Methods and compositions for evolving hydrogenase genes
View Patent ↗The invention provides methods and compositions for engineering microbes to generate Hydrogen. Some methods of the invention involve recoding of hydrogenase genes followed by subjecting the recoded genes to annealing-based recombination methods. The invention further provides methods of mating organisms that are transformed with recoded and recombined hydrogenase genes with other organisms containing different genome sequences.
1. A method of evolving an iron hydrogenase comprising:
a. substituting at least one amino acid in an iron hydrogenase within the segment FX 1 X 2 X 3 G 1 G 2 VMEA 1 A 2 X 4 R (SEQ ID NO: 347);
b. transforming an organism with a nucleic acid encoding the iron hydrogenase containing the at least one substitution; and
c. screening or selecting the transformed organism for the ability to produce hydrogen in the presence of more than 0.5% oxygen.
2. The method of claim 1 , wherein the amino acid F is substituted.
3. The method of claim 1 , wherein the amino acid X 1 is substituted.
4. The method of claim 1 , wherein the amino acid X 2 is substituted.
5. The method of claim 1 , wherein the amino acid X 3 is substituted.
6. The method of claim 1 , wherein the amino acid G 1 is substituted.
7. The method of claim 1 , wherein the amino acid G 2 is substituted.
8. The method of claim 1 , wherein the amino acid V is substituted.
9. The method of claim 1 , wherein the amino acid M is substituted.
10. The method of claim 1 , wherein the amino acid E is substituted.
11. The method of claim 1 , wherein the amino acid A 1 is substituted.
12. The method of claim 1 , wherein the amino acid A 2 is substituted.
13. The method of claim 1 , wherein the amino acid X 4 is substituted.
14. The method of claim 1 , wherein the amino acid R is substituted.
15. The method of claim 2 , wherein the substitution is conservative.
16. The method of claim 2 , wherein the substitution is non-conservative.
17. The method of claim 3 , wherein the substitution is conservative.
18. The method of claim 3 , wherein the substitution is non-conservative.
19. The method of claim 4 , wherein the substitution is conservative.
20. The method of claim 4 , wherein the substitution is non-conservative.
21. The method of claim 5 , wherein the substitution is conservative.
22. The method of claim 5 , wherein the substitution is non-conservative.
23. The method of claim 6 , wherein the substitution is conservative.
24. The method of claim 6 , wherein the substitution is non-conservative.
25. The method of claim 7 , wherein the substitution is conservative.
26. The method of claim 7 , wherein the substitution is non-conservative.
27. The method of claim 8 , wherein the substitution is conservative.
28. The method of claim 8 , wherein the substitution is non-conservative.
29. The method of claim 9 , wherein the substitution is conservative.
30. The method of claim 9 , wherein the substitution is non-conservative.
31. The method of claim 10 , wherein the substitution is conservative.
32. The method of claim 10 , wherein the substitution is non-conservative.
33. The method of claim 11 , wherein the substitution is conservative.
34. The method of claim 11 , wherein the substitution is non-conservative.
35. The method of claim 12 , wherein the substitution is conservative.
36. The method of claim 12 , wherein the substitution is non-conservative.
37. The method of claim 13 , wherein the substitution is conservative.
38. The method of claim 13 , wherein the substitution is non-conservative.
39. The method of claim 14 , wherein the substitution is conservative.
40. The method of claim 14 , wherein the substitution is non-conservative.
41. The method of claim 1 , wherein screening the transformed organism occurs in the presence of an oxygen concentration selected from the list consisting of 2%, 2.5%, 5%, 8%, 10%, 12%, 15%, 20% and approximately 21%.
42. The method of claim 1 , wherein at least one oligonucleotide encoding a variant of the FX 1 X 2 X 3 G 1 G 2 VMEA 1 A 2 X 4 R (SEQ ID NO: 347) segment of an iron hydrogenase is included in an annealing-based recombination reaction to generate the nucleic acid.
43. The method of claim 42 , wherein a plurality of oligonucleotides encoding variants of the FX 1 X 2 X 3 G 1 G 2 VMEA 2 X 4 R (SEQ ID NO: 347) segment are placed in the reaction in a higher proportion than other oligonucleotides encoding other regions of the iron hydrogenase.
44. The method of claim 42 , wherein at least one oligonucleotide encodes at least part of a FX 1 X 2 X 3 G 1 G 2 VMEA 1 A 2 X 4 R (SEQ ID NO: 347) segment that does not occur in SEQ ID NOs: 1–87.
45. The method of claim 42 , wherein oligonucleotides encoding all possible amino acids at one or more positions of the FX 1 X 2 X 3 G 1 G 2 VMEA 1 A 2 X 4 R (SEQ ID NO: 347) segment are included in the reaction.
46. The method of claim 42 , wherein the reaction includes at least one nucleotide sequence that has been recoded.
47. The method of claim 1 , wherein the screening or selecting occurs in liquid culture media.
48. The method of claim 47 , wherein the culture media is minimal media and the organism is photosynthetic.
49. The method of claim 1 , further comprising:
d. mating a first strain of the organism that (i) has been transformed with an iron hydrogenase containing a substitution in the FX 1 X 2 X 3 G 1 G 2 VMEA 1 A 2 X 4 R (SEQ ID NO: 347) segment; and (ii) has ability to generate hydrogen in more than 0.5% oxygen to at least a second strain of different genetic background; and
e. screening or selecting the progeny of the mating for the ability to generate more hydrogen than the first or second strain.
50. The method of claim 49 , wherein the mating reaction contains more than two strains of organisms that have different genetic backgrounds.
51. The method of claim 1 , wherein hydrogen production is measured at least two times under shifting light and temperature conditions.
52. The method of claim 46 , wherein the nucleotide sequence has been recoded to the most preferred codons of the organism.
53. The method of claim 1 , wherein the iron hydrogenase of step (a) is selected from SEQ ID NOs: 1–87.
54. The method of claim 1 , wherein the iron hydrogenase of step (a), when compared to SEQ ID NO:24 using the BLAST algorithm under the parameters:
(i) Matrix: BLOSUM62;
(ii) Gap Penalties: Existence: 11
(iii) Extension: 1
generates an E value of 4 e−05 or smaller.
55. The method of claim 1 , wherein hydrogen production is measured at a plurality of time points.
56. The method of claim 1 , wherein hydrogen production is measured under a higher temperature and light intensity than a previous period of time under which the organism has been cultured at a lower temperature and lower light intensity.
57. The method of claim 1 , wherein all possible amino acids at one or more positions of the FX 1 X 2 X 3 G 1 G 2 VMEA 1 A 2 X 4 R (SEQ ID NO: 347) segment are substituted.
58. The method of claim 1 , wherein the organism is selected from the list consisting of Chlamydomonas reinhardtii, Escherichia coli , photosynthetic bacteria, cyanobacteria, and bacteria.
59. The method of claim 58 , wherein the organism is Chlamydomonas reinhardtii.
60. The method of claim 1 , wherein the screening is performed using a chemochromic sensing film that contains a transition metal.
61. The method of claim 46 , wherein all nucleotide sequences in the reaction have been recoded to preferred codons of the organism.
62. The method of claim 61 , wherein all nucleotide sequences in the reaction have been recoded to most preferred codons of the organism.