Methods and compositions for evolving hydrogenase genes

Abstract

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 - 38 . (canceled)  
     
     
         39 . A method of hydrogen production comprising: 
 a. providing a cell that has been transformed with a nucleic acid encoding an iron hydrogenase, wherein the iron hydrogenase contains at least one substitution within 
 i. the segment FX 1 EX 2 MX 3 C 1 X 4 X 5 G 1 C 2 X 6 X 7 G 2 G 3 G 4 QP (SEQ ID NO: 350); and/or  
 ii. the segment FX 1 X 2 X 3 G 1 G 2 VMEA 1 A 2 X 4 R (SEQ ID NO: 347);  
   b. culturing the cell within a culture container; and    c. collecting gas from the culture container.    
     
     
         40 . A culture container containing at least one cell that has been transformed with a nucleic acid encoding an iron hydrogenase, wherein the iron hydrogenase contains at least one substitution within: 
 a. the segment FX 1 EX 2 MX 3 C 1 X 4 X 5  G 1 C 2 X 6 X 7 G 2 G 3 G 4 QP (SEQ ID NO: 350); and/or    b. the segment FX 1 X 2 X 3 G 1 G 2 VMEA 1 A 2 X 4 R (SEQ ID NO: 347).    
     
     
         41 . A method of evolving an iron hydrogenase comprising: 
 a. substituting at least one amino acid in an iron hydrogenase within the segment FX 1 EX 2 MX 3 C 1 X 4 X 5 G 1 C 2 X 6 X 7 G 2 G 3 G 4 QP (SEQ ID NO: 350);    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.    
     
     
         42 . The method of  claim 41 , wherein the amino acid F is substituted.  
     
     
         43 . The method of  claim 41 , wherein the amino acid X 1  is substituted.  
     
     
         44 . The method of  claim 41 , wherein the amino acid E is substituted.  
     
     
         45 . The method of  claim 41 , wherein the amino acid X 2  is substituted.  
     
     
         46 . The method of  claim 42 , wherein the substitution is conservative.  
     
     
         47 . The method of  claim 42 , wherein the substitution is non-conservative.  
     
     
         48 . The method of  claim 43 , wherein the substitution is conservative.  
     
     
         49 . The method of  claim 43 , wherein the substitution is non-conservative.  
     
     
         50 . The method of  claim 44 , wherein the substitution is conservative.  
     
     
         51 . The method of  claim 44 , wherein the substitution is non-conservative.  
     
     
         52 . The method of  claim 45 , wherein the substitution is conservative.  
     
     
         53 . The method of  claim 45 , wherein the substitution is non-conservative.  
     
     
         54 . The method of  claim 41 , 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%.  
     
     
         55 . The method of  claim 41 , wherein at least one oligonucleotide encoding a variant of the FX 1 EX 2 MX 3 C 1 X 4 XG 1 C 2 X 6 X 7 G 2 G 3 G 4 QP (SEQ ID NO: 350) segment of an iron hydrogenase is included in an annealing-based recombination reaction to generate the nucleic acid.  
     
     
         56 . The method of  claim 55 , wherein a plurality of oligonucleotides encoding variants of the FX 1 EX 2 MX 3 C 1 X 4 X 1 G 1 C 2 X 6 X 7 G 2 G 3 G 4 QP (SEQ ID NO: 350) segment are placed in the reaction in a higher proportion than other oligonucleotides encoding other regions of the iron hydrogenase.  
     
     
         57 . The method of  claim 55 , wherein at least one oligonucleotide encodes at least part of a FX 1 EX 2 MX 3 C 1 X 4 X 5 G 1 C 2 X 6 X 7 G 2 G 3 G 4 QP (SEQ ID NO: 350) segment that does not occur in SEQ ID NOs: 1-87.  
     
     
         58 . The method of  claim 55 , wherein oligonucleotides encoding all possible amino acids at one or more positions of the FX 1 EX 2 MX 3 C 1 X 4 X 5 G 1 C 2 X 6 X 7 G 2 G 3 G 4 QP (SEQ ID NO: 350) segment are included in the reaction.  
     
     
         59 . The method of  claim 55 , wherein the reaction includes at least one nucleotide sequence that has been recoded.  
     
     
         60 . The method of  claim 41 , wherein the screening or selecting occurs in liquid culture media.  
     
     
         61 . The method of  claim 60 , wherein the culture media is minimal media and the organism is photosynthetic.  
     
     
         62 . The method of  claim 41 , further comprising: 
 a. mating a first strain of the organism that (i) has been transformed with an iron hydrogenase containing a substitution in the FX 1 EX 2 MX 3 C 1 X 4 X 5 G 1 C 2 X 6 X 7 G 2 G 3 G 4 QP (SEQ ID NO: 350) segment; and (ii) has the ability to generate hydrogen in more than 0.5% oxygen to at least a second strain of different genetic background; and    b. screening or selecting the progeny of the mating for the ability to generate more hydrogen than the first or second strain.    
     
     
         63 . The method of  claim 62 , wherein the mating reaction contains more than two strains of organisms that have different genetic backgrounds.  
     
     
         64 . The method of  claim 41 , wherein hydrogen production is measured at least two times under shifting light and temperature conditions.  
     
     
         65 . The method of  claim 41 , wherein the nucleic acid has been recoded to the most preferred codons of the organism.  
     
     
         66 . The method of  claim 41 , wherein the iron hydrogenase of step (a) is selected from SEQ ID NOs: 1-87.  
     
     
         67 . The method of  claim 41 , 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.    
     
     
         68 . The method of  claim 41 , wherein hydrogen production is measured at a plurality of time points.  
     
     
         69 . The method of  claim 41 , 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.  
     
     
         70 . The method of  claim 41 , wherein all possible amino acids at one or more positions of the FX 1 EX 1 MX 3 C 1 X 4 X 1 G 1 C 2 X 6 X 7 G 2 G 3 G 4 QP (SEQ ID NO: 350) segment are substituted.  
     
     
         71 . The method of  claim 41 , wherein the organism is selected from the list consisting of  Chlamydomonas reinhardtii, Escherichia coli , photosynthetic bacteria, cyanobacteria, and bacteria.  
     
     
         72 . The method of  claim 71 , wherein the organism is  Chlamydomonas reinhardtii.    
     
     
         73 . The method of  claim 41 , wherein the screening is performed using a chemochromic sensing film that contains a transition metal.  
     
     
         74 . The method of  claim 59 , wherein all nucleotide sequences in the reaction have been recoded to preferred codons of the organism.  
     
     
         75 . The method of  claim 59 , wherein all nucleotide sequences in the reaction have been recoded to most preferred codons of the organism.