US2005186666A1PendingUtilityA1
Protein expression systems
Assignee: DOW GLOBAL TECHNOLOGIES INCPriority: Nov 19, 2003Filed: Nov 19, 2004Published: Aug 25, 2005
Est. expiryNov 19, 2023(expired)· nominal 20-yr term from priority
C12N 1/20C12P 21/00C12N 15/78C12N 15/52C12N 9/88C12Y 305/05001C12N 9/0028C12P 21/02
57
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Claims
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.
Claims
exact text as granted — not AI-modified1 ) 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 Δ 1 -pyrroline-5-carboxylate 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 Q, and lacI 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 Q, and lacI 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 Δ 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 Q, and lac 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 Q, and lacI 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 Q, and lacI 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 Δ 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 Q, and lacI 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 Δ 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 Q, and lacI 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.Join the waitlist — get patent alerts
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