Methods and compositions for cellular and metabolic engineering

• US 6,391,640 B1
• Filed: 11/09/1998
• Issued: 05/21/2002
• 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): Whisenant, Ethan C.
• Application Number:
  US09/188,777
• Publication Number:
  US 20020025517A1
• Time in Patent Office: 1,289 Days
• Field of Search: 435/440, 435/6, 435/91.2, 536/23.1, 536/24.3, 935/76, 935/77, 935/78
• US Class Current: 435/440
• 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 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.

2. The method of claim 1, wherein the selected optimized recombinant nucleic acid is further recombined with one or more additional nucleic acid to produce at least one further recombined nucleic acid, wherein the at least one further recombined nucleic acid is selected for the desired property, thereby producing a further optimized nucleic acid.

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

4. The method of claim 3, wherein the library of expressed recombinant polynucleotides is expressed in a population of cells, thereby producing a library of expressed recombinant polynucleotide cellular expression products.

5. The method of claim 4, wherein the library of expressed recombinant polynucleotide cellular expression products comprises a library of expressed peptides.

6. The method of claim 4, wherein the population of cells is screened for the desired property.

7. The method of claim 1, wherein at least one cycle of polynucleotide extension is performed in vitro.

8. The method of claim 1, the multi-cyclic polynucleotide extension process comprising:

cleaving the plurality of polynucleotide variants into fragments;

mixing and denaturing the fragments; and

,

incubating the resulting denatured fragments with a polymerase under conditions which result in annealing of the denatured fragments and formation of the population of recombinant polynucleotides.

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

10. The method of claim 9, wherein the population of recombinant polynucleotides are expressed in a population of host cells.

11. The method of claim 10, further comprising selecting the population of host cells for the desired property.

12. The method of claim 1, wherein the desired property further comprises an ability encoded by the recombinant polynucleotide to detect compound A, the method comprising:

recombining at least first and second DNA segments from at least one gene conferring an ability to detect a structurally related compound B, the segments differing from each other in at least two nucleotides, to produce the population of recombinant polynucleotides;

selecting or screening at least one recombinant gene from the population of recombinant polynucleotides that confers an optimized ability to detect compound A relative to a wildtype form of the gene;

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 population of recombinant genes;

selecting or screening at least one further recombinant gene from the further population of recombinant genes that confers optimized ability to detect compound A relative to a previous recombinant gene; and

,

repeating the steps as desired, until the further recombinant gene confers a desired level of optimized ability to detect compound A.

13. The method of claim 12, wherein compound A and compound B are different.

14. The method of claim 12, wherein compound A and compound B are the same.

15. The method of claim 12, wherein compound A and compound B are independently selected from:

atrazine, benzene, biphenyl, xylene, toluene, camphor, naphtalene, halogenated hydrocarbons, polychlorinated biphenyls, polycyclic aromatic hydrocarbons, trichlorethylene, pesticides, and herbicides.

16. The method of claim 1, wherein the desired property further comprises an ability encoded by the recombinant polynucleotide to catalyze a reaction in a cell, wherein the plurality of polynucleotide variants to be shuffled encodes one or more nucleic acid encoding a dioxygenase.

17. The method of claim 16, wherein the dioxygenase is from a Pseudomoas species.

18. The method of claim 1, wherein the desired property further comprises an ability encoded by the recombinant polynucleotide confer enhanced resistance to a heavy metal when the recombinant polynucleotide is expressed in a cell, wherein the heavy metal is selected from mercury, arsenate, chromate, cadmium, and silver.

19. The method of claim 1, wherein the desired property further comprises an ability encoded by the recombinant nucleic acid to produce a desired metabolite, the method further comprising detecting the desired metabolite by mass spectroscopy.

20. The method of claim 1, wherein the desired property further comprises an ability encoded by the recombinant polynucleotide to utilize a substrate as a nutrient source, wherein the nutrient source is selected from lactose, whey, galactose, mannitol, xylan, cellobiose, cellulose, and sucrose.

21. The method of claim 1, wherein the desired property further comprises an ability to synthesize an isoprenoid, a polyketide, a carotenoid, an antibiotic, an amino acid, a polymer, vitamin C, or indigo, and wherein:

the antibiotic is selected from;

a peptide, a peptidolactone, a thiopeptide, a beta-lactam, a glycopeptide, a lantibiotic, a microcin, a polyketide-derived antibiotic an anthracyclin, a tetracyclin, a macrolide, an avermectin, a polyether, an ansamycin, a chloramphenicol, an aminoglycoside, an aminocyclitol, a polyoxin, an agrocin, a cyclosporin, a pepstatin, an actinomycin, a gramicidin, a depsipeptide, a vancomycin and an isoprenoid;

the carotenoid is selected from;

myxobacton, spheroidene, spheroidenone, lutein, astaxanthin, violaxanthin, 4-ketorulene, myxoxanthrophyll, echinenone, lycopene, zeaxanthin and its mono- and di- glucosides, alpha-, beta-, gamma- and delta-carotene, beta-cryptoxanthin monoglucoside and neoxanthin; and

,

the amino acid is selected from phenylalanine, monosodium glutamate, glycine, lysine, threonine, tryptophan, and methionine.

22. The method of claim 1, wherein the desired property further comprises an ability to synthesize an isoprenoid, a polyketide, a carotenoid, an antibiotic, an amino acid, a polymer, vitamin C, or indigo, and wherein the plurality of polynucleotide variants comprises a nucleic acid subsequence derived from one or more of:

a gene encoding an isoprenoid synthesis gene, a gene encoding a trichodiene synthase from Fusarium sprorotrichioides, a gene encoding a pentalene synthase from Streptomyces, a gene encoding an aristolochene synthase from Penicillium roquefortii, and a gene encoding an epi-aristolochene synthase from N. tabacum.

23. The method of claim 1, wherein the desired property further comprises an ability to synthesize an isoprenoid, a polyketide, a carotenoid, an antibiotic, an amino acid, a polymer, vitamin C, or indigo, and wherein the plurality of polynucleotide variants comprises a nucleic acid subsequence derived from one or more of:

a gene encoding a penicillin N expandase, a gene encoding a penicillin transacylase, a gene encoding a penicillin amidase, a gene encoding a Penicillin G acylase, and a gene encoding a Polyketide synthase.

24. The method of claim 1, wherein the plurality of polynucleotide variants comprises a nucleic acid subsequence derived from one or more gene from:

Alcaligenes, Zoogloea, Rhizobium, Bacillus, or an Azobacter which produces a polyhydroxyalkanoates (PHAs), or a polyhyroxybutyrate (PHB).

25. The method of claim 1, wherein the plurality of polynucleotide variants comprises a nucleic acid subsequence derived from phbB or phbC.

26. The method of claim 1, wherein the plurality of polynucleotide variants comprises at least one subsequence derived from a gene in a cellulose biosynthesis pathway.

27. The method of claim 1, wherein the plurality of polynucleotide variants comprises at least one subsequence derived from a gene in an E. coli indigo synthesis pathway which synthesizes indigo from glucose via a tryptophan/indole pathway.

28. The method of claim 1, wherein the partially annealed polynucleotide strands are produced by providing overlapping single-stranded segments of the polynucleotide variants and incubating under annealing conditions whereby the single-stranded segments from different polynucleotide variants anneal to form the partially annealed polynucleotide strands.

29. The method of claim 28, wherein the overlapping single-stranded segments are random segments of the polynucleotide variants.

30. The method of claim 28, wherein the overlapping single-stranded segments are non-random segments of the polynucleotide variants.

31. The method of claim 28, wherein the plurality of overlapping single-stranded segments are produced by cleaving the population of polynucleotide variants to produce a population of overlapping double-stranded fragments and denaturing the double-stranded fragments to produce the overlapping single-stranded segments.

32. The method of claim 31, wherein the population of polynucleotide variants comprise one or more DNA and the cleavage is performed by DNase I digestion.

33. The method of claim 1, further comprising amplifying the plurality of polynucleotide variants.

34. The method of claim 1, further comprising performing error-prone PCR on at least one of the polynucleotide variants.

35. The method of claim 1, wherein the partially annealed polynucleotide strands are produced with a DNA synthesizer.

36. The method of claim 1, wherein the plurality of polynucleotide variants which are shuffled are naturally occurring variants of a polynucleotide.

37. The method of claim 36, wherein the naturally occurring variants of the polynucleotide encode natural polypeptides.

38. The method of claim 36, wherein the naturally occurring variants of the polynucleotide comprise one or more of:

a naturally occurring human polynucleotide or subsequence thereof, a naturally occurring mouse polynucleotide or subsequence thereof, a naturally occurring bacterial polynucleotide or subsequence thereof, a naturally occurring plant polynucleotide or subsequence thereof, a naturally occurring fungal polynucleotide or subsequence thereof, a naturally occurring animal polynucleotide or subsequence thereof, and a naturally occurring viral polynucleotide or subsequence thereof.

39. The method of claim 36, wherein the naturally occurring variants of the polynucleotide comprise allelic or non-allelic naturally occurring variants of the polynucleotide, wherein the allelic or non-allelic naturally occurring variants are from a single species.

40. The method of claim 36, further comprising shuffling a first recombinant polynucleotide encoding the desired activity with a naturally occurring polynucleotide and selecting any resulting secondary recombinant nucleic acids for the desired property.

41. The method of claim 40, wherein the secondary recombinant nucleic acids are more closely similar in sequence to the naturally occurring polynucleotide than to the first recombinant polynucleotide.

42. The method of claim 36, further comprising supplementing the plurality of natural variants of a polynucleotide with induced variants of a polynucleotide before the amplification process.

43. The method of claim 36, further comprising randomly fragmenting the population of natural variants of a polynucleotide before conducting the amplification process.

44. The method of claim 1, wherein the multi-cyclic polynucleotide extension process comprises PCR amplification of the annealed polynucleotide strands.

45. The method of claim 1, wherein the desired property further comprises an ability encoded by the optimized recombinant polynucleotide to degrade at least one halogenated hydrocarbon, wherein the plurality of chosen polynucleotide variants to be recombined encodes one or more hydrolytic enzyme.

46. The method of claim 45, wherein the ability encoded by the optimized recombinant polynucleotide to degrade at least one halogenated hydrocarbon occurs in a cell.

47. The method of claim 1, wherein the plurality of polynucleotide variants comprises at least two homologous polynucleotides.

48. The method of claim 1, wherein the plurality of polynucleotide variants comprises species variants.

49. The method of claim 1, wherein the partially annealed polynucleotide strands are produced by providing overlapping single-stranded segments of the plurality of polynucleotide variants and incubating under annealing conditions whereby the single-stranded segments from the plurality of polynucleotide variants anneal to form the partially annealed polynucleotide strands.

50. The method of claim 49, wherein the overlapping single-stranded segments comprise random segments of the polynucleotide variants.

51. The method of claim 49, wherein the overlapping single-stranded segments comprise selected segments of the polynucleotide variants.

52. The method of claim 49, wherein the overlapping single-stranded segments are synthesized.

172. The method of claim 31, wherein the population of polynucleotide variants comprise one or more DNAs and the cleavage is performed by DNase I digestion.

53. 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.

54. The method of claim 53, wherein the selected optimized recombinant nucleic acid is further recombined with one or more additional nucleic acid to produce at least one further recombined nucleic acid, wherein the at least one further recombined nucleic acid is selected for the desired property, thereby producing a further optimized nucleic acid.

55. The method of claim 53, wherein the population of recombinant polynucleotides is cloned into at least one expression vector, thereby producing a library of expressed recombinant polynucleotides.

56. The method of claim 55, wherein the library of expressed recombinant polynucleotides is expressed in a population of cells, thereby producing a library of expressed recombinant polynucleotide cellular expression products.

57. The method of claim 56, wherein the library of expressed recombinant polynucleotide cellular expression products comprises a library of expressed polypeptides.

58. The method of claim 56, wherein the population of cells is screened for the desired property.

59. The method of claim 53, wherein at least one cycle of polynucleotide extension is performed in vitro.

60. The method of claim 53, the multi-cyclic polynucleotide extension process comprising:

cleaving the plurality of polynucleotide variants into fragments;

mixing and denaturing the fragments; and

,

incubating the resulting denatured fragments with a polymerase under conditions which result in annealing of the denatured fragments and formation of the population of recombinant polynucleotides.

61. The method of claim 53, wherein at least a portion of the multi-cyclic polynucleotide extension process is performed in a host cell.

62. The method of claim 61, wherein the population of recombinant polynucleotides are expressed in a population of host cells.

63. The method of claim 62, further comprising selecting the population of host cells for the desired property.

64. The method of claim 53, wherein the desired property is the ability encoded by the recombinant polynucleotide conferring enhanced resistance to a heavy metal when the recombinant polynucleotide is expressed in a cell, wherein the heavy metal is selected from mercury, arsenate, chromate, cadmium, and silver.

65. The method of claim 53, wherein the desired property is the ability encoded by the recombinant polynucleotide to utilize a substrate as a nutrient source, wherein the nutrient source is selected from lactose, whey, galactose, mannitol, xylan, cellobiose, cellulose, and sucrose.

66. The method of claim 53, wherein the partially annealed polynucleotide strands are produced by providing overlapping single-stranded segments of the polynucleotide variants and incubating under annealing conditions whereby the single-stranded segments from different polynucleotide variants anneal to form the partially annealed polynucleotide strands.

67. The method of claim 66, wherein the overlapping single-stranded segments are random segments of the polynucleotide variants.

68. The method of claim 66, wherein the overlapping single-stranded segments are non-random segments of the polynucleotide variants.

69. The method of claim 66, wherein the plurality of overlapping single-stranded segments are produced by cleaving the population of polynucleotide variants to produce a population of overlapping double-stranded fragments and denaturing the double-stranded fragments to produce the overlapping single-stranded segments.

70. The method of claim 69, wherein the population of polynucleotide variants comprise one or more DNAs and the cleavage is performed by DNase I digestion.

71. The method of claim 53, further comprising amplifying the plurality of polynucleotide variants.

72. The method of claim 53, further comprising performing error-prone PCR on at least one of the polynucleotide variants.

73. The method of claim 53, wherein the partially annealed polynucleotide strands are produced with a DNA synthesizer.

74. The method of claim 53, wherein the plurality of polynucleotide variants which are shuffled are naturally occurring variants of a polynucleotide.

75. The method of claim 74, wherein the naturally occurring variants of the polynucleotide encode natural polypeptides.

76. The method of claim 74, wherein the naturally occurring variants of the polynucleotide comprise one or more of:

a naturally occurring human polynucleotide or subsequence thereof, a naturally occurring mouse polynucleotide or subsequence thereof, a naturally occurring bacterial polynucleotide or subsequence thereof, a naturally occurring plant polynucleotide or subsequence thereof, a naturally occurring fungal polynucleotide or subsequence thereof, a naturally occurring animal polynucleotide or subsequence thereof, and a naturally occurring viral polynucleotide or subsequence thereof.

77. The method of claim 74, wherein the naturally occurring variants of the polynucleotide comprise allelic or non-allelic naturally occurring variants of the polynucleotide, wherein the allelic or non-allelic naturally occurring variants are from a single species.

78. The method of claim 74, further comprising shuffling a first recombinant polynucleotide encoding the desired activity with a naturally occurring polynucleotide and selecting any resulting secondary recombinant nucleic acids for the desired property.

79. The method of claim 78, wherein the secondary recombinant nucleic acids are more closely similar in sequence to the naturally occurring polynucleotide than to the first recombinant polynucleotide.

80. The method of claim 74, further comprising supplementing the plurality of naural variants of a polynucleotide with induced variants of a polynucleotide before the amplification process.

81. The method of claim 74, further comprising randomly fragmenting the population of natural variants of a polynucleotide before conducting the amplification process.

82. The method of claim 53, wherein the multi-cyclic polynucleotide extension process comprises PCR amplification of the annealed polynucleotide strands.

83. The method of claim 53, wherein the desired property is an ability encoded by the optimized recombinant polynucleotide to degrade at least one halogenated hydrocarbon, wherein the plurality of chosen polynucleotide variants to be recombined encodes one or more hydrolytic enzyme.

84. The method of claim 83, wherein the ability encoded by the optimized recombinant polynucleotide to degrade at least one halogenated hydrocarbon occurs in a cell.

85. 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.

86. The method of claim 85, wherein the selected optimized recombinant nucleic acid is further recombined with one or more additional nucleic acid to produce at least one further recombined nucleic acid, wherein the at least one further recombined nucleic acid is selected for the desired property, thereby producing a further optimized nucleic acid.

87. The method of claim 85, wherein the population of recombinant polynucleotides is cloned into at least one expression vector, thereby producing a library of expressed recombinant polynucleotides.

88. The method of claim 87, wherein the library of expressed recombinant polynucleotides is expressed in a population of cells, thereby producing a library of expressed recombinant polynucleotide cellular expression products.

89. The method of claim 88, wherein the library of expressed recombinant polynucleotide cellular expression products comprises a library of expressed polypeptides.

90. The method of claim 88, wherein the population of cells is screened for the desired property.

91. The method of claim 85, wherein at least one cycle of polynucleotide extension is performed in vitro.

92. The method of claim 85, the multi-cyclic polynucleotide extension process comprising:

cleaving the plurality of polynucleotide variants into fragments;

mixing and denaturing the fragments; and

,

incubating the resulting denatured fragments with a polymerase under conditions which result in annealing of the denatured fragments and formation of the population of recombinant polynucleotides.

93. The method of claim 85, wherein at least a portion of the multi-cyclic polynucleotide extension process is performed in a host cell.

94. The method of claim 93, wherein the population of recombinant polynucleotides are expressed in a population of host cells.

95. The method of claim 94, further comprising selecting the population of host cells for the desired property.

96. The method of claim 85, wherein the plurality of polynucleotide variants to be shuffled encodes one or more nucleic acid encoding a dioxygenase.

97. The method of claim 96, wherein the dioxygenase is from a Pseudomonas species.

98. The method of claim 85, wherein the reaction of interest comprises an ability encoded by the recombinant polynucleotide to detect compound A, the method comprising:

recombining at least first and second DNA segments from at least one gene conferring an ability to detect a structurally related compound B, the segments differing from each other in at least two nucleotides, to produce the population of recombinant polynucleotides;

selecting or screening at least one recombinant gene from the population of recombinant polynucleotides that confers an optimized ability to detect compound A relative to a wildtype form of the gene;

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 population of recombinant genes;

selecting or screening at least one further recombinant gene from the further population of recombinant genes that confers optimized ability to detect compound A relative to a previous recombinant gene; and

,

repeating the steps as desired, until the further recombinant gene confers a desired level of optimized ability to detect compound A.

99. The method of claim 98, wherein compound A and compound B are different.

100. The method of claim 98, wherein compound A and compound B are the same.

101. The method of claim 98, wherein compound A and compound B are independently selected from:

atrazine, benzene, biphenyl, xylene, toluene, camphor, naphtalene, halogenated hydrocarbons, polychlorinated biphenyls, polycyclic aromatic hydrocarbons, trichlorethylene, pesticides, and herbicides.

102. The method of claim 85, wherein the the plurality of polynucleotide variants to be shuffled encodes one or more nucleic acid encoding a dioxygenase.

103. The method of claim 102, wherein the dioxygenase is from a Pseudomonas species.

104. The method of claim 85, wherein the reaction of interest confers enhanced resistance to a heavy metal when the recombinant polynucleotide is expressed in a cell, wherein the heavy metal is selected from mercury, arsenate, chromate, cadmium, and silver.

105. The method of claim 85, wherein the reaction of interest produces a desired metabolite, the method further comprising detecting the desired metabolite by mass spectroscopy.

106. The method of claim 85, wherein the reaction of interest provides utilization of a substrate as a nutrient source, wherein the nutrient source is selected from lactose, whey, galactose, mannitol, xylan, cellobiose, cellulose, and sucrose.

107. The method of claim 85, wherein the reaction of interest comprise synthesis of an isoprenoid, a polyketide, a carotenoid, an antibiotic, an amino acid, a polymer, vitamin C, or indigo, and wherein:

the antibiotic is selected from;

a peptide, a peptidolactone, a thiopeptide, a beta-lactam, a glycopeptide, a lantibiotic, a microcin, a polyketide-derived antibiotic an anthracyclin, a tetracyclin, a macrolide, an avermectin, a polyether, an ansamycin, a chloramphenicol, an aminoglycoside, an aminocyclitol, a polyoxin, an agrocin, a cyclosporin, a pepstatin, an actinomycin, a gramicidin, a depsipeptide, a vancomycin and an isoprenoid;

the carotenoid is selected from;

myxobacton, spheroidene, spheroidenone, lutein, astaxanthin, violaxanthin, 4-ketorulene, myxoxanthrophyll, echinenone, lycopene, zeaxanthin and its mono- and di- glucosides, alpha-, beta-, gamma- and delta-carotene, beta-cryptoxanthin monoglucoside and neoxanthin; and

,

the amino acid is selected from phenylalanine, monosodium glutamate, glycine, lysine, threonine, tryptophan, and methionine.

108. The method of claim 85, wherein the reaction of interest comprises synthesis of an isoprenoid, a polyketide, a carotenoid, an antibiotic, an amino acid, a polymer, vitamin C, or indigo, and wherein the plurality of polynucleotide variants comprises a nucleic acid subsequence derived from one or more of:

a gene encoding an isoprenoid synthesis gene, a gene encoding a trichodiene synthase from Fusarium sprorotrichioides, a gene encoding a pentalene synthase from Streptomyces, a gene encoding an aristolochene synthase from Penicillium roquefortii, and a gene encoding an epi-aristolochene synthase from N. tabacum.

109. The method of claim 85, wherein the reaction of interest comprises synthesis of an isoprenoid, a polyketide, a carotenoid, an antibiotic, an amino acid, a polymer, vitamin C, or indigo, and wherein the plurality of polynucleotide variants comprises a nucleic acid subsequence derived from one or more of:

a gene encoding a penicillin N expandase, a gene encoding a penicillin transacylase, a gene encoding a penicillin amidase, a gene encoding a Penicillin G acylase, and a gene encoding a Polyketide synthase.

110. The method of claim 85, wherein the plurality of polynucleotide variants comprises a nucleic acid subsequence derived from one or more gene from:

Alcaligenes, Zoogloea, Rhizobium, Bacillus, or an Azobacter which produces a polyhydroxyalkanoates (PHAs), or a polyhyroxybutyrate (PHB).

111. The method of claim 85, wherein the plurality of polynucleotide variants comprises a nucleic acid subsequence derived from phbB or phbC.

112. The method of claim 85, wherein the plurality of polynucleotide variants comprises at least one subsequence derived from a gene in a cellulose biosynthesis pathway.

113. The method of claim 85, wherein the plurality of polynucleotide variants comprises at least one subsequence derived from a gene in an E. coli indigo synthesis pathway which synthesizes indigo from glucose via a tryptophan/indole pathway.

114. The method of claim 85, wherein the partially annealed polynucleotide strands are produced by providing overlapping single-stranded segments of the polynucleotide variants and incubating under annealing conditions whereby the single-stranded segments from different polynucleotide variants anneal to form the partially annealed polynucleotide strands.

115. The method of claim 114, wherein the overlapping single-stranded segments are random segments of the polynucleotide variants.

116. The method of claim 114, wherein the overlapping single-stranded segments are non-random segments of the polynucleotide variants.

117. The method of claim 114, wherein the plurality of overlapping single-stranded segments are produced by cleaving the population of polynucleotide variants to produce a population of overlapping double-stranded fragments and denaturing the double-stranded fragments to produce the overlapping single-stranded segments.

118. The method of claim 117, wherein the population of polynucleotide variants comprise one or more DNAs and the cleavage is performed by DNase I digestion.

119. The method of claim 85, further comprising amplifying the plurality of polynucleotide variants.

120. The method of claim 85, further comprising performing error-prone PCR on at least one of the polynucleotide variants.

121. The method of claim 85, wherein the partially annealed polynucleotide strands are produced with a DNA synthesizer.

122. The method of claim 85, wherein the plurality of polynucleotide variants which are shuffled are naturally occurring variants of a polynucleotide.

123. The method of claim 122, wherein the naturally occurring variants of the polynucleotide encode natural polypeptides.

124. The method of claim 122, wherein the naturally occurring variants of the polynucleotide comprise one or more of:

a naturally occurring human polynucleotide or subsequence thereof, a naturally occurring mouse polynucleotide or subsequence thereof, a naturally occurring bacterial polynucleotide or subsequence thereof, a naturally occurring plant polynucleotide or subsequence thereof, a naturally occurring fungal polynucleotide or subsequence thereof, a naturally occurring animal polynucleotide or subsequence thereof, and a naturally occurring viral polynucleotide or subsequence thereof.

125. The method of claim 122, wherein the naturally occurring variants of the polynucleotide comprise allelic or non-allelic naturally occurring variants of the polynucleotide, wherein the allelic or non-allelic naturally occurring variants are from a single species.

126. The method of claim 122, further comprising shuffling a first recombinant polynucleotide encoding the desired activity with a naturally occurring polynucleotide and selecting any resulting secondary recombinant nucleic acids for the desired property.

127. The method of claim 126, wherein the secondary recombinant nucleic acids are more closely similar in sequence to the naturally occurring polynucleotide than to the first recombinant polynucleotide.

128. The method of claim 122, further comprising supplementing the plurality of naural variants of a polynucleotide with induced variants of a polynucleotide before the amplification process.

129. The method of claim 122, further comprising randomly fragmenting the population of natural variants of a polynucleotide before conducting the amplification process.

130. The method of claim 85, wherein the multi-cyclic polynucleotide extension process comprises PCR amplification of the annealed polynucleotide strands.

131. The method of claim 85, wherein the reaction of interest degrades at least one halogenated hydrocarbon, wherein the plurality of chosen polynucleotide variants to be recombined encodes one or more hydrolytic enzyme.

132. The method of claim 131, wherein the ability encoded by the optimized recombinant polynucleotide to degrade at least one halogenated hydrocarbon occurs in a cell.

133. The method of claim 85, wherein the plurality of polynucleotide variants comprises at least two homologous polynucleotides.

134. The method of claim 85, wherein the plurality of polynucleotide variants comprises species variants.

135. The method of claim 85, wherein the partially annealed polynucleotide strands are produced by providing overlapping single-stranded segments of the plurality of polynucleotide variants and incubating under annealing conditions whereby the single-stranded segments from the plurality of polynucleotide variants anneal to form the partially annealed polynucleotide strands.

136. The method of claim 135, wherein the overlapping single-stranded segments comprise random segments of the polynucleotide variants.

137. The method of claim 135, wherein the overlapping single-stranded segments comprise selected segments of the polynucleotide variants.

138. The method of claim 135, wherein the overlapping single-stranded segments are synthesized.

139. 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.

140. The method of claim 139, wherein the selected optimized recombinant nucleic acid is further recombined with one or more additional nucleic acid to produce at least one further recombined nucleic acid, wherein the at least one further recombined nucleic acid is selected for the desired property, thereby producing a further optimized nucleic acid.

141. The method of claim 139, wherein the population of recombinant polynucleotides is cloned into at least one expression vector, thereby producing a library of expressed recombinant polynucleotides.

142. The method of claim 141, wherein the library of expressed recombinant polynucleotides is expressed in a population of cells, thereby producing a library of expressed recombinant polynucleotide cellular expression products.

143. The method of claim 142, wherein the library of expressed recombinant polynucleotide cellular expression products comprises a library of expressed polypeptides.

144. The method of claim 142, wherein the population of cells is screened for the desired property.

145. The method of claim 139, wherein at least one cycle of polynucleotide extension is performed in vitro.

146. The method of claim 139, the multi-cyclic polynucleotide extension process comprising:

cleaving the plurality of polynucleotide variants into fragments;

mixing and denaturing the fragments; and

,

incubating the resulting denatured fragments with a polymerase under conditions which result in annealing of the denatured fragments and formation of the population of recombinant polynucleotides.

147. The method of claim 139, wherein at least a portion of the multi-cyclic polynucleotide extension process is performed in a host cell.

148. The method of claim 147, wherein the population of recombinant polynucleotides are expressed in a population of host cells.

149. The method of claim 148, further comprising selecting the population of host cells for the desired property.

150. The method of claim 139, wherein the desired property is an ability encoded by the recombinant polynucleotide to detect compound A, the method comprising:

recombining at least first and second DNA segments from at least one gene conferring an ability to detect a structurally related compound B, the segments differing from each other in at least two nucleotides, to produce the population of recombinant polynucleotides;

selecting or screening at least one recombinant gene from the population of recombinant polynucleotides that confers an optimized ability to detect compound A relative to a wildtype form of the gene;

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 population of recombinant genes;

selecting or screening at least one further recombinant gene from the further population of recombinant genes that confers optimized ability to detect compound A relative to a previous recombinant gene; and

,

repeating the steps as desired, until the further recombinant gene confers a desired level of optimized ability to detect compound A.

151. The method of claim 150, wherein compound A and compound B are different.

152. The method of claim 150, wherein compound A and compound B are the same.

153. The method of claim 150, wherein compound A and compound B are independently selected from:

atrazine, benzene, biphenyl, xylene, toluene, camphor, napthalene, halogenated hydrocarbons, polychlorinated biphenyls, polycyclic aromatic hydrocarbons, trichlorethylene, pesticides, and herbicides.

154. The method of claim 139, wherein the desired property is the ability encoded by the recombinant polynucleotide to catalyze a reaction in a cell, wherein the plurality of polynucleotide variants to be shuffled encodes one or more nucleic acid encoding a dioxygenase.

155. The method of claim 154, wherein the dioxygenase is from a Pseudomonas species.

156. The method of claim 139, wherein the reaction in the cell confers enhanced resistance of the cell to a heavy metal when the recombinant polynucleotide is expressed in the cell, wherein the heavy metal is selected from mercury, arsenate, chromate, cadmium, and silver.

157. The method of claim 139, wherein the reaction in the cell provides for production of a desired metabolite in the cell.

158. The method of claim 157, the method further comprising detecting the desired metabolite by mass spectroscopy.

159. The method of claim 139, wherein the reaction provides the cell with an ability to utilize a substrate as a nutrient source, wherein the nutrient source is selected from lactose, whey, galactose, mannitol, xylan, cellobiose, cellulose, and sucrose.

160. The method of claim 139, wherein the reaction provides the cell with an ability to synthesize an isoprenoid, a polyketide, a carotenoid, an antibiotic, an amino acid, a polymer, vitamin C, or indigo.

161. The method of claim 139, wherein the reaction provides the cell with an ability to synthesize an isoprenoid, a polyketide, a carotenoid, an antibiotic, an amino acid, a polymer, vitamin C, or indigo and wherein:

the antibiotic is selected from;

a peptide, a peptidolactone, a thiopeptide, a beta-lactam, a glycopeptide, a lantibiotic, a microcin, a polyketide-derived antibiotic an anthracyclin, a tetracyclin, a macrolide, an avermectin, a polyether, an ansamycin, a chloramphenicol, an aminoglycoside, an aminocyclitol, a polyoxin, an agrocin, a cyclosporin, a pepstatin, an actinomycin, a gramicidin, a depsipeptide, a vancomycin and an isoprenoid;

the carotenoid is selected from;

myxobacton, spheroidene, spheroidenone, lutein, astaxanthin, violaxanthin, 4-ketorulene, myxoxanthrophyll, echinenone, lycopene, zeaxanthin and its mono- and di- glucosides, alpha-, beta-, gamma- and delta-carotene, beta-cryptoxanthin monoglucoside and neoxanthin; and

,

the amino acid is selected from phenylalanine, monosodium glutamate, glycine, lysine, threonine, tryptophan, and methionine.

162. The method of claim 139, wherein the reaction provides the cell with an ability to synthesize an isoprenoid, a polyketide, a carotenoid, an antibiotic, an amino acid, a polymer, vitamin C, or indigo and, and wherein the plurality of polynucleotide variants comprises a nucleic acid subsequence derived from one or more of:

a gene encoding an isoprenoid synthesis gene, a gene encoding a trichodiene synthase from Fusarium sprorotrichioides, a gene encoding a pentalene synthase from Streptomyces, a gene encoding an aristolochene synthase from Penicillium roquefortii, and a gene encoding an epi-aristolochene synthase from N. tabacum.

163. The method of claim 139, wherein the reaction provides the cell with an ability to synthesize an isoprenoid, a polyketide, a carotenoid, an antibiotic, an amino acid, a polymer, vitamin C, or indigo, and wherein the plurality of polynucleotide variants comprises a nucleic acid subsequence derived from one or more of:

a gene encoding a penicillin N expandase, a gene encoding a penicillin transacylase, a gene encoding a penicillin amidase, a gene encoding a Penicillin G acylase, and a gene encoding a Polyketide synthase.

164. The method of claim 139, wherein the plurality of polynucleotide variants comprises a nucleic acid subsequence derived from one or more gene from:

Alcaligenes, Zoogloea, Rhizobium, Bacillus, or an Azobacter which produces a polyhydroxyalkanoates (PHAs), or a polyhyroxybutyrate (PHB).

165. The method of claim 139, wherein the plurality of polynucleotide variants comprises a nucleic acid subsequence derived from phbB or phbC.

166. The method of claim 139, wherein the plurality of polynucleotide variants comprises at least one subsequence derived from a gene in a cellulose biosynthesis pathway.

167. The method of claim 139, wherein the plurality of polynucleotide variants comprises at least one subsequence derived from a gene in an E. coli indigo synthesis pathway which synthesizes indigo from glucose via a tryptophan/indole pathway.

168. The method of claim 139, wherein the partially annealed polynucleotide strands are produced by providing overlapping single-stranded segments of the polynucleotide variants and incubating under annealing conditions whereby the single-stranded segments from different polynucleotide variants anneal to form the partially annealed polynucleotide strands.

169. The method of claim 168, wherein the overlapping single-stranded segments are random segments of the polynucleotide variants.

170. The method of claim 168, wherein the overlapping single-stranded segments are non-random segments of the polynucleotide variants.

171. The method of claim 168, wherein the plurality of overlapping single-stranded segments are produced by cleaving the population of polynucleotide variants to produce a population of overlapping double-stranded fragments and denaturing the double-stranded fragments to produce the overlapping single-stranded segments.

173. The method of claim 139, further comprising amplifying the plurality of polynucleotide variants.

174. The method of claim 139, further comprising performing error-prone PCR on at least one of the polynucleotide variants.

175. The method of claim 139, wherein the partially annealed polynucleotide strands are produced with a DNA synthesizer.

176. The method of claim 139, wherein the plurality of polynucleotide variants which are shuffled are naturally occurring variants of a polynucleotide.

177. The method of claim 176, wherein the naturally occurring variants of the polynucleotide encode natural polypeptides.

178. The method of claim 177, wherein the naturally occurring variants of the polynucleotide comprise one or more of:

a naturally occurring human polynucleotide or subsequence thereof, a naturally occurring mouse polynucleotide or subsequence thereof, a naturally occurring bacterial polynucleotide or subsequence thereof, a naturally occurring plant polynucleotide or subsequence thereof, a naturally occurring fungal polynucleotide or subsequence thereof, a naturally occurring animal polynucleotide or subsequence thereof, and a naturally occurring viral polynucleotide or subsequence thereof.

179. The method of claim 177, wherein the naturally occurring variants of the polynucleotide comprise allelic or non-allelic naturally occurring variants of the polynucleotide, wherein the allelic or non-allelic naturally occurring variants are from a single species.

180. The method of claim 177, further comprising shuffling a first recombinant polynucleotide encoding the desired activity with a naturally occurring polynucleotide and selecting any resulting secondary recombinant nucleic acids for the desired property.

181. The method of claim 180, wherein the secondary recombinant nucleic acids are more closely similar in sequence to the naturally occurring polynucleotide than to the first recombinant polynucleotide.

182. The method of claim 177, further comprising supplementing the plurality of naural variants of a polynucleotide with induced variants of a polynucleotide before the amplification process.

183. The method of claim 177, further comprising randomly fragmenting the population of natural variants of a polynucleotide before conducting the amplification process.

184. The method of claim 139, wherein the multi-cyclic polynucleotide extension process comprises PCR amplification of the annealed polynucleotide strands.

185. The method of claim 139, wherein the reaction confers an ability to degrade at least one halogenated hydrocarbon to the cell, wherein the plurality of chosen polynucleotide variants to be recombined encodes one or more hydrolytic enzyme.

186. The method of claim 185, wherein the ability encoded by the optimized recombinant polynucleotide to degrade at least one halogenated hydrocarbon occurs in a cell.

187. 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.

188. The method of claim 187, wherein the selected optimized recombinant nucleic acid is further recombined with one or more additional nucleic acid to produce at least one further recombined nucleic acid, wherein the at least one further recombined nucleic acid is selected for the desired property, thereby producing a further optimized nucleic acid.

189. The method of claim 187, wherein the population of recombinant polynucleotides is cloned into at least one expression vector, thereby producing a library of expressed recombinant polynucleotides.

190. The method of claim 189, wherein the library of expressed recombinant polynucleotides is expressed in a population of cells, thereby producing a library of expressed recombinant polynucleotide cellular expression products.

191. The method of claim 190, wherein the library of expressed recombinant polynucleotide cellular expression products comprises a library of expressed polypeptides.

192. The method of claim 190, wherein the population of cells is screened for the desired property.

193. The method of claim 187, wherein at least one cycle of polynucleotide extension is performed in vitro.

194. The method of claim 187, the multi-cyclic polynucleotide extension process comprising:

cleaving the plurality of polynucleotide variants into fragments;

mixing and denaturing the fragments; and

,

incubating the resulting denatured fragments with a polymerase under conditions which result in annealing of the denatured fragments and formation of the population of recombinant polynucleotides.

195. The method of claim 187, wherein at least a portion of the multi-cyclic polynucleotide extension process is performed in a host cell.

196. The method of claim 195, wherein the population of recombinant polynucleotides are expressed in a population of host cells.

197. The method of claim 196, further comprising selecting the population of host cells for the desired property.

198. The method of claim 187, wherein the desired property is the ability to synthesize an isoprenoid, a polyketide, a carotenoid, an antibiotic, an amino acid, a polymer, vitamin C, or indigo, and wherein:

the antibiotic is selected from;

a peptide, a peptidolactone, a thiopeptide, a beta-lactam, a glycopeptide, a lantibiotic, a microcin, a polyketide-derived antibiotic an anthracyclin, a tetracyclin, a macrolide, an avermectin, a polyether, an ansamycin, a chloramphenicol, an aminoglycoside, an aminocyclitol, a polyoxin, an agrocin, a cyclosporin, a pepstatin, an actinomycin, a gramicidin, a depsipeptide, a vancomycin and an isoprenoid;

the carotenoid is selected from;

myxobacton, spheroidene, spheroidenone, lutein, astaxanthin, violaxanthin, 4-ketorulene, myxoxanthrophyll, echinenone, lycopene, zeaxanthin and its mono- and di- glucosides, alpha-, beta-, gamma- and delta-carotene, beta-cryptoxanthin monoglucoside and neoxanthin; and

,

the amino acid is selected from phenylalanine, monosodium glutamate, glycine, lysine, threonine, tryptophan, and methionine.

199. The method of claim 187, wherein the desired property is the ability to synthesize an isoprenoid, a polyketide, a carotenoid, an antibiotic, an amino acid, a polymer, vitamin C, or indigo, and wherein the plurality of polynucleotide variants comprises a nucleic acid subsequence derived from one or more of:

a gene encoding an isoprenoid synthesis gene, a gene encoding a trichodiene synthase from Fusarium sprorotrichioides, a gene encoding a pentalene synthase from Streptomyces, a gene encoding an aristolochene synthase from Penicillium roquefortii, and a gene encoding an epi-aristolochene synthase from N. tabacum.

200. The method of claim 187, wherein the desired property is the ability to synthesize an isoprenoid, a polyketide, a carotenoid, an antibiotic, an amino acid, a polymer, vitamin C, or indigo, and wherein the plurality of polynucleotide variants comprises a nucleic acid subsequence derived from one or more of:

a gene encoding a penicillin N expandase, a gene encoding a penicillin transacylase, a gene encoding a penicillin amidase, a gene encoding a Penicillin G acylase, and a gene encoding a Polyketide synthase.

201. The method of claim 187, wherein the plurality of polynucleotide variants comprises a nucleic acid subsequence derived from one or more gene from:

Alcaligenes, Zoogloea, Rhizobium, Bacillus, or an Azobacter which produces a polyhydroxyalkanoates (PHAs), or a polyhydroxybutyrate (PHB).

202. The method of claim 187, wherein the plurality of polynucleotide variants comprises a nucleic acid subsequence derived from phbB or phbC.

203. The method of claim 187, wherein the plurality of polynucleotide variants comprises at least one subsequence derived from a gene in a cellulose biosynthesis pathway.

204. The method of claim 187, wherein the plurality of polynucleotide variants comprises at least one subsequence derived from a gene in an E. coli indigo synthesis pathway which synthesizes indigo from glucose via a tryptophan/indole pathway.

205. The method of claim 187, wherein the partially annealed polynucleotide strands are produced by providing overlapping single-stranded segments of the polynucleotide variants and incubating under annealing conditions whereby the single-stranded segments from different polynucleotide variants anneal to form the partially annealed polynucleotide strands.

206. The method of claim 205, wherein the overlapping single-stranded segments are random segments of the polynucleotide variants.

207. The method of claim 205, wherein the overlapping single-stranded segments are non-random segments of the polynucleotide variants.

208. The method of claim 205, wherein the plurality of overlapping single-stranded segments are produced by cleaving the population of polynucleotide variants to produce a population of overlapping double-stranded fragments and denaturing the double-stranded fragments to produce the overlapping single-stranded segments.

209. The method of claim 208, wherein the population of polynucleotide variants comprise one or more DNAs and the cleavage is performed by DNase I digestion.

210. The method of claim 187, further comprising amplifying the plurality of polynucleotide variants.

211. The method of claim 187, further comprising performing error-prone PCR on at least one of the chosen polynucleotide variants.

212. The method of claim 187, wherein the partially annealed polynucleotide strands are produced with a DNA synthesizer.

213. The method of claim 187, wherein the plurality of polynucleotide variants which are shuffled are naturally occurring variants of a polynucleotide.

214. The method of claim 213, wherein the naturally occurring variants of the polynucleotide encode natural polypeptides.

215. The method of claim 213, wherein the naturally occurring variants of the polynucleotide comprise one or more of:

a naturally occurring human polynucleotide or subsequence thereof, a naturally occurring mouse polynucleotide or subsequence thereof, a naturally occurring bacterial polynucleotide or subsequence thereof, a naturally occurring plant polynucleotide or subsequence thereof, a naturally occurring fungal polynucleotide or subsequence thereof, a naturally occurring animal polynucleotide or subsequence thereof, and a naturally occurring viral polynucleotide or subsequence thereof.

216. The method of claim 214, wherein the naturally occurring variants of the polynucleotide comprise allelic or non-allelic naturally occurring variants of the polynucleotide, wherein the allelic or non-allelic naturally occurring variants are from a single species.

217. The method of claim 214, further comprising shuffling a first recombinant polynucleotide encoding the desired activity with a naturally occurring polynucleotide and selecting any resulting secondary recombinant nucleic acids for the desired property.

218. The method of claim 217, wherein the secondary recombinant nucleic acids are more closely similar in sequence to the naturally occurring polynucleotide than to the first recombinant polynucleotide.

219. The method of claim 214, further comprising supplementing the plurality of naural variants of a polynucleotide with induced variants of a polynucleotide before the amplification process.

220. The method of claim 214, further comprising randomly fragmenting the population of natural variants of a polynucleotide before conducting the amplification process.

221. The method of claim 187, wherein the multi-cyclic polynucleotide extension process comprises PCR amplification of the annealed polynucleotide strands.

222. The method of claim 187, wherein the desired property is an ability encoded by the optimized recombinant polynucleotide to degrade at least one halogenated hydrocarbon, wherein the plurality of chosen polynucleotide variants to be recombined encodes one or more hydrolytic enzyme.

223. The method of claim 222, wherein the ability encoded by the optimized recombinant polynucleotide to degrade at least one halogenated hydrocarbon occurs in a cell.

224. 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.

225. The method of claim 224, further comprising the steps of:

conducting a multi-cyclic polynucleotide extension process on at least partially annealed polynucleotide strands comprising sequences from a plurality of chosen hydrolytic enzyme coding polynucleotide variants, the polynucleotide strands comprising 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 hydrolytic enzyme polynucleotides; and

,

selecting or screening a recombinant hydrolytic enzyme polynucleotide from the population of recombinant hydrolytic enzyme polynucleotides for an ability to degrade at least one halogenated hydrocarbon, thereby providing an optimized recombinant hydrolytic enzyme.