Patent Application
20240093246
This application is being filed electronically via EFS-Web and includes an informal Sequence Listing in .xml format. The contents of the electronic sequence listing (112624.01410.xml; Size: 24,842 bytes; and Date of Creation: Jul. 28, 2023) is herein incorporated by reference in its entirety.
Conversion of algal cells into solar-powered biofactories generating high-energy product molecules is a promising avenue for addressing the ever-increasing global energy demand, due to its environmental friendliness and cheap replication. Hydrogen (H2) is an attractive target product for several reasons. It is an important commodity with over 60 million tons produced globally, but about 95% of it is produced from steam reformation of fossil fuels, thus contributing to the rise of atmospheric CO2. The [FeFe] hydrogenase enzyme catalyzes the rapid and reversible reduction of protons (2H++2 e−⇄H2). The active site of the enzyme is a metallic cofactor that is O2-sensitive and must be inserted by maturation factors.
Photosynthesis has been driving biomass accumulation on Earth for over 3 billion years, using fleeting light energy conversion into stable chemical bonds by pigment-protein machinery. As the oxygenic photosynthesis became dominant, the light could do the most work by splitting water into oxygen, protons, and reducing electrons. In the thylakoid membranes of the chloroplast, the photosynthetic electron transport chain (PETC) performs light-driven electron transport from water to ferredoxin (Fd) and pumps protons across the membrane, ultimately providing metabolic energy (ATP) and low-potential reductant (NADPH) to drive CO2 fixation by the Calvin-Benson-Bassham (CBB) cycle.
Low redox potential electrons traverse energetically downhill via the PETC using energy differences to generate proton gradient. First, at acceptor side of Photosystem II the quinone is reduced to quinol taking two protons and two electrons from the stromal side and via the Q-cycle, involving cytochrome b6f, releasing 4 protons (per 2 electrons transported) at the luminal side of the membrane. Then, these electrons are “reenergized” by Photosystem-I becoming highly reducing (˜−520-590 mV at PSI acceptor side cofactors) and readily go to ferredoxin (Emo=−430 mV). Ferredoxins are a logistical hub for electrons in the stroma, with delivery based on the momentary needs of the cell. When electrons are delivered to ferredoxin-NADP reductase (FNR) to make NADPH, it is called linear electron flow. When electrons are delivered back to the quinone pool (via cytochrome b6f), it is called cyclic electron flow (CEF). The former is used for carbon fixation resulting in NADPH and ATP consumption, while the latter allows supplementary ATP synthesis via additional proton pumping.
Alternative pathways for reductant at ferredoxin level take place when the linear pathway is stalling or cyclic electron flow is impaired. To prevent overreduction and damage to PSI, electrons can follow the path of 02 reduction if 02 is available: either directly via Mehler reaction or flavodiiron proteins mediated pathway. In the absence of oxygen, some microalga developed a mechanism that involves hydrogenases—metalloenzymes catalyzing reversible proton reduction, thusly, reductant changes phase from liquid to gaseous storing energy as molecular hydrogen that can be recaptured later and converted back to reduced ferredoxin.
It has been shown that some of the reducing power can be captured and redirected from carbon fixation to proton reduction, hence, changing the energy storage molecule from carbohydrate to molecular hydrogen. In that work, endogenous [FeFe] hydrogenases of Chlamydomonas reinhardtii have been used, which are notoriously oxygen sensitive. In the two endogenous hydrogenases, function significantly suffers from oxygen inactivation, as active oxygen removal methods improve chimeric hydrogenase activity. Without a way to keep most of the chimeric protein active, photobiological hydrogen production cannot approach its theoretical maximum of solar-to-hydrogen conversion efficiency of ˜13% as electrons are diverted to alternative electron acceptors (O2) and become feasible for industrial scale. A wild-type Chlamydomonas light-to-hydrogen energy conversion efficiency is typically an order of magnitude lower and is around 1.61% under conditions of mitigating O2 by respiration. Accordingly, there remains a need in the art for improved efficient, scalable, and economically feasible methods of producing hydrogen in algae.
Fusion proteins, genetically engineered cells and expression cassettes comprising polynucleotides encoding the fusion proteins are provided herein as well as methods of using the same.
In an aspect, a genetically engineered cell comprising a polynucleotide encoding a fusion protein comprising a photosystem I (PSI) protein and a bacterial hydrogenase is provided. In embodiments, the PSI protein is PsaC. In embodiments, the hydrogenase is bacterial hydrogenase A (HydA). In embodiments, the hydrogenase is inserted in frame into the PSI protein. In embodiments, the hydrogenase is inserted in frame into the hinge region of PsaC.
In embodiments, 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.
In embodiments, 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.
In embodiments, the bacterial hydrogenase is a Megasphaera elsdenii hydrogenase or a Clostridium beijerincki hydrogenase. In embodiments, the hydrogenase F-domain is removed.
In embodiments, the cell is an algal cell. In embodiments, the cell is selected from Chlamydomonas reinhardtii, Chlorella vulgaris, Picochlorum soloecismus, Galdieria sulphuraria and Cyanidioschyzon merolae.
In another aspect, provided herein is an algal biomass comprising the genetically engineered cell described herein.
In another aspect, provided herein is 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. In embodiments, the PSI protein is PsaC. In embodiments, the polynucleotide comprises bacterial hydrogenase A inserted in frame into the 0-hairpin of PsaC. In embodiments, 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. In embodiments, 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.
In another aspect, provided herein is a method of increasing hydrogen (H2) production in a cell, the method comprising (a) introducing into the cell the expression cassette described herein 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 H2 production under such conditions relative to a control cell of the same species under the same conditions.
In another aspect, provided herein is a fusion protein comprising a bacterial FeFe hydrogenase or functional portion thereof inserted into a PsaC protein. In embodiments, 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.
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.
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.
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 (H2) production under certain light conditions relative to unmodified algal cells. This means for H2 production offers an ecologically-friendly, inexpensive renewable energy source.
The use of algal hydrogenases to produce H2 in previously described PSI-HydA fusion proteins is limited by their oxygen sensitivity. Also, the H2 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.
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.
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.
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.
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.
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.
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 O2 tolerant, exhibiting a ˜10-fold lower O2 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 Fe—Fe 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
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 β-hairpin in
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).
“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.
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.
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
MAHIVKIYDTCIGCTQCVRACPLDVLEMVPW
GG
ATATDAVPNDVDKVKAA
CPTDFLSVRVYLGSESTRSMGLSY.
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.
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:
The amino acid sequence of the PsaC-CbA5H fusion protein is:
MAHIVKIYDTCIGCTQCVRACPLDVLEMVPW
GG
ATATDAVPNTMLFLRDL
VYLGSESTRSMGLSY.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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 Fe—Fe 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.
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.
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.
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.
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.44 the hydrogenase can be expressed in chloroplasts using the psbD promoter/5′UTR to drive expression of the chloroplast-optimized hydA gene. A similar system could be used for expression of the fusion protein described herein.
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.
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.
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.
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.
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.
In a fourth aspect, provided herein are methods for increasing hydrogen (H2) 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.
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.
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.
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.”
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.
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.
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.
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.
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.
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
Experimental Design
Chimeric protein design and modeling: A protein sequence of PsaC-MeHydA is shown in
A model of the MeHydA was generated with Phyre2 server27. The first 80 residues of MeHydA correspond to the ferredoxin domain and residues past the last conserved residue among all 3 hydrogenases (highlighted in
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 vector32, in which the fusion gene replaced the psaC gene. Transformations were carried out in the PBC4-2H6 strain lacking psaC and having hexahistidine tag on exon 1 of psaA33 via biolistic transformation as previously described17. Positive transformants were selected on Tris-acetate-phosphate (TAP) plates with 100 mg L−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
Growth conditions: All algae strains were grown on liquid Trisacetate-phosphate (TAP) medium with revised mineral nutrient supplement34. Mutants on plates were kept in the dark. Liquid cultures were grown under low room light (5-10 μmol m−2 s−1 PAR).
Chlorophyll (Chl) measurement: Concentrations of Chl a and b were determined in 80% acetone as previously described35.
Thylakoid and PSI preparation: Thylakoid membranes and PSI preparations were done as previously described17 with minor modifications outlined below.
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 ½” (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 cells36. 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 described17.
French press lysis protocol was followed exactly as previously described17.
PSI isolation was done oxically using PROTEINDEX™ Ni-Penta™ agarose resin (Marvelgent Biosciences) as previously described33. 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 MgSO4, 10% glycerol, 0.03% n-dodecyl-β-D-maltoside (β-DDM)).
Laser-flash spectroscopy: Thylakoids were resuspended at ˜60 μg Chl ml−1 in 25 mM HEPES-KOH (pH 7.5), 300 mM KCl, 10% glycerol, 5 mM sodium ascorbate while PSI were resuspended at ˜6 μg Chl ml−1 in with the same buffer with 0.03% β-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 μs) using JTS-10 (Bio-Logic) kinetic spectrophotometer. Background transients were collected by running the same sequence with laser shutter closed.
Gas chromatography (GC) measurements: A model SRI 310 gas chromatograph with thermal conductivity detector and 5 Å molecular sieve prepacked column (91.4 cm long) was used. Gas tight syringes (1700 series) with non-coring needles were used for probing headspace.
Immunoblotting: Western blots were carried out as previously described17. Solubilized thylakoids were loaded on the same Chl (2 μg).
Membrane inlet mass spectrometry (MIMS) measurements: MIMS measurements were carried out as previously described18.
Flavodoxin photoreduction assay: The experiment was carried out as described in Kanygin et al.18, with one major difference—no oxygen was expected as it was set up in the anaerobic chamber filled with 5% H2/95% N2 (Coy).
In vivo P700 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 μg/mL Chl (P700+) or ˜9 μg Chl mL−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). P700+ signal was measured at 696 nm as previously described17. Chlorophyll steady state fluorescence was measured after 2 min of green LED (520 nm) of variable intensity as previously described 18.
Results and Discussion
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.37 and a multiple sequence alignment with CrHydAs (
The designed psaC-hydA gene was delivered to the chloroplast of the PBC4-2 strain, which lacks the psaC gene and harbors a His6-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 (
Expression of PsaC-MeHydA was confirmed by Western blot using solubilized thylakoids prepared from aerobically grown cells (
Spectroscopic Characterization of the PSI-MeHydA
A primary function of reaction centers—conversion of absorbed light energy into a stable charge separation—requires all cofactors of the electron transfer chain to be assembled. The PsaC domain of PsaC-MeHydA carries two terminal [Fe4S4] clusters: FA and FB. 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.
First, the amplitude of P700+ 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 WTH6 signal was observed (
In thylakoids prepared by French press (
Second, the decay of P700+ 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 P700+. Charge recombination of the P700+ (FA/FB)-state typically happens with a time constant(s) of 40-200 ms and if FAFB are not available (e.g., PsaC is absent or FA/FB is reduced), charge recombination from FX− would happen with a time constant of ≤1 ms38. The P700+ transients (see
Of note, all thylakoids prepared via sonication show signs of shorter decay time constants than expected from a typical P700+-(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 P700+ 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).
To get rid of dissolved oxygen, PSI prepared from sonicated thylakoids under anoxia were concentrated. In P700 bleaching and recovery kinetic experiment (
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 μM), 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 (
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 ridge41. Moreover, PSI lacking the FB cluster show no flavodoxin reduction from the remaining FA cluster of PsaC42. The PSI-HydA2 and PSI-HydA1 chimeras exhibit flavodoxin reduction in the presence of O2, 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 O2 take place and usually no significant accumulation of flavodoxin hydroquinone occurs43. 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 chimera—thus, there is no competition with proton reduction.
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 described18. With a single distant restraint between C14 (FB) and outermost C42 of Fd (limited to 20 Å), the most likely docking model places Fd in a different position than typical WT ferredoxin site (
In vivo photosynthetic activity of ΨH3H6 strain: When analyzing hydrogen production/oxidation activity of the ΨH3H6 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 H2 without added bicarbonate or acetate on a relatively short time scale was examined using MIMS (
Photobiological H2 production of the ΨH3H6 strain on a short time scale had a maximal rate of 37 μmol h−1 (mg Chl)−1, as measured by MIMS. Using the ratio of 3420 Chl per P700 determined for strain ΨH3H6-1, this would correspond to a turnover rate of ˜31H2 s−1, assuming that each PSI-MeHydA complex is active. This number is comparable to the rate obtained for PSI-HydA2 in the ΨH1 strain—˜52H2 s−1. The PSI-MeHydA turnover rate is about twice as high as PSI-HydA1 (16H2 s−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.
When bicarbonate was added to the cells at the beginning of the experiment (
The reduction in net H2 production rate upon bicarbonate addition indicates the likely involvement of endogenous hydrogenases in concomitant H2/CO2 uptake processes competing with H2 evolution processes by PSI-MeHydA. The H2/CO2 uptake eventually wins over as the CBB cycle activates, creating a large electron since, until H2 is completely consumed and O2 starts to build up (
Next, H2 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 (
Net O2 was initially evolved at 9.5±1.5 μmol O2 h−1 (mg Chl)−1 by ΨH3H6 for the first hour and then remained fairly stable at about 0.05% for the next 3 h. The amount of O2 doubled overnight. In contrast, WTH6 exhibited a linear rate of O2 evolution in the first 4 hours at an average rate of 10.7 μmol O2 h−1 (mg Chl)−1; O2 reached 0.2% in the headspace and did not significantly change overnight. (
It was also of interest to know how the ΨH3H6 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. ΨH3H6 strain saturates with relatively low light: at 67 μmol m−2 s−1 PAR (green LED at 520 nm) the quantum yield of PSII drops below 0.2. It takes almost 1500 μmol m−2 s−1 PAR for the WTH6 control for a comparable decrease in quantum yield (
PSII activity was blocked with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and the rate of P700+ decay was measured to estimate CEF around PSI (
ΨH3H6 doesn't show the same linearity to O2 production and that could be due to increased mitochondrial respiration needed to compensate for the significantly reduced CEF in ΨH3H6 (
This application claims priority to, and the benefit of, U.S. Application No. 63/359,666 filed Jul. 8, 2022, the content of which is incorporated herein by reference in its entirety
This invention was made with government support under 1706960 awarded by the National Science Foundation. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63359666 | Jul 2022 | US |
