Methods and compositions for cellular and metabolic engineering

• US 7,795,030 B2
• Filed: 01/28/2008
• Issued: 09/14/2010
• Est. Priority Date: 02/17/1994
• Status: Expired due to Fees

• Current Assignee: Codexis, Inc.
• Original Assignee: Maxygen Inc.
• Inventors: Minshull, Jeremy, Stemmer, Willem P. C.
• Primary Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US12/011,767
• Publication Number:
  US 20080227649A1
• Time in Patent Office: 960 Days
• Field of Search: 435/440, 435/6, 536/22.1, 536/23.1
• 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 first and second DNA subsequences derived from the sequence of at least one gene conferring ability to catalyze a reaction of interest, the subsequences differing from each other in at least two nucleotides, to produce a library of recombinant genes, and 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 subsequence derived from the at least one recombinant gene with a further DNA subsequence derived from the at least one gene, the same or different from the first and second subsequences, 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 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.

2. The method of claim 1, wherein the further recombinant gene in (e) encodes an enzyme for production of ethanol and/or butanol.

3. The method of claim 1, further comprising altering an organism with the further recombinant gene in (e), wherein the organism is used in production of ethanol and/or butanol.

4. The method of claim 1, wherein the further recombinant gene in (e) encodes an enzyme with increased specificity to cellulose.

5. The method of claim 1, wherein the further recombinant gene in (e) is used to increase production of enzymes for the metabolism of cellulose.

6. The method of claim 1, further comprising amplifying a mixture from (e).

7. The method of claim 6, further comprising cloning the amplified mixture into a vector.

8. The method of claim 1, wherein the recombining operation comprises:

cleaving the first and second DNA subsequences 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 further library of recombinant genes.

9. The method of claim 8, wherein the fragments arc between about 50 bp and 5 kb in length.

10. The method of claim 8, wherein the fragments are between about 10 bp and 1000 bp in length.

11. The method of claim 8, wherein a concentration of a fragment having a particular length or sequence is less than about 1% by weight of the first and second DNA subsequences.

12. The method of claim 8, wherein a number of the fragments is least about 100.

13. The method of claim 8, wherein the cleaving, mixing, and incubating are repeated between about 2 and 100 times.

14. The method of claim 1, wherein the further library of recombinant genes comprises double stranded polynucleotides that are between about 50 bp and 100 kb in length.