Designer proton-channel transgenic algae for photobiological hydrogen production

Abstract

A designer proton-channel transgenic alga for photobiological hydrogen production that is specifically designed for production of molecular hydrogen (H 2 ) through photosynthetic water splitting. The designer transgenic alga includes proton-conductive channels that are expressed to produce such uncoupler proteins in an amount sufficient to increase the algal H 2 productivity. In one embodiment the designer proton-channel transgene is a nucleic acid construct ( 300 ) including a PCR forward primer ( 302 ), an externally inducible promoter ( 304 ), a transit targeting sequence ( 306 ), a designer proton-channel encoding sequence ( 308 ), a transcription and translation terminator ( 310 ), and a PCR reverse primer ( 312 ). In various embodiments, the designer proton-channel transgenic algae are used with a gas-separation system ( 500 ) and a gas-products-separation and utilization system ( 600 ) for photobiological H 2 production.

1 . A transgenic alga comprising a first exogenous transgene encoding a proton conductive polypeptide in a photosynthetic membrane of the transgenic alga; and
 further comprising a second exogenous transgene encoding for at least one proton channel in at least one cellular membrane of the transgenic alga wherein the transgenic alga is not capable of reproduction.   
     
     
         2 . The transgenic alga of  claim 1  wherein the first exogenous transgene is expressed under H 2 -producing condition providing for the transgenic alga to have an increased photobiological H 2  productivity relative to alga without the first exogenous transgene. 
     
     
         3 . The transgenic alga of  claim 1  wherein the alga is selected from the group consisting of green alga, brown alga, red alga, blue-green alga, oxygen-tolerant-hydrogenase algal strains, H 2 -consuming-activity-deleted algal strains, uptake-hydrogenase-deleted algal strains, and combinations thereof. 
     
     
         4 . The transgenic alga of  claim 1  further comprising a genetic deletion of up-take hydrogenase of the transgenic alga. 
     
     
         5 . The transgenic alga of  claim 1  wherein the proton conductive polypeptide is a proton-conductive channel for passage of at least one proton therethrough. 
     
     
         6 . (canceled) 
     
     
         7 . The transgenic alga of  claim 1  wherein the proton-conductive polypeptide is a carrier protein for passage of at least one proton through the photosynthetic membrane. 
     
     
         8 . The transgenic alga of  claim 1  wherein the first exogenous transgene is a nucleic acid construct having an externally inducible promoter, and a transcription terminator. 
     
     
         9 . The transgenic alga of  claim 1  wherein the proton conductive polypeptide is a polynucleotide sequence for a designer proton-conductive polypeptide. 
     
     
         10 . The transgenic alga of  claim 8  wherein the expression of the first exogenous transgene is at least partially operatively linked to the externally inducible promoter. 
     
     
         11 . The transgenic alga of  claim 8  further comprising the expression of the first exogenous transgene at least partially operatively linked to a targeting transit-peptide DNA sequence. 
     
     
         12 . The transgenic alga of  claim 8  further comprising the expression of the first exogenous transgene is at least partially operatively linked to the inducible promoter and to a targeting transit-peptide DNA sequence. 
     
     
         13 . (canceled) 
     
     
         14 . (canceled) 
     
     
         15 . (canceled) 
     
     
         16 . The transgenic alga of  claim 11  wherein the transit-peptide DNA sequence is selected from the group consisting of: plastocyanin transit-peptide sequence (Pcy1); the LhcII transit-peptide sequences; OEE1 transit-peptide sequence (PsbO); OEE2 transit-peptide sequence (PsbP); OEE3 transit-peptide sequence (PsbQ); hydrogenase transit-peptide sequences (such as Hyd1); PSII-T transit-peptide sequence (PsbT); PSII-S transit-peptide sequence (PsbS); PSII-W transit-peptide sequence (PsbW); CFoCF1 subunit-γ transit-peptide sequence (AtpC);  CFoCF1 subunit-δ transit-peptide sequence (AtpD); CFoCF1 subunit-II transit-peptide sequence (AtpG); photosystem I (PSI) transit-peptide sequence of genes PsaD, PsaE, PsaF, PsaG, PasH, and PsaK; Rubisco SSU transit-peptide sequences; α-tubulin transit-peptide sequence (TubA); β-tubulin transit-peptide sequence (TubB2); mitochondrial carbonic anhydrase transit-peptide sequence (Ca1 and Ca2); mitochondrial transit-peptide sequences of nuclear genes; chloroplast transit-peptide sequences of nuclear genes; analogs; and modified designer sequences. 
     
     
         17 . The transgenic alga of  claim 8  wherein the inducible promoter is selected from the group consisting of hydrogenase promoters, Cytochrome C6 (Cyc6) promoter, Nia1 promoter, CabII-1 promoter, Ca1 promoter, Ca2 promoter, coprogen oxidase promoter, analogs, and modified designer sequences. 
     
     
         18 . (canceled) 
     
     
         19 . The transgenic alga of  claim 1  wherein the transgenic alga is selected from the group consisting of marine alga and freshwater alga wherein the transgenic alga has a corresponding tolerance to salt or freshwater for photobiological H 2  production. 
     
     
         20 . The transgenic alga of  claim 1  wherein the transgenic alga is selected from the group consisting of cold-tolerant algal strains and heat-tolerant algal strains wherein the transgenic alga has a corresponding tolerance to temperature for photobiological H 2  production. 
     
     
         21 . The transgenic alga of  claim 7  wherein the carrier protein includes a proton-conductive path. 
     
     
         22 . The transgenic alga of  claim 7  wherein the carrier protein includes a proton-conductive path created through molecular genetic engineering that uses the genetic sequences of CFoCF1 related structures such as CFoCF1 subunit-γ (AtpC), CFoCF1 subunit-δ (AtpD), CFoCF1 subunit-II (AtpG), and CFo protein. 
     
     
         23 . A transgenic alga comprising a first exogenous transgene encoding a proton conductive polypeptide in a photosynthetic membrane of the transgenic alga; and
 wherein the proton conductive polypeptide is a carrier protein for passage of at least one proton through the photosynthetic membrane; and   wherein the carrier protein includes a proton-conductive path.   
     
     
         24 . The transgenic alga of  claim 23  wherein the first exogenous transgene is expressed under H 2 -producing condition providing for the transgenic alga to have an increased photobiological H 2  productivity relative to alga without the first exogenous transgene. 
     
     
         25 . The transgenic alga of  claim 23  wherein the alga is selected from the group consisting of green alga, brown alga, red alga, blue-green alga, oxygen-tolerant-hydrogenase algal strains, H 2 -consuming-activity-deleted algal strains, uptake-hydrogenase-deleted algal strains, and combinations thereof. 
     
     
         26 . The transgenic alga of  claim 23  further comprising a genetic deletion of up-take hydrogenase of the transgenic alga. 
     
     
         27 . The transgenic alga of  claim 23  wherein the first exogenous transgene is a nucleic acid construct having an externally inducible promoter, and a transcription terminator. 
     
     
         28 . The transgenic alga of  claim 23  wherein the transgenic alga is selected from the group consisting of marine alga and freshwater alga wherein the transgenic alga has a corresponding tolerance to salt or freshwater for photobiological H 2  production. 
     
     
         29 . The transgenic alga of  claim 23  wherein the transgenic alga is selected from the group consisting of cold-tolerant algal strains and heat-tolerant algal strains wherein the transgenic alga has a corresponding tolerance to temperature for photobiological H 2  production. 
     
     
         30 . The transgenic alga of  claim 23  wherein the proton conductive polypeptide is selected from the group that consist of the structures of melittin, gramicidin, CF o  protein, F o  protein, and their analogs including artificially designed polypeptide proton channels. 
     
     
         31 . The transgenic alga of  claim 27  wherein the expression of the first exogenous transgene is at least partially operatively linked to the externally inducible promoter. 
     
     
         32 . The transgenic alga of  claim 27  further comprising the expression of the first exogenous transgene at least partially operatively linked to a targeting transit-peptide DNA sequence. 
     
     
         33 . The transgenic alga of  claim 32  wherein the transit-peptide DNA sequence is selected from the group consisting of: plastocyanin transit-peptide sequence (Pcy1); the LhcII transit-peptide sequences; OEE1 transit-peptide sequence (PsbO); OEE2 transit-peptide sequence (PsbP); OEE3 transit-peptide sequence (PsbQ); hydrogenase transit-peptide sequences (such as Hyd1); PSII-T transit-peptide sequence (PsbT); PSII-S transit-peptide sequence (PsbS); PSII-W transit-peptide sequence (PsbW); CFoCF1 subunit-γ transit-peptide sequence (AtpC); CFoCF1 subunit-δ transit-peptide sequence (AtpD); CFoCF1 subunit-II transit-peptide sequence (AtpG); photosystem I (PSI) transit-peptide sequence of genes PsaD, PsaE, PsaF, PsaG, PasH, and PsaK; Rubisco SSU transit-peptide sequences; α-tubulin transit-peptide sequence (TubA); β-tubulin transit-peptide sequence (TubB2); mitochondrial carbonic anhydrase transit-peptide sequence (Ca1 and Ca2); mitochondrial transit-peptide sequences of nuclear genes; chloroplast transit-peptide sequences of nuclear genes; analogs; and modified designer sequences. 
     
     
         34 . The transgenic alga of  claim 27  further comprising the expression of the first exogenous transgene is at least partially operatively linked to the inducible promoter and to a targeting transit-peptide DNA sequence. 
     
     
         35 . The transgenic alga of  claim 27  wherein the inducible promoter is selected from the group consisting of hydrogenase promoters, Cytochrome C6 (Cyc6) promoter, Nia1 promoter, CabII-1 promoter, Ca1 promoter, Ca2 promoter, coprogen oxidase promoter, analogs, and modified designer sequences.