Hydrogenase polypeptide and methods of use

Abstract

Provided herein are polypeptides having hydrogenase activity. The polypeptide may be multimeric, and may have hydrogenase activity of at least 0.05 micromoles H 2 produced min −1 mg protein −1 . Also provided herein are polynucleotides encoding the polypeptides, genetically modified microbes that include polynucleotides encoding one or more subunits of the multimeric polypeptide, and methods for making and using the polypeptides.

1 . (canceled) 
     
     
         2 . A tetrameric polypeptide comprising four subunits, wherein the amino acid sequence of the first subunit and the amino acid sequence of SEQ ID NO:6 have at least 80% identity, wherein the amino acid sequence of the second subunit and the amino acid sequence of SEQ ID NO:8 have at least 80% identity, wherein the amino acid sequence of the third subunit and the amino acid sequence of SEQ ID NO:2 have at least 80% identity, wherein the amino acid sequence of the fourth subunit and the amino acid sequence of SEQ ID NO:4 have at least 80% identity, wherein the tetrameric polypeptide has hydrogenase activity, and wherein the tetrameric polypeptide is present in a genetically modified microbial cell. 
     
     
         3 . A tetrameric polypeptide comprising four subunits, wherein the amino acid sequence of the first subunit and the amino acid sequence of SEQ ID NO:6 have at least 80% identity, wherein the amino acid sequence of the second subunit and the amino acid sequence of SEQ ID NO:8 have at least 80% identity, wherein the amino acid sequence of the third subunit and the amino acid sequence of SEQ ID NO:2 have at least 80% identity, wherein the amino acid sequence of the second subunit and the amino acid sequence of SEQ ID NO:4 have at least 80% identity, wherein the tetrameric polypeptide has hydrogenase activity, and wherein one at least one subunit of the tetrameric polypeptide is a fusion comprising a heterologous amino acid sequence. 
     
     
         4 . The tetrameric polypeptide of  claim 3  wherein the tetrameric polypeptide is isolated. 
     
     
         5 . (canceled) 
     
     
         6 . The tetrameric polypeptide of  claim 3  wherein the tetrameric polypeptide is present in a microbial cell. 
     
     
         7 . (canceled) 
     
     
         8 . The tetrameric polypeptide of  claim 3  wherein the hydrogenase activity is at least 0.05 micromoles H 2  produced min −1  mg protein −1  when isolated by whole cell extract, centrifugation of a whole cell extract at 100,000×g, heat-treatment at 80° C. for 30 minutes, and re-centrifugation at 100,000×g. 
     
     
         9 - 14 . (canceled) 
     
     
         15 . The polypeptide of  claim 3  wherein the polypeptide consists of the first subunit, the second subunit, the third subunit, and the fourth subunit. 
     
     
         16 . (canceled) 
     
     
         17 . A genetically modified microbe comprising an exogenous polypeptide, wherein the exogenous polypeptide comprises four subunits, wherein the first subunit comprises an amino acid sequence, and the amino acid sequence of the first subunit and the amino acid sequence of SEQ ID NO:6 have at least 80% identity, wherein the second subunit comprises an amino acid sequence, and the amino acid sequence of the second subunit and the amino acid sequence of SEQ ID NO:8 have at least 80% identity, wherein the third subunit comprises an amino acid sequence, and the amino acid sequence of the third subunit and the amino acid sequence of SEQ ID NO:2 have at least 80% identity, wherein the fourth subunit comprises an amino acid sequence, and the amino acid sequence of the fourth subunit and the amino acid sequence of SEQ ID NO:4 have at least 80% identity., and wherein the four subunits form a dimeric tetrameric polypeptide having hydrogenase activity. 
     
     
         18 . (canceled) 
     
     
         19 . The genetically modified microbe of  claim 17  wherein one at least one subunit is a fusion comprising a heterologous amino acid sequence. 
     
     
         20 - 27 . (canceled) 
     
     
         28 . A method for using a polypeptide comprising:
 providing   a) a tetrameric polypeptide comprising four subunits, wherein the amino acid sequence of the first subunit and the amino acid sequence of SEQ ID NO:6 have at least 80% identity, wherein the amino acid sequence of the second subunit and the amino acid sequence of SEQ ID NO:8 have at least 80% identity, wherein the amino acid sequence of the third subunit and the amino acid sequence of SEQ ID NO:2 have at least 80% identity, wherein the amino acid sequence of the fourth subunit and the amino acid sequence of SEQ ID NO:4 have at least 80% identity, wherein the tetrameric polypeptide has hydrogenase activity, and wherein the tetrameric polypeptide is present in a genetically modified microbial cell,   b) a tetrameric polypeptide comprising four subunits, wherein the amino acid sequence of the first subunit and the amino acid sequence of SEQ ID NO:6 have at least 80% identity, wherein the amino acid sequence of the second subunit and the amino acid sequence of SEQ ID NO:8 have at least 80% identity, wherein the amino acid sequence of the third subunit and the amino acid sequence of SEQ ID NO:2 have at least 80% identity, wherein the amino acid sequence of the fourth subunit and the amino acid sequence of SEQ ID NO:4 have at least 80% identity, wherein the tetrameric polypeptide has hydrogenase activity, and wherein one at least one subunit of the tetrameric polypeptide is a fusion comprising a heterologous amino acid sequence, or   c) a tetrameric polypeptide consisting of four subunits, wherein the amino acid sequence of the first subunit and the amino acid sequence of SEQ ID NO:2 have at least 80% identity, wherein the amino acid sequence of the second subunit and the amino acid sequence of SEQ ID NO:4 have at least 80% identity, wherein the amino acid sequence of the third subunit and the amino acid sequence of SEQ ID NO:6 have at least 80% identity, wherein the amino acid sequence of the fourth subunit and the amino acid sequence of SEQ ID NO:8 have at least 80% identity, wherein one at least one subunit of the tetrameric polypeptide is a fusion comprising a heterologous amino acid sequence, and wherein the tetrameric polypeptide has hydrogenase activity; and   incubating the polypeptide under conditions suitable for producing H 2  or NADPH.   
     
     
         29 . The method of  claim 28  further comprising collecting the produced H 2  or the produced NADPH. 
     
     
         30 . The method of  claim 28  wherein the tetrameric polypeptide of (a) or (b) is an isolated polypeptide. 
     
     
         31 . The method of  claim 30  wherein the tetrameric polypeptide is present on a surface. 
     
     
         32 . The method of  claim 31  wherein the surface conducts electricity. 
     
     
         33 . The method of  claim 32  wherein the surface is an anode. 
     
     
         34 . The method of  claim 28  wherein the tetrameric polypeptide of (a) or (b) is chemically modified. 
     
     
         35 . The method of  claim 28  wherein the incubating comprises conditions that comprise a polysaccharide. (Original) The method of  claim 35  wherein the polysaccharide comprises starch. 
     
     
         37 . The method of  claim 28  wherein the conditions comprise a temperature of at least 70° C. 
     
     
         38 - 49 . (canceled)