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US 6,391,640 B1Granted
Methods and compositions for cellular and metabolic engineering
AssigneeMaxygen, Inc.
InventorsJeremy Minshull, Willem P. C. Stemmer
Life of this patent
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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.
Claims225
A method of shuffling polynucleotides, the method comprising:
- (i) providing a plurality of polynucleotide variants to be shuffled;
- (ii) conducting a multi-cyclic polynucleotide extension process on at least partially annealed polynucleotide strands having sequences from the plurality of polynucleotide variants, the polynucleotide strands having regions of similarity and regions of heterology with each other, and being at least partially annealed through the regions of similarity under conditions whereby one strand serves as a template for extension of another strand with which it is partially annealed, to generate a population of recombinant polynucleotides;
- and,
- (iii) selecting or screening a recombinant polynucleotide from the population of recombinant polynucleotides for a desired property, thereby producing an optimized recombinant polynucleotide having the desired property;
- wherein the desired property comprises:
- an elevated expression of the recombinant polynucleotide in a first cell relative to an expression level of one or more of the plurality of polynucleotide variants to be shuffled in the first cell.
3 more elements
A method of shuffling polynucleotides, the method comprising:
- (i) providing a plurality of polynucleotide variants to be shuffled;
- (ii) conducting a multi-cyclic polynucleotide extension process on at least partially annealed polynucleotide strands having sequences from the plurality of polynucleotide variants, the polynucleotide strands having regions of similarity and regions of heterology with each other, and being at least partially annealed through the regions of similarity under conditions whereby one strand serves as a template for extension of another strand with which it is partially annealed, to generate a population of recombinant polynucleotides;
- and,
- (iii) selecting or screening a recombinant polynucleotide from the population of recombinant polynucleotides for a desired property, thereby producing an optimized recombinant polynucleotide having the desired property;
- wherein the desired property comprises one or more of:
- (a) an ability encoded by the recombinant polynucleotide to utilize a substrate as a nutrient source;
- (b) an ability encoded by the recombinant polynucleotide to detoxify a composition;
- or,
- (c) an ability encoded by the recombinant polynucleotide to confer enhanced resistance to a heavy metal when the recombinant polynucleotide is expressed in a cell.
6 more elements
A method of shuffling polynucleotides, the method comprising:
- (i) providing a plurality of polynucleotide variants to be shuffled;
- (ii) conducting a multi-cyclic polynucleotide extension process on at least partially annealed polynucleotide strands having sequences from the plurality of polynucleotide variants, the polynucleotide strands having regions of similarity and regions of heterology with each other, and being at least partially annealed through the regions of similarity under conditions whereby one strand serves as a template for extension of another strand with which it is partially annealed, to generate a population of recombinant polynucleotides;
- and,
- (iii) selecting or screening a recombinant polynucleotide from the population of recombinant polynucleotides for a desired property, thereby producing an optimized recombinant polynucleotide having the desired property;
- wherein the desired property comprises:
- an ability encoded by the recombinant polynucleotide to catalyze a reaction of interest, wherein the reaction of interest is not substantially catalyzed by the plurality of polynucleotide variants to be shuffled.
3 more elements
A method of shuffling polynucleotides, the method comprising:
- (i) providing a plurality of polynucleotide variants to be shuffled;
- (ii) conducting a multi-cyclic polynucleotide extension process on at least partially annealed polynucleotide strands having sequences from the plurality of polynucleotide variants, the polynucleotide strands having regions of similarity and regions of heterology with each other, and being at least partially annealed through the regions of similarity under conditions whereby one strand serves as a template for extension of another strand with which it is partially annealed, to generate a population of recombinant polynucleotides;
- and,
- (iii) selecting or screening a recombinant polynucleotide from the population of recombinant polynucleotides for a desired property, thereby producing an optimized recombinant polynucleotide having the desired property;
- wherein the desired property comprises one or more of:
- (a) an ability encoded by the recombinant polynucleotide to catalyze a reaction in a cell, or,
- (b) an ability encoded by the recombinant polynucleotide to detect a compound.
4 more elements
A method of shuffling polynucleotides, the method comprising:
- (i) providing a plurality of polynucleotide variants to be shuffled;
- (ii) conducting a multi-cyclic polynucleotide extension process on at least partially annealed polynucleotide strands having sequences from the plurality of polynucleotide variants, the polynucleotide strands having regions of similarity and regions of heterology with each other, and being at least partially annealed through the regions of similarity under conditions whereby one strand serves as a template for extension of another strand with which it is partially annealed, to generate a population of recombinant polynucleotides;
- and,
- (iii) selecting or screening a recombinant polynucleotide from the population of recombinant polynucleotides for a desired property, thereby producing an optimized recombinant polynucleotide having the desired property;
- wherein the desired property comprises one or more property selected from the group consisting of:
- (a) an ability encoded by the recombinant nucleic acid to produce a desired metabolite;
- and,
- (b) an ability to synthesize an isoprenoid, a polyketide, a carotenoid, an antibiotic, an amino acid, a polymer, vitamin C, or indigo.
5 more elements
A method of providing an optimized recombinant hydrolytic enzyme comprising shuffling two or more polynucleotides which are derived from one or more hydrolytic enzyme coding polynucleotides.
Dependencies are inferred from the claim text.