Methods and compositions for cellular and metabolic engineering

• US 8,048,674 B2
• Filed: 09/11/2009
• Issued: 11/01/2011
• Est. Priority Date: 02/17/1994
• Status: Expired due to Fees

• Current Assignee: Codexis, Inc.
• Original Assignee: Codexis Mayflower Holdings, LLC (Codexis, Inc.)
• Inventors: Minshull, Jeremy, Stemmer, Willem P. C.
• Primary Examiner(s): Whisenant; Ethan C
• Application Number:
  US12/557,812
• Publication Number:
  US 20100144547A1
• Time in Patent Office: 781 Days
• Field of Search: 435/440, 435/6, 536/22.1, 536/23.1, 536/24.3
• US Class Current: 435/440
• CPC Class Codes: C12N 15/1027 by DNA shuffling, e.g. RSR, STEP, RPR
• CPC Class Codes: C12N 15/1058 Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms

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.

Claims

1. A method of evolving a biocatalytic activity of a cell, comprising:

(a) recombining at least a first and a second DNA segment from at least one gene conferring ability to catalyze a reaction of interest by conducting a multi-cyclic polynucleotide extension process on at least partially annealed polynucleotide strands having sequences from the first and the second DNA segment, the first and the second DNA segment differing from each other in at least two nucleotides, to produce a library of recombinant genes, wherein the reaction of interest is the metabolism of cellulose; and

(b) screening 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,

wherein at least one of the first and the second DNA segment is produced by performing mutagenesis on one or more DNA molecules.

2. The method of claim 1, wherein the at least partially annealed polynucleotide strands are partially annealed through regions of similarity under conditions, whereby one strand serves as a template for extension of another strand, with which it is partially annealed, to produce the library of recombinant genes.

3. The method of claim 1, further comprising:

(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 the second DNA segment, 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 reaction of interest by the cell relative to the at least one 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.

4. The method of claim 1, further comprising expressing an industrial enzyme encoded by the at least one recombinant gene.

5. The method of claim 4, further comprising using the industrial enzyme to metabolize the cellulose.

6. The method of claim 4, further comprising using the industrial enzyme to produce ethanol and/or butanol.

7. The method of claim 1, wherein the reaction of interest is a part of production of ethanol and/or butanol.

8. The method of claim 1, further comprising altering an organism with the at least one recombinant gene, wherein the organism is used in production of ethanol and/or butanol.

9. The method of claim 1, wherein the at least one recombinant gene encodes an enzyme with increased specificity to cellulose relative to the wildtype form of the gene.

10. The method of claim 1, wherein the at least one recombinant gene increases production of enzymes for the metabolism of cellulose relative to the wildtype form of the gene.

11. The method of claim 1, wherein the library of recombinant genes is cloned into at least one expression vector, thereby producing a library of expressed recombinant genes.

12. The method of claim 11, further comprising expressing the library of expressed recombinant genes in a population of cells, thereby producing a library of expressed recombinant polynucleotide cellular expression products.

13. The method of claim 12, wherein the library of expressed recombinant polynucleotide cellular expression products comprises a library of expressed peptides.

14. The method of claim 1, wherein at least a portion of the multi-cyclic polynucleotide extension process is performed in a host cell.

15. The method of claim 14, wherein the library of recombinant genes is expressed in a population of host cells.

16. The method of claim 1, wherein mutagenesis comprises one or more techniques selected from the group consisting of:

error-prone PCR mutagenesis, cassette mutagenesis, passage through a bacterial mutator strain, and treatment with a chemical mutagen.

17. A method of evolving a biocatalytic activity of a cell, comprising:

(a) recombining at least a first and a second DNA segment from at least one gene conferring ability to catalyze a reaction of interest by conducting a multi-cyclic polynucleotide extension process on at least partially annealed polynucleotide strands having sequences from the first and the second DNA segment, the first and the second DNA segment differing from each other in at least two nucleotides, to produce a library of recombinant genes, wherein the reaction of interest is the metabolism of cellulose;

(b) screening 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 the first and the second DNA segment, 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 reaction of interest by the cell relative to the at least one recombinant gene; and

(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.

18. A method of evolving a biocatalytic activity of a cell, comprising:

(a) recombining at least a first and a second DNA segment from at least one gene conferring ability to catalyze a reaction of interest by conducting a multi-cyclic polynucleotide extension process on at least partially annealed polynucleotide strands having sequences from the first and the second DNA segment, the first and the second DNA segment differing from each other in at least two nucleotides, to produce a library of recombinant genes, wherein the reaction of interest is the metabolism of cellulose; and

(b) screening 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,

wherein at least a portion of the multi-cyclic polynucleotide extension process is performed in a host cell.

19. The method of claim 18, wherein the library of recombinant genes is expressed in a population of host cells.