US5837458A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Methods and compositions for cellular and metabolic engineering
Assignee: Enchira Biotechnology Corp
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Filing: 1996-05-20
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Granted: 1998-11-17
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Expires: 2016-05-20
Abstract
Abstract The present invention is generally directed to the evolution of new metabolic pathways and the enhancement of bioprocessing through a process herein termed recursive sequence recombination. Recursive sequence recombination entails performing iterative cycles of recombination and screening or selection to "evolve" individual genes, whole plasmids or viruses, multigene clusters, or even whole genomes. Such techniques do not require the extensive analysis and computation required by conventional methods for metabolic engineering.
Inventors
- 👤 Jeremy Minshull
- 👤 Willem P. C. Stemmer
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 01:42
CPC Classifications
C; C40; C40B; C40B40/00; C40B40/02; C07; C07K; C07K14/00; C07K14/435; C07K14/43504; C07K14/43595; C07K14/52; C07K14/54; C07K14/545; C07K16/00; C12; C12N; C12N15/00; C12N15/09; C12N15/10; C12N15/102; C12N15/1027; C12N15/1034; C12N15/1037; C12N15/1058; C12N15/1093; C12N15/11; C12N15/52; C12N15/63; C12N15/64; C12N9/00; C12N9/14; C12N9/24; C12N9/2402; C12N9/2468; C12N9/2471; C12N9/78; C12N9/86; C12Q; C12Q1/00; C12Q1/68; C12Q1/6811; C12Y; C12Y302/00; C12Y302/01; C12Y302/01023; B; B07; B07C; B07C2501/00; B07C2501/009; C07K2317/00; C07K2317/50; C07K2317/56; C07K2317/565; C07K2317/60; C07K2317/62; C07K2317/622
Claims Analysis
Total Claims: 50
Claim 1
Independent Claim
216 words
A method of evolving a biocatalytic activity of a cell, comprising: (a) recombining at least a first and second DNA segment from at least one gene conferring ability to catalyze a reaction of interest, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes; (b) screening the library to identify at least one recombinant gene from the library that confers enhanced ability to catalyze the reaction of interest by the cell relative to a wildtype form of the gene; (c) recombining at least a segment from the at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from said first and second DNA segments, to produce a second library of recombinant genes; (d) screening the second library of recombinant genes to identify at least one further recombinant gene from the second library of recombinant genes that confers enhanced ability to catalyze the reaction of interest by the cell relative to a previous recombinant gene; (e) repeating (c) and (d), as necessary, until the further recombinant gene confers a desired level of enhanced ability to catalyze the reaction of interest by the cell, wherein the reaction of interest is the ability to utilize a substrate as a nutrient source.
Claim 2
Independent Claim
216 words
A method of evolving a biocatalytic activity of a cell, comprising: (a) recombining at least a first and second DNA segment from at least one gene conferring ability to catalyze a reaction of interest, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes; (b) screening the library to identify at least one recombinant gene from the library that confers enhanced ability to catalyze the reaction of interest by the cell relative to a wildtype form of the gene; (c) recombining at least a segment from the at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from said first and second DNA segments, to produce a second library of recombinant genes; (d) screening the second library of recombinant genes to identify at least one further recombinant gene from the second library of recombinant genes that confers enhanced ability to catalyze the reaction of interest by the cell relative to a previous recombinant gene; (e) repeating (c) and (d), as necessary, until the further recombinant gene confers a desired level of enhanced ability to catalyze the reaction of interest by the cell, wherein the reaction of interest is the ability to utilize a substrate as a nutrient source.
Claim 3
Independent Claim
211 words
A method of evolving a biocatalytic activity of a cell, comprising: (a) recombining at least a first and second DNA segment from at least one gene conferring ability to catalyze a reaction of interest, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes; (b) screening the library to identify at least one recombinant gene from the library that confers enhanced ability to catalyze the reaction of interest by cell relative to a wildtype form of the gene; (c) recombining at least a segment from the at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from said first and second DNA segments, to produce a second library of recombinant genes; (d) screening the second library of recombinant genes to identify at least one further recombinant gene from the second library of recombinant genes that confers enhanced ability to catalyze the reaction of interest by the cell relative to a previous recombinant gene; (e) repeating (c) and (d), as necessary, until the further recombinant gene confers a desired level of enhanced ability to catalyze the reaction of interest by the cell, wherein the reaction of interest is the ability to detoxify a compound.
Claim 4
Independent Claim
211 words
A method of evolving a biocatalytic activity of a cell, comprising: (a) recombining at least a first and second DNA segment from at least one gene conferring ability to catalyze a reaction of interest, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes; (b) screening the library to identify at least one recombinant gene from the library that confers enhanced ability to catalyze the reaction of interest by cell relative to a wildtype form of the gene; (c) recombining at least a segment from the at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from said first and second DNA segments, to produce a second library of recombinant genes; (d) screening the second library of recombinant genes to identify at least one further recombinant gene from the second library of recombinant genes that confers enhanced ability to catalyze the reaction of interest by the cell relative to a previous recombinant gene; (e) repeating (c) and (d), as necessary, until the further recombinant gene confers a desired level of enhanced ability to catalyze the reaction of interest by the cell, wherein the reaction of interest is the ability to detoxify a compound.
Claim 5
Independent Claim
236 words
A method of evolving a biocatalytic activity of a cell, comprising: (a) recombining at least a first and second DNA segment from at least one gene conferring ability to catalyze a reaction of interest, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes; (b) screening the library to identify at least one recombinant gene from the library that confers enhanced ability to catalyze the reaction of interest by the cell relative to a wildtype form of the gene; (c) recombining at least a segment from the at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from said first and second DNA segments, to produce a second library of recombinant genes; (d) screening the second library of recombinant genes to identify at least one further recombinant gene from the second library of recombinant genes that confers enhanced ability to catalyze the reaction of interest by the cell relative to a previous recombinant gene; (e) repeating (c) and (d), as necessary, until the further recombinant gene confers a desired level of enhanced ability to catalyze the reaction of interest by the cell wherein the reaction of interest is the ability to synthesize a compound of interest and wherein the compound of interest is selected from the group consisting of an amino acid, a polymer, vitamin C, and indigo.
Claim 6
Independent Claim
236 words
A method of evolving a biocatalytic activity of a cell, comprising: (a) recombining at least a first and second DNA segment from at least one gene conferring ability to catalyze a reaction of interest, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes; (b) screening the library to identify at least one recombinant gene from the library that confers enhanced ability to catalyze the reaction of interest by the cell relative to a wildtype form of the gene; (c) recombining at least a segment from the at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from said first and second DNA segments, to produce a second library of recombinant genes; (d) screening the second library of recombinant genes to identify at least one further recombinant gene from the second library of recombinant genes that confers enhanced ability to catalyze the reaction of interest by the cell relative to a previous recombinant gene; (e) repeating (c) and (d), as necessary, until the further recombinant gene confers a desired level of enhanced ability to catalyze the reaction of interest by the cell wherein the reaction of interest is the ability to synthesize a compound of interest and wherein the compound of interest is selected from the group consisting of an amino acid, a polymer, vitamin C, and indigo.
Claim 7
Dependent Claim
44 words
The method of claim 1, 2 or 3, wherein at least one recombining step is performed in vitro, and the resulting library of recombinants is introduced into the cell whose biocatalytic activity is to be enhanced generating a library of cells containing different recombinants.
Claim 8
Dependent Claim
51 words
The method of claim 4, wherein the in vitro recombining step comprises: cleaving the first and second segments into fragments; mixing and denaturing the fragments; and incubating the denatured fragments with a polymerase under conditions which result in annealing of the denatured fragments and formation of the library of recombinant genes.
Claim 9
Dependent Claim
18 words
The method of claim 1, 2 or 3, wherein at least one recombining step is performed in vivo.
Claim 10
Dependent Claim
26 words
The method of claim 1, 2 or 3, wherein at least one recombining step is performed in the cell whose biocatalytic activity is to be enhanced.
Claim 11
Dependent Claim
28 words
The method of claim 1, 2 or 3, wherein at least one DNA segment comprises a cluster of genes collectively conferring ability to catalyze a reaction of interest.
Claim 12
Independent Claim
187 words
A method of generating a new biocatalytic activity in a cell, comprising: (a) recombining at least first and second DNA segments from at least one gene conferring ability to catalyze a first enzymatic reaction related to a second enzymatic reaction of interest, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes; (b) screening at least one recombinant gene from the library that confers a new ability to catalyze the second reaction of interest; (c) recombining at least a segment from at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from the first and second segments, to produce a further library of recombinant genes; (d) screening at least one further recombinant gene from the further library of recombinant genes that confers enhanced ability to catalyze the second reaction of interest in the cell relative to a previous recombinant gene; (e) repeating (c) and (d), as necessary, until the further recombinant gene confers a desired level of enhanced ability to catalyze the second reaction of interest in the cell.
Claim 13
Independent Claim
187 words
A method of generating a new biocatalytic activity in a cell, comprising: (a) recombining at least first and second DNA segments from at least one gene conferring ability to catalyze a first enzymatic reaction related to a second enzymatic reaction of interest, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes; (b) screening at least one recombinant gene from the library that confers a new ability to catalyze the second reaction of interest; (c) recombining at least a segment from at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from the first and second segments, to produce a further library of recombinant genes; (d) screening at least one further recombinant gene from the further library of recombinant genes that confers enhanced ability to catalyze the second reaction of interest in the cell relative to a previous recombinant gene; (e) repeating (c) and (d), as necessary, until the further recombinant gene confers a desired level of enhanced ability to catalyze the second reaction of interest in the cell.
Claim 14
Independent Claim
197 words
A method of optimizing expression of a gene product, the method comprising: (a) recombining at least a first and second DNA segments from at least one gene conferring ability to produce the gene product, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes; (b) screening the library to identify at least one recombinant gene from the library that confers optimized expression of the gene product relative to a wildtype form of the gene; (c) recombining at least a segment from the at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from said first and second DNA segments, to produce a second library of recombinant genes; (d) screening the second library of recombinant genes to identify at least one further recombinant gene from the second library of recombinant genes that confers optimized ability to produce the gene product relative to a previous recombinant gene; (e) repeating (c) and (d), as necessary, until the further recombinant gene confers a desired level of optimized ability to express the gene product wherein at least one recombining step is performed in vivo.
Claim 15
Independent Claim
197 words
A method of optimizing expression of a gene product, the method comprising: (a) recombining at least a first and second DNA segments from at least one gene conferring ability to produce the gene product, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes; (b) screening the library to identify at least one recombinant gene from the library that confers optimized expression of the gene product relative to a wildtype form of the gene; (c) recombining at least a segment from the at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from said first and second DNA segments, to produce a second library of recombinant genes; (d) screening the second library of recombinant genes to identify at least one further recombinant gene from the second library of recombinant genes that confers optimized ability to produce the gene product relative to a previous recombinant gene; (e) repeating (c) and (d), as necessary, until the further recombinant gene confers a desired level of optimized ability to express the gene product wherein at least one recombining step is performed in vivo.
Claim 16
Dependent Claim
21 words
The method of claim 10, wherein the recombining step is performed in a host cell wherein the gene product is expressed.
Claim 17
Dependent Claim
28 words
The method of claim 10, wherein the at least one gene is a host cell gene and wherein the host cell gene does not encode the gene product.
Claim 18
Dependent Claim
15 words
The method of claim 10, wherein optimization results in increased expression of the gene product.
Claim 19
Independent Claim
176 words
A method of evolving a biosensor for a compound A of interest, the method comprising: (a) recombining at least first and second DNA segments from at least one gene conferring ability to detect a structurally related compound B, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes; (b) screening at least one recombinant gene from the library that confers optimized ability to detect compound A relative to a wildtype form of the gene; (c) recombining at least a segment from the at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from the first and second segments, to produce a further library of recombinant genes; (d) screening at least one further recombinant gene from the further library of recombinant genes that confers optimized ability to detect compound A relative to a previous recombinant gene; (e) repeating (c) and (d), as necessary, until the further recombinant gene confers a desired level of optimized ability to detect compound A.
Claim 20
Independent Claim
176 words
A method of evolving a biosensor for a compound A of interest, the method comprising: (a) recombining at least first and second DNA segments from at least one gene conferring ability to detect a structurally related compound B, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes; (b) screening at least one recombinant gene from the library that confers optimized ability to detect compound A relative to a wildtype form of the gene; (c) recombining at least a segment from the at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from the first and second segments, to produce a further library of recombinant genes; (d) screening at least one further recombinant gene from the further library of recombinant genes that confers optimized ability to detect compound A relative to a previous recombinant gene; (e) repeating (c) and (d), as necessary, until the further recombinant gene confers a desired level of optimized ability to detect compound A.
Claim 21
Dependent Claim
16 words
The method of claim 14, wherein optimization results in increased amplitude of response by the biosensor.
Claim 22
Dependent Claim
13 words
The method of claim 14, wherein compound A and compound B are different.
Claim 23
Dependent Claim
13 words
The method of claim 14, wherein compound A and compound B are identical.
Claim 24
Dependent Claim
32 words
The method of claim 2, wherein the compound is selected from the group consisting of benzene, biphenyl, xylene, toluene, camphor, naphthalene, halogenated hydrocarbons, polychlorinated biphenyls, polycyclic aromatic hydrocarbons, trichlorethylene, pesticides, and herbicides.
Claim 25
Dependent Claim
10 words
The method of claim 18, wherein the compound is atrazine.
Claim 26
Independent Claim
203 words
A method of evolving a biocatalytic activity of a cell, comprising: (a) recombining at least a first and second DNA segment from at least one gene conferring ability of a dioxygenase to catalyze a reaction of interest, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes; (b) screening the library to identify at least one recombinant gene from the library that confers enhanced ability to catalyze the reaction of interest by the cell relative to a wildtype form of the gene; (c) recombining at least a segment from the at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from said first and second DNA segments, to produce a second library of recombinant genes; (d) screening the second library of recombinant genes to identify at least one further recombinant gene from the second library of recombinant genes that confers enhanced ability to catalyze the reaction of interest by the cell relative to a previous recombinant gene; (e) repeating (c) and (d), as necessary, until the further recombinant gene confers a desired level of enhanced ability to catalyze the reaction of interest by the cell.
Claim 27
Independent Claim
203 words
A method of evolving a biocatalytic activity of a cell, comprising: (a) recombining at least a first and second DNA segment from at least one gene conferring ability of a dioxygenase to catalyze a reaction of interest, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes; (b) screening the library to identify at least one recombinant gene from the library that confers enhanced ability to catalyze the reaction of interest by the cell relative to a wildtype form of the gene; (c) recombining at least a segment from the at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from said first and second DNA segments, to produce a second library of recombinant genes; (d) screening the second library of recombinant genes to identify at least one further recombinant gene from the second library of recombinant genes that confers enhanced ability to catalyze the reaction of interest by the cell relative to a previous recombinant gene; (e) repeating (c) and (d), as necessary, until the further recombinant gene confers a desired level of enhanced ability to catalyze the reaction of interest by the cell.
Claim 28
Dependent Claim
13 words
The method of claim 20, wherein the dioxygenase is from a Pseudomonas species.
Claim 29
Independent Claim
201 words
A method of evolving a biocatalytic activity of a cell, comprising: (a) recombining at least a first and second DNA segment from at least one gene conferring to the cell the ability to be resistant to a heavy metal, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes; (b) screening the library to identify at least one recombinant gene from the library that confers enhanced ability to catalyze the reaction of interest by the cell relative to a wildtype form of the gene; (c) recombining at least a segment from the at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from said first and second DNA segments, to produce a second library of recombinant genes; (d) screening the second library of recombinant genes to identify at least one further recombinant gene from the second library of recombinant genes that confers enhanced ability to catalyze the reaction of interest by the cell relative to a previous recombinant gene; (e) repeating (c) and (d), as necessary, until further recombinant gene confers a desired level of enhanced ability of resistance to the heavy metal.
Claim 30
Independent Claim
201 words
A method of evolving a biocatalytic activity of a cell, comprising: (a) recombining at least a first and second DNA segment from at least one gene conferring to the cell the ability to be resistant to a heavy metal, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes; (b) screening the library to identify at least one recombinant gene from the library that confers enhanced ability to catalyze the reaction of interest by the cell relative to a wildtype form of the gene; (c) recombining at least a segment from the at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from said first and second DNA segments, to produce a second library of recombinant genes; (d) screening the second library of recombinant genes to identify at least one further recombinant gene from the second library of recombinant genes that confers enhanced ability to catalyze the reaction of interest by the cell relative to a previous recombinant gene; (e) repeating (c) and (d), as necessary, until further recombinant gene confers a desired level of enhanced ability of resistance to the heavy metal.
Claim 31
Dependent Claim
22 words
The method of claim 22, wherein the heavy metal is selected from the group consisting of mercury, arsenate, chromate, cadmium, and silver.
Claim 32
Independent Claim
213 words
A method of evolving a biocatalytic activity of a cell, comprising: (a) recombining at least a first and second DNA segment from at least one gene conferring ability to catalyze a reaction of interest, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes; (b) screening the library to identify at least one recombinant gene from the library that confers enhanced ability to catalyze the reaction of interest by the cell relative to a wildtype form of the gene; (c) recombining at least a segment from the at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from said first and second DNA segments, to produce a second library of recombinant genes; (d) screening the second library of recombinant genes to identify at least one further recombinant gene from the second library of recombinant genes that confers enhanced ability to catalyze the reaction of interest by the cell relative to a previous recombinant gene; (e) repeating (c) and (d), as necessary, until the further recombinant gene confers a desired level of enhanced ability to catalyze the reaction of interest by the cell, wherein the reaction of interest is the ability to biocatalyze desulfurization of oil.
Claim 33
Independent Claim
213 words
A method of evolving a biocatalytic activity of a cell, comprising: (a) recombining at least a first and second DNA segment from at least one gene conferring ability to catalyze a reaction of interest, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes; (b) screening the library to identify at least one recombinant gene from the library that confers enhanced ability to catalyze the reaction of interest by the cell relative to a wildtype form of the gene; (c) recombining at least a segment from the at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from said first and second DNA segments, to produce a second library of recombinant genes; (d) screening the second library of recombinant genes to identify at least one further recombinant gene from the second library of recombinant genes that confers enhanced ability to catalyze the reaction of interest by the cell relative to a previous recombinant gene; (e) repeating (c) and (d), as necessary, until the further recombinant gene confers a desired level of enhanced ability to catalyze the reaction of interest by the cell, wherein the reaction of interest is the ability to biocatalyze desulfurization of oil.
Claim 34
Dependent Claim
18 words
The method of claim 24, wherein the first or second DNA segment is derived from a Rhodococcus species.
Claim 35
Independent Claim
185 words
A method of optimizing production of a metabolite by a metabolic pathway, the method comprising: (a) recombining at least a first and second DNA segments from at least one gene conferring ability to produce the metabolite, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes; (b) screening the library to identify at least one recombinant gene from the library that confers optimized production of the metabolite relative to a wildtype form of the gene; (c) recombining at least a segment from the at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from said first and second DNA segments, to produce a second library of recombinant genes; (d) screening the second library of recombinant genes to identify at least one further recombinant gene from the second library of recombinant genes that confers optimized ability to produce the metabolite relative to a previous recombinant gene; (e) repeating (c) and (d), as necessary, until the further recombinant gene confers a desired level of optimized production of the metabolite.
Claim 36
Independent Claim
185 words
A method of optimizing production of a metabolite by a metabolic pathway, the method comprising: (a) recombining at least a first and second DNA segments from at least one gene conferring ability to produce the metabolite, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes; (b) screening the library to identify at least one recombinant gene from the library that confers optimized production of the metabolite relative to a wildtype form of the gene; (c) recombining at least a segment from the at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from said first and second DNA segments, to produce a second library of recombinant genes; (d) screening the second library of recombinant genes to identify at least one further recombinant gene from the second library of recombinant genes that confers optimized ability to produce the metabolite relative to a previous recombinant gene; (e) repeating (c) and (d), as necessary, until the further recombinant gene confers a desired level of optimized production of the metabolite.
Claim 37
Dependent Claim
15 words
The method of claim 26, wherein at least one recombining step is performed in vivo.
Claim 38
Dependent Claim
15 words
The method of claim 26, wherein production of the metabolite is monitored by mass spectroscopy.
Claim 39
Dependent Claim
25 words
The method of claim 1, wherein the nutrient source is selected from the group consisting of lactose, whey, galactose, mannitol, xylan, cellobiose, cellulose and sucrose.
Claim 40
Dependent Claim
22 words
The method of claim 3, wherein the step of screening the library further comprises detecting the compound of interest by mass spectroscopy.
Claim 41
Dependent Claim
22 words
The method of claim 3, wherein the reaction of interest is the ability of an enzyme to synthesize the compound of interest.
Claim 42
Dependent Claim
23 words
The method of claim 6, wherein at least one recombining step is performed in the cell whose biocatalytic activity is to be enhanced.
Claim 43
Independent Claim
223 words
A method of evolving a biocatalytic activity of a cell, comprising: (a) recombining at least a first and second DNA segment from at least one gene conferring ability to catalyze a reaction of interest, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes; (b) screening the library to identify at least one recombinant gene from the library that confers enhanced ability to catalyze the reaction of interest by the cell relative to a wildtype form of the gene; (c) recombining at least a segment from the at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from said first and second DNA segments, to produce a second library of recombinant genes; (d) screening the second library of recombinant genes to identify at least one further recombinant gene from the second library of recombinant genes that confers enhanced ability to catalyze the reaction of interest by the cell relative to a previous recombinant gene; (e) repeating (c) and (d), as necessary, until the further recombinant gene confers a desired level of enhanced ability to catalyze the reaction of interest by the cell wherein the reaction of interest is the ability to synthesize a compound of interest and wherein the compound of interest is an isoprenoid.
Claim 44
Independent Claim
223 words
A method of evolving a biocatalytic activity of a cell, comprising: (a) recombining at least a first and second DNA segment from at least one gene conferring ability to catalyze a reaction of interest, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes; (b) screening the library to identify at least one recombinant gene from the library that confers enhanced ability to catalyze the reaction of interest by the cell relative to a wildtype form of the gene; (c) recombining at least a segment from the at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from said first and second DNA segments, to produce a second library of recombinant genes; (d) screening the second library of recombinant genes to identify at least one further recombinant gene from the second library of recombinant genes that confers enhanced ability to catalyze the reaction of interest by the cell relative to a previous recombinant gene; (e) repeating (c) and (d), as necessary, until the further recombinant gene confers a desired level of enhanced ability to catalyze the reaction of interest by the cell wherein the reaction of interest is the ability to synthesize a compound of interest and wherein the compound of interest is an isoprenoid.
Claim 45
Independent Claim
223 words
A method of evolving a biocatalytic activity of a cell, comprising: (a) recombining at least a first and second DNA segment from at least one gene conferring ability to catalyze a reaction of interest, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes; (b) screening the library to identify at least one recombinant gene from the library that confers enhanced ability to catalyze the reaction of interest by the cell relative to a wildtype form of the gene; (c) recombining at least a segment from the at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from said first and second DNA segments, to produce a second library of recombinant genes; (d) screening the second library of recombinant genes to identify at least one further recombinant gene from the second library of recombinant genes that confers enhanced ability to catalyze the reaction of interest by the cell relative to a previous recombinant gene; (e) repeating (c) and (d), as necessary, until the further recombinant gene confers a desired level of enhanced ability to catalyze the reaction of interest by the cell wherein the reaction of interest is the ability to synthesize a compound of interest and wherein the compound of interest is a polyketide.
Claim 46
Independent Claim
223 words
A method of evolving a biocatalytic activity of a cell, comprising: (a) recombining at least a first and second DNA segment from at least one gene conferring ability to catalyze a reaction of interest, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes; (b) screening the library to identify at least one recombinant gene from the library that confers enhanced ability to catalyze the reaction of interest by the cell relative to a wildtype form of the gene; (c) recombining at least a segment from the at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from said first and second DNA segments, to produce a second library of recombinant genes; (d) screening the second library of recombinant genes to identify at least one further recombinant gene from the second library of recombinant genes that confers enhanced ability to catalyze the reaction of interest by the cell relative to a previous recombinant gene; (e) repeating (c) and (d), as necessary, until the further recombinant gene confers a desired level of enhanced ability to catalyze the reaction of interest by the cell wherein the reaction of interest is the ability to synthesize a compound of interest and wherein the compound of interest is a polyketide.
Claim 47
Independent Claim
223 words
A method of evolving a biocatalytic activity of a cell, comprising: (a) recombining at least a first and second DNA segment from at least one gene conferring ability to catalyze a reaction of interest, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes; (b) screening the library to identify at least one recombinant gene from the library that confers enhanced ability to catalyze the reaction of interest by the cell relative to a wildtype form of the gene; (c) recombining at least a segment from the at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from said first and second DNA segments, to produce a second library of recombinant genes; (d) screening the second library of recombinant genes to identify at least one further recombinant gene from the second library of recombinant genes that confers enhanced ability to catalyze the reaction of interest by the cell relative to a previous recombinant gene; (e) repeating (c) and (d), as necessary, until the further recombinant gene confers a desired level of enhanced ability to catalyze the reaction of interest by the cell wherein the reaction of interest is the ability to synthesize a compound of interest and wherein the compound of interest is a carotenoid.
Claim 48
Independent Claim
223 words
A method of evolving a biocatalytic activity of a cell, comprising: (a) recombining at least a first and second DNA segment from at least one gene conferring ability to catalyze a reaction of interest, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes; (b) screening the library to identify at least one recombinant gene from the library that confers enhanced ability to catalyze the reaction of interest by the cell relative to a wildtype form of the gene; (c) recombining at least a segment from the at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from said first and second DNA segments, to produce a second library of recombinant genes; (d) screening the second library of recombinant genes to identify at least one further recombinant gene from the second library of recombinant genes that confers enhanced ability to catalyze the reaction of interest by the cell relative to a previous recombinant gene; (e) repeating (c) and (d), as necessary, until the further recombinant gene confers a desired level of enhanced ability to catalyze the reaction of interest by the cell wherein the reaction of interest is the ability to synthesize a compound of interest and wherein the compound of interest is a carotenoid.
Claim 49
Independent Claim
223 words
A method of evolving a biocatalytic activity of a cell, comprising: (a) recombining at least a first and second DNA segment from at least one gene conferring ability to catalyze a reaction of interest, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes; (b) screening the library to identify at least one recombinant gene from the library that confers enhanced ability to catalyze the reaction of interest by the cell relative to a wildtype form of the gene; (c) recombining at least a segment from the at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from said first and second DNA segments, to produce a second library of recombinant genes; (d) screening the second library of recombinant genes to identify at least one further recombinant gene from the second library of recombinant genes that confers enhanced ability to catalyze the reaction of interest by the cell relative to a previous recombinant gene; (e) repeating (c) and (d), as necessary, until the further recombinant gene confers a desired level of enhanced ability to catalyze the reaction of interest by the cell wherein the reaction of interest is the ability to synthesize a compound of interest and wherein the compound of interest is an antibiotic.
Claim 50
Independent Claim
223 words
A method of evolving a biocatalytic activity of a cell, comprising: (a) recombining at least a first and second DNA segment from at least one gene conferring ability to catalyze a reaction of interest, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes; (b) screening the library to identify at least one recombinant gene from the library that confers enhanced ability to catalyze the reaction of interest by the cell relative to a wildtype form of the gene; (c) recombining at least a segment from the at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from said first and second DNA segments, to produce a second library of recombinant genes; (d) screening the second library of recombinant genes to identify at least one further recombinant gene from the second library of recombinant genes that confers enhanced ability to catalyze the reaction of interest by the cell relative to a previous recombinant gene; (e) repeating (c) and (d), as necessary, until the further recombinant gene confers a desired level of enhanced ability to catalyze the reaction of interest by the cell wherein the reaction of interest is the ability to synthesize a compound of interest and wherein the compound of interest is an antibiotic.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 2001-11-16 | 2002-05-18 | 2002-11-17 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2005-11-16 | 2006-05-18 | 2006-11-17 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2009-11-16 | 2010-05-18 | 2010-11-17 | $7700.00 | Window Closed / Lapsed |