, T, V or W; a C-terminal addition (-318) of at
least one amino acid.
[0718] 24. A variant lipid acyltransferase enzyme according to any
one of paragraphs 20-23 wherein the enzyme comprises one or more of
the following amino acid modifications: S3T, S3N, S3Q, S3K, S3R,
S3P, S3M; D157 is substituted with a polar uncharged amino acid,
preferably with C, S, T or M, more preferably C; Q182 is
substituted with an aliphatic amino acid residue, preferably a
polar amino acid, more preferably a polar charged amino acid, more
preferably D or E, most preferably D; A309 is substituted with an
aliphatic residue, preferably a non-polar residue, preferably G, A,
or P, more preferably A; Y230 is substituted with an aliphatic
amino acid or one of the following amino acid residues G, D, T, V,
R or M, more preferably G, D, T, V, R or M, more preferably G or T;
a C-terminal addition comprising one or more of I, L or V.
[0719] 25. A variant lipid acyltransferase enzyme according to any
one of paragraphs 20-24 wherein the enzyme comprises one or more of
the following amino acid modifications: K187D, E309A, Y230T, Y230G,
S3Q.
[0720] 26. A variant lipid acyltransferase enzyme according to any
one of paragraphs 20-25 wherein the enzyme comprises one or more of
the following amino acid modifications: K187D, K187D, Y230G, Y230T,
Y230R, Y230M, Y230V, D157C, E309A, G2181.
[0721] 27. A variant lipid acyltransferase enzyme according to any
one of paragraphs 20-26 wherein the enzyme comprises one or more of
the following amino acid modifications: S3K, S3R, S3Q, S3N, S3P,
S3M.
[0722] 28. A variant lipid acyltransferase enzyme according to any
one of paragraphs 20-27 wherein the enzyme comprises one or more of
the following amino acid modifications: Y230T, K187D, Y230G,
E309A.
[0723] 29. A variant lipid acyltransferase enzyme according to any
one paragraphs 20-28 wherein the variant enzyme has an enhanced
ratio of activity on galactolipids to either phospholipids and/or
triglycerides when compared with the parent enzyme.
[0724] 30. A variant lipid acyltransferase enzyme according to any
one of paragraphs 20-29 wherein the variant enzyme is an enzyme
which comprises an amino acid sequence, which amino acid sequence
is shown as SEQ ID No. 2 or SEQ ID No. 28 except for one or more
amino acid modifications at any one or more of the following amino
acid residues: Ser3, Leu17, Ala114, Trp111, Tyr117, Leu118, Pro156,
Gly159, Gln160, Asn161, Pro162, Ser163, Ala164, Arg165, Ser166,
Gln167, Lys168, Val169, Val170, Glu171, Ala172, Tyr179, Gln182,
Lys187, His180, Asn181, Met209, Leu210, Arg211, Asn215, Lys284,
Met285, Gln289, Val290, Ala309, Ser310, Lys22, Met23, Gly40, Asn80,
Pro81, Lys82, Val112, Asn87, Asn88.
[0725] 31. Use of a variant lipolytic enzyme according to any one
of paragraphs 20-30 or obtained by the method according to any one
of paragraphs 1-19 in a substrate for preparing a lyso-glycolipid,
for example digalactosyl monoglyceride (DGMG) or monogalactosyl
monoglyceride (MGMG) by treatment of a glycolipid (e.g.
digalactosyl diglyceride (DGDG) or monogalactosyl diglyceride
(MGDG)) with the variant lipolytic enzyme according to the present
invention or obtained by a method according to the present
invention to produce the partial hydrolysis product, i.e. the
lyso-glycolipid.
[0726] 32. Use according to paragraph 31 wherein the substrate is a
foodstuff.
[0727] 33. A method of preparing a foodstuff the method comprising
adding a variant lipolytic enzyme according to any one of
paragraphs 20-30 or obtained by the method according to any one of
paragraphs 1-19 to one or more ingredients of the foodstuff.
[0728] 34. A method of preparing a baked product from a dough, the
method comprising adding a variant lipolytic enzyme according to
any one of paragraphs 20-30 or obtained by the method according to
any one of paragraphs 1-19 to the dough.
[0729] 35. Use of a variant lipolytic enzyme according to any one
of paragraphs 20-30 or obtained by the method according to any one
of paragraphs 1-19 in a process of treating egg or egg-based
products to produce lysophospholipids.
[0730] 36. A process of enzymatic degumming of vegetable or edible
oils, comprising treating the edible or vegetable oil with a
variant lipolytic enzyme according to any one of paragraphs 20-30
or obtained by the method according to any one of paragraphs 1-19
so as to hydrolyse a major part of the polar lipids (e.g.
phospholipid and/or glycolipid).
[0731] 37. Use of a variant lipolytic enzyme according to any one
of paragraphs 20-30 or obtained by the method according to any one
of paragraphs 1-19 in a process for reducing the content of a
phospholipid in an edible oil, comprising treating the oil with
said variant lipolytic enzyme so as to hydrolyse a major part of
the phospholipid, and separating an aqueous phase containing the
hydrolysed phospholipid from the oil.
[0732] 38. Use of a variant lipolytic enzyme according to any one
of paragraphs 20-30 or obtained by the method according to any one
of paragraphs 1-19 in the bioconversion of polar lipids (preferably
glycolipids) to make high value products, such as carbohydrate
esters and/or protein esters and/or protein subunit esters and/or a
hydroxy acid ester.
[0733] 39. An immobilised variant lipolytic enzyme according to any
one of paragraphs 20-30 or obtained by the method according to any
one of paragraphs 1-19.
[0734] 40. A variant lipolytic enzyme generally as described herein
with reference to the figures and examples.
[0735] All publications mentioned in the above specification are
herein incorporated by reference. Various modifications and
variations of the described methods and system of the present
invention will be apparent to those skilled in the art without
departing from the scope and spirit of the present invention.
Although the present invention has been described in connection
with specific preferred embodiments, it should be understood that
the invention as claimed should not be unduly limited to such
specific embodiments. Indeed, various modifications of the
described modes for carrying out the invention which are obvious to
those skilled in biochemistry and biotechnology or related fields
are intended to be within the scope of the following claims.
Sequence CWU 1
1
55 1 361 PRT Artificial Sequence Description of Artificial Sequence
Synthetic pfam00657 consensus sequence 1 Ile Val Ala Phe Gly Asp
Ser Leu Thr Asp Gly Glu Ala Tyr Tyr Gly 1 5 10 15 Asp Ser Asp Gly
Gly Gly Trp Gly Ala Gly Leu Ala Asp Arg Leu Thr 20 25 30 Ala Leu
Leu Arg Leu Arg Ala Arg Pro Arg Gly Val Asp Val Phe Asn 35 40 45
Arg Gly Ile Ser Gly Arg Thr Ser Asp Gly Arg Leu Ile Val Asp Ala 50
55 60 Leu Val Ala Leu Leu Phe Leu Ala Gln Ser Leu Gly Leu Pro Asn
Leu 65 70 75 80 Pro Pro Tyr Leu Ser Gly Asp Phe Leu Arg Gly Ala Asn
Phe Ala Ser 85 90 95 Ala Gly Ala Thr Ile Leu Pro Thr Ser Gly Pro
Phe Leu Ile Gln Val 100 105 110 Gln Phe Lys Asp Phe Lys Ser Gln Val
Leu Glu Leu Arg Gln Ala Leu 115 120 125 Gly Leu Leu Gln Glu Leu Leu
Arg Leu Leu Pro Val Leu Asp Ala Lys 130 135 140 Ser Pro Asp Leu Val
Thr Ile Met Ile Gly Thr Asn Asp Leu Ile Thr 145 150 155 160 Ser Ala
Phe Phe Gly Pro Lys Ser Thr Glu Ser Asp Arg Asn Val Ser 165 170 175
Val Pro Glu Phe Lys Asp Asn Leu Arg Gln Leu Ile Lys Arg Leu Arg 180
185 190 Ser Asn Asn Gly Ala Arg Ile Ile Val Leu Ile Thr Leu Val Ile
Leu 195 200 205 Asn Leu Gly Pro Leu Gly Cys Leu Pro Leu Lys Leu Ala
Leu Ala Leu 210 215 220 Ala Ser Ser Lys Asn Val Asp Ala Ser Gly Cys
Leu Glu Arg Leu Asn 225 230 235 240 Glu Ala Val Ala Asp Phe Asn Glu
Ala Leu Arg Glu Leu Ala Ile Ser 245 250 255 Lys Leu Glu Asp Gln Leu
Arg Lys Asp Gly Leu Pro Asp Val Lys Gly 260 265 270 Ala Asp Val Pro
Tyr Val Asp Leu Tyr Ser Ile Phe Gln Asp Leu Asp 275 280 285 Gly Ile
Gln Asn Pro Ser Ala Tyr Val Tyr Gly Phe Glu Thr Thr Lys 290 295 300
Ala Cys Cys Gly Tyr Gly Gly Arg Tyr Asn Tyr Asn Arg Val Cys Gly 305
310 315 320 Asn Ala Gly Leu Cys Asn Val Thr Ala Lys Ala Cys Asn Pro
Ser Ser 325 330 335 Tyr Leu Leu Ser Phe Leu Phe Trp Asp Gly Phe His
Pro Ser Glu Lys 340 345 350 Gly Tyr Lys Ala Val Ala Glu Ala Leu 355
360 2 317 PRT Aeromonas hydrophila 2 Ala Asp Ser Arg Pro Ala Phe
Ser Arg Ile Val Met Phe Gly Asp Ser 1 5 10 15 Leu Ser Asp Thr Gly
Lys Met Tyr Ser Lys Met Arg Gly Tyr Leu Pro 20 25 30 Ser Ser Pro
Pro Tyr Tyr Glu Gly Arg Phe Ser Asn Gly Pro Val Trp 35 40 45 Leu
Glu Gln Leu Thr Asn Glu Phe Pro Gly Leu Thr Ile Ala Asn Glu 50 55
60 Ala Glu Gly Gly Pro Thr Ala Val Ala Tyr Asn Lys Ile Ser Trp Asn
65 70 75 80 Pro Lys Tyr Gln Val Ile Asn Asn Leu Asp Tyr Glu Val Thr
Gln Phe 85 90 95 Leu Gln Lys Asp Ser Phe Lys Pro Asp Asp Leu Val
Ile Leu Trp Val 100 105 110 Gly Ala Asn Asp Tyr Leu Ala Tyr Gly Trp
Asn Thr Glu Gln Asp Ala 115 120 125 Lys Arg Val Arg Asp Ala Ile Ser
Asp Ala Ala Asn Arg Met Val Leu 130 135 140 Asn Gly Ala Lys Glu Ile
Leu Leu Phe Asn Leu Pro Asp Leu Gly Gln 145 150 155 160 Asn Pro Ser
Ala Arg Ser Gln Lys Val Val Glu Ala Ala Ser His Val 165 170 175 Ser
Ala Tyr His Asn Gln Leu Leu Leu Asn Leu Ala Arg Gln Leu Ala 180 185
190 Pro Thr Gly Met Val Lys Leu Phe Glu Ile Asp Lys Gln Phe Ala Glu
195 200 205 Met Leu Arg Asp Pro Gln Asn Phe Gly Leu Ser Asp Gln Arg
Asn Ala 210 215 220 Cys Tyr Gly Gly Ser Tyr Val Trp Lys Pro Phe Ala
Ser Arg Ser Ala 225 230 235 240 Ser Thr Asp Ser Gln Leu Ser Ala Phe
Asn Pro Gln Glu Arg Leu Ala 245 250 255 Ile Ala Gly Asn Pro Leu Leu
Ala Gln Ala Val Ala Ser Pro Met Ala 260 265 270 Ala Arg Ser Ala Ser
Thr Leu Asn Cys Glu Gly Lys Met Phe Trp Asp 275 280 285 Gln Val His
Pro Thr Thr Val Val His Ala Ala Leu Ser Glu Pro Ala 290 295 300 Ala
Thr Phe Ile Glu Ser Gln Tyr Glu Phe Leu Ala His 305 310 315 3 336
PRT Aeromonas salmonicida 3 Met Lys Lys Trp Phe Val Cys Leu Leu Gly
Leu Ile Ala Leu Thr Val 1 5 10 15 Gln Ala Ala Asp Thr Arg Pro Ala
Phe Ser Arg Ile Val Met Phe Gly 20 25 30 Asp Ser Leu Ser Asp Thr
Gly Lys Met Tyr Ser Lys Met Arg Gly Tyr 35 40 45 Leu Pro Ser Ser
Pro Pro Tyr Tyr Glu Gly Arg Phe Ser Asn Gly Pro 50 55 60 Val Trp
Leu Glu Gln Leu Thr Lys Gln Phe Pro Gly Leu Thr Ile Ala 65 70 75 80
Asn Glu Ala Glu Gly Gly Ala Thr Ala Val Ala Tyr Asn Lys Ile Ser 85
90 95 Trp Asn Pro Lys Tyr Gln Val Tyr Asn Asn Leu Asp Tyr Glu Val
Thr 100 105 110 Gln Phe Leu Gln Lys Asp Ser Phe Lys Pro Asp Asp Leu
Val Ile Leu 115 120 125 Trp Val Gly Ala Asn Asp Tyr Leu Ala Tyr Gly
Trp Asn Thr Glu Gln 130 135 140 Asp Ala Lys Arg Val Arg Asp Ala Ile
Ser Asp Ala Ala Asn Arg Met 145 150 155 160 Val Leu Asn Gly Ala Lys
Gln Ile Leu Leu Phe Asn Leu Pro Asp Leu 165 170 175 Gly Gln Asn Pro
Ser Ala Arg Ser Gln Lys Val Val Glu Ala Val Ser 180 185 190 His Val
Ser Ala Tyr His Asn Lys Leu Leu Leu Asn Leu Ala Arg Gln 195 200 205
Leu Ala Pro Thr Gly Met Val Lys Leu Phe Glu Ile Asp Lys Gln Phe 210
215 220 Ala Glu Met Leu Arg Asp Pro Gln Asn Phe Gly Leu Ser Asp Val
Glu 225 230 235 240 Asn Pro Cys Tyr Asp Gly Gly Tyr Val Trp Lys Pro
Phe Ala Thr Arg 245 250 255 Ser Val Ser Thr Asp Arg Gln Leu Ser Ala
Phe Ser Pro Gln Glu Arg 260 265 270 Leu Ala Ile Ala Gly Asn Pro Leu
Leu Ala Gln Ala Val Ala Ser Pro 275 280 285 Met Ala Arg Arg Ser Ala
Ser Pro Leu Asn Cys Glu Gly Lys Met Phe 290 295 300 Trp Asp Gln Val
His Pro Thr Thr Val Val His Ala Ala Leu Ser Glu 305 310 315 320 Arg
Ala Ala Thr Phe Ile Glu Thr Gln Tyr Glu Phe Leu Ala His Gly 325 330
335 4 295 PRT Streptomyces coelicolor 4 Met Pro Lys Pro Ala Leu Arg
Arg Val Met Thr Ala Thr Val Ala Ala 1 5 10 15 Val Gly Thr Leu Ala
Leu Gly Leu Thr Asp Ala Thr Ala His Ala Ala 20 25 30 Pro Ala Gln
Ala Thr Pro Thr Leu Asp Tyr Val Ala Leu Gly Asp Ser 35 40 45 Tyr
Ser Ala Gly Ser Gly Val Leu Pro Val Asp Pro Ala Asn Leu Leu 50 55
60 Cys Leu Arg Ser Thr Ala Asn Tyr Pro His Val Ile Ala Asp Thr Thr
65 70 75 80 Gly Ala Arg Leu Thr Asp Val Thr Cys Gly Ala Ala Gln Thr
Ala Asp 85 90 95 Phe Thr Arg Ala Gln Tyr Pro Gly Val Ala Pro Gln
Leu Asp Ala Leu 100 105 110 Gly Thr Gly Thr Asp Leu Val Thr Leu Thr
Ile Gly Gly Asn Asp Asn 115 120 125 Ser Thr Phe Ile Asn Ala Ile Thr
Ala Cys Gly Thr Ala Gly Val Leu 130 135 140 Ser Gly Gly Lys Gly Ser
Pro Cys Lys Asp Arg His Gly Thr Ser Phe 145 150 155 160 Asp Asp Glu
Ile Glu Ala Asn Thr Tyr Pro Ala Leu Lys Glu Ala Leu 165 170 175 Leu
Gly Val Arg Ala Arg Ala Pro His Ala Arg Val Ala Ala Leu Gly 180 185
190 Tyr Pro Trp Ile Thr Pro Ala Thr Ala Asp Pro Ser Cys Phe Leu Lys
195 200 205 Leu Pro Leu Ala Ala Gly Asp Val Pro Tyr Leu Arg Ala Ile
Gln Ala 210 215 220 His Leu Asn Asp Ala Val Arg Arg Ala Ala Glu Glu
Thr Gly Ala Thr 225 230 235 240 Tyr Val Asp Phe Ser Gly Val Ser Asp
Gly His Asp Ala Cys Glu Ala 245 250 255 Pro Gly Thr Arg Trp Ile Glu
Pro Leu Leu Phe Gly His Ser Leu Val 260 265 270 Pro Val His Pro Asn
Ala Leu Gly Glu Arg Arg Met Ala Glu His Thr 275 280 285 Met Asp Val
Leu Gly Leu Asp 290 295 5 295 PRT Streptomyces coelicolor 5 Met Pro
Lys Pro Ala Leu Arg Arg Val Met Thr Ala Thr Val Ala Ala 1 5 10 15
Val Gly Thr Leu Ala Leu Gly Leu Thr Asp Ala Thr Ala His Ala Ala 20
25 30 Pro Ala Gln Ala Thr Pro Thr Leu Asp Tyr Val Ala Leu Gly Asp
Ser 35 40 45 Tyr Ser Ala Gly Ser Gly Val Leu Pro Val Asp Pro Ala
Asn Leu Leu 50 55 60 Cys Leu Arg Ser Thr Ala Asn Tyr Pro His Val
Ile Ala Asp Thr Thr 65 70 75 80 Gly Ala Arg Leu Thr Asp Val Thr Cys
Gly Ala Ala Gln Thr Ala Asp 85 90 95 Phe Thr Arg Ala Gln Tyr Pro
Gly Val Ala Pro Gln Leu Asp Ala Leu 100 105 110 Gly Thr Gly Thr Asp
Leu Val Thr Leu Thr Ile Gly Gly Asn Asp Asn 115 120 125 Ser Thr Phe
Ile Asn Ala Ile Thr Ala Cys Gly Thr Ala Gly Val Leu 130 135 140 Ser
Gly Gly Lys Gly Ser Pro Cys Lys Asp Arg His Gly Thr Ser Phe 145 150
155 160 Asp Asp Glu Ile Glu Ala Asn Thr Tyr Pro Ala Leu Lys Glu Ala
Leu 165 170 175 Leu Gly Val Arg Ala Arg Ala Pro His Ala Arg Val Ala
Ala Leu Gly 180 185 190 Tyr Pro Trp Ile Thr Pro Ala Thr Ala Asp Pro
Ser Cys Phe Leu Lys 195 200 205 Leu Pro Leu Ala Ala Gly Asp Val Pro
Tyr Leu Arg Ala Ile Gln Ala 210 215 220 His Leu Asn Asp Ala Val Arg
Arg Ala Ala Glu Glu Thr Gly Ala Thr 225 230 235 240 Tyr Val Asp Phe
Ser Gly Val Ser Asp Gly His Asp Ala Cys Glu Ala 245 250 255 Pro Gly
Thr Arg Trp Ile Glu Pro Leu Leu Phe Gly His Ser Leu Val 260 265 270
Pro Val His Pro Asn Ala Leu Gly Glu Arg Arg Met Ala Glu His Thr 275
280 285 Met Asp Val Leu Gly Leu Asp 290 295 6 238 PRT Saccharomyces
cerevisiae 6 Met Asp Tyr Glu Lys Phe Leu Leu Phe Gly Asp Ser Ile
Thr Glu Phe 1 5 10 15 Ala Phe Asn Thr Arg Pro Ile Glu Asp Gly Lys
Asp Gln Tyr Ala Leu 20 25 30 Gly Ala Ala Leu Val Asn Glu Tyr Thr
Arg Lys Met Asp Ile Leu Gln 35 40 45 Arg Gly Phe Lys Gly Tyr Thr
Ser Arg Trp Ala Leu Lys Ile Leu Pro 50 55 60 Glu Ile Leu Lys His
Glu Ser Asn Ile Val Met Ala Thr Ile Phe Leu 65 70 75 80 Gly Ala Asn
Asp Ala Cys Ser Ala Gly Pro Gln Ser Val Pro Leu Pro 85 90 95 Glu
Phe Ile Asp Asn Ile Arg Gln Met Val Ser Leu Met Lys Ser Tyr 100 105
110 His Ile Arg Pro Ile Ile Ile Gly Pro Gly Leu Val Asp Arg Glu Lys
115 120 125 Trp Glu Lys Glu Lys Ser Glu Glu Ile Ala Leu Gly Tyr Phe
Arg Thr 130 135 140 Asn Glu Asn Phe Ala Ile Tyr Ser Asp Ala Leu Ala
Lys Leu Ala Asn 145 150 155 160 Glu Glu Lys Val Pro Phe Val Ala Leu
Asn Lys Ala Phe Gln Gln Glu 165 170 175 Gly Gly Asp Ala Trp Gln Gln
Leu Leu Thr Asp Gly Leu His Phe Ser 180 185 190 Gly Lys Gly Tyr Lys
Ile Phe His Asp Glu Leu Leu Lys Val Ile Glu 195 200 205 Thr Phe Tyr
Pro Gln Tyr His Pro Lys Asn Met Gln Tyr Lys Leu Lys 210 215 220 Asp
Trp Arg Asp Val Leu Asp Asp Gly Ser Asn Ile Met Ser 225 230 235 7
1005 DNA Aeromonas hydrophila 7 atgaaaaaat ggtttgtgtg tttattggga
ttggtcgcgc tgacagttca ggcagccgac 60 agccgtcccg ccttctcccg
gatcgtgatg tttggcgaca gcctctccga taccggcaag 120 atgtacagca
agatgcgcgg ttacctcccc tccagccccc cctactatga gggccgcttc 180
tccaacgggc ccgtctggct ggagcagctg accaacgagt tcccgggcct gaccatagcc
240 aacgaggcgg aaggcggacc gaccgccgtg gcttacaaca agatctcctg
gaatcccaag 300 tatcaggtca tcaacaacct ggactacgag gtcacccagt
tcctgcaaaa agacagcttc 360 aagccggacg atctggtgat cctctgggtc
ggcgccaacg actatctggc ctatggctgg 420 aacacagagc aggatgccaa
gcgggtgcgc gacgccatca gcgatgcggc caaccgcatg 480 gtgctgaacg
gcgccaagga gatactgctg ttcaacctgc cggatctggg ccagaacccc 540
tcggcccgca gccagaaggt ggtcgaggcg gccagccatg tctccgccta ccacaaccag
600 ctgctgctga acctggcacg ccagctggct cccaccggca tggtgaagct
gttcgagatc 660 gacaagcagt ttgccgagat gctgcgtgat ccgcagaact
tcggcctgag cgaccagagg 720 aacgcctgct acggtggcag ctatgtatgg
aagccgtttg cctcccgcag cgccagcacc 780 gacagccagc tctccgcctt
caacccgcag gagcgcctcg ccatcgccgg caacccgctg 840 ctggcccagg
ccgtcgccag ccccatggct gcccgcagcg ccagcaccct caactgtgag 900
ggcaagatgt tctgggatca ggtccacccc accactgtcg tgcacgccgc cctgagcgag
960 cccgccgcca ccttcatcga gagccagtac gagttcctcg cccac 1005 8 1011
DNA Aeromonas salmonicida 8 atgaaaaaat ggtttgtttg tttattgggg
ttgatcgcgc tgacagttca ggcagccgac 60 actcgccccg ccttctcccg
gatcgtgatg ttcggcgaca gcctctccga taccggcaaa 120 atgtacagca
agatgcgcgg ttacctcccc tccagcccgc cctactatga gggccgtttc 180
tccaacggac ccgtctggct ggagcagctg accaagcagt tcccgggtct gaccatcgcc
240 aacgaagcgg aaggcggtgc cactgccgtg gcttacaaca agatctcctg
gaatcccaag 300 tatcaggtct acaacaacct ggactacgag gtcacccagt
tcttgcagaa agacagcttc 360 aagccggacg atctggtgat cctctgggtc
ggtgccaatg actatctggc atatggctgg 420 aatacggagc aggatgccaa
gcgagttcgc gatgccatca gcgatgcggc caaccgcatg 480 gtactgaacg
gtgccaagca gatactgctg ttcaacctgc cggatctggg ccagaacccg 540
tcagcccgca gtcagaaggt ggtcgaggcg gtcagccatg tctccgccta tcacaacaag
600 ctgctgctga acctggcacg ccagctggcc cccaccggca tggtaaagct
gttcgagatc 660 gacaagcaat ttgccgagat gctgcgtgat ccgcagaact
tcggcctgag cgacgtcgag 720 aacccctgct acgacggcgg ctatgtgtgg
aagccgtttg ccacccgcag cgtcagcacc 780 gaccgccagc tctccgcctt
cagtccgcag gaacgcctcg ccatcgccgg caacccgctg 840 ctggcacagg
ccgttgccag tcctatggcc cgccgcagcg ccagccccct caactgtgag 900
ggcaagatgt tctgggatca ggtacacccg accactgtcg tgcacgcagc cctgagcgag
960 cgcgccgcca ccttcatcga gacccagtac gagttcctcg cccacggatg a 1011 9
888 DNA Streptomyces coelicolor 9 atgccgaagc ctgcccttcg ccgtgtcatg
accgcgacag tcgccgccgt cggcacgctc 60 gccctcggcc tcaccgacgc
caccgcccac gccgcgcccg cccaggccac tccgaccctg 120 gactacgtcg
ccctcggcga cagctacagc gccggctccg gcgtcctgcc cgtcgacccc 180
gccaacctgc tctgtctgcg ctcgacggcc aactaccccc acgtcatcgc ggacacgacg
240 ggcgcccgcc tcacggacgt cacctgcggc gccgcgcaga ccgccgactt
cacgcgggcc 300 cagtacccgg gcgtcgcacc ccagttggac gcgctcggca
ccggcacgga cctggtcacg 360 ctcaccatcg gcggcaacga caacagcacc
ttcatcaacg ccatcacggc ctgcggcacg 420 gcgggtgtcc tcagcggcgg
caagggcagc ccctgcaagg acaggcacgg cacctccttc 480 gacgacgaga
tcgaggccaa cacgtacccc gcgctcaagg aggcgctgct cggcgtccgc 540
gccagggctc cccacgccag ggtggcggct ctcggctacc cgtggatcac cccggccacc
600 gccgacccgt cctgcttcct gaagctcccc ctcgccgccg gtgacgtgcc
ctacctgcgg 660 gccatccagg cacacctcaa cgacgcggtc cggcgggccg
ccgaggagac cggagccacc 720 tacgtggact tctccggggt gtccgacggc
cacgacgcct gcgaggcccc cggcacccgc 780 tggatcgaac cgctgctctt
cgggcacagc ctcgttcccg tccaccccaa cgccctgggc 840 gagcggcgca
tggccgagca cacgatggac gtcctcggcc tggactga 888 10 888 DNA
Streptomyces coelicolor 10 tcagtccagg ccgaggacgt ccatcgtgtg
ctcggccatg cgccgctcgc ccagggcgtt 60 ggggtggacg ggaacgaggc
tgtgcccgaa gagcagcggt tcgatccagc gggtgccggg 120 ggcctcgcag
gcgtcgtggc cgtcggacac cccggagaag tccacgtagg tggctccggt 180
ctcctcggcg gcccgccgga ccgcgtcgtt gaggtgtgcc tggatggccc gcaggtaggg
240 cacgtcaccg gcggcgaggg ggagcttcag gaagcaggac gggtcggcgg
tggccggggt 300 gatccacggg tagccgagag ccgccaccct ggcgtgggga
gccctggcgc ggacgccgag 360 cagcgcctcc ttgagcgcgg ggtacgtgtt
ggcctcgatc tcgtcgtcga aggaggtgcc 420
gtgcctgtcc ttgcaggggc tgcccttgcc gccgctgagg acacccgccg tgccgcaggc
480 cgtgatggcg ttgatgaagg tgctgttgtc gttgccgccg atggtgagcg
tgaccaggtc 540 cgtgccggtg ccgagcgcgt ccaactgggg tgcgacgccc
gggtactggg cccgcgtgaa 600 gtcggcggtc tgcgcggcgc cgcaggtgac
gtccgtgagg cgggcgcccg tcgtgtccgc 660 gatgacgtgg gggtagttgg
ccgtcgagcg cagacagagc aggttggcgg ggtcgacggg 720 caggacgccg
gagccggcgc tgtagctgtc gccgagggcg acgtagtcca gggtcggagt 780
ggcctgggcg ggcgcggcgt gggcggtggc gtcggtgagg ccgagggcga gcgtgccgac
840 ggcggcgact gtcgcggtca tgacacggcg aagggcaggc ttcggcat 888 11 717
DNA Saccharomyces cerevisiae 11 atggattacg agaagtttct gttatttggg
gattccatta ctgaatttgc ttttaatact 60 aggcccattg aagatggcaa
agatcagtat gctcttggag ccgcattagt caacgaatat 120 acgagaaaaa
tggatattct tcaaagaggg ttcaaagggt acacttctag atgggcgttg 180
aaaatacttc ctgagatttt aaagcatgaa tccaatattg tcatggccac aatatttttg
240 ggtgccaacg atgcatgctc agcaggtccc caaagtgtcc ccctccccga
atttatcgat 300 aatattcgtc aaatggtatc tttgatgaag tcttaccata
tccgtcctat tataatagga 360 ccggggctag tagatagaga gaagtgggaa
aaagaaaaat ctgaagaaat agctctcgga 420 tacttccgta ccaacgagaa
ctttgccatt tattccgatg ccttagcaaa actagccaat 480 gaggaaaaag
ttcccttcgt ggctttgaat aaggcgtttc aacaggaagg tggtgatgct 540
tggcaacaac tgctaacaga tggactgcac ttttccggaa aagggtacaa aatttttcat
600 gacgaattat tgaaggtcat tgagacattc tacccccaat atcatcccaa
aaacatgcag 660 tacaaactga aagattggag agatgtgcta gatgatggat
ctaacataat gtcttga 717 12 347 PRT Ralstonia solanacearum 12 Met Asn
Leu Arg Gln Trp Met Gly Ala Ala Thr Ala Ala Leu Ala Leu 1 5 10 15
Gly Leu Ala Ala Cys Gly Gly Gly Gly Thr Asp Gln Ser Gly Asn Pro 20
25 30 Asn Val Ala Lys Val Gln Arg Met Val Val Phe Gly Asp Ser Leu
Ser 35 40 45 Asp Ile Gly Thr Tyr Thr Pro Val Ala Gln Ala Val Gly
Gly Gly Lys 50 55 60 Phe Thr Thr Asn Pro Gly Pro Ile Trp Ala Glu
Thr Val Ala Ala Gln 65 70 75 80 Leu Gly Val Thr Leu Thr Pro Ala Val
Met Gly Tyr Ala Thr Ser Val 85 90 95 Gln Asn Cys Pro Lys Ala Gly
Cys Phe Asp Tyr Ala Gln Gly Gly Ser 100 105 110 Arg Val Thr Asp Pro
Asn Gly Ile Gly His Asn Gly Gly Ala Gly Ala 115 120 125 Leu Thr Tyr
Pro Val Gln Gln Gln Leu Ala Asn Phe Tyr Ala Ala Ser 130 135 140 Asn
Asn Thr Phe Asn Gly Asn Asn Asp Val Val Phe Val Leu Ala Gly 145 150
155 160 Ser Asn Asp Ile Phe Phe Trp Thr Thr Ala Ala Ala Thr Ser Gly
Ser 165 170 175 Gly Val Thr Pro Ala Ile Ala Thr Ala Gln Val Gln Gln
Ala Ala Thr 180 185 190 Asp Leu Val Gly Tyr Val Lys Asp Met Ile Ala
Lys Gly Ala Thr Gln 195 200 205 Val Tyr Val Phe Asn Leu Pro Asp Ser
Ser Leu Thr Pro Asp Gly Val 210 215 220 Ala Ser Gly Thr Thr Gly Gln
Ala Leu Leu His Ala Leu Val Gly Thr 225 230 235 240 Phe Asn Thr Thr
Leu Gln Ser Gly Leu Ala Gly Thr Ser Ala Arg Ile 245 250 255 Ile Asp
Phe Asn Ala Gln Leu Thr Ala Ala Ile Gln Asn Gly Ala Ser 260 265 270
Phe Gly Phe Ala Asn Thr Ser Ala Arg Ala Cys Asp Ala Thr Lys Ile 275
280 285 Asn Ala Leu Val Pro Ser Ala Gly Gly Ser Ser Leu Phe Cys Ser
Ala 290 295 300 Asn Thr Leu Val Ala Ser Gly Ala Asp Gln Ser Tyr Leu
Phe Ala Asp 305 310 315 320 Gly Val His Pro Thr Thr Ala Gly His Arg
Leu Ile Ala Ser Asn Val 325 330 335 Leu Ala Arg Leu Leu Ala Asp Asn
Val Ala His 340 345 13 1044 DNA Ralstonia solanacearum 13
atgaacctgc gtcaatggat gggcgccgcc acggctgccc ttgccttggg cttggccgcg
60 tgcgggggcg gtgggaccga ccagagcggc aatcccaatg tcgccaaggt
gcagcgcatg 120 gtggtgttcg gcgacagcct gagcgatatc ggcacctaca
cccccgtcgc gcaggcggtg 180 ggcggcggca agttcaccac caacccgggc
ccgatctggg ccgagaccgt ggccgcgcaa 240 ctgggcgtga cgctcacgcc
ggcggtgatg ggctacgcca cctccgtgca gaattgcccc 300 aaggccggct
gcttcgacta tgcgcagggc ggctcgcgcg tgaccgatcc gaacggcatc 360
ggccacaacg gcggcgcggg ggcgctgacc tacccggttc agcagcagct cgccaacttc
420 tacgcggcca gcaacaacac attcaacggc aataacgatg tcgtcttcgt
gctggccggc 480 agcaacgaca ttttcttctg gaccactgcg gcggccacca
gcggctccgg cgtgacgccc 540 gccattgcca cggcccaggt gcagcaggcc
gcgacggacc tggtcggcta tgtcaaggac 600 atgatcgcca agggtgcgac
gcaggtctac gtgttcaacc tgcccgacag cagcctgacg 660 ccggacggcg
tggcaagcgg cacgaccggc caggcgctgc tgcacgcgct ggtgggcacg 720
ttcaacacga cgctgcaaag cgggctggcc ggcacctcgg cgcgcatcat cgacttcaac
780 gcacaactga ccgcggcgat ccagaatggc gcctcgttcg gcttcgccaa
caccagcgcc 840 cgggcctgcg acgccaccaa gatcaatgcc ctggtgccga
gcgccggcgg cagctcgctg 900 ttctgctcgg ccaacacgct ggtggcttcc
ggtgcggacc agagctacct gttcgccgac 960 ggcgtgcacc cgaccacggc
cggccatcgc ctgatcgcca gcaacgtgct ggcgcgcctg 1020 ctggcggata
acgtcgcgca ctga 1044 14 261 PRT Streptomyces coelicolor 14 Met Ile
Gly Ser Tyr Val Ala Val Gly Asp Ser Phe Thr Glu Gly Val 1 5 10 15
Gly Asp Pro Gly Pro Asp Gly Ala Phe Val Gly Trp Ala Asp Arg Leu 20
25 30 Ala Val Leu Leu Ala Asp Arg Arg Pro Glu Gly Asp Phe Thr Tyr
Thr 35 40 45 Asn Leu Ala Val Arg Gly Arg Leu Leu Asp Gln Ile Val
Ala Glu Gln 50 55 60 Val Pro Arg Val Val Gly Leu Ala Pro Asp Leu
Val Ser Phe Ala Ala 65 70 75 80 Gly Gly Asn Asp Ile Ile Arg Pro Gly
Thr Asp Pro Asp Glu Val Ala 85 90 95 Glu Arg Phe Glu Leu Ala Val
Ala Ala Leu Thr Ala Ala Ala Gly Thr 100 105 110 Val Leu Val Thr Thr
Gly Phe Asp Thr Arg Gly Val Pro Val Leu Lys 115 120 125 His Leu Arg
Gly Lys Ile Ala Thr Tyr Asn Gly His Val Arg Ala Ile 130 135 140 Ala
Asp Arg Tyr Gly Cys Pro Val Leu Asp Leu Trp Ser Leu Arg Ser 145 150
155 160 Val Gln Asp Arg Arg Ala Trp Asp Ala Asp Arg Leu His Leu Ser
Pro 165 170 175 Glu Gly His Thr Arg Val Ala Leu Arg Ala Gly Gln Ala
Leu Gly Leu 180 185 190 Arg Val Pro Ala Asp Pro Asp Gln Pro Trp Pro
Pro Leu Pro Pro Arg 195 200 205 Gly Thr Leu Asp Val Arg Arg Asp Asp
Val His Trp Ala Arg Glu Tyr 210 215 220 Leu Val Pro Trp Ile Gly Arg
Arg Leu Arg Gly Glu Ser Ser Gly Asp 225 230 235 240 His Val Thr Ala
Lys Gly Thr Leu Ser Pro Asp Ala Ile Lys Thr Arg 245 250 255 Ile Ala
Ala Val Ala 260 15 786 DNA Streptomyces coelicolor 15 gtgatcgggt
cgtacgtggc ggtgggggac agcttcaccg agggcgtcgg cgaccccggc 60
cccgacgggg cgttcgtcgg ctgggccgac cggctcgccg tactgctcgc ggaccggcgc
120 cccgagggcg acttcacgta cacgaacctc gccgtgcgcg gcaggctcct
cgaccagatc 180 gtggcggaac aggtcccgcg ggtcgtcgga ctcgcgcccg
acctcgtctc gttcgcggcg 240 ggcggcaacg acatcatccg gcccggcacc
gatcccgacg aggtcgccga gcggttcgag 300 ctggcggtgg ccgcgctgac
cgccgcggcc ggaaccgtcc tggtgaccac cgggttcgac 360 acccgggggg
tgcccgtcct caagcacctg cgcggcaaga tcgccacgta caacgggcac 420
gtccgcgcca tcgccgaccg ctacggctgc ccggtgctcg acctgtggtc gctgcggagc
480 gtccaggacc gcagggcgtg ggacgccgac cggctgcacc tgtcgccgga
ggggcacacc 540 cgggtggcgc tgcgcgcggg gcaggccctg ggcctgcgcg
tcccggccga ccctgaccag 600 ccctggccgc ccctgccgcc gcgcggcacg
ctcgacgtcc ggcgcgacga cgtgcactgg 660 gcgcgcgagt acctggtgcc
gtggatcggg cgccggctgc ggggcgagtc gtcgggcgac 720 cacgtgacgg
ccaaggggac gctgtcgccg gacgccatca agacgcggat cgccgcggtg 780 gcctga
786 16 260 PRT Streptomyces coelicolor 16 Met Gln Thr Asn Pro Ala
Tyr Thr Ser Leu Val Ala Val Gly Asp Ser 1 5 10 15 Phe Thr Glu Gly
Met Ser Asp Leu Leu Pro Asp Gly Ser Tyr Arg Gly 20 25 30 Trp Ala
Asp Leu Leu Ala Thr Arg Met Ala Ala Arg Ser Pro Gly Phe 35 40 45
Arg Tyr Ala Asn Leu Ala Val Arg Gly Lys Leu Ile Gly Gln Ile Val 50
55 60 Asp Glu Gln Val Asp Val Ala Ala Ala Met Gly Ala Asp Val Ile
Thr 65 70 75 80 Leu Val Gly Gly Leu Asn Asp Thr Leu Arg Pro Lys Cys
Asp Met Ala 85 90 95 Arg Val Arg Asp Leu Leu Thr Gln Ala Val Glu
Arg Leu Ala Pro His 100 105 110 Cys Glu Gln Leu Val Leu Met Arg Ser
Pro Gly Arg Gln Gly Pro Val 115 120 125 Leu Glu Arg Phe Arg Pro Arg
Met Glu Ala Leu Phe Ala Val Ile Asp 130 135 140 Asp Leu Ala Gly Arg
His Gly Ala Val Val Val Asp Leu Tyr Gly Ala 145 150 155 160 Gln Ser
Leu Ala Asp Pro Arg Met Trp Asp Val Asp Arg Leu His Leu 165 170 175
Thr Ala Glu Gly His Arg Arg Val Ala Glu Ala Val Trp Gln Ser Leu 180
185 190 Gly His Glu Pro Glu Asp Pro Glu Trp His Ala Pro Ile Pro Ala
Thr 195 200 205 Pro Pro Pro Gly Trp Val Thr Arg Arg Thr Ala Asp Val
Arg Phe Ala 210 215 220 Arg Gln His Leu Leu Pro Trp Ile Gly Arg Arg
Leu Thr Gly Arg Ser 225 230 235 240 Ser Gly Asp Gly Leu Pro Ala Lys
Arg Pro Asp Leu Leu Pro Tyr Glu 245 250 255 Asp Pro Ala Arg 260 17
783 DNA Streptomyces coelicolor 17 atgcagacga accccgcgta caccagtctc
gtcgccgtcg gcgactcctt caccgagggc 60 atgtcggacc tgctgcccga
cggctcctac cgtggctggg ccgacctcct cgccacccgg 120 atggcggccc
gctcccccgg cttccggtac gccaacctgg cggtgcgcgg gaagctgatc 180
ggacagatcg tcgacgagca ggtggacgtg gccgccgcca tgggagccga cgtgatcacg
240 ctggtcggcg ggctcaacga cacgctgcgg cccaagtgcg acatggcccg
ggtgcgggac 300 ctgctgaccc aggccgtgga acggctcgcc ccgcactgcg
agcagctggt gctgatgcgc 360 agtcccggtc gccagggtcc ggtgctggag
cgcttccggc cccgcatgga ggccctgttc 420 gccgtgatcg acgacctggc
cgggcggcac ggcgccgtgg tcgtcgacct gtacggggcc 480 cagtcgctgg
ccgaccctcg gatgtgggac gtggaccggc tgcacctgac cgccgagggc 540
caccgccggg tcgcggaggc ggtgtggcag tcgctcggcc acgagcccga ggaccccgag
600 tggcacgcgc cgatcccggc gacgccgccg ccggggtggg tgacgcgcag
gaccgcggac 660 gtccggttcg cccggcagca cctgctgccc tggataggcc
gcaggctgac cgggcgctcg 720 tccggggacg gcctgccggc caagcgcccg
gacctgctgc cctacgagga ccccgcacgg 780 tga 783 18 454 PRT
Streptomyces coelicolor 18 Met Thr Arg Gly Arg Asp Gly Gly Ala Gly
Ala Pro Pro Thr Lys His 1 5 10 15 Arg Ala Leu Leu Ala Ala Ile Val
Thr Leu Ile Val Ala Ile Ser Ala 20 25 30 Ala Ile Tyr Ala Gly Ala
Ser Ala Asp Asp Gly Ser Arg Asp His Ala 35 40 45 Leu Gln Ala Gly
Gly Arg Leu Pro Arg Gly Asp Ala Ala Pro Ala Ser 50 55 60 Thr Gly
Ala Trp Val Gly Ala Trp Ala Thr Ala Pro Ala Ala Ala Glu 65 70 75 80
Pro Gly Thr Glu Thr Thr Gly Leu Ala Gly Arg Ser Val Arg Asn Val 85
90 95 Val His Thr Ser Val Gly Gly Thr Gly Ala Arg Ile Thr Leu Ser
Asn 100 105 110 Leu Tyr Gly Gln Ser Pro Leu Thr Val Thr His Ala Ser
Ile Ala Leu 115 120 125 Ala Ala Gly Pro Asp Thr Ala Ala Ala Ile Ala
Asp Thr Met Arg Arg 130 135 140 Leu Thr Phe Gly Gly Ser Ala Arg Val
Ile Ile Pro Ala Gly Gly Gln 145 150 155 160 Val Met Ser Asp Thr Ala
Arg Leu Ala Ile Pro Tyr Gly Ala Asn Val 165 170 175 Leu Val Thr Thr
Tyr Ser Pro Ile Pro Ser Gly Pro Val Thr Tyr His 180 185 190 Pro Gln
Ala Arg Gln Thr Ser Tyr Leu Ala Asp Gly Asp Arg Thr Ala 195 200 205
Asp Val Thr Ala Val Ala Tyr Thr Thr Pro Thr Pro Tyr Trp Arg Tyr 210
215 220 Leu Thr Ala Leu Asp Val Leu Ser His Glu Ala Asp Gly Thr Val
Val 225 230 235 240 Ala Phe Gly Asp Ser Ile Thr Asp Gly Ala Arg Ser
Gln Ser Asp Ala 245 250 255 Asn His Arg Trp Thr Asp Val Leu Ala Ala
Arg Leu His Glu Ala Ala 260 265 270 Gly Asp Gly Arg Asp Thr Pro Arg
Tyr Ser Val Val Asn Glu Gly Ile 275 280 285 Ser Gly Asn Arg Leu Leu
Thr Ser Arg Pro Gly Arg Pro Ala Asp Asn 290 295 300 Pro Ser Gly Leu
Ser Arg Phe Gln Arg Asp Val Leu Glu Arg Thr Asn 305 310 315 320 Val
Lys Ala Val Val Val Val Leu Gly Val Asn Asp Val Leu Asn Ser 325 330
335 Pro Glu Leu Ala Asp Arg Asp Ala Ile Leu Thr Gly Leu Arg Thr Leu
340 345 350 Val Asp Arg Ala His Ala Arg Gly Leu Arg Val Val Gly Ala
Thr Ile 355 360 365 Thr Pro Phe Gly Gly Tyr Gly Gly Tyr Thr Glu Ala
Arg Glu Thr Met 370 375 380 Arg Gln Glu Val Asn Glu Glu Ile Arg Ser
Gly Arg Val Phe Asp Thr 385 390 395 400 Val Val Asp Phe Asp Lys Ala
Leu Arg Asp Pro Tyr Asp Pro Arg Arg 405 410 415 Met Arg Ser Asp Tyr
Asp Ser Gly Asp His Leu His Pro Gly Asp Lys 420 425 430 Gly Tyr Ala
Arg Met Gly Ala Val Ile Asp Leu Ala Ala Leu Lys Gly 435 440 445 Ala
Ala Pro Val Lys Ala 450 19 1365 DNA Streptomyces coelicolor 19
atgacccggg gtcgtgacgg gggtgcgggg gcgcccccca ccaagcaccg tgccctgctc
60 gcggcgatcg tcaccctgat agtggcgatc tccgcggcca tatacgccgg
agcgtccgcg 120 gacgacggca gcagggacca cgcgctgcag gccggaggcc
gtctcccacg aggagacgcc 180 gcccccgcgt ccaccggtgc ctgggtgggc
gcctgggcca ccgcaccggc cgcggccgag 240 ccgggcaccg agacgaccgg
cctggcgggc cgctccgtgc gcaacgtcgt gcacacctcg 300 gtcggcggca
ccggcgcgcg gatcaccctc tcgaacctgt acgggcagtc gccgctgacc 360
gtcacacacg cctcgatcgc cctggccgcc gggcccgaca ccgccgccgc gatcgccgac
420 accatgcgcc ggctcacctt cggcggcagc gcccgggtga tcatcccggc
gggcggccag 480 gtgatgagcg acaccgcccg cctcgccatc ccctacgggg
cgaacgtcct ggtcaccacg 540 tactccccca tcccgtccgg gccggtgacc
taccatccgc aggcccggca gaccagctac 600 ctggccgacg gcgaccgcac
ggcggacgtc accgccgtcg cgtacaccac ccccacgccc 660 tactggcgct
acctgaccgc cctcgacgtg ctgagccacg aggccgacgg cacggtcgtg 720
gcgttcggcg actccatcac cgacggcgcc cgctcgcaga gcgacgccaa ccaccgctgg
780 accgacgtcc tcgccgcacg cctgcacgag gcggcgggcg acggccggga
cacgccccgc 840 tacagcgtcg tcaacgaggg catcagcggc aaccggctcc
tgaccagcag gccggggcgg 900 ccggccgaca acccgagcgg actgagccgg
ttccagcggg acgtgctgga acgcaccaac 960 gtcaaggccg tcgtcgtcgt
cctcggcgtc aacgacgtcc tgaacagccc ggaactcgcc 1020 gaccgcgacg
ccatcctgac cggcctgcgc accctcgtcg accgggcgca cgcccgggga 1080
ctgcgggtcg tcggcgccac gatcacgccg ttcggcggct acggcggcta caccgaggcc
1140 cgcgagacga tgcggcagga ggtcaacgag gagatccgct ccggccgggt
cttcgacacg 1200 gtcgtcgact tcgacaaggc cctgcgcgac ccgtacgacc
cgcgccggat gcgctccgac 1260 tacgacagcg gcgaccacct gcaccccggc
gacaaggggt acgcgcgcat gggcgcggtc 1320 atcgacctgg ccgcgctgaa
gggcgcggcg ccggtcaagg cgtag 1365 20 340 PRT Streptomyces coelicolor
20 Met Thr Ser Met Ser Arg Ala Arg Val Ala Arg Arg Ile Ala Ala Gly
1 5 10 15 Ala Ala Tyr Gly Gly Gly Gly Ile Gly Leu Ala Gly Ala Ala
Ala Val 20 25 30 Gly Leu Val Val Ala Glu Val Gln Leu Ala Arg Arg
Arg Val Gly Val 35 40 45 Gly Thr Pro Thr Arg Val Pro Asn Ala Gln
Gly Leu Tyr Gly Gly Thr 50 55 60 Leu Pro Thr Ala Gly Asp Pro Pro
Leu Arg Leu Met Met Leu Gly Asp 65 70 75 80 Ser Thr Ala Ala Gly Gln
Gly Val His Arg Ala Gly Gln Thr Pro Gly 85 90 95 Ala Leu Leu Ala
Ser Gly Leu Ala Ala Val Ala Glu Arg Pro Val Arg 100 105 110 Leu Gly
Ser Val Ala Gln Pro Gly Ala Cys Ser Asp Asp Leu Asp Arg 115 120 125
Gln Val Ala Leu Val Leu Ala Glu Pro Asp Arg Val Pro Asp Ile Cys 130
135 140 Val Ile Met Val Gly Ala Asn Asp Val Thr His Arg Met Pro Ala
Thr 145 150 155 160 Arg Ser Val Arg His Leu Ser Ser Ala Val Arg Arg
Leu Arg Thr Ala 165 170 175 Gly Ala Glu Val Val Val Gly Thr Cys Pro
Asp Leu Gly Thr Ile Glu 180 185 190 Arg Val Arg Gln Pro Leu Arg Trp
Leu Ala Arg Arg Ala Ser Arg Gln 195 200
205 Leu Ala Ala Ala Gln Thr Ile Gly Ala Val Glu Gln Gly Gly Arg Thr
210 215 220 Val Ser Leu Gly Asp Leu Leu Gly Pro Glu Phe Ala Gln Asn
Pro Arg 225 230 235 240 Glu Leu Phe Gly Pro Asp Asn Tyr His Pro Ser
Ala Glu Gly Tyr Ala 245 250 255 Thr Ala Ala Met Ala Val Leu Pro Ser
Val Cys Ala Ala Leu Gly Leu 260 265 270 Trp Pro Ala Asp Glu Glu His
Pro Asp Ala Leu Arg Arg Glu Gly Phe 275 280 285 Leu Pro Val Ala Arg
Ala Ala Ala Glu Ala Ala Ser Glu Ala Gly Thr 290 295 300 Glu Val Ala
Ala Ala Met Pro Thr Gly Pro Arg Gly Pro Trp Ala Leu 305 310 315 320
Leu Lys Arg Arg Arg Arg Arg Arg Val Ser Glu Ala Glu Pro Ser Ser 325
330 335 Pro Ser Gly Val 340 21 1023 DNA Streptomyces coelicolor 21
atgacgagca tgtcgagggc gagggtggcg cggcggatcg cggccggcgc ggcgtacggc
60 ggcggcggca tcggcctggc gggagcggcg gcggtcggtc tggtggtggc
cgaggtgcag 120 ctggccagac gcagggtggg ggtgggcacg ccgacccggg
tgccgaacgc gcagggactg 180 tacggcggca ccctgcccac ggccggcgac
ccgccgctgc ggctgatgat gctgggcgac 240 tccacggccg ccgggcaggg
cgtgcaccgg gccgggcaga cgccgggcgc gctgctggcg 300 tccgggctcg
cggcggtggc ggagcggccg gtgcggctgg ggtcggtcgc ccagccgggg 360
gcgtgctcgg acgacctgga ccggcaggtg gcgctggtgc tcgccgagcc ggaccgggtg
420 cccgacatct gcgtgatcat ggtcggcgcc aacgacgtca cccaccggat
gccggcgacc 480 cgctcggtgc ggcacctgtc ctcggcggta cggcggctgc
gcacggccgg tgcggaggtg 540 gtggtcggca cctgtccgga cctgggcacg
atcgagcggg tgcggcagcc gctgcgctgg 600 ctggcccggc gggcctcacg
gcagctcgcg gcggcacaga ccatcggcgc cgtcgagcag 660 ggcgggcgca
cggtgtcgct gggcgacctg ctgggtccgg agttcgcgca gaacccgcgg 720
gagctcttcg gccccgacaa ctaccacccc tccgccgagg ggtacgccac ggccgcgatg
780 gcggtactgc cctcggtgtg cgccgcgctc ggcctgtggc cggccgacga
ggagcacccg 840 gacgcgctgc gccgcgaggg cttcctgccg gtggcgcgcg
cggcggcgga ggcggcgtcc 900 gaggcgggta cggaggtcgc cgccgccatg
cctacggggc ctcgggggcc ctgggcgctg 960 ctgaagcgcc ggagacggcg
tcgggtgtcg gaggcggaac cgtccagccc gtccggcgtt 1020 tga 1023 22 305
PRT Streptomyces coelicolor 22 Met Gly Arg Gly Thr Asp Gln Arg Thr
Arg Tyr Gly Arg Arg Arg Ala 1 5 10 15 Arg Val Ala Leu Ala Ala Leu
Thr Ala Ala Val Leu Gly Val Gly Val 20 25 30 Ala Gly Cys Asp Ser
Val Gly Gly Asp Ser Pro Ala Pro Ser Gly Ser 35 40 45 Pro Ser Lys
Arg Thr Arg Thr Ala Pro Ala Trp Asp Thr Ser Pro Ala 50 55 60 Ser
Val Ala Ala Val Gly Asp Ser Ile Thr Arg Gly Phe Asp Ala Cys 65 70
75 80 Ala Val Leu Ser Asp Cys Pro Glu Val Ser Trp Ala Thr Gly Ser
Ser 85 90 95 Ala Lys Val Asp Ser Leu Ala Val Arg Leu Leu Gly Lys
Ala Asp Ala 100 105 110 Ala Glu His Ser Trp Asn Tyr Ala Val Thr Gly
Ala Arg Met Ala Asp 115 120 125 Leu Thr Ala Gln Val Thr Arg Ala Ala
Gln Arg Glu Pro Glu Leu Val 130 135 140 Ala Val Met Ala Gly Ala Asn
Asp Ala Cys Arg Ser Thr Thr Ser Ala 145 150 155 160 Met Thr Pro Val
Ala Asp Phe Arg Ala Gln Phe Glu Glu Ala Met Ala 165 170 175 Thr Leu
Arg Lys Lys Leu Pro Lys Ala Gln Val Tyr Val Ser Ser Ile 180 185 190
Pro Asp Leu Lys Arg Leu Trp Ser Gln Gly Arg Thr Asn Pro Leu Gly 195
200 205 Lys Gln Val Trp Lys Leu Gly Leu Cys Pro Ser Met Leu Gly Asp
Ala 210 215 220 Asp Ser Leu Asp Ser Ala Ala Thr Leu Arg Arg Asn Thr
Val Arg Asp 225 230 235 240 Arg Val Ala Asp Tyr Asn Glu Val Leu Arg
Glu Val Cys Ala Lys Asp 245 250 255 Arg Arg Cys Arg Ser Asp Asp Gly
Ala Val His Glu Phe Arg Phe Gly 260 265 270 Thr Asp Gln Leu Ser His
Trp Asp Trp Phe His Pro Ser Val Asp Gly 275 280 285 Gln Ala Arg Leu
Ala Glu Ile Ala Tyr Arg Ala Val Thr Ala Lys Asn 290 295 300 Pro 305
23 918 DNA Streptomyces coelicolor 23 atgggtcgag ggacggacca
gcggacgcgg tacggccgtc gccgggcgcg tgtcgcgctc 60 gccgccctga
ccgccgccgt cctgggcgtg ggcgtggcgg gctgcgactc cgtgggcggc 120
gactcacccg ctccttccgg cagcccgtcg aagcggacga ggacggcgcc cgcctgggac
180 accagcccgg cgtccgtcgc cgccgtgggc gactccatca cgcgcggctt
cgacgcctgt 240 gcggtgctgt cggactgccc ggaggtgtcg tgggcgaccg
gcagcagcgc gaaggtcgac 300 tcgctggccg tacggctgct ggggaaggcg
gacgcggccg agcacagctg gaactacgcg 360 gtcaccgggg cccggatggc
ggacctgacc gctcaggtga cgcgggcggc gcagcgcgag 420 ccggagctgg
tggcggtgat ggccggggcg aacgacgcgt gccggtccac gacctcggcg 480
atgacgccgg tggcggactt ccgggcgcag ttcgaggagg cgatggccac cctgcgcaag
540 aagctcccca aggcgcaggt gtacgtgtcg agcatcccgg acctcaagcg
gctctggtcc 600 cagggccgca ccaacccgct gggcaagcag gtgtggaagc
tcggcctgtg cccgtcgatg 660 ctgggcgacg cggactccct ggactcggcg
gcgaccctgc ggcgcaacac ggtgcgcgac 720 cgggtggcgg actacaacga
ggtgctgcgg gaggtctgcg cgaaggaccg gcggtgccgc 780 agcgacgacg
gcgcggtgca cgagttccgg ttcggcacgg accagttgag ccactgggac 840
tggttccacc cgagtgtgga cggccaggcc cggctggcgg agatcgccta ccgcgcggtc
900 accgcgaaga atccctga 918 24 268 PRT Streptomyces rimosus 24 Met
Arg Leu Ser Arg Arg Ala Ala Thr Ala Ser Ala Leu Leu Leu Thr 1 5 10
15 Pro Ala Leu Ala Leu Phe Gly Ala Ser Ala Ala Val Ser Ala Pro Arg
20 25 30 Ile Gln Ala Thr Asp Tyr Val Ala Leu Gly Asp Ser Tyr Ser
Ser Gly 35 40 45 Val Gly Ala Gly Ser Tyr Asp Ser Ser Ser Gly Ser
Cys Lys Arg Ser 50 55 60 Thr Lys Ser Tyr Pro Ala Leu Trp Ala Ala
Ser His Thr Gly Thr Arg 65 70 75 80 Phe Asn Phe Thr Ala Cys Ser Gly
Ala Arg Thr Gly Asp Val Leu Ala 85 90 95 Lys Gln Leu Thr Pro Val
Asn Ser Gly Thr Asp Leu Val Ser Ile Thr 100 105 110 Ile Gly Gly Asn
Asp Ala Gly Phe Ala Asp Thr Met Thr Thr Cys Asn 115 120 125 Leu Gln
Gly Glu Ser Ala Cys Leu Ala Arg Ile Ala Lys Ala Arg Ala 130 135 140
Tyr Ile Gln Gln Thr Leu Pro Ala Gln Leu Asp Gln Val Tyr Asp Ala 145
150 155 160 Ile Asp Ser Arg Ala Pro Ala Ala Gln Val Val Val Leu Gly
Tyr Pro 165 170 175 Arg Phe Tyr Lys Leu Gly Gly Ser Cys Ala Val Gly
Leu Ser Glu Lys 180 185 190 Ser Arg Ala Ala Ile Asn Ala Ala Ala Asp
Asp Ile Asn Ala Val Thr 195 200 205 Ala Lys Arg Ala Ala Asp His Gly
Phe Ala Phe Gly Asp Val Asn Thr 210 215 220 Thr Phe Ala Gly His Glu
Leu Cys Ser Gly Ala Pro Trp Leu His Ser 225 230 235 240 Val Thr Leu
Pro Val Glu Asn Ser Tyr His Pro Thr Ala Asn Gly Gln 245 250 255 Ser
Lys Gly Tyr Leu Pro Val Leu Asn Ser Ala Thr 260 265 25 1068 DNA
Streptomyces rimosus 25 ttcatcacaa cgatgtcaca acaccggcca tccgggtcat
ccctgatcgt gggaatgggt 60 gacaagcctt cccgtgacga aagggtcctg
ctacatcaga aatgacagaa atcctgctca 120 gggaggttcc atgagactgt
cccgacgcgc ggccacggcg tccgcgctcc tcctcacccc 180 ggcgctcgcg
ctcttcggcg cgagcgccgc cgtgtccgcg ccgcgaatcc aggccaccga 240
ctacgtggcc ctcggcgact cctactcctc gggggtcggc gcgggcagct acgacagcag
300 cagtggctcc tgtaagcgca gcaccaagtc ctacccggcc ctgtgggccg
cctcgcacac 360 cggtacgcgg ttcaacttca ccgcctgttc gggcgcccgc
acaggagacg tgctggccaa 420 gcagctgacc ccggtcaact ccggcaccga
cctggtcagc attaccatcg gcggcaacga 480 cgcgggcttc gccgacacca
tgaccacctg caacctccag ggcgagagcg cgtgcctggc 540 gcggatcgcc
aaggcgcgcg cctacatcca gcagacgctg cccgcccagc tggaccaggt 600
ctacgacgcc atcgacagcc gggcccccgc agcccaggtc gtcgtcctgg gctacccgcg
660 cttctacaag ctgggcggca gctgcgccgt cggtctctcg gagaagtccc
gcgcggccat 720 caacgccgcc gccgacgaca tcaacgccgt caccgccaag
cgcgccgccg accacggctt 780 cgccttcggg gacgtcaaca cgaccttcgc
cgggcacgag ctgtgctccg gcgccccctg 840 gctgcacagc gtcacccttc
ccgtggagaa ctcctaccac cccacggcca acggacagtc 900 caagggctac
ctgcccgtcc tgaactccgc cacctgatct cgcggctact ccgcccctga 960
cgaagtcccg cccccgggcg gggcttcgcc gtaggtgcgc gtaccgccgt cgcccgtcgc
1020 gccggtggcc ccgccgtacg tgccgccgcc cccggacgcg gtcggttc 1068 26
335 PRT Aeromonas hydrophila 26 Met Lys Lys Trp Phe Val Cys Leu Leu
Gly Leu Val Ala Leu Thr Val 1 5 10 15 Gln Ala Ala Asp Ser Arg Pro
Ala Phe Ser Arg Ile Val Met Phe Gly 20 25 30 Asp Ser Leu Ser Asp
Thr Gly Lys Met Tyr Ser Lys Met Arg Gly Tyr 35 40 45 Leu Pro Ser
Ser Pro Pro Tyr Tyr Glu Gly Arg Phe Ser Asn Gly Pro 50 55 60 Val
Trp Leu Glu Gln Leu Thr Lys Gln Phe Pro Gly Leu Thr Ile Ala 65 70
75 80 Asn Glu Ala Glu Gly Gly Ala Thr Ala Val Ala Tyr Asn Lys Ile
Ser 85 90 95 Trp Asn Pro Lys Tyr Gln Val Ile Asn Asn Leu Asp Tyr
Glu Val Thr 100 105 110 Gln Phe Leu Gln Lys Asp Ser Phe Lys Pro Asp
Asp Leu Val Ile Leu 115 120 125 Trp Val Gly Ala Asn Asp Tyr Leu Ala
Tyr Gly Trp Asn Thr Glu Gln 130 135 140 Asp Ala Lys Arg Val Arg Asp
Ala Ile Ser Asp Ala Ala Asn Arg Met 145 150 155 160 Val Leu Asn Gly
Ala Lys Gln Ile Leu Leu Phe Asn Leu Pro Asp Leu 165 170 175 Gly Gln
Asn Pro Ser Ala Arg Ser Gln Lys Val Val Glu Ala Val Ser 180 185 190
His Val Ser Ala Tyr His Asn Gln Leu Leu Leu Asn Leu Ala Arg Gln 195
200 205 Leu Ala Pro Thr Gly Met Val Lys Leu Phe Glu Ile Asp Lys Gln
Phe 210 215 220 Ala Glu Met Leu Arg Asp Pro Gln Asn Phe Gly Leu Ser
Asp Val Glu 225 230 235 240 Asn Pro Cys Tyr Asp Gly Gly Tyr Val Trp
Lys Pro Phe Ala Thr Arg 245 250 255 Ser Val Ser Thr Asp Arg Gln Leu
Ser Ala Phe Ser Pro Gln Glu Arg 260 265 270 Leu Ala Ile Ala Gly Asn
Pro Leu Leu Ala Gln Ala Val Ala Ser Pro 275 280 285 Met Ala Arg Arg
Ser Ala Ser Pro Leu Asn Cys Glu Gly Lys Met Phe 290 295 300 Trp Asp
Gln Val His Pro Thr Thr Val Val His Ala Ala Leu Ser Glu 305 310 315
320 Arg Ala Ala Thr Phe Ile Ala Asn Gln Tyr Glu Phe Leu Ala His 325
330 335 27 1008 DNA Aeromonas hydrophila 27 atgaaaaaat ggtttgtgtg
tttattggga ttggtcgcgc tgacagttca ggcagccgac 60 agtcgccccg
ccttttcccg gatcgtgatg ttcggcgaca gcctctccga taccggcaaa 120
atgtacagca agatgcgcgg ttacctcccc tccagcccgc cctactatga gggccgtttc
180 tccaacggac ccgtctggct ggagcagctg accaaacagt tcccgggtct
gaccatcgcc 240 aacgaagcgg aaggcggtgc cactgccgtg gcttacaaca
agatctcctg gaatcccaag 300 tatcaggtca tcaacaacct ggactacgag
gtcacccagt tcttgcagaa agacagcttc 360 aagccggacg atctggtgat
cctctgggtc ggtgccaatg actatctggc ctatggctgg 420 aacacggagc
aggatgccaa gcgggttcgc gatgccatca gcgatgcggc caaccgcatg 480
gtactgaacg gtgccaagca gatactgctg ttcaacctgc cggatctggg ccagaacccg
540 tcagctcgca gtcagaaggt ggtcgaggcg gtcagccatg tctccgccta
tcacaaccag 600 ctgctgctga acctggcacg ccagctggcc cccaccggca
tggtaaagct gttcgagatc 660 gacaagcaat ttgccgagat gctgcgtgat
ccgcagaact tcggcctgag cgacgtcgag 720 aacccctgct acgacggcgg
ctatgtgtgg aagccgtttg ccacccgcag cgtcagcacc 780 gaccgccagc
tctccgcctt cagtccgcag gaacgcctcg ccatcgccgg caacccgctg 840
ctggcacagg ccgttgccag tcctatggcc cgccgcagcg ccagccccct caactgtgag
900 ggcaagatgt tctgggatca ggtacacccg accactgtcg tgcacgcagc
cctgagcgag 960 cgcgccgcca ccttcatcgc gaaccagtac gagttcctcg cccactga
1008 28 336 PRT Aeromonas salmonicida 28 Met Lys Lys Trp Phe Val
Cys Leu Leu Gly Leu Ile Ala Leu Thr Val 1 5 10 15 Gln Ala Ala Asp
Thr Arg Pro Ala Phe Ser Arg Ile Val Met Phe Gly 20 25 30 Asp Ser
Leu Ser Asp Thr Gly Lys Met Tyr Ser Lys Met Arg Gly Tyr 35 40 45
Leu Pro Ser Ser Pro Pro Tyr Tyr Glu Gly Arg Phe Ser Asn Gly Pro 50
55 60 Val Trp Leu Glu Gln Leu Thr Lys Gln Phe Pro Gly Leu Thr Ile
Ala 65 70 75 80 Asn Glu Ala Glu Gly Gly Ala Thr Ala Val Ala Tyr Asn
Lys Ile Ser 85 90 95 Trp Asn Pro Lys Tyr Gln Val Ile Asn Asn Leu
Asp Tyr Glu Val Thr 100 105 110 Gln Phe Leu Gln Lys Asp Ser Phe Lys
Pro Asp Asp Leu Val Ile Leu 115 120 125 Trp Val Gly Ala Asn Asp Tyr
Leu Ala Tyr Gly Trp Asn Thr Glu Gln 130 135 140 Asp Ala Lys Arg Val
Arg Asp Ala Ile Ser Asp Ala Ala Asn Arg Met 145 150 155 160 Val Leu
Asn Gly Ala Lys Gln Ile Leu Leu Phe Asn Leu Pro Asp Leu 165 170 175
Gly Gln Asn Pro Ser Ala Arg Ser Gln Lys Val Val Glu Ala Val Ser 180
185 190 His Val Ser Ala Tyr His Asn Lys Leu Leu Leu Asn Leu Ala Arg
Gln 195 200 205 Leu Ala Pro Thr Gly Met Val Lys Leu Phe Glu Ile Asp
Lys Gln Phe 210 215 220 Ala Glu Met Leu Arg Asp Pro Gln Asn Phe Gly
Leu Ser Asp Val Glu 225 230 235 240 Asn Pro Cys Tyr Asp Gly Gly Tyr
Val Trp Lys Pro Phe Ala Thr Arg 245 250 255 Ser Val Ser Thr Asp Arg
Gln Leu Ser Ala Phe Ser Pro Gln Glu Arg 260 265 270 Leu Ala Ile Ala
Gly Asn Pro Leu Leu Ala Gln Ala Val Ala Ser Pro 275 280 285 Met Ala
Arg Arg Ser Ala Ser Pro Leu Asn Cys Glu Gly Lys Met Phe 290 295 300
Trp Asp Gln Val His Pro Thr Thr Val Val His Ala Ala Leu Ser Glu 305
310 315 320 Arg Ala Ala Thr Phe Ile Glu Thr Gln Tyr Glu Phe Leu Ala
His Gly 325 330 335 29 1011 DNA Aeromonas salmonicida 29 atgaaaaaat
ggtttgtttg tttattgggg ttgatcgcgc tgacagttca ggcagccgac 60
actcgccccg ccttctcccg gatcgtgatg ttcggcgaca gcctctccga taccggcaaa
120 atgtacagca agatgcgcgg ttacctcccc tccagcccgc cctactatga
gggccgtttc 180 tccaacggac ccgtctggct ggagcagctg accaagcagt
tcccgggtct gaccatcgcc 240 aacgaagcgg aaggcggtgc cactgccgtg
gcttacaaca agatctcctg gaatcccaag 300 tatcaggtca tcaacaacct
ggactacgag gtcacccagt tcttgcagaa agacagcttc 360 aagccggacg
atctggtgat cctctgggtc ggtgccaatg actatctggc atatggctgg 420
aatacggagc aggatgccaa gcgagttcgc gatgccatca gcgatgcggc caaccgcatg
480 gtactgaacg gtgccaagca gatactgctg ttcaacctgc cggatctggg
ccagaacccg 540 tcagcccgca gtcagaaggt ggtcgaggcg gtcagccatg
tctccgccta tcacaacaag 600 ctgctgctga acctggcacg ccagctggcc
cccaccggca tggtaaagct gttcgagatc 660 gacaagcaat ttgccgagat
gctgcgtgat ccgcagaact tcggcctgag cgacgtcgag 720 aacccctgct
acgacggcgg ctatgtgtgg aagccgtttg ccacccgcag cgtcagcacc 780
gaccgccagc tctccgcctt cagtccgcag gaacgcctcg ccatcgccgg caacccgctg
840 ctggcacagg ccgttgccag tcctatggcc cgccgcagcg ccagccccct
caactgtgag 900 ggcaagatgt tctgggatca ggtacacccg accactgtcg
tgcacgcagc cctgagcgag 960 cgcgccgcca ccttcatcga gacccagtac
gagttcctcg cccacggatg a 1011 30 347 PRT Aeromonas hydrophila 30 Met
Phe Lys Phe Lys Lys Asn Phe Leu Val Gly Leu Ser Ala Ala Leu 1 5 10
15 Met Ser Ile Ser Leu Phe Ser Ala Thr Ala Ser Ala Ala Ser Ala Asp
20 25 30 Ser Arg Pro Ala Phe Ser Arg Ile Val Met Phe Gly Asp Ser
Leu Ser 35 40 45 Asp Thr Gly Lys Met Tyr Ser Lys Met Arg Gly Tyr
Leu Pro Ser Ser 50 55 60 Pro Pro Tyr Tyr Glu Gly Arg Phe Ser Asn
Gly Pro Val Trp Leu Glu 65 70 75 80 Gln Leu Thr Lys Gln Phe Pro Gly
Leu Thr Ile Ala Asn Glu Ala Glu 85 90 95 Gly Gly Ala Thr Ala Val
Ala Tyr Asn Lys Ile Ser Trp Asn Pro Lys 100 105 110 Tyr Gln Val Ile
Asn Asn Leu Asp Tyr Glu Val Thr Gln Phe Leu Gln 115 120 125 Lys Asp
Ser Phe Lys Pro Asp Asp Leu Val Ile Leu Trp Val Gly Ala 130 135 140
Asn Asp Tyr Leu Ala Tyr Gly Trp Asn Thr Glu Gln Asp Ala Lys Arg 145
150 155 160 Val Arg Asp Ala Ile Ser Asp Ala Ala Asn Arg Met Val Leu
Asn Gly 165 170 175 Ala Lys Gln Ile Leu Leu Phe Asn Leu Pro Asp
Leu
Gly Gln Asn Pro 180 185 190 Ser Ala Arg Ser Gln Lys Val Val Glu Ala
Val Ser His Val Ser Ala 195 200 205 Tyr His Asn Gln Leu Leu Leu Asn
Leu Ala Arg Gln Leu Ala Pro Thr 210 215 220 Gly Met Val Lys Leu Phe
Glu Ile Asp Lys Gln Phe Ala Glu Met Leu 225 230 235 240 Arg Asp Pro
Gln Asn Phe Gly Leu Ser Asp Val Glu Asn Pro Cys Tyr 245 250 255 Asp
Gly Gly Tyr Val Trp Lys Pro Phe Ala Thr Arg Ser Val Ser Thr 260 265
270 Asp Arg Gln Leu Ser Ala Phe Ser Pro Gln Glu Arg Leu Ala Ile Ala
275 280 285 Gly Asn Pro Leu Leu Ala Gln Ala Val Ala Ser Pro Met Ala
Arg Arg 290 295 300 Ser Ala Ser Pro Leu Asn Cys Glu Gly Lys Met Phe
Trp Asp Gln Val 305 310 315 320 His Pro Thr Thr Val Val His Ala Ala
Leu Ser Glu Arg Ala Ala Thr 325 330 335 Phe Ile Ala Asn Gln Tyr Glu
Phe Leu Ala His 340 345 31 1047 DNA Aeromonas hydrophila 31
atgtttaagt ttaaaaagaa tttcttagtt ggattatcgg cagctttaat gagtattagc
60 ttgttttcgg caaccgcctc tgcagctagc gccgacagcc gtcccgcctt
ttcccggatc 120 gtgatgttcg gcgacagcct ctccgatacc ggcaaaatgt
acagcaagat gcgcggttac 180 ctcccctcca gcccgcccta ctatgagggc
cgtttctcca acggacccgt ctggctggag 240 cagctgacca aacagttccc
gggtctgacc atcgccaacg aagcggaagg cggtgccact 300 gccgtggctt
acaacaagat ctcctggaat cccaagtatc aggtcatcaa caacctggac 360
tacgaggtca cccagttctt gcagaaagac agcttcaagc cggacgatct ggtgatcctc
420 tgggtcggtg ccaatgacta tctggcctat ggctggaaca cggagcagga
tgccaagcgg 480 gttcgcgatg ccatcagcga tgcggccaac cgcatggtac
tgaacggtgc caagcagata 540 ctgctgttca acctgccgga tctgggccag
aacccgtcag ctcgcagtca gaaggtggtc 600 gaggcggtca gccatgtctc
cgcctatcac aaccagctgc tgctgaacct ggcacgccag 660 ctggccccca
ccggcatggt aaagctgttc gagatcgaca agcaatttgc cgagatgctg 720
cgtgatccgc agaacttcgg cctgagcgac gtcgagaacc cctgctacga cggcggctat
780 gtgtggaagc cgtttgccac ccgcagcgtc agcaccgacc gccagctctc
cgccttcagt 840 ccgcaggaac gcctcgccat cgccggcaac ccgctgctgg
cacaggccgt tgccagtcct 900 atggcccgcc gcagcgccag ccccctcaac
tgtgagggca agatgttctg ggatcaggta 960 cacccgacca ctgtcgtgca
cgcagccctg agcgagcgcg ccgccacctt catcgcgaac 1020 cagtacgagt
tcctcgccca ctgatga 1047 32 1371 DNA Streptomyces coelicolor 32
acaggccgat gcacggaacc gtacctttcc gcagtgaagc gctctccccc catcgttcgc
60 cgggacttca tccgcgattt tggcatgaac acttccttca acgcgcgtag
cttgctacaa 120 gtgcggcagc agacccgctc gttggaggct cagtgagatt
gacccgatcc ctgtcggccg 180 catccgtcat cgtcttcgcc ctgctgctcg
cgctgctggg catcagcccg gcccaggcag 240 ccggcccggc ctatgtggcc
ctgggggatt cctattcctc gggcaacggc gccggaagtt 300 acatcgattc
gagcggtgac tgtcaccgca gcaacaacgc gtaccccgcc cgctgggcgg 360
cggccaacgc accgtcctcc ttcaccttcg cggcctgctc gggagcggtg accacggatg
420 tgatcaacaa tcagctgggc gccctcaacg cgtccaccgg cctggtgagc
atcaccatcg 480 gcggcaatga cgcgggcttc gcggacgcga tgaccacctg
cgtcaccagc tcggacagca 540 cctgcctcaa ccggctggcc accgccacca
actacatcaa caccaccctg ctcgcccggc 600 tcgacgcggt ctacagccag
atcaaggccc gtgcccccaa cgcccgcgtg gtcgtcctcg 660 gctacccgcg
catgtacctg gcctcgaacc cctggtactg cctgggcctg agcaacacca 720
agcgcgcggc catcaacacc accgccgaca ccctcaactc ggtgatctcc tcccgggcca
780 ccgcccacgg attccgattc ggcgatgtcc gcccgacctt caacaaccac
gaactgttct 840 tcggcaacga ctggctgcac tcactcaccc tgccggtgtg
ggagtcgtac caccccacca 900 gcacgggcca tcagagcggc tatctgccgg
tcctcaacgc caacagctcg acctgatcaa 960 cgcacggccg tgcccgcccc
gcgcgtcacg ctcggcgcgg gcgccgcagc gcgttgatca 1020 gcccacagtg
ccggtgacgg tcccaccgtc acggtcgagg gtgtacgtca cggtggcgcc 1080
gctccagaag tggaacgtca gcaggaccgt ggagccgtcc ctgacctcgt cgaagaactc
1140 cggggtcagc gtgatcaccc ctcccccgta gccgggggcg aaggcggcgc
cgaactcctt 1200 gtaggacgtc cagtcgtgcg gcccggcgtt gccaccgtcc
gcgtagaccg cttccatggt 1260 cgccagccgg tccccgcgga actcggtggg
gatgtccgtg cccaaggtgg tcccggtggt 1320 gtccgagagc accgggggct
cgtaccggat gatgtgcaga tccaaagaat t 1371 33 267 PRT Streptomyces
coelicolor 33 Met Arg Leu Thr Arg Ser Leu Ser Ala Ala Ser Val Ile
Val Phe Ala 1 5 10 15 Leu Leu Leu Ala Leu Leu Gly Ile Ser Pro Ala
Gln Ala Ala Gly Pro 20 25 30 Ala Tyr Val Ala Leu Gly Asp Ser Tyr
Ser Ser Gly Asn Gly Ala Gly 35 40 45 Ser Tyr Ile Asp Ser Ser Gly
Asp Cys His Arg Ser Asn Asn Ala Tyr 50 55 60 Pro Ala Arg Trp Ala
Ala Ala Asn Ala Pro Ser Ser Phe Thr Phe Ala 65 70 75 80 Ala Cys Ser
Gly Ala Val Thr Thr Asp Val Ile Asn Asn Gln Leu Gly 85 90 95 Ala
Leu Asn Ala Ser Thr Gly Leu Val Ser Ile Thr Ile Gly Gly Asn 100 105
110 Asp Ala Gly Phe Ala Asp Ala Met Thr Thr Cys Val Thr Ser Ser Asp
115 120 125 Ser Thr Cys Leu Asn Arg Leu Ala Thr Ala Thr Asn Tyr Ile
Asn Thr 130 135 140 Thr Leu Leu Ala Arg Leu Asp Ala Val Tyr Ser Gln
Ile Lys Ala Arg 145 150 155 160 Ala Pro Asn Ala Arg Val Val Val Leu
Gly Tyr Pro Arg Met Tyr Leu 165 170 175 Ala Ser Asn Pro Trp Tyr Cys
Leu Gly Leu Ser Asn Thr Lys Arg Ala 180 185 190 Ala Ile Asn Thr Thr
Ala Asp Thr Leu Asn Ser Val Ile Ser Ser Arg 195 200 205 Ala Thr Ala
His Gly Phe Arg Phe Gly Asp Val Arg Pro Thr Phe Asn 210 215 220 Asn
His Glu Leu Phe Phe Gly Asn Asp Trp Leu His Ser Leu Thr Leu 225 230
235 240 Pro Val Trp Glu Ser Tyr His Pro Thr Ser Thr Gly His Gln Ser
Gly 245 250 255 Tyr Leu Pro Val Leu Asn Ala Asn Ser Ser Thr 260 265
34 548 PRT Termobifida fusca 34 Met Leu Pro His Pro Ala Gly Glu Arg
Gly Glu Val Gly Ala Phe Phe 1 5 10 15 Ala Leu Leu Val Gly Thr Pro
Gln Asp Arg Arg Leu Arg Leu Glu Cys 20 25 30 His Glu Thr Arg Pro
Leu Arg Gly Arg Cys Gly Cys Gly Glu Arg Arg 35 40 45 Val Pro Pro
Leu Thr Leu Pro Gly Asp Gly Val Leu Cys Thr Thr Ser 50 55 60 Ser
Thr Arg Asp Ala Glu Thr Val Trp Arg Lys His Leu Gln Pro Arg 65 70
75 80 Pro Asp Gly Gly Phe Arg Pro His Leu Gly Val Gly Cys Leu Leu
Ala 85 90 95 Gly Gln Gly Ser Pro Gly Val Leu Trp Cys Gly Arg Glu
Gly Cys Arg 100 105 110 Phe Glu Val Cys Arg Arg Asp Thr Pro Gly Leu
Ser Arg Thr Arg Asn 115 120 125 Gly Asp Ser Ser Pro Pro Phe Arg Ala
Gly Trp Ser Leu Pro Pro Lys 130 135 140 Cys Gly Glu Ile Ser Gln Ser
Ala Arg Lys Thr Pro Ala Val Pro Arg 145 150 155 160 Tyr Ser Leu Leu
Arg Thr Asp Arg Pro Asp Gly Pro Arg Gly Arg Phe 165 170 175 Val Gly
Ser Gly Pro Arg Ala Ala Thr Arg Arg Arg Leu Phe Leu Gly 180 185 190
Ile Pro Ala Leu Val Leu Val Thr Ala Leu Thr Leu Val Leu Ala Val 195
200 205 Pro Thr Gly Arg Glu Thr Leu Trp Arg Met Trp Cys Glu Ala Thr
Gln 210 215 220 Asp Trp Cys Leu Gly Val Pro Val Asp Ser Arg Gly Gln
Pro Ala Glu 225 230 235 240 Asp Gly Glu Phe Leu Leu Leu Ser Pro Val
Gln Ala Ala Thr Trp Gly 245 250 255 Asn Tyr Tyr Ala Leu Gly Asp Ser
Tyr Ser Ser Gly Asp Gly Ala Arg 260 265 270 Asp Tyr Tyr Pro Gly Thr
Ala Val Lys Gly Gly Cys Trp Arg Ser Ala 275 280 285 Asn Ala Tyr Pro
Glu Leu Val Ala Glu Ala Tyr Asp Phe Ala Gly His 290 295 300 Leu Ser
Phe Leu Ala Cys Ser Gly Gln Arg Gly Tyr Ala Met Leu Asp 305 310 315
320 Ala Ile Asp Glu Val Gly Ser Gln Leu Asp Trp Asn Ser Pro His Thr
325 330 335 Ser Leu Val Thr Ile Gly Ile Gly Gly Asn Asp Leu Gly Phe
Ser Thr 340 345 350 Val Leu Lys Thr Cys Met Val Arg Val Pro Leu Leu
Asp Ser Lys Ala 355 360 365 Cys Thr Asp Gln Glu Asp Ala Ile Arg Lys
Arg Met Ala Lys Phe Glu 370 375 380 Thr Thr Phe Glu Glu Leu Ile Ser
Glu Val Arg Thr Arg Ala Pro Asp 385 390 395 400 Ala Arg Ile Leu Val
Val Gly Tyr Pro Arg Ile Phe Pro Glu Glu Pro 405 410 415 Thr Gly Ala
Tyr Tyr Thr Leu Thr Ala Ser Asn Gln Arg Trp Leu Asn 420 425 430 Glu
Thr Ile Gln Glu Phe Asn Gln Gln Leu Ala Glu Ala Val Ala Val 435 440
445 His Asp Glu Glu Ile Ala Ala Ser Gly Gly Val Gly Ser Val Glu Phe
450 455 460 Val Asp Val Tyr His Ala Leu Asp Gly His Glu Ile Gly Ser
Asp Glu 465 470 475 480 Pro Trp Val Asn Gly Val Gln Leu Arg Asp Leu
Ala Thr Gly Val Thr 485 490 495 Val Asp Arg Ser Thr Phe His Pro Asn
Ala Ala Gly His Arg Ala Val 500 505 510 Gly Glu Arg Val Ile Glu Gln
Ile Glu Thr Gly Pro Gly Arg Pro Leu 515 520 525 Tyr Ala Thr Phe Ala
Val Val Ala Gly Ala Thr Val Asp Thr Leu Ala 530 535 540 Gly Glu Val
Gly 545 35 3000 DNA Termobifida fusca 35 ggtggtgaac cagaacaccc
ggtcgtcggc gtgggcgtcc aggtgcaggt gcaggttctt 60 caactgctcc
agcaggatgc cgccgtggcc gtgcacgatg gccttgggca ggcctgtggt 120
ccccgacgag tacagcaccc atagcggatg gtcgaacggc agcggggtga actccagttc
180 cgcgccttcg cccgcggctt cgaactccgc ccaggacagg gtgtcggcga
cagggccgca 240 gcccaggtac ggcaggacga cggtgtgctg caggctgggc
atgccgtcgc gcagggcttt 300 gagcacgtca cggcggtcga agtccttacc
gccgtagcgg tagccgtcca cggccagcag 360 cactttcggt tcgatctgcg
cgaaccggtc gaggacgctg cgcaccccga agtcggggga 420 acaggacgac
caggtcgcac cgatcgcggc gcaggcgagg aatgcggccg tcgcctcggc 480
gatgttcggc aggtaggcca cgacccggtc gccggggccc accccgaggc tgcggagggc
540 cgcagcgatc gcggcggtgc gggtccgcag ttctccccag gtccactcgg
tcaacggccg 600 gagttcggac gcgtgccgga tcgccacggc tgatgggtca
cggtcgcgga agatgtgctc 660 ggcgtagttg agggtggcgc cggggaacca
gacggcgccg ggcatggcgt cggaggcgag 720 cactgtggtg tacggggtgg
cggcgcgcac ccggtagtac tcccagatcg cggaccagaa 780 tccttcgagg
tcggttaccg accagcgcca cagtgcctcg tagtccggtg cgtccacacc 840
gcggtgctcc cgcacccagc gggtgaacgc ggtgaggttg gcgcgttctt tgcgctcctc
900 gtcgggactc cacaggatcg gcggctgcgg cttgagtgtc atgaaacgcg
accccttcgt 960 ggacggtgcg gatgcggtga gcgtcgggtg cctcccctaa
cgctccccgg tgacggagtg 1020 ttgtgcacca catctagcac gcgggacgcg
gaaaccgtat ggagaaaaca cctacaaccc 1080 cggccggacg gtgggtttcg
gccacactta ggggtcgggt gcctgcttgc cgggcagggc 1140 agtcccgggg
tgctgtggtg cgggcgggag ggctgtcgct tcgaggtgtg ccggcgggac 1200
actccgggcc tcagccgtac ccgcaacggg gacagttctc ctcccttccg ggctggatgg
1260 tcccttcccc cgaaatgcgg cgagatctcc cagtcagccc ggaaaacacc
cgctgtgccc 1320 aggtactctt tgcttcgaac agacaggccg gacggtccac
gggggaggtt tgtgggcagc 1380 ggaccacgtg cggcgaccag acgacggttg
ttcctcggta tccccgctct tgtacttgtg 1440 acagcgctca cgctggtctt
ggctgtcccg acggggcgcg agacgctgtg gcgcatgtgg 1500 tgtgaggcca
cccaggactg gtgcctgggg gtgccggtcg actcccgcgg acagcctgcg 1560
gaggacggcg agtttctgct gctttctccg gtccaggcag cgacctgggg gaactattac
1620 gcgctcgggg attcgtactc ttcgggggac ggggcccgcg actactatcc
cggcaccgcg 1680 gtgaagggcg gttgctggcg gtccgctaac gcctatccgg
agctggtcgc cgaagcctac 1740 gacttcgccg gacacttgtc gttcctggcc
tgcagcggcc agcgcggcta cgccatgctt 1800 gacgctatcg acgaggtcgg
ctcgcagctg gactggaact cccctcacac gtcgctggtg 1860 acgatcggga
tcggcggcaa cgatctgggg ttctccacgg ttttgaagac ctgcatggtg 1920
cgggtgccgc tgctggacag caaggcgtgc acggaccagg aggacgctat ccgcaagcgg
1980 atggcgaaat tcgagacgac gtttgaagag ctcatcagcg aagtgcgcac
ccgcgcgccg 2040 gacgcccgga tccttgtcgt gggctacccc cggatttttc
cggaggaacc gaccggcgcc 2100 tactacacgc tgaccgcgag caaccagcgg
tggctcaacg aaaccattca ggagttcaac 2160 cagcagctcg ccgaggctgt
cgcggtccac gacgaggaga ttgccgcgtc gggcggggtg 2220 ggcagcgtgg
agttcgtgga cgtctaccac gcgttggacg gccacgagat cggctcggac 2280
gagccgtggg tgaacggggt gcagttgcgg gacctcgcca ccggggtgac tgtggaccgc
2340 agtaccttcc accccaacgc cgctgggcac cgggcggtcg gtgagcgggt
catcgagcag 2400 atcgaaaccg gcccgggccg tccgctctat gccactttcg
cggtggtggc gggggcgacc 2460 gtggacactc tcgcgggcga ggtggggtga
cccggcttac cgtccggccc gcaggtctgc 2520 gagcactgcg gcgatctggt
ccactgccca gtgcagttcg tcttcggtga tgaccagcgg 2580 cggggagagc
cggatcgttg agccgtgcgt gtctttgacg agcacacccc gctgcaggag 2640
ccgttcgcac agttctcttc cggtggccag agtcgggtcg acgtcgatcc cagcccacag
2700 gccgatgctg cgggccgcga ccacgccgtt gccgaccagt tggtcgaggc
gggcgcgcag 2760 cacgggggcg agggcgcgga catggtccag gtaagggccg
tcgcggacga ggctcaccac 2820 ggcagtgccg accgcgcagg cgagggcgtt
gccgccgaag gtgctgccgt gctggccggg 2880 gcggatcacg tcgaagactt
ccgcgtcgcc taccgccgcc gccacgggca ggatgccgcc 2940 gcccagcgct
ttgccgaaca ggtagatatc ggcgtcgact ccgctgtggt cgcaggcccg 3000 36 372
PRT Termobifida fusca 36 Val Gly Ser Gly Pro Arg Ala Ala Thr Arg
Arg Arg Leu Phe Leu Gly 1 5 10 15 Ile Pro Ala Leu Val Leu Val Thr
Ala Leu Thr Leu Val Leu Ala Val 20 25 30 Pro Thr Gly Arg Glu Thr
Leu Trp Arg Met Trp Cys Glu Ala Thr Gln 35 40 45 Asp Trp Cys Leu
Gly Val Pro Val Asp Ser Arg Gly Gln Pro Ala Glu 50 55 60 Asp Gly
Glu Phe Leu Leu Leu Ser Pro Val Gln Ala Ala Thr Trp Gly 65 70 75 80
Asn Tyr Tyr Ala Leu Gly Asp Ser Tyr Ser Ser Gly Asp Gly Ala Arg 85
90 95 Asp Tyr Tyr Pro Gly Thr Ala Val Lys Gly Gly Cys Trp Arg Ser
Ala 100 105 110 Asn Ala Tyr Pro Glu Leu Val Ala Glu Ala Tyr Asp Phe
Ala Gly His 115 120 125 Leu Ser Phe Leu Ala Cys Ser Gly Gln Arg Gly
Tyr Ala Met Leu Asp 130 135 140 Ala Ile Asp Glu Val Gly Ser Gln Leu
Asp Trp Asn Ser Pro His Thr 145 150 155 160 Ser Leu Val Thr Ile Gly
Ile Gly Gly Asn Asp Leu Gly Phe Ser Thr 165 170 175 Val Leu Lys Thr
Cys Met Val Arg Val Pro Leu Leu Asp Ser Lys Ala 180 185 190 Cys Thr
Asp Gln Glu Asp Ala Ile Arg Lys Arg Met Ala Lys Phe Glu 195 200 205
Thr Thr Phe Glu Glu Leu Ile Ser Glu Val Arg Thr Arg Ala Pro Asp 210
215 220 Ala Arg Ile Leu Val Val Gly Tyr Pro Arg Ile Phe Pro Glu Glu
Pro 225 230 235 240 Thr Gly Ala Tyr Tyr Thr Leu Thr Ala Ser Asn Gln
Arg Trp Leu Asn 245 250 255 Glu Thr Ile Gln Glu Phe Asn Gln Gln Leu
Ala Glu Ala Val Ala Val 260 265 270 His Asp Glu Glu Ile Ala Ala Ser
Gly Gly Val Gly Ser Val Glu Phe 275 280 285 Val Asp Val Tyr His Ala
Leu Asp Gly His Glu Ile Gly Ser Asp Glu 290 295 300 Pro Trp Val Asn
Gly Val Gln Leu Arg Asp Leu Ala Thr Gly Val Thr 305 310 315 320 Val
Asp Arg Ser Thr Phe His Pro Asn Ala Ala Gly His Arg Ala Val 325 330
335 Gly Glu Arg Val Ile Glu Gln Ile Glu Thr Gly Pro Gly Arg Pro Leu
340 345 350 Tyr Ala Thr Phe Ala Val Val Ala Gly Ala Thr Val Asp Thr
Leu Ala 355 360 365 Gly Glu Val Gly 370 37 300 PRT Corynebacterium
efficiens 37 Met Arg Thr Thr Val Ile Ala Ala Ser Ala Leu Leu Leu
Leu Ala Gly 1 5 10 15 Cys Ala Asp Gly Ala Arg Glu Glu Thr Ala Gly
Ala Pro Pro Gly Glu 20 25 30 Ser Ser Gly Gly Ile Arg Glu Glu Gly
Ala Glu Ala Ser Thr Ser Ile 35 40 45 Thr Asp Val Tyr Ile Ala Leu
Gly Asp Ser Tyr Ala Ala Met Gly Gly 50 55 60 Arg Asp Gln Pro Leu
Arg Gly Glu Pro Phe Cys Leu Arg Ser Ser Gly 65 70 75 80 Asn Tyr Pro
Glu Leu Leu His Ala Glu Val Thr Asp Leu Thr Cys Gln 85 90 95 Gly
Ala Val Thr Gly Asp Leu Leu Glu Pro Arg Thr Leu Gly Glu Arg 100 105
110 Thr Leu Pro Ala Gln Val Asp Ala Leu Thr Glu Asp Thr Thr Leu Val
115 120 125 Thr Leu Ser Ile Gly Gly Asn Asp Leu Gly Phe Gly Glu Val
Ala Gly 130 135 140 Cys Ile Arg Glu Arg Ile Ala Gly Glu Asn Ala Asp
Asp Cys Val Asp 145 150 155 160 Leu Leu Gly Glu Thr Ile Gly Glu Gln
Leu Asp Gln Leu Pro Pro Gln 165 170 175 Leu Asp Arg Val
His Glu Ala Ile Arg Asp Arg Ala Gly Asp Ala Gln 180 185 190 Val Val
Val Thr Gly Tyr Leu Pro Leu Val Ser Ala Gly Asp Cys Pro 195 200 205
Glu Leu Gly Asp Val Ser Glu Ala Asp Arg Arg Trp Ala Val Glu Leu 210
215 220 Thr Gly Gln Ile Asn Glu Thr Val Arg Glu Ala Ala Glu Arg His
Asp 225 230 235 240 Ala Leu Phe Val Leu Pro Asp Asp Ala Asp Glu His
Thr Ser Cys Ala 245 250 255 Pro Pro Gln Gln Arg Trp Ala Asp Ile Gln
Gly Gln Gln Thr Asp Ala 260 265 270 Tyr Pro Leu His Pro Thr Ser Ala
Gly His Glu Ala Met Ala Ala Ala 275 280 285 Val Arg Asp Ala Leu Gly
Leu Glu Pro Val Gln Pro 290 295 300 38 3000 DNA Corynebacterium
efficiens 38 ttctggggtg ttatggggtt gttatcggct cgtcctgggt ggatcccgcc
aggtggggta 60 ttcacggggg acttttgtgt ccaacagccg agaatgagtg
ccctgagcgg tgggaatgag 120 gtgggcgggg ctgtgtcgcc atgagggggc
ggcgggctct gtggtgcccc gcgacccccg 180 gccccggtga gcggtgaatg
aaatccggct gtaatcagca tcccgtgccc accccgtcgg 240 ggaggtcagc
gcccggagtg tctacgcagt cggatcctct cggactcggc catgctgtcg 300
gcagcatcgc gctcccgggt cttggcgtcc ctcggctgtt ctgcctgctg tccctggaag
360 gcgaaatgat caccggggag tgatacaccg gtggtctcat cccggatgcc
cacttcggcg 420 ccatccggca attcgggcag ctccgggtgg aagtaggtgg
catccgatgc gtcggtgacg 480 ccatagtggg cgaagatctc atcctgctcg
agggtgctca ggccactctc cggatcgata 540 tcgggggcgt ccttgatggc
gtccttgctg aaaccgaggt gcagcttgtg ggcttccaat 600 ttcgcaccac
ggagcgggac gaggctggaa tgacggccga agagcccgtg gtggacctca 660
acgaaggtgg gtagtcccgt gtcatcattg aggaacacgc cctccaccgc acccagcttg
720 tggccggagt tgtcgtaggc gctggcatcc agaagggaaa cgatctcata
tttgtcggtg 780 tgctcagaca tgatcttcct ttgctgtcgg tgtctggtac
taccacggta gggctgaatg 840 caactgttat ttttctgtta ttttaggaat
tggtccatat cccacaggct ggctgtggtc 900 aaatcgtcat caagtaatcc
ctgtcacaca aaatgggtgg tgggagccct ggtcgcggtt 960 ccgtgggagg
cgccgtgccc cgcaggatcg tcggcatcgg cggatctggc cggtaccccg 1020
cggtgaataa aatcattctg taaccttcat cacggttggt tttaggtatc cgcccctttc
1080 gtcctgaccc cgtccccggc gcgcgggagc ccgcgggttg cggtagacag
gggagacgtg 1140 gacaccatga ggacaacggt catcgcagca agcgcattac
tccttctcgc cggatgcgcg 1200 gatggggccc gggaggagac cgccggtgca
ccgccgggtg agtcctccgg gggcatccgg 1260 gaggaggggg cggaggcgtc
gacaagcatc accgacgtct acatcgccct cggggattcc 1320 tatgcggcga
tgggcgggcg ggatcagccg ttacggggtg agccgttctg cctgcgctcg 1380
tccggtaatt acccggaact cctccacgca gaggtcaccg atctcacctg ccagggggcg
1440 gtgaccgggg atctgctcga acccaggacg ctgggggagc gcacgctgcc
ggcgcaggtg 1500 gatgcgctga cggaggacac caccctggtc accctctcca
tcgggggcaa tgacctcgga 1560 ttcggggagg tggcgggatg catccgggaa
cggatcgccg gggagaacgc tgatgattgc 1620 gtggacctgc tgggggaaac
catcggggag cagctcgatc agcttccccc gcagctggac 1680 cgcgtgcacg
aggctatccg ggaccgcgcc ggggacgcgc aggttgtggt caccggttac 1740
ctgccgctcg tgtctgccgg ggactgcccc gaactggggg atgtctccga ggcggatcgt
1800 cgttgggcgg ttgagctgac cgggcagatc aacgagaccg tgcgcgaggc
ggccgaacga 1860 cacgatgccc tctttgtcct gcccgacgat gccgatgagc
acaccagttg tgcaccccca 1920 cagcagcgct gggcggatat ccagggccaa
cagaccgatg cctatccgct gcacccgacc 1980 tccgccggcc atgaggcgat
ggccgccgcc gtccgggacg cgctgggcct ggaaccggtc 2040 cagccgtagc
gccgggcgcg cgcttgtcga cgaccaaccc atgccaggct gcagtcacat 2100
ccgcacatag cgcgcgcggg cgatggagta cgcaccatag aggatgagcc cgatgccgac
2160 gatgatgagc agcacactgc cgaagggttg ttccccgagg gtgcgcagag
ccgagtccag 2220 acctgcggcc tgctccggat catgggccca accggcgatg
acgatcaaca cccccaggat 2280 cccgaaggcg ataccacggg cgacataacc
ggctgttccg gtgatgatga tcgcggtccc 2340 gacctgccct gaccccgcac
ccgcctccag atcctcccgg aaatcccggg tggccccctt 2400 ccagaggttg
tagacacccg cccccagtac caccagcccg gcgaccacaa ccagcaccac 2460
accccagggt tgggatagga cggtggcggt gacatcggtg gcggtctccc catcggaggt
2520 gctgccgccc cgggcgaagg tggaggtggt caccgccagg gagaagtaga
ccatggccat 2580 gaccgccccc ttggcccttt ccttgaggtc ctcgcccgcc
agcagctggc tcaattgcca 2640 gagtcccagg gccgccaggg cgatgacggc
aacccacagg aggaactgcc cacccggagc 2700 ctccgcgatg gtggccaggg
cacctgaatt cgaggcctca tcacccgaac cgccggatcc 2760 agtggcgatg
cgcaccgcga tccacccgat gaggatgtgc agtatgccca ggacaatgaa 2820
accacctctg gccagggtgg tcagcgcggg gtggtcctcg gcctggtcgg cagcccgttc
2880 gatcgtccgt ttcgcggatc tggtgtcgcc cttatccata gctcccattg
aaccgccttg 2940 aggggtgggc ggccactgtc agggcggatt gtgatctgaa
ctgtgatgtt ccatcaaccc 3000 39 284 PRT Novosphingobium
aromaticivorans 39 Met Gly Gln Val Lys Leu Phe Ala Arg Arg Cys Ala
Pro Val Leu Leu 1 5 10 15 Ala Leu Ala Gly Leu Ala Pro Ala Ala Thr
Val Ala Arg Glu Ala Pro 20 25 30 Leu Ala Glu Gly Ala Arg Tyr Val
Ala Leu Gly Ser Ser Phe Ala Ala 35 40 45 Gly Pro Gly Val Gly Pro
Asn Ala Pro Gly Ser Pro Glu Arg Cys Gly 50 55 60 Arg Gly Thr Leu
Asn Tyr Pro His Leu Leu Ala Glu Ala Leu Lys Leu 65 70 75 80 Asp Leu
Val Asp Ala Thr Cys Ser Gly Ala Thr Thr His His Val Leu 85 90 95
Gly Pro Trp Asn Glu Val Pro Pro Gln Ile Asp Ser Val Asn Gly Asp 100
105 110 Thr Arg Leu Val Thr Leu Thr Ile Gly Gly Asn Asp Val Ser Phe
Val 115 120 125 Gly Asn Ile Phe Ala Ala Ala Cys Glu Lys Met Ala Ser
Pro Asp Pro 130 135 140 Arg Cys Gly Lys Trp Arg Glu Ile Thr Glu Glu
Glu Trp Gln Ala Asp 145 150 155 160 Glu Glu Arg Met Arg Ser Ile Val
Arg Gln Ile His Ala Arg Ala Pro 165 170 175 Leu Ala Arg Val Val Val
Val Asp Tyr Ile Thr Val Leu Pro Pro Ser 180 185 190 Gly Thr Cys Ala
Ala Met Ala Ile Ser Pro Asp Arg Leu Ala Gln Ser 195 200 205 Arg Ser
Ala Ala Lys Arg Leu Ala Arg Ile Thr Ala Arg Val Ala Arg 210 215 220
Glu Glu Gly Ala Ser Leu Leu Lys Phe Ser His Ile Ser Arg Arg His 225
230 235 240 His Pro Cys Ser Ala Lys Pro Trp Ser Asn Gly Leu Ser Ala
Pro Ala 245 250 255 Asp Asp Gly Ile Pro Val His Pro Asn Arg Leu Gly
His Ala Glu Ala 260 265 270 Ala Ala Ala Leu Val Lys Leu Val Lys Leu
Met Lys 275 280 40 1500 DNA Novosphingobium aromaticivorans 40
tgccggaact caagcggcgt ctagccgaac tcatgcccga aagcgcgtgg cactatcccg
60 aagaccaggt ctcggacgcc agcgagcgcc tgatggccgc cgaaatcacg
cgcgaacagc 120 tctaccgcca gctccacgac gagctgccct atgacagtac
cgtacgtccc gagaagtacc 180 tccatcgcaa ggacggttcg atcgagatcc
accagcagat cgtgattgcc cgcgagacac 240 agcgtccgat cgtgctgggc
aagggtggcg cgaagatcaa ggcgatcgga gaggccgcac 300 gcaaggaact
ttcgcaattg ctcgacacca aggtgcacct gttcctgcat gtgaaggtcg 360
acgagcgctg ggccgacgcc aaggaaatct acgaggaaat cggcctcgaa tgggtcaagt
420 gaagctcttc gcgcgccgct gcgccccagt acttctcgcc cttgccgggc
tggctccggc 480 ggctacggtc gcgcgggaag caccgctggc cgaaggcgcg
cgttacgttg cgctgggaag 540 ctccttcgcc gcaggtccgg gcgtggggcc
caacgcgccc ggatcgcccg aacgctgcgg 600 ccggggcacg ctcaactacc
cgcacctgct cgccgaggcg ctcaagctcg atctcgtcga 660 tgcgacctgc
agcggcgcga cgacccacca cgtgctgggc ccctggaacg aggttccccc 720
tcagatcgac agcgtgaatg gcgacacccg cctcgtcacc ctgaccatcg gcggaaacga
780 tgtgtcgttc gtcggcaaca tcttcgccgc cgcttgcgag aagatggcgt
cgcccgatcc 840 gcgctgcggc aagtggcggg agatcaccga ggaagagtgg
caggccgacg aggagcggat 900 gcgctccatc gtacgccaga tccacgcccg
cgcgcctctc gcccgggtgg tggtggtcga 960 ttacatcacg gtcctgccgc
catcaggcac ttgcgctgcc atggcgattt cgccggaccg 1020 gctggcccag
agccgcagcg ccgcgaaacg gcttgcccgg attaccgcac gggtcgcgcg 1080
agaagagggt gcatcgctgc tcaagttctc gcatatctcg cgccggcacc atccatgctc
1140 tgccaagccc tggagcaacg gcctttccgc cccggccgac gacggcatcc
cggtccatcc 1200 gaaccggctc ggacatgctg aagcggcagc ggcgctggtc
aagcttgtga aattgatgaa 1260 gtagctactg cactgatttc aaatagtatt
gcctgtcagc tttccagccc ggattgttgc 1320 agcgcaacag aaacttgtcc
gtaatggatt gatggtttat gtcgctcgca aattgccgtc 1380 gaagggaacg
ggcgcgtcgc tcgttaacgt cctgggtgca gcagtgacgg agcgcgtgga 1440
tgagtgatac tggcggtgtc atcggtgtac gcgccgccat tcccatgcct gtacgcgccg
1500 41 268 PRT Streptomyces coelicolor 41 Met Arg Arg Phe Arg Leu
Val Gly Phe Leu Ser Ser Leu Val Leu Ala 1 5 10 15 Ala Gly Ala Ala
Leu Thr Gly Ala Ala Thr Ala Gln Ala Ala Gln Pro 20 25 30 Ala Ala
Ala Asp Gly Tyr Val Ala Leu Gly Asp Ser Tyr Ser Ser Gly 35 40 45
Val Gly Ala Gly Ser Tyr Ile Ser Ser Ser Gly Asp Cys Lys Arg Ser 50
55 60 Thr Lys Ala His Pro Tyr Leu Trp Ala Ala Ala His Ser Pro Ser
Thr 65 70 75 80 Phe Asp Phe Thr Ala Cys Ser Gly Ala Arg Thr Gly Asp
Val Leu Ser 85 90 95 Gly Gln Leu Gly Pro Leu Ser Ser Gly Thr Gly
Leu Val Ser Ile Ser 100 105 110 Ile Gly Gly Asn Asp Ala Gly Phe Ala
Asp Thr Met Thr Thr Cys Val 115 120 125 Leu Gln Ser Glu Ser Ser Cys
Leu Ser Arg Ile Ala Thr Ala Glu Ala 130 135 140 Tyr Val Asp Ser Thr
Leu Pro Gly Lys Leu Asp Gly Val Tyr Ser Ala 145 150 155 160 Ile Ser
Asp Lys Ala Pro Asn Ala His Val Val Val Ile Gly Tyr Pro 165 170 175
Arg Phe Tyr Lys Leu Gly Thr Thr Cys Ile Gly Leu Ser Glu Thr Lys 180
185 190 Arg Thr Ala Ile Asn Lys Ala Ser Asp His Leu Asn Thr Val Leu
Ala 195 200 205 Gln Arg Ala Ala Ala His Gly Phe Thr Phe Gly Asp Val
Arg Thr Thr 210 215 220 Phe Thr Gly His Glu Leu Cys Ser Gly Ser Pro
Trp Leu His Ser Val 225 230 235 240 Asn Trp Leu Asn Ile Gly Glu Ser
Tyr His Pro Thr Ala Ala Gly Gln 245 250 255 Ser Gly Gly Tyr Leu Pro
Val Leu Asn Gly Ala Ala 260 265 42 2000 DNA Streptomyces coelicolor
42 cccggcggcc cgtgcaggag cagcagccgg cccgcgatgt cctcgggcgt
cgtcttcatc 60 aggccgtcca tcgcgtcggc gaccggcgcc gtgtagttgg
cccggacctc gtcccaggtg 120 cccgcggcga tctggcgggt ggtgcggtgc
gggccgcgcc gaggggagac gtaccagaag 180 cccatcgtca cgttctccgg
ctgcggttcg ggctcgtccg ccgctccgtc cgtcgcctcg 240 ccgagcacct
tctcggcgag gtcggcgctg gtcgccgtca ccgtgacgtc ggcgccccgg 300
ctccagcgcg agatcagcag cgtccagccg tcgccctccg ccagcgtcgc gctgcggtcg
360 tcgtcgcggg cgatccgcag cacgcgcgcg ccgggcggca gcagcgtggc
gccggaccgt 420 acgcggtcga tgttcgccgc gtgcgagtac ggctgctcac
ccgtggcgaa acggccgagg 480 aacagcgcgt cgacgacgtc ggacggggag
tcgctgtcgt ccacgttgag ccggatcggc 540 agggcttcgt gcgggttcac
ggacatgtcg ccatgatcgg gcacccggcc gccgcgtgca 600 cccgctttcc
cgggcacgca cgacaggggc tttctcgccg tcttccgtcc gaacttgaac 660
gagtgtcagc catttcttgg catggacact tccagtcaac gcgcgtagct gctaccacgg
720 ttgtggcagc aatcctgcta agggaggttc catgagacgt ttccgacttg
tcggcttcct 780 gagttcgctc gtcctcgccg ccggcgccgc cctcaccggg
gcagcgaccg cccaggcggc 840 ccaacccgcc gccgccgacg gctatgtggc
cctcggcgac tcctactcct ccggggtcgg 900 agcgggcagc tacatcagct
cgagcggcga ctgcaagcgc agcacgaagg cccatcccta 960 cctgtgggcg
gccgcccact cgccctccac gttcgacttc accgcctgtt ccggcgcccg 1020
tacgggtgat gttctctccg gacagctcgg cccgctcagc tccggcaccg gcctcgtctc
1080 gatcagcatc ggcggcaacg acgccggttt cgccgacacc atgacgacct
gtgtgctcca 1140 gtccgagagc tcctgcctgt cgcggatcgc caccgccgag
gcgtacgtcg actcgacgct 1200 gcccggcaag ctcgacggcg tctactcggc
aatcagcgac aaggcgccga acgcccacgt 1260 cgtcgtcatc ggctacccgc
gcttctacaa gctcggcacc acctgcatcg gcctgtccga 1320 gaccaagcgg
acggcgatca acaaggcctc cgaccacctc aacaccgtcc tcgcccagcg 1380
cgccgccgcc cacggcttca ccttcggcga cgtacgcacc accttcaccg gccacgagct
1440 gtgctccggc agcccctggc tgcacagcgt caactggctg aacatcggcg
agtcgtacca 1500 ccccaccgcg gccggccagt ccggtggcta cctgccggtc
ctcaacggcg ccgcctgacc 1560 tcaggcggaa ggagaagaag aaggagcgga
gggagacgag gagtgggagg ccccgcccga 1620 cggggtcccc gtccccgtct
ccgtctccgt cccggtcccg caagtcaccg agaacgccac 1680 cgcgtcggac
gtggcccgca ccggactccg cacctccacg cgcacggcac tctcgaacgc 1740
gccggtgtcg tcgtgcgtcg tcaccaccac gccgtcctgg cgcgagcgct cgccgcccga
1800 cgggaaggac agcgtccgcc accccggatc ggagaccgac ccgtccgcgg
tcacccaccg 1860 gtagccgacc tccgcgggca gccgcccgac cgtgaacgtc
gccgtgaacg cgggtgcccg 1920 gtcgtgcggc ggcggacagg cccccgagta
gtgggtgcgc gagcccacca cggtcacctc 1980 caccgactgc gctgcggggc 2000 43
269 PRT Streptomyces avermitilis 43 Met Arg Arg Ser Arg Ile Thr Ala
Tyr Val Thr Ser Leu Leu Leu Ala 1 5 10 15 Val Gly Cys Ala Leu Thr
Gly Ala Ala Thr Ala Gln Ala Ser Pro Ala 20 25 30 Ala Ala Ala Thr
Gly Tyr Val Ala Leu Gly Asp Ser Tyr Ser Ser Gly 35 40 45 Val Gly
Ala Gly Ser Tyr Leu Ser Ser Ser Gly Asp Cys Lys Arg Ser 50 55 60
Ser Lys Ala Tyr Pro Tyr Leu Trp Gln Ala Ala His Ser Pro Ser Ser 65
70 75 80 Phe Ser Phe Met Ala Cys Ser Gly Ala Arg Thr Gly Asp Val
Leu Ala 85 90 95 Asn Gln Leu Gly Thr Leu Asn Ser Ser Thr Gly Leu
Val Ser Leu Thr 100 105 110 Ile Gly Gly Asn Asp Ala Gly Phe Ser Asp
Val Met Thr Thr Cys Val 115 120 125 Leu Gln Ser Asp Ser Ala Cys Leu
Ser Arg Ile Asn Thr Ala Lys Ala 130 135 140 Tyr Val Asp Ser Thr Leu
Pro Gly Gln Leu Asp Ser Val Tyr Thr Ala 145 150 155 160 Ile Ser Thr
Lys Ala Pro Ser Ala His Val Ala Val Leu Gly Tyr Pro 165 170 175 Arg
Phe Tyr Lys Leu Gly Gly Ser Cys Leu Ala Gly Leu Ser Glu Thr 180 185
190 Lys Arg Ser Ala Ile Asn Asp Ala Ala Asp Tyr Leu Asn Ser Ala Ile
195 200 205 Ala Lys Arg Ala Ala Asp His Gly Phe Thr Phe Gly Asp Val
Lys Ser 210 215 220 Thr Phe Thr Gly His Glu Ile Cys Ser Ser Ser Thr
Trp Leu His Ser 225 230 235 240 Leu Asp Leu Leu Asn Ile Gly Gln Ser
Tyr His Pro Thr Ala Ala Gly 245 250 255 Gln Ser Gly Gly Tyr Leu Pro
Val Met Asn Ser Val Ala 260 265 44 1980 DNA Streptomyces
avermitilis 44 ccaccgccgg gtcggcggcg agtctcctgg cctcggtcgc
ggagaggttg gccgtgtagc 60 cgttcagcgc ggcgccgaac gtcttcttca
ccgtgccgcc gtactcgttg atcaggccct 120 tgcccttgct cgacgcggcc
ttgaagccgg tgcccttctt gagcgtgacg atgtagctgc 180 ccttgatcgc
ggtgggggag ccggcggcga gcaccgtgcc ctcggccggg gtggcctggg 240
cgggcagtgc ggtgaatccg cccacgaggg cgccggtcgc cacggcggtt atcgcggcga
300 tccggatctt cttgctacgc agctgtgcca tacgagggag tcctcctctg
ggcagcggcg 360 cgcctgggtg gggcgcacgg ctgtgggggg tgcgcgcgtc
atcacgcaca cggccctgga 420 gcgtcgtgtt ccgccctggg ttgagtaaag
cctcggccat ctacgggggt ggctcaaggg 480 agttgagacc ctgtcatgag
tctgacatga gcacgcaatc aacggggccg tgagcacccc 540 ggggcgaccc
cggaaagtgc cgagaagtct tggcatggac acttcctgtc aacacgcgta 600
gctggtacga cggttacggc agagatcctg ctaaagggag gttccatgag acgttcccga
660 attacggcat acgtgacctc actcctcctc gccgtcggct gcgccctcac
cggggcagcg 720 acggcgcagg cgtccccagc cgccgcggcc acgggctatg
tggccctcgg cgactcgtac 780 tcgtccggtg tcggcgccgg cagctacctc
agctccagcg gcgactgcaa gcgcagttcg 840 aaggcctatc cgtacctctg
gcaggccgcg cattcaccct cgtcgttcag tttcatggct 900 tgctcgggcg
ctcgtacggg tgatgtcctg gccaatcagc tcggcaccct gaactcgtcc 960
accggcctgg tctccctcac catcggaggc aacgacgcgg gcttctccga cgtcatgacg
1020 acctgtgtgc tccagtccga cagcgcctgc ctctcccgca tcaacacggc
gaaggcgtac 1080 gtcgactcca ccctgcccgg ccaactcgac agcgtgtaca
cggcgatcag cacgaaggcc 1140 ccgtcggccc atgtggccgt gctgggctac
ccccgcttct acaaactggg cggctcctgc 1200 ctcgcgggcc tctcggagac
caagcggtcc gccatcaacg acgcggccga ctatctgaac 1260 agcgccatcg
ccaagcgcgc cgccgaccac ggcttcacct tcggcgacgt caagagcacc 1320
ttcaccggcc atgagatctg ctccagcagc acctggctgc acagtctcga cctgctgaac
1380 atcggccagt cctaccaccc gaccgcggcc ggccagtccg gcggctatct
gccggtcatg 1440 aacagcgtgg cctgagctcc cacggcctga atttttaagg
cctgaatttt taaggcgaag 1500 gtgaaccgga agcggaggcc ccgtccgtcg
gggtctccgt cgcacaggtc accgagaacg 1560 gcacggagtt ggacgtcgtg
cgcaccgggt cgcgcacctc gacggcgatc tcgttcgaga 1620 tcgttccgct
cgtgtcgtac gtggtgacga acacctgctt ctgctgggtc tttccgccgc 1680
tcgccgggaa ggacagcgtc ttccagcccg gatccgggac ctcgcccttc ttggtcaccc
1740 agcggtactc cacctcgacc ggcacccggc ccaccgtgaa ggtcgccgtg
aacgtgggcg 1800 cctgggcggt gggcggcggg caggcaccgg agtagtcggt
gtgcacgccg gtgaccgtca 1860 ccttcacgga ctgggccggc ggggtcgtcg
taccgccgcc gccaccgccg cctcccggag 1920 tggagcccga gctgtggtcg
cccccgccgt cggcgttgtc gtcctcgggg gttttcgaac 1980 45 267 PRT
Streptomyces coelicolor 45 Met Arg Leu Thr Arg Ser Leu Ser Ala Ala
Ser Val Ile Val Phe Ala 1 5 10 15 Leu Leu Leu Ala Leu Leu Gly Ile
Ser Pro Ala Gln Ala Ala Gly Pro 20 25 30 Ala Tyr Val Ala Leu Gly
Asp Ser Tyr Ser Ser Gly Asn Gly Ala Gly 35 40 45 Ser Tyr Ile Asp
Ser Ser Gly Asp Cys His Arg Ser Asn Asn Ala Tyr 50 55 60 Pro Ala
Arg Trp Ala Ala Ala Asn Ala Pro Ser Ser Phe Thr Phe Ala 65
70 75 80 Ala Cys Ser Gly Ala Val Thr Thr Asp Val Ile Asn Asn Gln
Leu Gly 85 90 95 Ala Leu Asn Ala Ser Thr Gly Leu Val Ser Ile Thr
Ile Gly Gly Asn 100 105 110 Asp Ala Gly Phe Ala Asp Ala Met Thr Thr
Cys Val Thr Ser Ser Asp 115 120 125 Ser Thr Cys Leu Asn Arg Leu Ala
Thr Ala Thr Asn Tyr Ile Asn Thr 130 135 140 Thr Leu Leu Ala Arg Leu
Asp Ala Val Tyr Ser Gln Ile Lys Ala Arg 145 150 155 160 Ala Pro Asn
Ala Arg Val Val Val Leu Gly Tyr Pro Arg Met Tyr Leu 165 170 175 Ala
Ser Asn Pro Trp Tyr Cys Leu Gly Leu Ser Asn Thr Lys Arg Ala 180 185
190 Ala Ile Asn Thr Thr Ala Asp Thr Leu Asn Ser Val Ile Ser Ser Arg
195 200 205 Ala Thr Ala His Gly Phe Arg Phe Gly Asp Val Arg Pro Thr
Phe Asn 210 215 220 Asn His Glu Leu Phe Phe Gly Asn Asp Trp Leu His
Ser Leu Thr Leu 225 230 235 240 Pro Val Trp Glu Ser Tyr His Pro Thr
Ser Thr Gly His Gln Ser Gly 245 250 255 Tyr Leu Pro Val Leu Asn Ala
Asn Ser Ser Thr 260 265 46 1371 DNA Streptomyces coelicolor 46
acaggccgat gcacggaacc gtacctttcc gcagtgaagc gctctccccc catcgttcgc
60 cgggacttca tccgcgattt tggcatgaac acttccttca acgcgcgtag
cttgctacaa 120 gtgcggcagc agacccgctc gttggaggct cagtgagatt
gacccgatcc ctgtcggccg 180 catccgtcat cgtcttcgcc ctgctgctcg
cgctgctggg catcagcccg gcccaggcag 240 ccggcccggc ctatgtggcc
ctgggggatt cctattcctc gggcaacggc gccggaagtt 300 acatcgattc
gagcggtgac tgtcaccgca gcaacaacgc gtaccccgcc cgctgggcgg 360
cggccaacgc accgtcctcc ttcaccttcg cggcctgctc gggagcggtg accacggatg
420 tgatcaacaa tcagctgggc gccctcaacg cgtccaccgg cctggtgagc
atcaccatcg 480 gcggcaatga cgcgggcttc gcggacgcga tgaccacctg
cgtcaccagc tcggacagca 540 cctgcctcaa ccggctggcc accgccacca
actacatcaa caccaccctg ctcgcccggc 600 tcgacgcggt ctacagccag
atcaaggccc gtgcccccaa cgcccgcgtg gtcgtcctcg 660 gctacccgcg
catgtacctg gcctcgaacc cctggtactg cctgggcctg agcaacacca 720
agcgcgcggc catcaacacc accgccgaca ccctcaactc ggtgatctcc tcccgggcca
780 ccgcccacgg attccgattc ggcgatgtcc gcccgacctt caacaaccac
gaactgttct 840 tcggcaacga ctggctgcac tcactcaccc tgccggtgtg
ggagtcgtac caccccacca 900 gcacgggcca tcagagcggc tatctgccgg
tcctcaacgc caacagctcg acctgatcaa 960 cgcacggccg tgcccgcccc
gcgcgtcacg ctcggcgcgg gcgccgcagc gcgttgatca 1020 gcccacagtg
ccggtgacgg tcccaccgtc acggtcgagg gtgtacgtca cggtggcgcc 1080
gctccagaag tggaacgtca gcaggaccgt ggagccgtcc ctgacctcgt cgaagaactc
1140 cggggtcagc gtgatcaccc ctcccccgta gccgggggcg aaggcggcgc
cgaactcctt 1200 gtaggacgtc cagtcgtgcg gcccggcgtt gccaccgtcc
gcgtagaccg cttccatggt 1260 cgccagccgg tccccgcgga actcggtggg
gatgtccgtg cccaaggtgg tcccggtggt 1320 gtccgagagc accgggggct
cgtaccggat gatgtgcaga tccaaagaat t 1371 47 335 PRT Aeromonas
hydrophila 47 Met Lys Lys Trp Phe Val Cys Leu Leu Gly Leu Val Ala
Leu Thr Val 1 5 10 15 Gln Ala Ala Asp Ser Arg Pro Ala Phe Ser Arg
Ile Val Met Phe Gly 20 25 30 Asp Ser Leu Ser Asp Thr Gly Lys Met
Tyr Ser Lys Met Arg Gly Tyr 35 40 45 Leu Pro Ser Ser Pro Pro Tyr
Tyr Glu Gly Arg Phe Ser Asn Gly Pro 50 55 60 Val Trp Leu Glu Gln
Leu Thr Asn Glu Phe Pro Gly Leu Thr Ile Ala 65 70 75 80 Asn Glu Ala
Glu Gly Gly Pro Thr Ala Val Ala Tyr Asn Lys Ile Ser 85 90 95 Trp
Asn Pro Lys Tyr Gln Val Ile Asn Asn Leu Asp Tyr Glu Val Thr 100 105
110 Gln Phe Leu Gln Lys Asp Ser Phe Lys Pro Asp Asp Leu Val Ile Leu
115 120 125 Trp Val Gly Ala Asn Asp Tyr Leu Ala Tyr Gly Trp Asn Thr
Glu Gln 130 135 140 Asp Ala Lys Arg Val Arg Asp Ala Ile Ser Asp Ala
Ala Asn Arg Met 145 150 155 160 Val Leu Asn Gly Ala Lys Glu Ile Leu
Leu Phe Asn Leu Pro Asp Leu 165 170 175 Gly Gln Asn Pro Ser Ala Arg
Ser Gln Lys Val Val Glu Ala Ala Ser 180 185 190 His Val Ser Ala Tyr
His Asn Gln Leu Leu Leu Asn Leu Ala Arg Gln 195 200 205 Leu Ala Pro
Thr Gly Met Val Lys Leu Phe Glu Ile Asp Lys Gln Phe 210 215 220 Ala
Glu Met Leu Arg Asp Pro Gln Asn Phe Gly Leu Ser Asp Gln Arg 225 230
235 240 Asn Ala Cys Tyr Gly Gly Ser Tyr Val Trp Lys Pro Phe Ala Ser
Arg 245 250 255 Ser Ala Ser Thr Asp Ser Gln Leu Ser Ala Phe Asn Pro
Gln Glu Arg 260 265 270 Leu Ala Ile Ala Gly Asn Pro Leu Leu Ala Gln
Ala Val Ala Ser Pro 275 280 285 Met Ala Ala Arg Ser Ala Ser Thr Leu
Asn Cys Glu Gly Lys Met Phe 290 295 300 Trp Asp Gln Val His Pro Thr
Thr Val Val His Ala Ala Leu Ser Glu 305 310 315 320 Pro Ala Ala Thr
Phe Ile Glu Ser Gln Tyr Glu Phe Leu Ala His 325 330 335 48 335 PRT
Aeromonas salmonicida 48 Met Lys Lys Trp Phe Val Cys Leu Leu Gly
Leu Ile Ala Leu Thr Val 1 5 10 15 Gln Ala Ala Asp Thr Arg Pro Ala
Phe Ser Arg Ile Val Met Phe Gly 20 25 30 Asp Ser Leu Ser Asp Thr
Gly Lys Met Tyr Ser Lys Met Arg Gly Tyr 35 40 45 Leu Pro Ser Ser
Pro Pro Tyr Tyr Glu Gly Arg Phe Ser Asn Gly Pro 50 55 60 Val Trp
Leu Glu Gln Leu Thr Lys Gln Phe Pro Gly Leu Thr Ile Ala 65 70 75 80
Asn Glu Ala Glu Gly Gly Ala Thr Ala Val Ala Tyr Asn Lys Ile Ser 85
90 95 Trp Asn Pro Lys Tyr Gln Val Ile Asn Asn Leu Asp Tyr Glu Val
Thr 100 105 110 Gln Phe Leu Gln Lys Asp Ser Phe Lys Pro Asp Asp Leu
Val Ile Leu 115 120 125 Trp Val Gly Ala Asn Asp Tyr Leu Ala Tyr Gly
Trp Asn Thr Glu Gln 130 135 140 Asp Ala Lys Arg Val Arg Asp Ala Ile
Ser Asp Ala Ala Asn Arg Met 145 150 155 160 Val Leu Asn Gly Ala Lys
Gln Ile Leu Leu Phe Asn Leu Pro Asp Leu 165 170 175 Gly Gln Asn Pro
Ser Ala Arg Ser Gln Lys Val Val Glu Ala Val Ser 180 185 190 His Val
Ser Ala Tyr His Asn Lys Leu Leu Leu Asn Leu Ala Arg Gln 195 200 205
Leu Ala Pro Thr Gly Met Val Lys Leu Phe Glu Ile Asp Lys Gln Phe 210
215 220 Ala Glu Met Leu Arg Asp Pro Gln Asn Phe Gly Leu Ser Asp Val
Glu 225 230 235 240 Asn Pro Cys Tyr Asp Gly Gly Tyr Val Trp Lys Pro
Phe Ala Thr Arg 245 250 255 Ser Val Ser Thr Asp Arg Gln Leu Ser Ala
Phe Ser Pro Gln Glu Arg 260 265 270 Leu Ala Ile Ala Gly Asn Pro Leu
Leu Ala Gln Ala Val Ala Ser Pro 275 280 285 Met Ala Arg Arg Ser Ala
Ser Pro Leu Asn Cys Glu Gly Lys Met Phe 290 295 300 Trp Asp Gln Val
His Pro Thr Thr Val Val His Ala Ala Leu Ser Glu 305 310 315 320 Arg
Ala Ala Thr Phe Ile Glu Thr Gln Tyr Glu Phe Leu Ala His 325 330 335
49 367 PRT Artificial Sequence Description of Artificial Sequence
Synthetic Pfam00657.11 consensus sequence 49 Ile Val Ala Phe Gly
Asp Ser Leu Thr Asp Gly Gly Gly Ala Tyr Tyr 1 5 10 15 Gly Asp Ser
Asp Gly Gly Gly Trp Gly Ala Gly Leu Ala Asp Arg Leu 20 25 30 Thr
Ser Leu Ala Arg Leu Arg Ala Arg Gly Arg Gly Val Asp Val Phe 35 40
45 Asn Arg Gly Ile Ser Gly Arg Thr Ser Asp Gly Arg Leu Val Val Asp
50 55 60 Ala Arg Leu Val Ala Thr Leu Leu Phe Leu Ala Gln Phe Leu
Gly Leu 65 70 75 80 Asn Leu Pro Pro Tyr Leu Ser Gly Asp Phe Leu Arg
Gly Ala Asn Phe 85 90 95 Ala Ser Ala Gly Ala Thr Ile Leu Gly Thr
Ser Leu Ile Pro Phe Leu 100 105 110 Asn Ile Gln Val Gln Phe Lys Asp
Phe Lys Ser Lys Val Leu Glu Leu 115 120 125 Arg Gln Ala Leu Gly Leu
Leu Gln Glu Leu Leu Arg Leu Val Pro Val 130 135 140 Leu Asp Ala Lys
Ser Pro Asp Leu Val Thr Ile Met Ile Gly Thr Asn 145 150 155 160 Asp
Leu Ile Thr Val Ala Lys Phe Gly Pro Lys Ser Thr Lys Ser Asp 165 170
175 Arg Asn Val Ser Val Pro Glu Phe Arg Asp Asn Leu Arg Lys Leu Ile
180 185 190 Lys Arg Leu Arg Ser Ala Asn Gly Ala Arg Ile Ile Ile Leu
Ile Thr 195 200 205 Leu Val Leu Leu Asn Leu Pro Leu Pro Leu Gly Cys
Leu Pro Gln Lys 210 215 220 Leu Ala Leu Ala Leu Ala Ser Ser Lys Asn
Val Asp Ala Thr Gly Cys 225 230 235 240 Leu Glu Arg Leu Asn Glu Ala
Val Ala Asp Tyr Asn Glu Ala Leu Arg 245 250 255 Glu Leu Ala Glu Ile
Glu Lys Leu Gln Ala Gln Leu Arg Lys Asp Gly 260 265 270 Leu Pro Asp
Leu Lys Glu Ala Asn Val Pro Tyr Val Asp Leu Tyr Ser 275 280 285 Ile
Phe Gln Asp Leu Asp Gly Ile Gln Asn Pro Ser Ala Tyr Val Tyr 290 295
300 Gly Phe Glu Glu Thr Lys Ala Cys Cys Gly Tyr Gly Gly Arg Tyr Asn
305 310 315 320 Tyr Asn Arg Val Cys Gly Asn Ala Gly Leu Cys Lys Val
Thr Ala Lys 325 330 335 Ala Cys Asp Ala Ser Ser Tyr Leu Leu Ala Thr
Leu Phe Trp Asp Gly 340 345 350 Phe His Pro Ser Glu Lys Gly Tyr Lys
Ala Val Ala Glu Ala Leu 355 360 365 50 230 PRT Aspergillus
aculeatus 50 Thr Thr Val Tyr Leu Ala Gly Asp Ser Thr Met Ala Lys
Asn Gly Gly 1 5 10 15 Gly Ser Gly Thr Asn Gly Trp Gly Glu Tyr Leu
Ala Ser Tyr Leu Ser 20 25 30 Ala Thr Val Val Asn Asp Ala Val Ala
Gly Arg Ser Ala Arg Ser Tyr 35 40 45 Thr Arg Glu Gly Arg Phe Glu
Asn Ile Ala Asp Val Val Thr Ala Gly 50 55 60 Asp Tyr Val Ile Val
Glu Phe Gly His Asn Asp Gly Gly Ser Leu Ser 65 70 75 80 Thr Asp Asn
Gly Arg Thr Asp Cys Ser Gly Thr Gly Ala Glu Val Cys 85 90 95 Tyr
Ser Val Tyr Asp Gly Val Asn Glu Thr Ile Leu Thr Phe Pro Ala 100 105
110 Tyr Leu Glu Asn Ala Ala Lys Leu Phe Thr Ala Lys Gly Ala Lys Val
115 120 125 Ile Leu Ser Ser Gln Thr Pro Asn Asn Pro Trp Glu Thr Gly
Thr Phe 130 135 140 Val Asn Ser Pro Thr Arg Phe Val Glu Tyr Ala Glu
Leu Ala Ala Glu 145 150 155 160 Val Ala Gly Val Glu Tyr Val Asp His
Trp Ser Tyr Val Asp Ser Ile 165 170 175 Tyr Glu Thr Leu Gly Asn Ala
Thr Val Asn Ser Tyr Phe Pro Ile Asp 180 185 190 His Thr His Thr Ser
Pro Ala Gly Ala Glu Val Val Ala Glu Ala Phe 195 200 205 Leu Lys Ala
Val Val Cys Thr Gly Thr Ser Leu Lys Ser Val Leu Thr 210 215 220 Thr
Thr Ser Phe Glu Gly 225 230 51 184 PRT Escherichia coli 51 Ala Asp
Thr Leu Leu Ile Leu Gly Asp Ser Leu Ser Ala Gly Tyr Arg 1 5 10 15
Met Ser Ala Ser Ala Ala Trp Pro Ala Leu Leu Asn Asp Lys Trp Gln 20
25 30 Ser Lys Thr Ser Val Val Asn Ala Ser Ile Ser Gly Asp Thr Ser
Gln 35 40 45 Gln Gly Leu Ala Arg Leu Pro Ala Leu Leu Lys Gln His
Gln Pro Arg 50 55 60 Trp Val Leu Val Glu Leu Gly Gly Asn Asp Gly
Leu Arg Gly Phe Gln 65 70 75 80 Pro Gln Gln Thr Glu Gln Thr Leu Arg
Gln Ile Leu Gln Asp Val Lys 85 90 95 Ala Ala Asn Ala Glu Pro Leu
Leu Met Gln Ile Arg Leu Pro Ala Asn 100 105 110 Tyr Gly Arg Arg Tyr
Asn Glu Ala Phe Ser Ala Ile Tyr Pro Lys Leu 115 120 125 Ala Lys Glu
Phe Asp Val Pro Leu Leu Pro Phe Phe Met Glu Glu Val 130 135 140 Tyr
Leu Lys Pro Gln Trp Met Gln Asp Asp Gly Ile His Pro Asn Arg 145 150
155 160 Asp Ala Gln Pro Phe Ile Ala Asp Trp Met Ala Lys Gln Leu Gln
Pro 165 170 175 Leu Val Asn His Asp Ser Leu Glu 180 52 232 PRT
Aspergillus aculeatus 52 Thr Thr Val Tyr Leu Ala Gly Asp Ser Thr
Met Ala Lys Asn Gly Gly 1 5 10 15 Gly Ser Gly Thr Asn Gly Trp Gly
Glu Tyr Leu Ala Ser Tyr Leu Ser 20 25 30 Ala Thr Val Val Asn Asp
Ala Val Ala Gly Arg Ser Ala Arg Ser Tyr 35 40 45 Thr Arg Glu Gly
Arg Phe Glu Asn Ile Ala Asp Val Val Thr Ala Gly 50 55 60 Asp Tyr
Val Ile Val Glu Phe Gly His Asn Asp Gly Gly Ser Leu Ser 65 70 75 80
Thr Asp Asn Gly Arg Thr Asp Cys Ser Gly Thr Gly Ala Glu Val Cys 85
90 95 Tyr Ser Val Tyr Asp Gly Val Asn Glu Thr Ile Leu Thr Phe Pro
Ala 100 105 110 Tyr Leu Glu Asn Ala Ala Lys Leu Phe Thr Ala Lys Gly
Ala Lys Val 115 120 125 Ile Leu Ser Ser Gln Thr Pro Asn Asn Pro Trp
Glu Thr Gly Thr Phe 130 135 140 Val Asn Ser Pro Thr Arg Phe Val Glu
Tyr Ala Glu Leu Ala Ala Glu 145 150 155 160 Val Ala Gly Val Glu Tyr
Val Asp His Trp Ser Tyr Val Asp Ser Ile 165 170 175 Tyr Glu Thr Leu
Gly Asn Ala Thr Val Asn Ser Tyr Phe Pro Ile Asp 180 185 190 His Thr
His Thr Ser Pro Ala Gly Ala Glu Val Val Ala Glu Ala Phe 195 200 205
Leu Lys Ala Val Val Cys Thr Gly Thr Ser Leu Lys Ser Val Leu Thr 210
215 220 Thr Thr Ser Phe Glu Gly Thr Cys 225 230 53 4 PRT Artificial
Sequence Description of Artificial Sequence Synthetic peptide motif
53 Gly Asp Ser Leu 1 54 5 PRT Artificial Sequence Description of
Artificial Sequence Synthetic peptide motif 54 Gly Ala Asn Asp Tyr
1 5 55 6 PRT Artificial Sequence Description of Artificial Sequence
Synthetic 6xHis tag 55 His His His His His His 1 5
* * * * *
References