PHOTOSYSTEM I-BACTERIAL HYDROGENASE CHIMERAS FOR HYDROGEN PRODUCTION
20240093246 ยท 2024-03-21
Inventors
Cpc classification
International classification
C12P3/00
CHEMISTRY; METALLURGY
Abstract
Provided herein, in some embodiments, are engineered cells and use of the same for increased hydrogen production. In particular, provided herein are genetically engineered cells comprising a polynucleotide encoding a fusion protein comprising a photosystem I (PSI) protein and a bacterial hydrogenase, as well as methods for producing such genetically engineered cells. Also provided herein are methods for increasing hydrogen (H.sub.2) production in cells.
Claims
1. A genetically engineered cell comprising a polynucleotide encoding a fusion protein comprising a photosystem I (PSI) protein and a bacterial hydrogenase.
2. The cell of claim 1, wherein the PSI protein is PsaC.
3. The cell of claim 1, wherein the polynucleotide comprises bacterial hydrogenase A (HydA).
4. The cell of claim 1, wherein the hydrogenase is inserted in frame into the PSI protein.
5. The cell of claim 2, wherein the hydrogenase is inserted in frame in the hinge region of PsaC.
6. The cell of claim 2, wherein the polynucleotide further comprises a nucleic acid linker encoding at least one amino acid at the junction between the PsaC protein and the hydrogenase protein at the N-terminal end of the fusion protein, the C-terminal end of the fusion protein, or both ends of the fusion protein.
7. The cell of claim 1, wherein the polynucleotide encodes the polypeptide of SEQ ID NO: 1 or 12 or a polypeptide having 95% identity to SEQ ID NO: 1 or 12.
8. The cell of claim 1, wherein the bacterial hydrogenase is a Megasphaera elsdenii hydrogenase or a Clostridium beijerincki hydrogenase.
9. The cell of claim 1, wherein the hydrogenase F-domain is removed.
10. The cell of claim 1, wherein the cell is an algal cell.
11. The cell of claim 10, wherein the cell is selected from Chlamydomonas reinhardtii, Chlorella vulgaris, Picochlorum soloecismus, Galdieria sulphuraria and Cyanidioschyzon merolae.
12. An algal biomass comprising the genetically engineered cell of claim 1.
13. An expression cassette comprising a polynucleotide encoding a fusion protein comprising a PSI protein and a bacterial hydrogenase, wherein the polynucleotide is operably linked to a promoter that drives expression of the fusion protein.
14. The expression cassette of claim 13, wherein the PSI protein is PsaC.
15. The expression cassette of claim 13, wherein the polynucleotide comprises bacterial hydrogenase A inserted in frame into the ?-hairpin of PsaC.
16. The expression cassette of claim 13, wherein the polynucleotide further comprises a nucleic acid linker encoding at least one amino acid at the junction between the PSA protein and the hydrogenase protein at the N-terminal end of the fusion protein, the C-terminal end of the fusion protein, or both ends of the fusion protein.
17. The expression cassette of claim 13, wherein the polynucleotide encodes the polypeptide of SEQ ID NO: 1 or 12 or a polypeptide having at least 95% identity to SEQ ID NO: 1 or 12.
18. A method of increasing hydrogen (H.sub.2) production in a cell, the method comprising (a) introducing into the cell the expression cassette of claim 12 to produce a genetically engineered cell; and (b) culturing the genetically engineered cell under continuous illumination, wherein the genetically engineered cell exhibits at least a 4-fold increase in H.sub.2 production under such conditions relative to a control cell of the same species under the same conditions.
19. A fusion protein comprising a bacterial FeFe hydrogenase or functional portion thereof inserted into a PsaC protein.
20. The fusion protein of claim 20, wherein the fusion protein comprises SEQ ID NO: 1 or 12 or a polypeptide having at least 95% identity to SEQ ID NO: 1 or 12.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
[0020]
[0021]
[0022]
[0023]
[0024]
[0025]
[0026]
[0027]
[0028]
[0029]
[0030]
[0031]
[0032]
[0033]
[0034]
[0035]
[0036] While the present invention is susceptible to various modifications and alternative forms, exemplary embodiments thereof are shown by way of example in the drawings and are herein described in detail. It should be understood, however, that the description of exemplary embodiments is not intended to limit the invention to the particular forms disclosed, but on the contrary, the intention is to cover all modifications, equivalents and alternatives falling within the spirit and scope of the invention as defined by the appended claims.
DETAILED DESCRIPTION
[0037] The compositions and methods described herein are based, at least in part, on the inventor's development of a fusion of a photosystem I (PSI) protein and a heterologous iron-iron hydrogenase, created by insertion of a nucleotide sequence encoding a bacterial hydrogenase (e.g. HydA) sequence into or adjacent to a nucleotide sequence encoding a PSI protein (e.g. PsaC), and in vivo co-assembly of the PSI and hydrogenase portions. When expressed in algal cells, this PSI-HydA fusion protein increases molecular hydrogen (H.sub.2) production under certain light conditions relative to unmodified algal cells. This means for H.sub.2 production offers an ecologically-friendly, inexpensive renewable energy source.
[0038] The use of algal hydrogenases to produce H.sub.2 in previously described PSI-HydA fusion proteins is limited by their oxygen sensitivity. Also, the H.sub.2 production of PSI-HydA fusion proteins made with algal hydrogenases is also limited due to observed electron escape to ferredoxin/flavodoxin, which is thought to be due to escape from the hydrogenase domain. Given that algal hydrogenases must be able to bind and oxidize/reduce the algal ferredoxin, it was hypothesized that replacement with a bacterial hydrogenase that has not evolved to interact with chloroplast ferredoxin might result in less electron escape.
[0039] In a first aspect, this disclosure provides a genetically engineered cell comprising a polynucleotide encoding a fusion protein, the fusion protein comprising a photosystem I (PSI) protein, or a portion thereof, and a bacterial hydrogenase, or a portion thereof.
[0040] The engineered cell may be an algal cell or any cell capable of photosynthesis having PSI and a bacterial iron-iron hydrogenase. As used herein, the terms genetically engineered and genetically modified are used interchangeably and refer to a cell that includes an exogenous polynucleotide. In some cases, the cell has been engineered to comprise a non-naturally occurring nucleic acid molecule that has been created or modified by the hand of man (e.g., using recombinant DNA technology) or is derived from such a molecule (e.g., by transcription, translation, etc.). A cell that contains an exogenous, recombinant, synthetic, and/or otherwise modified polynucleotide is considered to be an engineered cell.
[0041] An engineered cell may be produced by introducing a recombinant nucleic acid molecule that encodes a PSI-HydA fusion protein of this disclosure. As used herein, the term recombinant nucleic acid or recombinant polynucleotide refers to a polynucleotide that is manipulated by human intervention. A recombinant nucleic acid molecule can contain two or more nucleotide sequences that are linked in a manner such that the product is not found in a cell in nature. In particular, the two or more nucleotide sequences can be operatively linked and, for example, can encode a fusion polypeptide. A recombinant nucleic acid molecule also can be based on, but manipulated so as to be different, from a naturally occurring polynucleotide, for example, a polynucleotide having one or more nucleotide changes such that a first codon, which normally is found in the polynucleotide, is biased for chloroplast codon usage, or such that a sequence of interest is introduced into the polynucleotide, for example, a restriction endonuclease recognition site or a splice site, a promoter, a DNA origin of replication, or the like.
[0042] As used herein, the terms polynucleotide, polynucleotide sequence, nucleic acid and nucleic acid sequence refer to a nucleotide, oligonucleotide, polynucleotide (which terms may be used interchangeably), or any fragment thereof. These phrases also refer to DNA or RNA of natural or synthetic origin (which may be single-stranded or double-stranded and may represent the sense or the antisense strand). The polynucleotides may be cDNA or genomic DNA.
[0043] Polynucleotides homologous to the polynucleotides described herein are also provided. Those of skill in the art understand the degeneracy of the genetic code and that a variety of polynucleotides can encode the same polypeptide. In some embodiments, the polynucleotides (i.e., polynucleotides encoding the fusion polypeptides) may be codon-optimized for expression in a particular cell including, without limitation, a plant cell, bacterial cell, or algal cell. While particular polynucleotide sequences which are found in particular algae are disclosed herein any polynucleotide sequences may be used which encode a desired form of the polypeptides described herein. These represent non-naturally occurring sequences. Computer programs for generating degenerate coding sequences are available and can be used for this purpose. Pencil, paper, the genetic code, and a human hand can also be used to generate degenerate coding sequences.
[0044] The bacterial hydrogenase may be from Megasphaera elsdenii (MeHydA) or Clostridium beijerinckii hydrogenase (Cb5Ah). Compared to algal hydrogenases, the hydrogenase of Megasphaera elsdenii has been shown to be relatively O.sub.2 tolerant, exhibiting a ?10-fold lower O.sub.2 inactivation rate. The PSI protein may comprise one or more subunits of PSI (e.g., PsaA, PsaB, PsaC, PsaD, PsaE). In exemplary embodiments, the PSI protein is PsaC. The bacterial hydrogenase may be hydrogenase A (HydA). Any of the PSI subunits may be fused to HydA. Other bacterial FeFe hydrogenases, and other oxygen-tolerant hydrogenases may be used. Other PsaC proteins may also be used. For example, 4 PsaC proteins from various algal species are provided in
[0045] A fusion protein comprising a bacterial hydrogenase or a functional portion thereof inserted into a PSI protein is also provided herein. Fusion proteins or chimeric proteins are proteins created through the joining of two or more genes that originally coded for separate proteins. Translation of this fusion gene results in a single or multiple polypeptides with functional properties derived from each of the original proteins. The fusion protein provided herein may be generated by inserting the nucleotide sequence encoding the hydrogenase (or a portion thereof) into or adjacent to the nucleotide sequence of one or more of the PSI subunits, thereby producing a recombinant nucleic acid molecule encoding a PSI-hydrogenase fusion protein. The nucleotide sequence encoding the hydrogenase may be inserted into or adjacent to a sequence encoding the PsaC subunit. The hydrogenase may be inserted in frame into the hinge region (shown as the ?-hairpin in
[0046] A deletion in a fusion polypeptide refers to a change in the amino acid sequence resulting in the absence of one or more amino acid residues. A deletion may remove at least 1, 2, 3, 4, 5, 10, 20, or more amino acids residues. A deletion may include an internal deletion and/or a terminal deletion (e.g., an N-terminal truncation, a C-terminal truncation or both of a reference polypeptide).
[0047] Insertions and additions in a fusion polypeptide refers to changes in an amino acid sequence resulting in the addition of one or more amino acid residues. An insertion or addition may refer to 1, 2, 3, 4, 5, 10, 20, 30, 40, or more amino acid residues. A variant of a polypeptide may have N-terminal insertions, C-terminal insertions, internal insertions, or any combination of N-terminal insertions, C-terminal insertions, and internal insertions.
[0048] One of skill in the art will appreciate that hydrogenases may be inserted into various PSI subunit proteins, e.g. PsaC hinge regions, and that the linking regions between the two protein portions could have altered amino acids, or the linkers could be of differing lengths.
[0049] In an exemplary embodiment, the fusion protein comprises the PsaC protein and the Megasphaera elsdenii hydrogenase (PsaC-MeHydA). The sequence of the PsaC-MeHydA fusion protein
TABLE-US-00001 (SEQIDNO:1) MAHIVKIYDTCIGCTQCVRACPLDVLEMVPWGGATATDAVPNDVDKVKAA LKDPEKIVIFQTAPAVRVGLGEAFGMDPGTFVEGKMVAALRTLGADYVED TDFGADLTIMEEATELLHRLQSEEIPIPQFTSCCPAWVEFAETFYPDLLQ HLSSTKSPISILSPVIKTYFAQQKNIDPKKIVNVCVTPCTAKKAEIRRPE LSASGLFWDEPEIRDTDICITTRELAQWIQDENIDFASLEDSKFDKAFGE ASGGGRIFGNSGGVMEAAIRTAYHMFTGRPAPKDFIPFEPVRGLQGVKKA TVIFGHFVLHVAAISGLGNARAFIDDLIKNDAFEDYSFIEVMACPGGCIG GGGQPKVKLPQVKKVQEARTASIYKSDEETDIKASWQNPEIETLYEAFLD EPLSEMAEFTLHTHYSAGSGGGGSGAGGASQMASAPRTEDCVGCKRCETA CPTDFLSVRVYLGSESTRSMGLSY.
[0050] The singly underlined residues indicate the PsaC fragments; the doubly underlined residues are the N-terminal junction; the italicized residues are a HydA2 N-terminus linker; and the bold residues are a C-terminal linker.
[0051] In another exemplary embodiment, the fusion protein comprises the PsaC protein and the Clostridium beijerincki hydrogenase PsaC-CbA5H. The sequence of the Clostridium beijerincki hydrogenase CbA5H protein is:
TABLE-US-00002 (SEQIDNO:11) MGDNKKSFIQSALGSVFSVFSEEELKELSNGRKIAICGKVNNPGIIEVPE GATLNEIIQLCGGLINKSNFKAAQIGLPFGGFLTEDSLDKEFDFGIFYEN IARTIIVLSQEDCIIQFEKFYIEYLLAKIKDGSYKNYEVVKEDITEMFNI LNRISKGVSNMREIYLLRNLAVTVKSKMNQKHNIMEEIIDKFYEEIEEHI EEKKCYTSQCNHLVKLTITKKCIGCGACKRACPVDCINGELKKKHEIDYN RCTHCGACVSACPVDAISAGDNTMLFLRDLATPNKVVITQMAPAVRVAIG EAFGFEPGENVEKKIAAGLRKLGVDYVFDTSWGADLTIMEEAAELQERLE RHLAGDESVKLPILTSCCPSWIKFIEQNYGDMLDVPSSAKSPMEMFAIVA KEIWAKEKGLSRDEVTSVAIMPCIAKKYEASRAEFSVDMNYDVDYVITTR ELIKIFENSGINLKEIEDEEIDTVMGEYTGAGIIFGRTGGVIEAATRTAL EKMTGERFDNIEFEGLRGWDGFRVCELEAGDIKLRIGVAHGLREAAKMLD KIRSGEEFFHAIEIMACVGGCIGGGGQPKTKGNKQAALQKRAEGLNNIDR SKTLRRSNENPEVLAIYEKYLDHPLSNKAHELLHTVYFPRVKKD.
[0052] The amino acid sequence of the PsaC-CbA5H fusion protein is:
TABLE-US-00003 (SEQIDNO:12) MAHIVKIYDTCIGCTQCVRACPLDVLEMVPWGGATATDAVPNTMLFLRDL ATPNKVVITQMAPAVRVAIGEAFGFEPGENVEKKIAAGLRKLGVDYVFDT SWGADLTIMEEAAELQERLERHLAGDESVKLPILTSCCPSWIKFIEQNYG DMLDVPSSAKSPMEMFAIVAKEIWAKEKGLSRDEVTSVAIMPCIAKKYEA SRAEFSVDMNYDVDYVITTRELIKIFENSGINLKEIEDEEIDTVMGEYTG AGIIFGRTGGVIEAATRTALEKMTGERFDNIEFEGLRGWDGFRVCELEAG DIKLRIGVAHGLREAAKMLDKIRSGEEFFHAIEIMACVGGCIGGGGQPKT KGNKQAALQKRAEGLNNIDRSKTLRRSNENPEVLAIYEKYLDHPLSNKAH ELLHTVYFPRVKKDSGAGGASQMASAPRTEDCVGCKRCETACPTDFLSVR VYLGSESTRSMGLSY.
[0053] The singly underlined residues indicate the PsaC fragments; the doubly underlined residues are the N-terminal junction; the italicized residues are a N-terminus linker.
[0054] The fusion protein/polypeptide may comprise SEQ ID NO: 1 or 12, or a sequence having at least 90, 92, 94, 95, 96, 97, 98, 99 percent identity to the fusion protein/polypeptide of SEQ ID NO: 1 or 12. In particular the amino acids in bold, italics or double underlined in SEQ ID NO: 1 or 12 may be altered to provide additional linking regions between the portions of the fusion protein.
[0055] Protein and nucleic acid sequence identities may be evaluated using the Basic Local Alignment Search Tool (BLAST) which is well known in the art (Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2267-2268; Altschul et al., 1997, Nucl. Acids Res. 25: 3389-3402). The BLAST programs identify homologous sequences by identifying similar segments, which are referred to herein as high-scoring segment pairs, between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from a protein or nucleic acid sequence database. Preferably, the statistical significance of a high-scoring segment pair is evaluated using the statistical significance formula (Karlin and Altschul, 1990), the disclosure of which is incorporated by reference in its entirety. The BLAST programs can be used with the default parameters or with modified parameters provided by the user. Methods of amino acid sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.
[0056] The phrases % sequence identity, percent identity, or % identity refer to the percentage of residue matches between at least two amino acid sequences aligned using a standardized algorithm. For example, reference to at least 95% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 12, refers to alternative embodiments with at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 12. Percent identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
[0057] Polypeptide sequence identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, may be used to describe a length over which percentage identity may be measured.
[0058] Any appropriate technique for introducing recombinant nucleic acid molecules into algal cells may be used. Techniques for nuclear and chloroplast transformation are known and include, without limitation, electroporation, biolistic transformation (also referred to as micro-projectile/particle bombardment), agitation in the presence of glass beads, and Agrobacterium-based transformation. Accordingly, a recombinant nucleic acid molecule encoding a PsaC-MeHydA fusion protein may be introduced into an algal cell by, for example, electroporation, by particle bombardment, by agitation in the presence of glass beads, or by Agrobacterium-based transformation. With chloroplast transformation, transgenes can be easily directed to integrate via homologous recombination. Nuclear transformation usually results in random integration events. In some embodiments the native psaC and hydA genes are mutated and/or replaced with the fusion protein in the cells such that all copies of PSI in the cell contain the fusion protein.
[0059] By way of example, described herein is introduction of a nucleic acid encoding apsaC-hydA fusion protein into an algal cell's chloroplast genome by particle-mediated gene transfer. In this example, flanking sequences were used to direct homologous recombination such that the recombinant nucleic acid replaces the endogenous psaC gene.
[0060] Any appropriate method can be performed to confirm introduction and expression of recombinant nucleic acids in the modified cell. For instance, polymerase chain reaction (PCR) or PCR-based methods can be used to verify replacement of psaC by psaC-MeHydA. Amplified products may be sequenced to ensure that no mutations are found in the recombinant nucleic acid as a result of the cloning process.
[0061] After the PSI-HydA fusion protein is expressed in the cell, hydrogenase activity may be measured. Any appropriate means of measuring hydrogenase activity may be used. Samples may be collected for analysis of hydrogenase activity by gas chromatography (GC). Those of skill in the art are aware of methods to measure hydrogenase activity and such methods are provided in the Examples.
[0062] The PSI-HydA fusion protein may be expressed in any cell comprising a chloroplast or using photosystem I for photosynthesis. For example, plant cells, algal cells, or cyanobacteria may be used. The cell must also have or be engineered to have the maturase proteins, HydE/F/G. The FeFe hydrogenases for use in the methods suitably have a structure similar to MeHydA, in which the N-terminal and C-terminal ends of the protein are in proximity to each other or the hydrogenases are modified via truncation such that the N-terminal and C-terminal ends of the protein are in proximity to each other. Those of skill in the art can use protein modeling programs or crystal structures of hydrogenases to determine appropriate hydrogenases for use in the methods, cells and constructs provided herein.
[0063] The terms algal cell or algae as used herein refer to unicellular, photosynthetic, oxygenic algae. They are non-parasitic plants without roots, stems or leaves; they contain chlorophyll and have a great variety in size, from microscopic to large seaweeds. Green algae, belonging to Eukaryota-Viridiplantae-Chlorophyta-Chlorophyceae, can be used. Blue-green, red, or brown algae may also be used. Exemplary algae for which the compositions and methods described herein includes those of the genus Chlamydomonas. In some embodiments, the engineered algal cells are unicellular green alga of the species Chlamydomonas reinhardtii, for which the sequence of all three genomes (nuclear, chloroplast and mitochondria) has been determined. Algal cells of the genus Chlorella and other genera may be used in other embodiments. For example, the PsaC of Chlorella vulgaris, Picochlorum soloecismus, or Cyanidioschyzon merolae may be used. These PsaC proteins are described in U.S. Publication No. US20220204996A1, which is incorporated by reference in its entirety. Algal cells of Galdieria sulphuraria may also be used. Iron-Iron hydrogenases from any of these algal species may be inserted into the hinge region of the PsaC in the same or similar manner as shown in the Examples for Chlamydomonas.
[0064] In the Examples, the polynucleotide encoding the fusion protein was integrated into the native PsaC gene within the chloroplast genome via homologous recombination. A similar fusion protein can be recombined into the chloroplast genome of any cell containing a chloroplast, such as algal cell or plant cells. A similar fusion protein and polynucleotide encoding the same may be used in the cyanobacteria to engineer cyanobacteria cells capable of generating increased hydrogen.
[0065] In a second aspect, provided herein is algal biomass comprising genetically engineered algal cells of the disclosure. In particular, provided herein is algal biomass that contains genetically engineered algal cells that exhibit increased hydrogen production on particular growth (e.g., light) conditions relative to genetically unmodified algal cells or other controls when cultured under the same conditions. As used herein, the term algal biomass refers to the amount or density of algae in a given area or volume (e.g., of water or other liquid) at a given time. Algal biomass encompasses algae grown in various cultivation systems such as photoreactors and open ponds, but also algal material obtained from different types of waste from industry and sewage plants.
[0066] In a third aspect, provided herein is an expression cassette that drives expression of a fusion protein comprising a PSI protein and a bacterial hydrogenase. The expression cassette may comprise a promoter operably linked to a nucleic acid sequence that encodes a fusion protein comprising a PSI protein and a bacterial hydrogenase. In preferred embodiments, the nucleic acid is a recombinant nucleic acid that encodes a PSI-MeHydA fusion protein, such as the fusion protein of SEQ ID NO: 1. While PSI is encoded in the chloroplast, the gene and thus the fusion protein may be encoded in the nucleus, added as an additional copy in the chloroplast or even encoded on an extra chromosomal expression cassette such as a plasmid or artificial chromosome. In these alternative expression cassettes, the promoters may be selected based on the expression system being contemplated by those of skill in the art. For example, as demonstrated in Reifschneider-Wegner et al..sup.44 the hydrogenase can be expressed in chloroplasts using the psbD promoter/5UTR to drive expression of the chloroplast-optimized hydA gene. A similar system could be used for expression of the fusion protein described herein.
[0067] Constructs are also provided herein. As used herein, the term construct refers to recombinant polynucleotides including, without limitation, DNA and RNA, which may be single-stranded or double-stranded and may represent the sense or the antisense strand. The constructs provided herein may be prepared by methods available to those of skill in the art. The constructs and expression cassettes provided herein are recombinant molecules and as such do not occur in nature. Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, and recombinant DNA techniques that are well known and commonly employed in the art. Standard techniques available to those skilled in the art may be used for cloning, DNA and RNA isolation, amplification and purification. Such techniques are thoroughly explained in the literature.
[0068] The constructs and expression cassettes provided herein may include a promoter operably linked to any one of the polynucleotides described herein but need not have a promoter and may be used for homologous recombination into the native site of psaC in the algae. Alternatively, the constructs may include a promoter and the promoter may be a heterologous promoter or an endogenous promoter associated with the PsaC polypeptide.
[0069] As used herein, the terms heterologous promoter, promoter, promoter region, or promoter sequence refer generally to transcriptional regulatory regions of a gene, which may be found at the 5 or 3 side of the polynucleotides described herein, or within the coding region of the polynucleotides, or within introns in the polynucleotides. Typically, a promoter is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3 direction) coding sequence. The typical 5 promoter sequence is bounded at its 3 terminus by the transcription initiation site and extends upstream (5 direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence is a transcription initiation site (conveniently defined by mapping with nuclease 51), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
[0070] In some embodiments, the disclosed polynucleotides are operably connected to the promoter. As used herein, a polynucleotide is operably connected or operably linked when it is placed into a functional relationship with a second polynucleotide sequence. For instance, a promoter is operably linked to a polynucleotide if the promoter is connected to the polynucleotide such that it may affect transcription of the polynucleotides. The polynucleotides may be operably linked to at least 1, at least 2, at least 3, at least 4, at least 5, or at least 10 promoters.
[0071] Heterologous promoters useful in the practice of the present invention include, but are not limited to, constitutive, inducible, temporally-regulated, developmentally regulated, chemically regulated, tissue-preferred and tissue-specific promoters. The heterologous promoter may be a plant, animal, bacterial, fungal, or synthetic promoter.
[0072] In a fourth aspect, provided herein are methods for increasing hydrogen (H.sub.2) production in a cell. The method comprises introducing into the cell a polynucleotide or an expression cassette comprising the polynucleotide encoding a fusion protein comprising a PSI protein and a bacterial hydrogenase described herein, and culturing the cell under continuous illumination (e.g. saturating light conditions). The cell may be cultured in a bioreactor growth system and the gas released during growth can be collected, removed from the bioreactor, and the hydrogen can be separated and collected from the remaining air in the bioreactor. The cell may be an algal cell.
[0073] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.
[0074] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.
[0075] The indefinite articles a and an, as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean at least one.
[0076] The phrase and/or, as used herein in the specification and in the claims, should be understood to mean either or both of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with and/or should be construed in the same fashion, i.e., one or more of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the and/or clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to A and/or B, when used in conjunction with open-ended language such as comprising can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
[0077] As used herein in the specification and in the claims, or should be understood to have the same meaning as and/or as defined above. For example, when separating items in a list, or or and/or shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as only one of or exactly one of, or, when used in the claims, consisting of, will refer to the inclusion of exactly one element of a number or list of elements. In general, the term or as used herein shall only be interpreted as indicating exclusive alternatives (i.e. one or the other but not both) when preceded by terms of exclusivity, such as either, one of only one of or exactly one of Consisting essentially of when used in the claims, shall have its ordinary meaning as used in the field of patent law.
[0078] As used herein, the terms approximately or about in reference to a number are generally taken to include numbers that fall within a range of 5% in either direction (greater than or less than) the number unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). Where ranges are stated, the endpoints are included within the range unless otherwise stated or otherwise evident from the context.
[0079] As used herein, the terms optional or optionally mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.
[0080] The present invention has been described in terms of one or more preferred embodiments, and it should be appreciated that many equivalents, alternatives, variations, and modifications, aside from those expressly stated, are possible and within the scope of the invention.
EXAMPLES
Example 1In Vivo Fusion of Photosystem I (PSI) and Bacterial Hydrogenase
[0081] Using a unicellular green alga (Chlamydomonas reinhardtii) as an experimental system, an in vivo fusion of PSI and the [FeFe] hydrogenase expressed by Megasphaera elsdenii was created (see
[0082] Experimental Design
[0083] Chimeric protein design and modeling: A protein sequence of PsaC-MeHydA is shown in
[0084] A model of the MeHydA was generated with Phyre.sup.2 server.sup.27. The first 80 residues of MeHydA correspond to the ferredoxin domain and residues past the last conserved residue among all 3 hydrogenases (highlighted in
[0085] Generation of PSI-MeHydA algal mutants: The psaC-MeHydA fusion gene sequence was codon optimized for chloroplast expression and synthesized by Genscript (Piscataway, NJ USA). The gene construct was delivered on pBS-EP 5.8 vector.sup.32, in which the fusion gene replaced the psaC gene. Transformations were carried out in the PBC4-2.sup.H6 strain lacking psaC and having hexahistidine tag on exon 1 of psaA.sup.33 via biolistic transformation as previously described.sup.17. Positive transformants were selected on Tris-acetate-phosphate (TAP) plates with 100 mg L.sup.?1-spectinomycin in the dark. Positive transformants were confirmed by PCR with region specific primers (PsaC5: TAATATGGAGATGACATATTTAG (SEQ ID NO: 3) and PsaC3: GATCTCAC CAAGATACTCCC (SEQ ID NO: 4). See
[0086] Growth conditions: All algae strains were grown on liquid Trisacetate-phosphate (TAP) medium with revised mineral nutrient supplement.sup.34. Mutants on plates were kept in the dark. Liquid cultures were grown under low room light (5-10 ?mol m.sup.?2 s.sup.?1 PAR).
[0087] Chlorophyll (Chl) measurement: Concentrations of Chl a and b were determined in 80% acetone as previously described.sup.35.
[0088] Thylakoid and PSI preparation: Thylakoid membranes and PSI preparations were done as previously described.sup.17 with minor modifications outlined below.
[0089] In the cell lysis step, ultrasonic cell disruption or French pressure cell was used. For ultrasonic cell disruption, cell pellet (from 10 L mid-late log phase cultures, equivalent of 80-100 mg of Chl) was resuspended in breaking buffer (50 mM HEPES-KOH pH 7.5, 0.3 M sucrose, 10 mM EDTA-KOH, 1 mM phenylmethyl sulfonyl fluoride (PMSF)) to a final volume of 200 mL. Ultrasonic lysis was done in a thin-walled aluminum cup on ice with continuous stirring in the dark. Temperature was continuously monitored during sonication and was always at 4? C. Branson sonifier S-450 was operated with ? (12.7 mm) catenoidal horn at amplitude level 3 (10% duty cycle) for 60 min. It approximately corresponded to 800 J per 10 mL of lysate as recommended for complete breakage of algal cells.sup.36. The lysate was spun down at 64000?g at 4? C. for 15 min, washed with H2 buffer (5 mM HEPES-KOH pH 7.5, 0.3 M sucrose, 10 mM EDTA-KOH) and resuspended in buffer H3 (5 mM HEPES-KOH, 1.8 M sucrose, 10 mM EDTA-KOH). All the following steps of discontinuous sucrose gradient were as previously described.sup.17.
[0090] French press lysis protocol was followed exactly as previously described.sup.17.
[0091] PSI isolation was done oxically using PROTEINDEX? Ni-Penta? agarose resin (Marvelgent Biosciences) as previously described.sup.33. For oxygen removal, PSI elution samples were concentrated and exchanged buffer using Amicon? stirred ultrafiltration cell (furnished with 100 kDa MWCO regenerated cellulose disc) inside the glovebox with a degassed solubilization buffer (25 mM HEPES-KOH, pH 7.5, 300 mM KCl, 5 mM MgSO.sub.4, 10% glycerol, 0.03% n-dodecyl-?-D-maltoside (?-DDM)).
[0092] Laser-flash spectroscopy: Thylakoids were resuspended at ?60 ?g Chl ml.sup.?1 in 25 mM HEPES-KOH (pH 7.5), 300 mM KCl, 10% glycerol, 5 mM sodium ascorbate while PSI were resuspended at ?6 ?g Chl ml.sup.?1 in with the same buffer with 0.03% ?-DDM. Samples were kept on ice in the dark before measurements. Absorbance changes pumped by a saturating laser flash (532 nm, 6 ns, 20-25 mJ) were monitored at 696 nm with weak LED pulses (10 ?s) using JTS-10 (Bio-Logic) kinetic spectrophotometer. Background transients were collected by running the same sequence with laser shutter closed.
[0093] Gas chromatography (GC) measurements: A model SRI 310 gas chromatograph with thermal conductivity detector and 5 ? molecular sieve prepacked column (91.4 cm long) was used. Gas tight syringes (1700 series) with non-coring needles were used for probing headspace.
[0094] Immunoblotting: Western blots were carried out as previously described.sup.17. Solubilized thylakoids were loaded on the same Chl (2 ?g).
[0095] Membrane inlet mass spectrometry (MIMS) measurements: MIMS measurements were carried out as previously described.sup.18.
[0096] Flavodoxin photoreduction assay: The experiment was carried out as described in Kanygin et al..sup.18, with one major differenceno oxygen was expected as it was set up in the anaerobic chamber filled with 5% H2/95% N2 (Coy).
[0097] In vivo P.sub.700 photobleaching and fluorescence measurements: Early log phase cells were washed and then resuspended in 10 mM sodium phosphate (pH 7.0), 2 mM sodium bicarbonate and 20% Ficoll? PM400 (GE Healthcare) to ?30 ?g/mL Chl (P.sub.700.sup.+) or ?9 ?g Chl mL.sup.?1 (for Chl fluorescence measurements). Cells were dark adapted for 5 min. Aerobic conditions were ensured by occasional mixing and brief sparging with air (in between the measurements). P.sub.700.sup.+ signal was measured at 696 nm as previously described.sup.17. Chlorophyll steady state fluorescence was measured after 2 min of green LED (520 nm) of variable intensity as previously described 18.
[0098] Results and Discussion
[0099] Design and expression of PSI-MeHydA: To generate a reasonable model of PSI-MeHydA, the hydrogenase domain of the MeHydA homology model was isolated based on the work of Caserta et al..sup.37 and a multiple sequence alignment with CrHydAs (
[0100] The designed psaC-hydA gene was delivered to the chloroplast of the PBC4-2 strain, which lacks the psaC gene and harbors a His.sub.6-tagged version of psaA, by particle bombardment. Transformants were selected for spectinomycin/streptomycin resistance. Homologous recombination with the chloroplast chromosome should result in replacement of psaC with the psaC-hydA gene. Positive transformants were confirmed by PCR with region-specific primers (
[0101] Expression of PsaC-MeHydA was confirmed by Western blot using solubilized thylakoids prepared from aerobically grown cells (
[0102] Spectroscopic Characterization of the PSI-MeHydA
[0103] A primary function of reaction centersconversion of absorbed light energy into a stable charge separationrequires all cofactors of the electron transfer chain to be assembled. The PsaC domain of PsaC-MeHydA carries two terminal [Fe4S4] clusters: F.sub.A and F.sub.B. They can only be inserted upon proper folding of the PsaC domain from the first 30 and last 46 residues of the chimeric polypeptide. To test assembly and accumulation of PSI-MeHydA, thylakoids were prepared oxically via 2 methods: ultrasonic cell disruption and French Press. The former has the advantage of potentially being used under complete anoxia (inside the glovebox), while the latter is a standard thylakoid preparation.
[0104] First, the amplitude of P.sub.700.sup.+ formation was observed in thylakoids prepared via sonication. After a saturating laser flash in the presence of ascorbate (to ensure P700 is reduced before the flash), accumulation of PSI-MeHydA relative to the WT.sup.H6 signal was observed (
[0105] In thylakoids prepared by French press (
[0106] Second, the decay of P.sub.700.sup.+ signal in the dark was examined. Upon charge separation in the absence of available electron acceptors, the reduced iron sulfur clusters of PSI harbored by PsaC will eventually backreact with P.sub.700.sup.+. Charge recombination of the P.sub.700.sup.+ (F.sub.A/F.sub.B)-state typically happens with a time constant(s) of 40-200 ms and if F.sub.AF.sub.B are not available (e.g., PsaC is absent or F.sub.A/F.sub.B is reduced), charge recombination from F.sub.X? would happen with a time constant of ?1 ms.sup.38. The P.sub.700.sup.+ transients (see
TABLE-US-00004 TABLE 1 Fitting coefficients of P700+ decay in thylakoids Sonication protocol French press protocol Parameter WT.sup.H6 JVD-1b.sup.H6 ?H3.sup.H6-1 ?H3.sup.H6-1 ?H3.sup.H6-2 ?.sub.1 (ms) 2.3 ? 0.3 2.3 ? 0.4 2.0 ? 0.3 23.4 ? 4.5 13.9 ? 3.1 A.sub.1 (%) 30 ? 1.4 16.5 ? 1.2 41 ? 1.8 11.4 ? 1.5 8.0 ? 0.9 ?.sub.2 (ms) 47 ? 3 52 ? 2.5 76 ? 9 192 ? 21 184 ? 13 A.sub.2 (%) 50 ? 1.3 61.5 ? 1.2 32 ? 1.4 19 ? 1.4 22.4 ? 1 ?.sub.3 (s) 2.9 ? 0.4 0.85 ? 0.2 5.5 ? 0.9 15.2 ? 0.6 16.4 ? 0.6 A.sub.3 (%) 9.9 ? 0.6 9.6 ? 1 14 ? 0.8 60 ? 1 69 ? 1 A.sub.0 (%) 10.1 ? 0.3 12.6 ? 0.2 12.8 ? 0.6 8.1 ? 1.1 0.3 ? 1.2 R.sup.2 0.99895 0.99941 0.9968 0.99964 0.9997
[0107] Of note, all thylakoids prepared via sonication show signs of shorter decay time constants than expected from a typical P.sub.700.sup.+-(FA/FB)- and likely result from some fraction of reaction centers having lost PsaC-MeHydA. The slowest decay component (on the order of seconds) is due to P.sub.700.sup.+ being reduced by ascorbate, because electrons had escaped from the iron sulfur clusters to exogenous acceptors (such as 02). This phase was especially prevalent in the French press preparations (60-70% amplitude) while also present in the ultrasonically disrupted cells (10-15% amplitude).
[0108] To get rid of dissolved oxygen, PSI prepared from sonicated thylakoids under anoxia were concentrated. In P.sub.700 bleaching and recovery kinetic experiment (
TABLE-US-00005 TABLE 2 Fitting coefficients of P.sub.700.sup.+ decay in purified PSI under anoxia Sonication protocol Parameter WT.sup.H6 ?H3.sup.H6-1 ?.sub.1 (ms) 24.3 ? 1.4 51.4 ? 2.5 A.sub.1 (%) 48.9 ? 3.4 54.9 ? 2.9 ?.sub.2 (ms) 98.1 ? 5.7 210.7 ? 16.3 A.sub.2 (%) 46.4 ? 3.4 31.8 ? 2.9 ?.sub.3 (s) 13.4 ? 5.5 11.0 ? 1.3 A.sub.3 (%) 3.3 ? 0.5 9.7 ? 0.3 A.sub.0 (%) 1.6 ? 0.6 3.6 ? 0.4 R.sup.2 0.99989 0.99986
[0109] In vitro activity toward flavodoxin photoreduction: An ideal PSI-HydA chimera would direct all electrons to the hydrogenase domain. In such a situation, the rate of reduction of its normal electron acceptor would approach zero. To test the ability of PSI-MeHydA to reduce cyanobacterial flavodoxin, purified PSI or PSI-MeHydA complexes (?0.1 ?M), reduced plastocyanin (50-fold excess) and ascorbate (5 mM) were used as electron donors, under anoxia with a ?50-fold excess of flavodoxin exposed to brief illuminations of saturating actinic light. The linear rate determined over a ?1 s interval (
[0110] Direct reduction of flavodoxin (or ferredoxin) by PSI-MeHydA in vitro could be viewed as a reciprocal function of proton reduction activity by the chimeric complex since the 2 processes are in competition when the hydrogenase active site is functional. Flavodoxin and ferredoxin share the binding interface on PSI, which requires the collaboration of PsaC, PsaD and PsaE forming a stromal ridge.sup.41. Moreover, PSI lacking the F.sub.B cluster show no flavodoxin reduction from the remaining FA cluster of PsaC.sup.42. The PSI-HydA2 and PSI-HydA1 chimeras exhibit flavodoxin reduction in the presence of O.sub.2, hence, the rates for the semiquinone formation by WT PSI are an order of magnitude lower than are shown here, despite similar setup. Under oxic conditions and with a large excess of flavodoxin relative to PSI, competing processes of flavodoxin semiquinone reoxidation by O.sub.2 take place and usually no significant accumulation of flavodoxin hydroquinone occurs.sup.43. For that reason, only the initial 4 points were used to determine steady-state rate of flavodoxin reduction. Thusly, 6-8 times drop in flavodoxin steady-state photoreduction rate (per PSI complex) by PSI-MeHydA is comparable to previously made constructs. This suggests that flavodoxin binding does not involve interaction with hydrogenase domain of the chimeric complex and perhaps is limited to PsaC domain and linking regions. It is important to remember that this in vitro experiment is not representative of what is occurring in vivo, as the hydrogenase active site is damaged during purification of the PSI-HydA chimerathus, there is no competition with proton reduction.
[0111] To find a probable site for Fd binding on PSI-MeHydA, the generated PsaC-MeHydA with PsaA, PsaB, PsaD, and PsaF subunits was used as a receptor for Fd hard docking as previously described.sup.18. With a single distant restraint between C14 (F.sub.B) and outermost C42 of Fd (limited to 20 ?), the most likely docking model places Fd in a different position than typical WT ferredoxin site (
TABLE-US-00006 TABLE 3 Important pairs of residues involved in Fd1 docking to PSI-MeHydA (FIG. 16). Ferredoxin 1 PsaC-MeHydA Distance residue residue (in ?) S43 G32 2.6 Y94 G33 2.5 Y94 L359 2.4 PsaD residue H88 A163 3.2
[0112] In vivo photosynthetic activity of ?H3.sup.H6 strain: When analyzing hydrogen production/oxidation activity of the ?H3.sup.H6 strain, it is important to keep in mind that it has 3 hydrogenases enzymes: two endogenous enzymes (HydA1 and HydA2) and the chimeric PSI-MeHydA. This mutant's ability to produce H.sub.2 without added bicarbonate or acetate on a relatively short time scale was examined using MIMS (
[0113] Photobiological H.sub.2 production of the ?H3.sup.H6 strain on a short time scale had a maximal rate of 37 ?mol h.sup.?1 (mg Chl).sup.?1, as measured by MIMS. Using the ratio of 3420 Chl per P.sub.700 determined for strain ?H3.sup.H6-1, this would correspond to a turnover rate of ?31H.sub.2 s.sup.?1, assuming that each PSI-MeHydA complex is active. This number is comparable to the rate obtained for PSI-HydA2 in the ?H1 strain?52H.sub.2 s.sup.?1. The PSI-MeHydA turnover rate is about twice as high as PSI-HydA1 (16H.sub.2 s.sup.?1). A reciprocal relation of turnover number vs accumulation level of the PSI-HydA chimera may indicate that inactivation of the hydrogenase domain occurs faster and to a greater degree in a higher accumulating chimera.
[0114] When bicarbonate was added to the cells at the beginning of the experiment (
[0115] The reduction in net H.sub.2 production rate upon bicarbonate addition indicates the likely involvement of endogenous hydrogenases in concomitant H.sub.2/CO.sub.2 uptake processes competing with H.sub.2 evolution processes by PSI-MeHydA. The H.sub.2/CO.sub.2 uptake eventually wins over as the CBB cycle activates, creating a large electron since, until H.sub.2 is completely consumed and O.sub.2 starts to build up (
[0116] Next, H.sub.2 production was assessed on a longer scale. A sealed bottle experiment was set up with anaerobically adapted cultures and the headspace was periodically probed using GC (
[0117] Net O.sub.2 was initially evolved at 9.5?1.5 ?mol O.sub.2 h.sup.?1 (mg Chl).sup.?1 by ?H3.sup.H6 for the first hour and then remained fairly stable at about 0.05% for the next 3 h. The amount of O.sub.2 doubled overnight. In contrast, WT.sup.H6 exhibited a linear rate of O.sub.2 evolution in the first 4 hours at an average rate of 10.7 ?mol O.sub.2 h.sup.?1 (mg Chl).sup.?1; O.sub.2 reached 0.2% in the headspace and did not significantly change overnight. (
[0118] It was also of interest to know how the ?H3.sup.H6 strain would perform under fully aerobic conditions, as it must initially be grown this way. Chl fluorescence of Photosystem II was used to characterize its photosynthetic capacity for linear electron flow. ?H3.sup.H6 strain saturates with relatively low light: at 67 ?mol m.sup.?2 s.sup.?1 PAR (green LED at 520 nm) the quantum yield of PSII drops below 0.2. It takes almost 1500 ?mol m.sup.?2 s.sup.?1 PAR for the WT.sup.H6 control for a comparable decrease in quantum yield (
[0119] PSII activity was blocked with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and the rate of P.sub.700.sup.+ decay was measured to estimate CEF around PSI (
[0120] ?H3.sup.H6 doesn't show the same linearity to O.sub.2 production and that could be due to increased mitochondrial respiration needed to compensate for the significantly reduced CEF in ?H3.sup.H6 (
REFERENCES
[0121] [1] Hohmann-Marriott M F, Blankenship R E. Evolution of Photosynthesis. Annu Rev Plant Biol 2011; 62:515-48. https://doi.org/10.1146/annurev-arplant-042110-103811. [0122] [2] Nelson N, Ben-Shem A. The complex architecture of oxygenic photosynthesis. Nat Rev Mol Cell Biol 2004; 5:971. [0123] [3] Nelson N, Junge W. Structure and Energy Transfer in Photosystems of Oxygenic Photosynthesis. Annu Rev Biochem 2015; 84:659-83. https://doi.org/10.1146/annurev-biochem-092914-041942. [0124] [4] Nawrocki W J, Bailleul B, Picot D, Cardol P, Rappaport F, Wollman F-A, et al. The mechanism of cyclic electron flow. Biochim Biophys ActaBioenerg 2019; 1860:433-8. https://doi.org/10.1016/j.bbabio.2018.12.005. [0125] [5] Milrad Y, Schweitzer S, Feldman Y, Yacoby I. Green Algal Hydrogenase Activity Is Outcompeted by Carbon Fixation before Inactivation by Oxygen Takes Place. Plant Physiol 2018; 177:918-26. https://doi.org/10.1104/pp. 18.00229. [0126] [6] Pinto T S, Malcata F X, Arrabaca J D, Silva J M, Spreitzer R J, Esquivel M G. Rubisco mutants of Chlamydomonas reinhardtii enhance photosynthetic hydrogen production. Appl Microbiol Biotechnol 2013; 97:5635-43. https://doi.org/10.1007/s00253-013-4920-z. [0127] [7] Sun Y, Chen M, Yang H, Zhang J, Kuang T, Huang F. Enhanced H2 photoproduction by down-regulation of ferredoxin-NADP+ reductase (FNR) in the green alga Chlamydomonas reinhardtii. Int J Hydrogen Energy 2013; 38:16029-37. https://doi.org/10.1016/J.IJHYDENE.2013.10.011. [0128] [8] Dang K-V, Plet J, Tolleter D, Jokel M, Cuin? S, Carrier P, et al. Combined Increases in Mitochondrial Cooperation and Oxygen Photoreduction Compensate for Deficiency in Cyclic Electron Flow in Chlamydomonas reinhardtii. Plant Cell 2014; 26:3036-50. https://doi.org/10.1105/tpc.114.126375. [0129] [9] Elman T, Hoai Ho T T, Milrad Y, Hippler M, Yacoby I. Enhanced chloroplast-mitochondria crosstalk promotes ambient algal-H2 production. Cell Reports Phys Sci 2022; 3:100828. https://doi.org/10.1016/j.xcrp.2022.100828. [0130] [10] Kozuleva M, Petrova A, Milrad Y, Semenov A, Ivanov B, Redding K E, et al. Phylloquinone is the principal Mehler reaction site within photosystem I in high light. Plant Physiol 2021; 186:1848-58. https://doi.org/10.1093/plphys/kiab221. [0131] [11] Chaux F, Burlacot A, Mekhalfi M, Auroy P, Blangy S, Richaud P, et al. Flavodiiron Proteins Promote Fast and Transient 02 Photoreduction in Chlamydomonas. Plant Physiol 2017; 174:1825 LP-1836. https://doi.org/10.1104/pp. 17.00421. [0132] [12] Lubitz W, Ogata H, R?diger O, Reijerse E. Hydrogenases. Chem Rev 2014; 114:4081-148. https://doi.org/10.1021/cr4005814. [0133] [13] Engelbrecht V, Liedtke K, Rutz A, Yadav S, Gunzel A, Happe T. One isoform for one task? The second hydrogenase of Chlamydomonas reinhardtii prefers hydrogen uptake. Int J Hydrogen Energy 2021; 46:7165-75. https://doi.org/10.1016/j.ijhydene.2020.11.231. [0134] [14] Eroglu E, Melis A. Microalgal hydrogen production research. Int J Hydrogen Energy 2016; 41:12772-98. https://doi.org/https://doi.org/10.1016/j.ijhydene.2016.05.115. [0135] [15] Cournac L, Mus F, Bernard L, Guedeney G, Vignais P M, Peltier G. Limiting steps of hydrogen production in Chlamydomonas reinhardtii and Synechocystis PCC 6803 as analysed by light-induced gas exchange transients. Int J Hydrogen Energy 2002; 27:1229-37. https://doi.org/10.1016/S0360-3199(02)00105-2. [0136] [16] Milrad Y, Schweitzer S, Feldman Y, Yacoby I. Bi-directional electron transfer between H.sub.2 and NADPH mitigates light fluctuation responses in green algae. Plant Physiol 2021; 186:168-79. https://doi.org/10.1093/plphys/kiab051. [0137] [17] Kanygin A, Milrad Y, Thummala C, Reifschneider K, Baker P, Marco P, et al. Rewiring photosynthesis: a photosystem I-hydrogenase chimera that makes H2 in vivo. Energy Environ Sci 2020; 13:2903-14. https://doi.org/10.1039/C9EE03859K. [0138] [18] Kanygin A, Smith A, Nagy V, T6th S Z, Redding K E. Interplay between Hydrogen Production and Photosynthesis in a Green Alga Expressing an Active Photosystem I-hydrogenase Chimera. Int J Hydrogen Energy 2022; (accepted for publication on Mar. 11, 2022) [0139] [19] Swanson K D, Ratzloff M W, Mulder D W, Artz J H, Ghose S, Hoffman A, et al. [FeFe]-Hydrogenase Oxygen Inactivation Is Initiated at the H Cluster 2Fe Subcluster. J Am Chem Soc 2015; 137:1809-16. https://doi.org/10.1021/ja510169s. [0140] [20] Ghirardi M L, Togasaki R K, Seibert M. Oxygen sensitivity of algal H2-production. Appl Biochem Biotechnol 1997; 67:182. https://doi.org/10.1007/BF02787851. [0141] [21] Nagy V, Podmaniczki A, Vidal-Meireles A, Tengolics R, Kovacs L, R?khely G, et al. Water-splitting-based, sustainable and efficient H2 production in green algae as achieved by substrate limitation of the Calvin-Benson-Bassham cycle. Biotechnol Biofuels 2018; 11:69. https://doi.org/10.1186/s13068-018-1069-0. [0142] [22] Torzillo G, Scoma A, Faraloni C, Giannelli L. Advances in the biotechnology of hydrogen production with the microalga Chlamydomonas reinhardtii. Crit Rev Biotechnol 2015; 35:485-96. https://doi.org/10.3109/07388551.2014.900734. [0143] [23] Redding K E, Appel J, Boehm M, Schuhmann W, Nowaczyk M M, Yacoby I, et al. Advances and challenges in photosynthetic hydrogen production. Trends Biotechnol 2022. https://doi.org/10.1016/j.tibtech.2022.04.007. [0144] [24] Hwang J-H, Lee W H. Continuous photosynthetic biohydrogen production from acetate-rich wastewater: Influence of light intensity. Int J Hydrogen Energy 2021; 46:21812-21. https://doi.org/10.1016/j.ijhydene.2021.04.052. [0145] [25] Atta M, Meyer J. Characterization of the gene encoding the [Fe]-hydrogenase from Megasphaera elsdenii. Biochim Biophys ActaProtein Struct Mol Enzymol 2000; 1476:368-71. https://doi.org/10.1016/50167-4838(99)00245-9. [0146] [26] Sievers F, Wilm A, Dineen D, Gibson T J, Karplus K, Li W, et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 2011; 7:539. https://doi.org/10.1038/msb.2011.75. [0147] [27] Kelley L A, Mezulis S, Yates C M, Wass M N, Sternberg M J E. The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 2015; 10:845-58. https://doi.org/10.1038/nprot.2015.053. [0148] [28] Schr?dinger L. The {PyMOL} Molecular Graphics System, Version 2.4.0. 2015. [0149] [29] Suga M, Ozawa S-I, Yoshida-Motomura K, Akita F, Miyazaki N, Takahashi Y. Structure of the green algal photosystem I supercomplex with a decameric light-harvesting complex I. Nat Plants 2019; 5:626-36. https://doi.org/10.1038/s41477-019-0438-4. [0150] [30] Kozakov D, Hall D R, Xia B, Porter K A, Padhorny D, Yueh C, et al. The ClusPro web server for protein-protein docking. Nat Protoc 2017; 12:255. https://doi.org/10.1038/nprot.2016.169. [0151] [31] Song Y, DiMaio F, Wang R Y-R, Kim D, Miles C, Brunette T, et al. High-Resolution Comparative Modeling with Rosetta C M. Structure 2013; 21:1735-42. https://doi.org/10.1016/j.str.2013.08.005. [0152] [32] Fischer N, S?tif P, Rochaix J D. Targeted Mutations in the psaC Gene of Chlamydomonas reinhardtii: Preferential Reduction of F B at Low Temperature Is Not Accompanied by Altered Electron Flow from Photosystem I to Ferredoxin. Biochemistry 1997; 36:93-102. https://doi.org/10.1021/bi962244v. [0153] [33] Gulis G, Narasimhulu K V, Fox L N, Redding K E. Purification of His6-tagged Photosystem I from Chlamydomonas reinhardtii. Photosynth Res 2008; 96:51-60. https://doi.org/10.1007/s11120-007-9283-9. [0154] [34] Kropat J, Hong-Hermesdorf A, Casero D, Ent P, Castruita M, Pellegrini M, et al. A revised mineral nutrient supplement increases biomass and growth rate in Chlamydomonas reinhardtii. Plant J 2011; 66:770-80. https://doi.org/10.1111/j.1365-313X.2011.04537.x. [0155] [35] Porra R J, Thompson W A, Kriedemann P E. Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectrosco. Biochim Biophys ActaBioenerg 1989; 975:384-94. https://doi.org/10.1016/S0005-2728(89)80347-0. [0156] [36] Gerde J A, Montalbo-Lomboy M, Yao L, Grewell D, Wang T. Evaluation of microalgae cell disruption by ultrasonic treatment. Bioresour Technol 2012; 125:175-81. https://doi.org/10.1016/j.biortech.2012.08.110. [0157] [37] Caserta G, Papini C, Adamska-Venkatesh A, Pecqueur L, Sommer C, Reijerse E, et al. Engineering an [FeFe]-Hydrogenase: Do Accessory Clusters Influence O 2 Resistance and Catalytic Bias? J Am Chem Soc 2018; 140:5516-26. https://doi.org/10.1021/jacs.8b01689. [0158] [38] Brettel K. Electron transfer and arrangement of the redox cofactors in photosystem I. Biochim Biophys ActaBioenerg 1997; 1318:322-73. https://doi.org/10.1016/S0005-2728(96)00112-0. [0159] [39] Sawyer A, Bai Y, Lu Y, Hemschemeier A, Happe T. Compartmentalisation of [FeFe]-hydrogenase maturation in Chlamydomonas reinhardtii. Plant J 2017; 90:1134-43. https://doi.org/10.1111/tpj.13535. [0160] [40] Suslick K S. Sonochemistry. Science (80-) 1990; 247:1439-45. https://doi.org/10.1126/science.247.4949.1439. [0161] [41] S?tif P. Ferredoxin and flavodoxin reduction by photosystem I. Biochim Biophys ActaBioenerg 2001; 1507:161-79. https://doi.org/https://doi.org/10.1016/50005-2728(01)00205-5. [0162] [42] Vassiliev I R, Jung Y-S, Yang F, Golbeck J H. PsaC Subunit of Photosystem I Is Oriented with Iron-Sulfur Cluster F B as the Immediate Electron Donor to Ferredoxin and Flavodoxin. Biophys J 1998; 74:2029-35. https://doi.org/10.1016/S0006-3495(98)77909-3. [0163] [43] M?hlenhoff U, S?tif P. Laser Flash Absorption Spectroscopy Study of Flavodoxin Reduction by Photosystem I in Synechococcus sp. PCC 7002. Biochemistry 1996; 35:1367-74. [0164] [44] Reifschneider-Wegner et al. Expression of the [FeFe] hydrogenase in the chloroplast of Chlamydomonas reinhardtii. Int J of Hydrogen Energy 39: 3657-3665 (2014).