U.S. patent application number 10/994138 was filed with the patent office on 2005-08-25 for protein expression systems.
This patent application is currently assigned to Dow Global Technologies Inc.. Invention is credited to Badgley, Anne Kathryn, Chew, Lawrence C., Ramseier, Thomas Martin, Schneider, Jane C..
Application Number | 20050186666 10/994138 |
Document ID | / |
Family ID | 34636466 |
Filed Date | 2005-08-25 |
United States Patent
Application |
20050186666 |
Kind Code |
A1 |
Schneider, Jane C. ; et
al. |
August 25, 2005 |
Protein expression systems
Abstract
The present invention provides an improved expression system for
the production of recombinant polypeptides utilizing auxotrophic
selectable markers. In addition, the present invention provides
improved recombinant protein production in host cells through the
improved regulation of expression.
Inventors: |
Schneider, Jane C.; (San
Diego, CA) ; Chew, Lawrence C.; (San Diego, CA)
; Badgley, Anne Kathryn; (Poway, CA) ; Ramseier,
Thomas Martin; (Poway, CA) |
Correspondence
Address: |
KING & SPALDING LLP
191 PEACHTREE STREET, N.E.
45TH FLOOR
ATLANTA
GA
30303-1763
US
|
Assignee: |
Dow Global Technologies
Inc.
Midland
MI
|
Family ID: |
34636466 |
Appl. No.: |
10/994138 |
Filed: |
November 19, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60523420 |
Nov 19, 2003 |
|
|
|
60537147 |
Jan 16, 2004 |
|
|
|
Current U.S.
Class: |
435/108 ;
435/252.34; 435/471 |
Current CPC
Class: |
C12N 9/0028 20130101;
C12N 15/52 20130101; C12P 21/00 20130101; C12P 21/02 20130101; C12N
9/88 20130101; C12Y 305/05001 20130101; C12N 15/78 20130101 |
Class at
Publication: |
435/108 ;
435/252.34; 435/471 |
International
Class: |
C12P 013/22; C12N
015/74; C12N 001/21 |
Claims
We claim:
1) An auxotrophic Pseudomonad cell for use in a bacterial
expression system that comprises a nucleic acid construct
comprising: a. a nucleic acid encoding a recombinant polypeptide;
and, b. a nucleic acid encoding at least one polypeptide that
restores prototrophy to the auxotrophic host cell.
2) The cell of claim 1, wherein the Pseudomonad is Pseudomonas
fluorescens.
3) The cell of claim 1, wherein the cell is auxotrophic for
uracil.
4) The cell of claim 1, wherein the cell is auxotrophic for
proline.
5) The cell of claim 1, wherein the auxotrophic cell is auxotrophic
for more than one metabolite.
6) The cell of claim 5, wherein the cell is auxotrophic for uracil
and proline.
7) The cell of claim 1, wherein the prototrophy restoring
polypeptide is an enzyme active in the biosynthesis of a metabolite
required for cell survival.
8) The cell of claim 7, wherein the enzyme is
orotodine-5'-phosphate decarboxylase.
9) The cell of claim 8, wherein the enzyme is encoded by the
nucleic acid sequence selected from the group consisting of SEQ.
ID. 1 and 3.
10) The cell of claim 7, wherein the enzyme comprises the amino
acid sequence of SEQ ID No. 2.
11) The cell of claim 7, wherein the enzyme is
.DELTA..sup.1-pyrroline-5-c- arboxylate reductase.
12) The cell of claim 11, wherein the enzyme is encoded by the
nucleic acid sequence selected from the group consisting of SEQ.
ID. NO. 6 and 8.
13) The cell of claim 11, wherein the enzyme comprises the amino
acid sequence of SEQ. ID. No. 7.
14) The cell of claim 1, wherein the auxotrophic cell is produced
by disabling a pyrF gene.
15) The cell of claim 14, wherein the disabled pyrF gene comprises
the nucleic acid selected from the group consisting of SEQ. ID. No.
1 and SEQ. ID. No. 3.
16) The cell of claim 1, wherein the auxotrophic cell is produced
by disabling a proC gene.
17) The cell of claim 16, wherein the disabled proC gene comprises
the nucleic acid selected from the group consisting of SEQ. ID. No.
6 and SEQ. ID. No. 8.
18) The cell of claim 1, wherein the auxotrophic cell is produced
by disabling a pyrF gene and a proC gene.
19) The cell of claim 18, wherein the disabled pyrF gene comprises
the nucleic acid selected from the group consisting of SEQ. ID. No.
1 and SEQ. ID. No.3, and the disabled proC gene comprises the
nucleic acid selected from the group consisting of SEQ. ID. No. 6
and SEQ. ID. NO. 9.
20) The cell of claim 1, wherein the cell also contains a
chromosomal lacI insert that is other than as part of a
PlacI-lacI-lacZYA operon.
21) The cell of claim 20, wherein the lacI gene is selected from
the group consisting of lacI, lacI.sup.Q, and lacI.sup.Q1.
22) The cell of claim 1, wherein the nucleic acid construct further
comprises at least one lacOid sequence.
23) The cell of claim 22, wherein the lacOid sequence is selected
from the group consisting of SEQ. ID. NO. 14 and SEQ. ID. NO.
59.
24) The cell of claim 1, wherein the nucleic acid construct further
comprising more than one lac operator sequences.
25) The cell of claim 24, wherein at least one lac operator
sequence is located 5' of a promoter, and at least one lac operator
sequence is located 3' of a promoter.
26) The cell of claim 25, wherein at least one lac operator
sequence is a lacOid sequence.
27) The cell of claim 26, wherein the lacOid sequence is selected
from the group consisting of SEQ. ID. NO. 14 and SEQ. ID. NO.
59.
28) A genetically modified Pseudomonad cell for use in a bacterial
expression system, wherein the modification comprises at least one
chromosomal insertion of a lacI gene, wherein the lacI gene is
other than as part of a whole or truncated PlacI-lacI-lacZYA
operon.
29) The cell of claim 28, wherein the Pseudomonad is Pseudomonas
fluorescens.
30) The cell of claim 28, wherein the lacI gene is selected from
the group consisting of lacI, lacI.sup.Q, and lacI.sup.Q1.
31) The cell of claim 28, wherein the lacI gene is inserted in the
levansucrase locus.
32) The cell of claim 28, wherein the cell has been further
modified to create an auxotrophy for at least one metabolite in the
cell.
33) The cell of claim 32, wherein the auxotrophy is created by
modification to a gene selected from the group consisting of pyrF
and proC.
34) The cell of claim 32, wherein the auxotrophy is created by
modification to both the pyrF and the proC gene.
35) The cell of claim 28, further comprising a nucleic acid
comprising at least one lacOid sequence.
36) The cell of claim 35, wherein the lacOid sequence is selected
from the group consisting of SEQ. ID. NO. 14 and SEQ. ID. NO.
59.
37) The cell of claim 28 further comprising a nucleic acid
comprising more than one lac operator sequence.
38) The cell of claim 37, wherein at least one lac operator
sequence is a lacOid sequence.
39) The cell of claim 38, wherein the lacOid sequence is selected
from the group consisting of SEQ. ID. NO. 14 and SEQ. ID. NO.
59.
40) A Pseudomonad cell for use in a bacterial expression system
comprising a nucleic acid construct comprising at least one lacOid
operator sequence.
41) The cell of claim 40, wherein the Pseudomonad is a Pseudomonas
fluorescens.
42) The cell of claim 40, wherein the lacOid sequence is located 3'
of a promoter.
43) The cell of claim 40, wherein the lacOid sequence is located 5'
of a promoter.
44) The cell of claim 40, wherein lacOid sequences are located 3'
and 5' of a promoter.
45) The cell of claim 40, wherein the cell has been further
modified to create an auxotrophy for at least one metabolite in the
cell.
46) The cell of claim 45, wherein the auxotrophy is created by
modification to a gene selected from the group consisting of pyrF
and proC.
47) The cell of claim 45, wherein the auxotrophy is created by
modification to both the pyrF and the proC gene.
48) The cell of claim 40, wherein the cell contains a chromosomal
insertion of a lacI gene, wherein the lacI gene is other than as
part of a whole or truncated Plac-lacI-lacZYA operon.
49) The cell of claim 40, wherein the lacOid sequence is selected
from the group consisting of SEQ ID NO. 14 and SEQ. ID. NO. 59.
50) A Pseudomonad cell for use in a bacterial expression system
comprising a nucleic acid construct comprising more than one lac
operator sequence.
51) The cell of claim 50, wherein at least one lac operator is a
lacOid sequence.
52) The cell of claim 51, wherein the lacOid sequence is selected
from the group consisting of SEQ. ID. NO. 14 and SEQ. ID. NO.
59.
53) The cell of claim 51, wherein the lacOid sequence is 5' or 3'
of the promoter.
54) The cell of claim 51 wherein the lacOid sequence is 5' and 3'
of the promoter.
55) The cell of claim 50, wherein the cell has been further
modified to create an auxotrophy for at least one metabolite in the
cell.
56) The cell of claim 55, wherein the auxotrophy is created by
modification to a gene selected from the group consisting of pyrF
and proC.
57) The cell of claim 55, wherein the auxotrophy is created by
modification to both the pyrF and the proC gene.
58) The cell of claim 50, wherein the cell contains a chromosomal
insertion of a lacI gene, wherein the lacI gene is other than as
part of a whole or truncated Plac-lacI-lacZYA operon.
59) The cell of claim 50, wherein the Pseudomonad is Pseudomonas
fluorescens.
60) A process for producing a recombinant polypeptide comprising:
a. expressing a nucleic acid encoding the recombinant polypeptide
in a Pseudomonad cell that has been genetically modified to create
an auxotrophy for at least one metabolite; b. expressing a nucleic
acid encoding a polypeptide that restores prototrophy to the
auxotrophic cell; and, c. growing the cell on a medium that lacks
the auxotrophic metabolite.
61) The process of claim 60, wherein the Pseudomonad is Pseudomonas
fluorescens.
62) The process of claim 60, wherein the cell is auxotrophic for
uracil.
63) The process of claim 60, wherein the cell is auxotrophic for
proline.
64) The process of claim 60, wherein the auxotrophic cell is
auxotrophic for more than one metabolite.
65) The process of claim 64, wherein the cell is auxotrophic for
uracil and proline.
66) The process of claim 60, wherein the prototrophy restoring
polypeptide is an enzyme active in the biosynthesis of a metabolite
required for cell survival.
67) The process of claim 66, wherein the enzyme is
orotodine-5'-phosphate decarboxylase.
68) The process of claim 67, wherein the enzyme is encoded by the
nucleic acid sequence selected from the group consisting of SEQ.
ID. 1 and 3.
69) The process of claim 68, wherein the enzyme comprises the amino
acid sequence of SEQ ID No. 2.
70) The process of claim 66, wherein the enzyme is
.DELTA..sup.1-pyrroline- -5-carboxylate reductase.
71) The process of claim 70, wherein the enzyme is encoded by the
nucleic acid sequence selected from the group consisting of SEQ.
ID. NO. 6 and 8.
72) The process of claim 70, wherein the enzyme comprises the amino
acid sequence of SEQ. ID. No. 7.
73) The process of claim 60, wherein the auxotrophic cell is
produced by disabling a pyrF gene.
74) The process of claim 73, wherein the disabled pyrF gene
comprises the nucleic acid selected from the group consisting of
SEQ. ID. No. 1 and SEQ. ID. No. 3.
75) The process of claim 60, wherein the auxotrophic cell is
produced by disabling a proC gene.
76) The process of claim 75, wherein the disabled proC gene
comprises the nucleic acid selected from the group consisting of
SEQ. ID. No. 6 and SEQ. ID. No. 8.
77) The process of claim 60, wherein the auxotrophic cell is
produced by disabling a pyrF gene and a proC gene.
78) The process of claim 77, wherein the disabled pyrF gene
comprises the nucleic acid selected from the group consisting of
SEQ. ID. No. 1 and SEQ. ID. No. 3, and the disabled proC gene
comprises the nucleic acid selected from the group consisting of
SEQ. ID. No. 6 and SEQ. ID. NO. 9.
79) The process of claim 60, wherein the cell also contains a
chromosomal lacI insert that is other than as part of a
PlacI-lacI-lacZYA operon.
80) The process of claim 79, wherein the lacI gene is selected from
the group consisting of lacI, lacI.sup.Q, and lac.sup.Q1.
81) The process of claim 60, wherein the nucleic acid encoding the
recombinant polypeptide further comprises at least one lacOid
sequence.
82) The proves of claim 81, wherein the lacOid sequence is selected
from the group consisting of SEQ. ID. NO. 14 and SEQ. ID. NO.
59.
83) The process of claim 60, wherein the nucleic acid encoding the
recombinant polypeptide further comprises more than one lac
operator sequences.
84) The process of claim 83, wherein at least one lac operator
sequence is located 5' of a promoter, and at least one lac operator
sequence is located 3' of a promoter.
85) The process of claim 84, wherein at least one lac operator
sequence is a lacOid sequence.
86) The process of claim 85, wherein the lacOid sequence is
selected from the group consisting of SEQ. ID. NO. 14 and SEQ. ID.
NO. 59.
87) A process for producing a recombinant polypeptide comprising
expressing a nucleic acid encoding the recombinant polypeptide in a
Pseudomonad that comprises at least one chromosomal insertion of a
lacI gene, wherein the lacI gene is other than as part of a whole
or truncated PlacI-lacI-lacZYA operon.
88) The process of claim 87, wherein the Pseudomonad is Pseudomonas
fluorescens.
89) The process of claim 87, wherein the lacI gene is selected from
the group consisting of lacI, lacI.sup.Q, and lacI.sup.Q1.
90) The process of claim 87, wherein the lacI gene is inserted in
the levansucrase locus.
91) The process of claim 87, wherein the cell has been further
modified to create an auxotrophy for at least one metabolite in the
cell.
92) The process of claim 91, wherein the auxotrophy is created by
modification to a gene selected from the group consisting of pyrF
and proC.
93) The process of claim 91, wherein the auxotrophy is created by
modification to both the pyrF and the proC gene.
94) The process of claim 87, wherein the nucleic acid encoding the
recombinant polypeptide comprises at least one lacOid sequence.
95) The process of claim 94, wherein the lacOid sequence is
selected from the group consisting of SEQ. ID. NO. 14 and SEQ. ID.
NO. 59.
96) The process of claim 87, wherein the nucleic acid encoding the
recombinant polypeptide comprises more than one lac operator
sequence.
97) The process of claim 96, wherein at least one lac operator
sequence is a lacOid sequence.
98) The process of claim 97, wherein the lacOid sequence is
selected from the group consisting of SEQ. ID. NO. 14 and SEQ. ID.
NO. 59.
99) A process for producing a recombinant polypeptide comprising
expressing a nucleic acid encoding the recombinant polypeptide in a
Pseudomonad cell, wherein the nucleic acid further comprises at
least one lac operator sequence, wherein the lac operator sequence
is a lacOid sequence.
100) The process of claim 99, wherein the Pseudomonad is a
Pseudomonas fluorescens.
101) The process of claim 99, wherein the lacOid sequence is
selected from the group consisting of SEQ. ID. NO. 14 and SEQ. ID.
NO. 59.
102) The process of claim 99, wherein at least one lacOid sequence
is located 3' of a promoter.
103) The process of claim 99, wherein at least one lacOid sequence
is located 5' of a promoter.
104) The process of claim 99, wherein at least one lacOid sequence
is located 3' and 5' of a promoter.
105) The process of claim 99, wherein the cell has been further
modified to create an auxotrophy for at least one metabolite in the
cell.
106) The process of claim 105, wherein the auxotrophy is created by
modification to a gene selected from the group consisting of pyrF
and proC.
107) The process of claim 105, wherein the auxotrophy is created by
modification to both the pyrF and the proC gene.
108) The process of claim 99, wherein the cell contains a
chromosomal insertion of a lacI gene, wherein the lacI gene is
other than as part of a whole or truncated Plac-lacI-lacZYA
operon.
109) A process for producing a recombinant polypeptide comprising
expressing a nucleic acid encoding the recombinant polypeptide in a
Pseudomonad cell, wherein the nucleic acid further comprises more
than one lac operator sequence.
110) The process of claim 109, wherein at least one lac operator is
a lacOid sequence.
111) The process of claim 110, wherein the lacOid sequence is
selected from the group consisting of SEQ. ID. NO. 14 and SEQ. ID.
NO. 59.
112) The process of claim 110, wherein the lacOid sequence is 5' or
3' of the promoter.
113) The process of claim 110, wherein the lacOid sequence is 5'
and 3' of the promoter.
114) The process of claim 109, wherein the cell has been further
modified to create an auxotrophy for at least one metabolite in the
cell.
115) The process of claim 114, wherein the auxotrophy is created by
modification to a gene selected from the group consisting of pyrF
and proC.
116) The process of claim 114, wherein the auxotrophy is created by
modification to both the pyrF and the proC gene.
117) The process of claim 109, wherein the cell contains a
chromosomal insertion of a lacI gene, wherein the lacI gene is
other than as part of a whole or truncated Plac-lacI-lacZYA
operon.
118) The process of claim 109, wherein the Pseudomonad is
Pseudomonas fluorescens
119) A process for modulating the expression of a recombinant
polypeptide in a host cell comprising: a. selecting a Pseudomonad
cell, wherein the cell has been genetically modified by
chromosomally inserting a lacI gene into the cell, wherein the lacI
gene is other than as part of a whole or truncated
PlacI-lacI-lacZYA operon; and, b. introducing into the cell a
nucleic acid construct comprising a LacI protein promoter operably
attached to a nucleic acid encoding the recombinant
polypeptide.
120) The process of claim 119, wherein the Pseudomonad is
Pseudomonas fluorescens.
121) The process of claim 119, wherein the lacI gene is selected
from the group consisting of lacI, lacI.sup.Q, and lacI.sup.Q1.
122) The process of claim 119, wherein the lacI gene is inserted in
the levansucrase locus.
123) The process of claim 119, wherein the cell has been further
modified to create an auxotrophy for at least one metabolite in the
cell.
124) The process of claim 123, wherein the auxotrophy is created by
modification to a gene selected from the group consisting of pyrF
and proC.
125) The process of claim 123, wherein the auxotrophy is created by
modification to both the pyrF and the proC gene.
126) The process of claim 119, wherein the nucleic acid encoding
the recombinant polypeptide comprises at least one lacOid
sequence.
127) The process of claim 126, wherein the lacOid sequence is
selected from the group consisting of SEQ. ID. NO. 14 and SEQ. ID.
NO. 59.
128) The process of claim 126, wherein at least one lacOid sequence
is located 3' of the promoter.
129) The process of claim 126, wherein at least one lacOid sequence
is located 5' of the promoter.
130) The process of claim 126, wherein lacOid sequence are located
5' and 3' of the promoter.
131) The process of claim 119, wherein the nucleic acid encoding
the recombinant polypeptide comprises more than one lac operator
sequence.
132) The process of claim 131, wherein at least one lac operator
sequence is a lacOid sequence.
133) The process of claim 132, wherein the lacOid sequence is
selected from the group consisting of SEQ. ID. NO. 14 and SEQ. ID.
NO. 59.
134) A process for modulating the expression of a recombinant
polypeptide in a host cell comprising: a. selecting a Pseudomonad
cell; and b. introducing a nucleic acid construct comprising: i. a
nucleic acid encoding the recombinant polypeptide, and, ii. at
least one lacOid operator sequence.
135) The process of claim 134, wherein the Pseudomonad is a
Pseudomonas fluorescens.
136) The process of claim 134, wherein the lacOid sequence is
located 3' of a promoter.
137) The process of claim 134, wherein the lacOid sequence is
located 5' of a promoter.
138) The process of claim 134, wherein lacOid sequences are located
3' and 5' of a promoter.
139) The process of claim 134, wherein the cell has been further
modified to create an auxotrophy for at least one metabolite in the
cell.
140) The process of claim 139, wherein the auxotrophy is created by
modification to a gene selected from the group consisting of pyrF
and proC.
141) The process of claim 139, wherein the auxotrophy is created by
modification to both the pyrF and the proC gene.
142) The process of claim 134, wherein the cell contains a
chromosomal insertion of a lacI gene, wherein the lacI gene is
other than as part of a whole or truncated Plac-lacI-lacZYA
operon.
143) The process of claim 134, wherein the lacOid sequence is
selected from the group consisting of SEQ. ID. NO. 14 and SEQ. ID.
NO. 59.
144) A process for modulating the expression of a recombinant
polypeptide in a host cell comprising: a. selecting a Pseudomonad
cell; and b. introducing a nucleic acid construct comprising: i. a
nucleic acid encoding the recombinant polypeptide, and, ii. more
than one lac operator sequence.
145) The process of claim 144, wherein at least one lac operator is
a lacOid sequence.
146) The process of claim 145, wherein the lacOid sequence is
selected from the group consisting of SEQ. ID. NO.14 and SEQ. ID.
NO. 59.
147) The process of claim 145, wherein the lacOid sequence is 5' or
3' of the promoter.
148) The process of claim 145 wherein the lacOid sequence is 5' and
3' of the promoter.
149) The process of claim 144, wherein the cell has been further
modified to create an auxotrophy for at least one metabolite in the
cell.
150) The process of claim 149, wherein the auxotrophy is created by
modification to a gene selected from the group consisting of pyrF
and proC.
151) The process of claim 149, wherein the auxotrophy is created by
modification to both the pyrF and the proC gene.
152) The process of claim 144, wherein the cell contains a
chromosomal insertion of a lacI gene, wherein the lacI gene is
other than as part of a whole or truncated Plac-lacI-lacZYA
operon.
153) The process of claim 144, wherein the Pseudomonad is
Pseudomonas fluorescens.
154) A process for the production of a recombinant polypeptide in
the absence of antibiotics comprising: a. selecting a Pseudomonad
cell, wherein the cell has been genetically modified to induce an
auxotrophy for at least one metabolite, thereby creating an
auxotrophic cell; b. introducing into the cell a nucleic acid
construct comprising i. a nucleic acid encoding the recombinant
polypeptide; and ii. a nucleic acid encoding a polypeptide that
restores prototrophy to the auxotrophic host cell; c. expressing
the recombinant polypeptide and prototrophy restoring polypeptide
in the cell; and, d. growing the cell on a medium that lacks the
auxotrophic metabolite.
155) The process of claim 154, wherein the Pseudomonad is
Pseudomonas fluorescens.
156) The process of claim 154, wherein the cell is auxotrophic for
uracil.
157) The process of claim 154, wherein the cell is auxotrophic for
proline.
158) The process of claim 154, wherein the auxotrophic cell is
auxotrophic for more than one metabolite.
159) The process of claim 158, wherein the cell is auxotrophic for
uracil and proline.
160) The process of claim 154, wherein the prototrophy restoring
polypeptide is an enzyme active in the biosynthesis of a metabolite
required for cell survival.
161) The process of claim 160, wherein the enzyme is
orotodine-5'-phosphate decarboxylase.
162) The process of claim 161, wherein the enzyme is encoded by the
nucleic acid sequence selected from the group consisting of SEQ.
ID. 1 and 3.
163) The process of claim 160, wherein the enzyme comprises the
amino acid sequence of SEQ ID No. 2.
164) The process of claim 160, wherein the enzyme is
.DELTA..sup.1-pyrroline-5-carboxylate reductase.
165) The process of claim 164, wherein the enzyme is encoded by the
nucleic acid sequence selected from the group consisting of SEQ.
ID. NO. 6 and 8.
166) The process of claim 164, wherein the enzyme comprises the
amino acid sequence of SEQ. ID. No. 7.
167) The process of claim 154, wherein the auxotrophic cell is
produced by disabling a pyrF gene.
168) The process of claim 167, wherein the disabled pyrF gene
comprises the nucleic acid selected from the group consisting of
SEQ. ID. No.1 and SEQ. ID. No. 3.
169) The process of claim 154, wherein the auxotrophic cell is
produced by disabling a proC gene.
170) The process of claim 169, wherein the disabled proC gene
comprises the nucleic acid selected from the group consisting of
SEQ. ID. No. 6 and SEQ. ID. No. 8.
171) The process of claim 154, wherein the auxotrophic cell is
produced by disabling a pyrF gene and a proC gene.
172) The process of claim 171, wherein the disabled pyrF gene
comprises the nucleic acid selected from the group consisting of
SEQ. ID. No.1 and SEQ. ID. No. 3, and the disabled proC gene
comprises the nucleic acid selected from the group consisting of
SEQ. ID. No. 6 and SEQ. ID. NO.9.
173) The process of claim 154, wherein the cell also contains a
chromosomal lacI insert that is other than as part of a
PlacI-lacI-lacZYA operon.
174) The process of claim 173, wherein the lacI gene is selected
from the group consisting of lacI, lacI.sup.Q, and lacI.sup.Q1.
175) The process of claim 154, wherein the nucleic acid construct
further comprises at least one lacOid sequence.
176) The process of claim 154, wherein the nucleic acid construct
further comprising more than one lac operator sequences.
177) The process of claim 176, wherein at least one lac operator
sequence is located 5' of a promoter, and at least one lac operator
sequence is located 3' of a promoter.
178) The process of claim 177, wherein at least one lac operator
sequence is a lacOid sequence.
179) A process for the production of a recombinant polypeptide in
the absence of antibiotics wherein cross feeding inhibition is
minimized during selection comprising: a. selecting a Pseudomonad
cell, wherein the cell has been genetically modified to induce an
auxotrophy for at least one metabolite, thereby creating an
auxotrophic cell; b. introducing into the cell a nucleic acid
construct comprising i. a nucleic acid encoding a recombinant
polypeptide; and ii. a nucleic acid encoding a polypeptide that
restores prototrophy to the auxotrophic host cell; c. expressing
the recombinant polypeptide and the prototrophy restoring
polypeptide in the cell; and, d. growing the cell on a medium that
lacks the auxotrophic metabolite.
180) The process of claim 179, wherein the Pseudomonad is
Pseudomonas fluorescens.
181) The process of claim 179, wherein the cell is auxotrophic for
uracil.
182) The process of claim 179, wherein the cell is auxotrophic for
proline.
183) The process of claim 179, wherein the auxotrophic cell is
auxotrophic for more than one metabolite.
184) The process of claim 183, wherein the cell is auxotrophic for
uracil and proline.
185) The process of claim 179, wherein the prototrophy restoring
polypeptide is an enzyme active in the biosynthesis of a metabolite
required for cell survival.
186) The process of claim 185, wherein the enzyme is
orotodine-5'-phosphate decarboxylase.
187) The process of claim 186, wherein the enzyme is encoded by the
nucleic acid sequence selected from the group consisting of SEQ.
ID. 1 and 3.
188) The process of claim 185, wherein the enzyme comprises the
amino acid sequence of SEQ ID No. 2.
189) The process of claim 185, wherein the enzyme is
.DELTA..sup.1-pyrroline-5-carboxylate reductase.
190) The process of claim 189, wherein the enzyme is encoded by the
nucleic acid sequence selected from the group consisting of SEQ.
ID. NO. 6 and 8.
191) The process of claim 189, wherein the enzyme comprises the
amino acid sequence of SEQ. ID. No. 7.
192) The process of claim 179, wherein the auxotrophic cell is
produced by disabling a pyrF gene.
193) The process of claim 192, wherein the disabled pyrF gene
comprises the nucleic acid selected from the group consisting of
SEQ. ID. No. 1 and SEQ. ID. No. 3.
194) The process of claim 179, wherein the auxotrophic cell is
produced by disabling a proC gene.
195) The process of claim 194, wherein the disabled proC gene
comprises the nucleic acid selected from the group consisting of
SEQ. ID. No. 6 and SEQ. ID. No. 8.
196) The process of claim 179, wherein the auxotrophic cell is
produced by disabling a pyrF gene and a proC gene.
197) The process of claim 196, wherein the disabled pyrF gene
comprises the nucleic acid selected from the group consisting of
SEQ. ID. No. 1 and SEQ. ID. No.3, and the disabled proC gene
comprises the nucleic acid selected from the group consisting of
SEQ. ID. No. 6 and SEQ. ID. NO. 9.
198) The process of claim 179, wherein the cell also contains a
chromosomal lacI insert that is other than as part of a
PlacI-lacI-lacZYA operon.
199) The process of claim 198, wherein the lacI gene is selected
from the group consisting of lacI, lacI.sup.Q, and lacI.sup.Q1.
200) The process of claim 199, wherein the nucleic acid construct
further comprises at least one lacOid sequence.
201) The process of claim 179, wherein the nucleic acid construct
further comprising more than one lac operator sequences.
202) The process of claim 201, wherein at least one lac operator
sequence is located 5' of a promoter, and at least one lac operator
sequence is located 3' of a promoter.
203) The process of claim 202, wherein at least one lac operator
sequence is a lacOid sequence.
204) A Pseudomonas fluorescens pyrF gene, or nucleic acid that
hybridizes with the pyrF gene, comprising the nucleic acid sequence
selected from the group consisting of SEQ. ID. No. 1 or 3.
205) The gene of claim 204, wherein the nucleic acid comprises the
sequence of SEQ. ID. No. 1.
206) The gene of claim 204, wherein the nucleic acid comprises the
sequence of SEQ. ID. No. 3
207) A Pseudomonas fluorescens proC gene, or nucleic acid that
hybridizes with the proC gene, comprising the nucleic acid sequence
selected from the group consisting of SEQ. ID. No. 6 or 8.
208) The gene of claim 207, wherein the nucleic acid comprises the
sequence of SEQ ID No. 6.
209) The gene of claim 207, wherein the nucleic acid comprises the
sequence of SEQ ID No. 8.
210) A nucleic acid construct comprising: a. a nucleic acid
encoding a recombinant polypeptide; and b. a nucleic acid encoding
a pyrF gene isolated from a Pseudomonas fluorescens.
211) The construct of claim 210, wherein the pyrF gene comprises
the nucleic acid sequence of SEQ. ID No. 1.
212) The construct of claim 210, wherein the pyrF gene comprises
the nucleic acid sequence of SEQ. ID No. 3.
213) A nucleic acid construct comprising: a. a nucleic acid
encoding a recombinant polypeptide; and b. a nucleic acid encoding
a proC gene isolated from a Pseudomonas fluorescens.
214) The construct of claim 213, wherein the proC gene comprises
the nucleic acid sequence of SEQ ID No. 6.
215) The construct of claim 213, wherein the proC gene comprises
the nucleic acid sequence of SEQ ID No. 8.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional patent
application Ser. No. 60/523,420 filed Nov. 19, 2003, entitled
"Improved Pseudomonas Expression Systems with Auxotrophic Selection
Markers," and U.S. Provisional patent application 60/537,147 filed
Jan. 16, 2004, and entitled "Bacterial Expression Systems with
Improved Repression."
FIELD OF THE INVENTION
[0002] The present invention provides an improved expression system
for the production of recombinant polypeptides utilizing
auxotrophic selectable markers. In addition, the present invention
provides improved recombinant protein production in host cells
through the improved regulation of expression.
BACKGROUND OF THE INVENTION
[0003] The use of bacterial cells to produce protein based
therapeutics is increasing in commercial importance. One of the
goals in developing a bacterial expression system is the production
of high quality target polypeptides quickly, efficiently, and
abundantly. An ideal host cell for such an expression system would
be able to efficiently utilize a carbon source for the production
of a target polypeptide, quickly grow to high cell densities in a
fermentation reaction, express the target polypeptide only when
induced, and grow on a medium that is devoid of regulatory and
environmental concerns.
[0004] There are many hurdles to the creation of a superior host
cell. First, in order to produce a recombinant polypeptide, an
expression vector encoding the target protein must be inserted into
the host cell. Many bacteria are capable of reverting back into an
untransformed state, wherein the expression vector is eliminated
from the host. Such revertants can decrease the fermentation
efficiency of the production of the desired recombinant
polypeptide.
[0005] Expression vectors encoding a target peptide typically
include a selection marker in the vector. Often, the selection
marker is a gene whose product is required for survival during the
fermentation process. Host cells lacking the selection marker, such
as revertants, are unable to survive. The use of selection markers
during the fermentation process is intended to ensure that only
bacteria containing the expression vector survive, eliminating
competition between the revertants and transformants and reducing
the efficiency of fermentation.
[0006] The most commonly used selection markers are antibiotic
resistance genes. Host cells are grown in a medium supplemented
with an antibiotic capable of being degraded by the selected
antibiotic resistance gene product. Cells that do not contain the
expression vector with the antibiotic resistance gene are killed by
the antibiotic. Typical antibiotic resistance genes include
tetracycline, neomycin, kanamycin, and ampicillin. The presence of
antibiotic resistance genes in a bacterial host cell, however,
presents environmental, regulatory, and commercial problems. For
example, antibiotic resistance gene-containing products (and
products produced by the use of antibiotic resistance gene) have
been identified as potential biosafety risks for environmental,
human, and animal health. For example, see M. Droge et al.,
Horizontal Gene Transfer as a Biosafety issue: A natural phenomenon
of public concern, J. Biotechnology. 64(1): 75-90 (17 Sept. 1998);
Gallagher, D. M., and D. P. Sinn. 1983. Penicillin-induced
anaphylaxis in a patient under hypotensive anaesthesia. Oral Surg.
Oral Med. Oral Pathol. 56:361-364; Jorro, G., C. Morales, J. V.
Braso, and A. Pelaez. 1996. Anaphylaxis to erythromycin. Ann.
Allergy Asthma Immunol. 77:456-458; F. Gebhard & K. Smalla,
Transformation of Acinetobacter sp. strain BD413 by transgenic
sugar beet DNA, Appl. & Environ. Microbiol. 64(4):1550-54 (Apr.
1998); T. Hoffinann et al., Foreign DNA sequences are received by a
wild type strain of Aspergillus niger after co-culture with
transgenic higher plants, Curr. Genet. 27(1): 70-76 (Dec. 1994); DK
Mercer et al., Fate of free DNA and transformation of the oral
bacterium Streptococcus gordonoii DL1 by plasmid DNA in human
saliva, Appl. & Environ. Microbiol. 65(1):6-10 (Jan 1999); R.
Schubbert et al., Foreign (M13) DNA ingested by mice reaches
peripheral leukocytes, spleen, and liver via the intestinal wall
mucosa and can be covalently linked to mouse DNA, PNAS USA
94:961-66 (Feb. 4, 1997); and AA Salyers, Gene transfer in the
mammalian intestinal tract, Curr. Opin. in Biotechnol. 4(3):294-98
(Jun. 1993).
[0007] As a result of these concerns, many governmental food, drug,
health, and environmental regulatory agencies, as well as many end
users, require that antibiotic resistance gene nucleic acid be
removed from products or be absent from organisms for use in
commerce. In addition, evidence demonstrating clearance of the
selection antibiotics from the final product must be provided in
order to secure regulatory clearance. The United Kingdom, Canada,
France, the European Community, and the United States have all
addressed the use of antibiotic resistance genes in foods, animal
feeds, drugs and drug production, including recombinant drug
production. Clearance of these agents, and especially demonstrating
such clearance, is expensive, time consuming, and often only
minimally effective.
[0008] Because of the concerns inherent in the use of antibiotic
resistance genes for selection in the production of recombinant
polypeptides, alternative selection methods have been examined.
[0009] Auxotrophic Selection Markers
[0010] Auxotrophic selection markers have been utilized as an
alternative to antibiotic selection in some systems. For example,
auxotrophic markers have been widely utilized in yeast, due largely
to the inefficiency of antibiotic resistance selection markers in
these host cells. See, for example, JT Pronk, (2002) "Auxotrophic
yeast strains in fundamental and applied research," App. &
Envirn. Micro. 68(5): 2095-2100; Boeke et al., (1984) "A positive
selection for mutants lacking orotodine-5'-phosphate decarboxylase
activity in yeast; 5-fluoro-orotic acid resistance," Mol. Gen.
Genet. 197: 345-346; Botstein & Davis, (1982) "Principles and
practice of recombinant DNA research with yeast," p.607-636, in J N
Strathern, E W Jones. And JR Broach (ed.), The molecular biology of
the yeast Saccharomyces cerevisiae, Metabolism and gene expression,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Cost
& Boeke, (1996) "A useful colony color phenotype associated
with the yeast selectable/counter selectable marker MET15," Yeast
12: 939-941. However, yeast expression systems due not provide the
potential speed and efficiency for producing target proteins that
bacterial systems do.
[0011] Auxotrophic marker selection in bacteria has also previously
been described. See, for example, U.S. Pat. Nos. 4,920,048,
5,691,185, 6,291,245, 6,413,768, 6,752,994, Struhl et al. (1976)
PNAS USA 73; 1471-1475;; MacCormick, C. A., et al., (1995)
"Construction of a food-grade host/vector system for Lactococcus
lactis based on the lactose operon," FEMS Microbiol. Lett.
127:105-109; Dickely et al. (1995), "Isolation of Lactococcus
lactis nonsense suppressors and construction of a food-grade
cloning vector," Mol. Microbiol. 15:839-847; S.o slashed.rensen et
al., (2000) "A food-grade cloning system for industrial strains of
Lactococcus lactis," Appl. Environ. Microbiol 66:1253-1258; Fiedler
& Skerra, (2001) "proBA complementation of an auxotrophic
E.coli strain improves plasmid stability and expression yield
during fermenter production of a recombinant antibody fragment,"
Gene 274: 111-118.
[0012] The use of auxotrophic selection markers in the previously
described commercial scale bacterial fermentation systems has
drawbacks that limit their use. A major drawback, as noted in U.S.
Pat. No. 6,413,768, is that nutritional auxotrophic selection
marker systems generally suffer from cross feeding. The term cross
feeding refers to the ability of a first cell, auxotrophic for a
particular metabolite, to survive in the absence of the metabolite
by obtaining its supply of that metabolite from its environment,
and typically, from the medium for which the cell is auxotrophic by
utilizing excreted intermediates of the metabolite, the metabolite
itself, or a prototrophic enabling molecule produced by a second
cell, prototrophic for the metabolite absent from the medium. See
also G R Barker et al., Biochem. J. 157(1):221-27 (1976) (cross
feeding of thymine in E.coli): T J Kerr & G J Tritz, J. Bact.
115(3):982-86 (Sep. 1973) (cross feeding of NAD in E.coli
auxotrophic for NAD synthesis); G A Sprenger et al., FEMS
Microbiol. Lett. 37(3):299-304 (1986) (selection of nalidixic acid
to avoid the cross feeding problem).
[0013] Because cross feeding allows revertant bacteria to survive,
cross feeding decreases the overall capacity of the fermentation
process to produce the desired product at efficient and maximized
levels due to the presence of fewer target protein producing host
cells.
[0014] Expression Vector Control
[0015] Another hurdle to the creation of the ideal host cell is the
inefficient and low level production of target polypeptides in the
fermentation process. Controlling expression of the target protein
until optimal host cell densities and fermentation conditions are
reached allows for a more efficient and larger yield of
polypeptide. The reasons for this are several fold, including a
more efficient utilization of a particular carbon source and the
reduction of extended metabolic stresses on the host cell.
[0016] In many cases, however, repression of expression of the
target protein during cell growth can be imperfect, resulting in a
significant amount of expression prior to the particular induction
phase. This "leaky" repression results in host cell stress,
inefficient utilization of carbon source due to metabolic energy
being diverted from normal cell growth to transgene, and a delay in
reaching optimal cell density induction points, resulting in a more
lengthy and costly fermentation run, and often, a reduced yield of
the target protein.
[0017] Therefore, it is an object of the present invention to
provide an improved expression system for the production of target
proteins, wherein the production is efficient, regulatable, and
performed in a medium that minimizes of regulatory and
environmental concerns.
[0018] It is another object of the present invention to provide
organisms for use as host cells in an improved expression system
for the production of target proteins.
[0019] It is still another object of the present invention to
provide processes for the improved production of target
proteins.
[0020] It is yet another object of the present invention to provide
novel constructs and nucleic acids for use in an improved
expression system for the production of target proteins.
SUMMARY OF THE INVENTION
[0021] It has been discovered that bacterial protein production can
be improved by selecting as a host cell a Pseudomonad organism that
is capable of non-antibiotic resistant, auxotrophic selection,
and/or contains a chromosomal insert of a lacI gene or
derivative.
[0022] Specifically, it has been discovered that the Pseudomonad
organism Pseudomonas fluorescens is particularly well suited for
this purpose. To this end, it has been surprisingly discovered that
Pseudomonas fluorescens does not exhibit adverse cross feeding
inhibition under auxotrophic selection during the high-cell density
fermentation of recombinant polypeptides. Such a discovery allows
for the use of auxotrophic Pseudomonas fluorescens as host cells in
the efficient production of high levels of recombinant
polypeptides, overcoming the drawbacks inherent with the use of
antibiotic resistance selection markers and the problems of
auxotrophic cross feeding present in other bacterial expression
systems.
[0023] It has also been surprisingly discovered that the use of a
LacI-encoding gene other than as part of a whole or truncated
Plac-lacI-lacZYA operon in Pseudomonads surprisingly resulted in
substantially improved repression of pre-induction recombinant
protein expression, higher cell densities in commercial-scale
fermentation, and higher yields of the desired product in
comparison with previously taught lacI-lacZYA Pseudomonad
chromosomal insertion (U.S. Pat. No. 5,169,760). This lacI
insertion is as effective in repressing Plac-Ptac family
promoter-controlled transgenes as a multi-copy plasmid encoding a
LacI repressor protein in Pseudomonas fluorescens, thereby
eliminating the need to maintain a separate plasmid encoding a LacI
repressor protein in the cell and reducing potential production
inefficiencies caused by such maintenance.
[0024] It has also been discovery that the use of dual lac operator
sequences provides superior repression of recombinant protein
expression prior to induction without a concomitant reduction in
subsequent induction yields in Pseudomonas fluorescens
[0025] Therefore, in one aspect of the present invention,
Pseudomonad organisms are provided for use as host cells in the
improved production of proteins.
[0026] In one embodiment, the Pseudomonad organisms have been
genetically modified to induce an auxotrophy. In a particular
embodiment, the Pseudomonad organism is Pseudomonas fluorescens. In
one embodiment, the auxotrophy is a result of genetic modifications
to at least one nitrogenous base compound biosynthesis gene, or at
least one amino acid biosynthesis gene. In a further embodiment,
the genetic modification is to a gene encoding an enzyme active in
the uracil biosynthetic pathway, the thymidine biosynthetic
pathway, or the proline biosynthetic pathway. In still a further
embodiment, the genetic modification is to the pyrF gene encoding
orotidine-5'-phosphate decarboxylase, the thyA gene encoding
thymidylate synthase, or the proC gene encoding
.DELTA..sup.1-pyrroline-5-carboxylate reductase.
[0027] In another embodiment, the present invention provides
Pseudomonad organisms that have been genetically modified to
provide at least one copy of a LacI-encoding gene inserted into the
genome, other than as part of the whole or truncated
Plac-lacI-lacZYA operon. In a particular embodiment, the
Pseudomonad host cell is Pseudomonas fluorescens. In one
embodiment, the Pseudomonad contains a native E.coli lacI gene
encoding the LacI repressor protein. In another embodiment, the
Pseudomonad cell contains the lacI.sup.Q gene. In still another
embodiment, the Pseudomonad cell contains the lacI.sup.Q1 gene.
[0028] In another embodiment, a Pseudomonad organism is provided
comprising a nucleic acid construct containing a nucleic acid
comprising at least one lacO sequence involved in the repression of
transgene expression. In a particular embodiment, the Pseudomonad
host cell is Pseudomonas fluorescens. In one embodiment, the
nucleic acid construct comprises more than one lacO sequence. In
another embodiment, the nucleic acid construct comprises at least
one, and preferably more than one, lacOid sequence. In one
embodiment, the nucleic acid construct comprises a lacO sequence,
or derivative thereof, located 3' of a Plac family promoter, and a
lacO sequence, or derivative thereof, located 5' of a Plac family
promoter. In a particular embodiment, the lacO derivative is a
lacOid sequence.
[0029] In a further embodiment, the present invention provides
Pseudomonad organisms that have been genetically modified to induce
an auxotrophy and further modified to contain a chromosomal
insertion of a native E.coli lacI gene, lacI.sup.Q gene, or
lacI.sup.Q1 gene other than as part of a whole or truncated
Plac-lacI-lacZYA operon. In another embodiment, the Pseudomonad
organism is further modified to contain a nucleic acid construct
comprising at least one lacO sequence involved in the repression of
transgene expression. In a particular embodiment, the Pseudomonad
organism is a Pseudomonas fluorescens.
[0030] In another aspect of the present invention, nucleic acid
sequences are provided for use in the improved production of
proteins.
[0031] In one embodiment, nucleic acid sequences encoding
prototrophy-restoring enzymes for use in an auxotrophic Pseudomonad
host cells are provided. In a particular embodiment, nucleic acid
sequences encoding nitrogenous base compound biosynthesis enzymes
purified from the organism Pseudomonas fluorescens are provided. In
one embodiment, nucleic acid sequences encoding the pyrF gene in
Pseudomonas fluorescens is provided (SEQ. ID No.s 1 and 3). In
another embodiment, a nucleic acid sequence encoding the thyA gene
in Pseudomonas fluorescens is provided (SEQ. ID. No. 4). In still
another embodiment, nucleic acid sequences encoding an amino acid
biosynthetic compound purified from the organism Pseudomonas
fluorescens are provided. In a particular embodiment, a nucleic
acid sequence encoding the proC gene in Pseudomonas fluorescens is
provided (SEQ. ID No.s 6 and 8).
[0032] In another aspect, the present invention produces novel
amino acid sequences which are the products of the novel nucleic
acid expression.
[0033] In still another aspect of the present invention, nucleic
acid constructs are provided for use in the improved production of
peptides.
[0034] In one embodiment, a nucleic acid construct for use in
transforming a Pseudomonad host cell comprising a) a nucleic acid
sequence encoding a recombinant polypeptide, and b) a nucleic acid
sequence encoding a prototrophy-enabling enzyme is provided. In
another embodiment, the nucleic acid construct further comprises c)
a Plac-Ptac family promoter. In still another embodiment, the
nucleic acid construct further comprises d) at least one lacO
sequence, or derivative, 3' of a lac or tac family promoter. In yet
another embodiment, the nucleic acid construct further comprises e)
at least one lacO sequence, or derivative, 5' of a lac or tac
family promoter. In one embodiment, the derivative lacO sequence
can be a lacOid sequence. In a particular embodiment, the
Pseudomonad organism is Pseudomonas fluorescens.
[0035] In one embodiment of the present invention, nucleic acid
constructs are provided for use as expression vectors in
Pseudomonad organisms comprising a) a nucleic acid sequence
encoding a recombinant polypeptide, b) a Plac-Ptac family promoter,
c) at least one lacO sequence, or derivative, 3' of a lac or tac
family promoter, d) at least one lacO sequence, or derivative, 5'
of a lac or tac family promoter. In one embodiment, the derivative
lacO sequence can be a lacOid sequence. In one embodiment, the
nucleic acid construct further comprises e) a prototrophy-enabling
selection marker for use in an auxotrophic Pseudomonad cell. In a
particular embodiment, the Pseudomonad organism is Pseudomonas
fluorescens.
[0036] In another aspect of the present invention, modified cells
are provided for use in the improved production of proteins.
[0037] In one embodiment, an auxotrophic Pseudomonad cell is
provided that has a nucleic acid construct comprising i) a
recombinant polypeptide, and ii) a prototrophy-enabling nucleic
acid. In another embodiment, the nucleic acid construct further
comprises iii) a Plac-Ptac family promoter. In still another
embodiment, the nucleic acid construct further comprises iv) more
than one lacO sequence. In one embodiment, the Pseudomonad is an
auxotrophic Pseudomonas fluorescens cell. In a further embodiment,
the invention further comprises auxotrophic Pseudomonad organisms,
including Pseudomonas fluorescens, that have been further
genetically modified to contain a chromosomal insertion of a native
E.coli lacI gene, lacI.sup.Q gene, or lacI.sup.Q1 gene other than
as part of a whole or truncated Plac-lacI-lacZYA operon.
[0038] In another embodiment, a Pseudomonad cell is provided that
comprises a lacI transgene, or derivative thereof, other than as
part of a whole or truncated Plac-lacI-lacZYA operon, inserted into
the chromosome, and b) a nucleic acid construct comprising i) a
recombinant polypeptide, and ii) a Plac-Ptac family promoter. In
still another embodiment, the nucleic acid construct further
comprises iii) at least one lacO sequence, and preferably, more
than one lacO sequence. In one embodiment, the lacO sequence is a
lacOid sequence. In one embodiment, the Pseudomonad has been
further modified to induce auxotrophy. In one embodiment, the
Pseudomonad cell is a Pseudomonas fluorescens.
[0039] In one aspect of the present invention, processes of
expressing recombinant polypeptides for use in improved protein
production are provided.
[0040] In one embodiment, the process provides expression of a
nucleic acid construct comprising nucleic acids encoding a) a
recombinant polypeptide, and b) a prototrophy-restoring enzyme in a
Pseudomonad that is auxotrophic for at least one metabolite. In an
alternative embodiment, the Pseudomonad is auxotrophic for more
than one metabolite. In one embodiment, the Pseudomonad is a
Pseudomonas fluorescens cell. In a particular embodiment, a
recombinant polypeptide is expressed in a Pseudomonad that is
auxotrophic for a metabolite, or combination of metabolites,
selected from the group consisting of a nitrogenous base compound
and an amino acid. In a more particular embodiment, recombinant
polypeptides are expressed in a Pseudomonad that is auxotrophic for
a metabolite selected from the group consisting of uracil, proline,
and thymidine. In another embodiment, the auxotrophy can be
generated by the knock-out of the host pyrF, proC, or thyA gene,
respectively. An alternative embodiment, recombinant polypeptides
are expressed in an auxotrophic Pseudomonad cell that has been
genetically modified through the insertion of a native E.coli lacI
gene, lacI.sup.Q gene, or lacI.sup.Q1 gene, other than as part of
the PlacI-lacI-lacZYA operon, into the host cell's chromosome. In
one particular embodiment, the vector containing the recombinant
polypeptide expressed in the auxotroph comprises at least one
lacOid operator sequences. In one particular embodiment, the vector
containing the recombinant polypeptide expressed in the auxotrophic
host cell comprises at least two lac operator sequences, or
derivatives thereof. In still a further embodiment, the recombinant
polypeptide is driven by a Plac family promoter.
[0041] In another embodiment, the process involves the use of
Pseudomonad host cells that have been genetically modified to
provide at least one copy of a LacI encoding gene inserted into the
Pseudomonad host cell's genome, wherein the lacI encoding gene is
other than as part of the PlacI-lacI-lacZYA operon. In one
embodiment, the gene encoding the Lac repressor protein is
identical to that of native E.coli lacI gene. In another
embodiment, the gene encoding the Lac repressor protein is the
lacI.sup.Q gene. In still another embodiment, the gene encoding the
Lac repressor protein is the lacI.sup.Q1 gene. In a particular
embodiment, the Pseudomonad host cell is Pseudomonas fluorescens.
In another embodiment, the Pseudomonad is further genetically
modified to produce an auxotrophic cell. In another embodiment, the
process produces recombinant polypeptide levels of at least about 3
g/L, 4 g/L, 5 g/L 6 g/L, 7 g/L, 8 g/L, 9 g/L or at least about 10
g/L. In another embodiment, the recombinant polypeptide is
expressed in levels of between 3 g/L and 100 g/L.
BRIEF DESCRIPTION OF THE FIGURES
[0042] FIG. 1 represents a comparison of the performance of P.
fluorescens dual-plasmid expression systems using a pyrF marker
(.DELTA. and .quadrature.) against the performance of P.
fluorescens dual-plasmid expression systems using only antibiotic
resistance markers (.diamond-solid.). All data shown are averages
of 9-multiple, representative 20-L fermentations, with IPTG being
added to induce target enzyme expression during mid-exponential
phase. The upper set of three curves presents relative cell density
data, which is read with reference to the left vertical axis. The
lower set of three curves presents relative enzyme activity data
for the target enzyme produced in the corresponding fermentations,
and is read with reference to the right vertical axis.
.diamond-solid.--P. fluorescens containing pMYC plasmid having a
tac promoter-controlled target enzyme expression cassette and a
tetracycline resistance marker gene and containing a pCN plasmid
having a lacI repressor expression cassette and a kanamycin
resistance marker gene. Variance bars shown are for these data
points (n=4), and represent the normal variance typically observed
for this expression system among different fermentation runs.
.DELTA.--P. fluorescens strain with inactivated genomic pyrF
containing pMYC plasmid having a tac promoter-controlled target
enzyme expression cassette and a pyrF auxotrophic marker gene and
containing pCN plasmid having a lacI repressor expression cassette
and a kanamycin resistance marker gene. .quadrature.--P.
fluorescens strain with inactivated genomic pyrF and proC
containing pMYC plasmid having a tac promoter-controlled target
enzyme expression cassette and a pyrF auxotrophic marker gene and
containing pCN plasmid having a lacI repressor expression cassette
and a proC auxotrophic marker gene.
[0043] FIG. 2 represents a map of the plasmid pDOW1249-2.
[0044] FIG. 3 represents a map of the plasmid pDOW1269-2.
[0045] FIG. 4 represents a schematic of lac operator constructs.
LacZ represents the positions of the native E.coli lacO sequences.
tac DC239, DC240 represents the position of the native E.coli lac
operator on a construct comprising a tac promoter and a nitrilase
encoding nucleic acid. Opt lacO DC281 represents the position of
the lacOid operator sequence on a construct comprising a tac
promoter and a nitrilase encoding nucleic acid. Dual lacO DC262
represents the position of a lacOid operator sequence 5', and wild
type lac operator sequence 3' of a tac promoter on a construct
further comprising a nitrilase encoding nucleic acid.
[0046] FIG. 5 represents a Western Blot analysis (UnBlot) of LacI
protein accumulation in the lacI integrant strains grown in a shake
flask gene expression medium. Broth samples were normalized to
OD.sub.600, combined with LDS NuPAGE sample buffer (Invitrogen),
50mM DTT and heated at 95.degree. C. for 40 min, then centrifuged
briefly. Aliquots of 20 uL were loaded on a 10%, 1 mm NuPAGE
Bis-Tris gel run in MOPS with antioxidant in the inner chamber.
Detection of the LacI protein was accomplished with an in-gel
hybridization method ("UnBlot", Pierce), using a polyclonal rabbit
antibody to LacI (Stratagene cat. no. 217449-51) at 1:1000 and the
secondary antibody, Stabilized Goat Anti-rabbit Horseradish
Peroxidase Conjugated Antibody (Pierce) at 1:500. The horseradish
peroxidase was visualized with UnBlot Stable Peroxide and UnBlot
Luminol Enhancer as according to the UnBlot kit.
[0047] FIG. 6 represents the composite of nitrilase accumulation
profiles of DC 140, DC239 and DC240. Data were compiled from DC140
(n=5), DC239 (n=5) and DC240 (n=4) runs. Dc140 is represented by
.box-solid.. DC239 is represented by .quadrature.. DC240 is
represented by .quadrature.. Fermentation runs were performed over
a 48 hour period.
DETAILED DESCRIPTION OF THE INVENTION
[0048] In one embodiment, the Pseudomonad organisms have been
genetically modified to induce an auxotrophy. In a particular
embodiment, the Pseudomonad organism is Pseudomonas fluorescens. In
one embodiment, the auxotrophy is a result of genetic modifications
to at least one nitrogenous base compound biosynthesis gene, or at
least one amino acid biosynthesis gene. In a further embodiment,
the genetic modification is to a gene encoding an enzyme active in
the uracil biosynthetic pathway, the thymidine biosynthetic
pathway, or the proline biosynthetic pathway. In still a further
embodiment, the genetic modification is to the pyrF gene encoding
orotidine-5'-phosphate decarboxylase, the thyA gene encoding
thymidilate synthase, or the proC gene encoding
.DELTA..sup.1-pyrroline-5-carboxylate reductase.
[0049] In another embodiment, the present invention provides
Pseudomonad organisms that have been genetically modified to
provide at least one copy of a LacI-encoding gene inserted into the
genome, other than as part of the PlacI-lacI-lacZYA operon. In a
particular embodiment, the Pseudomonad host cell is Pseudomonas
fluorescens. In one embodiment, the Pseudomonad contains a native
E.coli lacI gene encoding the LacI repressor protein.. In another
embodiment, the Pseudomonad cell contains the lacI.sup.Q gene. In
still another embodiment, the Pseudomonad cell contains the
lacI.sup.Q1 gene.
[0050] In another embodiment, a Pseudomonad organism is provided
comprising a nucleic acid construct containing a nucleic acid
comprising at least one lacO sequence involved in the repression of
transgene expression. In a particular embodiment, the Pseudomonad
host cell is Pseudomonad fluorescens. In one embodiment, the
nucleic acid construct comprises more than one lacO sequence. In
another embodiment, the nucleic acid construct comprises at least
one, and preferably more than one, lacOid sequence. In one
embodiment, the nucleic acid construct comprises a lacO sequence,
or derivative thereof, located 3' of a Plac family promoter, and a
lacO sequence, or derivative thereof, located 5' of a Plac family
promoter. In a particular embodiment, the lacO derivative is a
lacOid sequence.
[0051] In a further embodiment, the present invention provides
Pseudomonad organisms that have been genetically modified to induce
an auxotrophy and further modified to contain a chromosomal
insertion of a native E.coli lacI gene, lacI Q gene, or lacIQ1 gene
other than as part of a whole or truncated Plac-lacI-lacZYA operon.
In another embodiment, the Pseudomonad organism is further modified
to contain a nucleic acid construct comprising at least one lacO
sequence involved in the repression of transgene expression. In a
particular embodiment, the Pseudomonad organism is a Pseudomonas
fluorescens.
[0052] The host cell provided by the present invention for use in
an expression system producing recombinant polypeptides can be
selected from the "Pseudomonads and closely related bacteria" or
from a Subgroup thereof, as defined below. In one embodiment, the
host cell is selected from the genus Pseudomonas. In a particular
embodiment, the particular species of Pseudomonas is P.
fluorescens. In a particular embodiment, the host cell is
Pseudomonas fluorescens biotype A or biovar I.
[0053] Definitions
[0054] The term "isolated" refers to nucleic acid, protein, or
peptide that is substantially or essentially free from other
material components, for example, which can be cellular
components.
[0055] The term "fragment" means a portion or partial sequence of a
nucleotide, protein, or peptide sequence.
[0056] As used herein, the term "percent total cell protein" means
the amount of protein or peptide in the host cell as a percentage
of aggregate cellular protein.
[0057] The term "operably attached," as used herein, refers to any
configuration in which the transcriptional and any translational
regulatory elements are covalently attached to the encoding
sequence in such disposition(s), relative to the coding sequence,
that in and by action of the host cell, the regulatory elements can
direct the expression of the coding sequence.
[0058] The term "auxotrophic," as used herein, refers to a cell
which has been modified to eliminate or reduce its ability to
produce a specific substance required for growth and
metabolism.
[0059] As used herein, the term "percent total cell protein" means
a measure of the fraction of total cell protein that represents the
relative amount of a given protein expressed by the cell.
[0060] The term "prototrophy," as used herein, refers to a cell
that is capable of producing a specific substance required for
growth and metabolism.
[0061] As used herein, the term "homologous" or means either i) a
protein or peptide that has an amino acid sequence that is
substantially similar (i.e., at least 70, 75, 80, 85, 90, 95, or
98%) to the sequence of a given original protein or peptide and
that retains a desired function of the original protein or peptide
or ii) a nucleic acid that has a sequence that is substantially
similar (i.e., at least 70, 75, 80, 5, 90, 95, or 98%) to the
sequence of a given nucleic acid and that retains a desired
function of the original nucleic acid sequence. In all of the
embodiments of this invention and disclosure, any disclosed
protein, peptide or nucleic acid can be substituted with a
homologous or substantially homologous protein, peptide or nucleic
acid that retains a desired function. In all of the embodiments of
this invention and disclosure, when any nucleic acid is disclosed,
it should be assumed that the invention also includes all nucleic
acids that hybridize to the disclosed nucleic acid.
[0062] In one non-limiting embodiment, the non-identical amino acid
sequence of the homologous polypeptide can be amino acids that are
members of any one of the 15 conservative or semi-conservative
groups shown in Table 1.
1TABLE 1 SIMILAR AMINO ACID SUBSTITUTION GROUPS Semi-Conservative
Conservative Groups (8) Groups (7) Arg, Lys Arg, Lys, His Asp, Glu
Asn, Asp, Glu, Gln Asn, Gln Ile, Leu, Val Ile, Leu, Val, Met, Phe
Ala, Gly Ala, Gly, Pro, Ser, Thr Ser, Thr Ser, Thr, Tyr Phe, Tyr
Phe, Trp, Tyr Cys (non-cystine), Ser Cys (non-Cystine), Ser,
Thr
[0063] Amino acid sequences provided herein are represented by the
following abbreviations:
2 A Ala alanine P Pro proline B aspartate or asparagine Q Gln
glutamine C Cys cysteine R Arg arginine D Asp aspartate S Ser
serine E Glu glutamate T Thr threonine F Phe phenylalanine G Gly
glycine V Val valine H His histidine W Trp tryptophan I Ile
isoleucine Y Tyr tyrosine Z glutamate or glutamine K Lys lysine L
Leu leucine M Met methionine N Asn asparagine
[0064] I. Selection of Pseudomonads and Related Bacteria as Host
Cells
[0065] The present invention provides the use of Pseudomonads and
related bacteria as host cells in the improved production of
proteins.
[0066] Auxotrophic Selection Efficiency
[0067] It has been discovered that Pseudomonads have the ability to
utilize auxotrophic selection markers for the maintenance of
protein expressing plasmids without the drawbacks typically
associated with other systems, such as plasmid instability and
cross-feeding.
[0068] Auxotrophic markers, in other host cell systems, are not
always sufficient to maintain plasmids in every cell, especially
during fermentations where loss of the plasmid may give
plasmid-less cells a selective advantage, resulting in the
accumulation of a large fraction of nonproductive cells, reducing
product formation. Such revertant strains are especially
troublesome if they have the ability to cross-feed the auxotrophic
metabolite from prototrophic enabled bacteria. For example, use of
the trp operon on a plasmid in an E.coli tryptophan auxotroph was
not sufficient to prevent a large proportion of plasmid-less cells
from accumulating, until combined with the valS gene (encoding
valyl t-RNA synthetase) in a valS.sup.ts host ( Skogman, S. G.;
Nilsson, J., Temperature-dependent retention of a
tryptophan-operon-bearing plasmid in Escherichia coli. Gene 1984,
31, (1-3), 117-22.) Presumably, the cells containing the trp operon
on a plasmid secreted enough tryptophan or related molecules to
allow growth of plasmid-less cells. Likewise, using the LEU2 gene
on a xylitol-reductase production plasmid in leu2 mutant yeast
resulted in plasmid loss; up to 80% of a fed-batch culture was made
up of cells without a production plasmid, because leucine was
secreted by plasmid-containing cells into the broth (Meinander, N.
Q.; Hahn-Haegerdal, B., Fed-batch xylitol production with two
recombinant Saccharomyces cerevisiae strains expressing XYL1 at
different levels, using glucose as a cosubstrate: a comparison of
production parameters and strain stability. Biotechnology and
Bioengineering 1997, 54, (4), 391-399).
[0069] It has been discovered that Pseudomonas fluorescens (Pf)
does not exhibit the inherent problems associated with
cross-feeding observed in other host cell systems, for example,
E.coli and yeast. While not wanting to be bound by any particular
theory, it is thought that auxotrophic Pseudomonas fluorescens is a
particularly suitable organism for use as a host cell because of
the observed inability of a Pf auxotrophic cell to out compete a
auxotrophic cell containing a prototrophic-enabling plasmid on a
supplemented medium that contains the auxotrophic metabolite,
indicating an innate difficulty of an Pf auxotroph to import the
required metabolite. Because of this, Pf auxotrophic cells that
lose the selection marker plasmid do not gain a selective advantage
over Pf auxotrophic cells containing the selection marker, even in
the presence of a supplemental metabolite, greatly reducing any
potential effects of cross-feeding. Because of the reduced effects
of cross-feeding, production yields of the recombinant polypeptide
in a fermentation run are not reduced due to the presence of
non-recombinant polypeptide producing cells.
[0070] LacI Insert
[0071] It has been discovered that Pseudomonads are able to use a
single-copy lacI transgene, other than as part of a whole or
truncated Plac-lacI-lacZYA operon, chromosomal insert to
effectively repress protein expression until induction.
[0072] Transcription initiation from regulated promoters by RNA
polymerase is activated or deactivated by the binding or releasing
of a regulatory protein. Thus, regulated promoters include those
that participate in negative control (i.e. repressible promoters),
wherein the gene encoding the target polypeptide of interest is
expressed only when the promoter is free of the regulator protein
(i.e. a "repressor" protein), and those that participate in
positive control, wherein the gene is expressed only when the
promoter is bound by the regulator protein (i.e. an "activator"
protein).
[0073] One of the most common classes of repressible promoters used
in bacterial expression systems is the family of Plac-based
promoters. The family of Plac-based promoters originates with the
native E.coli lactose operon, referred to as the "lac" operon, also
symbolized as "lacZYA," the expression of which is regulated by the
expression product of the lacI gene. The native E.coli structure of
the operon is "PlacI-lacI-PlacZ-lacZYA," wherein the native E.coli
Plac promoter is represented by "PlacZ" (also called "PlacZYA").
"PlacI" represents the native promoter for the lacI gene, and
"lacI" represents the gene encoding the lac repressor, i.e. the
LacI protein. "lacZYA" represents the operon encoding the lactose
utilization pathway.
[0074] The LacI-regulated promoters include, among others, the
native E.coli lactose operon promoter ("Plac"). In addition,
improved mutants have also been discovered, as have intra promoter
hybrids of Plac, such as the "Ptac" promoter, "Ptrc" promoter, and
the "PtacII" promoters. The Ptac promoter in E.coli, for example,
is 3-fold stronger than the Plac promoter when fully derepressed.
Therefore, it is frequently used for promoting high level regulated
gene expression in E.coli. However, while the Plac promoter is
1,000-fold repressed by LacI, while the Ptac promoter is only
50-fold repressed under similar conditions (Lanzer, M. & H.
Bujard. 1988. Proc. Natl. Acad. Sci. USA. 85:8973). Repression of
the E. coli Ptac promoter or other lac related promoters, depends
upon the concentration of the repressor, LacI. (De Boer, et al.,
1983. Proc. Natl. Acad. Sci. USA. 78:21-25). As set forth above,
release from repression can occur through the addition of an
inducer which reduces the affinity of the repressor for its
specific DNA binding site, in this case, the lac operator (lacO).
Alternatively, a reduction in the concentration of the repressor
relative to the molar concentration of specific DNA binding sites
on the plasmid can also derepress the promoter. If the lacI gene is
located on a high copy number cloning plasmid, then a large amount
of inducer is required to initiate expression because of the large
amount of repressor produced in such a system.
[0075] In commercial production systems, the lac repressor is
typically encoded by a gene whose expression is constitutive, i.e.
non-regulated, thus providing an intracellular environment in which
the desired transgene, encoding the desired target protein, is
repressed until a desired host cell biomass or cell density is
achieved. At that time, a quantity of a small molecule known as an
inducer whose presence is effective to dissociate the repressor
from the transgene, is added to the cell culture and taken up by
the host cell, thereby permitting transcription of the transgene.
In the case of lac repressor proteins, the inducer can be lactose
or a non-metabolized, gratuitous inducer such as
isopropyl-beta-D-thio-galactoside ("IPTG"). The selected point in
time at which the inducer is to be added is referred to as the
"induction phase."
[0076] A variety of lac repressor genes have been identified as
useful for the repression of Plac family promoters present on
recombinant polypeptide expression vectors. These include the
native E.coli lacI gene and/or by variants thereof, including the
lacI.sup.Q and lacI.sup.Q1 genes that encode the same LacI protein,
but at a higher expression level. For example, the lacI.sup.Q
mutation is a single CG to TA change at -35 of the promoter region
of lacI (Calos, M. 1978. Nature 274:762) which causes a 10-fold
increase in LacI expression in E.coli (Mueller-Hill, B., et al.
1968. Proc. Natl. Acad. Sci. USA. 59:1259). Wild-type E.coli cells
have a concentration of LacI of 10.sup.-8 M or about 10 molecules
per cell, with 99% of the protein present as a tetramer (Fickert,
R. & B. Mueller-Hill 1992. J. Mol. Biol. 226:59). Cells
containing the lacI.sup.Q mutation contain about 100 molecules per
cell or 10.sup.-7 M LacI. As a result, a number of bacterial
expression systems have been developed in which Plac family
promoter controlled transgenes, resident in plasmids, are
maintained in host cells expressing LacI proteins at different
levels, thereby repressing the desired transgene until a chosen
"induction phase" of cell growth.
[0077] In many cases, however, repression of expression of the
target protein during cell growth can be imperfect, resulting in a
significant amount of expression prior to the particular induction
phase. This "leaky" repression results in host cell stress,
inefficient utilization of carbon source due to metabolic energy
being diverted from normal cell growth to transgene, and a delay in
reaching optimal cell density induction points, resulting in a more
lengthy and costly fermentation run, and often, a reduced yield of
the target protein.
[0078] One common strategy for improving repression of Plac-family
promoter-driven transgenes has been to place a lacI or a lacI.sup.Q
gene on the plasmid bearing the Plac-family promoter-driven target
gene (e.g. see MJR Stark in Gene 51:255-67 (1987) and E Amann et
al. in Gene:301-15 (1988)). However, this often results in
overproduction of the Lac repressor protein, which then requires
use of an even higher inducer concentration to restore induction
levels of the transgene to overcome the decrease in recombinant
protein production. Moreover, the use of a second plasmid
containing the lacI gene, separate from the plasmid containing the
Plac-family promoter-driven target gene, requires the use of two
different selection marker genes in order to maintain both plasmids
in the expression host cell: one selection marker gene for each of
the two different plasmids. The presence of the second selection
marker gene, i.e. the selection marker gene for the second plasmid,
in turn requires the use of either: 1) a separate antibiotic in the
case of an antibiotic-resistance selection marker gene, which is
costly and disadvantageous from a health/safety regulatory
perspective; or 2) a separate metabolic deficiency in the host cell
genome, in the case of an auxotrophic selection marker gene, which
requires the additional work of mutating the host cell.
[0079] It has surprisingly been discovered that a lacI insertion,
other than as part of a whole or truncated Plac-lacI-lacZYA operon,
is as effective in repressing Plac-Ptac family promoter-controlled
transgenes as a multi-copy plasmid encoding a LacI repressor
protein in Pseudomonas fluorescens. This surprising discovery
eliminates the need to maintain a separate plasmid encoding a LacI
repressor protein in the cell, or eliminates the need to define an
additional auxotrophic selection marker, and further reduces the
potential production inefficiencies caused by such maintenance of a
lacI containing plasmid.
[0080] In a previous attempt to regulate transgene expression in
Pseudomonas, an E.coli PlacI-lacI-lacZYA operon that has been
deleted of the lacZ promoter region, but that retains the
constitutive PlacI promoter, was chromosomally inserted (See U.S.
Pat. No. 5,169,760). The deletion allows for constitutive
expression of the gene products of the lac operon. However, the
inserted operon contains the E.coli lacy gene, which encodes for
the lactose transporter protein lactose permease. Lactose permease
is capable of transporting lactose, or similar derivatives, into
the host cell from the medium. The presence of lactose permease may
lead to increased importation of lactose-like contaminants from the
medium, ultimately resulting in derepression of the Plac family
promoter prior to induction. Furthermore, expression of the lac
operon lacZ, lacY, and lacA gene products may result in the
inefficient dedication of carbon utilization resources to these
products, resulting in increased metabolic stress on the cells, and
delaying the establishment of a high cell density for induction. In
addition, the larger lacI-lacZYA fusion operon may produce
increased message instability compared to a lacI insert alone in a
host cell.
[0081] It has been surprisingly discovered that the use of a
LacI-encoding gene other than as part of a whole or truncated
PlacI-lacI-lacZYA operon in Pseudomonads surprisingly resulted in
substantially improved repression of pre-induction recombinant
protein expression, higher cell densities in commercial-scale
fermentation, and higher yields of the desired product in
comparison with previously taught lacI-lacZYA Pseudomonad
chromosomal insertion (U.S. Pat. No. 5,169,760).
[0082] Additional attempts to utilize derivative lacI genes, such
as lacI.sup.Q and lacI.sup.Q1, which are expressed at greater
levels than lacI due to promoter modifications, have also been
described. C G Glascock & M J Weickert describe E.coli strains
in which a separate LacI protein-encoding gene was present in the
chromosome of the host cell in an attempt to assess the level of
control of a plasmid-borne Ptac-driven target gene. See C G
Glascock & M J Weickert, "Using chromosomal lacI.sup.Q1 to
control expression of genes on high-copy number plasmids in
Escherichia coli," Gene 223(1-2):221-31(1998); See also WO
97/04110. Among the LacI protein-encoding genes tested were lacI,
lacI.sup.Q, and lacI.sup.Q1. The results obtained for the lacI gene
and the lacI.sup.Q gene demonstrated inferior levels of repression
of the Ptac-driven target gene when present on a high-copy number
plasmid, resulting in substantial levels of pre-induction target
gene expression. Only the high expressing lacI.sup.Q1 gene provided
sufficient repression in that system.
[0083] Such a strategy, however, has the potential to increase
costs by increasing the amount of inducer required to sufficiently
derepress the promoter at induction, and decreasing yields due to
the inability of the inducer to sufficiently bind all of the
constitutively expressed repressor protein.
[0084] Comparatively, it has surprisingly been discovered that a
single-copy lacI chromosomal insert was sufficient to repress
Plac-Ptac family promoter driven transgene expression. Such a
discovery allows potential cost saving measures on the amount of
inducer used, and provides additional flexibility in the
development of Pseudomonas fluorescens as a host cell in the
improved production of proteins.
[0085] Pseudomonas Organisms
[0086] Pseudomonads and closely related bacteria, as used herein,
is co-extensive with the group defined herein as "Gram(-)
Proteobacteria Subgroup 1.""Gram(-) Proteobacteria Subgroup 1" is
more specifically defined as the group of Proteobacteria belonging
to the families and/or genera described as falling within that
taxonomic "Part" named "Gram-Negative Aerobic Rods and Cocci" by R.
E. Buchanan and N. E. Gibbons (eds.), Bergey's Manual of
Determinative Bacteriology, pp. 217-289 (8th ed., 1974) (The
Williams & Wilkins Co., Baltimore, Md., USA) (hereinafter
"Bergey (1974)"). Table 4 presents the families and genera of
organisms listed in this taxonomic "Part."
3TABLE 1 FAMILIES AND GENERA LISTED IN THE PART, "GRAM- NEGATIVE
AEROBIC RODS AND COCCI" (IN BERGEY (1974)) Family I.
Pseudomonadaceae Gluconobacter Pseudomonas Xanthomonas Zoogloea
Family II. Azotobacteraceae Azomonas Azotobacter Beijerinckia
Derxia Family III. Rhizobiaceae Agrobacterium Rhizobium Family IV.
Methylomonadaceae Methylococcus Methylomonas Family V.
Halobacteriaceae Halobacterium Halococcus Other Genera Acetobacter
Alcaligenes Bordetella Brucella Francisella Thermus
[0087] "Gram(-) Proteobacteria Subgroup 1"contains all
Proteobacteria classified there under, as well as all
Proteobacteria that would be classified according to the criteria
used in forming that taxonomic "Part." As a result, "Gram(-)
Proteobacteria Subgroup 1" excludes, e.g.: all Gram-positive
bacteria; those Gram-negative bacteria, such as the
Enterobacteriaceae, which fall under others of the 19 "Parts" of
this Bergey (1974) taxonomy; the entire "Family V.
Halobacteriaceae" of this Bergey (1974) "Part," which family has
since been recognized as being a non-bacterial family of Archaea;
and the genus, Thermus, listed within this Bergey (1974) "Part,"
which genus which has since been recognized as being a
non-Proteobacterial genus of bacteria. "Gram(-) Proteobacteria
Subgroup 1" further includes those Proteobacteria belonging to (and
previously called species of) the genera and families defined in
this Bergey (1974) "Part," and which have since been given other
Proteobacterial taxonomic names. In some cases, these re-namings
resulted in the creation of entirely new Proteobacterial genera.
For example, the genera Acidovorax, Brevundimonas, Burkholderia,
Hydrogenophaga, Oceanimonas, Ralstonia, and Stenotrophomonas, were
created by regrouping organisms belonging to (and previously called
species of) the genus Pseudomonas as defined in Bergey (1974).
Likewise, e.g., the genus Sphingomonas (and the genus Blastomonas,
derived therefrom) was created by regrouping organisms belonging to
(and previously called species of) the genus Xanthomonas as defined
in Bergey (1974). Similarly, e.g., the genus Acidomonas was created
by regrouping organisms belonging to (and previously called species
of) the genus Acetobacter as defined in Bergey (1974). Such
subsequently reassigned species are also included within "Gram(-)
Proteobacteria Subgroup 1" as defined herein.
[0088] In other cases, Proteobacterial species falling within the
genera and families defined in this Bergey (1974) "Part" were
simply reclassified under other, existing genera of Proteobacteria.
For example, in the case of the genus Pseudomonas, Pseudomonas
enalia (ATCC 14393), Pseudomonas nigrifaciens (ATCC 19375), and
Pseudomonas putrefaciens (ATCC 8071) have since been reclassified
respectively as Alteromonas haloplanktis, Alteromonas nigrifaciens,
and Alteromonas putrefaciens. Similarly, e.g., Pseudomonas
acidovorans (ATCC 15668) and Pseudomonas testosteroni (ATCC 11996)
have since been reclassified as Comamonas acidovorans and Comamonas
testosteroni, respectively; and Pseudomonas nigrifaciens (ATCC
19375) and Pseudomonas piscicida (ATCC 15057) have since been
reclassified respectively as Pseudoalteromonas nigrifaciens and
Pseudoalteromonas piscicida. Such subsequently reassigned
Proteobacterial species are also included within "Gram(-)
Proteobacteria Subgroup 1" as defined herein. "Gram(-)
Proteobacteria Subgroup 1" also includes Proteobacterial species
that have since been discovered, or that have since been
reclassified as belonging, within the Proteobacterial families
and/or genera of this Bergey (1974) "Part." In regard to
Proteobacterial families, "Gram(-) Proteobacteria Subgroup 1" also
includes Proteobacteria classified as belonging to any of the
families: Pseudomonadaceae, Azotobacteraceae (now often called by
the synonym, the "Azotobacter group" of Pseudomonadaceae),
Rhizobiaceae, and Methylomonadaceae (now often called by the
synonym, "Methylococcaceae"). Consequently, in addition to those
genera otherwise described herein, further Proteobacterial genera
falling within "Gram(-) Proteobacteria Subgroup 1" include: 1)
Azotobacter group bacteria of the genus Azorhizophilus; 2)
Pseudomonadaceae family bacteria of the genera Cellvibrio,
Oligella, and Teredinibacter; 3) Rhizobiaceae family bacteria of
the genera Chelatobacter, Ensifer, Liberibacter (also called
"Candidatus Liberibacter"), and Sinorhizobium; and 4)
Methylococcaceae family bacteria of the genera Methylobacter,
Methylocaldum, Methylomicrobium, Methylosarcina, and
Methylosphaera.
[0089] In one embodiment, the host cell is selected from "Gram(-)
Proteobacteria Subgroup 1,"as defined above.
[0090] In another embodiment, the host cell is selected from
"Gram(-) Proteobacteria Subgroup 2." "Gram(-) Proteobacteria
Subgroup 2" is defined as the group of Proteobacteria of the
following genera (with the total numbers of catalog-listed,
publicly-available, deposited strains thereof indicated in
parenthesis, all deposited at ATCC, except as otherwise indicated):
Acidomonas (2); Acetobacter (93); Gluconobacter (37); Brevundimonas
(23); Beijerinckia (13); Derxia (2); Brucella (4); Agrobacterium
(79); Chelatobacter (2); Ensifer (3); Rhizobium (144);
Sinorhizobium (24); Blastomonas (1); Sphingomonas (27); Alcaligenes
(88); Bordetella (43); Burkholderia (73); Ralstonia (33);
Acidovorax (20); Hydrogenophaga (9); Zoogloea (9); Methylobacter
(2); Methylocaldum (1 at NCIMB); Methylococcus (2);
Methylomicrobium (2); Methylomonas (9); Methylosarcina (1);
Methylosphaera; Azomonas (9); Azorhizophilus (5); Azotobacter (64);
Cellvibrio (3); Oligella (5); Pseudomonas (1139); Francisella (4);
Xanthomonas (229); Stenotrophomonas (50); and Oceanimonas (4).
[0091] Exemplary host cell species of "Gram(-) Proteobacteria
Subgroup 2" include, but are not limited to the following bacteria
(with the ATCC or other deposit numbers of exemplary strain(s)
thereof shown in parenthesis): Acidomonas methanolica (ATCC 43581);
Acetobacter aceti (ATCC 15973); Gluconobacter oxydans (ATCC 19357);
Brevundimonas diminuta (ATCC 11568); Bejerinckia indica (ATCC 9039
and ATCC 19361); Derxia gummosa (ATCC 15994); Brucella melitensis
(ATCC 23456), Brucella abortus (ATCC 23448); Agrobacterium
tumefaciens (ATCC 23308), Agrobacterium radiobacter (ATCC 19358),
Agrobacterium rhizogenes (ATCC 11325); Chelatobacter heintzii (ATCC
29600); Ensifer adhaerens (ATCC 33212); Rhizobium leguminosarum
(ATCC 10004); Sinorhizobium fredii (ATCC 35423); Blastomonas
natatoria (ATCC 35951); Sphingomonas paucimobilis (ATCC 29837);
Alcaligenes faecalis (ATCC 8750); Bordetella pertussis (ATCC 9797);
Burkholderia cepacia (ATCC 25416); Ralstonia pickettii (ATCC
27511); Acidovoraxfacilis (ATCC 11228); Hydrogenophaga flava (ATCC
33667); Zoogloea ramigera (ATCC 19544); Methylobacter luteus (ATCC
49878); Methylocaldum gracile (NCIMB 11912); Methylococcus
capsulatus (ATCC 19069); Methylomicrobium agile (ATCC 35068);
Methylomonas methanica (ATCC 35067); Methylosarcina fibrata (ATCC
700909); Methylosphaera hansonii (ACAM 549); Azomonas agilis (ATCC
7494); Azorhizophilus paspali (ATCC 23833); Azotobacter chroococcum
(ATCC 9043); Cellvibrio mixtus (UQM 2601); Oligella urethralis
(ATCC 17960); Pseudomonas aeruginosa (ATCC 10145), Pseudomonas
fluorescens (ATCC 35858); Francisella tularensis (ATCC 6223);
Stenotrophomonas maltophilia (ATCC 13637); Xanthomonas campestris
(ATCC 33913); and Oceanimonas doudoroffii (ATCC 27123).
[0092] In another embodiment, the host cell is selected from
"Gram(-) Proteobacteria Subgroup 3." "Gram(-) Proteobacteria
Subgroup 3" is defined as the group of Proteobacteria of the
following genera: Brevundimonas; Agrobacterium; Rhizobium;
Sinorhizobium; Blastomonas; Sphingomonas ; Alcaligenes;
Burkholderia; Ralstonia; Acidovorax; Hydrogenophaga; Methylobacter;
Methylocaldum; Methylococcus; Methylomicrobium; Methylomonas;
Methylosarcina; Methylosphaera; Azomonas; Azorhizophilus;
Azotobacter; Cellvibrio; Oligella; Pseudomonas; Teredinibacter;
Francisella; Stenotrophomonas; Xanthomonas; and Oceanimonas.
[0093] In another embodiment, the host cell is selected from
"Gram(-) Proteobacteria Subgroup 4." "Gram(-) Proteobacteria
Subgroup 4" is defined as the group of Proteobacteria of the
following genera: Brevundimonas; Blastomonas; Sphingomonas;
Burkholderia; Ralstonia; Acidovorax; Hydrogenophaga; Methylobacter;
Methylocaldum; Methylococcus; Methylomicrobium; Methylomonas;
Methylosarcina; Methylosphaera; Azomonas; Azorhizophilus;
Azotobacter; Cellvibrio; Oligella; Pseudomonas; Teredinibacter;
Francisella; Stenotrophomonas; Xanthomonas; and Oceanimonas.
[0094] In an embodiment, the host cell is selected from "Gram(-)
Proteobacteria Subgroup 5." "Gram(-) Proteobacteria Subgroup 5" is
defined as the group of Proteobacteria of the following genera:
Methylobacter; Methylocaldum; Methylococcus; Methylomicrobium;
Methylomonas; Methylosarcina; Methylosphaera; Azomonas;
Azorhizophilus; Azotobacter; Cellvibrio; Oligella; Pseudomonas;
Teredinibacter; Francisella; Stenotrophomonas; Xanthomonas; and
Oceanimonas.
[0095] The host cell can be selected from "Gram(-) Proteobacteria
Subgroup 6." "Gram(-) Proteobacteria Subgroup 6" is defined as the
group of Proteobacteria of the following genera: Brevundimonas;
Blastomonas; Sphingomonas; Burkholderia; Ralstonia; Acidovorax;
Hydrogenophaga; Azomonas; Azorhizophilus; Azotobacter; Cellvibrio;
Oligella; Pseudomonas; Teredinibacter; Stenotrophomonas;
Xanthomonas; and Oceanimonas.
[0096] The host cell can be selected from "Gram(-) Proteobacteria
Subgroup 7." "Gram(-) Proteobacteria Subgroup 7" is defined as the
group of Proteobacteria of the following genera: Azomonas;
Azorhizophilus; Azotobacter; Cellvibrio; Oligella; Pseudomonas;
Teredinibacter; Stenotrophomonas; Xanthomonas; and Oceanimonas.
[0097] The host cell can be selected from "Gram(-) Proteobacteria
Subgroup 8." "Gram(-) Proteobacteria Subgroup 8" is defined as the
group of Proteobacteria of the following genera: Brevundimonas;
Blastomonas; Sphingomonas; Burkholderia; Ralstonia; Acidovorax;
Hydrogenophaga; Pseudomonas; Stenotrophomonas; Xanthomonas; and
Oceanimonas.
[0098] The host cell can be selected from "Gram(-) Proteobacteria
Subgroup 9." "Gram(-) Proteobacteria Subgroup 9" is defined as the
group of Proteobacteria of the following genera: Brevundimonas;
Burkholderia; Ralstonia; Acidovorax; Hydrogenophaga; Pseudomonas;
Stenotrophomonas; and Oceanimonas.
[0099] The host cell can be selected from "Gram(-) Proteobacteria
Subgroup 10." "Gram(-) Proteobacteria Subgroup 10" is defined as
the group of Proteobacteria of the following genera: Burkholderia;
Ralstonia; Pseudomonas; Stenotrophomonas; and Xanthomonas.
[0100] The host cell can be selected from "Gram(-) Proteobacteria
Subgroup 11." "Gram(-) Proteobacteria Subgroup 11" is defined as
the group of Proteobacteria of the genera: Pseudomonas;
Stenotrophomonas; and Xanthomonas.
[0101] The host cell can be selected from "Gram(-) Proteobacteria
Subgroup 12." "Gram(-) Proteobacteria Subgroup 12" is defined as
the group of Proteobacteria of the following genera: Burkholderia;
Ralstonia; Pseudomonas.
[0102] The host cell can be selected from "Gram(-) Proteobacteria
Subgroup 13." "Gram(-) Proteobacteria Subgroup 13" is defined as
the group of Proteobacteria of the following genera: Burkholderia;
Ralstonia; Pseudomonas; and Xanthomonas.
[0103] The host cell can be selected from "Gram(-) Proteobacteria
Subgroup 14." "Gram(-) Proteobacteria Subgroup 14" is defined as
the group of Proteobacteria of the following genera: Pseudomonas
and Xanthomonas.
[0104] The host cell can be selected from "Gram(-) Proteobacteria
Subgroup 15." "Gram(-) Proteobacteria Subgroup 15" is defined as
the group of Proteobacteria of the genus Pseudomonas.
[0105] The host cell can be selected from "Gram(-) Proteobacteria
Subgroup 16. " "Gram(-) Proteobacteria Subgroup 16" is defined as
the group of Proteobacteria of the following Pseudomonas species
(with the ATCC or other deposit numbers of exemplary strain(s)
shown in parenthesis): Pseudomonas abietaniphila (ATCC 700689);
Pseudomonas aeruginosa (ATCC 10145); Pseudomonas alcaligenes (ATCC
14909); Pseudomonas anguilliseptica (ATCC 33660); Pseudomonas
citronellolis (ATCC 13674); Pseudomonasflavescens (ATCC 51555);
Pseudomonas mendocina (ATCC 25411); Pseudomonas nitroreducens (ATCC
33634); Pseudomonas oleovorans (ATCC 8062); Pseudomonas
pseudoalcaligenes (ATCC 17440); Pseudomonas resinovorans (ATCC
14235); Pseudomonas straminea (ATCC 33636); Pseudomonas agarici
(ATCC 25941); Pseudomonas alcaliphila; Pseudomonas alginovora;
Pseudomonas andersonii; Pseudomonas asplenii (ATCC 23835);
Pseudomonas azelaica (ATCC 27162); Pseudomonas beijerinckii (ATCC
19372); Pseudomonas borealis; Pseudomonas boreopolis (ATCC 33662);
Pseudomonas brassicacearum; Pseudomonas butanovora (ATCC 43655);
Pseudomonas cellulosa (ATCC 55703); Pseudomonas aurantiaca (ATCC
33663); Pseudomonas chlororaphis (ATCC 9446, ATCC 13985, ATCC
17418, ATCC 17461); Pseudomonas fragi (ATCC 4973); Pseudomonas
lundensis (ATCC 49968); Pseudomonas taetrolens (ATCC 4683);
Pseudomonas cissicola (ATCC 33616); Pseudomonas coronafaciens;
Pseudomonas diterpeniphila; Pseudomonas elongata (ATCC 10144);
Pseudomonas flectens (ATCC 12775); Pseudomonas azotoformans;
Pseudomonas brenneri; Pseudomonas cedrella; Pseudomonas corrugata
(ATCC 29736); Pseudomonas extremorientalis; Pseudomonas fluorescens
(ATCC 35858); Pseudomonas gessardii; Pseudomonas libanensis;
Pseudomonas mandelii (ATCC 700871); Pseudomonas marginalis (ATCC
10844); Pseudomonas migulae; Pseudomonas mucidolens (ATCC 4685);
Pseudomonas orientalis; Pseudomonas rhodesiae; Pseudomonas
synxantha (ATCC 9890); Pseudomonas tolaasii (ATCC 33618);
Pseudomonas veronii (ATCC 700474); Pseudomonas frederiksbergensis;
Pseudomonas geniculata (ATCC 19374); Pseudomonas gingeri;
Pseudomonas graminis; Pseudomonas grimontii; Pseudomonas
halodenitrificans; Pseudomonas halophila; Pseudomonas hibiscicola
(ATCC 19867); Pseudomonas huttiensis (ATCC 14670); Pseudomonas
hydrogenovora; Pseudomonas jessenii (ATCC 700870); Pseudomonas
kilonensis; Pseudomonas lanceolata (ATCC 14669); Pseudomonas lini;
Pseudomonas marginata (ATCC 25417); Pseudomonas mephitica (ATCC
33665); Pseudomonas denitrificans (ATCC 19244); Pseudomonas
pertucinogena (ATCC 190); Pseudomonas pictorum (ATCC 23328);
Pseudomonas psychrophila; Pseudomonas fulva (ATCC 31418);
Pseudomonas monteilii (ATCC 700476); Pseudomonas mosselii;
Pseudomonas oryzihabitans (ATCC 43272); Pseudomonas plecoglossicida
(ATCC 700383); Pseudomonas putida (ATCC 12633); Pseudomonas
reactans; Pseudomonas spinosa (ATCC 14606); Pseudomonas balearica;
Pseudomonas luteola (ATCC 43273); Pseudomonas stutzeri (ATCC
17588); Pseudomonas amygdali (ATCC 33614); Pseudomonas avellanae
(ATCC 700331); Pseudomonas caricapapayae (ATCC 33615); Pseudomonas
cichorii (ATCC 10857); Pseudomonas fcuserectae (ATCC 35104);
Pseudomonas fuscovaginae; Pseudomonas meliae (ATCC 33050);
Pseudomonas syringae (ATCC 19310); Pseudomonas viridiflava (ATCC
13223); Pseudomonas thermocarboxydovorans (ATCC 35961); Pseudomonas
thermotolerans; Pseudomonas thivervalensis; Pseudomonas
vancouverensis (ATCC 700688); Pseudomonas wisconsinensis; and
Pseudomonas xiamenensis.
[0106] The host cell can be selected from "Gram(-) Proteobacteria
Subgroup 17." "Gram(-) Proteobacteria Subgroup 17" is defined as
the group of Proteobacteria known in the art as the "fluorescent
Pseudomonads" including those belonging, e.g., to the following
Pseudomonas species: Pseudomonas azotoformans; Pseudomonas
brenneri; Pseudomonas cedrella; Pseudomonas corrugata; Pseudomonas
extremorientalis; Pseudomonas fluorescens; Pseudomonas gessardii;
Pseudomonas libanensis; Pseudomonas mandelii; Pseudomonas
marginalis; Pseudomonas migulae; Pseudomonas mucidolens;
Pseudomonas orientalis; Pseudomonas rhodesiae; Pseudomonas
synxantha; Pseudomonas tolaasii; and Pseudomonas veronii.
[0107] The host cell can be selected from "Gram(-) Proteobacteria
Subgroup 18." "Gram(-) Proteobacteria Subgroup 18" is defined as
the group of all subspecies, varieties, strains, and other
sub-special units of the species Pseudomonas fluorescens, including
those belonging, e.g., to the following (with the ATCC or other
deposit numbers of exemplary strain(s) shown in parenthesis):
Pseudomonas fluorescens biotype A, also called biovar 1 or biovar I
(ATCC 13525); Pseudomonas fluorescens biotype B, also called biovar
2 or biovar II (ATCC 17816); Pseudomonas fluorescens biotype C,
also called biovar 3 or biovar III (ATCC 17400); Pseudomonas
fluorescens biotype F, also called biovar 4 or biovar IV (ATCC
12983); Pseudomonas fluorescens biotype G, also called biovar 5 or
biovar V (ATCC 17518); Pseudomonas fluorescens biovar VI;
Pseudomonas fluorescens Pf0-1; Pseudomonas fluorescens Pf-5 (ATCC
BAA-477); Pseudomonas fluorescens SBW25; and Pseudomonas
fluorescens subsp. cellulosa (NCIMB 10462).
[0108] The host cell can be selected from "Gram(-) Proteobacteria
Subgroup 19." "Gram(-) Proteobacteria Subgroup 19" is defined as
the group of all strains of Pseudomonas fluorescens biotype A. A
particularly particular strain of this biotype is P. fluorescens
strain MB101 (see U.S. Pat. No. 5,169,760 to Wilcox), and
derivatives thereof.
[0109] In one embodiment, the host cell is any of the
Proteobacteria of the order Pseudomonadales. In a particular
embodiment, the host cell is any of the Proteobacteria of the
family Pseudomonadaceae.
[0110] In a particular embodiment, the host cell is selected from
"Gram(-) Proteobacteria Subgroup 1. " In a particular embodiment,
the host cell is selected from "Gram(-) Proteobacteria Subgroup 2.
" In a particular embodiment, the host cell is selected from
"Gram(-) Proteobacteria Subgroup 3. " In a particular embodiment,
the host cell is selected from "Gram(-) Proteobacteria Subgroup 5.
" In a particular embodiment, the host cell is selected from
"Gram(-) Proteobacteria Subgroup 7. " In a particular embodiment,
the host cell is selected from "Gram(-) Proteobacteria Subgroup 12.
" In a particular embodiment, the host cell is selected from
"Gram(-) Proteobacteria Subgroup 15. " In a particular embodiment,
the host cell is selected from "Gram(-) Proteobacteria Subgroup 17.
" In a particular embodiment, the host cell is selected from
"Gram(-) Proteobacteria Subgroup 18. " In a particular embodiment,
the host cell is selected from "Gram(-) Proteobacteria Subgroup 19.
"
[0111] Additional P. fluorescens strains that can be used in the
present invention include Pseudomonas fluorescens Migula and
Pseudomonas fluorescens Loitokitok, having the following ATCC
designations: [NCIB 8286]; NRRL B-1244; NCIB 8865 strain COI; NCIB
8866 strain CO2; 1291 [ATCC 17458; IFO 15837; NCIB 8917; LA; NRRL
B-1864; pyrrolidine; PW2 [ICMP 3966; NCPPB 967; NRRL B-899]; 13475;
NCTC 10038; NRRL B-1603 [6; IFO 15840]; 52-1C; CCEB 488-A [BU 140];
CCEB 553 [IEM 15/47]; IAM 1008 [AHH-27]; IAM 1055 [AHH-23]; 1 [IFO
15842]; 12 [ATCC 25323; NIH 11; den Dooren de Jong 216]; 18 [IFO
15833; WRRL P-7]; 93 [TR-10]; 108[52-22; IFO 15832]; 143 [IFO
15836; PL]; 149 [2-40-40; IFO 15838]; 182 [IFO 3081; PJ 73]; 184
[IFO 15830]; 185[W2 L-1]; 186 [IFO 15829; PJ 79]; 187 [NCPPB 263];
188 [NCPPB 316]; 189 [PJ227; 1208]; 191 [IFO 15834; PJ 236; 22/1];
194 [Klinge R-60; PJ 253]; 196 [PJ 288]; 197 [PJ 290]; 198[PJ 302];
201 [PJ 368]; 202 [PJ 372]; 203 [PJ 376]; 204 [IFO 15835; PJ 682];
205[PJ686]; 206 [PJ 692]; 207 [PJ 693]; 208 [PJ 722]; 212 [PJ 832];
215 [PJ 849]; 216 [PJ885]; 267 [B-9]; 271 [B-1612]; 401 [C71A; IFO
15831; PJ 187]; NRRL B-3178 [4; IFO 15841]; KY8521; 3081; 30-21;
[IFO 3081]; N; PYR; PW; D946-B83 [BU 2183; FERM-P 3328]; P-2563
[FERM-P 2894; IFO 13658]; IAM-1126 [43F]; M-1; A506 [A5-06];
A505[A5-05-1 ]; A526 [A5-26]; B69; 72; NRRL B4290; PMW6 [NCIB
11615]; SC 12936; Al [IFO 15839]; F 1847 [CDC-EB]; F 1848 [CDC 93];
NCIB 10586; P17; F-12; AmMS 257; PRA25; 6133D02; 6519E01; Ni;
SC15208; BNL-WVC; NCTC 2583 [NCIB 8194]; H13; 1013 [ATCC 11251;
CCEB 295]; IFO 3903; 1062; or Pf-5.
II. Auxotrophic Selection Markers
[0112] The present invention provides Pseudomonads and related
cells that have been genetically modified to induce auxotrophy for
at least one metabolite. The genetic modification can be to a gene
or genes encoding an enzyme that is operative in a metabolic
pathway, such as an anabolic biosynthetic pathway or catabolic
utilization pathway. Preferably, the host cell has all operative
genes encoding a given biocatalytic activity deleted or inactivated
in order to ensure removal of the biocatalytic activity. In a
particular embodiment, the Pseudomonad is a Pseudomonas fluorescens
cell.
[0113] One or more than one metabolic activity may be selected for
knock-out or replacement. In the case of native auxotrophy(ies),
additional metabolic knockouts or replacements can be provided.
Where multiple activities are selected, the auxotrophy-restoring
selection markers can be of a biosynthetic-type (anabolic), of a
utilization-type (catabolic), or may be chosen from both types. For
example, one or more than one activity in a given biosynthetic
pathway for the selected compound may be knocked-out; or more than
one activity, each from different biosynthetic pathways, may be
knocked-out. The corresponding activity or activities are then
provided by at least one recombinant vector which, upon
transformation into the cell, restores prototrophy to the cell.
[0114] Compounds and molecules whose biosynthesis or utilization
can be targeted to produce auxotrophic host cells include: lipids,
including, for example, fatty acids; mono- and disaccharides and
substituted derivatives thereof, including, for example, glucose,
fructose, sucrose, glucose-6-phosphate, and glucuronic acid, as
well as Entner-Doudoroff and Pentose Phosphate pathway
intermediates and products; nucleosides, nucleotides,
dinucleotides, including, for example, ATP, dCTP, FMN, FAD, NAD,
NADP, nitrogenous bases, including, for example, pyridines,
purines, pyrimidines, pterins, and hydro-, dehydro-, and/or
substituted nitrogenous base derivatives, such as cofactors, for
example, biotin, cobamamide, riboflavine, thiamine; organic acids
and glycolysis and citric acid cycle intermediates and products,
including, for example, hydroxyacids and amino acids; storage
carbohydrates and storage poly(hydroxyalkanoate) polymers,
including, for example, cellulose, starch, amylose, amylopectin,
glycogen, poly-hydroxybutyrate, and polylactate.
[0115] In one embodiment, the biocatalytic activity(ies) knocked
out to produce the auxotrophic host cell is selected from the group
consisting of: the lipids; the nucleosides, nucleotides,
dinucleotides, nitrogenous bases, and nitrogenous base derivatives;
and the organic acids and glycolysis and citric acid cycle
intermediates and products. Preferably, the biocatalytic
activity(ies) knocked out is selected from the group consisting of:
the nucleosides, nucleotides, dinucleotides, nitrogenous bases, and
nitrogenous base derivatives; and the organic acids and glycolysis
and citric acid cycle intermediates and products. More preferably,
the biocatalytic activity(ies) knocked out is selected from the
group consisting of: the pyrimidine nucleosides, nucleotides,
dinucleotides, nitrogenous bases, and nitrogenous base derivatives;
and the amino acids.
[0116] A given transgenic host cell may use one or more than one
selection marker or selection marker system. For example, one or
more biosynthesis selection marker(s) or selection marker system(s)
according to the present invention may be used together with each
other, and/or may be used in combination with a utilization-type
selection marker or selection marker system according to the
present invention. In any one of these prototrophy-enabling
embodiments, the host cell may also contain one or more
non-auxotrophic selection marker(s) or selection marker system(s).
Examples of non-auxotrophic selection marker(s) and system(s)
include, for example: toxin-resistance marker genes such as
antibiotic-resistance genes that encode an enzymatic activity that
degrades an antibiotic; toxin-resistant marker genes, such as, for
example, imidazolinone-resistant mutants of acetolactate synthase
("ALS;" EC 2.2.1.6) in which mutation(s) are expressed that make
the enzyme insensitive to toxin-inhibition exhibited by versions of
the enzyme that do not contain such mutation(s). The compound(s)
may exert this effect directly; or the compound(s) may exert this
effect indirectly, for example, as a result of metabolic action of
the cell that converts the compound(s) into toxin form or as a
result of combination of the compound(s) with at least one further
compound(s).
[0117] Bacterial-host-operative genes encoding such marker enzymes
can be obtained from the bacterial host cell strain chosen for
construction of the knock-out cell, from other bacteria, or from
other organisms, and may be used in native form or modified (e.g.,
mutated or sequence recombined) form. For example, a DNA coding
sequence for an enzyme exhibiting the knocked out biocatalytic
activity may be obtained from one or more organisms and then
operatively attached to DNA regulatory elements operative within
the host cell. In specific, all of the chosen host's intracellular
genes that encode a selected enzymatic activity are knocked-out;
the bacterial knock-out host is then transformed with a vector
containing at least one operative copy of a native or non-native
gene encoding an enzyme exhibiting the activity lost by the
bacterial knockout.
[0118] Bacterial and other genes encoding such enzymes can be
selected and obtained through various resources available to one of
ordinary skill in the art. These include the nucleotide sequences
of enzyme coding sequences and species-operative DNA regulatory
elements. Useful on-line InterNet resources include, e.g.,: (1) the
ExPASy proteomics facility (see the ENZYME and BIOCHEMICAL PATHWAYS
MAPS features) of the Swiss Institute of Bioinformatics (Batiment
Ecole de Pharmacie, Room 3041; Universitde Lausanne; 1015
Lausanne-Dorigny; Switzerland) available at, e.g.,
http://us.expasy.org/; and (2) the GenBank facility and other
Entrez resources (see the PUBMED, PROTEIN, NUCLEOTIDE, STRUCTURE,
GENOME, et al. features) offered by the National Center for
Biotechnology Information (NCBI, National Library of Medicine,
National Institutes of Health, U.S. Dept. of Health & Human
Services; Building 38A; Bethesda, Md., USA) and available at
http://www.ncbi.nlm.nih.gov/entrez/guery.fcgi.
[0119] The selected coding sequence may be modified by altering the
genetic code thereof to match that employed by the bacterial host
cell, and the codon sequence thereof may be enhanced to better
approximate that employed by the host. Genetic code selection and
codon frequency enhancement may be performed according to any of
the various methods known to one of ordinary skill in the art,
e.g., oligonucleotide-directed mutagenesis. Useful on-line InterNet
resources to assist in this process include, e.g.: (1) the Codon
Usage Database of the Kazusa DNA Research Institute (2-6-7
Kazusa-kamatari, Kisarazu, Chiba 292-0818 Japan) and available at
http://www.kazusa.or.jp/codon/; and (2) the Genetic Codes tables
available from the NCBI Taxonomy database at
http://www.ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc.cgi?mode=c. For
example, Pseudomonas species are reported as utilizing Genetic Code
Translation Table 11 of the NCBI Taxonomy site, and at the Kazusa
site as exhibiting the codon usage frequency of the table shown at
http://www.kazusa.or.jp/codon/cgibin/.
[0120] In a particular embodiment, Pseudomonas fluorescens can be
used as the host cell. In one embodiment, Pseudomonas fluorescens
provides at least one auxotrophic selection marker gene. In an
alternative embodiment, Pseudomonas fluorescens provides all
auxotrophic selection marker genes. In a particular embodiment,
Pseudomonas fluorescens can both be the host cell and provide at
least one, and preferably all, auxotrophic selection marker
genes.
[0121] Biosynthetic Nucleoside and Nitrogenous Base Selection
Markers
[0122] In one embodiment, a biosynthetic enzyme involved in
anabolic metabolism can be chosen as the auxotrophic selection
marker. In particular, the biosynthetic enzyme can be selected from
those involved in biosynthesis of the nucleosides, nucleotides,
dinucleotides, nitrogenous bases, and nitrogenous base
derivatives.
[0123] In a particular embodiment at least one purine-type
biosynthetic enzyme can be chosen as an auxotrophic selection
marker. Such purine biosynthetic enzymes include, for example,
adenine phosphoribosyltransferases, adenylosuccinate lyases,
adenylosuccinate synthases, GMP synthases, IMP cyclohydrolases, IMP
dehydrogenases, phosphoribosylamine-glycine ligases,
phosphoribosyl-aminoimidazolecarboxa- mide formyltransferases,
phosphoribosylaminoimidazole carboxylases, phosphoribosyl
aminoimidazolesuccinocarboxamide synthases,
phosphoribosyl-formylglycinamidine cyclo ligases,
phosphoribosyl-formylgl- ycinamidine synthases,
phosphoribosyl-glycinamide formyltransferases, ribose-phosphate
diphosphokinases, and ribose-5-phosphate-ammonia ligases.
[0124] In another particular embodiment, a pyrimidine-type
biosynthetic enzyme can be chosen as an auxotrophic selection
marker. Such pyrimidine-type biosynthetic include enzymes involved
in biosynthesis of UMP, such as carbamate kinase (EC 2.7.2.2),
carbamoyl-phosphate synthase (EC 6.3.5.5), aspartate
carbamoyltransferase (EC 2.1.3.2), dihydroorotase (EC 3.5.2.3),
dihydroorotate dehydrogenase (EC 1.3.3.1), orotate
phosphoribosyltransferase ("OPRT;" EC 2.4.2.10), and
orotidine-5'-phosphate decarboxylase ("ODCase;" EC 4.1.1.23).
[0125] Examples of genes encoding pyrimidine-type biosynthetic
enzymes are well known. In the case of bacterial synthesis of UMP,
examples of useful genes include: arcC genes, encoding carbamate
kinases; carA and carB genes, collectively encoding
carbamoyl-phosphate synthases; pyrB genes, encoding aspartate
carbamoytransferases; pyrC genes, encoding dihydroorotases; pyrD
genes, singly or collectively encoding dihydroorotate
dehydrogenases; pyrE genes encoding orotate
phosphoribosyltransferases; and pyrF genes, encoding
orotidine-5'-phosphate decarboxylases.
[0126] In a particular embodiment, an expression system according
to the present invention will utilize a pyrF auxotrophic selection
marker gene. pyrF genes encode ODCase, an enzyme required for the
bacterial pyrimidine nucleotide biosynthesis pathway, by which the
cell performs de novo synthesis of pyrimidine nucleotides proper
(UTP, CTP), as well as pyrimidine deoxynucleotides (dTTP, dCTP).
The pathway's initial reactants are ATP, an amino group source
(i.e. ammonium ion or L-glutamine), and a carboxyl group source
(i.e. carbon dioxide or bicarbonate ion); the pathway's ultimate
product is dTTP, with dCTP, UTP, and CTP also being formed in the
process. Specifically, the bacterial de novo pyrimidine nucleotide
biosynthesis pathway begins with the formation of carbamoyl
phosphate. Carbamoyl phosphate is synthesized either: (a) by action
of carbamate kinase (EC 2.7.2.2), encoded by the arcC gene; or,
more commonly, (b) by action of the glutamine-hydrolyzing,
carbamoyl-phosphate synthase (EC 6.3.5.5), whose small and large
subunits are encoded by the carA and carB genes, respectively.
Carbamoyl phosphate is then converted to UDP by the following
six-step route: 1) conversion of carbamoyl phosphate to
N-carbamoyl-L-aspartate, by aspartate carbamoyltransferase (EC
2.1.3.2), encoded by pyrB; then 2) conversion thereof to
(S)-dihydroorotate, by dihydroorotase (EC 3.5.2.3), encoded by
pyrC; then 3) conversion thereof to orotate, by dihydroorotate
dehydrogenase (EC 1.3.3.1), encoded by pyrD gene(s); then 4)
conversion thereof to orotidine-5'-monophosphate ("OMP"), by
orotate phosphoribosyltransferase ("OPRT;" EC 2.4.2.10), encoded by
pyrE; and then 5) conversion thereof to uridine-5'-monophosphate
("UMP"), by orotidine-5'-phosphate decarboxylase ("ODCase;" EC
4.1.1.23), encoded by pyrF. The UMP is then utilized by a variety
of pathways for synthesis of pyrimidine nucleotides (UTP, CTP,
dTTP, dCTP), nucleic acids, nucleoproteins, and other cellular
metabolites.
[0127] In bacteria in which one or more of the carA, carB, or
pyrB-pyrF genes has become inactivated or lost, or mutated to
encode a non-functional enzyme, the cell can still thrive if uracil
is added to the medium, provided that the cell contains a
functioning uracil salvage pathway. Most bacteria contain a native
uracil salvage pathway, including the Pseudomonads and related
species. In a uracil salvage pathway, the cell imports and converts
exogenous uracil into UMP, to synthesize the required pyrimidine
nucleotides. In this, uracil is reacted with
5-phosphoribosyl-1-pyrophosphate to form UMP, by the action of
either uracil phosphoribosyltransferase (EC 2.4.2.9), encoded by
the upp gene, or by the bifunctional, pyrimidine operon regulatory
protein ("pyrR bifunctional protein"), encoded by pyrR. The
resulting UMP is then converted to UDP, and then the subsequent
pyrimidine nucleotides, as described above.
[0128] Consequently, a pyrF(-) Pseudomonad or related cell can be
maintained on uracil-containing medium. After a pyrF
gene-containing DNA construct is transfected into the pyrF(-) cell
and expressed to form a functioning ODCase enzyme, the resulting
combined pyrF(+) plasmid-host cell system can be maintained in a
medium lacking uracil.
[0129] The coding sequence of the pyrF gene for use in a
Pseudomonad or related host cell can be provided by any gene
encoding an orotidine-5'-phosphate decarboxylase enzyme ("ODCase"),
provided that the coding sequence can be transcribed, translated,
and otherwise processed by the selected Pseudomonad or related host
cell to form a functioning ODCase. The pyrF coding sequence may be
a native sequence, or it may be an engineered sequence resulting
from, for example, application of one or more sequence-altering,
sequence-combining, and/or sequence-generating techniques known in
the art. Before use as part of a pyrF selection marker gene, the
selected coding sequence may first be improved or optimized in
accordance with the genetic code and/or the codon usage frequency
of a selected Pseudomonad or related host cell. Expressible coding
sequences will be operatively attached to a transcription promoter
capable of functioning in the chosen host cell, as well as all
other required transcription and translation regulatory elements. A
native coding sequence for a pyrF gene as described above may be
obtained from a bacterium or from any other organism, provided that
it meets the above-described requirements.
[0130] In one embodiment, the pyrF coding sequence is isolated from
the Pseudomonad or related host cell in which it is intended to be
used as a selection marker. The entire pyrF gene (including the
coding sequence and surrounding regulatory regions) can be isolated
there from. In a particular embodiment, a bacterium providing the
pyrF gene or coding sequence will be selected from the group
consisting of a member of the order Pseudomonadales, a member of
the suborder Pseudomonadineae, a member of the family
Pseudomonadaceae, a member of the tribe Pseudomonadeae, a member of
the genus Pseudomonas, and a member of the Pseudomonas fluorescens
species group (i.e. the "fluorescent pseudomonads"). In a
particular embodiment, the bacterium will belong to the species,
Pseudomonas fluorescens.
[0131] In a particular embodiment, the pyrF gene contains the
nucleic acid sequence of SEQ ID NO. 1 (Table 2), or a variant
thereof. Alternatively, the ODCase encoded by the pyrF gene
contains the amino acid sequence of SEQ ID NO. 2 (Table 3), a
variant thereof, or a variant having a codon sequence redundant
therewith, in accordance with the genetic code used by a given host
cell according to the present invention.
[0132] Alternatively, the pyrF gene contains a nucleic acid
sequence encoding an ODCase enzyme selected from the group
consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%,
88%, 90%, and 95% homologous to SEQ ID No. 1. Likewise, the pyrF
gene encodes an ODCase selected from the group consisting of an
amino acid sequence at least 70%, 75%, 80%, 85%, 88%, 90%, and 95%
homologous to SEQ ID No. 2.
[0133] In another embodiment, the pyrF gene can contain a coding
sequence having a nucleotide sequence at least 90%, 93%, 95%, 96%,
97%, 98% or 99% homologous to the nucleotide sequence of
nucleotides 974-1669 of SEQ ID NO: 1.
[0134] In a particular embodiment, the pyrF gene can contain a
coding sequence having a codon sequence that hybridizes to the
anti-codon sequence of SEQ ID NO:3 (Table 4), when hybridization
has been performed under highly stringent hybridization conditions,
or can have a codon sequence redundant therewith. In a particularly
particular embodiment, the pyrF gene will contain the nucleotide
sequence of SEQ ID No. 3
4TABLE 2 PSEUDOMONAS FLUORESCENS PYRF NUCLEIC ACID SEQUENCE
gatcagttgcggagccttggggtcatcccccagtttctgac-
gcaggcgcgacaccagcaagtcgatgctgcggtcga SEQ ID NO. 1
aagcctcgatggaacgcccacgggccgcgtccagcagctgttcgcggctcagcacacgccgcgggcgttcgat-
aaac acccacaacaaacgaaactcggcgttggacagcggcaccaccaggccgtcatc-
ggccaccagctggcgcagtacgct gttcaggcgccaagtgtcgaaacggatattggc-
ccgctgttcggtgcggtcatcacgcacccggcgcaggatggtct
ggatacgcgcgaccagttcccggggttcgaacggcttggacatatagtcgtctgcccccagttccaggccgat-
gatg cggtcggtgggttcgcagcgggcggtgagcatcaggatcggaatgtccgattc-
ggcgcgcagccagcggcacaatgt cagcccgtcttcgcccggcagcatcaggtcgag-
caccaccacatcgaaggtctccgcttgcatggcctggcgcatgg
cgatgccgtcggtgacgcctgaggcgagaatattgaagcgtgccaggtagtcgatcagcagttcgcggatcgg-
cacg tcgtcgtcgacaatcagcgcgcgggtgttccagcgcttgtcttcggcgatcac-
cgcgtcttttggcgcttcgtttac agggtcgcaaggggtatgcatagcgaggtcatc-
tgcctggttgtggctgtcagcataggcgcccagttccagggctg
gaagtgctgggcgggcggtcatgtgcgcgaggctagccgggcggcgtattgggggcgtgtcgtgaatgtatcg-
ggct tgaaacaattgccttgaatcgccggtattgggcgcttgatcggtatttaccga-
tcatcggatcccgcaacggcgctg cttgcgctacaatccgcgccgatttcgacttgc-
ctgagagcccattccaatgtccgtctgccagactcctatcatcg
tcgccctggattaccccacccgtgacgccgcactgaagctggctgaccagttggaccccaagctttgccgggt-
caag gtcggcaaggaattgttcaccagttgcgcggcggaaatcgtcggcaccctgcg-
ggacaaaggcttcgaagtgttcct cgacctcaaattccatgacatccccaacaccac-
ggcgatggccgtcaaagccgcggccgagatgggcgtgtggatgg
tcaatgtgcactgctccggtggcctgcgcatgatgagcgcctgccgcgaagtgctggaacagcgcagcggccc-
caaa ccgttgttgatcggcgtgaccgtgctcaccagcatggagcgcgaagacctggc-
gggcattggcctggatatcgagcc gcaggtgcaagtgttgcgcctggcagccctggc-
gcagaaagccggcctcgacggcctggtgtgctcagccctggaag
cccaggccctgaaaaacgcacatccgtcgctgcaactggtgacaccgggtatccgtcctaccggcagcgccca-
ggat gaccagcgccgtatcctgaccccgcgccaggccctggatgcgggctctgacta-
cctggtgatcggccggccgatcag ccaggcggcggatcctgcaaaagcgttggcagc-
ggtcgtcgccgagatcgcctgatttttagagtgagcaaaaaatg
tgggagctggcttgcctgcgatagtatcaactcggtatcacttagaaaccgagttgcttgcatcgcaggcaag-
ccag ctcccacatttgtttttgtggtgtgtcagctgactttgagcaccaacttcccg-
aagttctcgccgttgaacagcttc atcagcgtttccgggaatgtctccagcccttcg-
acaatatcttccttgctcttgagcttgccctgggccatccagcc
ggccatttcctgacccgccgccgcgaagttcgccgcgtggtccatcaccacaaagccttccatacgcgcacgg-
ttga ccagcaatgacaggtagttcgccgggcctttgaccgcttccttgttgttgtac-
tggctgattgcaccgcaaatcacc acgcgggctttgagcgccaggcggctgagcacc-
gcgtcgagaatatcgccgccgacgttatcgaaatacacgtccac
gcctttggggcactcgcgcttgagggcggcgggcacgtcttcgcttttgtagtcgatggcggcgtcgaagccc-
agct catcgaccaggaacttgcacttctcggcgccaccggcgatccccactacgcga-
cagcctttgagcttagcgatctgc ccggcgatgctgcccacggcaccggcggcgccg-
gagatcaccacggtgtcaccggctttcggtgcgccggtctccag
cagagcaaagtaggccgtcatgccggtcatgcccagggcggacaggtagcggggcaggggcgccagcttgggg-
tcca ccttatagaaaccacggggctcgccaaggaagtaatcctgcacgcccagtgca-
ccgttcacgtagtcccccaccgcg aagttcggatggttcgaggcaagcaccttgcct-
acgcccagggcgcgcatcacttcgccgatgcctaccggtgggat
gtaggacttgccttcattcatccagccacgca
[0135]
5TABLE 3 PSEUDOMONAS FLUORESCENS ODCASE AMINO ACID SEQUENCE Met Ser
Val Cys Gln Thr Pro Ile Ile Val Ala Leu Asp Tyr Pro Thr Arg Asp SEQ
ID NO. 2 Ala Ala Leu Lys Leu Ala Asp Gln Leu Asp Pro Lys Leu Cys
Arg Val Lys Val Gly Lys Glu Leu Phe Thr Ser Cys Ala Ala Glu Ile Val
Gly Thr Leu Arg Asp Lys Gly Phe Glu Val Phe Leu Asp Leu Lys Phe His
Asp Ile Pro Asn Thr Thr Ala Met Ala Val Lys Ala Ala Ala Glu Met Gly
Val Trp Met Val Asn Val His Cys Ser Gly Gly Leu Arg Met Met Ser Ala
Cys Arg Glu Val Leu Glu Gln Arg Ser Gly Pro Lys Pro Leu Leu Ile Gly
Val Thr Val Leu Thr Ser Met Glu Arg Glu Asp Leu Ala Gly Ile Gly Leu
Asp Ile Glu Pro Gln Val Gln Val Leu Arg Leu Ala Ala Leu Ala Gln Lys
Ala Gly Leu Asp Gly Leu Val Cys Ser Ala Leu Glu Ala Gln Ala Leu Lys
Asn Ala His Pro Ser Leu Gln Leu Val Thr Pro Gly Ile Arg Pro Thr Gly
Ser Ala Gln Asp Asp Gln Arg Arg Ile Leu Thr Pro Arg Gln Ala Leu Asp
Ala Gly Ser Asp Tyr Leu Val Ile Gly Arg Pro Ile Ser Gln Ala Ala Asp
Pro Ala Lys Ala Leu Ala Ala Val Val Ala Glu Ile Ala
[0136]
6TABLE 4 PSEUDOMONAS FLUORESCENS PYRF NUCLEIC ACID SEQUENCE
atgtccgtctgccagactcctatcatcgtcgccctggatta-
ccccacccgtgacgccgcactgaag SEQ. ID No. 3
ctggctgaccagttggaccccaagctttgccgggtcaaggtcggcaaggaattgttcaccagttgc
gcggcggaaatcgtcggcaccctgcgggacaaaggcttcgaagtgttcctcgacctcaaattcc-
at gacatccccaacaccacggcgatggccgtcaaagccgcggccgagatgggcgtgt-
ggatggtcaat gtgcactgctccggtggcctgcgcatgatgagcgcctgccgcgaag-
tgctggaacagcgcagcggc cccaaaccgttgttgatcggcgtgaccgtgctcacca-
gcatggagcgcgaagacctggcgggcatt ggcctggatatcgagccgcaggtgcaag-
tgttgcgcctggcagccctggcgcagaaagccggcctc
gacggcctggtgtgctcagccctggaagcccaggccctgaaaaacgcacatccgtcgctgcaactg
gtgacaccgggtatccgtcctaccggcagcgcccaggatgaccagcgccgtatcctgaccccgc-
gc caggccctggatgcgggctctgactacctggtgatcggccggccgatcagccagg-
cggcggatcct gcaaaagcgttggcagcggtcgtcgccgagatcgcc
[0137] In an alternate embodiment, an expression system according
to the present invention will utilize a thyA auxotrophic selection
marker gene. thyA genes encode thymidylate synthase (EC 2.1.1.45),
an enzyme required for the bacterial pyrimidine nucleotide
biosynthesis pathway. Since DNA contains thymine (5-methyluracil)
as a major base instead of uracil, the synthesis of thymidine
monophospate (dTMP or thymidylate) is essential to provide dTTP
(thymidine triphosphate) needed for DNA replication together with
dATP, dGTP, and dCTP. Methylation of dUMP by thymidylate synthase
utilizing 5,10-methylenetetrahydrofolate as the source of the
methyl group generates thymidylate. Thymidylate synthesis can be
interrupted, and consequently the synthesis of DNA arrested, by the
removal, inhibition, or disruption of thymidylate synthase.
[0138] In bacteria in which the thyA gene has become inactivated or
lost, or mutated to encode a non-functional enzyme, the cell can
still thrive if exogenous thymidine is added to the medium.
[0139] In Pseudomonas fluorescens, the addition of an E.coli tdk
gene, encoding thymidine kinase, is required for survival on
exogenous thymidine. Therefore, prior to selection, a plasmid
comprising a tdk gene can be used to transform thyA(-) P.
fluorescens host cells, generating a thyA(-)/ptdk cell, allowing
survival on a thymidine containing medium. Alternatively, a tdk
gene producing a functional thymidylate synthase enzyme capable of
utilizing exogenous thymidine in Pseudomonas fluorescens can be
inserted into the genome, producing a thyA(-)/tdk(+) host cell.
After a thyA gene-containing DNA construct is transfected into the
thyA(-)/ptdk cell and expressed to form a functioning thymidylate
synthase enzyme, the resulting combined thyA(+) plasmid-host cell
system can be maintained in a medium lacking thymidine.
[0140] The coding sequence of the thyA gene for use in a
Pseudomonad or related host cell can be provided by any gene
encoding a thymidylate synthase enzyme ("TS"), provided that the
coding sequence can be transcribed, translated, and otherwise
processed by the selected Pseudomonad or related host cell to form
a functioning TS. The thyA coding sequence may be a native
sequence, or it may be an engineered sequence resulting from, for
example, application of one or more sequence-altering,
sequence-combining, and/or sequence-generating techniques known in
the art. Before use as part of a thyA selection marker gene, the
selected coding sequence may first be improved or optimized in
accordance with the genetic code and/or the codon usage frequency
of a selected Pseudomonad or related host cell. Expressible coding
sequences will be operatively attached to a transcription promoter
capable of functioning in the chosen host cell, as well as all
other required transcription and translation regulatory elements. A
native coding sequence for a thyA gene as described above may be
obtained from a bacterium or from any other organism, provided that
it meets the above-described requirements.
[0141] In one embodiment, the thyA coding sequence is isolated from
the Pseudomonad or related host cell in which it is intended to be
used as a selection marker. The entire thyA gene (including the
coding sequence and surrounding regulatory regions) can be isolated
there from. In a particular embodiment, a bacterium providing the
thyA gene or coding sequence will be selected from the group
consisting of a member of the order Pseudomonadales, a member of
the suborder Pseudomonadineae, a member of the family
Pseudomonadaceae, a member of the tribe Pseudomonadeae, a member of
the genus Pseudomonas, and a member of the Pseudomonas fluorescens
species group (i.e. the "fluorescent pseudomonads"). In a
particular embodiment, the bacterium will belong to the species,
Pseudomonas fluorescens.
[0142] In a particular embodiment, the thyA gene contains the
nucleic acid sequence of SEQ ID NO. 4 (Table 5). Alternatively, the
TS encoded by the thyA gene contains the amino acid sequence of SEQ
ID NO. 5 (Table 6), a variant thereof, or a variant having a codon
sequence redundant therewith, in accordance with the genetic code
used by a given host cell according to the present invention.
7TABLE 5 PSEUDOMONAS FLUORESCENS THYA NUCLEIC ACID SEQUENCE
atgaagcaatatctcgaactactgaacgacgtcgtgaccaa- tggattgaccaagggcgatcgcac
SEQ ID NO. 4
cggcaccggcaccaaagccgtatttgcccgtcagtatcggcataacttggccgacggcttcccgc
tgctgaccaccaagaagcttcatttcaaaagtatcgccaacgagttgatctggatgttgagcggc
aacaccaacatcaagtggctcaacgaaaatggcgtgaaaatctgggacgagtgggcc- accgaaga
cggcgacctgggcccggtgtacggcgagcaatggaccgcctggccgacc- aaggacggcggcaaga
tcaaccagatcgactacatggtccacaccctcaaaaccaac- cccaacagccgccgcatcctgttt
catggctggaacgtcgagtacctgccggacgaa- accaagagcccgcaggagaacgcgcgcaacgg
caagcaagccttgccgccgtgccat- ctgttgtaccaggcgttcgtgcatgacgggcatctgtcga
tgcagttgtatatccgcagctccgacgtcttcctcggcctgccgtacaacaccgccgcgttggcc
ttgctgactcacatgctggctcagcaatgcgacctgatccctcacgagatcatcgtcaccaccgg
cgacacccatgcttacagcaaccacatggaacagatccgcacccagctggcgcgtac- gccgaaaa
agctgccggaactggtgatcaagcgtaaacctgcgtcgatctacgatta- caagtttgaagacttt
gaaatcgttggctacgacgccgacccgagcatcaaggctga- cgtggctatctga
[0143]
8TABLE 6 PSEUDOMONAS FLUORESCENS TS AMINO ACID SEQUENCE
MKQYLELLNDVVTNGLTKGDRTGTGTKAVFARQYRHNLADG- FPLLTTKKLHFKSIANELIWMLSG
SEQ ID NO. 5
NTNIKWLNENGVKIWDEWATEDGDLGPVYGEQWTAWPTKDGGKINQIDYMVHTLKTNPNSRRILF
HGWNVEYLPDETKSPQENARNGKQALPPCHLLYQAFVHDGHLSMQLYIRSSDVFLGLPYNTAALA
LLTHMLAQQCDLIPHEIIVTTGDTHAYSNHMEQIRTQLARTPKKLPELVIKRKPASI- YDYKFEDF
EIVGYDADPSIKADVAI
[0144] Biosynthetic Amino Acid Selection Markers
[0145] In an alternative embodiment, the biosynthetic enzyme
involved in anabolic metabolism chosen as the auxotrophic selection
marker can be selected from those involved in the biosynthesis of
amino acids. In particular embodiments, the biosynthetic amino acid
enzymes are selected from the group consisting of enzymes active in
the biosynthesis of: the Glutamate Family (Glu; Gln, Pro, and Arg);
the Aspartate Family (Asp; Asn, Met, Thr, Lys, and Ile); the Serine
Family (Ser; Gly and Cys); the Pyruvate Family (Ala, Val, and Leu);
the Aromatic Family (Trp, Phe, and Tyr); and the Histidine Family
(His). Examples of genes and enzymes involved in these biosynthetic
pathways include: the Glutamate Family member arg, gdh, gln, and,
pro genes, including, for example, argA-argH, gdhA, glnA, proA,
proC; the Aspartate Family member asd, asn, asp, dap, lys, met, and
thr genes, including, for example, asnA, asnB, aspC, dapA, dapB,
dapD-dapF, lysA, lysC, metA-metC, metE, metH, metL, thrA-thrC; the
Serine Family member cys, gly, and ser genes, including, for
example, cysE, cysK, glyA, serA-serC; the Aromatic Family member
aro, phe, trp, and tyr genes, including, for example, aroA-aroH,
aroK, aroL, trpAtrpE, tyrA, and tyrB; and the Histidine Family
member his genes, including hisA-hisD, hisF-hisH.
[0146] In a further particular embodiment, the auxotrophic
selection marker can be selected from enzymes involved in the
biosynthesis of members of the Glutamate Family. Examples of useful
Glutamate Family auxotrophic selection markers include the
following, listed with representative examples of their encoding
genes: argA, encoding N-acetylglutamate synthases, amino acid
acetyltransferases; argB, encoding acetylglutamate kinases; argC,
encoding N-acetyl-gammaglutamylph- osphate reductases; argD,
encoding acetylornithine delta-aminotransferases- ; argE, encoding
acetylornithine deacetylases; argF and argI, encoding ornithine
carbamoyltransferases; argG, encoding argininosuccinate
synthetases; argH, encoding argininosuccinate lyases; gdhA,
encoding glutamate dehydrogenases; glnA, encoding glutamine
synthetases; proA, encoding gamma-glutamylphosphate reductases;
proB, encoding gamma-glutamate kinases; and proC, encoding
pyrroline-5-carboxylate reductases.
[0147] In one embodiment, an amino acid biosynthesis selection
marker gene can be at least one member of the proline biosynthesis
family, in particular proA, proB, or proC. In a particular
embodiment, the proline biosynthesis selection marker gene can
comprise a proC gene. proC genes encode an enzyme catalyzing the
final step of the proline biosynthesis pathway. In bacteria, the
proline (i.e. L-proline) biosynthesis pathway comprises a
three-enzyme process, beginning with L-glutamic acid. The steps of
this process are: 1) conversion of L-glutamic acid to
L-glutamyl-5-phosphate, by glutamate-5-kinase ("GK;" EC 2.7.2.11),
encoded by proB; then 2a) conversion thereof to
L-glutamate-5-semialdehyd- e, by glutamate-5-semialdehyde
dehydrogenase (EC 1.2.1.41), also known as glutamyl-5-phosphate
reductase ("GPR"), encoded by proA, followed by 2b) spontaneous
cyclization thereof to form .1-pyrroline-5-carboxylate; and then 3)
conversion thereof to L-proline, by .DELTA..sup.1-pyrroline-5-car-
boxylate reductase ("P5CR;" EC 1.5.1.2), encoded by proC. In most
bacteria, proC encodes the P5CR subunit, with the active P5CR
enzyme being a homo-multimer thereof.
[0148] In bacteria in which one or more of the proA, proB, or proC
genes has become inactivated or lost, or mutated to encode a
non-functional enzyme, the cell can still thrive if proline is
added to the medium. Consequently, a proC(-) Pseudomonad or related
cell can be maintained on a proline-containing medium. After a proC
gene-containing DNA construct is transfected into the proC(-) cell
and expressed to form a functioning P5CR enzyme, the resulting
combined proC(+) plasmid-host cell system can be maintained in a
medium lacking proline.
[0149] The coding sequence of the proC gene for use in a
Pseudomonad or related host cell can be provided by any gene
encoding an .DELTA..sup.1-pyrroline-5-carboxylate reductase enzyme
(P5CR), provided that the coding sequence can be transcribed,
translated, and otherwise processed by the selected Pseudomonad or
related host cell to form a functioning P5CR. The proC coding
sequence may be a native sequence, or it may be an engineered
sequence resulting from, for example, application of one or more
sequence-altering, sequence-combining, and/or sequence-generating
techniques known in the art. Before use as part of a proC selection
marker gene, the selected coding sequence may first be improved or
optimized in accordance with the genetic code and/or the codon
usage frequency of a selected Pseudomonad or related host cell.
Expressible coding sequences will be operatively attached to a
transcription promoter capable of functioning in the chosen host
cell, as well as all other required transcription and translation
regulatory elements. A native coding sequence for a proC gene as
described above may be obtained from a bacterium or from any other
organism, provided that it meets the above-described
requirements.
[0150] In one embodiment, the proC coding sequence is isolated from
the Pseudomonad or related host cell in which it is intended to be
used as a selection marker. The entire proC gene (including the
coding sequence and surrounding regulatory regions) can be isolated
therefrom. In a particular embodiment, a bacterium providing the
proC gene or coding sequence will be selected from the group
consisting of a member of the order Pseudomonadales, a member of
the suborder Pseudomonadineae, a member of the family
Pseudomonadaceae, a member of the tribe Pseudomonadeae, a member of
the genus Pseudomonas, and a member of the Pseudomonas fluorescens
species group (i.e. the "fluorescent pseudomonads"). In a
particular embodiment, the bacterium will belong to the species,
Pseudomonas fluorescens.
[0151] In a particular embodiment, the proC gene contains the
nucleic acid sequence of SEQ ID NO. 6 (Table 7), or a variant
thereof. Alternatively, the P5CR encoded by the proC gene contains
the amino acid sequence of SEQ ID NO. 7 (Table 8), a variant
thereof, or a variant having a codon sequence redundant therewith,
in accordance with the genetic code used by a given host cell
according to the present invention.
[0152] Alternatively, the proC gene contains a nucleic acid
sequence encoding an P5CR enzyme that is at least 70%, 75%, 80%,
85%, 88%, 90%, and 95% homologous to SEQ ID No. 6. Likewise, the
proC gene encodes an ODCase that is at least 70%, 75%, 80%, 85%,
88%, 90%, and 95% homologous to SEQ ID No. 7.
[0153] In another embodiment, the proC gene can contain a coding
sequence at least 90%, 93%, 95%, 96%, 97%, 98% or 99% homologous to
the nucleotide sequence of SEQ. ID NO. 8 (Table 9).
[0154] In a particular embodiment, the proC gene can contain a
coding sequence having a codon sequence that hybridizes to the
anti-codon sequence of SEQ ID NO. 8, when hybridization has been
performed under stringent hybridization conditions, or can have a
codon sequence redundant therewith. In a particularly particular
embodiment, the proC gene will contain the nucleotide sequence of
SEQ ID NO. 8.
9TABLE 7 PSEUDOMONAS FLUORESCENS PROC NUCLEIC ACID SEQUENCE
gcccttgagttggcacttcatcggccccattcaatcgaaca-
agactcgtgccatcgccgagcacttcgcttgg SEQ ID NO. 6
gtgcactccgtggaccgcctgaaaatcgcacaacgcctgtccgaacaacgcccggccgacctgccgccgctca
atatctgcatccaggtcaatgtcagtggcgaagccagcaagtccggctgcacgcccg-
ctgacctgccggccct ggccacagcgatcagcgccctgccgcgcttgaagctgcggg-
gcttgatggcgattcccgagccgacgcaagac cgggcggagcaggatgcggcgttcg-
ccacggtgcgcgacttgcaagccagcttgaacctggcgctggacacac
tttccatgggcatgagccacgaccttgagtcggccattgcccaaggcgccacctgggtgcggatcggtaccgc
cctgtttggcgcccgcgactacggccagccgtgaaatggctgacatccctcgaaata-
aggacctgtcatgagc aacacgcgtattgcctttatcggcgccggtaacatggcggc-
cagcctgatcggtggcctgcgggccaagggcc tggacgccgagcagatccgcgccag-
cgaccccggtgccgaaacccgcgagcgcgtcagagccgaacacggtat
ccagaccttcgccgataacgccgaggccatccacggcgtcgatgtgatcgtgctggcggtcaagccccaggcc
atgaaggccgtgtgcgagagcctgagcccgagcctgcaaccccatcaactggtggtg-
tcgattgccgctggca tcacctgcgccagcatgaccaactggctcggtgcccagccc-
attgtgcgctgcatgcccaacaccccggcgct gctgcgccagggcgtcagcggtttg-
tatgccactggcgaagtcaccgcgcagcaacgtgaccaggcccaggaa
ctgctgtctgcggtgggcatcgccgtgtggctggagcaggaacagcaactggatgcggtcaccgccgtctccg
gcagcggcccggcttacttcttcctgttgatcgaggccatgacggccgcaggcgtca-
agctgggcctgcccca cgacgtggccgagcaactggcggaacaaaccgccctgggcg-
ccgccaagatggcggtcggcagcgaggtggat gccgccgaactgcgccgtcgcgtca-
cctcgccaggtggtaccacacaagcggctattgagtcgttccaggccg
ggggctttgaagccctggtggaaacagcactgggtgccgccgcacatcgttcagccgagatggctgagcaact
gggcaaatagtcgtcccttaccaaggtaatcaaacatgctcggaatcaatgacgctg-
ccattttcatcatcca gaccctgggcagcctgtacctgctgatcgtactgatgcgct-
ttatcctgcaactggtgcgtgcgaacttctac aacccgctgtgccagttcgtggtga-
aggccacccaaccgctgctcaagccgctgcgccgggtgatcccgagcc
tgttcggcctggacatgtcgtcgctggtgctggcgctgttgctgcagattttgctgttcgtggtgatcctgat
gctcaatggataccaggccttcaccgtgctgctgttgccatggggcctgatcgggat-
tttctcgctgttcctg aagatcattttctggtcgatgatcatcagcgtgatcctgtc-
ctgggtcgcaccgggtagccgtagcccgggtg ccgaattggtggctcagatcaccga-
gccggtgctggcacccttccgtcgcctgattccgaacctgggtggcct
ggatatctcgccgatcttcgcgtttatc
[0155]
10TABLE 8 PSEUDOMONAS FLUORESCENS P5CR AMINO ACID SEQUENCE Met Ser
Asn Thr Arg Ile Ala Phe Ile Gly Ala Gly Asn Met Ala Ala Ser Leu SEQ
ID NO. 7 Ile Gly Gly Leu Arg Ala Lys Gly Leu Asp Ala Glu Gln Ile
Arg Ala Ser Asp Pro Gly Ala Glu Thr Arg Glu Arg Val Arg Ala Glu His
Gly Ile Gln Thr Phe Ala Asp Asn Ala Glu Ala Ile His Gly Val Asp Val
Ile Val Leu Ala Val Lys Pro Gln Ala Met Lys Ala Val Cys Glu Ser Leu
Ser Pro Ser Leu Gln Pro His Gln Leu Val Val Ser Ile Ala Ala Gly Ile
Thr Cys Ala Ser Met Thr Asn Trp Leu Gly Ala Gln Pro Ile Val Arg Cys
Met Pro Asn Thr Pro Ala Leu Leu Arg Gln Gly Val Ser Gly Leu Tyr Ala
Thr Gly Glu Val Thr Ala Gln Gln Arg Asp Gln Ala Gln Glu Leu Leu Ser
Ala Val Gly Ile Ala Val Trp Leu Glu Gln Glu Gln Gln Leu Asp Ala Val
Thr Ala Val Ser Gly Ser Gly Pro Ala Tyr Phe Phe Leu Leu Ile Glu Ala
Met Thr Ala Ala Gly Val Lys Leu Gly Leu Pro His Asp Val Ala Glu Gln
Leu Ala Glu Gln Thr Ala Leu Gly Ala Ala Lys Met Ala Val Gly Ser Glu
Val Asp Ala Ala Glu Leu Arg Arg Arg Val Thr Ser Pro Gly Gly Thr Thr
Gln Ala Ala Ile Glu Ser Phe Gln Ala Gly Gly Phe Glu Ala Leu Val Glu
Thr Ala Leu Gly Ala Ala Ala His Arg Ser Ala Glu Met Ala Glu Gln Leu
Gly Lys
[0156]
11TABLE 9 PSEUDOMONAS FLUORESCENS PROC NUCLEIC ACID SEQUENCE
atgagcaacacgcgtattgcctttatcggcgccggtaacat- ggcggccagcctgatcggtggc
SEQ ID NO. 8
ctgcgggccaagggcctggacgccgagcagatccgcgccagcgaccccggtgccgaaacccgc
gagcgcgtcagagccgaacacggtatccagaccttcgccgataacgccgaggccatccacggc
gtcgatgtgatcgtgctggcggtcaagccccaggccatgaaggccgtgtgcgagagcctg- agc
ccgagcctgcaaccccatcaactggtggtgtcgattgccgctggcatcacctgc- gccagcatg
accaactggctcggtgcccagcccattgtgcgctgcatgcccaacacc- ccggcgctgctgcgc
cagggcgtcagcggtttgtatgccactggcgaagtcaccgcg- cagcaacgtgaccaggcccag
gaactgctgtctgcggtgggcatcgccgtgtggctg- gagcaggaacagcaactggatgcggtc
accgccgtctccggcagcggcccggcttac- ttcttcctgttgatcgaggccatgacggccgca
ggcgtcaagctgggcctgccccac- gacgtggccgagcaactggcggaacaaaccgccctgggc
gccgccaagatggcggtcggcagcgaggtggatgccgccgaactgcgccgtcgcgtcacctcg
ccaggtggtaccacacaagcggctattgagtcgttccaggccgggggctttgaagccctggtg
gaaacagcactgggtgccgccgcacatcgttcagccgagatggctgagcaactgggcaaa
[0157] Utilization Selection Markers
[0158] In one embodiment, an enzyme involved in the catabolic
utilization of metabolites can be chosen as the auxotrophic
selection marker. In particular, the enzymes can be selected from
those involved in the utilization of a carbon source. Examples of
such enzymes include, for example, sucrases, lactases, maltases,
starch catabolic enzymes, glycogen catabolic enzymes, cellulases,
and poly(hydroxyalkanoate)depolymerases. If the bacterial host cell
exhibits native catabolic activity of the selected type, it can be
knocked-out before transformation with the prototrophy-restoring
vector. Bacteria exhibiting native auxotrophy for these compounds
can also be used in their native state for such transformation. In
those embodiments in which a compound not importable or diffusible
into the cell can be selected and supplied to the medium, the
prototrophy restoring or prototrophy-enabling enzyme(s) can be
secreted for use. In that case, the secreted enzyme(s) can degrade
the compound extracellularly to produce smaller compounds, for
example glucose, that are diffusible or importable into the cell,
by selecting or designing the coding sequence of the enzyme(s) to
include a coding sequence for a secretion signal peptide operative
within the chosen host cell. In these embodiments, the
prototrophy-restorative gene can be selected or be engineered to
include a coding sequence for a secretion signal peptide operative
within the chosen host cell to obtaining transport of the enzyme
across the cytoplasmic membrane. In either of these embodiments, or
those in which the selected compound is importable or diffusible
into the cell, the cell will be grown in medium supplying no other
carbon source apart from the selected compound.
[0159] In a carbon-source-utilization-based marker system, every
prototrophy-restorative or prototrophy-enabling carbon-source
utilization enzyme can be involved in utilization of only one
carbon source. For example, two genes from the same catabolic
pathway may be expressed together on one vector or may be
co-expressed separately on different vectors in order to provide
the prototrophy. Specific examples of such multi-gene
carbon-source-utilization-based marker systems include, for
example, the use of glycogen as the sole carbon source with
transgenic expression of both a glycogen phosphorylase and an
(alpha-1,4)glucantransferase; and the use of starch as the sole
carbon source with transgenic expression of both an alpha-amylase,
and an alpha(1->6) glucosidase. However, the selected single- or
multi-gene carbon-source marker system can be used simultaneously
with other types of marker system(s) in the same host cell,
provided that the only carbon source provided to the cell is the
compound selected for use in the carbon-source catabolic selection
marker system.
[0160] Other examples of useful enzymes for
biochemical-utilization-type activities are well known in the art,
and can include racemases and epimerases that are capable of
converting a non-utilizable D-carbon source, supplied to the cell,
to a nutritive L-carbon source. Examples of these systems include,
for example: a D-acid or a D-acyl compound used with trangenic
expression of the corresponding racemase; and lactate used with
transgenically expressed lactate racemase.
[0161] Similarly, where an amino acid biosynthetic activity has
been selected for use in the marker system, the auxotrophy may also
be overcome by supplying the cell with both a non-utilizable
R-amino acid and an R-amino acid racemase or epimerase (EC 5.1.1)
that converts the R-amino acid into the corresponding L-amino acid
for which the cell is auxotrophic.
[0162] Trait Stacking
[0163] A plurality of phenotypic changes can also be made to a host
cell, before or after insertion of an auxotrophic selection marker
gene, for target gene expression, according to the present
invention. For example, the cell can be genetically engineered,
either simultaneously or sequentially, to exhibit a variety of
enhancing phenotypic traits. This process is referred to as "trait
stacking." A pryF deletion may be present as one such phenotypic
trait. In such a strain, a pyrF gene, according to the present
invention, can be used on a suicide vector as both a selectable
marker and a counterselectable marker (in the presence of
5'-fluoroorotic acid) in order to effect a cross-in/cross-out
allele exchange of other desirable traits, Thus, a pyrF gene
according to the present invention may be used in a process for
"trait stacking" a host cell. In such a process, a suicide vector
containing such a pyrF gene can be transformed into the host cell
strain in a plurality of separate transformations; in each such
procedure the re-establishment of the pyrF phenotype can be used to
create, ad infinitum, subsequent genetically-enhancing phenotypic
change. Thus, not only can the pyrF gene itself provide a trait, it
can be used to obtain additional phenotypic traits in a process of
trait-stacking.
[0164] In one embodiment, the present invention provides
auxotrophic Pseudomonads and related bacteria that have been
further genetically modified to induce additional auxotrophies. For
example, a pyrF(-) auxotroph can be further modified to inactivate
another biosynthetic enzyme present in an anabolic or catabolic
pathway, such as through the inactivation of a proC gene or a thyA
gene. In this way, multiple auxotrophies in the host cell can be
produced.
[0165] In another embodiment, genetic alterations can be made to
the host cell in order to improve the expression of recombinant
polypeptides in the host cell. Further modifications can include
genetic alterations that allow for a more efficient utilization of
a particular carbon source, thereby optimizing the overall
efficiency of the entire fermentation.
[0166] In one particular embodiment, auxotrophic host cells are
further modified by the insertion of a lacI containing transgene
into the host chromosome. Preferably, the lacI transgene, or
derivate thereof, is other than part of a whole or truncated
structural gene containing PlacI-lacI-lacZYA construct.
[0167] Modifications to Induce Auxotrophism
[0168] A Pseudomonad or related host cell selected for use in an
expression system according to the present invention can be
deficient in its ability to express any functional biocatalyst
exhibiting the selected auxotrophic activity. For example, where an
orotidine-5'-phosphate decarboxylase activity is selected, the host
cell can be deficient in its ability to express a) any pyrF gene
product (i.e. any functional ODCase enzyme), and b) any effective
replacement therefore (i.e. any other biocatalyst having ODCase
activity). In a one embodiment, the host cell will be made
biocatalytically-deficient for the selected activity by altering
its genomic gene(s) so that the cell cannot express, from its
genome, a functional enzyme involved in the targeted auxotrophy
(i.e. ODCase). In other words, the prototrophic cell (activity(+)
cell) will become auxotrophic through the "knock-out" of a
functional enzymatic encoding gene involved in the targeted
prototrophic pathway (i.e. an activity(-) cell). This alteration
can be done by altering the cell's genomic coding sequence(s) of
the gene(s) encoding the selected activty(ies). In one embodiment,
the coding sequence alteration(s) will be accomplished by
introducing: insertion or deletion mutation(s) that change the
coding sequence reading frame(s); substitution or inversion
mutations that alter a sufficient number of codons; and/or deletion
mutations that delete a sufficiently large group of contiguous
codons there from capable of producing a non-functional enzyme.
[0169] In a one embodiment in which the host cell strain has also
provided the auxotrophic gene(s) for use as selection marker(s)
therein, preferably each of the selected gene's transcription
promoter and/or transcription terminator element(s) can also be
inactivated by introduction of mutation(s), including deletion
mutations. For example, the transcription element inactivation can
be optionally performed in addition to the coding sequence
alteration(s) described above. In a one embodiment in which the
host cell strain has also provided the auxotrophic selection marker
gene(s), all of the selected gene(s)'s DNA can be deleted from the
host cell genome.
[0170] Such knock-out strains can be prepared according to any of
the various methods known in the art as effective. For example,
homologous recombination vectors containing homologous targeted
gene sequences 5' and 3' of the desired nucleic acid deletion
sequence can be transformed into the host cell. Ideally, upon
homologous recombination, a desired targeted enzymatic gene
knock-out can be produced.
[0171] Specific examples of gene knock-out methodologies include,
for example: Gene inactivation by insertion of a polynucleotide has
been previously described. See, e.g., D L Roeder & A Collmer,
Marker-exchange mutagenesis of a pectate lyase isozyme gene in
Erwinia chrysanthemi, J Bacteriol. 164(1):51-56 (1985).
Alternatively, transposon mutagenesis and selection for desired
phenotype (such as the inability to metabolize benzoate or
anthranilate) can be used to isolate bacterial strains in which
target genes have been insertionally inactivated. See, e.g., K Nida
& P P Cleary, Insertional inactivation of streptolysin S
expression in Streptococcus pyogenes, J Bacteriol. 155(3):1156-61
(1983). Specific mutations or deletions in a particular gene can be
constructed using cassette mutagenesis, for example, as described
in J A Wells et al., Cassette mutagenesis: an efficient method for
generation of multiple mutations at defined sites, Gene
34(2-3):315-23 (1985); whereby direct or random mutations are made
in a selected portion of a gene, and then incorporated into the
chromosomal copy of the gene by homologous recombination.
[0172] In one embodiment, both the organism from which the
selection marker gene(s) is obtained and the host cell in which the
selection marker gene(s) is utilized can be selected from a
prokaryote. In a particular embodiment, both the organism from
which the selection marker gene(s) is obtained and the host cell in
which a selection marker gene(s) is utilized can be selected from a
bacteria. In another embodiment, both the bacteria from which the
selection marker gene(s) is obtained and the bacterial host cell in
which a selection marker gene(s) is utilized, will be selected from
the Proteobacteria. In still another embodiment, both the bacteria
from which the selection marker gene(s) is obtained and the
bacterial host cells in which a selection marker gene(s) is
utilized, can be selected from the Pseudomonads and closely related
bacteria or from a Subgroup thereof, as defined below.
[0173] In a particular embodiment, both the selection marker
gene(s) source organism and the host cell can be selected from the
same species. Preferably, the species will be a prokaryote; more
preferably a bacterium, still more preferably a Proteobacterium. In
another particular embodiment, both the selection marker gene(s)
source organism and the host cell can be selected from the same
species in a genus selected from the Pseudomonads and closely
related bacteria or from a Subgroup thereof, as defined below. In
one embodiment, both the selection marker gene(s) source organism
and the host cell can be selected from a species of the genus
Pseudomonas, particularly the species Pseudomonas fluorescens, and
preferably the species Pseudomonas fluorescens biotype A.
III. LacI Insertion
[0174] The present invention provides Pseudomonads and related
cells that have been genetically modified to contain a
chromosomally insert lacI transgene or derivative, other than as
part of a whole or truncated PlacI-lacI-lacZYA operon. In one
embodiment, the lacI insert provides stringent expression vector
control through the expression of the LacI repressor protein which
binds to the lacO sequence or derivative on the vector, and
inhibits a Plac-Ptac family promoter on the vector. The result is
reduced basal levels of recombinant polypeptide expression prior to
induction.
[0175] In one embodiment, Pseudomonad host cells containing a
chromosomal insertion of a native E.coli lacI gene, or lacI gene
derivative such as lacI.sup.Q or lacI.sup.Q 1, are provided wherein
the lacI insert is other than part of a whole or truncated,
structural gene-containing PlacI-lacI-lacZYA construct. Other
derivative lacI transgenes useful in the present invention include:
lacI derivatives that have altered codon sequences different from a
native lacI gene (for example, the native E.coli lacI gene contains
a `gtg` initiation codon, and this may be replaced by an
alternative initiation codon effective for translation initiation
in the selected expression host cell, e.g., `atg`); lacI
derivatives that encode LacI proteins having mutated amino acid
sequences, including temperature-sensitive lacI mutants, such as
that encoded by lacI.sup.ts (or "lacI(Ts)"), which respond to a
shift in temperature in order to achieve target gene induction,
e.g., a shift up to 42.degree. C. (see, e.g., Bukrinsky et al.,
Gene 70:415-17 (1989); N Hasan & W Szybalski, Gene 163(1):35-40
(1995); H Adari et al., DNA Cell Biol. 14:945-50 (1995)); LacI
mutants that respond to the presence of alternative sugars other
than lactose in order to achieve induction, e.g., arabinose,
ribose, or galactose (see, e.g., WO 99/27108 for Lac Repressor
Proteins with Altered Responsivity); and LacI mutants that exhibit
at least wild-type binding to lac operators, but enhanced
sensitivity to an inducer (e.g., IPTG), or that exhibit enhanced
binding to lac operators, but at least wild-type de-repressibility
(see, e.g., L Swint-Kruse et al., Biochemistry 42(47):14004-16
(2003)).
[0176] In a particular embodiment, the gene encoding the Lac
repressor protein inserted into the chromosome is identical to that
of native E.coli lacI gene, and has the nucleic acid sequence of
SEQ ID NO. 9 (Table 10). In another embodiment, the gene inserted
into the host chromosome encodes the Lac repressor protein having
the amino acid sequence of SEQ ID NO. 10 (Table 1 1).
12TABLE 10 NUCLEIC ACID SEQUENCE OF NATIVE E. COLI LACI GENE
Gacaccatcgaatggcgcaaaacctttcgcggtatggcat- gatagcgcccggaagagagtca
SEQ ID NO 9
attcagggtggtgaatgtgaaaccagtaacgttatacgatgtcgcagagtatgccggtgtct
cttatcagaccgtttcccgcgtggtgaaccaggccagccacgtttctgcgaaaacgcgggaa
aaagtggaagcggcgatggcggagctgaattacattcccaaccgcgtggcacaacaactggc
gggcaaacagtcgttgctgattggcgttgccacctccagtctggccctgcacgcgcc- gtcgc
aaattgtcgcggcgattaaatctcgcgccgatcaactgggtgccagcgtggt- ggtgtcgatg
gtagaacgaagcggcgtcgaagcctgtaaagcggcggtgcacaatct- tctcgcgcaacgcgt
cagtgggctgatcattaactatccgctggatgaccaggatgc- cattgctgtggaagctgcct
gcactaatgttccggcgttatttcttgatgtctctga- ccagacacccatcaacagtattatt
ttctcccatgaagacggtacgcgactgggcgt- ggagcatctggtcgcattgggtcaccagca
aatcgcgctgttagcgggcccattaag- ttctgtctcggcgcgtctgcgtctggctggctggc
ataaatatctcactcgcaatcaaattcagccgatagcggaacgggaaggcgactggagtgcc
atgtccggttttcaacaaaccatgcaaatgctgaatgagggcatcgttcccactgcgatgct
ggttgccaacgatcagatggcgctgggcgcaatgcgcgccattaccgagtccgggctgcgcg
ttggtgcggatatctcggtagtgggatacgacgataccgaagacagctcatgttata- tcccg
ccgtcaaccaccatcaaacaggattttcgcctgctggggcaaaccagcgtgg- accgcttgct
gcaactctctcagggccaggcggtgaagggcaatcagctgttgcccg- tctcactggtgaaaa
gaaaaaccaccctggcgcccaatacgcaaaccgcctctcccc- gcgcgttggccgattcatta
atgcagctggcacgacaggtttcccgactggaaagcg- ggcagtgagcgcaacgcaattaatg
tgagttagctcactcattaggcaccccaggct- ttacactttatgcttccggctcgtatgttg
tgtggaattgtgagcggataacaattt- cacacaggaaacagctatgaccatgattacggatt
cactggccgtcgttttacaacgtcgtga
[0177]
13TABLE 11 AMINO ACID SEQUENCE OF LACI REPRESSOR Met Lys Pro Val
Thr Leu Tyr Asp Val Ala Glu Tyr Ala Gly Val SEQ ID NO. 10 Ser Tyr
Gln Thr Val Ser Arg Val Val Asn Gln Ala Ser His Val Ser Ala Lys Thr
Arg Glu Lys Val Glu Ala Ala Met Ala Glu Leu Asn Tyr Ile Pro Asn Arg
Val Ala Gln Gln Leu Ala Gly Lys Gln Ser Leu Leu Ile Gly Val Ala Thr
Ser Ser Leu Ala Leu His Ala Pro Ser Gln Ile Val Ala Ala Ile Lys Ser
Arg Ala Asp Gln Leu Gly Ala Ser Val Val Val Ser Met Val Glu Arg Ser
Gly Val Glu Ala Cys Lys Ala Ala Val His Asn Leu Leu Ala Gln Arg Val
Ser Gly Leu Ile Ile Asn Tyr Pro Leu Asp Asp Gln Asp Ala Ile Ala Val
Glu Ala Ala Cys Thr Asn Val Pro Ala Leu Phe Leu Asp Val Ser Asp Gln
Thr Pro Ile Asn Ser Ile Phe Ser His Glu Asp Gly Thr Arg Leu Gly Val
Glu His Leu Val Ala Leu Gly His Gln Gln Ile Ala Leu Leu Ala Gly Pro
Leu Ser Ser Val Ser Ala Arg Leu Arg Leu Ala Gly Trp His Lys Tyr Leu
Thr Arg Asn Gln Ile Gln Pro Ile Ala Glu Arg Glu Gly Asp Trp Ser Ala
Met Ser Gly Phe Gln Gln Thr Met Gln Met Leu Asn Glu Gly Ile Val Pro
Thr Ala Met Leu Val Ala Asn Asp Gln Met Ala Leu Gly Ala Met Arg Ala
Ile Thr Glu Ser Gly Leu Arg Val Gly Ala Asp Ile Ser Val Val Gly Tyr
Asp Asp Thr Glu Asp Ser Ser Cys Tyr Ile Pro Pro Ser Thr Thr Ile Lys
Gln Asp Phe Arg Leu Leu Gly Gln Thr Ser Val Asp Arg Leu Leu Gln Leu
Ser Gln Gly Gln Ala Val Lys Gly Asn Gln Leu Leu Pro Val Ser Leu Val
Lys Arg Lys Thr Thr Leu Ala Pro Asn Thr Gln Thr Ala Ser Pro Arg Ala
Leu Ala Asp Ser Leu Met Gln Leu Ala Arg Gln Val Ser Arg Leu Glu Ser
Gly Gln
[0178] In an alternative embodiment, the inserted lacI transgene is
a derivative of the native E.coli lacI gene. In one particular
embodiment, the lacI derivative gene is the lacI.sup.Q gene having
the nucleic acid sequence of SEQ ID NO. 11 (Table 12). The
lacI.sup.Q variant is identical to the native E.coli lacI gene
except that it has a single point mutation in the -35 region of the
promoter which increases the level of lacI repressor by 10-fold in
E.coli . See, for example, M P Calos, Nature 274 (5673): 762-65
(1978).
14TABLE 12 NUCLEIC ACID SEQUENCE OF LACI.sup.Q GENE
gacaccatcgaatggtgcaaaacctttcgcggtatggcatgatagcgccc- ggaagagagtca
SEQ ID NO. 11 attcagggtggtgaatgtgaaaccagtaac-
gttatacgatgtcgcagagtatgccggtgtct cttatcagaccgtttcccgcgtggt-
gaaccaggccagccacgtttctgcgaaaacgcgggaa
aaagtggaagcggcgatggcggagctgaattacattcccaaccgcgtggcacaacaactggc
gggcaaacagtcgttgctgattggcgttgccacctccagtctggccctgcacgcgccgtcgc
aaattgtcgcggcgattaaatctcgcgccgatcaactgggtgccagcgtggtggtgtcgatg
gtagaacgaagcggcgtcgaagcctgtaaagcggcggtgcacaatcttctcgcgcaa- cgcgt
cagtgggctgatcattaactatccgctggatgaccaggatgccattgctgtg- gaagctgcct
gcactaatgttccggcgttatttcttgatgtctctgaccagacaccc- atcaacagtattatt
ttctcccatgaagacggtacgcgactgggcgtggagcatctg- gtcgcattgggtcaccagca
aatcgcgctgttagcgggcccattaagttctgtctcg- gcgcgtctgcgtctggctggctggc
ataaatatctcactcgcaatcaaattcagccg- atagcggaacgggaaggcgactggagtgcc
atgtccggttttcaacaaaccatgcaa- atgctgaatgagggcatcgttcccactgcgatgct
ggttgccaacgatcagatggcgctgggcgcaatgcgcgccattaccgagtccgggctgcgcg
ttggtgcggatatctcggtagtgggatacgacgataccgaagacagctcatgttatatcccg
ccgtcaaccaccatcaaacaggattttcgcctgctggggcaaaccagcgtggaccgcttgct
gcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcactggt- gaaaa
gaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttggc- cgattcatta
atgcagctggcacgacaggtttcccgactggaaagcgggcagtgagc- gcaacgcaattaatg
tgagttagctcactcattaggcaccccaggctttacacttta- tgcttccggctcgtatgttg
tgtggaattgtgagcggataacaatttcacacaggaa- acagctatgaccatgattacggatt
cactggccgtcgttttac
[0179] In still another embodiment, the lacI derivate gene is the
lacI.sup.Q1 gene having the nucleic acid sequence of SEQ ID NO. 12
(Table 13). The lacI.sup.Q1 variant has a rearrangement which
substitutes a -35 region whose nucleotide sequence exactly matches
that of the E.coli-35 region consensus sequence, resulting in
expression that is 100-fold higher than the native promoter in
E.coli. See, for example, M P Colas & J H Miller, Mol. &
Gen. Genet. 183(3): 559-60(1980).
15TABLE 13 NUCLEIC ACID SEQUENCE OF LACI.sup.Q1 GENE
agcggcatgcatttacgttgacaccacctttcgcggtatggcatg- atagcgcccggaagaga
SEQ ID NO. 12 gtcaattcagggtggtgaatgtgaa-
accagtaacgttatacgatgtcgcagagtatgccggt
gtctcttatcagaccgtttcccgcgtggtgaaccaggccagccacgtttctgcgaaaacgcg
ggaaaaagtggaagcggcgatggcggagctgaattacattcccaaccgcgtggcacaacaac
tggcgggcaaacagtcgttgctgattggcgttgccacctccagtctggccctgcacgcgccg
tcgcaaattgtcgcggcgattaaatctcgcgccgatcaactgggtgccagcgtggtg- gtgtc
gatggtagaacgaagcggcgtcgaagcctgtaaagcggcggtgcacaatctt- ctcgcgcaac
gcgtcagtgggctgatcattaactatccgctggatgaccaggatgcc- attgctgtggaagct
gcctgcactaatgttccggcgttatttcttgatgtctctgac- cagacacccatcaacagtat
tattttctcccatgaagacggtacgcgactgggcgtg- gagcatctggtcgcattgggtcacc
agcaaatcgcgctgttagcgggcccattaagt- tctgtctcggcgcgtctgcgtctggctggc
tggcataaatatctcactcgcaatcaa- attcagccgatagcggaacgggaaggcgactggag
tgccatgtccggttttcaacaaaccatgcaaatgctgaatgagggcatcgttcccactgcga
tgctggttgccaacgatcagatggcgctgggcgcaatgcgcgccattaccgagtccgggctg
cgcgttggtgcggatatctcggtagtgggatacgacgataccgaagacagctcatgttatat
cccgccgtcaaccaccatcaaacaggattttcgcctgctggggcaaaccagcgtgga- ccgct
tgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgt- ctcactggtg
aaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccg- cgcgttggccgattc
attaatgcagctggcacgacaggtttcccgactggaaagcgg- gcagtgagcgcaacgcaatt
aatgtgagttagctcactcattaggcaccccaggctt- tacactttatgcttccggctcgtat
gttgtgtggaattgtgagcggataacaatttc- acacaggaaacagctatgaccatgattacg
gattcactggccgtcgttttac
[0180] In the present invention, the host cell chromosome can be
modified by insertion of at least one nucleic acid sequence
containing at least one copy of a gene encoding a LacI protein, the
gene being capable of use by the cell to, preferably,
constitutively express the encoded LacI protein, and the
polynucleotide containing the gene being other than a
PlacI-lacI-lacZYA nucleic acid sequence (i.e. a Plac(-) version of
the PlacI-lacI-lacZYA operon) or a PlacI-lacI-lacZ polynucleotide
(i.e. a structural lac utilization operon gene-containing portion
of such a Plac(-) operon, such as an at least partially truncated
version of a PlacI-lacI-lacZYA nucleic acid sequence).
[0181] The gene encoding the chosen LacI protein is preferably
constitutively expressed. This may be accomplished by use of any
promoter that is constitutively expressed in the selected
expression host cell. For example, a native E.coli PlacI may be
operably attached to the selected LacI coding sequence, or a
different constitutively expressed promoter may be operably
attached thereto. In some cases, a regulated promoter may be used,
provided that the regulated promoter is maintained throughout
fermentation in a state wherein the LacI protein is continually
expressed there from. In a particular embodiment, a lac or tac
family promoter is utilized in the present invention, including
Plac, Ptac, Ptrc, PtacII, PlacUV5, 1 pp-PlacUV5, lpp-lac, nprM-lac,
T71ac, T5lac, T3lac, and Pmac.
[0182] Genomic Insertion Sites
[0183] Chromosomal insertion may be performed according to any
technique known in the art. For example, see: D S Toder, "Gene
replacement in Pseudomonas aeruginosa," Methods in Enzymology
235:466-74 (1994); and J Quandt & M F Hynes, "Versatile suicide
vectors which allow direct selection for gene replacement in Gram
negative bacteria," Gene 127(1):15-21 (1993). Transposon-type
insertion techniques such as are known in the art, followed by
selection, may also be used; see, e.g., I Y Goryshin & W S
Reznikoff, "Tn5 in vitro transposition," Journal of Biological
Chemistry 273(13):7367-74 (1998). Alternatively, gene transfection
by (non-lytic) phage transduction may also be used for chromosomal
insertion; see, e.g., J H Miller, Experiments in Molecular Genetics
(1972) (Cold Spring Harbor Lab., N.Y.).
[0184] Sites within the bacterial expression host cell chromosome
that are useful places in which to insert the lacI gene(s), or
derivative thereof, include any location that is are not required
for cell function under the fermentation conditions used, for
example within any gene whose presence, transcription, or
expression is important for the healthy functioning of the cell
under the fermentation conditions used. Illustrative examples of
such insertion sites include, but are not limited to: sucrose
import and metabolism genes (e.g., sacB), fructose import and
catabolism genes (e.g., fructokinase genes, 1-phosphofructokinase
genes), aromatic carbon source import and utilization genes (e.g.,
anthranilate operon genes, such as antABC genes, benzoate operon
genes, as benABCD genes), beta-lactamase genes (e.g., ampC, bll1,
blc genes, blo genes, blp genes), alkaline phosphatase genes (e.g.,
phoA), nucleobase or nucleotide biosynthetic genes (e.g., pyrBCDEF
genes), amino acid biosynthetic genes (e.g., proABC genes),
aspartate semi-aldehyde dehydrogenase genes (e.g., asd),
3-isopropylmalate dehydrogenase genes (e.g., leuB), and
anthranilate synthase genes (e.g., trpE).
[0185] In any embodiment in which the genomic insertion has
resulted in or is concomitant with an auxotrophy, then either the
host cell will be grown in media supplying an effective replacement
metabolite to the cell to overcome (and avoid) the lethal effect,
or a replacement gene will be provided in the host cell that
expresses a biocatalyst effective to restore the corresponding
prototrophy, e.g., as a selection marker gene. The gene or genes
selected for deletion or inactivation (i.e. "knock-out") in
constructing a metabolic auxotroph can be any gene encoding an
enzyme that is operative in a metabolic pathway. The enzyme can be
one that is involved in the anabolic biosynthesis of molecules that
are necessary for cell survival. Alternatively, the enzyme can be
one that is involved in the catabolic utilization of molecules that
are necessary for cell survival. Preferably, all operative genes
encoding a given biocatalytic activity are deleted or inactivated
in order to ensure removal of the targeted enzymatic activity from
the host cell in constructing the auxotrophic host cell.
Alternatively, the host cell can exhibit a pre-existing auxotrophy
(i.e. native auxotrophy), wherein no further genetic modification
via deletion or inactivation (knock-out) need be performed.
[0186] For example, an amino acid biosynthetic gene (e.g., a proA,
proB, or proC gene) or a nucleobase or nucleotide biosynthetic gene
(e.g., pyrB, pyrC, pyrD, pyrE, or pyrF) may be used as the
insertion site, in which case a necessary biosynthetic activity is
normally disrupted, thus producing an auxotrophy. In such a case,
either: 1) the medium is supplemented to avoid metabolic reliance
on the biosynthetic pathway, as with a proline or uracil
supplement; or 2) the auxotrophic host cell is transformed with a
further gene that is expressed and thus replaces the biocatalyst(s)
missing from the biosynthetic pathway, thereby restoring
prototrophy to the cell, as with a metabolic selection marker gene
such as proC, pyrF, or thyA. In a particular embodiment, the lacI
transgene, or variant thereof, is inserted into a cell that is
concomitantly or subsequently auxotrophically induced through the
knock-out of a gene, or combination of genes, selected from the
group consisting of pyrF, thyA, and proC. In a specific embodiment,
a native E.coli lacI, lacI.sup.Q, or lacI.sup.Q1 transgene is
inserted into a cell that is concomitantly or subsequently rendered
auxotrophic through the knock-out of pyrF. In another specific
embodiment, a native E.coli lacI, lacI.sup.Q ,or lacI.sup.Q1
transgene is inserted into a cell that is concomitantly of
subsequently rendered auxotrophic through the knock-out of proC. In
still a further embodiment, a native E.coli lacI, lacI.sup.Q, or
lacI.sup.Q1 transgene is inserted into a cell that is concomitantly
or subsequently rendered auxotrophic through the knock-out of pyrF
and proC.
[0187] In another embodiment, a native E.coli lacI, lacI.sup.Q, or
lacI.sup.Q1 transgene, or derivative thereof, can be inserted into
the Levansucrase locus of the host cell. For example, in one
particular embodiment, a native E.coli lacI, lacI.sup.Q, or
lacI.sup.Q1 transgene, or derivative thereof, can be inserted in
the Levansucrase gene locus of Pseudomonas fluorescens. In
particular, a native E.coli lacI, lacI.sup.Q, or lacI.sup.Q1
transgene, or derivative thereof, can be inserted into the
Levansucrase gene locus of Pseudomonas fluorescens having the
nucleic acid sequence of SEQ ID. NO. 13 (Table 14).
16TABLE 14 OPEN READING FRAME OF PF LEVAN SUCRASE GENE LOCUS
ctacccagaacgaagatcagcgcctcaatggcctcaagg- ttctactggtcgatgattcagcc
SEQ ID NO. 13
gaagtcgttgaggtgctgaacatgctgctggaaatggaaggcgcccaagtgagcgccttcag
cgaccctttgagcgcgcttgaaacagcccgggatgcccattacgacgtgattatttcggaca
tcggcatgccgaaaatgaatggccatgagctgatgcagaagctgcgtaaagtaggccacctt
cgacaggctcccgccatcgccttaacgggctatggcgctggcaatgaccagaaaaag- gcgac
tgaatcgggctttaatgcgcatgtcagcaaacccgttggccatgattcgctc- atcaccttga
tcgaaaaactgtgccgctcccgcccctaggcgtggggcaggcgttca- agggtagatgaactg
agaaaagcgcacggacgcgcccgtttctggtcgcgacacctg- ggtatccacgctgcccaccg
tgtcgctgcgcaaggtcaggtacaacacggcctggcc- ggcgctgtcactcagcatccagacg
ctcacaccctccccggccgccctggccttgag- cggctgaggctgcagcatctcgatattgaa
accgcgcagcagctcaccgctcaactc- gacctccaggggttcctgggccttaccttgcacat
gaatcaccagcccatcggaggcgccattgcgcaaaaagcgttggtactccacgcgcaactgc
ccatcggcactgcgcacctcgcggctgctcagcggcccgctggaaaacagccctgccaagct
caagccgatcagcaccagcagcgcgtaccaacccacccgctcaaagcgccagaccttgcgct
gcaaggccatgttttcctgcaccggataattgcggctgtgtaagtcgtcagggtctg- ggttg
ttcatagcggggcccggactcaacccttgctgtgctcgggagaagacggccc- cttggtgaca
ccccgtgggccggcaatcgcccatatcgcagcgcccagaaacggcag- caccacgactaccgc
actccagcctgccttgctggccgaggcgttatcgctgcgcca- gatgctgttgatgatccacg
catcgagcagtacgaggatcactgccaggcctatcca- gaagtaagtggtttgcatgatgcac
ctccaggttatgtaacttttggtgcgcgggtg- cgggcagggttcattatttttaggttctct
gcctggcgcttggtttgccgccatcat- gcgggcaacttcgccgatctacttaatgatcgaac
ctcttcaaacaagacaagctgaaacgtctcagctcctataaaaagccaaatcatgcacaaat
gcattttttgccttgaccacgggaatcgagtcttctaaagtcaaatcactgtatatgaatac
agtaatttgattcccttcatggacgagacttactatgaaaagcaccccttcgaaatttggca
aaacaccccatcaacccagcctgtggacccgcgccgatgcgcttaaagtgcatgcgg- acgac
cccaccaccacccagccgctggtcagcgcgaacttcccggtattgagtgacg- aggtgtttat
ctgggacaccatgccgctgcgtgatatcgacggcaacatcacctccg- tcgatggctggtcgg
tgatcttcaccctcaccgcggatcgccacccgaacgacccgc- aatacctcgatcagaatggc
aactacgacgtcatccgcgactggaacgatcgccatg- gccgggcaaagatgtactactggtt
ctcccgcaccggcaaagactggaagctcggcg- gccgagtgatggctgaaggggtttcgccca
ccgtgcgcgaatgggccggcacgccga- tcctgttgaacgagcaaggcgaagtagacctgtac
tacaccgccgtcacgcccggcgcgaccatcgtcaaggtgcgtggccgcgtggtgaccaccga
gcatggcgtcagcctggtgggctttgagaaggtcaagccgctgttcgaggcggacggcaaga
tgtaccagaccgaagcgcaaaatgcgttctggggctttcgcgatccatggccgttccgcgac
ccgaaagacggcaagctgtacatgctgttcgaaggtaacgtggccggcgaacgcggc- tcgca
caaggtcggtaaagccgaaatcggcgacgtgccgccaggttatgaagacgtc- ggtaactcgc
gcttccagactgcctgcgtcggtatcgccgtggcccgcgacgaagac- ggcgacgactgggaa
atgctgccaccgctgctgaccgcggtgggcgtcaacgaccag- accgaacgcccgcacttcgt
gttccaggacggcaagtactacctgttcaccatcagc- cacaccttcacctacgccgacggcg
tgaccggcccggacggcgtgtacggcttcgtc- gccgattcgctgttcggtccgtatgtgccg
ttgaacggctctggtctggtactgggc- aacccgtcctcccaaccgttccagacctactcgca
ctgcgtcatgcccaacggcctggtgacctccttcatcgacagcgtaccgaccgacgacaccg
gcacgcagatccgtatcggcggcaccgaagcaccgacggtgggcatcaagatcaaagggcag
caaacgtttgtggtcgctgagtatgactacggttacatcccgccgatgctcgacgttacgct
caagtaaccggaggctatgaggtagcggctttgagctcgatgacaaacccgcggtga- atatt
cgctgcacctgtggcgagggagcttgctcccggttgggccggacagccgcca- tcgcaggcaa
gccagctcccacattttggttcctggggcgtcagggaggtatgtgtc- ggctgaggggccgtc
acgggagcaagctccctcgccacaggttcaacagcccattgg- gtggatattcaggaaataga
aatgcctgcaccattgagttgagtc
IV. LacO Sequences
[0188] Attempts to repress the leakiness of a promoter must be
balanced by the potential concomitant reduction in target
recombinant polypeptide expression. One approach to further repress
a promoter and reduce the leakiness of the promoters is to modify
regulatory elements known as operator sequences, to increase the
capacity of the associated repressor protein to bind to the
operator sequence without reducing the potential expression of the
target recombinant polypeptide upon induction.
[0189] It has been discovered that the use of a dual lac operator
in Pseudomonas fluorescens offers superior repression of
pre-induction recombinant protein expression without concomitant
reductions in induced protein yields.
[0190] In one embodiment, a Pseudomonad organism is provided
comprising a nucleic acid construct containing a nucleic acid
comprising at least one lacO sequence involved in the repression of
transgene expression. In a particular embodiment, the Pseudomonad
host cell is Pseudomonad fluorescens. In one embodiment, the
nucleic acid construct comprises more than one lacO sequence. In
another embodiment, the nucleic acid construct comprises at least
one, and preferably more than one, lacOid sequence. In one
embodiment, the nucleic acid construct comprises a lacO sequence,
or derivative thereof, located 3' of a promoter, and a lacO
sequence, or derivative thereof, located 5' of a promoter. In a
particular embodiment, the lacO derivative is a lacOid
sequence.
[0191] In another embodiment of the present invention, nucleic acid
constructs comprising more than one lac operator sequence, or
derivative thereof for use in a Pseudomonad host cell is provided.
In one embodiment, at least one lac operator sequence may be a
lacO.sub.id sequence.
[0192] The native E.coli lac operator acts to down regulate
expression of the lac operon in the absence of an inducer. To this
end, the lac operator is bound by the LacI repressor protein,
inhibiting transcription of the operon. It has been determined that
the LacI protein can bind simultaneously to two lac operators on
the same DNA molecule. See, for example, Muller et al., (1996)
"Repression of lac promoter as a function of distance, phase, and
quality of an auxiliary lac operator," J.Mol.Biol. 257: 21-29. The
repression is mediated by the promoter-proximal operator O.sub.1
and the two auxiliary operators O.sub.2 and O.sub.3, located 401
base pairs downstream of O.sub.1 within the coding region of the
lacZ gene and 92 bp upstream of O.sub.1, respectively (See FIG. 4).
Replacement of the native E.coli lac operator sequences with an
ideal lac operator (O.sub.id) results in increased repression of
the native lac operon in E.coli . See Muller et al., (1996)
"Repression of lac promoter as a function of distance, phase, and
quality of an auxiliary lac operator," J.Mol.Biol. 257: 21-29.
[0193] The lacO sequence or derivative can be positioned in the
E.coli native O.sub.1 position with respect to a promoter.
Alternatively, the lacO sequence or derivative can be positioned in
the E.coli O.sub.3 position with respect to a promoter. In another
embodiment, the lacO sequence or derivative can be located in the
E.coli native O.sub.1 position, the native O.sub.3 position, or
both with respect to a promoter. In one embodiment, the nucleic
acid construct contains at least one lacOid sequence either 5' to
the promoter sequence or 3' to the promoter sequence. In a
particular embodiment, the nucleic acid construct contains a lacOid
sequence 3' of a promoter, and at least one lacO sequence, or
derivative, 5' of a promoter. In an alternative embodiment, the
nucleic acid construct contains a lacOid sequence 5' of a promoter,
and at least one lacO sequence, or derivative, 3' of a promoter. In
still another embodiment, the nucleic acid construct contains a
lacOid sequence both 5' and 3' of a promoter.
[0194] In a particular embodiment, the laco sequence is lacOid
represented by SEQ ID NO. 14, or a sequence substantially
homologous. In another embodiment, a lacOid sequence of SEQ. ID.
NO. 59, or a sequence substantially homologous to SEQ ID NO. 59 is
employed.
17TABLE 15 LACOID SEQUENCE 5'-AATTGTGAGCGCTCACAATT-3' SEQ ID NO. 14
5'-tgtgtggAATTGTGAGCGCTCACAATTccaca SEQ ID NO. 59 ca-3'
[0195] V. Isolated Nucleic Acids and Amino Acids
[0196] In another aspect of the present invention, nucleic acid
sequences are provided for use in the improved production of
proteins.
[0197] In one embodiment, nucleic acid sequences encoding
prototrophy-restoring enzymes for use in an auxotrophic Pseudomonad
host cells are provided. In a particular embodiment, nucleic acid
sequences encoding nitrogenous base compound biosynthesis enzymes
purified from the organism Pseudomonas fluorescens are provided. In
one embodiment, nucleic acid sequences encoding the pyrF gene in
Pseudomonas fluorescens is provided (SEQ. ID No.s 1 and 3). In
another embodiment, a nucleic acid sequence encoding the thyA gene
in Pseudomonas fluorescens is provided (SEQ. ID. No. 4). In still
another embodiment, nucleic acid sequences encoding an amino acid
biosynthetic compound purified from the organism Pseudomonas
fluorescens are provided. In a particular embodiment, a nucleic
acid sequence encoding the proC gene in Pseudomonas fluorescens is
provided (SEQ. ID No.s 6 and 8).
[0198] In another aspect, the present invention provides novel
amino acid sequences for use in the improved production of
proteins.
[0199] In one embodiment, amino acid sequences of nitrogenous base
compound biosynthesis enzymes purified from the organism
Pseudomonas fluorescens are provided. In one embodiment, the amino
acid sequence containing SEQ. ID No. 2 is provided. In another
embodiment, an amino acid sequence containing SEQ. ID. No. 5 is
provided. In still another embodiment, amino acid sequences of an
amino acid biosynthetic compound purified from the organism
Pseudomonas fluorescens is provided. In a particular embodiment, an
amino acid sequence containing SEQ. ID No. 7 is provided.
[0200] One embodiment of the present invention is novel isolated
nucleic acid sequences of the Pseudomonas fluorescens pyrF gene
(Table 2, Seq. ID No. 1; Table 4, Seq. ID No. 3). Another aspect of
the present invention provides isolated peptide sequences of the
Pseudomonas fluorescens pyrF gene (Table 3, Seq. ID No. 2). Nucleic
and amino acid sequences containing at least 90, 95, 98 or 99%
homologous to Seq. ID Nos. 1, 2, or 3 are provided. In addition,
nucleotide and peptide sequences that contain at least 10, 15, 17,
20 or 25, 30, 40, 50, 75, 100, 150, 250, 350, 500, or 1000
contiguous nucleic or amino acids of Seq ID Nos 1, 2, or 3 are also
provided. Further provided are fragments, derivatives and analogs
of Seq. ID Nos. 1, 2, or 3. Fragments of Seq. ID Nos. 1, 2, or 3
can include any contiguous nucleic acid or peptide sequence that
includes at least about 10 bp, 15 bp, 17 bp, 20 bp, 50 bp, 100 bp,
500 bp, 1 kbp, 5 kbp or 10 kpb.
[0201] Another embodiment of the present invention is novel
isolated nucleic acid sequences of the Pseudomonas fluorescens thyA
gene (Table 5, Seq. ID No. 4). Another aspect of the present
invention provides isolated peptide sequences of the Pseudomonas
fluorescens thyA gene (Table 6, Seq. ID No. 5). Nucleic and amino
acid sequences containing at least 90, 95, 98 or 99% homologous to
Seq. ID Nos. 4 or 5 are provided. In addition, nucleotide and
peptide sequences that contain at least 10, 15, 17, 20 or 25, 30,
40, 50, 75, 100, 150, 250, 350, 500, or 1000 contiguous nucleic or
amino acids of Seq ID Nos 4 or 5 are also provided. Further
provided are fragments, derivatives and analogs of Seq. ID Nos. 4
or 5. Fragments of Seq. ID Nos. 4 or 5 can include any contiguous
nucleic acid or peptide sequence that includes at least about 10
bp, 15 bp, 17 bp, 20 bp, 50 bp, 100 bp, 500 bp, 1 kbp, 5 kbp or
10
[0202] Another embodiment of the present invention is novel
isolated nucleic acid sequences of the Pseudomonas fluorescens proC
gene (Table 7, Seq. ID No. 6; Table 9, Seq. ID. No. 8). Another
aspect of the present invention provides isolated peptide sequences
of the Pseudomonas fluorescens proC gene (Table 8, Seq. ID No. 7).
Nucleic and amino acid sequences containing at least 90, 95, 98 or
99% homologous to Seq. ID Nos. 6, 7, or 8 are provided. In
addition, nucleotide and peptide sequences that contain at least
10, 15, 17, 20 or 25, 30, 40, 50, 75, 100, 150, 250, 350, 500, or
1000 contiguous nucleic or amino acids of Seq ID Nos 6, 7, or 8 are
also provided. Further provided are fragments, derivatives and
analogs of Seq. ID Nos. 6, 7, or 8. Fragments of Seq. ID Nos. 6, 7,
or 8 can include any contiguous nucleic acid or peptide sequence
that includes at least about 10 bp, 15 bp, 17 bp, 20 bp, 50 bp, 100
bp, 500 bp, 1 kbp, 5 kbp or 10 kpb.
[0203] Sequence Homology
[0204] Sequence homology is determined according to any of various
methods well known in the art. Examples of useful sequence
alignment and homology determination methodologies include those
described below.
[0205] Alignments and searches for homologous sequences can be
performed using the U.S. National Center for Biotechnology
Information (NCBI) program, MegaBLAST (currently available at
http://www.ncbi.nlm.nih.gov/BL- AST/). Use of this program with
options for percent identity set at 70% for amino acid sequences,
or set at 90% for nucleotide sequences, will identify those
sequences with 70%, or 90%, or greater homology to the query
sequence. Other software known in the art is also available for
aligning and/or searching for homologous sequences, e.g., sequences
at least 70% or 90% homologous to an information string containing
a promoter base sequence or activator-protein-encoding base
sequence according to the present invention. For example, sequence
alignments for comparison to identify sequences at least 70% or 90%
homologous to a query sequence can be performed by use of, e.g.,
the GAP, BESTFIT, BLAST, FASTA, and TFASTA programs available in
the GCG Sequence Analysis Software Package (available from the
Genetics Computer Group, University of Wisconsin Biotechnology
Center, 1710 University Avenue, Madison, Wis. 53705), with the
default parameters as specified therein, plus a parameter for the
extent of homology set at 70% or 90%. Also, for example, the
CLUSTAL program (available in the PC/Gene software package from
Intelligenetics, Mountain View, Calif.) may be used.
[0206] These and other sequence alignment methods are well known in
the art and may be conducted by manual alignment, by visual
inspection, or by manual or automatic application of a sequence
alignment algorithm, such as any of those embodied by the
above-described programs. Various useful algorithms include, e.g.:
the similarity search method described in W. R. Pearson & D. J.
Lipman, Proc. Nat'l Acad. Sci. USA 85:2444-48 (Apr 1988); the local
homology method described in T. F. Smith & M. S. Waterman, in
Adv. Appl. Math. 2:482-89 (1981) and in J. Molec. Biol. 147:195-97
(1981); the homology alignment method described in S. B. Needleman
& C. D. Wunsch, J. Molec. Biol. 48(3):443-53 (Mar 1970); and
the various methods described, e.g., by W. R. Pearson, in Genomics
11(3):635-50 (Nov 1991); by W. R. Pearson, in Methods Molec. Biol.
24:307-31 and 25:365-89 (1994); and by D. G. Higgins & P. M.
Sharp, in Comp. Appl'ns in Biosci. 5:151-53 (1989) and in Gene
73(1):237-44 (15 Dec 1988).
[0207] Nucleic acid hybridization performed under highly stringent
hybridization conditions is also a useful technique for obtaining
sufficiently homologous sequences for use herein.
VI. Nucleic Acid Constructs
[0208] In still another aspect of the present invention, nucleic
acid constructs are provided for use in the improved production of
peptides.
[0209] In one embodiment, a nucleic acid construct for use in
transforming a Pseudomonad host cell comprising a) a nucleic acid
sequence encoding a recombinant polypeptide, and b) a nucleic acid
sequence encoding a prototrophy-enabling enzyme is provided. In
another embodiment, the nucleic acid construct further comprises c)
a Plac-Ptac family promoter. In still another embodiment, the
nucleic acid construct further comprises d) at least one lacO
sequence, or derivative, 3' of a lac or tac family promoter. In yet
another embodiment, the nucleic acid construct further comprises e)
at least one lacO sequence, or derivative, 5' of a lac or tac
family promoter. In one embodiment, the derivative lacO sequence
can be a lacOid sequence. In a particular embodiment, the
Pseudomonad organism is Pseudomonas fluorescens.
[0210] In one embodiment of the present invention, nucleic acid
constructs are provided for use as expression vectors in
Pseudomonad organisms comprising a) a nucleic acid sequence
encoding a recombinant polypeptide, b) a Plac-Ptac family promoter,
c) at least one lacO sequence, or derivative, 3' of a lac or tac
family promoter, d) at least one lacO sequence, or derivative, 5'
of a lac or tac family promoter. In one embodiment, the derivative
lacO sequence can be a lacOid sequence. In one embodiment, the
nucleic acid construct further comprises e) a prototrophy-enabling
selection marker for use in an auxotrophic Pseudomonad cell. In a
particular embodiment, the Pseudomonad organism is Pseudomonas
fluorescens.
[0211] In one embodiment of the present invention, a nucleic acid
construct is provided comprising nucleic acids that encode at least
one biosynthetic enzyme capable of transforming an auxotrophic host
cell to prototrophy. The biosynthetic enzyme can be any enzyme
capable of allowing an auxotrophic host cell to survive on a
selection medium that, without the expression of the biosynthetic
enzyme, the host cell would be incapable of survival due to the
auxotrophic metabolic deficiency. As such, the biosynthetic enzyme
can be an enzyme that complements the metabolic deficiency of the
auxotrophic host by restoring prototrophic ability to grow on
non-auxotrophic metabolite supplemented media.
[0212] In one particular embodiment, the present invention provides
a nucleic acid construct that encodes a functional
orotodine-5'-phosphate decarboxylase enzyme that complements an
pyrF(-) auxotrophic host. In a particular embodiment, the nucleic
acid construct contains the nucleic acid sequence of SEQ ID NO. 1
or 3. In an alternative embodiment, the nucleic acid construct
contains a nucleic acid sequence that encodes the amino acid
sequence of SEQ ID NO. 2.
[0213] In another particular embodiment, the present invention
provides a nucleic acid construct that encodes a functional
thymidylate synthase enzyme that complements a thyA (-) auxotrophic
host. In a particular embodiment, the nucleic acid construct
contains the nucleic acid sequence of SEQ ID NO. 4. In an
alternative embodiment, the nucleic acid construct contains a
nucleic acid sequence that encodes the amino acid sequence of SEQ
ID NO. 5.
[0214] In a further particular embodiment, the present invention
provides a nucleic acid construct that encodes a functional
.DELTA..sup.1-pyrroline-5-carboxylate reductase enzyme that
complements a proC (-) auxotrophic host. In a particular
embodiment, the nucleic acid construct contains the nucleic acid
sequence of SEQ ID NO. 6 or 8. In an alternative embodiment, the
nucleic acid construct contains the nucleic acid sequence that
encodes the amino acid sequence of SEQ ID NO. 7.
[0215] In an alternative embodiment, the present invention provides
a nucleic acid construct that encodes at least one biosynthetic
enzyme capable of transforming an auxotrophic host cell to
prototrophy and an additional non-auxotrophic selection marker.
Examples of non-auxotrophic selection markers are well known in the
art, and can include markers that give rise to
colorimetric/chromogenic or a luminescent reaction such as lacZ
gene, the GUS gene, the CAT gene, the luxAB gene, antibiotic
resistance selection markers such as amphotericin B, bacitracin,
carbapenem, cephalosporin, ethambutol, fluoroquinolones, isonizid,
cephalosporin, methicillin, oxacillin, vanomycin, streptomycin,
quinolines, rifampin, rifampicin, sulfonamides, ampicillin,
tetracycline, neomycin, cephalothin, erythromycin, streptomycin,
kanamycin, gentamycin, penicillin, and chloramphenicol resistance
genes, or other commonly used non-auxotrophic selection
markers.
[0216] In another embodiment, the expression vector can comprise
more than one biosynthetic enzyme capable of transforming an
auxotrophic host cell to prototrophy. The biosynthetic enzymes can
be any enzymes capable of allowing an auxotrophic host cell to
survive on a selection medium that, without the expression of the
biosynthetic enzyme, the host cell would be incapable of survival
due to the auxotrophic metabolic deficiency. As such, the
biosynthetic enzymes can be enzymes that complement the metabolic
deficiencies of the auxotrophic host by restoring prototrophic
ability to grow on non-auxotrophic metabolite supplemented media.
For example, an expression vector comprise a first and second
prototrophy-enabling selection marker gene, allowing the host cell
containing the construct to be maintained under either or both of
the conditions in which host cell survival requires the presence of
the selection marker gene(s). When only one of the marker-gene
dependent survival conditions is present, the corresponding marker
gene must be expressed, and the other marker gene(s) may then be
either active or inactive, though all necessary nutrients for which
the cell remains auxotrophic will still be supplied by the medium.
This permits the same target gene, or the same set of covalently
linked target genes, encoding the desired transgenic product(s)
and/or desired transgenic activity(ies), to be maintained in the
host cell continuously as the host cell is transitioned between or
among different conditions. The coding sequence of each of the
chosen selection marker genes independently can be operatively
attached to either a constitutive or a regulated promoter.
[0217] In a particular embodiment, the nucleic acid vector
comprises a nucleic acid construct that encodes a functional
orotodine-5'-phosphate decarboxylase enzyme and a functional
.DELTA..sup.1-pyrroline-5-carboxyla- te reductase enzyme that can
complement a pyrF(-) auxotrophic host cell, a proC(-) auxotrophic
host cell, or a pyrF(-)/proC(-) dual-auxotrophic host cell. In a
particular embodiment, the nucleic acid construct comprises the
nucleic acid sequences of SEQ ID NO. 1 or 3, and SEQ ID. NO. 6 or
8. In an alternative embodiment, the nucleic acid construct
contains a nucleic acid sequence that encodes the amino acid
sequences of SEQ ID NO. 2 and 7.
[0218] In an alternative particular embodiment, the nucleic acid
vector comprises a nucleic acid construct that encodes a functional
orotodine-5'-phosphate decarboxylase enzyme and a functional
thymidylate synthase enzyme that can complement a pyrF(-)
auxotrophic host cell, a thyA(-) auxotrophic host cell, or a
pyrF(-)/thyA(-) dual-auxotrophic host cell. In a particular
embodiment, the nucleic acid construct comprises the nucleic acid
sequences of SEQ ID NO. 1 or 3, and SEQ ID. NO. 4. In an
alternative embodiment, the nucleic acid construct contains a
nucleic acid sequence that encodes the amino acid sequences of SEQ
ID NO. 2 and 5.
[0219] In a particular embodiment, the nucleic acid vector
comprises a nucleic acid construct that encodes a functional
.DELTA..sup.1-pyrroline-- 5-carboxylate reductase enzyme and a
thymidylate synthase enzyme that can complement a proC(-)
auxotrophic host cell, a thyA(-) auxotrophic host cell, or a
proC(-)/thyA(-) dual-auxotrophic host cell. In a particular
embodiment, the nucleic acid construct comprises the nucleic acid
sequences of SEQ ID NO. 4, and SEQ ID. NO. 6 or 8. In an
alternative embodiment, the nucleic acid construct contains a
nucleic acid sequence that encodes the amino acid sequences of SEQ
ID NO. 5 and 7.
[0220] Promoters
[0221] In a fermentation process, once expression of the target
recombinant polypeptide is induced, it is ideal to have a high
level of production in order to maximize efficiency of the
expression system. The promoter initiates transcription and is
generally positioned 10-100 nucleotides upstream of the ribosome
binding site. Ideally, a promoter will be strong enough to allow
for recombinant polypeptide accumulation of around 50% of the total
cellular protein of the host cell, subject to tight regulation, and
easily (and inexpensively) induced.
[0222] The promoters used in accordance with the present invention
may be constitutive promoters or regulated promoters. Examples of
commonly used inducible promoters and their subsequent inducers
include lac (IPTG), lacUV5 (IPTG), tac (IPTG), trc (IPTG),
P.sub.syn (IPTG), trp (tryptophan starvation), araBAD
(1-arabinose), 1pp.sup.a (IPTG), 1pp-lac (IPTG), phoA (phosphate
starvation), recA (nalidixic acid), proU (osmolarity), cst-1
(glucose starvation), teta (tretracylin), cada (pH), nar (anaerobic
conditions), PL (thermal shift to 42.degree. C.), cspA (thermal
shift to 20.degree. C.), T7 (thermal induction), T7-lac operator
(IPTG), T3-lac operator (IPTG), T5-lac operator (IPTG), T4 gene32
(T4 infection), nprM-lac operator (IPTG), Pm (alkyl- or
halo-benzoates), Pu (alkyl- or halo-toluenes), Psa1 (salicylates),
and VHb (oxygen). See, for example, Makrides, S. C. (1996)
Microbiol. Rev. 60, 512-538; Hannig G. & Makrides, S. C. (1998)
TIBTECH 16, 54-60; Stevens, R. C. (2000) Structures 8, R177-R185.
See, e.g.: J. Sanchez-Romero & V. De Lorenzo, Genetic
Engineering of Nonpathogenic Pseudomonas strains as Biocatalysts
for Industrial and Environmental Processes, in Manual of Industrial
Microbiology and Biotechnology (A. Demain & J. Davies, eds.)
pp.460-74 (1999) (ASM Press, Washington, D.C.); H. Schweizer,
Vectors to express foreign genes and techniques to monitor gene
expression for Pseudomonads, Current-Opinion in Biotechnology,
12:439-445 (2001); and R. Slater & R. Williams, The Expression
of Foreign DNA in Bacteria, in Molecular Biology and Biotechnology
(J. Walker & R. Rapley, eds.) pp.125-54 (2000) (The Royal
Society of Chemistry, Cambridge, UK).
[0223] A promoter having the nucleotide sequence of a promoter
native to the selected bacterial host cell can also be used to
control expression of the transgene encoding the target
polypeptide, e.g, a Pseudomonas anthranilate or benzoate operon
promoter (Pant, Pben). Tandem promoters may also be used in which
more than one promoter is covalently attached to another, whether
the same or different in sequence, e.g., a Pant-Pben tandem
promoter (interpromoter hybrid) or a Plac-Plac tandem promoter.
[0224] Regulated promoters utilize promoter regulatory proteins in
order to control transcription of the gene of which the promoter is
a part. Where a regulated promoter is used herein, a corresponding
promoter regulatory protein will also be part of an expression
system according to the present invention. Examples of promoter
regulatory proteins include: activator proteins, e.g., E.coli
catabolite activator protein, MalT protein; AraC family
transcriptional activators ; repressor proteins, e.g., E.coli LacI
proteins; and dual-faction regulatory proteins, e.g., E.coli NagC
protein. Many regulated-promoter/promoter-regulatory-protein pairs
are known in the art.
[0225] Promoter regulatory proteins interact with an effector
compound, i.e. a compound that reversibly or irreversibly
associates with the regulatory protein so as to enable the protein
to either release or bind to at least one DNA transcription
regulatory region of the gene that is under the control of the
promoter, thereby permitting or blocking the action of a
transcriptase enzyme in initiating transcription of the gene.
Effector compounds are classified as either inducers or
co-repressors, and these compounds include native effector
compounds and gratuitous inducer compounds. Many
regulated-promoter/promoter-regulatory-protein/ef- fector-compound
trios are known in the art. Although an effector compound can be
used throughout the cell culture or fermentation, in a particular
embodiment in which a regulated promoter is used, after growth of a
desired quantity or density of host cell biomass, an appropriate
effector compound is added to the culture in order to directly or
indirectly result in expression of the desired target gene(s).
[0226] By way of example, where a lac family promoter is utilized,
a lacI gene, or derivative thereof such as a lacI.sup.Q or
lacI.sup.Q1 gene, can also be present in the system. The lacI gene,
which is (normally) a constitutively expressed gene, encodes the
Lac repressor protein (LacI protein) which binds to the lac
operator of these promoters. Thus, where a lac family promoter is
utilized, the lacI gene can also be included and expressed in the
expression system. In the case of the lac promoter family members,
e.g., the tac promoter, the effector compound is an inducer,
preferably a gratuitous inducer such as IPTG
(isopropyl-.beta.-D-1-thiogalactopyranoside, also called
"isopropylthiogalactoside").
[0227] In a particular embodiment, a lac or tac family promoter is
utilized in the present invention, including Plac, Ptac, Ptrc,
PtacII, PlacUV5, Ipp-PlacUV5, Ipp-lac, nprM-lac, T71ac, T51ac,
T31ac, and Pmac.
[0228] Other Elements
[0229] Other regulatory elements can be included in an expression
construct, including lacO sequences and derivatives, as discussed
above. Such elements include, but are not limited to, for example,
transcriptional enhancer sequences, translational enhancer
sequences, other promoters, activators, translational start and
stop signals, transcription terminators, cistronic regulators,
polycistronic regulators, tag sequences, such as nucleotide
sequence "tags" and "tag" peptide coding sequences, which
facilitates identification, separation, purification, or isolation
of an expressed polypeptide, including His-tag, Flag-tag, T7-tag,
S-tag, HSV-tag, B-tag, Strep-tag, polyarginine, polycysteine,
polyphenylalanine, polyaspartic acid, (Ala-Trp-Trp-Pro)n,
thioredoxin, beta-galactosidase, chloramphenicol acetyltransferase,
cyclomaltodextrin gluconotransferase,
CTP:CMP-3-deoxy-D-manno-octulosonate cytidyltransferase, trpE or
trpLE, avidin, streptavidin, T7 gene 10, T4 gp55, Staphylococcal
protein A, streptococcal protein G, GST, DHFR, CBP, MBP, galactose
binding domain, Calmodulin binding domain, GFP, KSI, c-myc, ompT,
ompA, pelB, , NusA, ubiquitin, and hemosylin A.
[0230] At a minimum, a protein-encoding gene according to the
present invention can include, in addition to the protein coding
sequence, the following regulatory elements operably linked
thereto: a promoter, a ribosome binding site (RBS), a transcription
terminator, translational start and stop signals. Useful RBSs can
be obtained from any of the species useful as host cells in
expression systems according to the present invention, preferably
from the selected host cell. Many specific and a variety of
consensus RBSs are known, e.g., those described in and referenced
by D. Frishman et al., Starts of bacterial genes: estimating the
reliability of computer predictions, Gene 234(2):257-65 (8 Jul.
1999); and B. E. Suzek et al., A probabilistic method for
identifying start codons in bacterial genomes, Bioinformatics
17(12):1123-30 (December 2001). In addition, either native or
synthetic RBSs may be used, e.g., those described in: EP 0207459
(synthetic RBSs); 0. Ikehata et al., Primary structure of nitrile
hydratase deduced from the nucleotide sequence of a Rhodococcus
species and its expression in Escherichia coli, Eur. J. Biochem.
181(3):563-70 (1989) (native RBS sequence of AAGGAAG). Further
examples of methods, vectors, and translation and transcription
elements, and other elements useful in the present invention are
described in, e.g.: U.S. Pat. No. 5,055,294 to Gilroy and U.S. Pat.
No. 5,128,130 to Gilroy et al.; U.S. Pat. No. 5,281,532 to Rammler
et al.; U.S. Pat. Nos. 4,695,455 and 4,861,595 to Barnes et al.;
U.S. Pat. No.4,755,465 to Gray et al.; and U.S. Pat. No. 5,169,760
to Wilcox.
[0231] Vectors
[0232] Transcription of the DNA encoding the enzymes of the present
invention by a Pseudomonad host can further be increased by
inserting an enhancer sequence into the vector or plasmid. Typical
enhancers are cis-acting elements of DNA, usually about from 10 to
300 bp in size that act on the promoter to increase its
transcription.
[0233] Generally, the recombinant expression vectors will include
origins of replication and selectable markers permitting
transformation of the Pseudomonad host cell, e.g., the prototrophy
restoring genes of the present invention, and a promoter derived
from a highly-expressed gene to direct transcription of a
downstream structural sequence. Such promoters have been described
above. The heterologous structural sequence is assembled in
appropriate phase with translation initiation and termination
sequences, and in certain embodiments, a leader sequence capable of
directing secretion of the translated polypeptide. Optionally, and
in accordance with the present invention, the heterologous sequence
can encode a fusion polypeptide including an N-terminal
identification peptide imparting desired characteristics, e.g.,
stabilization or simplified purification of expressed recombinant
product.
[0234] Useful expression vectors for use with P. fluorescens in
expressing enzymes are constructed by inserting a structural DNA
sequence encoding a desired target polypeptide together with
suitable translation initiation and termination signals in operable
reading phase with a functional promoter. The vector will comprise
one or more phenotypic selectable markers and an origin of
replication to ensure maintenance of the vector and to, if
desirable, provide amplification within the host. Suitable hosts
for transformation in accordance with the present disclosure
include various species within the genera Pseudomonas, and
particularly particular is the host cell strain of Pseudomonas
fluorescens.
[0235] Vectors are known in the art as useful for expressing
recombinant proteins in host cells, and any of these may be
modified and used for expressing the genes according to the present
invention. Such vectors include, e.g., plasmids, cosmids, and phage
expression vectors. Examples of useful plasmid vectors that can be
modified for use on the present invention include, but are not
limited to, the expression plasmids pBBR1MCS, pDSK519, pKT240,
pML122, pPS10 , RK2, RK6, pRO1600, and RSF1010. Further examples
can include pALTER-Ex1, pALTER-Ex2, pBAD/His, pBAD/Myc-His,
pBAD/gIII, pCal-n, pCal-n-EK, pCal-c, pCal-Kc, pcDNA 2.1, pDUAL,
pET-3a-c, pET 9a-d, pET-11a-d, pET-12a-c, pET-14b, pET15b, pET-16b,
pET-17b, pET-19b, pET-20b(+), pET-21a-d(+), pET-22b(+),
pET-23a-d(+), pET24a-d(+), pET-25b(+), pET-26b(+), pET-27b(+),
pET28a-c(+), pET-29a-c(+), pET-30a-c(+), pET31b(+), pET-32a-c(+),
pET-33b(+), pET-34b(+), pET35b(+), pET-36b(+), pET-37b(+),
pET-38b(+), pET-39b(+), pET-40b(+), pET41la-c(+),
pET-42a-c(+pET43a-c(+), pETBlue-1, pETBlue-2, pETBlue-3, pGEMEX-1,
pGEMEX-2, pGEX1.lambda.T, pGEX-2T, pGEX-2TK, pGEX-3X, pGEX4T,
pGEX-5X, pGEX-6P, pHAT10/11/12, pHAT20, pHAT-GFPuv, pKK223-3, pLEX,
pMAL-c2X, pMAL-c2E, pMAL-c2g, pMAL-p2X, pMAL-p2E, pMAL-p2G, pProEX
HT, pPROLar.A, pPROTet.E, pQE-9, pQE-16, pQE-30/31/32, pQE40,
pQE-50, pQE-70, pQE-80/81/82L, pQE-100, pRSET, and pSE280, pSE380,
pSE420, pThioHis, pTrc99A, pTrcHis, pTrcHis2, pTriEx-1, pTriEx-2,
pTrxFus. Other examples of such useful vectors include those
described by, e.g.: N. Hayase, in Appl. Envir. Microbiol.
60(9):3336-42 (September 1994); A. A. Lushnikov et al., in Basic
Life Sci. 30:657-62 (1985); S. Graupner & W. Wackernagel, in
Biomolec. Eng. 17(1):11-16. (October 2000); H. P. Schweizer, in
Curr. Opin. Biotech. 12(5):439-45 (October 2001); M. Bagdasarian
&. K. N. Timmis, in Curr. Topics Microbiol. Immunol. 96:47-67
(1982); T. Ishii et al., in FEMS Microbiol. Lett. 116(3):307-13
(Mar 1, 1994); I. N. Olekhnovich & Y. K. Fomichev, in Gene
140(1):63-65 (Mar 11, 1994); M. Tsuda & T. Nakazawa, in Gene
136(1-2):257-62 (Dec. 22, 1993); C. Nieto et al., in Gene
87(1):145-49 (Mar 1, 1990); J. D. Jones & N. Gutterson, in Gene
61(3):299-306 (1987); M. Bagdasarian et al., in Gene 16(1-3):237-47
(December 1981); H. P. Schweizer et al., in Genet. Eng. (NY)
23:69-81 (2001); P. Mukhopadhyay et al., in J. Bact. 172(1):477-80
(January 1990); D. O. Wood et al., in J. Bact. 145(3):1448-51
(March 1981); Holtwick et al., in Microbiology 147(Pt 2):337-44
(Febuary 2001).
[0236] Further examples of expression vectors that can be useful in
Pseudomonas host cells include those listed in Table 16 as derived
from the indicated replicons.
18TABLE 16 SOME EXAMPLES OF USEFUL EXPRESSION VECTORS Replicon
Vector(s) .sub.PPS10 .sub.PCN39, .sub.PCN51 RSF1010 .sub.PKT261-3
.sub.PMMB66EH .sub.PEB8 .sub.PPLGN1 .sub.PMYC1050 RK2/RP1
.sub.PRK415 .sub.PJB653 .sub.PRO1600 .sub.PUCP .sub.PBSP
[0237] The expression plasmid, RSF1010, is described, e.g., by F.
Heffron et al., in Proc. Nat'l Acad. Sci. USA 72(9):3623-27
(September 1975), and by K. Nagahari & K. Sakaguchi, in J.
Bact. 133(3):1527-29 (March 1978). Plasmid RSF1010O and derivatives
thereof are particularly useful vectors in the present invention.
Exemplary, useful derivatives of RSF1010, which are known in the
art, include, e.g., pKT212, pKT214, pKT231 and related plasmids,
and pMYC1050 and related plasmids (see, e.g., U.S. Pat, Nos.
5,527,883 and 5,840,554 to Thompson et al.), such as, e.g.,
pMYC1803. Plasmid pMYC1803 is derived from the RSF1010-based
plasmid pTJS260 (see U.S. Pat. No. 5,169,760 to Wilcox), which
carries a regulated tetracycline resistance marker and the
replication and mobilization loci from the RSF1010 plasmid. Other
exemplary useful vectors include those described in U.S. Pat. No.
4,680,264 to Puhler et al.
[0238] In a one embodiment, an expression plasmid is used as the
expression vector. In another embodiment, RSF1010 or a derivative
thereof is used as the expression vector. In still another
embodiment, pMYC1050 or a derivative thereof, or pMYC1803 or a
derivative thereof, is used as the expression vector.
VII. Expression of Recombinant Polypeptides in an Pseudomonad Host
Cells
[0239] In one aspect of the present invention, processes of
expressing recombinant polypeptides for use in improved protein
production are provided.
[0240] In one embodiment, the process provides expression of a
nucleic acid construct comprising nucleic acids encoding a) a
recombinant polypeptide, and b) a prototrophy-restoring enzyme in a
Pseudomonad that is auxotrophic for at least one metabolite. In an
alternative embodiment, the Pseudomonad is auxotrophic for more
than one metabolite. In one embodiment, the Pseudomonad is a
Pseudomonas fluorescens cell. In a particular embodiment, a
recombinant polypeptide is expressed in a Pseudomonad that is
auxotrophic for a metabolite, or combination of metabolites,
selected from the group consisting of a nitrogenous base compound
and an amino acid. In a more particular embodiment, recombinant
polypeptides are expressed in a Pseudomonad that is auxotrophic for
a metabolite selected from the group consisting of uracil, proline,
and thymidine. In another embodiment, the auxotrophy can be
generated by the knock-out of the host pyrF, proC, or thyA gene,
respectively. An alternative embodiment, recombinant polypeptides
are expressed in an auxotrophic Pseudomonad cell that has been
genetically modified through the insertion of a native E.coli lacI
gene, lacI.sup.Q gene, or lacI.sup.Q1 gene, other than as part of
the PlacI-lacI-lacZYA operon, into the host cell's chromosome. In
one particular embodiment, the vector containing the recombinant
polypeptide expressed in the auxotrophic host cell comprises at
least two lac operator sequences, or derivatives thereof. In still
a further embodiment, the recombinant polypeptide is driven by a
Plac family promoter.
[0241] In another embodiment, the process involves the use of
Pseudomonad host cells that have been genetically modified to
provide at least one copy of a LacI encoding gene inserted into the
Pseudomonad host cell's genome, wherein the lacI encoding gene is
other than as part of the PlacI-lacI-lacZYA operon. In one
embodiment, the gene encoding the Lac repressor protein is
identical to that of native E.coli lacI gene. In another
embodiment, the gene encoding the Lac repressor protein is the
lacI.sup.Q gene. In still another embodiment, the gene encoding the
Lac repressor protein is the lacI.sup.Q1 gene. In a particular
embodiment, the Pseudomonad host cell is Pseudomonas fluorescens.
In another embodiment, the Pseudomonad is further genetically
modified to produce an auxotrophic cell. In another embodiment, the
process produces recombinant polypeptide levels of at least about 3
g/L, 4 g/L, 5 g/L 6 g/L, 7 g/L, 8 g/L, 9 g/L or at least about 10
g/L. In another embodiment, the recombinant polypeptide is
expressed in levels of between 3 g/L and 100 g/L.
[0242] The method generally includes:
[0243] a) providing a Pseudomonad host cell, preferably a
Pseudomonas fluorescens, as described in the present invention,
[0244] b) transfecting the host cell with at least one nucleic acid
expression vector comprising i) a target recombinant polypeptide of
interest, and, in the case of the utilization of an auxotrophic
host, ii) a gene encoding a prototrophy enabling enzyme that, when
expressed, overcomes the auxotrophy of the host cell;
[0245] c) growing the host cell in a growth medium that provides a
selection pressure effective for maintaining the nucleic acid
expression vector containing the recombinant polypeptide of
interest in the host cell; and
[0246] d) expressing the target recombinant polypeptide of
interest.
[0247] The method can further comprise transfecting the host cell
with at least once nucleic acid expression vector further
comprising iii) a Plac family promoter, and optionally iv) more
than one lac operator sequences. In one embodiment, at least one
lac operator sequence may be a lac.sub.Oid sequence. Preferably,
the expression system is capable of expressing the target
polypeptide at a total productivity of polypeptide of at least 1
g/L to at least 80 g/L. In a particular embodiment, the recombinant
polypeptide is expressed at a level of at least 3 g/L, 4g/L, 5g/L,
6 g/L, 7 g/L, 8 g/L, 9 g/L, 10 g/L, 12 g/L, 15 g/L, 20 g/L, 25 gL,
30 g/L, 35 g/L, 40 g/L, 45 g/L, 50 g/L, 60 g/L, 70 g/L, or at least
80 g/L. In a particular embodiment, a lac or tac family promoter is
utilized in the present invention, including Plac, Ptac, Ptrc,
PtacII, PlacUV5, 1pp-PlacUV5, 1pp-lac, nprM-lac, T71ac, T51ac,
T31ac, and Pmac.
[0248] In one embodiment, at least one recombinant polypeptide can
be expressed in a Pseudomonad cell that is auxotrophic for one
metabolite, wherein the auxotrophy serves as a selection marker for
the maintenance of the nucleic acid expression vector encoding the
polypeptide of interest and the prototrophy-enabling enzyme.
Alternatively, more than one recombinant polypeptide can be
expressed in a Pseudomonad cell that is auxotrophic for one
metabolite, wherein the nucleic acids encoding the recombinant
polypeptides can be contained on the same vector, or alternatively,
on multiple vectors.
[0249] In yet another embodiment, more than one expression vector
encoding different target polypeptides can be maintained in a
Pseudomonad host cell auxotrophic for at least one metabolite,
wherein one expression vector contains a nucleic acid encoding a
prototrophic-enabling enzyme and a first target polypeptide of
interest, and a second expression vector contains a nucleic acid
encoding an alternative, non-auxotrophic selection marker and a
second polypeptide of interest.
[0250] In another embodiment, at least one recombinant polypeptide
can be expressed in a Pseudomonad cell that is auxotrophic for more
than one metabolite, wherein the multiple auxotrophies serve as
selection markers for the maintenance of nucleic acid expression
vectors. For example, an expression vector may be utilized in which
a first and second prototrophy-enabling selection marker gene are
present. If both marker genes are located on the same DNA
construct, the host cell containing the construct may be maintained
under either or both of the conditions in which host cell survival
requires the presence of the selection marker gene(s). When only
one of the marker-gene dependent survival conditions is present,
the corresponding marker gene must be expressed, and the other
marker gene(s) can then be either active or inactive, though all
necessary nutrients for which the cell remains auxotrophic will
still be supplied by the medium. This permits the same target gene,
or the same set of covalently linked target genes, encoding the
desired transgenic product(s) and/or desired transgenic
activity(ies), to be maintained in the host cell continuously as
the host cell is transitioned between or among different
conditions. If each of the two selection marker genes is located on
a different DNA construct, then, in order to maintain both of the
DNA constructs in the host cell, both of the marker-gene dependent
survival conditions are present, and both of the corresponding
marker gene must be expressed. This permits more than one
non-covalently linked target gene or set of target gene(s) to be
separately maintained in the host cell. The coding sequence of each
of the chosen selection marker genes independently can be
operatively attached to either a constitutive or a regulated
promoter.
[0251] Dual-target-gene examples of such a multi-target-gene system
include, but are not limited to: (1) systems in which the
expression product of one of the target genes interacts with the
other target gene itself; (2) systems in which the expression
product of one of the target genes interacts with the other target
gene's expression product, e.g., a protein and its binding protein
or the .alpha.- and .beta.- polypeptides of an .alpha.n-.beta.n
protein; (3) systems in which the two expression products of the
two genes both interact with a third component, e.g., a third
component present in the host cell; (4) systems in which the two
expression products of the two genes both participate in a common
biocatalytic pathway; and (5) systems in which the two expression
products of the two genes function independently of one another,
e.g., a bi-clonal antibody expression system.
[0252] In one example of a dual-target-gene system of the
above-listed type (1), a first target gene can encode a desired
target protein, wherein the first target gene is under the control
of a regulated promoter; the second target gene may then encode a
protein involved in regulating the promoter of the first target
gene, e.g., the second target gene may encode the first target
gene's promoter activator or repressor protein. In an example in
which the second gene encodes a promoter regulatory protein for the
first gene, the coding sequence of the second gene can be under the
control of a constitutive promoter. In one embodiment, the second
gene will be part of a separate DNA construct that is a maintained
in the cell as a high-copy-number construct with a copy number of
at least 10, 20, 30, 40, 50, or more than 50 copies being
maintained in the host cell.
[0253] In another embodiment, the present invention provides the
use of more than one lacO sequence on an expression vector in the
production of recombinant polypeptides in Pseudomonads,
particularly in Pseudomonas fluorescens
[0254] In another aspect, the present invention provides a method
of producing a recombinant polypeptide comprising transforming a
bacterial host cell that is a member of the Pseudomonads and
closely related bacteria having at least one chromosomally inserted
copy of a Lac repressor protein encoding a lacI transgene, or
derivative thereof such as lacI.sup.Q1 or lacI.sup.Q1, which
transgene is other than part of a whole or truncated structural
gene containing PlacI-lacI-lacZYA construct with a nucleic acid
construct encoding at least one target recombinant polypeptide. The
nucleic acid encoding at least one target recombinant polypeptide
can be operably linked to a Plac family promoter, in which all of
the Plac family promoters present in the host cell are regulated by
Lac repressor proteins expressed solely from the lacI transgene
inserted in the chromosome. Optionally, the expression system is
capable of expressing the target polypeptide at a total
productivity of at least 3 g/L to at least 10 g/L. Preferably, the
expression system is capable of expressing the target polypeptide
at a total productivity of polypeptide of at least 3 g/L, 4g/L,
5g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, or at least 10 g/L.
[0255] In one embodiment, the present invention provides a method
of expressing recombinant polypeptides in an expression system
utilizing auxotrophic Pseudomonads or related bacteria that have
been further genetically modified to provide at least one copy of a
LacI encoding gene inserted into the cell's genome, other than as
part of the PlacI-lacI-lacZYA operon. In a particular embodiment, a
recombinant polypeptide is expressed in an auxotrophic Pseudomonas
fluorescens host cell containing a lacI transgene insert. In
another particular embodiment, a recombinant polypeptide is
expressed in an auxotrophic Pseudomonas fluorescens host cell
containing a lacI.sup.Q1 transgene insert. In still another
particular embodiment, a recombinant polypeptide is expressed in an
auxotrophic Pseudomonas fluorescens host cell containing a
lacI.sup.Q1 transgene insert. The Pseudomonas fluorescens host can
be auxotrophic for a biochemical required by the cell for survival.
In a particular embodiment, the Pseudomonas fluorescens cell is
auxotrophic for a nitrogenous base. In a particular embodiment, the
Pseudomonas fluorescens is auxotrophic for a nitrogenous base
selected from the group consisting of thymine and uracil. In a
particularly particular embodiment, the Pseudomonas fluorescens
host cell's auxotrophy is induced by a genetic modification to a
pyrF or thyA gene rendering the associated encoded product
non-functional. In an alternative embodiment, the Pseudomonas
fluorescens cell is auxotrophic for an amino acid. In a particular
embodiment, the Pseudomonas fluorescens is auxotrophic for the
amino acid proline. In a particularly particular embodiment, the
Pseudomonas fluorescens host cell's auxotrophy is induced by a
genetic modification to a proC gene rendering the associated
encoded product non-functional.
[0256] Transformation
[0257] Transformation of the Pseudomonad host cells with the
vector(s) may be performed using any transformation methodology
known in the art, and the bacterial host cells may be transformed
as intact cells or as protoplasts (i.e. including cytoplasts).
Exemplary transformation methodologies include poration
methodologies, e.g., electroporation, protoplast fusion, bacterial
conjugation, and divalent cation treatment, e.g., calcium chloride
treatment or CaCl/Mg.sup.2+ treatment, or other well known methods
in the art. See, e.g., Morrison, J. Bact., 132:349-351 (1977);
Clark-Curtiss & Curtiss, Methods in Enzymology, 101:347-362 (Wu
et al., eds, 1983), Sambrook et al., Molecular Cloning, A
Laboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer and
Expression: A Laboratory Manual (1990); and Current Protocols in
Molecular Biology (Ausubel et al., eds., 1994)).
[0258] Selection
[0259] Preferably, cells that are not successfully transformed are
selected against following transformation, and continuously during
the fermentation. The selection marker can be an auxotrophic
selection marker or a traditional antibiotic selection marker. When
the cell is auxotrophic for multiple nutrient compounds, the
auxotrophic cell can be grown on medium supplemented with all of
those nutrient compounds until transformed with the
prototrophy-restoring vector. Where the host cell is or has been
made defective for multiple biosynthetic activities, the
prototrophy-restorative marker system(s) can be selected to restore
one or more or all of the biosynthetic activities, with the
remainder being compensated for by continuing to provide, in the
medium, the still-lacking nutrients. In selection marker systems in
which more than one biosynthetic activity, and/or more than one
prototrophy, is restored, the plurality of selection marker genes
may be expressed together on one vector or may be co-expressed
separately on different vectors. Even where a single metabolite is
the target of the selection marker system, multiple biosynthetic
activities may be involved in the selection marker system. For
example, two or more genes encoding activities from the same
anabolic pathway may be expressed together on one vector or may be
co-expressed separately on different vectors, in order to restore
prototrophy in regard to biosynthesis of the compound that is the
product of the pathway.
[0260] Where the selection marker is an antibiotic resistance gene,
the associated antibiotic can be added to the medium to select
against non transformed and revertant cells, as well known in the
art.
[0261] Fermentation
[0262] As used herein, the term "fermentation" includes both
embodiments in which literal fermentation is employed and
embodiments in which other, non-fermentative culture modes are
employed. Fermentation may be performed at any scale. In one
embodiment, the fermentation medium may be selected from among rich
media, minimal media, a mineral salts media; a rich medium may be
used, but is preferably avoided. In another embodiment either a
minimal medium or a mineral salts medium is selected. In still
another embodiment, a minimal medium is selected. In yet another
embodiment, a mineral salts medium is selected. Mineral salts media
are particularly particular.
[0263] Prior to transformation of the host cell with a nucleic acid
construct encoding a prototrophic enabling enzyme, the host cell
can be maintained in a media comprising a supplemental metabolite,
or analogue thereof, that complements the auxotrophy. Following
transformation, the host cell can be grown in a media that is
lacking the complementary metabolite that the host cell is
auxotrophic for. In this way, host cells that do not contain the
selection marker enabling prototrophy are selected against.
Likewise cells expressing recombinant proteins from expression
vectors containing an antibiotic resistance selection marker gene
can be maintained prior to transformation on a medium lacking the
associated antibiotic used for selection. After transformation and
during the fermentation, an antibiotic can be added to the medium,
at concentrations known in the art, to select against
non-transformed and revertant cells.
[0264] Mineral salts media consists of mineral salts and a carbon
source such as, e.g., glucose, sucrose, or glycerol. Examples of
mineral salts media include, e.g., M9 medium, Pseudomonas medium
(ATCC 179), Davis and Mingioli medium (see, B D Davis & E S
Mingioli, in J. Bact. 60:17-28 (1950)). The mineral salts used to
make mineral salts media include those selected from among, e.g.,
potassium phosphates, ammonium sulfate or chloride, magnesium
sulfate or chloride, and trace minerals such as calcium chloride,
borate, and sulfates of iron, copper, manganese, and zinc. No
organic nitrogen source, such as peptone, tryptone, amino acids, or
a yeast extract, is included in a mineral salts medium. Instead, an
inorganic nitrogen source is used and this may be selected from
among, e.g., ammonium salts, aqueous ammonia, and gaseous ammonia.
A particular mineral salts medium will contain glucose as the
carbon source. In comparison to mineral salts media, minimal media
can also contain mineral salts and a carbon source, but can be
supplemented with, e.g., low levels of amino acids, vitamins,
peptones, or other ingredients, though these are added at very
minimal levels.
[0265] In one embodiment, media can be prepared using the
components listed in Table 16 below. The components can be added in
the following order: first (NH.sub.4)HPO.sub.4, KH.sub.2PO.sub.4
and citric acid can be dissolved in approximately 30 liters of
distilled water; then a solution of trace elements can be added,
followed by the addition of an antifoam agent, such as Ucolub N
115. Then, after heat sterilization (such as at approximately
121.degree. C.), sterile solutions of glucose MgSO.sub.4 and
thiamine-HCL can be added. Control of pH at approximately 6.8 can
be achieved using aqueous ammonia. Sterile distilled water can then
be added to adjust the initial volume to 371 minus the glycerol
stock (123 mL). The chemicals are commercially available from
various suppliers, such as Merck. This media can allow for high
cell density cultivation (HCDC) for growth of Pseudomonas species
and related bacteria. The HCDC can start as a batch process which
is followed by two-phase fed-batch cultivation. After unlimited
growth in the batch part, growth can be controlled at a reduced
specific growth rate over a period of 3 doubling times in which the
biomass concentration can increased several fold. Further details
of such cultivation procedures is described by Riesenberg, D.;
Schulz, V.; Knorre, W. A.; Pohl, H. D.; Korz, D.; Sanders, E. A.;
Ross, A.; Deckwer, W. D. (1991) "High cell density cultivation of
Escherichia coli at controlled specific growth rate" J Biotechnol:
20(1) 17-27.
[0266] The expression system according to the present invention can
be cultured in any fermentation format. For example, batch,
fed-batch, semi-continuous, and continuous fermentation modes may
be employed herein.
[0267] The expression systems according to the present invention
are useful for transgene expression at any scale (i.e. volume) of
fermentation. Thus, e.g., microliter-scale, centiliter scale, and
deciliter scale fermentation volumes may be used; and 1 Liter scale
and larger fermentation volumes can be used. In one embodiment, the
fermentation volume will be at or above 1 Liter. In another
embodiment, the fermentation volume will be at or above 5 Liters,
10 Liters, 15 Liters, 20 Liters, 25 Liters, 50 Liters, 75 Liters,
100 Liters, 200 Liters, 50 Liters, 1,000 Liters, 2,000 Liters,
5,000 Liters, 10,000 Liters or 50,000 Liters.
[0268] In the present invention, growth, culturing, and/or
fermentation of the transformed host cells is performed within a
temperature range permitting survival of the host cells, preferably
a temperature within the range of about 4.degree. C. to about
55.degree. C., inclusive.
[0269] Cell Density
[0270] An additional advantage in using Pseudomonas fluorescens in
expressing recombinant proteins includes the ability of Pseudomonas
fluorescens to be grown in high cell densities compared to E.coli
or other bacterial expression systems. To this end, Pseudomonas
fluorescens expressions systems according to the present invention
can provide a cell density of about 20 g/L or more. The Pseudomonas
fluorescens expressions systems according to the present invention
can likewise provide a cell density of at least about 70 g/L, as
stated in terms of biomass per volume, the biomass being measured
as dry cell weight.
[0271] In one embodiment, the cell density will be at least 20 g/L.
In another embodiment, the cell density will be at least 25 g/L, 30
g/L, 35 g/L, 40 g/L, 45 g/L, 50 g/L, 60 g/L, 70 g/L 80 g/L, 90
g/L., 100 g/L, 110 g/L, 120 g/L, 130 g/L, 140 g/L, or at least 150
g/L.
[0272] In another embodiments, the cell density at induction will
be between 20 g/L and 150 g/L;, 20 g/L and 120 g/L; 20 g/L and 80
g/L; 25 g/L and 80 g/L; 30 g/L and 80 g/L; 35 g/L and 80 g/L; 40
g/L and 80 g/L; 45 g/L and 80 g/L; 50 g/L and 80 g/L; 50 g/L and 75
g/L; 50 g/L and 70 g/L; 40 g/L and 80 g/L.
[0273] Expression Levels of Recombinant Protein
[0274] The expression systems according to the present invention
can express transgenic polypeptides at a level at between 5% and
80% total cell protein (%tcp). In one embodiment, the expression
level will be at or above 5%, 8%, 10%, 12%, 15%, 20%, 25%, 30%,
35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, or 80% tcp.
[0275] Isolation and Purification
[0276] The recombinant proteins produced according to this
invention may be isolated and purified to substantial purity by
standard techniques well known in the art, including, but not
limited to, ammonium sulfate or ethanol precipitation, acid
extraction, anion or cation exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, nickel chromatography,
hydroxylapatite chromatography, reverse phase chromatography,
lectin chromatography, preparative electrophoresis, detergent
solubilization, selective precipitation with such substances as
column chromatography, immunopurification methods, and others. For
example, proteins having established molecular adhesion properties
can be reversibly fused a ligand. With the appropriate ligand, the
protein can be selectively adsorbed to a purification column and
then freed from the column in a relatively pure form. The fused
protein is then removed by enzymatic activity. In addition, protein
can be purified using immunoaffinity columns or Ni-NTA columns.
General techniques are further described in, for example, R.
Scopes, Protein Purification: Principles and Practice,
Springer-Verlag: N.Y. (1982); Deutscher, Guide to Protein
Purification, Academic Press (1990); U.S. Pat. No. 4,511,503; S.
Roe, Protein Purification Techniques: A Practical Approach
(Practical Approach Series), Oxford Press (2001); D. Bollag, et
al., Protein Methods, Wiley-Lisa, Inc. (1996); A K Patra et al.,
Protein Expr Purif, 18(2): p/ 182-92 (2000); and R. Mukhija, et
al., Gene 165(2): p. 303-6 (1995). See also, for example, Ausubel,
et al. (1987 and periodic supplements); Deutscher (1990) "Guide to
Protein Purification," Methods in Enzymology vol. 182, and other
volumes in this series; Coligan, et al. (1996 and periodic
Supplements) Current Protocols in Protein Science Wiley/Greene, NY;
and manufacturer's literature on use of protein purification
products, e.g., Pharmacia, Piscataway, N.J., or Bio-Rad, Richmond,
Calif. Combination with recombinant techniques allow fusion to
appropriate segments, e.g., to a FLAG sequence or an equivalent
which can be fused via a protease-removable sequence. See also, for
example., Hochuli (1989) Chemische Industrie 12:69-70; Hochuli
(1990) "Purification of Recombinant Proteins with Metal Chelate
Absorbent" in Setlow (ed.) Genetic Engineering, Principle and
Methods 12:87-98, Plenum Press, NY; and Crowe, et al. (1992)
QIAexpress: The High Level Expression & Protein Purification
System QUIAGEN, Inc., Chatsworth, Calif.
[0277] Detection of the expressed protein is achieved by methods
known in the art and includes, for example, radioimmunoassays,
Western blotting techniques or immunoprecipitation.
[0278] The recombinantly produced and expressed enzyme can be
recovered and purified from the recombinant cell cultures by
numerous methods, for example, high performance liquid
chromatography (HPLC) can be employed for final purification steps,
as necessary.
[0279] Certain proteins expressed in this invention may form
insoluble aggregates ("inclusion bodies"). Several protocols are
suitable for purification of proteins from inclusion bodies. For
example, purification of inclusion bodies typically involves the
extraction, separation and/or purification of inclusion bodies by
disruption of the host cells, e.g., by incubation in a buffer of 50
mM TRIS/HCL pH 7.5, 50 mM NaCl, 5 mM MgCl.sub.2, 1 mM DTT, 0.1 mM
ATP, and 1 mM PMSF. The cell suspension is typically lysed using
2-3 passages through a French Press. The cell suspension can also
be homogenized using a Polytron (Brinknan Instruments) or sonicated
on ice. Alternate methods of lysing bacteria are apparent to those
of skill in the art (see, e.g., Sambrook et al., supra; Ausubel et
al., supra).
[0280] If necessary, the inclusion bodies can be solubilized, and
the lysed cell suspension typically can be centrifuged to remove
unwanted insoluble matter. Proteins that formed the inclusion
bodies may be renatured by dilution or dialysis with a compatible
buffer. Suitable solvents include, but are not limited to urea
(from about 4 M to about 8 M), formamide (at least about 80%,
volume/volume basis), and guanidine hydrochloride (from about 4 M
to about 8 M). Although guanidine hydrochloride and similar agents
are denaturants, this denaturation is not irreversible and
renaturation may occur upon removal (by dialysis, for example) or
dilution of the denaturant, allowing re-formation of
immunologically and/or biologically active protein. Other suitable
buffers are known to those skilled in the art.
[0281] Alternatively, it is possible to purify the recombinant
proteins or peptides from the host periplasm. After lysis of the
host cell, when the recombinant protein is exported into the
periplasm of the host cell, the periplasmic fraction of the
bacteria can be isolated by cold osmotic shock in addition to other
methods known to those skilled in the art. To isolate recombinant
proteins from the periplasm, for example, the bacterial cells can
be centrifuged to form a pellet. The pellet can be resuspended in a
buffer containing 20% sucrose. To lyse the cells, the bacteria can
be centrifuged and the pellet can be resuspended in ice-cold 5 mM
MgSO.sub.4 and kept in an ice bath for approximately 10 minutes.
The cell suspension can be centrifuged and the supernatant decanted
and saved. The recombinant proteins present in the supernatant can
be separated from the host proteins by standard separation
techniques well known to those of skill in the art.
[0282] An initial salt fractionation can separate many of the
unwanted host cell proteins (or proteins derived from the cell
culture media) from the recombinant protein of interest. One such
example can be ammonium sulfate. Ammonium sulfate precipitates
proteins by effectively reducing the amount of water in the protein
mixture. Proteins then precipitate on the basis of their
solubility. The more hydrophobic a protein is, the more likely it
is to precipitate at lower ammonium sulfate concentrations. A
typical protocol includes adding saturated ammonium sulfate to a
protein solution so that the resultant ammonium sulfate
concentration is between 20-30%. This concentration will
precipitate the most hydrophobic of proteins. The precipitate is
then discarded (unless the protein of interest is hydrophobic) and
ammonium sulfate is added to the supernatant to a concentration
known to precipitate the protein of interest. The precipitate is
then solubilized in buffer and the excess salt removed if
necessary, either through dialysis or diafiltration. Other methods
that rely on solubility of proteins, such as cold ethanol
precipitation, are well known to those of skill in the art and can
be used to fractionate complex protein mixtures.
[0283] The molecular weight of a recombinant protein can be used to
isolated it from proteins of greater and lesser size using
ultrafiltration through membranes of different pore size (for
example, Amicon or Millipore membranes). As a first step, the
protein mixture can be ultrafiltered through a membrane with a pore
size that has a lower molecular weight cut-off than the molecular
weight of the protein of interest. The retentate of the
ultrafiltration can then be ultrafiltered against a membrane with a
molecular cut off greater than the molecular weight of the protein
of interest. The recombinant protein will pass through the membrane
into the filtrate. The filtrate can then be chromatographed as
described below.
[0284] Recombinant proteins can also be separated from other
proteins on the basis of its size, net surface charge,
hydrophobicity, and affinity for ligands. In addition, antibodies
raised against proteins can be conjugated to column matrices and
the proteins immunopurified. All of these methods are well known in
the art. It will be apparent to one of skill that chromatographic
techniques can be performed at any scale and using equipment from
many different manufacturers (e.g., Pharmacia Biotech).
[0285] Renaturation and Refolding
[0286] Insoluble protein can be renatured or refolded to generate
secondary and tertiary protein structure conformation. Protein
refolding steps can be used, as necessary, in completing
configuration of the recombinant product. Refolding and
renaturation can be accomplished using an agent that is known in
the art to promote dissociation/association of proteins. For
example, the protein can be incubated with dithiothreitol followed
by incubation with oxidized glutathione disodium salt followed by
incubation with a buffer containing a refolding agent such as
urea.
[0287] Recombinant protein can also be renatured, for example, by
dialyzing it against phosphate-buffered saline (PBS) or 50 mM
Na-acetate, pH 6 buffer plus 200 mM NaCl. Alternatively, the
protein can be refolded while immobilized on a column, such as the
Ni NTA column by using a linear 6M-7M urea gradient in 500 mM NaCl,
20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease
inhibitors. The renaturation can be performed over a period of 1.5
hours or more. After renaturation the proteins can be eluted by the
addition of 250 mM immidazole. Immidazole can be removed by a final
dialyzing step against PBS or 50 mM sodium acetate pH 6 buffer plus
200 mM NaCl. The purified protein can be stored at 4.degree. C. or
frozen at -80.degree. C.
[0288] Other methods include, for example, those that may be
described in M H Lee et al., Protein Expr. Purif., 25(1): p. 166-73
(2002), W. K. Cho et al., J. Biotechnology, 77(2-3): p. 169-78
(2000), Ausubel, et al. (1987 and periodic supplements), Deutscher
(1990) "Guide to Protein Purification," Methods in Enzymology vol.
182, and other volumes in this series, Coligan, et al. (1996 and
periodic Supplements) Current Protocols in Protein Science
Wiley/Greene, NY, S. Roe, Protein Purification Techniques: A
Practical Approach (Practical Approach Series), Oxford Press
(2001); D. Bollag, et al., Protein Methods, Wiley-Lisa, Inc.
(1996).
VI. Recombinant Polypeptides
[0289] The present invention provides improved protein production
in bacterial expression systems. Examples of recombinant
polypeptides that can be used in the present invention include
polypeptides derived from prokaryotic and eukaryotic organisms.
Such organisms include organisms from the domain Archea, Bacteria,
Eukarya, including organisms from the Kingdom Protista, Fungi,
Plantae, and Animalia.
[0290] Types of proteins that can be utilized in the present
invention include non-limiting examples such as enzymes, which are
responsible for catalyzing the thousands of chemical reactions of
the living cell; keratin, elastin, and collagen, which are
important types of structural, or support, proteins; hemoglobin and
other gas transport proteins; ovalbumin, casein, and other nutrient
molecules; antibodies, which are molecules of the immune system;
protein hormones, which regulate metabolism; and proteins that
perform mechanical work, such as actin and myosin, the contractile
muscle proteins.
[0291] Other specific non-limiting polypeptides include molecules
such as, e.g., renin, a growth hormone, including human growth
hormone; bovine growth hormone; growth hormone releasing factor;
parathyroid hormone; thyroid stimulating hormone; lipoproteins;
alpha. 1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin;
thrombopoietin; follicle stimulating hormone; calcitonin;
luteinizing hormone; glucagon; clotting factors such as factor
VIIIC, factor IX, tissue factor, and von Willebrands factor;
anti-clotting factors such as Protein C; atrial naturietic factor;
lung surfactant; a plasminogen activator, such as urokinase or
human urine or tissue-type plasminogen activator (t-PA); bombesin;
thrombin; hemopoietic growth factor; tumor necrosis factor-alpha
and -beta; enkephalinase; a serum albumin such as human serum
albumin; mullerian-inhibiting substance; relaxin A-chain; relaxin
B-chain; prorelaxin; mouse gonadotropin-associated peptide; a
microbial protein, such as beta-lactamase; Dnase; inhibin; activin;
vascular endothelial growth factor (VEGF); receptors for hormones
or growth factors; integrin; protein A or D; rheumatoid factors; a
neurotrophic factor such as brain-derived neurotrophic factor
(BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT4, NT-5, or NT-6),
or a nerve growth factor such as NGF-.beta.; cardiotrophins
(cardiac hypertrophy factor) such as cardiotrophin-1 (CT-1);
platelet-derived growth factor (PDGF); fibroblast growth factor
such as aFGF and bFGF; epidermal growth factor (EGF); transforming
growth factor (TGF) such as TGF-alpha and TGF-beta, including
TGF-.beta.1, TGF-.beta.2, TGF-.beta.3, TGF-.beta.4, or TGF-.beta.5;
insulin-like growth factor-I and -II (IGF-I and IGF-II);
des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor binding
proteins; CD proteins such as CD-3, CD-4, CD-8, and CD-19;
erythropoietin; osteoinductive factors; immunotoxins; a bone
morphogenetic protein (BMP); an interferon such as
interferon-alpha, -beta, and -gamma; colony stimulating factors
(CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g.,
IL-1 to IL-10; anti-HER-2 antibody; superoxide dismutase; T-cell
receptors; surface membrane proteins; decay accelerating factor;
viral antigen such as, for example, a portion of the AIDS envelope;
transport proteins; homing receptors; addressins; regulatory
proteins; antibodies; and fragments of any of the above-listed
polypeptides.
[0292] The recombinant peptides to be expressed by according to the
present invention can be expressed from polynucleotides in which
the target polypeptide coding sequence is operably attached to
transcription and translation regulatory elements to form a
functional gene from which the host cell can express the protein or
peptide. The coding sequence can be a native coding sequence for
the target polypeptide, if available, but will more preferably be a
coding sequence that has been selected, improved, or optimized for
use in the selected expression host cell: for example, by
synthesizing the gene to reflect the codon use bias of a
Pseudomonas species such as Pseudomonas fluorescens. The gene(s)
that result will have been constructed within or will be inserted
into one or more vector, which will then be transformed into the
expression host cell. Nucleic acid or a polynucleotide said to be
provided in an "expressible form" means nucleic acid or a
polynucleotide that contains at least one gene that can be
expressed by the selected bacterial expression host cell.
[0293] Extensive sequence information required for molecular
genetics and genetic engineering techniques is widely publicly
available. Access to complete nucleotide sequences of mammalian, as
well as human, genes, cDNA sequences, amino acid sequences and
genomes can be obtained from GenBank at the URL address
http://www.ncbi.nlm.nih.gov/Entrez. Additional information can also
be obtained from GeneCards, an electronic encyclopedia integrating
information about genes and their products and biomedical
applications from the Weizmann Institute of Science Genome and
Bioinformatics (http:/Ibioinformatics.weizmann.ac.il/cards/),
nucleotide sequence information can be also obtained from the EMBL
Nucleotide Sequence Database ( http://www.ebi.ac.uk/embl/) or the
DNA Databank or Japan (DDBJ, http://www.ddbi.nig.ac.jp/; additional
sites for information on amino acid sequences include Georgetown's
protein information resource website
(http://www-nbrf.georgetown.edu/pir/) and Swiss-Prot
(http://au.expasy.org/sprot/sprot-top.html).
EXAMPLES
Example 1
Construction of a pyrF Selection Marker System in a P. fluorescens
Host Cell Expression System
[0294] Reagents were acquired from Sigma-Aldrich (St. Louis Mo.)
unless otherwise noted. LB is 10 g/L tryptone, 5 g/L yeast extract
and 5 g/L NaCI in a gelatin capsule (BIO 101). When required,
uracil (from BIO101, Carlsbad Calif.) or L-proline was added to a
final concentration of 250 ug/mL, and tetracycline was added to 15
ug/mL. LB/5-FOA plates contain LB with 250 mM uracil and 0.5 mg/mL
5-fluoroorotic acid (5-FOA). M9 media consists of 6 g/L
Na.sub.2HPO.sub.4, 3 g/L KH.sub.2PO.sub.4, 1 g/L NH.sub.4Cl, 0.5
g/L NaCl, 10 mM MgSO.sub.4, 1.times. HoLe Trace Element Solution,
pH7. Glucose was added to a final concentration of 1%. The
1000.times. HoLe Trace Element Solution is 2.85 g/L
H.sub.3BO.sub.3, 1.8 g/L MnCl.sub.2 . 4H2O, 1.77 g/L sodium
tartrate, 1.36 g/L FeSO.sub.4. 7H2O, 0.04 g/L CoCl.sub.2.
6H.sub.2O, 0.027 g/L CuCl.sub.2. 2H.sub.2O, 0.025 g/L
Na.sub.2MoO.sub.4. 2H.sub.2O, 0.02 g/L ZnCl.sub.2.
Oligonucleotides Used Herein
[0295]
19 MB214pyrF1 (NotI site in bold) 5'-GCGGCCGCTTTGGCGCTTCGT-
TTACAGG-3' (SEQ ID NO: 14) MB214pyrR1 (PvuI site in bold; KpnI site
in underlined bold) 5'-CGATCGGGTACCTGTCGAAGGGCTGG- AGACA (SEQ ID
NO: 15) T-3' pyrFPstF (PstI site in bold)
5'-AACTGCAGGATCAGTTGCGGAGCCTTGG-3' (SEQ ID NO: 16) pyrFoverlap
5'-TGCTCACTCTAAAAATCTGGAATGGGCTCTC (SEQ ID NO: 17) AGGC-3'
pyrFXbaR2 (XbaI site in bold) 5'-GCTCTAGATGCGTGGCTGGATGAATGAA-3'
(SEQ ID NO: 18) pyrana1F 5'-GGCGTCGAACAGGTAGCCTT-3' (SEQ ID NO: 19)
pyrana1R 5'-CTCGCCTCCTGCCACATCAA-3' (SEQ ID NO: 20) M13F(-40)
5'-CAGGGTTTTCCCAGTCACGA-3' (SEQ ID NO: 21)
Cloning of a pyrF gene from P. fluorescens
[0296] The pyrF gene was cloned from P. fluorescens by polymerase
chain reaction (PCR) amplification, using primers MB214pyrF1 and
MB214pyrR1 that bind 297 bp upstream from the pyrF gene start codon
and 212 bp downstream of its stop codon, respectively. Restriction
sites were included at the 5' ends of the primers to facilitate
further cloning reactions The amplified region upstream of the pyrF
open reading frame (ORF) was estimated as long enough to include
the native promoter upstream of pyrF. A strong stem-loop structure
at 14-117 bp downstream of the pyrF ORF, which may be a
transcription terminator, was also included in the downstream
flanking region.
[0297] To PCR-amplify the pyrF gene, the high-fidelity PROOFSTART
DNA polymerase was mixed in a 50 uL reaction volume containing
buffer provided by the manufacturer (Qiagen, Valencia Calif.) 0.3
mM dNTPs (Promega, Madison, Wis.), 1 uM each of MB214pyrF1 and
MB214pyrR1 primers, and about 0.3 .mu.g of genomic DNA from P.
fluorescens MB214. The amplification conditions were 5 min at
95.degree. C., followed by 35 cycles of a 30 sec denaturation at
94.degree. C., 30 sec annealing at 57.degree. C., and a 2 min
extension at 72.degree. C., followed by a final step a 72.degree.
C. for 10 min. The reaction was separated on a 1% gel of SEAKEM GTG
agarose (from BioWhittaker Molecular Applications , Rockland Me.).
The expected 1.2 kb band was excised from the gel and purified by
extraction on a ULTRAFREE-DA centrifugal gel nebulizer from
Millipore (Bedford Mass.) column and de-salted into Tris-HCI buffer
with a MICRoBIoSPIN 6 P-6 polyacrylamide spin column (from Bio-Rad,
Hercules Calif.).
[0298] The cloned gene contained a single ORF, encoding orotidine
5' phosphate decarboxylase. The identity of the gene was further
confirmed as pyrF by its high similarity (P-value of
3.3.times.10.sup.-78) along the entire length of the gene (209 out
of 232 residues) to the pyrF gene from P. aeruginosa, which had
been previously reported (Strych et al., 1994). The P. fluorescens
strain used was found to contain no other copies of anypyrF
genes.
[0299] Sequencing was performed by The Dow Chemical Company. The
pyrF sequence is presented within SEQ ID NO:1.
Construction of a pyrF(-) P. fluorescens
[0300] To construct a pyrF(-) P. fluorescens , the cell's genomic
pyrF gene was altered by deleting of the ORF between and including
the gene's start and stop codons. The deletion was made by fusing
in vitro the upstream and downstream regions flanking the pyrF
region on a nonreplicating plasmid, then using allele exchange,
i.e. homologous recombination, to replace the endogenous pyrF gene
in MB101 with the deletion allele.
[0301] To construct the fusion of the flanking regions, the
"Megaprimer" method (Barik 1997) was used, whereby the region
upstream and then downstream of the desired deletion were
subsequently amplified by PCR using an overlapping primer with
homology on both sides of the desired deletion, so that the
flanking regions become linked, leaving out the pyrF ORF. The
upstream region was amplified from MB214 genomic DNA using the
Proofstart polymerase (Qiagen) as described above, with the primers
pyrFPstF and pyrFoverlap, and an extension time of 1 minute. After
gel purification using binding to glass milk (GENECLEAN Spin Kit
from Bio101, Carlsbad, Calif., USA), the 1 kB product was used as
the "Megaprimer" for the second amplification.
[0302] Because there was difficulty amplifying the desired product
in this second step, a template containing the genomic pyrF region
was made by PCR amplification in order to increase the template
quantity. HOTSTARTAQ DNA polymerase (from Qiagen, Valencia Calif.)
was used with P. fluorescens genomic DNA and the pyrFPstF and
pyrFXbaR2 primers. The Megaprimer and the pyrFXbaR2 primer were
then used with this template and HOTSTARTAQ polymerase, to amplify
the deletion product by PCR, using amplification conditions of 15
min at 95.degree. C., followed by 30 cycles of a 30 sec
denaturation at 94.degree. C., 30 sec annealing at 59.degree. C.,
and a 2 min extension at 72.degree. C., followed by a final step at
72.degree. C. for 3 min. The expected 2 kB band was separated from
a number of other products by gel electrophoresis, and then gel
purified as above and cloned into plasmid pCR2.1Topo (from
Invitrogen, Carlsbad CA) according to instructions from the
manufacturer, to form pDOW1215-7 Sequencing the PCR-amplified
region of pDOW1215-7 showed that there were 3 mutations introduced
by the amplification process; all three changes were within 112 bp
downstream of the stop codon for pyrF. Sequencing through this area
was difficult, because the process of the reaction stopped in this
area. Analysis by M-FOLD (GCG) of the secondary structure of RNA
that would be encoded by this area showed the presence of a very
stable stem-loop structure and a run of uridine residues that is
characteristic of a rho-independent transcription terminator. None
of the mutations occurred in the open reading frame. pDOW 1215-7
was used to delete the chromosomal pyrF gene in MB 101. To do this,
first, electrocompetent P. fluorescens cells made according to the
procedure of Artiguenave et al. (1997), were transformed with 0.5
.mu.g of the purified plasmid. Transformants were selected by
plating on LB medium with kanamycin at 50 .mu.g/mL. This plasmid
cannot replicate in P. fluorescens , therefore kanamycin resistant
colonies result from the plasmid integrating into the chromosome.
The site of integration of the plasmid was analyzed by PCR using
the HOTSTARTAQ polymerase and primers pryanalF and M13F(-40),
annealing at 57.degree. C. and with an extension time of 4 min. One
out of the 10 isolates (MB101::pDOW1215-7#2) contained an insertion
of pDOW1215-7 into the downstream region (2.8 kB analytical
product) and in the other nine were in the upstream region (2.1 kb
analytical product).
[0303] Second, to identify strains that had lost the integrated
plasmid by recombination between the homologous regions the
following analytical PCR procedure was used: MB1010::pDOW1215-7#2
was inoculated from a single colony into LB supplemented with 250
mM uracil, grown overnight, and then plated onto LB-uracil and 500
.mu.g/mL 5-fluoroorotic acid (5-FOA--Zymo Research, Orange Calif.).
Eight colonies were analyzed by PCR with HOTSTARTAQ and primers
pyranalF and pyranalR, annealing at 57.degree. C. and extending for
4 min. The expected size of the amplified product from the parent
MB 101 was 3.2 kB, or if the pyrF gene was deleted, then 2.5 kB.
Each of the colonies gave rise to the 2.5 kB band expected from a
deletion of pyrF. The first three isolates were purified and named
PFG116, PFG117, and PFG118 (also known as DC36). The three isolates
exhibit the phenotype expected from a pyrF deletion, i.e. they are
sensitive to kanamycin, uracil is required for growth, and they are
resistant to 5-FOA. The DNA sequence of PFG118 was identical to
that of the amplified regions in pDOW1215-7; i.e. the three
mutations in the stem-loop structure immediately downstream from
pyrF were incorporated into the PFG 118 genome, along with the pyrF
deletion.
Use of the pyrF Gene as a Selection Marker in P. fluorescens
Expression System
[0304] The ability of the pyrF gene to act as a selectable marker
was tested by cloning it into a pMYC expression plasmid containing
both an existing tetracycline resistance marker and the target
enzyme coding sequence under the control of the tac promoter. For
this, the plasmid pMYC5088 was digested at 37.degree. C. for 2 hr
with SnaBI in a 50 uL reaction using NEB Buffer 4 and 0.1 mg/mL of
bovine serum albumen (BSA) (from New England Biolabs, Beverly
Mass.). The reaction mixture was then treated at 70.degree. C. for
20 min to inactivate the enzyme, then gel-purified as described
above. 60 ng of the SnaBI-digested pMYC5088 was ligated to 50 ng of
the MB214pyrF1- MB214pyrR1 PCR product using the FAST-LINK DNA
Ligation Kit (Epicentre Technologies, Madison Wis.). After 1 hr at
25.degree. C., the reaction was stopped by treating the mixture at
70.degree. C. for 20 min. The result was then transformed into
chemically-competent JM109 E.coli cells (Promega Corp., Madison
Wis.) using conditions recommended by the manufacturer.
[0305] Transformants were selected on LB medium containing
tetracycline at 15 .mu.g/mL. Plasmid DNA was prepared from 12
isolates using the QiaPrep Spin Miniprep Kit (Qiagen, Valencia
Calif.) and screened with NotI and EcoRI, which indicated that one
isolate contained the desired clone, pDOW1249-2 (FIG. 2). The
plasmid pDOW1249-2 was transformed into pyrF(-) P. fluorescens
containing a pCN plasmid containing a lacI repressor expression
cassette and a kanamycin resistance marker gene. Isolates were
tested in shake flasks and in 20-L fernentors.
[0306] Isolates were grown in minimal salts medium and kanamycin,
but no tetracycline, so that the only selective pressure for the
pDOW1249-2 plasmid was provided by the ability of the pyrF gene on
the plasmid to complement the pyrF deletion in the chromosome. As
determined by SDS-PAGE analysis, the amount of target protein
produced by the new strain in the shake flask test was similar to
that of the control strain, a genomically pyrF(+) P. fluorescens
control system containing the same two plasmids, but for the
absence of the pyrF gene in pDOW1249-2, and grown on the same
medium but further supplemented with tetracycline in order to
maintain the plasmid (data not shown). Two strains were chosen for
further analysis at the 20-L scale, based on the amount of target
protein seen on the SDS-PAGE gel and OD.sub.575 values in shake
flasks. Both strains showed a level of accumulation of target
protein within the normal range observed for the control strain
(FIG. 1).
Example 2
Construction of a pyrF--proC Dual Auxotrophic Selection Marker
System in a P. fluorescens Host Cell Expression System
[0307] Oligonucleotides Used Herein
20 proC1 5'-ATATGAGCTCCGACCTTGAGTCGGCCATTG- (SEQ ID NO: 22) 3'
proC2 5'-ATATGAGCTCGGATCCAGTACGATCAG- CAGG (SEQ ID NO: 23) TACAG-3'
proC3 5'-AGCAACACGCGTATTGCCTT-3' (SEQ ID NO: 24) proC5
5'-GCCCTTGAGTTGGCACTTCATCG-3' (SEQ ID NO: 25) proC6
5'-GATAAACGCGAAGATCGGCGAGATA-3' (SEQ ID NO: 26) proC7
5'-CCGAGCATGTTTGATTAGACAGGTCCTTATT (SEQ ID NO: 27) TCGA-3' proC8
5'-TGCAACGTGACGCAAGCAGCATCCA-3' (SEQ ID NO: 28) proC9
5'-GGAACGATCAGCACAAGCCATGCTA-3' (SEQ ID NO: 29) genF2
5'-ATATGAGCTCTGCCGTGATCGAAATCCAGA- (SEQ ID NO: 30) 3' genR2
5'-ATATGGATCCCGGCGTTGTG- ACAATTTACC- (SEQ ID NO: 31) 3' XbaNotDraU2
linker 5'-TCTAGAGCGGCCGCGTT-3' (SEQ ID NO: 32) XbaNotDraL linker
5'-GCGGCCGCTCTAGAAAC-3' (SEQ ID NO: 33)
Cloning of proC from P. fluorescens and Formation of a pCN
Expression Plasmid Containing proC
[0308] Replacing Antibiotic Resistant Gene in pCN51lacI with
proC
[0309] The proC ORF and about 100 bp of adjacent upstream and
downstream sequence was amplified from MB101 genomic DNA using
proC1 and proC2, an annealing temperature of 56.degree. C. and a 1
min extension. After gel purification of the 1 kB product and
digestion with SacI, the fragment was cloned into SacI-digested
pDOW1243 (a plasmid derived from pCN51lacI by addition of a
polylinker and replacement of kanR with the gentamycin resistance
gene), to create pDOW1264-2. This plasmid was tested in the proC(-)
mutant strain PFG932 for its ability to regulate amylase synthesis
from pDOW1249-2. Expressed target enzyme production levels at the
20-L scale was similar to that of the dual-antibiotic-resistance
marker control strain DC88 (data not shown).
[0310] The genR antibiotic marker gene was then removed from the
pDOW 1264-2 (FIG. 3) to create an antibiotic-marker-free plasmid
with proC and lacI. Removing the genR gene was accomplished by
restriction digestion of pDOW1264-2 with BamHI, purification of the
6.1 kB fragment, ligation to itself, and electroporation into the
proC(-) P. fluorescens host PFG1016. Isolates were checked by
restriction digestion using EcoRI. The resulting plasmid was named
pDOW1306-6. Analytical restriction digests with EcoRI and
sequencing across the BamHI junction verified the identity of the
plasmid and the proper orientations of the genes therein.
[0311] Sequencing was performed by The Dow Chemical Company. The
proC sequence is presented within SEQ ID NO:4.
Construction of Target Enzyme Expression Plasmid Containing a pyrF
Marker in Place of an Antibiotic Resistance Marker
[0312] The antibiotic-marker-free production plasmid, pDOW1269-2,
containing a target enzyme-encoding gene under control of a tac
promoter, was constructed by restriction digestion of pDOW1249-2
with PvuI to remove the tetR/tetA genes. Derived from pMYC5088 by
insertion of the pyrF gene from MB214, pDOW1249-2 was prepared as
described in Example 1. The 10.6 kB PvuI fragment was gel-purified,
ligated to itself, transformed into PFG118/pCN51lacI by
electroporation and spread on M9 glucose medium containing
kanamycin (to retain the pCN51lacI). Plasmid DNA was isolated and
analytical restriction digests with NcoI were carried out; two
isolates showed a restriction digest that was consistent with the
expected bands. Both isolates were sequenced across the PvuI
junction, which verified the identity of the plasmids and the
proper orientations of the genes therein.
Construction of a Pseudomonas fluorescens Strain with Genomic
Deletions of pyrF and proC
[0313] PFG118, a P. fluorescens MB 101 strain with a deletion of
pyrF, was described in Example 1.
Construction of pDOW 1261-2, a Vector for Gene Replacement and
Deletion
[0314] The vector pDOW1261-2 was designed to create clean deletions
of genomic DNA, using marker exchange by the cross-in/cross-out
method (Toder 1994; Davison 2002), by combining the following
properties:
[0315] a ColEI replication origin that functions only in E.coli and
not in P. fluorescens;
[0316] a selectable marker (tetR/tetA) for integration of the
plasmid into the chromosome;
[0317] a counterselectable marker (pyrF) that allows for selection
for loss of the inserted plasmid (as long as the host strain is
pyrF-); cells that lose the pyrF gene are resistant to 5-FOA;
and
[0318] a blunt-end cloning site, SrfI, which has an uncommon 8 bp
recognition site - if the desired insert lacks the site, the
efficiency of insertion can be increased by adding SrfI
(Stratagene, La Jolla Calif.) to the ligation reaction to re-cleave
vectors that ligate without an insert.
[0319] To construct this vector, a 5 kB PstI to EcoRI fragment
containing the tetR, tetA, and pyrF genes was cloned into
pCRScriptCAM (Stratagene, La Jolla Calif.) that had been digested
with PstI and EcoRI, creating pDOW 1261-2.
Construction of a Vector to Delete proC from the Chromosome
[0320] To construct a deletion of proC, the copies of the flanking
regions upstream and downstream of the proC gene were joined
together by PCR, and then cloned into the pDOW1261-2 gene
replacement vector. The proC7 primer, which bridges the proC ORF,
was designed to delete the entire coding sequence from the ATG
start codon to the TAG stop codon. An additional 16 bp downstream
of the stop codon was also included in the deletion.
[0321] To make the PCR fusion of regions upstream and downstream
from proC, the Megaprimer method of PCR amplification was used
(Barik 1997). To make the megaprimer, the 0.5 kB region directly
upstream of the proC open reading frame was amplified by PCR from
MB214 genomic DNA, using primers proC5 and proC7. Primer proC7
overlaps the regions upstream and downstream of the proC ORF. The
polymerase chain reaction was carried out with 1 uM of primers, 200
uM each of the four dNTPs, and Herculase high-fidelity polymerase
(Stratagene, La Jolla Calif.) in the buffer recommended by the
vendor. Herculase is a high-fidelity enzyme that consists mostly of
Pfu polymerase, which leaves blunt ends. The amplification program
was 95.degree. C. for 2 min, 30 cycles of 95.degree. C. for 30 sec,
50.degree. C. for 30 sec, and 72.degree. C. for 1 min per kB,
followed by 10 min at 72.degree. C. The amplified products were
separated by 1% agarose gel electrophoresis in TBE and visualized
using ethidium bromide. A gel slice containing the DNA was cut from
the gel and purified as above The 1.3 kB region downstream from the
proC gene was amplified using primers proC3 and proC6, to serve as
a template for subsequent reactions. The same amplification
protocol was used, except for an annealing temperature of
60.degree. C. The reaction was checked on an agarose gel, and then
purified using the StrataPrep PCR Purification Kit (Stratagene, La
Jolla Calif.).
[0322] In the second step to make the deletion fusion, the
megaprimer was used as one of the primers in a PCR reaction along
with primer proC6, and with the proC3-proC6 PCR reaction as the
template. An annealing temperature of 61.degree. C. and extension
time of 2 min was used. The 1 kB PCR product was purified and
blunt-end ligated into the suicide vector pDOW1261-2 that had been
digested with SrfI. SrfI was included in the ligation in order to
decrease background caused by re-ligation of the vector, as
according to instructions from the manufacturer (pCRScriptCam
Cloning Kit--Stratagene, La Jolla Calif.). The ligation was
transformed into DH10 .beta. (Gibco BRL Life Technologies, now
Invitrogen, Carlsbad Calif.) by electroporation (2 mM gap cuvette,
25 .mu.F, 2.25 kV, 200 Ohms) (Artiguenave et al. 1997), and
isolates were screened using the DraIII restriction enzyme. The PCR
amplified region of each isolate was sequenced by The Dow Chemical
Company; isolate pDOW1305-6 was verified as containing the correct
genomic DNA sequence.
[0323] Formation of the P. fluorescens pvrF-proC Double
Deletion
[0324] To make a doubly deleted strain, PFG118 was transformed with
pDOW1305-6 by electroporation as described above. Analytical PCR on
the colonies with primers proC8 and the M13/pUC Reverse Sequencing
Primer (-48) (which hybridizes to the plasmid only) (New England
Biolabs, Beverly Mass.), using HotStarTaq (Qiagen, Valencia
Calif.), an annealing temperature of 59.degree. C. and an extension
time of 4 min, showed that 9 out of 22 isolates had the plasmid
integrated into the region upstream from proC, and 7 out of 22 had
the plasmid integrated downstream (data not shown). Three of each
orientation were purified to single colonies. The three isolates
PFG118::1305-6.1, -6.8, -6.10 have an insertion in the region
upstream, and the three isolates PFG118::1305-6.2, -6.3, -6.9 have
an insertion in the region downstream.
[0325] To select for cells that have carried out a homologous
recombination between the plasmid and the chromosome genes thereby
leaving a deletion, PFG118::1305-6.1 and -6.2 were grown to
stationary phase in 50 mL of LB with uracil and proline
supplementation and then plated on LB-5-FOA with uracil and proline
supplementation. Cells that lose the integrated plasmid by
recombination also lose the pyrF gene, and are therefore expected
to be resistant to 5-FOA which would otherwise be converted into a
toxic compound. PCR analysis with proC8 and proC9 was carried out
to distinguish between cells that had lost the plasmid and
regenerated the original sequence, and those that had left the
deletion. Two isolates with the expected 1.3 kB band were chosen
from each of the two selections and named PFG1013, PFG1014, PFG1015
and PFG1016 (also known as DC164). All four isolates were unable to
grow on M9 glucose unless both proline and uracil were added, and
were tetracycline-sensitive. The genomic region of PFG118 (wild
type proC) and PFG1016 (proC deletion) was amplified by PCR
(primers proC8 and proC9, HotStarTaq polymerase, 63.degree. C.
annealing and 3 min extension) and sequenced. The region between
proC5 and proC6 of strain PFG1016 was identical to the parent,
except for the expected 835 bp deletion.
Construction of a Dual Auxotrophic Selection Marker Expression
System PFG 1016/pDOW 1306-6 pDOW 1269-2
[0326] Plasmids were isolated from strain PFG118 pCN51lacI
pDOW1269-2 by HISPEED Plasmid Midi Kit (Qiagen, Valencia Calif.).
The pDOW1269-2 was partially purified from the pCN51lacI by agarose
gel electrophoresis and then electroporated into PFG1016 pDOW1306-6
. Transformants were selected on M9/glucose without
supplementation. Because there was a possibility that some of the
pCN51lacI contaminating the pDOW1269-2 preparation would also be
cotransformed into the cells, three isolates from each
transformation were tested for sensitivity to kanamycin, the
antibiotic marker carried on pCN51lac; all six were found
sensitive. All six strains were found to express the target enzyme,
in a test of target enzyme activity. PCR analysis showed that all
six also contained the chromosomal proC deletion.
[0327] Restriction digestion of plasmids isolated from the
transformants was consistent with the expected pattern.
Performance Testing of the Dual Auxotrophic Marker Expression
System in Shake Flasks
[0328] The six strains were then tested in shake flasks as
described above in Example 1. Induction of target enzyme expression
was initiated at 26 hours by addition of IPTG. The OD.sub.575 for
all six strains was comparable to that of the
dual-antibiotic-resistance marker expression system control, DC88.
Target enzyme production levels in all six were also comparable to
that of the control, as assessed by SDS-PAGE. The two strains that
achieved the highest OD.sub.575, strains 1046 and 1048, were
selected for further characterization.
Performance Testing of the Dual Auxotrophic Marker Expression
System in 20-L Bioreactors
[0329] Strains 1046 and 1048 were subsequently tested in 20-L
bioreactors. Induction of target enzyme expression was initiated at
26 hours by addition of IPTG. Both strains achieved performance
levels within the normal range for the DC88 control strain, for
both OD.sub.575 and target enzyme activity. The performance
averages of these two strains are shown in FIG. 1. Restriction
digests of plasmids prepared from samples taken at the seed stage
and at a time just before the 26-hour start of induction showed a
pattern consistent with that expected. Analytical PCR of genomic
DNA carried out on the same samples demonstrated the retention of
the proC deletion and the pyrF deletion. Aliquots of the 25 hr
samples were plated on tetracycline-, gentamycin-, or
kanamycin-supplemented media; no cell growth was observed, thus
confirming the absence of antibiotic resistance gene activity.
[0330] Analysis of strain 1046 (also known as DC167) in 20-L
bioreactors was repeated twice with similar results. Plasmid
stability at the seed stage and after 25 hours of fermentation
(immediately before induction) was tested by replica plating from
samples that had been diluted and plated on complete media. Both
plasmids were present in more than 97% of the colonies examined,
indicating the lack of cross feeding revertants able to survive
without the plasmid and the stable maintenance of the expression
vector in Pseudomonas fluorescens.
Results
[0331] Both of the pyrF expression systems performed as well as the
control system in which only antibiotic resistance markers were
used (FIG. 1). For the control strain, there is no negative effect
of cross-feeding, since any importation of exogenous metabolites
from lysed cells does not decrease or remove the selection
pressures provided by the antibiotics in the medium. The expected
decreases in performance expected as a result of cross-feeding on
the two pyrF expression systems were surprisingly not observed.
Example 3
Chromosomal Integration of lacI. lacI.sup.Q and lacI.sup.Q1 in P.
fluorescens
[0332] Three P. fluorescens strains have been constructed, each
with one of three different Escherichia coli lacI alleles, lacI
(SEQ ID NO:9), lacI.sup.Q (SEQ ID NO: 11), and lacI.sup.Q1 (SEQ ID
NO:12), integrated into the chromosome. The three strains exhibit
differing amounts of LacI repressor accumulation. Each strain
carries a single copy of its lacI gene at the levansucrase locus
(SEQ ID NO:13) of P. fluorescens DC36, which is an MB101 derivative
(see TD Landry et al., "Safety evaluation of an .alpha.-amylase
enzyme preparation derived from the archaeal order Thermococcales
as expressed in Pseudomonas fluorescens biovar I," Regulatory
Toxicology and Pharmacology 37(1): 149-168(2003)) formed by
deleting the pyrF gene thereof, as described above.
[0333] No vector or other foreign DNA sequences remain in the
strains. The strains are antibiotic-resistance-gene free and also
contain a pyrF deletion, permitting maintenance, during growth in
uracil un-supplemented media, of an expression plasmid carrying a
pyrF+ gene. Protein production is completely free of antibiotic
resistance genes and antibiotics.
[0334] MB214 contains the lacI-lacZYA chromosomal insert described
in U.S. Pat. No. 5,169,760. MB214 also contains a duplication in
the C-terminus of the LacI protein, adding about 10 kDa to the
molecular weight of the LacI repressor.
Construction of Vector pDOW1266-1 for Insertion of Genes into the
Levansucrase Locus
[0335] Plasmid pDOW1266-1 was constructed by PCR amplification of
the region upstream of and within the P. fluorescens gene for
levansucrase (SEQ ID NO:13), replacing the start codon with an XbaI
site, using the Megaprimer method, see A Barik, "Mutagenesis and
Gene Fusion by Megaprimer PCR," in BA White, PCR Cloning Protocols
173-182 (1997) (Humana). PCR was performed using HERCULASE
polymerase (Stratagene, Madison Wis., USA) using primers LEV1 (SEQ
ID NO:34) and LEV2 (SEQ ID NO:35), and P. fluorescens MB214 genomic
DNA as a template (see below for oligonucleotide sequences). Primer
LEV2 (SEQ ID NO:35) contains the sequence that inserts an XbaI
site. The reaction was carried out at 95.degree. C. for 2 min, 35
cycles of [95.degree. C. for 30 sec, 58.degree. C. for 30 sec,
72.degree. C. for 1 min], followed by 10 min at 72.degree. C. The 1
kB product was gel purified and used as one of the primers in the
next reaction, along with LEV3 (SEQ ID NO:36), using MB214 genomic
DNA as a template and the same conditions except that extension
time was 2 min. The 2 kB product was gel purified and re-amplified
with LEV2 (SEQ ID NO: 35) and LEV3 (SEQ ID NO. 36) in order to
increase the quantity.
[0336] Oligonucleotides Used
21 LEV1 5'-TTCGAAGGGGTGCTTTTTCTAGAAGTAAGTC (SEQ ID NO: 34)
TCGTCCATGA LEV2 5'-CGCAAGGTCAGGTACAACAC (SEQ ID NO: 35) LEV3
5'-TACCAGACCAGAGCCGTTCA (SEQ ID NO: 36) LEV7
5'-CTACCCAGAACGAAGATCAG (SEQ ID NO: 37) LEV8
5'-GACTCAACTCAATGGTGCAGG (SEQ ID NO: 38) BglXbaLacF
5'-AGATCTCTAGAGAAGGCGAAGCGGCATGCAT (SEQ ID NO: 39) TTACG lacIR4
5'-ATATTCTAGAGACAACTCGCGCTAACTTACA (SEQ ID NO: 40) TTAATTGC Lacpro9
5'-ATATTCTAGAATGGTGCAAAACCTTTCGCGG (SEQ ID NO: 41) TATGGCATGA
LacIQF 5'-GCTCTAGAAGCGGCATGCATTTACGTTGACA (SEQ ID NO: 42) CC
LacINXR 5'-AGCTAGCTCTAGAAAGTTGGGTAACGCCAGG (SEQ ID NO: 43) GT
lacIQ1 5'-AGTAAGCGGCCGCAGCGGCATGCATTTACGT (SEQ ID NO: 44)
TGACACCACCTTTCGCGGTATGGCATG The Oligos Below were Used for
Analytical Sequencing Only lacIF1 5'-ACAATCTTCTCGCGCAACGC (SEQ ID
NO: 45) lacIF2 5'-ATGTTATATCCCGCCGTTAA (SEQ ID NO: 46) lacIR1
5'-CCGCTATCGGCTGAATTTGA (SEQ ID NO: 47) lacIR2
5'-TGTAATTCAGCTCCGCCATC (SEQ ID NO: 48) SeqLev5AS
5'-TATCGAGATGCTGCAGCCTC (SEQ ID NO: 49) SeqLev3S
5'-ACACCTTCACCTACGCCGAC (SEQ ID NO: 50) LEV10
5'-TCTACTTCGCCTTGCTCGTT (SEQ ID NO: 51)
[0337] The LEV2 - LEV3 amplification product was cloned into the
SrfI site of pDOW1261-2, a suicide vector that contains P.
fluorescens pyrF+ gene as a selection marker to facilitate
selection for cross-outs. The new plasmid was named pDOW1266-1. The
amplified region was sequenced.
Cloning the lacI Genes into Insertion Vector pDOW1266-1
[0338] The E.coli lacI gene was amplified from pCN51lacI with
primers BglXbaLacF (SEQ ID 10 NO:39) and lacIR4 (SEQ ID NO. 40),
using HERCULASE polymerase (annealing at 62.degree. C. and
extension time of 2 min). After gel purification and digestion with
XbaI, the lacI gene was cloned into the XbaI site of pDOW1266-1, to
make pDOW1310. The lacI.sup.Q gene was created by PCR amplification
using pCN51lacI as a template with 15 primers lacpro9 (SEQ ID NO.
41) and lacIR4 (SEQ ID NO. 40), using HERCULASE polymerase
(annealing at 62.degree. C. and extension time of 2 min). After gel
purification and digestion with XbaI, it was cloned into the XbaI
site of pDOW1266-1, to make pDOW1311.
[0339] The lacI.sup.Q1 gene was created by amplifying the lacI gene
from E.coli K12 (ATCC47076) using primers lacIQ1 (SEQ ID NO. 44)
and lacINXR (SEQ ID NO. 43) and cloned into pCR2.1 Topo
(Invitrogen, Carlsbad, Calif., USA), to make pCR2-lacIQ1. The
lacI.sup.Q1 gene was reamplified from pCR2-lacIQ1 using primers
lacIQF (SEQ ID NO. 42) and lacINXR (SEQ ID NO. 43) with Herculase
polymerase (61.degree. C. annealing, 3 min extension time, 35
cycles). After gel purification and digestion with XbaI, the PCR
product was cloned into the XbaI site of pDOW1266-1, to make
pDOW1309.
[0340] The PCR amplified inserts in pCR2-lacIQ1, pDOW1310,
pDOW1311, and pDOW1309 were sequenced (using primers lacIF1 (SEQ ID
NO:45), lacIF2 (SEQ ID NO. 46), lacIR1 (SEQ ID NO. 47), lacIR2 (SEQ
ID NO. 48), SeqLev5AS (SEQ ID NO. 49), SeqLev3S (SEQ ID NO. 50),
and LEV10 (SEQ ID NO. 51)) to insure that no mutations had been
introduced by the PCR reaction. In each case, an orientation was
chosen in which the lacI was transcribed in the same direction as
the levansucrase gene. Although the levansucrase promoter is
potentially able to control transcription of lacI, the promoter
would only be active in the presence of sucrose, which is absent in
the fermentation conditions used.
Construction of P. fluorescens Strains with Integrated lacI Genes
at the Levansucrase Locus
[0341] The vectors pDOW1309, pDOW1310, and pDOW1311 were introduced
into DC36 by electroporation, first selecting for integration of
the vector into the genome with tetracycline resistance. Colonies
were screened to determine that the vector had integrated at the
levansucrase locus by PCR with primers LEV7 (SEQ ID NO. 37) and
M13R (from New England Biolabs, Gloucester Mass., USA). To select
for the second cross-over which would leave the lacI gene in the
genome, the isolates were grown in the presence of 5'-fluoroorotic
acid and in the absence of tetracycline. Recombination between the
duplicated regions in the genome either restores the parental
genotype, or leaves the lacI gene. The resulting isolates were
screened for sensitivity to tetracycline, growth in the absence of
uracil, and by PCR with primers LEV7 (SEQ ID NO. 37) and LEV8 (SEQ
ID NO. 38). The names of the new strains are shown in Table 17. To
obtain sequence information for genomic regions, PCR products were
sequenced directly, see E Werle, "Direct sequencing of polymerase
chain reaction products, "Laboratory Methods for the Detection of
Mutations and Polymorphisms in DNA 163-174 (1997). For each strain,
the sequencing confirmed the identity of the promoter, the
orientation of the lacI variant relative to the flanking regions,
and whether there were any point mutations relative to the parental
sequence. The sequences of DC202 and DC206 were as expected. The
sequence of DC204 showed a point mutation within the levansucrase
open reading frame, downstream of lacl.sup.Q, which did not change
any coding sequence and therefore is inconsequential.
22TABLE 17 P. FLUORESCENS STRAINS WITH LACI ALLELES INTEGRATED INTO
THE GENOME Plasmid used to make Strain Designation the lacI
insertion Genotype DC202 pDOW1310-1 pyrF lev::lacI DC204 pDOW1311-4
pyrF lev::lacI.sup.Q DC206 pDOW1309oriA pyrF lev::lacI.sup.Q1
Analysis of Relative Concentration of LacI in the lacI Integrants,
Compared to pCN51lacI
[0342] UnBlot is a method analogous to Western analysis, in which
proteins are detected in the gel without the need for transfer to a
filter. The technique was carried out following the directions from
Pierce Biotechnology (Rockford, Ill., USA), the manufacturer.
Analysis using UnBlot showed that the amount of LacI in each of the
new integrant strains was higher than in MB214. MB214 contains the
lacI-lacZYA insert described in U.S. Pat. 5,169,760. The relative
concentration of LacI in the lacI.sup.Q and lacI.sup.Q1 integrants
was about the same as in strains carrying pCN51lacI, the multi-copy
plasmid containing lacI. See FIG. 5.
[0343] A dilution series was carried out in order to assess more
precisely the relative difference in LacI concentration in MB214,
DC202 (lacI integrated) and DC206 (lacI.sup.Q1 integrated).
MB101pCN51lacI, DC204 and DC206 have about 100 times more LacI than
MB214, whereas DC202 has about 5 times more.
Example 4
Nitrilase Expression and Transcription
[0344] Strain DC140 was constructed by introducing into P.
fluorescens MB214 a tetracycline- resistant broad-host-range
plasmid, pMYC1803 (WO 2003/068926), into which a nitrilase gene (G
DeSanthis et al., J Amer. Chem. Soc. 125:11476-77 (2003)), under
the control of the Ptac promoter, had been inserted. In order to
compare regulation of un-induced expression of the target gene in
DC202 and DC206 with MB214, the same nitrilase gene was cloned onto
a pMYC1803 derivative where the tetracycline-resistance gene has
been replaced by a pyrF selection marker. The new plasmid,
pDOW2415, was then electroporated into DC202 and DC204, resulting
in DC239 and DC240, respectively. DC140, DC239 and DC240 were
cultured in 20 L fermentors by growth in a mineral salts medium fed
with glucose or glycerol, ultimately to cell densities providing
biomasses within the range of about 20 g/L to more than 70 g/L dry
cell weight (See WO 2003/068926). The gratuitous inducer of the
Ptac promoter, IPTG, was added to induce expression.
[0345] The ratio of pre-induction nitrilase activities of DC140 to
DC239 to DC240 was 6:2:1. RNA analysis by Northern blots of the
same samples revealed the same ranking of derepression. Based on
densitometric measurements, the ratio of un-induced transcript
levels of DC140:DC239:DC240 was 2.4:1.4:1.0. Shortly after
induction (30 min) with 0.3mM IPTG, the levels of transcript of all
the strains were the same. Post-induction nitrilase productivity
rates were also comparable. This indicated that the concentration
of inducer used was sufficient to fully induce the Ptac promoter in
these three strains despite their different LacI protein levels.
However, fermentations of the most derepressed strain, DC140,
suffered significant cell lysis accompanied with loss of nitrilase
activity after approximately 24 hours post-induction. Induction of
the improved, more tightly regulated strains, DC239 and DC240,
could be extended to more than 48 hours post induction, while
maintaining high nitrilase productivity, with the ultimate result
of a doubling of nitrilase yields. See FIG. 6.
Results
[0346] The examples indicate It that the use of a LacI-encoding
gene other than as part of a whole or truncated Plac-lacI-lacZYA
operon in Pseudomonads resulted in substantially improved
repression of pre-induction recombinant protein expression, higher
cell densities in commercial-scale fermentation, and higher yields
of the desired product in comparison with previously taught
lacI-lacZYA Pseudomonad chromosomal insertion (U.S. Pat. No.
5,169,760). The results also indicated that the lacI insertion is
as effective in producing LacI repressor protein in Pseudomonas
fluorescens , thereby eliminating the need to maintain a separate
plasmid encoding a LacI repressor protein in the cell and reducing
potential production inefficiencies caused by such maintenance.
[0347] In addition to being antibiotic free, derepression during
the growth stage in DC239 and DC240 was up to 10 fold less than the
MB214 strain. Pre-induction nitrilase activity levels of DC239 and
DC240 averaged 0.4 U/ml in more than 13 separate fermentations, and
cell density and nitrilase expression in DC239 and DC240 did not
decay during extended induction phase, as it did in DC140. Given
the higher derepression, DC239 and DC240 fermentation runs
decreased the time of the growth phase by more than 30%, reducing
fermentation costs.
Example 5
Construction of tac Promoter with a Single Optimal lac Operator and
with Two lac Operators
[0348] The native tac promoter only has a single native lac
operator, AATTGTGAGCGGATAACAATT, at the O1 position (FIG. 4). In
the first construct, pDOW1418, the native operator was replaced by
the more symmetrical lacOid operator sequence 5'-AATTGTGAGC
GCTCACAATT - 3' (SEQ. ID. NO. 14) (J R. Sadler, H. Sasmor and J L.
Betz. PNAS. 1983 Nov.; 80 (22): 6785-9). A 289 bp HindIII/ SpeI
fragment containing the tac promoter and the native lacO sequence
was removed from a pMYC1803 derivative, pDOW2118, and replaced by a
HindIII/SpeI fragment isolated from an SOE PCR amplification
product containing the symmetrical lacOid sequence. The SOE PCR
primers (RC-3 and RC-9) incorporated 4 nucleotide changes that
produced the optimized/symmetrical lacO sequence (three base pair
substitutions and one base pair deletion). The HindIII/SpeI
promoter fragment of the resulting plasmid, pDOW2201, was cloned
into the nitrilase expression plasmid based on pMYC1803, to replace
the native tac promoter, resulting in pDOW1414. This expression
cassette was then transferred onto the pyrF(+) plasmid pDOW1269,
resulting in pDOW1418 by exchanging DraI/XhoI fragments. Plasmid
pDOW1418 was then transformed into host strain DC206, resulting in
strain DC281 (See FIG. 4).
[0349] Oligonucleotides Used
23 RC-3 5'-GTGAGCGCTCACAATTCCACACAGGAAA (SEQ ID NO: 52) ACAG RC-4
5'-TTCGGGTGGAAGTCCAGGTAGTTGGCGG (SEQ ID NO: 53) TGTA RC-9
5'-GAATTGTGAGCGCTCACAATTCCACACA (SEQ ID NO: 54) TTATACGAGC RC-10
5'-ATTCAGCGCATGTTCAACGG (SEQ ID NO: 55)
[0350] In the second construct, pDOW1416, the lacOid operator,
5'-AATTGTGAGC GCTCACAATT-3' (SEQ ID. No. 14), was inserted 52
nucleotides up-stream (5') of the existing native lacO1 by PCR. PCR
amplification of the promoter region using the Megaprimer method
was performed using a pMYC1803 derivative, pMYC5088, and the
following primers AKB-1 and AKB-2 as a first step. The resulting
PCR product was combined with primer AKB-3 in a second round of PCR
amplification using the same template. After purification and
digestion with HindIII and SpeI, the promoter fragment containing
the dual operators was cloned into the HindIII and SpeI sites of
plasmid pMYC5088 resulting in pDOW1411. Introduction of the second
operator introduced a unique MfeI site immediately upstream of the
optimal operator. The XhoI/SpeI vector fragment with promoter
regions of pDOW1411 was then ligated with the compatible fragment
of the pMYC1803 derivative bearing the nitrilase gene, forming
pDOW1413. Subsequent ligation of the MfeI/XhoI expression cassette
fragment of pDOW1413 to the compatible vector fragment of pDOW1269
resulted in pDOW1416; which when transformed into DC206, formed the
strain DC262.
[0351] Oligonucleotides Used
24 AKB-1 5'-ACGGTTCTGGCAAACAATTGTGAGCGCTCAC (SEQ ID NO: 56)
AATTTATTCTGAAATGAGC AKB-2 5'-GCGTGGGCGGTGTTTATCATGTTC (SEQ ID NO:
57) AKB-3 5'-TACTGCACGCACAAGCCTGAACA (SEQ ID NO: 58)
Nitrilase Derepression
[0352] Northern blot analysis was performed pre and post induction
on MB214, DC202, and DC206. MB214, DC202, and DC206 were
transformed with a nitrilase expression vector containing the wild
type lacO sequence in the O.sub.1 position 3' of the tac promoter,
creating MB214 wtO.sub.1, DC202wtO.sub.1 (DC239), and
DC206wtO.sub.1 (DC240), as described above. DC206 was transformed
with a nitrilase expression vector containing a lacOid sequence in
place of the wild type lacO sequence at the O.sub.1 position 3' of
the tac promoter as described above, creating DC206Oid (DC281).
DC206 was also transformed with a nitrilase expression vector
containing a wild type lacO sequence at the O.sub.1 position 3' of
the tac promoter and a lacOid sequence at the O.sub.3 position 5'
of the tac promoter, creating the dual lacO containing
DC206wtO.sub.1 O.sub.3id (DC263).
[0353] Northern blot analysis indicated a greater repression by the
strain containing the Dual lacO sequence (DC206wtO.sub.1 O.sub.3id
(DC263)) cassette prior to induction. The greater repression of
pre-induction expression is especially useful when producing toxic
proteins, since basal levels of pre-induction toxic proteins result
in the delayed entry of the cell into the growth phase, and,
potentially, lower overall yields of the product.
Sequence CWU 1
1
60 1 2650 DNA Pseudomonas fluorescens 1 gatcagttgc ggagccttgg
ggtcatcccc cagtttctga cgcaggcgcg acaccagcaa 60 gtcgatgctg
cggtcgaaag cctcgatgga acgcccacgg gccgcgtcca gcagctgttc 120
gcggctcagc acacgccgcg ggcgttcgat aaacacccac aacaaacgaa actcggcgtt
180 ggacagcggc accaccaggc cgtcatcggc caccagctgg cgcagtacgc
tgttcaggcg 240 ccaagtgtcg aaacggatat tggcccgctg ttcggtgcgg
tcatcacgca cccggcgcag 300 gatggtctgg atacgcgcga ccagttcccg
gggttcgaac ggcttggaca tatagtcgtc 360 tgcccccagt tccaggccga
tgatgcggtc ggtgggttcg cagcgggcgg tgagcatcag 420 gatcggaatg
tccgattcgg cgcgcagcca gcggcacaat gtcagcccgt cttcgcccgg 480
cagcatcagg tcgagcacca ccacatcgaa ggtctccgct tgcatggcct ggcgcatggc
540 gatgccgtcg gtgacgcctg aggcgagaat attgaagcgt gccaggtagt
cgatcagcag 600 ttcgcggatc ggcacgtcgt cgtcgacaat cagcgcgcgg
gtgttccagc gcttgtcttc 660 ggcgatcacc gcgtcttttg gcgcttcgtt
tacagggtcg caaggggtat gcatagcgag 720 gtcatctgcc tggttgtggc
tgtcagcata ggcgcccagt tccagggctg gaagtgctgg 780 gcgggcggtc
atgtgcgcga ggctagccgg gcggcgtatt gggggcgtgt cgtgaatgta 840
tcgggcttga aacaattgcc ttgaatcgcc ggtattgggc gcttgatcgg tatttaccga
900 tcatcggatc ccgcaacggc gctgcttgcg ctacaatccg cgccgatttc
gacttgcctg 960 agagcccatt ccaatgtccg tctgccagac tcctatcatc
gtcgccctgg attaccccac 1020 ccgtgacgcc gcactgaagc tggctgacca
gttggacccc aagctttgcc gggtcaaggt 1080 cggcaaggaa ttgttcacca
gttgcgcggc ggaaatcgtc ggcaccctgc gggacaaagg 1140 cttcgaagtg
ttcctcgacc tcaaattcca tgacatcccc aacaccacgg cgatggccgt 1200
caaagccgcg gccgagatgg gcgtgtggat ggtcaatgtg cactgctccg gtggcctgcg
1260 catgatgagc gcctgccgcg aagtgctgga acagcgcagc ggccccaaac
cgttgttgat 1320 cggcgtgacc gtgctcacca gcatggagcg cgaagacctg
gcgggcattg gcctggatat 1380 cgagccgcag gtgcaagtgt tgcgcctggc
agccctggcg cagaaagccg gcctcgacgg 1440 cctggtgtgc tcagccctgg
aagcccaggc cctgaaaaac gcacatccgt cgctgcaact 1500 ggtgacaccg
ggtatccgtc ctaccggcag cgcccaggat gaccagcgcc gtatcctgac 1560
cccgcgccag gccctggatg cgggctctga ctacctggtg atcggccggc cgatcagcca
1620 ggcggcggat cctgcaaaag cgttggcagc ggtcgtcgcc gagatcgcct
gatttttaga 1680 gtgagcaaaa aatgtgggag ctggcttgcc tgcgatagta
tcaactcggt atcacttaga 1740 aaccgagttg cttgcatcgc aggcaagcca
gctcccacat ttgtttttgt ggtgtgtcag 1800 ctgactttga gcaccaactt
cccgaagttc tcgccgttga acagcttcat cagcgtttcc 1860 gggaatgtct
ccagcccttc gacaatatct tccttgctct tgagcttgcc ctgggccatc 1920
cagccggcca tttcctgacc cgccgccgcg aagttcgccg cgtggtccat caccacaaag
1980 ccttccatac gcgcacggtt gaccagcaat gacaggtagt tcgccgggcc
tttgaccgct 2040 tccttgttgt tgtactggct gattgcaccg caaatcacca
cgcgggcttt gagcgccagg 2100 cggctgagca ccgcgtcgag aatatcgccg
ccgacgttat cgaaatacac gtccacgcct 2160 ttggggcact cgcgcttgag
ggcggcgggc acgtcttcgc ttttgtagtc gatggcggcg 2220 tcgaagccca
gctcatcgac caggaacttg cacttctcgg cgccaccggc gatccccact 2280
acgcgacagc ctttgagctt agcgatctgc ccggcgatgc tgcccacggc accggcggcg
2340 ccggagatca ccacggtgtc accggctttc ggtgcgccgg tctccagcag
agcaaagtag 2400 gccgtcatgc cggtcatgcc cagggcggac aggtagcggg
gcaggggcgc cagcttgggg 2460 tccaccttat agaaaccacg gggctcgcca
aggaagtaat cctgcacgcc cagtgcaccg 2520 ttcacgtagt cccccaccgc
gaagttcgga tggttcgagg caagcacctt gcctacgccc 2580 agggcgcgca
tcacttcgcc gatgcctacc ggtgggatgt aggacttgcc ttcattcatc 2640
cagccacgca 2650 2 232 PRT Pseudomonas fluorescens 2 Met Ser Val Cys
Gln Thr Pro Ile Ile Val Ala Leu Asp Tyr Pro Thr 1 5 10 15 Arg Asp
Ala Ala Leu Lys Leu Ala Asp Gln Leu Asp Pro Lys Leu Cys 20 25 30
Arg Val Lys Val Gly Lys Glu Leu Phe Thr Ser Cys Ala Ala Glu Ile 35
40 45 Val Gly Thr Leu Arg Asp Lys Gly Phe Glu Val Phe Leu Asp Leu
Lys 50 55 60 Phe His Asp Ile Pro Asn Thr Thr Ala Met Ala Val Lys
Ala Ala Ala 65 70 75 80 Glu Met Gly Val Trp Met Val Asn Val His Cys
Ser Gly Gly Leu Arg 85 90 95 Met Met Ser Ala Cys Arg Glu Val Leu
Glu Gln Arg Ser Gly Pro Lys 100 105 110 Pro Leu Leu Ile Gly Val Thr
Val Leu Thr Ser Met Glu Arg Glu Asp 115 120 125 Leu Ala Gly Ile Gly
Leu Asp Ile Glu Pro Gln Val Gln Val Leu Arg 130 135 140 Leu Ala Ala
Leu Ala Gln Lys Ala Gly Leu Asp Gly Leu Val Cys Ser 145 150 155 160
Ala Leu Glu Ala Gln Ala Leu Lys Asn Ala His Pro Ser Leu Gln Leu 165
170 175 Val Thr Pro Gly Ile Arg Pro Thr Gly Ser Ala Gln Asp Asp Gln
Arg 180 185 190 Arg Ile Leu Thr Pro Arg Gln Ala Leu Asp Ala Gly Ser
Asp Tyr Leu 195 200 205 Val Ile Gly Arg Pro Ile Ser Gln Ala Ala Asp
Pro Ala Lys Ala Leu 210 215 220 Ala Ala Val Val Ala Glu Ile Ala 225
230 3 696 DNA Pseudomonas fluorescens 3 atgtccgtct gccagactcc
tatcatcgtc gccctggatt accccacccg tgacgccgca 60 ctgaagctgg
ctgaccagtt ggaccccaag ctttgccggg tcaaggtcgg caaggaattg 120
ttcaccagtt gcgcggcgga aatcgtcggc accctgcggg acaaaggctt cgaagtgttc
180 ctcgacctca aattccatga catccccaac accacggcga tggccgtcaa
agccgcggcc 240 gagatgggcg tgtggatggt caatgtgcac tgctccggtg
gcctgcgcat gatgagcgcc 300 tgccgcgaag tgctggaaca gcgcagcggc
cccaaaccgt tgttgatcgg cgtgaccgtg 360 ctcaccagca tggagcgcga
agacctggcg ggcattggcc tggatatcga gccgcaggtg 420 caagtgttgc
gcctggcagc cctggcgcag aaagccggcc tcgacggcct ggtgtgctca 480
gccctggaag cccaggccct gaaaaacgca catccgtcgc tgcaactggt gacaccgggt
540 atccgtccta ccggcagcgc ccaggatgac cagcgccgta tcctgacccc
gcgccaggcc 600 ctggatgcgg gctctgacta cctggtgatc ggccggccga
tcagccaggc ggcggatcct 660 gcaaaagcgt tggcagcggt cgtcgccgag atcgcc
696 4 834 DNA Pseudomonas fluorescens 4 atgaagcaat atctcgaact
actgaacgac gtcgtgacca atggattgac caagggcgat 60 cgcaccggca
ccggcaccaa agccgtattt gcccgtcagt atcggcataa cttggccgac 120
ggcttcccgc tgctgaccac caagaagctt catttcaaaa gtatcgccaa cgagttgatc
180 tggatgttga gcggcaacac caacatcaag tggctcaacg aaaatggcgt
gaaaatctgg 240 gacgagtggg ccaccgaaga cggcgacctg ggcccggtgt
acggcgagca atggaccgcc 300 tggccgacca aggacggcgg caagatcaac
cagatcgact acatggtcca caccctcaaa 360 accaacccca acagccgccg
catcctgttt catggctgga acgtcgagta cctgccggac 420 gaaaccaaga
gcccgcagga gaacgcgcgc aacggcaagc aagccttgcc gccgtgccat 480
ctgttgtacc aggcgttcgt gcatgacggg catctgtcga tgcagttgta tatccgcagc
540 tccgacgtct tcctcggcct gccgtacaac accgccgcgt tggccttgct
gactcacatg 600 ctggctcagc aatgcgacct gatccctcac gagatcatcg
tcaccaccgg cgacacccat 660 gcttacagca accacatgga acagatccgc
acccagctgg cgcgtacgcc gaaaaagctg 720 ccggaactgg tgatcaagcg
taaacctgcg tcgatctacg attacaagtt tgaagacttt 780 gaaatcgttg
gctacgacgc cgacccgagc atcaaggctg acgtggctat ctga 834 5 277 PRT
Pseudomonas fluorescens 5 Met Lys Gln Tyr Leu Glu Leu Leu Asn Asp
Val Val Thr Asn Gly Leu 1 5 10 15 Thr Lys Gly Asp Arg Thr Gly Thr
Gly Thr Lys Ala Val Phe Ala Arg 20 25 30 Gln Tyr Arg His Asn Leu
Ala Asp Gly Phe Pro Leu Leu Thr Thr Lys 35 40 45 Lys Leu His Phe
Lys Ser Ile Ala Asn Glu Leu Ile Trp Met Leu Ser 50 55 60 Gly Asn
Thr Asn Ile Lys Trp Leu Asn Glu Asn Gly Val Lys Ile Trp 65 70 75 80
Asp Glu Trp Ala Thr Glu Asp Gly Asp Leu Gly Pro Val Tyr Gly Glu 85
90 95 Gln Trp Thr Ala Trp Pro Thr Lys Asp Gly Gly Lys Ile Asn Gln
Ile 100 105 110 Asp Tyr Met Val His Thr Leu Lys Thr Asn Pro Asn Ser
Arg Arg Ile 115 120 125 Leu Phe His Gly Trp Asn Val Glu Tyr Leu Pro
Asp Glu Thr Lys Ser 130 135 140 Pro Gln Glu Asn Ala Arg Asn Gly Lys
Gln Ala Leu Pro Pro Cys His 145 150 155 160 Leu Leu Tyr Gln Ala Phe
Val His Asp Gly His Leu Ser Met Gln Leu 165 170 175 Tyr Ile Arg Ser
Ser Asp Val Phe Leu Gly Leu Pro Tyr Asn Thr Ala 180 185 190 Ala Leu
Ala Leu Leu Thr His Met Leu Ala Gln Gln Cys Asp Leu Ile 195 200 205
Pro His Glu Ile Ile Val Thr Thr Gly Asp Thr His Ala Tyr Ser Asn 210
215 220 His Met Glu Gln Ile Arg Thr Gln Leu Ala Arg Thr Pro Lys Lys
Leu 225 230 235 240 Pro Glu Leu Val Ile Lys Arg Lys Pro Ala Ser Ile
Tyr Asp Tyr Lys 245 250 255 Phe Glu Asp Phe Glu Ile Val Gly Tyr Asp
Ala Asp Pro Ser Ile Lys 260 265 270 Ala Asp Val Ala Ile 275 6 1853
DNA Pseudomonas fluorescens 6 gcccttgagt tggcacttca tcggccccat
tcaatcgaac aagactcgtg ccatcgccga 60 gcacttcgct tgggtgcact
ccgtggaccg cctgaaaatc gcacaacgcc tgtccgaaca 120 acgcccggcc
gacctgccgc cgctcaatat ctgcatccag gtcaatgtca gtggcgaagc 180
cagcaagtcc ggctgcacgc ccgctgacct gccggccctg gccacagcga tcagcgccct
240 gccgcgcttg aagctgcggg gcttgatggc gattcccgag ccgacgcaag
accgggcgga 300 gcaggatgcg gcgttcgcca cggtgcgcga cttgcaagcc
agcttgaacc tggcgctgga 360 cacactttcc atgggcatga gccacgacct
tgagtcggcc attgcccaag gcgccacctg 420 ggtgcggatc ggtaccgccc
tgtttggcgc ccgcgactac ggccagccgt gaaatggctg 480 acatccctcg
aaataaggac ctgtcatgag caacacgcgt attgccttta tcggcgccgg 540
taacatggcg gccagcctga tcggtggcct gcgggccaag ggcctggacg ccgagcagat
600 ccgcgccagc gaccccggtg ccgaaacccg cgagcgcgtc agagccgaac
acggtatcca 660 gaccttcgcc gataacgccg aggccatcca cggcgtcgat
gtgatcgtgc tggcggtcaa 720 gccccaggcc atgaaggccg tgtgcgagag
cctgagcccg agcctgcaac cccatcaact 780 ggtggtgtcg attgccgctg
gcatcacctg cgccagcatg accaactggc tcggtgccca 840 gcccattgtg
cgctgcatgc ccaacacccc ggcgctgctg cgccagggcg tcagcggttt 900
gtatgccact ggcgaagtca ccgcgcagca acgtgaccag gcccaggaac tgctgtctgc
960 ggtgggcatc gccgtgtggc tggagcagga acagcaactg gatgcggtca
ccgccgtctc 1020 cggcagcggc ccggcttact tcttcctgtt gatcgaggcc
atgacggccg caggcgtcaa 1080 gctgggcctg ccccacgacg tggccgagca
actggcggaa caaaccgccc tgggcgccgc 1140 caagatggcg gtcggcagcg
aggtggatgc cgccgaactg cgccgtcgcg tcacctcgcc 1200 aggtggtacc
acacaagcgg ctattgagtc gttccaggcc gggggctttg aagccctggt 1260
ggaaacagca ctgggtgccg ccgcacatcg ttcagccgag atggctgagc aactgggcaa
1320 atagtcgtcc cttaccaagg taatcaaaca tgctcggaat caatgacgct
gccattttca 1380 tcatccagac cctgggcagc ctgtacctgc tgatcgtact
gatgcgcttt atcctgcaac 1440 tggtgcgtgc gaacttctac aacccgctgt
gccagttcgt ggtgaaggcc acccaaccgc 1500 tgctcaagcc gctgcgccgg
gtgatcccga gcctgttcgg cctggacatg tcgtcgctgg 1560 tgctggcgct
gttgctgcag attttgctgt tcgtggtgat cctgatgctc aatggatacc 1620
aggccttcac cgtgctgctg ttgccatggg gcctgatcgg gattttctcg ctgttcctga
1680 agatcatttt ctggtcgatg atcatcagcg tgatcctgtc ctgggtcgca
ccgggtagcc 1740 gtagcccggg tgccgaattg gtggctcaga tcaccgagcc
ggtgctggca cccttccgtc 1800 gcctgattcc gaacctgggt ggcctggata
tctcgccgat cttcgcgttt atc 1853 7 272 PRT Pseudomonas fluorescens 7
Met Ser Asn Thr Arg Ile Ala Phe Ile Gly Ala Gly Asn Met Ala Ala 1 5
10 15 Ser Leu Ile Gly Gly Leu Arg Ala Lys Gly Leu Asp Ala Glu Gln
Ile 20 25 30 Arg Ala Ser Asp Pro Gly Ala Glu Thr Arg Glu Arg Val
Arg Ala Glu 35 40 45 His Gly Ile Gln Thr Phe Ala Asp Asn Ala Glu
Ala Ile His Gly Val 50 55 60 Asp Val Ile Val Leu Ala Val Lys Pro
Gln Ala Met Lys Ala Val Cys 65 70 75 80 Glu Ser Leu Ser Pro Ser Leu
Gln Pro His Gln Leu Val Val Ser Ile 85 90 95 Ala Ala Gly Ile Thr
Cys Ala Ser Met Thr Asn Trp Leu Gly Ala Gln 100 105 110 Pro Ile Val
Arg Cys Met Pro Asn Thr Pro Ala Leu Leu Arg Gln Gly 115 120 125 Val
Ser Gly Leu Tyr Ala Thr Gly Glu Val Thr Ala Gln Gln Arg Asp 130 135
140 Gln Ala Gln Glu Leu Leu Ser Ala Val Gly Ile Ala Val Trp Leu Glu
145 150 155 160 Gln Glu Gln Gln Leu Asp Ala Val Thr Ala Val Ser Gly
Ser Gly Pro 165 170 175 Ala Tyr Phe Phe Leu Leu Ile Glu Ala Met Thr
Ala Ala Gly Val Lys 180 185 190 Leu Gly Leu Pro His Asp Val Ala Glu
Gln Leu Ala Glu Gln Thr Ala 195 200 205 Leu Gly Ala Ala Lys Met Ala
Val Gly Ser Glu Val Asp Ala Ala Glu 210 215 220 Leu Arg Arg Arg Val
Thr Ser Pro Gly Gly Thr Thr Gln Ala Ala Ile 225 230 235 240 Glu Ser
Phe Gln Ala Gly Gly Phe Glu Ala Leu Val Glu Thr Ala Leu 245 250 255
Gly Ala Ala Ala His Arg Ser Ala Glu Met Ala Glu Gln Leu Gly Lys 260
265 270 8 816 DNA Pseudomonas fluorescens 8 atgagcaaca cgcgtattgc
ctttatcggc gccggtaaca tggcggccag cctgatcggt 60 ggcctgcggg
ccaagggcct ggacgccgag cagatccgcg ccagcgaccc cggtgccgaa 120
acccgcgagc gcgtcagagc cgaacacggt atccagacct tcgccgataa cgccgaggcc
180 atccacggcg tcgatgtgat cgtgctggcg gtcaagcccc aggccatgaa
ggccgtgtgc 240 gagagcctga gcccgagcct gcaaccccat caactggtgg
tgtcgattgc cgctggcatc 300 acctgcgcca gcatgaccaa ctggctcggt
gcccagccca ttgtgcgctg catgcccaac 360 accccggcgc tgctgcgcca
gggcgtcagc ggtttgtatg ccactggcga agtcaccgcg 420 cagcaacgtg
accaggccca ggaactgctg tctgcggtgg gcatcgccgt gtggctggag 480
caggaacagc aactggatgc ggtcaccgcc gtctccggca gcggcccggc ttacttcttc
540 ctgttgatcg aggccatgac ggccgcaggc gtcaagctgg gcctgcccca
cgacgtggcc 600 gagcaactgg cggaacaaac cgccctgggc gccgccaaga
tggcggtcgg cagcgaggtg 660 gatgccgccg aactgcgccg tcgcgtcacc
tcgccaggtg gtaccacaca agcggctatt 720 gagtcgttcc aggccggggg
ctttgaagcc ctggtggaaa cagcactggg tgccgccgca 780 catcgttcag
ccgagatggc tgagcaactg ggcaaa 816 9 1330 DNA Escherichia coli 9
gacaccatcg aatggcgcaa aacctttcgc ggtatggcat gatagcgccc ggaagagagt
60 caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga
gtatgccggt 120 gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca
gccacgtttc tgcgaaaacg 180 cgggaaaaag tggaagcggc gatggcggag
ctgaattaca ttcccaaccg cgtggcacaa 240 caactggcgg gcaaacagtc
gttgctgatt ggcgttgcca cctccagtct ggccctgcac 300 gcgccgtcgc
aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg 360
gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt gcacaatctt
420 ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca
ggatgccatt 480 gctgtggaag ctgcctgcac taatgttccg gcgttatttc
ttgatgtctc tgaccagaca 540 cccatcaaca gtattatttt ctcccatgaa
gacggtacgc gactgggcgt ggagcatctg 600 gtcgcattgg gtcaccagca
aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg 660 cgtctgcgtc
tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg 720
gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat
780 gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct
gggcgcaatg 840 cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata
tctcggtagt gggatacgac 900 gataccgaag acagctcatg ttatatcccg
ccgtcaacca ccatcaaaca ggattttcgc 960 ctgctggggc aaaccagcgt
ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag 1020 ggcaatcagc
tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg 1080
caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
1140 cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca
ctcattaggc 1200 accccaggct ttacacttta tgcttccggc tcgtatgttg
tgtggaattg tgagcggata 1260 acaatttcac acaggaaaca gctatgacca
tgattacgga ttcactggcc gtcgttttac 1320 aacgtcgtga 1330 10 359 PRT
Escherichia coli 10 Met Lys Pro Val Thr Leu Tyr Asp Val Ala Glu Tyr
Ala Gly Val Ser 1 5 10 15 Tyr Gln Thr Val Ser Arg Val Val Asn Gln
Ala Ser His Val Ser Ala 20 25 30 Lys Thr Arg Glu Lys Val Glu Ala
Ala Met Ala Glu Leu Asn Tyr Ile 35 40 45 Pro Asn Arg Val Ala Gln
Gln Leu Ala Gly Lys Gln Ser Leu Leu Ile 50 55 60 Gly Val Ala Thr
Ser Ser Leu Ala Leu His Ala Pro Ser Gln Ile Val 65 70 75 80 Ala Ala
Ile Lys Ser Arg Ala Asp Gln Leu Gly Ala Ser Val Val Val 85 90 95
Ser Met Val Glu Arg Ser Gly Val Glu Ala Cys Lys Ala Ala Val His 100
105 110 Asn Leu Leu Ala Gln Arg Val Ser Gly Leu Ile Ile Asn Tyr Pro
Leu 115 120 125 Asp Asp Gln Asp Ala Ile Ala Val Glu Ala Ala Cys Thr
Asn Val Pro 130 135 140 Ala Leu Phe Leu Asp Val Ser Asp Gln Thr Pro
Ile Asn Ser Ile Phe 145 150 155 160 Ser His Glu Asp Gly Thr Arg Leu
Gly Val Glu His Leu Val Ala Leu 165 170 175 Gly His Gln Gln Ile Ala
Leu Leu Ala Gly Pro Leu Ser Ser Val Ser 180 185 190 Ala Arg Leu Arg
Leu Ala Gly Trp His Lys Tyr Leu Thr Arg Asn Gln 195 200 205 Ile Gln
Pro Ile Ala Glu Arg Glu Gly Asp Trp Ser Ala Met Ser Gly 210 215 220
Phe Gln Gln Thr Met Gln Met Leu Asn Glu Gly Ile Val Pro Thr Ala 225
230 235 240 Met Leu Val Ala Asn Asp Gln Met Ala Leu Gly Ala Met Arg
Ala Ile 245 250 255 Thr Glu Ser Gly Leu Arg Val Gly Ala Asp Ile Ser
Val Val Gly Tyr
260 265 270 Asp Asp Thr Glu Asp Ser Ser Cys Tyr Ile Pro Pro Ser Thr
Thr Ile 275 280 285 Lys Gln Asp Phe Arg Leu Leu Gly Gln Thr Ser Val
Asp Arg Leu Leu 290 295 300 Gln Leu Ser Gln Gly Gln Ala Val Lys Gly
Asn Gln Leu Leu Pro Val 305 310 315 320 Ser Leu Val Lys Arg Lys Thr
Thr Leu Ala Pro Asn Thr Gln Thr Ala 325 330 335 Ser Pro Arg Ala Leu
Ala Asp Ser Leu Met Gln Leu Ala Arg Gln Val 340 345 350 Ser Arg Leu
Glu Ser Gly Gln 355 11 1320 DNA Escherichia coli 11 gacaccatcg
aatggtgcaa aacctttcgc ggtatggcat gatagcgccc ggaagagagt 60
caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga gtatgccggt
120 gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc
tgcgaaaacg 180 cgggaaaaag tggaagcggc gatggcggag ctgaattaca
ttcccaaccg cgtggcacaa 240 caactggcgg gcaaacagtc gttgctgatt
ggcgttgcca cctccagtct ggccctgcac 300 gcgccgtcgc aaattgtcgc
ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg 360 gtggtgtcga
tggtagaacg aagcggcgtc gaagcctgta aagcggcggt gcacaatctt 420
ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca ggatgccatt
480 gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc
tgaccagaca 540 cccatcaaca gtattatttt ctcccatgaa gacggtacgc
gactgggcgt ggagcatctg 600 gtcgcattgg gtcaccagca aatcgcgctg
ttagcgggcc cattaagttc tgtctcggcg 660 cgtctgcgtc tggctggctg
gcataaatat ctcactcgca atcaaattca gccgatagcg 720 gaacgggaag
gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat 780
gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg
840 cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt
gggatacgac 900 gataccgaag acagctcatg ttatatcccg ccgtcaacca
ccatcaaaca ggattttcgc 960 ctgctggggc aaaccagcgt ggaccgcttg
ctgcaactct ctcagggcca ggcggtgaag 1020 ggcaatcagc tgttgcccgt
ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg 1080 caaaccgcct
ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc 1140
cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc
1200 accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg
tgagcggata 1260 acaatttcac acaggaaaca gctatgacca tgattacgga
ttcactggcc gtcgttttac 1320 12 1324 DNA Escherichia coli 12
agcggcatgc atttacgttg acaccacctt tcgcggtatg gcatgatagc gcccggaaga
60 gagtcaattc agggtggtga atgtgaaacc agtaacgtta tacgatgtcg
cagagtatgc 120 cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag
gccagccacg tttctgcgaa 180 aacgcgggaa aaagtggaag cggcgatggc
ggagctgaat tacattccca accgcgtggc 240 acaacaactg gcgggcaaac
agtcgttgct gattggcgtt gccacctcca gtctggccct 300 gcacgcgccg
tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag 360
cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa
420 tcttctcgcg caacgcgtca gtgggctgat cattaactat ccgctggatg
accaggatgc 480 cattgctgtg gaagctgcct gcactaatgt tccggcgtta
tttcttgatg tctctgacca 540 gacacccatc aacagtatta ttttctccca
tgaagacggt acgcgactgg gcgtggagca 600 tctggtcgca ttgggtcacc
agcaaatcgc gctgttagcg ggcccattaa gttctgtctc 660 ggcgcgtctg
cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat 720
agcggaacgg gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct
780 gaatgagggc atcgttccca ctgcgatgct ggttgccaac gatcagatgg
cgctgggcgc 840 aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg
gatatctcgg tagtgggata 900 cgacgatacc gaagacagct catgttatat
cccgccgtca accaccatca aacaggattt 960 tcgcctgctg gggcaaacca
gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt 1020 gaagggcaat
cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa 1080
tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
1140 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
ctcactcatt 1200 aggcacccca ggctttacac tttatgcttc cggctcgtat
gttgtgtgga attgtgagcg 1260 gataacaatt tcacacagga aacagctatg
accatgatta cggattcact ggccgtcgtt 1320 ttac 1324 13 3001 DNA
Pseudomonas fluorescens 13 ctacccagaa cgaagatcag cgcctcaatg
gcctcaaggt tctactggtc gatgattcag 60 ccgaagtcgt tgaggtgctg
aacatgctgc tggaaatgga aggcgcccaa gtgagcgcct 120 tcagcgaccc
tttgagcgcg cttgaaacag cccgggatgc ccattacgac gtgattattt 180
cggacatcgg catgccgaaa atgaatggcc atgagctgat gcagaagctg cgtaaagtag
240 gccaccttcg acaggctccc gccatcgcct taacgggcta tggcgctggc
aatgaccaga 300 aaaaggcgac tgaatcgggc tttaatgcgc atgtcagcaa
acccgttggc catgattcgc 360 tcatcacctt gatcgaaaaa ctgtgccgct
cccgccccta ggcgtggggc aggcgttcaa 420 gggtagatga actgagaaaa
gcgcacggac gcgcccgttt ctggtcgcga cacctgggta 480 tccacgctgc
ccaccgtgtc gctgcgcaag gtcaggtaca acacggcctg gccggcgctg 540
tcactcagca tccagacgct cacaccctcc ccggccgccc tggccttgag cggctgaggc
600 tgcagcatct cgatattgaa accgcgcagc agctcaccgc tcaactcgac
ctccaggggt 660 tcctgggcct taccttgcac atgaatcacc agcccatcgg
aggcgccatt gcgcaaaaag 720 cgttggtact ccacgcgcaa ctgcccatcg
gcactgcgca cctcgcggct gctcagcggc 780 ccgctggaaa acagccctgc
caagctcaag ccgatcagca ccagcagcgc gtaccaaccc 840 acccgctcaa
agcgccagac cttgcgctgc aaggccatgt tttcctgcac cggataattg 900
cggctgtgta agtcgtcagg gtctgggttg ttcatagcgg ggcccggact caacccttgc
960 tgtgctcggg agaagacggc cccttggtga caccccgtgg gccggcaatc
gcccatatcg 1020 cagcgcccag aaacggcagc accacgacta ccgcactcca
gcctgccttg ctggccgagg 1080 cgttatcgct gcgccagatg ctgttgatga
tccacgcatc gagcagtacg aggatcactg 1140 ccaggcctat ccagaagtaa
gtggtttgca tgatgcacct ccaggttatg taacttttgg 1200 tgcgcgggtg
cgggcagggt tcattatttt taggttctct gcctggcgct tggtttgccg 1260
ccatcatgcg ggcaacttcg ccgatctact taatgatcga acctcttcaa acaagacaag
1320 ctgaaacgtc tcagctccta taaaaagcca aatcatgcac aaatgcattt
tttgccttga 1380 ccacgggaat cgagtcttct aaagtcaaat cactgtatat
gaatacagta atttgattcc 1440 cttcatggac gagacttact atgaaaagca
ccccttcgaa atttggcaaa acaccccatc 1500 aacccagcct gtggacccgc
gccgatgcgc ttaaagtgca tgcggacgac cccaccacca 1560 cccagccgct
ggtcagcgcg aacttcccgg tattgagtga cgaggtgttt atctgggaca 1620
ccatgccgct gcgtgatatc gacggcaaca tcacctccgt cgatggctgg tcggtgatct
1680 tcaccctcac cgcggatcgc cacccgaacg acccgcaata cctcgatcag
aatggcaact 1740 acgacgtcat ccgcgactgg aacgatcgcc atggccgggc
aaagatgtac tactggttct 1800 cccgcaccgg caaagactgg aagctcggcg
gccgagtgat ggctgaaggg gtttcgccca 1860 ccgtgcgcga atgggccggc
acgccgatcc tgttgaacga gcaaggcgaa gtagacctgt 1920 actacaccgc
cgtcacgccc ggcgcgacca tcgtcaaggt gcgtggccgc gtggtgacca 1980
ccgagcatgg cgtcagcctg gtgggctttg agaaggtcaa gccgctgttc gaggcggacg
2040 gcaagatgta ccagaccgaa gcgcaaaatg cgttctgggg ctttcgcgat
ccatggccgt 2100 tccgcgaccc gaaagacggc aagctgtaca tgctgttcga
aggtaacgtg gccggcgaac 2160 gcggctcgca caaggtcggt aaagccgaaa
tcggcgacgt gccgccaggt tatgaagacg 2220 tcggtaactc gcgcttccag
actgcctgcg tcggtatcgc cgtggcccgc gacgaagacg 2280 gcgacgactg
ggaaatgctg ccaccgctgc tgaccgcggt gggcgtcaac gaccagaccg 2340
aacgcccgca cttcgtgttc caggacggca agtactacct gttcaccatc agccacacct
2400 tcacctacgc cgacggcgtg accggcccgg acggcgtgta cggcttcgtc
gccgattcgc 2460 tgttcggtcc gtatgtgccg ttgaacggct ctggtctggt
actgggcaac ccgtcctccc 2520 aaccgttcca gacctactcg cactgcgtca
tgcccaacgg cctggtgacc tccttcatcg 2580 acagcgtacc gaccgacgac
accggcacgc agatccgtat cggcggcacc gaagcaccga 2640 cggtgggcat
caagatcaaa gggcagcaaa cgtttgtggt cgctgagtat gactacggtt 2700
acatcccgcc gatgctcgac gttacgctca agtaaccgga ggctatgagg tagcggcttt
2760 gagctcgatg acaaacccgc ggtgaatatt cgctgcacct gtggcgaggg
agcttgctcc 2820 cggttgggcc ggacagccgc catcgcaggc aagccagctc
ccacattttg gttcctgggg 2880 cgtcagggag gtatgtgtcg gctgaggggc
cgtcacggga gcaagctccc tcgccacagg 2940 ttcaacagcc cattgggtgg
atattcagga aatagaaatg cctgcaccat tgagttgagt 3000 c 3001 14 20 DNA
artificial lacOid 14 aattgtgagc gctcacaatt 20 15 32 DNA artificial
MB214pyrR1 15 cgatcgggta cctgtcgaag ggctggagac at 32 16 28 DNA
artificial pyrFPstF 16 aactgcagga tcagttgcgg agccttgg 28 17 35 DNA
artificial pyrFoverlap 17 tgctcactct aaaaatctgg aatgggctct caggc 35
18 28 DNA artificial pyrFXbaR2 18 gctctagatg cgtggctgga tgaatgaa 28
19 20 DNA artificial pyranalF 19 ggcgtcgaac aggtagcctt 20 20 20 DNA
artificial pyranalR 20 ctcgcctcct gccacatcaa 20 21 20 DNA
artificial M13F(-40) 21 cagggttttc ccagtcacga 20 22 30 DNA
artificial proC1 22 atatgagctc cgaccttgag tcggccattg 30 23 36 DNA
artificial proC2 23 atatgagctc ggatccagta cgatcagcag gtacag 36 24
20 DNA artificial proC3 24 agcaacacgc gtattgcctt 20 25 23 DNA
artificial proC5 25 gcccttgagt tggcacttca tcg 23 26 25 DNA
artificial proC6 26 gataaacgcg aagatcggcg agata 25 27 35 DNA
artificial proC7 27 ccgagcatgt ttgattagac aggtccttat ttcga 35 28 25
DNA artificial proC8 28 tgcaacgtga cgcaagcagc atcca 25 29 25 DNA
artificial proC9 29 ggaacgatca gcacaagcca tgcta 25 30 30 DNA
artificial genF2 30 atatgagctc tgccgtgatc gaaatccaga 30 31 30 DNA
artificial genR2 31 atatggatcc cggcgttgtg acaatttacc 30 32 17 DNA
artificial XbaNotDraU2 linker 32 tctagagcgg ccgcgtt 17 33 17 DNA
artificial XbaNotDraL linker 33 gcggccgctc tagaaac 17 34 41 DNA
artificial LEV1 34 ttcgaagggg tgctttttct agaagtaagt ctcgtccatg a 41
35 20 DNA artificial LEV2 35 cgcaaggtca ggtacaacac 20 36 20 DNA
artificial LEV3 36 taccagacca gagccgttca 20 37 20 DNA artificial
LEV7 37 ctacccagaa cgaagatcag 20 38 21 DNA artificial LEV8 38
gactcaactc aatggtgcag g 21 39 36 DNA artificial BglXbaLacF 39
agatctctag agaaggcgaa gcggcatgca tttacg 36 40 39 DNA artificial
lacIR4 40 atattctaga gacaactcgc gctaacttac attaattgc 39 41 41 DNA
artificial Lacpro9 41 atattctaga atggtgcaaa acctttcgcg gtatggcatg a
41 42 33 DNA artificial LacIQF 42 gctctagaag cggcatgcat ttacgttgac
acc 33 43 33 DNA artificial LacINXR 43 agctagctct agaaagttgg
gtaacgccag ggt 33 44 58 DNA artificial lacIQ1 44 agtaagcggc
cgcagcggca tgcatttacg ttgacaccac ctttcgcggt atggcatg 58 45 20 DNA
artificial lacIF1 45 acaatcttct cgcgcaacgc 20 46 20 DNA artificial
lacIF2 46 atgttatatc ccgccgttaa 20 47 20 DNA artificial lacIR1 47
ccgctatcgg ctgaatttga 20 48 20 DNA artificial lacIR2 48 tgtaattcag
ctccgccatc 20 49 20 DNA artificial SeqLev5AS 49 tatcgagatg
ctgcagcctc 20 50 20 DNA artificial SeqLev3S 50 acaccttcac
ctacgccgac 20 51 20 DNA artificial LEV10 51 tctacttcgc cttgctcgtt
20 52 32 DNA artificial RC-3 52 gtgagcgctc acaattccac acaggaaaac ag
32 53 32 DNA artificial RC-4 53 ttcgggtgga agtccaggta gttggcggtg ta
32 54 38 DNA artificial RC-9 54 gaattgtgag cgctcacaat tccacacatt
atacgagc 38 55 20 DNA artificial RC-10 55 attcagcgca tgttcaacgg 20
56 50 DNA artificial AKB-1 56 acggttctgg caaacaattg tgagcgctca
caatttattc tgaaatgagc 50 57 24 DNA artificial AKB-2 57 gcgtgggcgg
tgtttatcat gttc 24 58 23 DNA artificial AKB-3 58 tactgcacgc
acaagcctga aca 23 59 34 DNA artificial lacOid sequence 59
tgtgtggaat tgtgagcgct cacaattcca caca 34 60 28 DNA artificial
MB214pyrF1 60 gcggccgctt tggcgcttcg tttacagg 28
* * * * *
References