Methods and compositions for cellular and metabolic engineering

• US 5,837,458 A
• Filed: 05/20/1996
• Issued: 11/17/1998
• Est. Priority Date: 02/17/1994
• Status: Expired due to Term

• Current Assignee: Codexis, Inc.
• Original Assignee: Maxygen Inc.
• Inventors: Minshull, Jeremy, Stemmer, Willem P. C.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US08/650,400
• Time in Patent Office: 911 Days
• Field of Search: 435/6, 435/91.2, 435/172.3, 935/76, 935/77, 935/78
• US Class Current: 435/6.14
• CPC Class Codes: B07C 2501/009 Sorting of fruit
• CPC Class Codes: C07K 14/43595 from coelenteratae, e.g. medusae
• CPC Class Codes: C07K 14/545 IL-1
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 2317/565 Complementarity determining region [CDR]
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C12N 15/1027 by DNA shuffling, e.g. RSR, STEP, RPR
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• 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
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/64 General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
• CPC Class Codes: C12N 9/2471 Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
• CPC Class Codes: C12N 9/86 acting on amide bonds in cyclic amides, e.g. penicillinase (3.5.2)
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Y 302/01023 Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
• CPC Class Codes: C40B 40/02 Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors

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 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.

4. 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.

5. 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.

6. The method of claim 1, 2 or 3, wherein at least one recombining step is performed in vivo.

7. 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.

8. 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.

29. 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.

32. The method of claim 6, wherein at least one recombining step is performed in the cell whose biocatalytic activity is to be enhanced.

2. 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.

18. 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.

19. The method of claim 18, wherein the compound is atrazine.

3. 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.

30. The method of claim 3, wherein the step of screening the library further comprises detecting the compound of interest by mass spectroscopy.

31. The method of claim 3, wherein the reaction of interest is the ability of an enzyme to synthesize the compound of interest.

9. 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.

10. 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.

11. The method of claim 10, wherein the recombining step is performed in a host cell wherein the gene product is expressed.

12. 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.

13. The method of claim 10, wherein optimization results in increased expression of the gene product.

14. 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.

15. The method of claim 14, wherein optimization results in increased amplitude of response by the biosensor.

16. The method of claim 14, wherein compound A and compound B are different.

17. The method of claim 14, wherein compound A and compound B are identical.

20. 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.

21. The method of claim 20, wherein the dioxygenase is from a Pseudomonas species.

22. 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.

23. The method of claim 22, wherein the heavy metal is selected from the group consisting of mercury, arsenate, chromate, cadmium, and silver.

24. 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.

25. The method of claim 24, wherein the first or second DNA segment is derived from a Rhodococcus species.

26. 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.

27. The method of claim 26, wherein at least one recombining step is performed in vivo.

28. The method of claim 26, wherein production of the metabolite is monitored by mass spectroscopy.

33. 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.

34. 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.

35. 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.

36. 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.