Expression of nitrogenase polypeptides in plant cells
AssigneeCommonwealth Scientific and Industrial Research Organisation
InventorsEma Jayne JOHNSTON, Robert Silas ALLEN, Christina Maria GREGG, Shoko OKADA, Amratha MENON, Andrew Charles WARDEN, Matthew Craig TAYLOR, Craig Christopher WOOD
Life of this patent
Estimating the term…
Abstract
The present invention relates to methods and means for producing nitrogenase polypeptides in the mitochondria of plant cells.
Claims83
A plant cell comprising mitochondria and exogenous polynucleotides which encode at least 8 or at least 9 Nif fusion polypeptides, wherein the exogenous polynucleotides each comprise a promoter which is operably linked to a nucleotide sequence which encodes one of the Nif fusion polypeptides and which expresses the nucleotide sequence in the plant cell, wherein each Nif fusion polypeptide independently comprises a mitochondrial targeting peptide (MTP), wherein the Nif fusion polypeptides comprise (i) NifH, NifB, NifF, NifJ, NifS, NifU and NifV fusion polypeptides and either (ii) a NifD fusion polypeptide and a NifK fusion polypeptide or (iii) a NifD-linker-NifK fusion polypeptide which comprises a NifD sequence having a C-terminus, an oligopeptide linker and a NifK sequence having a N-terminus, wherein the oligopeptide linker is translationally fused to the C-terminus of the NifD sequence and the N-terminus of the NifK sequence, wherein mitochondrial processing protease (MPP)-cleaved products of at least the NifH, NifF, NifS and NifU fusion polypeptides are each at least partially soluble in mitochondria of a plant cell, wherein MPP-cleaved products of the NifD and NifK fusion polypeptides of (ii) if present in the plant cell are at least partially soluble in mitochondria of a plant cell, or a MPP-cleaved product of the NifD-linker-NifK fusion polypeptide of (iii) if present in the plant cell is at least partially soluble in mitochondria of a plant cell, and wherein the NifV fusion polypeptide and/or a MPP-cleaved product thereof produces homocitrate in the plant cell and is at least partially soluble in mitochondria of a plant cell.
A plant cell comprising mitochondria and exogenous polynucleotides which encode at least 2, at least 3, at least 4, at least 5 or at least 6 Nif fusion polypeptides, wherein the exogenous polynucleotides each comprise a promoter which is operably linked to a nucleotide sequence which encodes one of the Nif fusion polypeptides and which expresses the nucleotide sequence in the plant cell, wherein each Nif fusion polypeptide independently comprises a mitochondrial targeting peptide (MTP), wherein the Nif fusion polypeptides comprise (i) one or more than one or all of NifW, NifX, NifY, and NifZ fusion polypeptides, and either (ii) a NifD fusion polypeptide and a NifK fusion polypeptide or (iii) a NifD-linker-NifK fusion polypeptide which comprises a NifD sequence having a C-terminus, an oligopeptide linker and a NifK sequence having a N-terminus, wherein the oligopeptide linker is translationally fused to the C-terminus of the NifD sequence and the N-terminus of the NifK sequence, wherein mitochondrial processing protease (MPP)-cleaved products of at least the NifW, NifX, NifY and NifZ fusion polypeptides if present in the plant cell are each at least partially soluble in mitochondria of a plant cell, wherein either MPP-cleaved products of the NifD and NifK fusion polypeptides of (ii) if present in the plant cell are at least partially soluble in mitochondria of a plant cell, or a MPP-cleaved product of the NifD-linker-NifK fusion polypeptide of (iii) if present in the plant cell is at least partially soluble in mitochondria of a plant cell, and wherein the MPP-cleaved products of the NifD fusion polypeptide and NifK fusion polypeptide of ii) or the MPP-cleaved product of the NifD-linker-NifK fusion polypeptide of iii) is present in the plant cell in greater amount than the amount of the MPP-cleaved products of the NifD fusion polypeptide and NifK fusion polypeptide or the MPP-cleaved product of the NifD-linker-NifK fusion polypeptide present in a corresponding plant cell lacking the exogenous polynucleotides encoding the one or more than one or all of NifW, NifX, NifY and NifZ fusion polypeptides of (i).
A plant cell comprising mitochondria and exogenous polynucleotides which encode at least 5, at least 6, at least 7, at least 8 or at least 9 Nif fusion polypeptides, wherein the exogenous polynucleotides each comprise a promoter which is operably linked to a nucleotide sequence which encodes one of the Nif fusion polypeptides and which expresses the nucleotide sequence in the plant cell, wherein each Nif fusion polypeptide independently comprises a mitochondrial targeting peptide (MTP), wherein the Nif fusion polypeptides comprise (i) NifH, NifS and NifU fusion polypeptides and optionally a NifM polypeptide, (ii) one or more than one or all of NifW, NifX, NifY, and NifZ fusion polypeptides and either (iii) a NifD fusion polypeptide and a NifK fusion polypeptide or (iv) a NifD-linker-NifK fusion polypeptide which comprises a NifD sequence having a C-terminus, an oligopeptide linker and a NifK sequence having a N-terminus, wherein the oligopeptide linker is translationally fused to the C-terminus of the NifD sequence and the N-terminus of the NifK sequence, wherein mitochondrial processing protease (MPP)-cleaved products of the NifS and NifU fusion polypeptides are at least partially soluble in mitochondria of a plant cell, wherein MPP-cleaved products of the NifW, NifX, NifY and NifZ fusion polypeptides, if present in the plant cell, are at least partially soluble in mitochondria of a plant cell, wherein MPP-cleaved products of the NifD and NifK fusion polypeptides of (iii), if present in the plant cell, are at least partially soluble in mitochondria of a plant cell, wherein a MPP-cleaved product of the NifD-linker-NifK fusion polypeptide of (iv), if present in the plant cell, is at least partially soluble in mitochondria of a plant cell, and wherein either the MPP-cleaved products of the NifD fusion polypeptide and NifK fusion polypeptide of iii) or the MPP-cleaved product of the NifD-linker-NifK fusion polypeptide of iv) are present in the plant cell as a complex with P-cluster.
A plant cell comprising mitochondria and exogenous polynucleotides which encode at least 2, at least 3 or 4 Anf fusion polypeptides, wherein the exogenous polynucleotides each comprise a promoter which is operably linked to a nucleotide sequence which encodes one of the Anf fusion polypeptides and which expresses the nucleotide sequence in the plant cell, wherein each Anf fusion polypeptide independently comprises a mitochondrial targeting peptide (MTP), wherein the Anf fusion polypeptides comprise (i) an AnfG fusion polypeptide or AnfG and AnfH fusion polypeptides, and either (ii) an AnfD fusion polypeptide and an AnfK fusion polypeptide or (iii) an AnfD-linker-AnfK fusion polypeptide which comprises an AnfD sequence having a C-terminus, an oligopeptide linker and an AnfK sequence having a N-terminus, wherein the oligopeptide linker is translationally fused to the C-terminus of the AnfD sequence and the N-terminus of the AnfK sequence, wherein mitochondrial processing protease (MPP)-cleaved products of at least the AnfG and AnfH fusion polypeptides if present in the plant cell are each at least partially soluble in mitochondria of a plant cell, wherein either MPP-cleaved products of the AnfD and AnfK fusion polypeptides of (ii) if present in the plant cell are at least partially soluble in mitochondria of a plant cell, or a MPP-cleaved product of the AnfD-linker-AnfK fusion polypeptide of (iii) if present in the plant cell is at least partially soluble in mitochondria of a plant cell, and wherein the MPP-cleaved products of the AnfD fusion polypeptide and the AnfK fusion polypeptide of ii) or the MPP-cleaved product of the AnfD-linker-AnfK fusion polypeptide of iii) if present in the plant cell form a protein complex in the plant cell with the MPP-cleaved product of the AnfG fusion polypeptide.
A plant cell comprising mitochondria and an exogenous polynucleotide which encodes a NifV polypeptide (NV), wherein the exogenous polynucleotide comprises a promoter which is operably linked to a nucleotide sequence which encodes the NV and which expresses said nucleotide sequence in the plant cell, wherein the NV produces homocitrate in the plant cell and is at least partially soluble in mitochondria of a plant cell, wherein the exogenous polynucleotide is preferably integrated into the nuclear genome of the plant cell and/or is expressed in the nucleus of the plant cell, and optionally wherein the NV comprises a mitochondrial targeting peptide (MTP).
A plant cell comprising an exogenous polynucleotide which encodes a NifD polypeptide (ND) which is (a) resistant to protease cleavage at a site within an amino acid sequence corresponding to amino acids 97-100 of SEQ ID NO:18, and/or (b) comprises an amino acid sequence other than RRNY (SEQ ID NO:101) at positions corresponding to amino acids 97-100 of SEQ ID NO:18, wherein the exogenous polynucleotide comprises a promoter which is operably linked to a nucleotide sequence which encodes the ND and which expresses said nucleotide sequence in the plant cell, and wherein the NifD polypeptide preferably comprises a MTP.
A plant cell comprising an exogenous polynucleotide which encodes a NifH fusion polypeptide (NH), wherein the exogenous polynucleotide comprises a promoter which is operably linked to a nucleotide sequence which encodes the NH and which expresses said nucleotide sequence in the plant cell, wherein the NH comprises a mitochondrial targeting peptide (MTP), wherein a MPP-cleaved product of the NH is at least partially soluble in mitochondria of a plant cell, and optionally wherein the exogenous polynucleotide is integrated into the nuclear genome of the plant cell and/or is expressed in the nucleus of the plant cell.
A NifD fusion polypeptide comprising a mitochondrial targeting peptide (MTP) translationally fused to a NifD polypeptide (ND), or a cleaved product thereof which comprises the ND and optionally a scar peptide, wherein the NifD fusion polypeptide or the cleaved product thereof is (a) resistant to protease cleavage at a site within an amino acid sequence corresponding to amino acids 97-100 of SEQ ID NO:18 and/or (b) comprises an amino acid sequence other than RRNY (SEQ ID NO:101) at positions corresponding to amino acids 97-100 of SEQ ID NO:18.
A NifK fusion polypeptide comprising a mitochondrial targeting peptide (MTP) translationally fused to a NifK polypeptide (NK), wherein the NifK fusion polypeptide or a cleaved product thereof is at least partially soluble in mitochondria of a plant cell when the NifK fusion polypeptide or the cleaved product thereof is produced in the plant cell.
An AnfD fusion polypeptide comprising a mitochondrial targeting peptide (MTP) and an AnfD polypeptide (AD), or a cleaved product thereof comprising the AD and optionally a scar peptide, preferably which is at least partially soluble in mitochondria of a plant cell when the AnfD fusion polypeptide or the cleaved product thereof is produced in the plant cell.
An AnfH fusion polypeptide comprising a mitochondrial targeting peptide (MTP) and an AnfH polypeptide (AH), or a cleaved product thereof comprising the AH and optionally a scar peptide, preferably which is at least partially soluble in mitochondria of a plant cell when the AnfH fusion polypeptide or the cleaved product thereof is produced in the plant cell.
An AnfG fusion polypeptide comprising a mitochondrial targeting peptide (MTP) and an AnfG polypeptide (AG), or a cleaved product thereof comprising the AG and optionally a scar peptide, preferably which is at least partially soluble in mitochondria of a plant cell when the AnfG fusion polypeptide or the cleaved product thereof is produced in the plant cell.
An AnfD-linker-AnfK fusion polypeptide or a cleaved product thereof, comprising an AnfD polypeptide (AD), an oligopeptide linker and an AnfK polypeptide (AK) which are translationally fused, wherein the AD comprises an N-terminus and a C-terminus, and the AK comprises an N-terminus, wherein the oligopeptide linker is translationally fused to the C-terminus of the AD and the N-terminus of the AK, preferably wherein the fusion polypeptide comprises a mitochondrial targeting peptide (MTP) or the cleaved product comprises a scar peptide translationally fused to the N-terminus of the AD.
An isolated or recombinant NifV polypeptide, or a NifV fusion polypeptide comprising a mitochondrial targeting peptide (MTP) translationally fused to a NifV polypeptide (NV), or a cleaved product thereof which comprises the NV and optionally a scar peptide, wherein the NifV polypeptide and/or the NifV fusion polypeptide and/or the cleaved product thereof is at least partially soluble in a plant cell when produced in the plant cell, preferably is at least partially soluble in mitochondria of the plant cell.
A NifH fusion polypeptide comprising a mitochondrial targeting peptide (MTP) translationally fused to a NifH polypeptide (NH), or a cleaved product thereof which comprises the NH and optionally a scar peptide, wherein the NifH fusion polypeptide and/or the cleaved product thereof is at least partially soluble in mitochondria of a plant cell.
A method of increasing the amount of a NifD, NifK or NifD-linker-NifK fusion polypeptide in a plant cell, the method comprising expressing one or more or all of NifW, NifX, NifY and NifZ fusion polypeptides in the plant cell, wherein each Nif fusion polypeptide independently comprises a mitochondrial targeting peptide (MTP), wherein the amount of the NifD, NifK or NifD-linker-NifK fusion polypeptide in the plant cell is increased relative to a corresponding plant cell not expressing one or more or all of the NifW, NifX, NifY and NifZ fusion polypeptides.
A method of increasing the amount of a NifY polypeptide in a plant cell, the method comprising expressing one or more or all of NifW, NifX and NifZ fusion polypeptides in the plant cell, wherein each Nif fusion polypeptide independently comprises a mitochondrial targeting peptide (MTP), wherein the amount of the NifY polypeptide in the plant cell is increased relative to a corresponding plant cell not expressing one or more or all of the NifW, NifX and NifZ fusion polypeptides.
Use of one or more polynucleotides encoding one or more or all of NifW, NifX and NifZ fusion polypeptides to increase the amount of a NifY polypeptide in a plant cell.
Dependencies are inferred from the claim text.