EP4771133A1 - Nif variants - Google Patents

Nif variants

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Publication number
EP4771133A1
EP4771133A1 EP24857521.9A EP24857521A EP4771133A1 EP 4771133 A1 EP4771133 A1 EP 4771133A1 EP 24857521 A EP24857521 A EP 24857521A EP 4771133 A1 EP4771133 A1 EP 4771133A1
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Prior art keywords
polypeptide
nifh
amino acid
modified
seq
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EP24857521.9A
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German (de)
French (fr)
Inventor
Christina Maria GREGG
Shoko Okada
Robert Silas Allen
Xueqin Wang
Jessica Kate BILYJ
Trevor Douglas RAPSON
Robert Charles De Feyter
Craig Christopher Wood
Barnabas GALL
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Commonwealth Scientific and Industrial Research Organisation
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Commonwealth Scientific and Industrial Research Organisation
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Priority claimed from AU2023902807A external-priority patent/AU2023902807A0/en
Application filed by Commonwealth Scientific and Industrial Research Organisation filed Critical Commonwealth Scientific and Industrial Research Organisation
Publication of EP4771133A1 publication Critical patent/EP4771133A1/en
Pending legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0095—Oxidoreductases (1.) acting on iron-sulfur proteins as donor (1.18)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/142—Amino acids; Derivatives thereof
    • A23K20/147—Polymeric derivatives, e.g. peptides or proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y118/00—Oxidoreductases acting on iron-sulfur proteins as donors (1.18)
    • C12Y118/06—Oxidoreductases acting on iron-sulfur proteins as donors (1.18) with dinitrogen as acceptor (1.18.6)
    • C12Y118/06001—Nitrogenase (1.18.6.1)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/07—Fusion polypeptide containing a localisation/targetting motif containing a mitochondrial localisation signal

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
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  • Polymers & Plastics (AREA)
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  • Food Science & Technology (AREA)
  • Animal Husbandry (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Botany (AREA)
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  • Physiology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

The present invention relates, in part, to modified NifH polypeptides and NifH fusion polypeptides with improved solubility.

Description

NIF VARIANTS
FIELD OF THE INVENTION
The present invention relates, in part, to modified NifH polypeptides and NifH fusion polypeptides with improved solubility.
BACKGROUND OF THE INVENTION
Diazotrophic bacteria produce ammonia from N2 gas via biological nitrogen fixation (BNF), catalysed by the enzyme complex, nitrogenase. Yet the demands of modern agriculture yr outstrip this source of fixed nitrogen, and consequently industrially-produced nitrogenous fertiliser is used extensively in agriculture (Smil, 2002). However, both fertiliser production and application are causes of pollution (Good and Beatty, 2011) and considered unsustainable (Rockstrom et al., 2009). The majority of fertilizer applied worldwide is not taken up by crops (Cui et al., 2013; de Bruijn, 2015), leading to fertilizer runoff, promotion of weeds and eutrophication of waterways (Good and Beatty, 2011). Resultant algal blooms reduce oxygen levels, causing environmental damage locally and offshore throughout coral reefs (De'ath et al., 2012; Glibert et al., 2014; Sutton et al., 2008). Furthermore, although over fertilization is a problem in many developed countries, in certain regions nitrogen availability limits crop yields (Mueller et al., 2012). The production of fertilizer itself requires substantial energy inputs, and costs an estimated $100 USD billon/yr.
Clearly strategies to reduce industrially-produced nitrogenous dependence are required. To this end, the notion of engineering plants capable of biological nitrogen fixation has long attracted considerable interest (Merrick and Dixon, 1984), and has been the focus of recent reviews (de Bruijn, 2015; Oldroyd and Dixon, 2014). Potential approaches include i) extending the symbiotic relationship of diazotrophs from legumes to cereals (Santi et al., 2013), ii) re-engineering endosymbiotic microorganisms to be capable of nitrogen fixation (Geddes et al., 2015), and iii) genetic engineering of nitrogenase into plant cells (Curatti and Rubio, 2014). All of these approaches are ambitious and speculative due to the technical difficulty.
Nitrogenase, the enzyme complex capable of biological nitrogen fixation in diazotrophic bacteria, requires a multigene assembly pathway for its biosynthesis and function, reviewed extensively (Hu and Ribbe, 2013; Rubio and Ludden, 2008; Seefeldt et al., 2009). The components of the canonical iron-molybdenum nitrogenase include the catalytic proteins designated NifD and NifK and the electron donor NifH. About 12 other proteins are involved in nitrogenase assembly in diazotrophic bacteria including in the maturation, scaffolding and co-factor insertion of the complex, specifically NifM, NifS, NifU, NifE, NifN, NifX, NifV, NifJ, NifY, NifF, NifZ and NifQ. Genetic lesions, complementation assays between diazotrophs to non-diazotrophic prokaryotes and phylogenetic analyses (Dos Santos et al., 2012; Temme et al., 2012; Wang et al., 2013) have led to a subset of Nif proteins (NifD, NifK, NifB, NifE and NifN) being considered as the core components, whilst others are thought to be required for optimised activity and are considered auxiliary. Specific biochemical conditions are also required for nitrogenase assembly and function. Foremost among these, nitrogenase is extremely oxygen sensitive (Robson and Postgate, 1980). Furthermore, large amounts of ATP, reductant, readily available Fe, Mo, S-adenosylmethionine and homocitrate are required for biosynthesis and function of the metalloprotein catalytic centre (Hu and Ribbe, 2013; Rubio and Ludden, 2008). All of these factors contribute to the technical difficulty of producing a functional nitrogenase complex in plant cells.
SUMMARY OF THE INVENTION
The present inventors have identified NifH variants, such as AnfH variants, with improved solubility in mitochondria of a plant cell.
Thus, in a first aspect the present invention provides a plant cell comprising a modified NifH polypeptide, wherein the modified NifH polypeptide comprises at least one amino acid substitution when compared to a corresponding wild-type NifH polypeptide, and wherein the modified NifH polypeptide is more soluble in mitochondria of the cell than the corresponding wild-type NifH polypeptide.
In an embodiment, the at least one amino acid substitution is at an amino acid position selected from the group consisting of amino acid positions: 2, 5, 7, 19, 23, 24, 26 to 35, 45, 48, 49, 51, 53, 54, 56 to 59, 61, 62, 64 to 74, 76 to 78, 80 to 84, 102, 105, 107, 111 to 114, 116 to 118, 121 to 124, 139, 145, 147, 149, 158, 165, 166, 168, 169, 171, 179, 182, 183, 188, 191, 193 to 197, 200 to 203, 205 to 211, 214, 216, 219, 223 to 226, 228 to 235, 237, 238, 241, 242, 244 to 246, 248, 249, 251 to 253, 257, 259 to 264, 266 to 271 and 273 to 275 with reference to SEQ ID NO: 37, or at a corresponding amino acid position in the modified NifH polypeptide, preferably a modified AnfH polypeptide, when its sequence is aligned with SEQ ID NO:37. Alternately, or in addition, in an embodiment, the at least one amino acid substitution is at an amino acid position selected from the group consisting of amino acid positions: 3, 6, 8, 20, 24, 25, 27 to 35, 45, 48, 49, 51, 53, 54, 56 to 59, 61, 62, 64 to 75, 77 to 79, 81 to 85, 103, 106, 108, 112 to 115, 117 to 119, 122 to 125, 140, 146, 148, 150, 159, 166, 167, 169, 170, 172, 180, 183, 184, 189, 192, 194 to 198, 201 to 204, 206 to 212, 215, 217, 220, 224 to 227, 229 to 236, 238, 239, 242, 243, 245 to 247, 249, 250, 252 to 254, 258, 260 to 265, 267 to 272 and 274 to 276 with reference to SEQ ID NO:39, or at a corresponding amino acid position in the modified NifH polypeptide when its sequence is aligned with SEQ ID NO:39.
In a further aspect the present invention provides a plant cell comprising a modified NifH polypeptide, wherein the modified NifH polypeptide, preferably a modified AnfH polypeptide, comprises at least one amino acid substitution when compared to a corresponding wild-type NifH polypeptide, and wherein the at least one amino acid substitution is at an amino acid position selected from (i) the group consisting of amino acid positions: 2, 5, 7, 19, 23, 24, 26 to 35, 45, 48, 49, 51, 53, 54, 56 to 59, 61, 62, 64 to 74, 76 to 78, 80 to 84, 102, 105, 107, 111 to 114, 116 to 118, 121 to 124, 139, 145, 147, 149, 158, 165, 166, 168, 169, 171, 179, 182, 183, 188, 191, 193 to 197, 200 to 203, 205 to 211, 214, 216, 219, 223 to 226, 228 to 235, 237, 238, 241, 242, 244 to 246, 248, 249, 251 to 253, 257, 259 to 264, 266 to 271 and 273 to 275 with reference to SEQ ID NO: 37, or at a corresponding amino acid position in the modified NifH polypeptide when its sequence is aligned with SEQ ID NO:37, and/or (ii) the group consisting of amino acid positions: 3, 6, 8, 20, 24, 25, 27 to 35, 45, 48, 49, 51, 53, 54, 56 to 59, 61, 62, 64 to 75, 77 to 79, 81 to 85, 103, 106, 108, 112 to 115, 117 to 119, 122 to 125, 140, 146, 148, 150, 159, 166, 167, 169, 170, 172, 180, 183, 184, 189, 192, 194 to 198, 201 to 204, 206 to 212, 215, 217, 220, 224 to 227, 229 to 236, 238, 239, 242, 243, 245 to 247, 249, 250, 252 to 254, 258, 260 to 265, 267 to 272 and 274 to 276 with reference to SEQ ID NO: 39, or at a corresponding amino acid position in the modified NifH polypeptide when its sequence is aligned with SEQ ID NO:39. In an embodiment of this aspect, the modified NifH polypeptide is more soluble in mitochondria of the cell than the corresponding wild-type NifH polypeptide.
In an embodiment of the above aspects, at least 15%, at least 20%, at least 35%, at least 40%, at least 45%, at least 50%, or 15% to 90%, 15% to 80%, 15% to 70% or 15% to 60%, of the modified NifH polypeptide, preferably the modified AnfH polypeptide, in mitochondria of the cell is soluble.
In an embodiment of the above aspects, at least 2-fold, preferably at least 3 -fold, at least 4-fold or at least 5-fold, or between 2-fold and 10-fold, more of the NifH polypeptide in mitochondria of the cell is soluble when compared to the corresponding wild-type NifH polypeptide.
In an embodiment of the above aspects, the modified NifH polypeptide has a lower free energy than the wild-type NifH polypeptide, and/or wherein the amino acid substitution reduces the free energy of the modified NifH polypeptide relative to the wild-type NifH polypeptide. In an embodiment of the above aspects, the modified NifH polypeptide with the substituted amino acid has a free energy which is reduced by at least 0.5, at least 1.0, at least 1.5, at least 2, at least 3, at least 4, at least 5, or between 2 and 6 units relative to a corresponding NifH polypeptide which is identical in amino acid sequence to the modified NifH polypeptide except for the substituted amino acid. In an embodiment, the change in free energy from the amino acid substitution is determined by the Rosetta energy function.
In an embodiment of the above aspects, the modified NifH polypeptide comprises at least one, preferably at least two or at least three, amino acid substitution(s) when compared to the corresponding wild-type NifH polypeptide, wherein each substituted amino acid reduces the free energy of the modified NifH polypeptide by at least 0.5, at least 1.0, at least 1.5, at least 2, at least 3, at least 4, at least 5 units, or between 2 and 6 units, and/or wherein the amino acid substitutions, in sum, reduce the free energy of the modified NifH polypeptide by at least 4.0, at least 5.0, at least 6.0, at least 7.0, at least 8.0, at least 9.0, at least 10.0, at least 12.0 units, or between 4.0 and 15.0, between 4.0 and 13.0, or between 4.0 and 12.0 units.
In an embodiment of the above aspects, the modified NifH polypeptide, preferably a modified AnfH polypeptide, comprises at least one amino acid substitution, or two or three amino acid substitutions, or four or more amino acid substitutions, selected from the group of amino acid substitutions listed in one or more of Tables 4, 5, 9, 10 or 11, or corresponding amino acid substitutions when the modified NifH polypeptide sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment of the above aspects, the modified NifH polypeptide, preferably a modified AnfH polypeptide, comprises at least one amino acid substitution, or preferably two or three amino acid substitutions, or four or more amino acid substitutions, at amino acid position(s) selected from the group consisting of amino acid positions 69, 168, 200, 201, 224, 228, 234, 241, 252 and 263 with reference to SEQ ID NO: 37, or at a corresponding amino acid position in the modified NifH polypeptide when its sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment of the above aspects, the at least one amino acid substitution, or preferably two or three amino acid substitutions, or four or more amino acid substitutions, are selected from the group consisting of 69N, 1681, 200A, 20 IK, 224R, either 2281 or 228V, either 234H or 234C, either 241R or 241 A, 252M and 263E, wherein the amino acid positions correspond to the amino acid sequence provided as SEQ ID NO: 37, or at a corresponding amino acid position in the modified NifH polypeptide, preferably a modified AnfH polypeptide, when its sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment of the above aspects, the at least one amino acid substitution, or preferably two or three amino acid substitutions, or four or more amino acid substitutions, are selected from the group consisting of positions corresponding to amino acids 69, 168, 200, 201, 224, 228, 234, 252 and 263 of SEQ ID NO:37, or at a corresponding amino acid position in the modified NifH polypeptide, preferably a modified AnfH polypeptide, when its sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment of the above aspects, the at least one amino acid substitution, or two or three amino acid substitutions, or four or more amino acid substitutions, are selected from the group consisting of 69N, 1681, 200A, 20 IK, 224R, either 2281 or 228V, either 234H or 234C, 252M and 263E, wherein the amino acid positions correspond to the amino acid sequence provided as SEQ ID NO: 37, or at a corresponding amino acid position in the modified NifH polypeptide, preferably a modified AnfH polypeptide, when its sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment of the above aspects, the modified NifH polypeptide, preferably a modified AnfH polypeptide, has one, two, three, four, five, six, seven, eighth, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 1 to 20, 1 to 15, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 2 to 20, 2 to 15, 2 to 11, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 or 3, 3 to 20, 3 to 15, 3 to 11, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 or 4, preferably 1 to 3, 1 to 4, or 1 to 5, more preferably 2 to 4 or 2 to 5, most preferably 3 to 5, amino acid substitution(s) when compared to the corresponding wild-type NifH polypeptide.
In an embodiment of the above aspects, the modified NifH polypeptide, preferably a modified AnfH polypeptide, comprises at least one amino acid substitution which is 228V with reference to SEQ ID NO:37, or the same amino acid substitution at a corresponding amino acid position when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment of the above aspects, the modified NifH polypeptide, preferably a modified AnfH polypeptide, comprises at least one amino acid substitution which is 2281 with reference to SEQ ID NO:37, or the same amino acid substitution at a corresponding amino acid position when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment of the above aspects, the modified NifH polypeptide, preferably a modified AnfH polypeptide, comprises at least one amino acid substitution which is 200A with reference to SEQ ID NO:37, or the same amino acid substitution at a corresponding amino acid position when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment of the above aspects, the modified NifH polypeptide, preferably a modified AnfH polypeptide, comprises at least one amino acid substitution which is 234H with reference to SEQ ID NO:37, or the same amino acid substitution at a corresponding amino acid position when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment of the above aspects, the modified NifH polypeptide, preferably a modified AnfH polypeptide, comprises at least two amino acid substitutions which are 200 A and 228V with reference to SEQ ID NO: 37, or the same amino acid substitutions at corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment of the above aspects, the modified NifH polypeptide, preferably a modified AnfH polypeptide, comprises at least two amino acid substitutions which are 200 A and 2281 with reference to SEQ ID NO: 37, or the same amino acid substitutions at corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment of the above aspects, the modified NifH polypeptide, preferably a modified AnfH polypeptide, comprises at least two amino acid substitutions which are 228V and 234H with reference to SEQ ID NO:37, or the same amino acid substitutions at corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment of the above aspects, the modified NifH polypeptide, preferably a modified AnfH polypeptide, comprises at least two amino acid substitutions which are 2281 and 234H with reference to SEQ ID NO:37, or the same amino acid substitutions at corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment of the above aspects, the modified NifH polypeptide, preferably a modified AnfH polypeptide, comprises at least three amino acid substitutions which are 200A, 228V and 234H with reference to SEQ ID NO:37, or the same amino acid substitutions at corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment of the above aspects, the modified NifH polypeptide, preferably a modified AnfH polypeptide, comprises at least three amino acid substitutions which are 200A, 2281 and 234H with reference to SEQ ID NO:37, or the same amino acid substitutions at corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment of the above aspects, the modified NifH polypeptide, preferably a modified AnfH polypeptide, comprises at least four amino acid substitutions which are 200A, 228V or 2281, 234H and 241R with reference to SEQ ID NO:37, or the same amino acid substitutions at corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment of the above aspects, the modified NifH polypeptide, preferably a modified AnfH polypeptide, comprises at least four amino acid substitutions which are 1681, 200A, 2281 or 228V, and 234H with reference to SEQ ID NO:37, or the same amino acid substitutions at corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment of the above aspects, the modified NifH polypeptide, preferably a modified AnfH polypeptide, comprises at least seven amino acid substitutions which are 69N, 1681, 200A, 228V or 2281, 234H, 252M and 263E with reference to SEQ ID NO:37, or the same amino acid substitutions at corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment of the above aspects, the modified NifH polypeptide, preferably a modified AnfH polypeptide, comprises at least eight amino acid substitutions which are 69N, 1681, 200A, 20 IK, 228V or 2281, 234H, 252M and 263E with reference to SEQ ID NO:37, or the same amino acid substitutions at corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment of the above aspects, the modified NifH polypeptide, preferably a modified AnfH polypeptide, comprises at least nine amino acid substitutions which are 69N, 1681, 200A, 201K, 224R, 2281 or 228V, 234H, 252M and 263E with reference to SEQ ID NO:37, or the same amino acid substitutions at corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment of the above aspects, the modified NifH polypeptide, preferably a modified AnfH polypeptide, comprises at least five amino acid substitutions which are 1681, 200A, 2281 or 228V, 234H and 241R with reference to SEQ ID NO:37, or the same amino acid substitutions at corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39. In an embodiment of the above aspects, the modified NifH polypeptide, preferably a modified AnfH polypeptide, comprises at least eight amino acid substitutions which are 69N, 1681, 200A, 228V or 2281, 234H, 241R, 252M and 263E with reference to SEQ ID NO:37, or the same amino acid substitutions at corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment of the above aspects, the modified NifH polypeptide, preferably a modified AnfH polypeptide, comprises at least nine amino acid substitutions which are 69N, 1681, 200A, 201K, 228V or 2281, 234H, 241R, 252M and 263E with reference to SEQ ID NO:37, or the same amino acid substitutions at corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment of the above aspects, the modified NifH polypeptide, preferably a modified AnfH polypeptide, comprises at least ten amino acid substitutions which are 69N, 1681, 200A, 20 IK, 224R, 2281 or 228V, 234H, 241R, 252M and 263E with reference to SEQ ID NO:37, or the same amino acid substitutions at corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment of the above aspects, the modified NifH polypeptide, preferably a modified AnfH polypeptide, comprises at least five amino acid substitutions which are 112L, 200A, 228V or 2281, 234H and 241R with reference to SEQ ID NO:37, or the same amino acid substitutions at corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment of the above aspects, the modified NifH polypeptide, preferably a modified AnfH polypeptide, comprises at least six amino acid substitutions which are 112L, 1681, 200A, 2281 or 228V, 234H and 241R with reference to SEQ ID NO:37, or the same amino acid substitutions at corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment of the above aspects, the modified NifH polypeptide, preferably a modified AnfH polypeptide, comprises at least nine amino acid substitutions which are 69N, 112L, 1681, 200A, 228V or 2281, 234H, 241R, 252M and 263E with reference to SEQ ID NO:37, or the same amino acid substitutions at corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment of the above aspects, the modified NifH polypeptide, preferably a modified AnfH polypeptide, comprises at least ten amino acid substitutions which are 69N, 112L, 1681, 200A, 201K, 228V or 2281, 234H, 241R, 252M and 263E with reference to SEQ ID NO:37, or the same amino acid substitutions at corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment of the above aspects, the modified NifH polypeptide, preferably a modified AnfH polypeptide, comprises at least eleven amino acid substitutions which are 69N, 112L, 1681, 200A, 201K, 224R, 2281 or 228V, 234H, 241R, 252M and 263E with reference to SEQ ID NO:37, or the same amino acid substitutions at corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
As the skilled person would appreciate, any and all combinations of the amino acid substitutions are contemplated. It would also be understood that the desired amino acid may already be present at any one or more of the indicated position(s) in a wild-type NifH polypeptide, preferably an AnfH polypeptide, and therefore does not need to be substituted, provided that the modified NifH polypeptide comprises at least one amino acid substitution relative to the corresponding wild-type NifH polypeptide.
Therefore, in embodiments, the modified NifH polypeptide, preferably a modified AnfH polypeptide, comprises at least one of the following sets of amino acids at the indicated positions, which amino acids are either present as a result of a substitution or already present in the corresponding wild-type NifH polypeptide, provided that at least one of the listed amino acids is present as a result of a substitution: i) 228V, ii) 2281, iii) 200A, or iv) 234H, v) 200A and 228V, vi) 200A and 2281, vii) 228V and 234H, viii) 2281 and 234H, ix) 200A, 228V and 234H, x) 200A, 2281 and 234H, xi) 200A, 228V or 2281, 234H and 241R, xii) 1681, 200A, 2281 or 228V and 234H, xiii) 69N, 1681, 200A, 228V or 2281, 234H, 252M and 263E, xiv) 69N, 1681, 200A, 20 IK, 228V or 2281, 234H, 252M and 263E, xv) 69N, 1681, 200A, 20 IK, 224R, 2281 or 228V, 234H, 252M and 263E, xvi) 1681, 200A, 2281 or 228V, 234H and 241R, xvii) 69N, 1681, 200A, 228V or 2281, 234H, 241R, 252M and 263E, xviii) 69N, 1681, 200A, 20 IK, 228V or 2281, 234H, 241R, 252M and 263E, xix) 69N, 1681, 200A, 20 IK, 224R, 2281 or 228V, 234H, 241R, 252M and 263E, xx) 112L, 200A, 228V or 2281, 234H and 241R, xxi) 112L, 1681, 200A, 2281 or 228V, 234H and 241R, xxii) 69N, 112L, 1681, 200A, 228V or 2281, 234H, 241R, 252M and 263E, xxiii) 69N, 112L, 1681, 200A, 201K, 228V or 2281, 234H, 241R, 252M and 263E, or xxiv) 69N, 112L, 1681, 200A, 201K, 224R, 2281 or 228V, 234H, 241R, 252M and 263E,
In a preferred embodiment of the above aspects, the modified NifH polypeptide, preferably a modified AnfH polypeptide, comprises amino acids 200A, 228V and 234H with reference to SEQ ID NO:37, or the same amino acids at the corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment of the above aspects, preferably in combination with one of the above-mentioned embodiments, the modified NifH polypeptide, preferably a modified AnfH polypeptide, comprises one or more or all of the following motifs: YGKGGIGKSTTXQN (SEQ ID NO:61), IXGCDPKAD (SEQ ID NO: 62), CXESGGPEPGVGCAGRG (SEQ ID NO:63), DVLGDVVCGGFAMP (SEQ ID NO:43), VXSGEMMAXYAANNI (SEQ ID NO:64), and CNSRXXD (motif VII, SEQ ID NO:65), preferably at least DVLGDVVCGGFAMP (SEQ ID NO:43), where each X independently represents any amino acid.
In an embodiment of the above aspects, preferably in combination with one of the above-mentioned embodiments, the modified NifH polypeptide, preferably a modified AnfH polypeptide, comprises one or more or all of the following motifs: YGKGGIGKSTTXQNT (SEQ ID NO:40), IHGCDPKAD (SEQ ID NO:41), CVESGGPEPGVGCAGRG (SEQ ID NO:42), DVLGDVVCGGFAMP (SEQ ID NO:43), VASGEMMAXYAANNI (SEQ ID NO:44), QSGVR (SEQ ID NO:45) and CNSRXVD (SEQ ID NO:46), preferably at least DVLGDVVCGGFAMP (SEQ ID NO:43), where each X independently represents any amino acid.
In an embodiment of the above aspects, preferably in combination with one of the above-mentioned embodiments, the modified NifH polypeptide, preferably a modified AnfH polypeptide, has 12, 13, 14, 15 or all of following amino acids: 4K, 22T, 37H, 52G, 60D, 63R, 108L, 109M, 142G, 151 A, 174Q, 189V, 198E, 199F, 222F and 2471, with reference to SEQ ID NO:37, or the same amino acids at the corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment of the above aspects, preferably in combination with one of the above-mentioned embodiments, the modified NifH polypeptide, preferably a modified AnfH polypeptide, has 130, 131, 132, 133, 134, 135, 136 or all 137 of the following amino acids: 3R, 4K, 6A, 8Y, 9G, 10K, 11G, 12G, 131, 14G, 15K, 16S, 17T, 18T, 20Q, 21N, 22T, 25A, 361, 37H, 38G, 39C, 40D, 41P, 42K, 43A, 44D, 46T, 47R, 50L, 52G, 55Q, 60D, 63R, 75V, 79G, 85C, 86V, 87E, 88S, 89G, 90G, 91P, 92E, 93P, 94G, 95V, 96G, 97C, 98A, 99G, 100R, 101G, 1031, 104T, 1061, 108L, 109M, 110E, 115Y, 119L, 120D, 125D, 126V, 127L, 128G, 129D, 130 V, 131V, 132C, 133G, 134G, 135F, 136A,
137M, 138P, MOR, 142G, 143K, 144A, 146E, 148Y, 150V, 151A, 152S, 153G, 154E,
155M, 156M, 157A, 159Y, 160A, 161A, 162N, 163N, 1641, 167G, 170K, 172A, 174Q, 175S, 176G, 177 V, 178R, 180G, 181G, 184C, 185N, 186S, 187R, 189 V, 190D, 192E,
198E, 199F, 204G, 212P, 213R, 215N, 217V, 218Q, 220A, 221E, 222F, 227V, 236Q,
239E, 240Y, 243L, 2471, 250N, 254V, 2551, 256P, 258P, 265E and 272G, with reference to SEQ ID NO: 37, or the same amino acids at the corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment of the above aspects, the modified NifH polypeptide, preferably a modified AnfH polypeptide, has one or both of the following amino acids: 141D and 173K, with reference to SEQ ID NO:37, or the same amino acids at the corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment of the above aspects, the modified NifH polypeptide, preferably a modified AnfH polypeptide, has an amino acid sequence which is at least 60% identical, preferably at least 70% identical or at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to the amino acid sequence provided as SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment of the above aspects, the modified NifH polypeptide is a modified AnfH polypeptide.
In an embodiment, the modified AnfH polypeptide has an amino acid sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to the amino acid sequence provided as SEQ ID NO:37. In an embodiment of the above aspects, the modified AnfH polypeptide comprises at least amino acids 2-275 of the amino acid sequence provided as SEQ ID NO:78, or comprises SEQ ID NO:78.
In an embodiment of the above aspects, the modified NifH polypeptide, preferably a modified AnfH polypeptide, comprises an Fe-S cluster.
In an embodiment of the above aspects, the modified NifH polypeptide is a cleavage product of a NifH fusion polypeptide, preferably an AnfH fusion polypeptide, which comprises a mitochondrial targeting peptide (MTP) translationally fused to the modified NifH polypeptide, wherein the MTP is preferably translationally fused at the N-terminus of the modified NifH polypeptide, wherein the modified NifH polypeptide is produced in the plant cell by protease cleavage of the NifH fusion polypeptide within or immediately adjacent to the MTP. In this embodiment, the plant cell comprises an exogenous polynucleotide which encodes the NifH fusion polypeptide, which is also referred to herein as the encoded polypeptide.
In an embodiment, therefore, the invention provides a plant cell comprising an exogenous polynucleotide which encodes a NifH fusion polypeptide, preferably an AnfH fusion polypeptide, wherein the exogenous polynucleotide is operably linked to a promoter which directs expression of the exogenous polynucleotide to produce the NifH fusion polypeptide (encoded polypeptide) in the plant cell, wherein the encoded polypeptide comprises an MTP translationally fused at the N-terminus of a modified NifH polypeptide, optionally with an intervening oligopeptide linker, wherein the modified NifH polypeptide, preferably modified AnfH polypeptide, is produced in the plant cell by protease cleavage of the NifH fusion polypeptide within or immediately adjacent to the MTP, and wherein the modified NifH polypeptide, preferably modified AnfH polypeptide, has increased solubility in the plant cell. Preferably, the modified NifH polypeptide comprises at least one of the sets of amino acids listed in the above- mentioned embodiments. Preferably, the exogenous polynucleotide is integrated into the genome of the plant cell.
In an embodiment of the above aspects, the NifH fusion polypeptide is cleaved within the MTP by mitochondrial processing protease (MPP) to produce the modified NifH polypeptide, wherein the modified NifH polypeptide comprises either (i) at its N- terminal end, a C-terminal peptide from the MTP (scar peptide), or (ii) does not comprise a C-terminal peptide from the MTP.
In an embodiment of the above aspects, the modified NifH polypeptide comprises two NifH polypeptides which are covalently linked by an oligopeptide linker in the N- terminal to C-terminal order NifH::linker::NifH, optionally further comprising at its N- terminal end, a C-terminal peptide (scar peptide) from a mitochondrial targeting peptide (MTP), optionally with an intervening oligopeptide linker between the scar peptide and the first NifH polypeptide.
In an embodiment of the above aspects, the modified NifH polypeptide is a modified AnfH polypeptide which comprises two modified AnfH polypeptides which are covalently linked by an oligopeptide linker in the N-terminal to C-terminal order AnfH: dinker: AnfH, optionally further comprising at its N-terminal end, a C-terminal peptide from a mitochondrial targeting peptide (MTP), optionally with an intervening oligopeptide linker between the MTP and the first AnfH polypeptide.
In an embodiment of the above aspects, the linker between the two NifH or AnfH polypeptides has a length of 10-50 residues, preferably 16-50 residues in length or 20-35 residues in length, more preferably about 25 or about 30 residues in length, or most preferably is 25 or 30 residues in length. In an embodiment, the linker between the MTP or scar peptide and the first NifH/ AnfH polypeptide is less than 20 amino acids in length, for example only two amino acids in length, preferably a GlyGly di-peptide.
In an embodiment of the above aspects, the modified NifH polypeptide is capable, in combination with NifD and NifK polypeptides, or in combination with AnfD, AnfK and AnfG polypeptides, of reducing (i) N2 gas to produce ammonia and/or (ii) acetylene to ethylene, preferably both (i) and (ii). In this context, the capability may be shown for the polypeptide, when isolated from the plant cell, without in vitro treatment with a NifU polypeptide having Fe-S clusters, or with such treatment, or both.
In a preferred embodiment of the above aspects and embodiments, the modified NifH polypeptide is a modified AnfH polypeptide.
In an embodiment of the above aspects, the plant cell further comprises exogenous polynucleotides encoding (a) a NifM polypeptide, a NifS polypeptide, a NifU polypeptide and either (i) a NifD and a NifK polypeptide or (ii) a NifD-NifK fusion polypeptide, or (b) a NifS polypeptide, a NifU polypeptide, and AnfG polypeptide and either (iii) a AnfD and a AnfK polypeptide, or (iv) a AnfD-AnfK fusion polypeptide. In a preferred embodiment, the plant cell further comprises a NifB polypeptide, a NifF polypeptide, a NifI polypeptide and a NifV polypeptide, and optionally a FdxN polypeptide. In a most preferred embodiment, at least some of each of the polypeptides is soluble in mitochondria of the plant cell.
In an embodiment, the plant cell further comprises an exogenous polynucleotide encoding a NifM polypeptide.
In a further aspect, the present invention provides a plant cell comprising a NifH fusion polypeptide which comprises two NifH polypeptides, preferably two AnfH polypeptides, which are covalently linked by an oligopeptide linker in the N-terminal to C-terminal order NifH: linker: :NifH, preferably in the order AnfH: linker: AnfH, optionally further comprising at its N-terminal end, a C-terminal peptide from a mitochondrial targeting peptide (MTP).
In an embodiment, the two NifH polypeptides, preferably the two AnfH polypeptides, are the same or where they differ only by the presence of a methionine at amino acid position 1 of one of the NifH polypeptides.
In an embodiment, the NifH fusion polypeptide, preferably the AnfH fusion polypeptide, is more soluble in mitochondria of the cell than a corresponding NifH or AnfH polypeptide which has only a single NifH or AnfH polypeptide.
In an embodiment, the two NifH polypeptides, preferably the two AnfH polypeptides, are each at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to an amino acid sequence provided as SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment, the NifH fusion polypeptide is a cleavage product of an encoded polypeptide which comprises a mitochondrial targeting peptide (MTP) translationally fused to the NifH: linker: :NifH polypeptide, wherein the MTP is preferably translationally fused at the N-terminus of a NifH polypeptide, wherein the NifH fusion polypeptide is produced in the plant cell by protease cleavage of the encoded polypeptide within or immediately adjacent to the MTP, wherein NifH fusion polypeptide comprises either (i) at its N-terminal end, a C-terminal peptide from the MTP, or (ii) does not comprise a C-terminal peptide from the MTP .
In an embodiment of the above aspects, the modified NifH polypeptide or the NifH fusion polypeptide is a cleavage product of a polypeptide encoded by an exogenous polynucleotide in the plant cell, i.e. a precursor polypeptide comprising the modified NifH polypeptide or the NifH fusion polypeptide, wherein the encoded polypeptide comprises a mitochondrial targeting peptide (MTP) translationally fused to a modified NifH polypeptide or the NifH: linker: :NifH polypeptide, wherein the MTP is preferably translationally fused at the N-terminus of the modified NifH polypeptide or the NifH: linker: :NifH polypeptide, wherein the modified NifH polypeptide or the NifH fusion polypeptide is produced in the plant cell by protease cleavage of the encoded polypeptide within or immediately adjacent to the MTP.
In an embodiment, therefore, the invention provides a plant cell comprising an exogenous polynucleotide which encodes a NifH fusion polypeptide, preferably an AnfH fusion polypeptide, wherein the exogenous polynucleotide is operably linked to a promoter which directs expression of the exogenous polynucleotide in the plant cell, wherein the encoded polypeptide comprises a MTP translationally fused at the N- terminus of a NifH: inker: NifH polypeptide, preferably a modified NifH: linker: :modified NifH polypeptide comprising at least one amino acid substitution in each of the NifH sequences as described in the embodiments above, optionally with an intervening oligopeptide linker between the MTP and the first NifH sequence, wherein the NifH fusion polypeptide, preferably AnfH fusion polypeptide, is produced in the plant cell by protease cleavage of the encoded polypeptide within or immediately adjacent to the MTP, and wherein the cleavage product NifH fusion polypeptide has increased solubility in the plant cell. In the case where the protease cleaves within the MTP sequence, the NifH fusion polypeptide has the structure: scar: NifH: linker: NifH, or scar:: AnfH: linker:: AnfH, optionally with an intervening oligopeptide linker between the scar sequence and the first NifH/ AnfH sequence. Preferably, both NifH sequences or both AnfH sequences comprise at least one of the sets of amino acids listed in the above-mentioned embodiments i.e. both are modified NifH/ AnfH sequences.
In an embodiment, the exogenous polypeptide is integrated into the genome, preferably into the nuclear genome, of the plant cell, and/or wherein the modified NifH polypeptide is a modified AnfH polypeptide and/or the NifH fusion polypeptide is an AnfH fusion polypeptide.
In an embodiment, the modified NifH polypeptide, NifH fusion polypeptide or encoded polypeptide is capable of transferring electrons to NifDK to reduce acetylene and/or N2 gas. In an embodiment, the modified NifH polypeptide, NifH fusion polypeptide or encoded polypeptide is capable of transferring electrons to NifDK to reduce acetylene and/or N2 gas in a plant cell.
Furthermore provided is a modified NifH polypeptide as defined herein.
Also provided is a NifH fusion polypeptide as defined herein.
In addition, provided is an encoded polypeptide as defined herein.
In an embodiment, the MTP of the encoded polypeptide is translationally fused at the N-terminus of the modified NifH polypeptide or the NifH: linker: NifH polypeptide.
In an embodiment, the modified NifH polypeptide, NifH fusion polypeptide or encoded polypeptide is a modified AnfH polypeptide, an AnfH fusion polypeptide or an encoded polypeptide which comprises one or two AnfH polypeptides, respectively.
In an embodiment, the encoded polypeptide comprises an AnfH polypeptide and a mitochondrial targeting peptide (MTP) which is translationally fused to the AnfH polypeptide. In an embodiment, the MTP is translationally fused at the N-terminus of the AnfH polypeptide. In another aspect the present invention provides an exogenous polynucleotide, comprising a promoter operably linked to a nucleotide sequence which encodes the modified NifH polypeptide of the invention, a NifH fusion polypeptide of the invention, or an encoded polypeptide of the invention, wherein the promoter directs expression of the nucleotide sequence in a cell, preferably a plant cell.
In an embodiment, the modified NifH polypeptide is a modified AnfH polypeptide.
In an embodiment, the NifH fusion polypeptide is an AnfH fusion polypeptide.
In an embodiment, the encoded polypeptide is an AnfH polypeptide.
In an embodiment, the exogenous polynucleotide is integrated into the genome of the cell, preferably into the nuclear genome of a plant cell.
In an embodiment, the protein coding region of the polynucleotide has been codon-modified for expression in a plant cell.
In a further aspect, the present invention provides a vector comprising the exogenous polynucleotide of the invention.
In another aspect, the present invention provides a transgenic plant or part thereof, comprising one or more of a plant cell of the invention, a polypeptide of the invention or a polynucleotide of the invention, or preferably which is transgenic for an exogenous polynucleotide of the invention.
In an embodiment, the modified NifH polypeptide, NifH fusion polypeptide or encoded polypeptide is a modified AnfH polypeptide, an AnfH fusion polypeptide or an encoded polypeptide which comprises one or two AnfH polypeptides, respectively.
In an embodiment, the part is a transgenic seed.
In an embodiment, the plant is a cereal plant or part thereof. Examples include, but are not limited to, a wheat, rice, maize, triticale, oat or barley plant.
In a further aspect, the present invention provides a method of selecting a modified NifH polypeptide, preferably a modified AnfH polypeptide, or a NifH fusion polypeptide, preferably an AnfH fusion polypeptide, of the invention, the method comprising i) expressing an exogenous polynucleotide of the invention in a plant cell, ii) extracting proteins comprising the modified NifH polypeptide or NifH fusion polypeptide from the mitochondria of the plant cell, iii) determining the level of solubility of the modified NifH polypeptide or NifH fusion polypeptide in the extracted proteins, and iv) selecting the modified NifH polypeptide or NifH fusion polypeptide, wherein at least 15%, preferably at least 50%, of the modified NifH polypeptide or NifH fusion polypeptide in the cell is soluble.
In an embodiment of the above aspects, the modified NifH has one or more of the features of the modified NifH polypeptide as defined herein, or the NifH fusion polypeptide has one or more of the features of the NifH fusion polypeptide as defined herein. In an embodiment, the NifH fusion polypeptide comprises two NifH polypeptides, preferably two AnfH polypeptides, which are covalently linked by an oligopeptide linker in the N-terminal to C-terminal order NifH: : linker ::NifH, preferably in the order AnfH: dinker: AnfH, optionally further comprising at its N-terminal end, a C-terminal peptide from a mitochondrial targeting peptide (MTP).
In an embodiment, the modified NifH polypeptide has at least one amino acid substitution which confers onto the modified NifH polypeptide a lower free energy when compared to a corresponding NifH polypeptide which lacks the at least one amino acid substitution.
In a further aspect, the present invention provides a method of producing a modified NifH polypeptide, preferably a modified AnfH polypeptide, or a NifH fusion polypeptide, preferably an AnfH fusion polypeptide, of the invention, the method comprising expressing an exogenous polynucleotide of the invention in a plant cell or a transgenic plant.
In a further aspect, the present invention provides a method of selecting a plant cell which produces a modified NifH polypeptide, preferably a modified AnfH polypeptide, or a NifH fusion polypeptide, preferably an AnfH fusion polypeptide, of the invention, the method comprising i) expressing an exogenous polynucleotide of the invention in a plant cell, ii) determining whether the modified NifH polypeptide or NifH fusion polypeptide is produced at a desired level and/or has a desired activity, iii) selecting the plant cell on the basis of the results of step ii), iv) optionally producing a transgenic plant from the selected plant cell, and v) optionally producing transgenic progeny plants and/or transgenic seed from the transgenic plant.
In a further aspect, the present invention provides a method of selecting a plant which produces a modified NifH polypeptide, preferably a modified AnfH polypeptide, or a NifH fusion polypeptide, preferably an AnfH fusion polypeptide, of the invention, the method comprising i) expressing an exogenous polynucleotide of the invention in a plant or in multiple plants, ii) determining whether the modified NifH polypeptide or NifH fusion polypeptide is produced at a desired level and/or has a desired activity in the plant or multiple plants, iii) selecting a plant from step ii) which produces the modified NifH polypeptide or NifH fusion polypeptide at a desired level and/or has a desired activity in the plant or a part thereof, and iv) optionally producing transgenic progeny plants and/or transgenic seed from the transgenic plant.
In an embodiment of the above aspects, at least 15%, preferably at least 50%, of the modified NifH polypeptide or NifH fusion polypeptide in the cell or plant or part thereof is soluble.
In an embodiment of the above aspects, the modified NifH polypeptide or NifH fusion polypeptide comprises an MTP, preferably at the N-terminus of the polypeptide, for localisation to mitochondria of the plant cell or plant.
In an embodiment of the above aspects, the NifH fusion polypeptide comprises two NifH polypeptides, preferably two AnfH polypeptides, which are covalently linked by an oligopeptide linker in the N-terminal to C-terminal order NifH::linker::NifH, preferably in the order AnfH: dinker: AnfH, optionally further comprising at its N- terminal end, a C-terminal peptide from a mitochondrial targeting peptide (MTP). In an embodiment, the linker between the two NifH or AnfH polypeptides has a length of 10- 50 residues, preferably 16-50 residues in length or 20-35 residues in length, more preferably about 25 or about 30 residues in length, or most preferably is 25 or 30 residues in length.
In an embodiment of the above aspects, the exogenous polynucleotide is integrated into the genome of the plant cell or plants, preferably into the nuclear genome of the plant cell or plant.
Also provided is the use of an exogenous polynucleotide of the invention, and/or a vector of the invention, for producing a transgenic plant cell.
Additionally, provided is a method of producing a transgenic plant, the method comprising the steps of i) introducing one or more exogenous polynucleotides of the invention, and/or a vector of the invention, into a plant cell, ii) from the cell of step i), regenerating a transgenic plant of the invention, and iii) optionally, producing transgenic seed and/or progeny plants from the transgenic plant regenerated in step ii).
In a further aspect, the present invention provides a method of producing transgenic seed, comprising i) harvesting seed from a transgenic plant of the invention, and/or ii) harvesting seed from one or more transgenic progeny plants produced by a method of the invention.
In a further aspect, the present invention provides a method of producing flour, wholemeal, starch, oil, seedmeal or other product obtained from seed, the method comprising extracting flour, wholemeal, starch, oil or other product, or producing the seedmeal, from the seed of the invention.
In a further aspect, the present invention provides a product produced from the transgenic plant or part thereof of the invention such as seed of the invention, wherein the product comprises one or more or all of a modified NifH polypeptide of the invention, a NifH fusion polypeptide of the invention, an encoded polypeptide of the invention, and an exogenous polynucleotide of the invention.
In a further aspect, the present invention provides a method of preparing a food product, the method comprising mixing seed of the invention, or flour, wholemeal, starch, oil or other product from the seed, with another food ingredient.
In a further aspect, the present invention provides a process of feeding an animal, comprising providing to the animal the plant or part thereof of the invention such as seed of the invention, or the product of the invention.
Any embodiment herein shall be taken to apply mutatis mutandis to any other embodiment unless specifically stated otherwise.
The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.
Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.
The invention is hereinafter described by way of the following non-limiting Examples and with reference to the accompanying figures. BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
Figure 1. AlphaFold prediction of the tertiary structure of wild-type vinelandii NifU protein, showing the N-terminal, central and C-terminal domains.
Figure 2. Structural model of the FeFe nitrogenase enzyme. The polypeptide subunits forming the AnfDKGH complex are labelled with their Anf letter. Members of polypeptide pairs are shown, for example, as K and K’ . A part of AnfD that appears to reach across AnfEC to AnfD is a section for which there was no corresponding structure over which to construct a model, so is possibly not an accurate depiction of these residues, but has been included for completeness.
Figure 3. Western blot analysis of HA-tagged AnfH fusion polypeptides produced in plant cells and targeted to the mitochondrial matrix. The lanes marked T were loaded with total protein extracts, whereas those marked S were loaded with soluble protein extracts from the same infiltrations. WT refers to the wild-type AnfH fusion polypeptide, and the A labels correspond to variants V1-V5. The upper panel shows the signal after probing for HA-tagged polypeptides, the lower panel shows the staining of the same gel with Coomassie blue for total protein.
Figure 4. Coomassie stained gel and Western blot analysis from different steps of the purification process of the scar: :TS: :AnfH variant VI fusion polypeptide after targeting to plant mitochondria. The samples were (left to right): Protein size markers, total protein extract, the supernatant loaded onto the column, the pellet post-centrifugation of the total sample, the flowthrough from the column and the eluted protein. The Western blot shows the TwinStrep-tagged AnfH-Vl polypeptide in those fractions, arrowed at the position indicated (AnfH) between 33 and 40 kDa.
Figure 5. Upper panel: A scatter plot of the log-likelihood of amino acid substitutions in the AnfH polypeptide, for residues that are non-conserved and not already the highest log-likelihood substitution at the wild-type position. On the x-axis is the change in Rosetta energy units from wild type to substituted AnfH and on the y-axis is the loglikelihood of the substitution. All potential substitutions are shown as circles. Of the potential substitutions, those selected by PROSS across all five variants from Table 4 are shown with triangles. Lower panel: A scatter plot of the likely stabilising substitutions, the top-left hand quadrant of the upper panel, where A Rosetta energy units from wildtype to single substituted AnfH is negative, and the probability of substitution is positive. Potential substitutions are shown as circles, and the selected substitutions for variant VI are shown as triangles.
Figure 6. Upper panel: Western blot and Coomassie stained gel of total (T) and soluble (S) extracts of wild-type and variant AnfH fusion polypeptides after expression in N. benthamiana leaves, for two of the biological repeats. The lanes are labelled for the construct encoding the fusion polypeptide as follows: SN675 (WT), SN676 (A5), SN710 (A3R), SN708 (A4) and SN709 (A3H). Lower panel: a box-and-whisker plot of the integrated signal intensity of the bands in the Western blot above.
Figure 7. Coomassie stained gel and Western blot analysis of samples from different steps of the purification process of the scar: :TS: AnfH wild-type (A), variant V7 from SN709 (B) and variant V8 from SN710 (C) fusion polypeptides after targeting to plant mitochondria. The samples were (left to right): total protein extract (T), the pellet postcentrifugation of the total sample (P), the supernatant loaded onto the column (S), the flowthrough from the column (FT) and the eluted protein (E). The scar::TS::AnfH polypeptide in those fractions is labelled. The position of molecular weight markers are shown (kDa).
Figure 8: Average acetylene reduction (A and B) and delta-15N values for N2 reduction (C) activities of AnfH-V7 and -V8 polypeptides isolated from plant mitochondria, when combined in vitro with NifDK from A. vinelandii, without or with in vitro treatment with NifU polypeptide purified from E. coli. Symbols show data for replicates and bars show the averages across multiple experiments. A. The AnfH proteins were assayed ‘as isolated’ without treatment with NifU; B. Acetylene reduction activity of wild-type AnfH and AnfH variants isolated from N. benthamiana after treatment with NifU. C. Delta-15N values for N2 reduction assays of isolated wild-type AnfH and AnfH-V7 and -V8 polypeptides when combined in vitro with purified AnfDKG, after treatment with NifU. For wildtype AnfH, only one experiment was carried out due to the limited availability of isolated polypeptide. Labels: Av AnfH refers to the presence of purified, wild-type A. vinelandii AnfH, AvNifDK refers to purified, wild-type A. vinelandii NifD, and AnfDKG refers to purified, wild-type vinelandii AnfDKG, all used in positive control reactions; EcNifU refers to the TS::NifU protein purified from E. coli, which had been re-loaded with Fe-S clusters prior to use, added in the ratios indicated; NbAnfH-wt, NbAnfH-V7 and -V8 refer to the wild-type, V7 and V8 AnfH polypeptides, respectively, isolated from N benthamiana mitochondria and added in the ratios as indicated. Figure 9: Comparison of the structure of methyl viologen (MV, a)) and a sulfonated derivative (S2V, b)).
Figure 10: Time-course of an in vitro S2V-based colourimetric assay to measure the activity of combinations of bacterial components of purified AvNifH, AvAnfH, AvNifD- NifK (NifDK) and AvAnfD-AnfK-AnfG (AnfDKG). The ratio of AvNifH/AvNifDK was maintained at 20: 1, the ratio of AvAnfH/ Av AnfDKG at 30: 1 and the ratio of AvAnfH/AvNifDK at 30: 1.
Figure 11. Delta-15N of triplicate plant samples as determined by IRMS. A. Increasing amounts of non-purified 15N2 gas were added to plants in sealed containers at the indicated percentages and sampled after 24 hr in the dark. B. Non-purified or purified 15N2 gas was added to leaf disks in sealed containers and sampled after 24 hr, before IRMS. C. Leaves were infiltrated (P samples) with live A. vinelandii cells or infiltrated with non-nitrogen fixing Agrobacterium (A samples) and analysed by IRMS after 24 hr in the dark.
Figure 12. DNA modules used for construction of expression vectors, indicating the order of elements for A. Non-replicating vector with single transcription terminator (Tm), B. Non-replicating vector with hybrid transcription terminator (TTm), and C. Replicative vector with GV replication regions. The MTP, protein coding region (CDS) and epitope TAG sequences are selected as desired. Restriction enzyme Bsal cuts just within the ends of the modules to provide compatible ends for ligation into a T-DNA of a binary vector to provide the genetic construct for introduction into plant cells.
Figure 13. Western blot and Coomassie-stained gel of Nif fusion polypeptides expressed from a standard non-replicative vector (C), a GV-based replicative vector (GVc or GVt). The genetic constructs encoded a MTP::NifD::HA, MTP::HA:NifH, MTP::NifU::HA (SN466, SN499, SN500) or MTP::GFP::HA (SN493) or MTP::GFP (pRAl) fusion polypeptide as indicated. Molecular weights for polypeptides are shown for the Western blot (kDa). The lower left and right panels show gels after Coomassie staining. The bands for the NifD and NifH fusion polypeptides expressed from the GVc and GVt vectors are boxed. The position of the dominant protein band for rubisco is arrowed. Figure 14. Gel electrophoresis and Western blot of a NifH: linker: :NifH fusion polypeptide expressed from a standard non-replicative vector with single terminator (SN655), a non-replicative vector using a hybrid terminator (TTm, SN656) and a GV- based replicative vector (SN657). Each genetic construct was co-infiltrated with a construct (SN360) encoding a MTP::NifM fusion polypeptide. The extracts from N. benthamiana leaves after 4 days were processed for total protein (T), soluble protein (S) and insoluble protein (I) as described herein. The lower inset panel shows the gel after Coomassie staining and the outer lanes show molecular weight marker proteins.
Figure 15. Analysis by Western blot (upper panel) of the solubility of scar: NifH: linker: :NifH dimer fusion polypeptide after production in plant cells and localisation to the mitochondria. A genetic construct (SN283) to produce the MTP::NifH: linker: :NifH::HA dimer polypeptide (SEQ ID NO: 103) was introduced into N benthamiana leaf cells without constructs to express accessory proteins (lanes 1-3, HH only), or with MTP::GFP as a control (lanes 4-6), or with MTP::NifM (lanes 7-9). Lanes marked T were from the total protein extracts, lanes marked S from the soluble protein extracts, and lanes marked I from the insoluble protein extracts. Lane 7 shows polypeptide size markers (kDa). The Western blot was probed with anti-HA antibody. The lower panel shows the Coomassie stained gel as a control for protein loading in each lane.
Figure 16. Analysis by Western blot (upper panel) of the processing in plant mitochondria of MTP: ifH: linker: ifH dimer fusion polypeptide, probed with anti- HA antibody. Genetic construct (SN283) encoding the MTP::KoNifH: linker: :KoNifH::HA dimer polypeptide (SEQ ID NO: 103), based on the K. oxytoca NifH sequence, was introduced into N. benthamiana leaf cells with MTP- KoNifM. Left-hand lane shows polypeptide size markers (kDa). Lane 1, 35S-pl9 only; 2, MTP-FAy51 ::KoNifH: linker: :KoNifH::HA (SN283); 3, non-processed alaMTP- FAy51::KoNifH: linker: :KoNifH::HA (SN807); 4, 6xHis::KoNifH: linker::
KoNifH::HA (SN303); 5, MTP-FAy51::HA::KoNifH::linker(TS)::KoNifH (SN656); 6, non-processed alaMTP-FAy51HA::KoNifH::linker(TS)::KoNifH (SN808). Open triangle denotes bands corresponding to unprocessed MTP::NifH: linker: :NifH, solid triangle denotes bands corresponding to MPP-processed scar: :NifH: linker: :NifH. The lower panel shows the Coomassie stained gel as a control for protein loading in each lane. Lanes 1 and 2 had less protein loaded than lanes 3-6. Figure 17. Analysis by Western blot (upper panel) of the solubility of scar: :AvNifH: linker: :NifH dimer fusion polypeptide after production in plant cells and localisation to the mitochondria. A genetic construct (SN678) to produce the MTP::NifH: linker: :NifH dimer polypeptide (SEQ ID NO: 109), based on the A. vinelandii NifH sequence, was introduced into N. benthamiana leaf cells without NifM (lanes 2-4, -AvNifM), or with MTP::NifM (lanes 5-7) from SN605. Lanes marked T were from the total protein extracts, lanes marked S from the soluble protein extracts, and lanes marked I from the insoluble protein extracts. Lane 1 shows polypeptide size markers (kDa). The lower panel shows the Coomassie stained gel as a control for protein loading in each lane.
Figure 18. Amount of ethylene produced in an in vitro acetylene reduction assay (ARA) when NifD and NifK from A. vinelandii were combined with isolated scar::TS::KoNifH::linker::KoNifH produced from SN638 (bars 1 and 2) or SN650 (bars 3-5) or scar::HA::KoNifH::linker(TS)::KoNifH produced from SN656 (bars 6 and 7), in each case isolated from mitochondria of plant cells. The constructs had been coexpressed with genetic constructs, as indicated in the lower panel: encoding MTP::KoNifM (KoNifM), MTP::AvNifS and MTP::AvNifU (AvNifSU), MTP::AvFdxN::HA (AvFdxN), and MTP::KoNifD::linker(HA)::NifK (KoNifD::HA::K). Some leaves were harvested after a growth period in the dark (dark harvest). Fe and cysteine were added to some isolation buffers. The ratio of the amounts of NifH dimer: AvNifDK proteins in the assay are indicated.
Figure 19. A dimeric NifH protein, scar: :HA::NifH: linker (TS)::NifH, expressed and isolated from plant mitochondria is functional when tested for acetylene reduction in vitro with purified bacterial NifD and NifK (AvNifDK). The graph shows the amount of ethylene produced by bacterial NifD and NifK combined with bacterial NifH (AvNifH- AnNifDK) as the positive control for fully functional NifH, bacterial NifD and NifK without bacterial NifH (AnNifDK only) as the negative control, or plant-expressed scar::HA::NifH::linker(TS)::NifH which was produced in the presence of NifM (KoHH_KoM) or additionally in the presence of NifM, NifS and NifU (KoHH KoM AvSU). The three right-hand bars show ethylene produced after treatment of NifH polypeptides in vitro with Fe-S cluster-loaded NifU protein purified from E. coh. either the scar::HA::NifH::linker(TS)::NifH protein or apoNifH from A. vinelandii as a positive control for the Fe-S re-constitution. Each bar represents a biological replicate. Figure 20. Analysis by Western blot (left panels) or Coomassie staining (right panels) of the solubility of scar: :AnfH-V7: linker: :AnfH-V7 dimer fusion polypeptide after production in plant cells and localisation to the mitochondria. The genetic constructs SN775 (e35S lanes), SN776 (TTm) and SN777 (GV) having three different expression systems were expressed in plant leaves to produce the scar: :AnfH-V7: linker: :AnfH-V7 dimer polypeptide. Protein extracts from the leaves were run on the gels, for total protein extracts (T), soluble protein extracts (S) and insoluble protein preparations (I). Lanes 1 (upper) or 4 (lower) on the Western blots shows polypeptide size markers (kDa). The Western blot was probed with anti-Strep antibody. The righthand panels show the Coomassie stained gels as a control for protein loading in each lane.
Figure 21. Western blot and Coomassie-stained gel of samples from different steps of the affinity purification process of the scar::AnfH-V7::linker(TS)::AnfH-V7 polypeptide produced from SN776 after targeting to plant mitochondria. The samples were from (left to right): total protein extract (T), the pellet post-centrifugation of the total sample (P), the supernatant loaded onto the column (I), the flowthrough from the column (FT) and the eluted protein (E). The position of molecular weight markers are shown (kDa).
Figure 22. Upper panel: In vitro ethylene production from acetylene by scar::AnfH- V7::linker(TS)::AnfH-V7 polypeptide produced from SN776 after targeting to plant mitochondria, in combination with NifDK from A. vinelandii. Lower panel: delta-15N in 15N2 reduction assays for the same V7 polypeptide in combination with AnfDKG from A. vinelandii. Lanes marked with AvNifDK had purified, bacterial AvNifDK added; lanes marked with Av AnfDKG had purified, bacterial Av AnfDKG added; lanes marked with AvAnfH had purified, bacterial AvAnfH added as a positive control at the 20: 1 or 40: 1 ratio of AvAnfH: AvNifDK/ Av AnfDKG; lanes marked with SN776AvNifDK/AvAnfDKG had the plant produced and isolated AnfH-V7 dimer polypeptide added without NifU treatment in combination with the purified, bacterial AvNifDK or Av AnfDKG at the ratio of 20: 1 or 10: 1; and the lanes marked SN776AvNifDKU/AvAnfDKGU had the isolated AnfH-V7 dimer polypeptide added after NifU treatment in combination with the purified, bacterial AvNifDK or Av AnfDKG at the ratio of 20: 1 or 10: 1.
Figure 23. Amino acid sequence variation between wild-type NifH sequences from natural sources for thermophiles compared to mesophiles. The number at the top of each panel is for the position number in a consensus alignment. Clear differences were seen, for example, at positions shown in panels (A) 71 and 75; (B) 125; (C) 153; and (D) 200 and 204 with reference to the consensus sequence.
Figure 24. Western blot analysis of transgenic N. benthamiana plants transformed with the T-DNA from SL149. The blot was probed with anti-TS antibody to detect the scar::TS::AvAnfH-V7 polypeptide of about 38 kDa. The faint, upper band represents binding of the anti-TS antibody to a non-specific, endogenous protein.
KEY TO THE SEQUENCE LISTING
SEQ ID NO:1 Amino acid sequence of NifH polypeptide from K. oxyloca. 293 aa.
SEQ ID NO:2 Amino acid sequence of wild-type NifD polypeptide from K. oxytoca, according to Accession No. X13303.1; 483aa.
SEQ ID NO:3 Amino acid sequence of NifK polypeptide from K. oxytoca, according to Temme et al. (2012); 520aa.
SEQ ID NO:4 Amino acid sequence ofNifB polypeptide firom X. oxyloca. 468aa.
SEQ ID NO:5 Amino acid sequence of NifE polypeptide from K. oxyloca. 457aa.
SEQ ID NO:6 Amino acid sequence ofNifF polypeptide from ". oxyloca. 176 aa; NCBI Accession No. X03214.
SEQ ID NO:7 Amino acid sequence of NifI polypeptide from K. oxytoca, 1171 aa; NCBI Accession No. WP_064371580, Cannon et al., (1988).
SEQ ID NO:8 Amino acid sequence of NifM polypeptide from K. oxyloca. 266 aa; NCBI Accession No. X05887; Paul and Merrick (1987).
SEQ ID NO:9 Amino acid sequence of NifN polypeptide from K. oxyloca. NCBI Accession No. P08738; 461aa; (Arnold et al., 1988).
SEQ ID NO: 10 Amino acid sequence of NifQ polypeptide from Klebsiella. NCBI Accession No. WP_004138772. SEQ ID NO: 11 Amino acid sequence of NifS polypeptide from T. oxytoca, 400aa.
SEQ ID NO: 12 Amino acid sequence of NifU polypeptide from K. oxytoca, 274aa. NCBI Accession No. P05343.2 (Arnold et al., 1988).
SEQ ID NO: 13 Amino acid sequence of NifV polypeptide from K. oxytoca, 381aa. NCBI Accession No. CAA31119.1 (Arnold et al., 1988).
SEQ ID NO: 14 Amino acid sequence of NifX polypeptide from K. oxytoca, 156aa (Accession No. P09136).
SEQ ID NO: 15 Amino acid sequence of NifY polypeptide from K. oxytoca, 220aa; NCBI Accession No. CAA31670 (Arnold et al., 1988).
SEQ ID NO: 16 Amino acid sequence of NifZ polypeptide from K. oxytoca, 148aa; NCBI Accession No. P0A3U2 (Arnold et al., 1988).
SEQ ID NO: 17. Amino acid sequence ofNifW polypeptide from T. oxytoca.
SEQ ID NO: 18. Amino acid sequence of wild-type K. oxytoca NifD according to Temme et al. (2012)
SEQ ID NO: 19. Amino acid sequence of wild-type K. oxytoca according to Temme et al. (2012).
SEQ ID NO:20. Amino acid sequence of the MTP-FAy51 : :NifH: :HA fusion polypeptide encoded by SN18 and SN27. Amino acids 1-54 correspond to the MTP-FAy51 with an additional start methionine and a C-terminal GG, amino acids 55-347 are the K. oxytoca NifH amino acids (SEQ ID NO: 1) and amino acids 348-358 include the HA epitope.
SEQ ID NO:21. Amino acid sequence of linker for the NifD: linker: :NifK fusion polypeptide. The linker is 30 residues in length and consists of an 11-residue section from Hypocrea jecorina cellobiohydrolase II (Accession no. AAG39980.1; SEQ ID NO:38) with the final arginine replaced by an alanine, then a 9-residue HA epitope (SEQ ID NO: 39) followed by another copy of the 11-residue section with the arginine replaced by an alanine. SEQ ID NO:22. Peptide sequence.
SEQ ID NO:23. Peptide sequence.
SEQ ID NO:24. Amino acid sequence of MTP-FAy51::NifD(Y100Q)::linker(HA) : :NifK fusion polypeptide encoded by SN159. Amino acids 1-54 correspond to the MTP - FAy51 with GG at its C-terminus, amino acids 55-536 correspond to K. oxytoca NifD with the Y 100Q substitution, amino acids 537-566 correspond to the linker including the HA epitope, and amino acids 567-1085 correspond to NifK (SEQ ID NO:3) without its N-terminal Met and with its wild-type C-terminus.
SEQ ID NO:25. Amino acid sequence of the NifV polypeptide from A. vinelandii (AvNifV; Accession no WP_012698855).
SEQ ID NO:26. Amino acid sequence of the MTP-FAy51 ::AnfD::HA polypeptide encoded by SN81. Amino acids 1-54 correspond to the MTP- FAy51 sequence including a GG linker at its C-terminus, amino acids 55-572 correspond to the AnfD sequence from A. vinelandii, and amino acids 573-583 correspond to the HA epitope.
SEQ ID NO:27. Amino acid sequence of the MTP-FAy51 ::HA::AnfK polypeptide encoded by SN129. Amino acids 1-53 correspond to the MTP- FAy51 sequence including a GG linker at its C-terminus, amino acids 54-64 correspond to the HA epitope, and amino acids 65-526 correspond to the AnfK sequence from A. vinelandii.
SEQ ID NO:28. Amino acid sequence of the MTP-FAy51 ::HA::AnfH polypeptide encoded by SL48 and SL79. Amino acids 1-53 correspond to the MTP-FAy51 sequence including a GG linker at its C-terminus, amino acids 54-64 correspond to the HA epitope with a GG linker at its C-terminus, and amino acids 65-339 correspond to the AnfH sequence from A. vinelandii.
SEQ ID NO:29. Amino acid sequence of the MTP-FAy51 ::HA::AnfG polypeptide encoded by SL48 and SL79. Amino acids 1-53 correspond to the MTP-FAy51 sequence including a GG linker at its C-terminus, amino acids 54-64 correspond to the HA epitope with a GG linker at its C-terminus, and amino acids 65-196 correspond to the AnfG sequence from A. vinelandii. SEQ ID NO:30. Amino acid sequence of the MTP-FAy51 ::HA::AnfD polypeptide encoded by SN161. Amino acids 1-53 correspond to the MTP-FAy51 sequence including a GG linker at its C-terminus, amino acids 54-64 correspond to the HA epitope with a GG linker at its C-terminus, and amino acids 65-582 correspond to the AnfD sequence from A. vinelandii.
SEQ ID NO:31. Amino acid sequence of the MTP-FAy51 ::AnfD::Twin Strep polypeptide encoded by SN177. Amino acids 1-54 correspond to the MTP-FAy51 sequence including a GG linker at its C-terminus, amino acids 55-572 correspond to the AnfD sequence from A. vinelandii, and amino acids 573-604 correspond to the TwinStrep epitope.
SEQ ID NO:32. Amino acid sequence of the MTP-CoxIV::TwinStrep::AnfK polypeptide encoded by SN195. Amino acids 1-41 correspond to the MTP-CoxIV sequence including a GG linker at its C-terminus, amino acids 42-61 correspond to the TwinStrep epitope including a GG at the C-terminus, and amino acids 62-523 correspond to the AnfK sequence from A. vinelandii.
SEQ ID NO:33. Amino acid sequence of the MTP-FAy51 : : AnfD: :linker26(HA) : : AnfK polypeptide encoded by SN272. Amino acids 1-64 correspond to the MTP-FAy51 ::HA sequence including the GG at its C-terminus, amino acids 65-581 correspond to the AnfD sequence (A. vinelandii), amino acids 582-607 correspond to the 26-amino acid linker (Linker26(HA)), and amino acids 608-1068 to AnfK (A. vinelandii).
SEQ ID NO:34. Amino acid sequence of the MTP-CoxIV::AnfD::linker26(HA):: AnfK polypeptide encoded by SL48 and SL79. Amino acids 1-61 correspond to the MTP- CoxIV sequence including the GG at its C-terminus, amino acids 62-578 correspond to the AnfD sequence (A. vinelandii), amino acids 579-604 correspond to the 26-amino acid linker (Linker26(HA)), and amino acids 605-1065 to AnfK (A. vinelandii).
SEQ ID NO:35. Amino acid sequence of AnfD from A. vinelandii (Accession No. WP_012703361); 518aa.
SEQ ID NO:36. Amino acid sequence of AnfK from A. vinelandii (Accession No. WP_012703359); 462aa. SEQ ID NO :37. Amino acid sequence of AnfH from A. vinelandii (Accession No. WP_012703362); 275aa.
SEQ ID NO:38. Amino acid sequence of AnfG from A. vinelandii (Accession No. WP_012703360); 132aa.
SEQ ID NO:39 Amino acid sequence of the NifH polypeptide from A. vinelandii (AvNifH; Accession No. WP_012698831); 290aa.
SEQ ID NO:40. Peptide sequence, AnfH motif I, where X represents any amino acid.
SEQ ID NO:41. Peptide sequence, AnfH motif II.
SEQ ID NO:42. Peptide sequence, AnfH motif III.
SEQ ID NO:43. Peptide sequence, AnfH motif IV.
SEQ ID NO:44. Peptide sequence, AnfH motif V, where X represents any amino acid.
SEQ ID NO:45. Peptide sequence, AnfH motif VI.
SEQ ID NO:46. Peptide sequence, AnfH motif VII, where X represents any amino acid.
SEQ ID NO:47 Amino acid sequence of the FdxN protein of A. vinelandii,' Accession No. WP_012703542; 92aa.
SEQ ID NO:48 Amino acid sequence of the NafY polypeptide from A. vinelandii (AvNafY; Accession No. AGK13761).
SEQ ID NO:49-53. C-terminal amino acid sequences of NifK polypeptides.
SEQ ID NO:54-58. C-terminal amino acid sequences of AnfK polypeptides.
SEQ ID NO:59 Amino acid sequence of wild-type A. vinelandii NifU polypeptide, Genbank Accession No. WP_012698853.1; 312aa. SEQ ID NO:60 Amino acid sequence of wild-type A. vinelandii NifS polypeptide (Johnson et al., 2005; Yuvaniyama et al., 2000); NCBI Reference Sequence: WP_012698854.1; 402aa.
SEQ ID NO:61. Peptide sequence, NifH motif I, where X represents any amino acid.
SEQ ID NO:62. Peptide sequence, NifH motif II, where X represents any amino acid.
SEQ ID NO:63. Peptide sequence, NifH motif III, where X represents any amino acid.
SEQ ID NO:64. Peptide sequence, NifH motif V, where X represents any amino acid.
SEQ ID NO:65. Peptide sequence, NifH motif VII, where X represents any amino acid.
SEQ ID NO:66. Amino acid sequence of the MTP-FAy51 ::HA::MiNifB fusion polypeptide encoded by SL78. Amino acids 1-53 correspond to the MTP-FAy51 with GG at its C-terminus, amino acids 54-64 include the HA epitope with GG, and amino acids 65-366 correspond to Methanocaldococcus infernus NifB with its initiator Met, 366aa.
SEQ ID NO:67 Amino acid sequence of the TS::NifU fusion polypeptide, comprising the A. vinelandii NifU sequence, produced in E. coli. Amino acids 10-41 correspond to the TwinStrep epitope followed by GG, and amino acids 42-353 correspond to the A. vinelandii NifU sequence; 353aa.
SEQ ID NO:68. Amino acid sequence of the MTP-FAy51 ::NifS fusion polypeptide encoded by SL122. Amino acids 1-54 correspond to the MTP-FAy51 with GG at its C- terminus, while amino acids 55-456 correspond to d. vinelandii NifS (SEQ ID NO:60), including its initiator Met; 456aa.
SEQ ID NO:69. Amino acid sequence of the scar::NifS fusion polypeptide after processing in mitochondria by MPP. Amino acids 1-11 correspond to the scar sequence from MTP-FAy51, while amino acids 12-413 correspond to d. vinelandii NifS (SEQ ID NO: 60); 4 Baa. SEQ ID NO:70. Amino acid sequence of the MTP-CoxIV::TwinStrep::NifU fusion polypeptide encoded by SL122. Amino acids 1-29 correspond to the MTP-CoxIV, amino acids 30-61 correspond to the TwinStrep epitope with GG at its C-terminus, while amino acids 62-373 correspond to A. vinelandii NifU (SEQ ID NO:59) including its initiator Met; 373aa.
SEQ ID NO:71. Amino acid sequence of the scar:: TwinStrep ::NifU fusion polypeptide after processing in mitochondria by MPP. Amino acids 1-4 correspond to the scar sequence from MTP-CoxIV, amino acids 5-36 correspond to the TwinStrep epitope with GG at its C-terminus, while amino acids 37-348 correspond to vinelandii NifU (SEQ ID NO:59); 348aa.
SEQ ID NO :72. Amino acid sequence of variant VI of AnfH from A. vinelandii, with 5 amino acid substitutions compared to wild-type AnAnfH (Table 4); 275aa.
SEQ ID NO :73. Amino acid sequence of variant V2 of AnfH from A. vinelandii, with 6 amino acid substitutions compared to wild-type AnAnfH (Table 4); 275aa.
SEQ ID NO:74. Amino acid sequence of variant V3 of AnfH from A. vinelandii, with 9 amino acid substitutions compared to wild-type AnAnfH (Table 4); 275aa.
SEQ ID NO :75. Amino acid sequence of variant V4 of AnfH from A. vinelandii, with
10 amino acid substitutions compared to wild-type AnAnfH (Table 4); 275aa.
SEQ ID NO :76. Amino acid sequence of variant V5 of AnfH from A. vinelandii, with
11 amino acid substitutions compared to wild-type AnAnfH (Table 4); 275aa.
SEQ ID NO :77. Amino acid sequence of variant V6 of AnfH from A. vinelandii, with 4 amino acid substitutions compared to wild-type AnAnfH (Table 4); 275aa.
SEQ ID NO:78. Amino acid sequence of variant V7 of AnfH from A. vinelandii, with 3 amino acid substitutions compared to wild-type AnAnfH (Table 4); 275aa.
SEQ ID NO :79. Amino acid sequence of variant V8 of AnfH from A. vinelandii, with 3 amino acid substitutions compared to wild-type AnAnfH (Table 4); 275aa. SEQ ID NO:80. Amino acid sequence of the MTP-CoxIV::TS::AvAnfH fusion polypeptide encoded by SN675. Amino acids 1-29 are the MTP-CoxIV from S. cerevisiae cytochrome c oxidase subunit 4 which is cleaved by MPP between amino acids 25 and 26, amino acids 30-61 are the TwinStrep epitope tag including a C-terminal GG, amino acids 62-336 are the wild-type vinelandii AnfH sequence; 336aa.
SEQ ID NO:81. Amino acid sequence of the MTP-CoxIV: :TS:: Av AnfH-Vl fusion polypeptide encoded by SN676. Amino acids 1-29 are the MTP-CoxIV, amino acids 30- 61 are the TwinStrep epitope tag including a C-terminal GG, and amino acids 62-336 are the Av AnfH-Vl sequence; 336aa.
SEQ ID NO:82. Amino acid sequence of the MTP-CoxIV: :TS:: Av AnfH-V6 fusion polypeptide encoded by SN708. Amino acids 1-29 are the MTP-CoxIV, amino acids 30- 61 are the TwinStrep epitope tag including a C-terminal GG, and amino acids 62-336 are the AvAnfH-V6 sequence; 336aa.
SEQ ID NO:83. Amino acid sequence of the MTP-CoxIV: :TS:: Av AnfH-V7 fusion polypeptide encoded by SN709. Amino acids 1-29 are the MTP-CoxIV, amino acids 30- 61 are the TwinStrep epitope tag including a C-terminal GG, and amino acids 62-336 are the AvAnfH-V7 sequence; 336aa.
SEQ ID NO:84. Amino acid sequence of the MTP-CoxIV: :TS:: Av AnfH-V8 fusion polypeptide encoded by SN710. Amino acids 1-29 are the MTP-CoxIV, amino acids 30- 61 are the TwinStrep epitope tag including a C-terminal GG, and amino acids 62-336 are the AvAnfH-V8 sequence; 336aa.
SEQ ID NO:85. Amino acid sequence of the scar::TS::AvAnfH-Vl fusion polypeptide produced from SN676. Amino acids 1-4 are the scar sequence, the C-terminal amino acids from the MTP-CoxIV after MPP-processing in mitochondria, amino acids 5-34 are the TwinStrep epitope tag including a C-terminal GG, and amino acids 35-311 are the Av AnfH- VI sequence; 311aa.
SEQ ID NO:86. Amino acid sequence of the scar::TS::AvAnfH-V6 fusion polypeptide produced from SN708. Amino acids 1-4 are the scar sequence, the C-terminal amino acids from the MTP-CoxIV after MPP-processing in mitochondria, amino acids 5-34 are the TwinStrep epitope tag including a C-terminal GG, and amino acids 35-311 are the AvAnfH-V6 sequence; 311aa.
SEQ ID NO:87. Amino acid sequence of the scar::TS::AvAnfH-V7 fusion polypeptide produced from SN709. Amino acids 1-4 are the scar sequence, the C-terminal amino acids from the MTP-CoxIV after MPP-processing in mitochondria, amino acids 5-34 are the TwinStrep epitope tag including a C-terminal GG, and amino acids 35-311 are the AvAnfH-V7 sequence; 311aa.
SEQ ID NO:88. Amino acid sequence of the scar::TS::AvAnfH-V8 fusion polypeptide produced from SN710. Amino acids 1-4 are the scar sequence, the C-terminal amino acids from the MTP-CoxIV after MPP-processing in mitochondria, amino acids 5-34 are the TwinStrep epitope tag including a C-terminal GG, and amino acids 35-311 are the AvAnfH-V8 sequence; 311aa.
SEQ ID NO:89. Amino acid sequence of the MTP-CPN60::AvNifS fusion polypeptide encoded by SL133. Amino acids 1-33 are the MTP-CPN60 which is cleaved by MPP between amino acids 31 and 32, with a C-terminal GG, and amino acids 34-435 are the sequence; 435aa.
SEQ ID NO:90. Amino acid sequence of the MTP-SU9::AvNifU fusion polypeptide encoded by SL133. Amino acids 1-68 are the MTP-SU9 which is cleaved by MPP between amino acids 66 and 67, followed by GG, and amino acids 71-382 are the A. vinelandii NifU sequence; 336aa.
SEQ ID NO:91 Amino acid sequence of the MTP-FAy51 ::AvFdxN fusion polypeptide encoded by SN291. Amino acids 1-52 are the MTP-FAy51 which is cleaved by MPP between amino acids 43 and 44, followed by GG, and amino acids 55-145 are the A. vinelandii FdxN sequence without its start methionine; 145aa.
SEQ ID NO:92. Nucleotide sequence of the DNA fragment for the P module comprising the LIR from BeYDV; Fragment number EN38510. Bsal restriction sites are present at nucleotides 9-14 and 319-324, flanking the LIR region, including the invariant 9 nucleotides of the LIR at positions 179-187; 332nt. SEQ ID NO:93 Nucleotide sequence of the DNA fragment for the U module comprising the CaMV e35S promoter and TMV 5’UTR regions, Fragment number EN38509. Bsal restriction sites are present at nucleotides 9-14 and 864-869, the e35S promoter at nucleotides 13-767 and the TMV 5’UTR at positions 796-852; the translation start ATG for the S module polypeptide is at nucleotides 860-862; 877nt.
SEQ ID NO:94 Nucleotide sequence of the DNA fragment for the T module comprising the CaMV 35S Tm transcription terminator and SIR/Rep/RepA/LIR regions from Gemini Virus BeYDV, used in GV vectors herein, number EN38511. Bsal restriction sites are present at nucleotides 9-14 and 1766-1771, the 35S Tm at nucleotides 20-223, the SIR at positions 224-375, the Rep/RepA coding region in reverse orientation from nucleotides 1466 to 376, and an LIR at positions 1467-1760. The thymidine at nucleotide position 1469 was replaced with a guanosine. Nucleotide position 1469 is a G in GVc and an A for GVt; 1779nt.
SEQ ID NO:95. Nucleotide sequence of the DNA fragment for the T module comprising the TTm hybrid transcription terminator. Bsal restriction sites are present at nucleotides 9-14 and 1155-1160, the EU Tm at nucleotides 20-499, the 35S Tm at positions 500-710, and the reduced Rb7 MAR sequence at positions 711-1149; 1168nt.
SEQ ID NO:96. Amino acid sequence of the MTP-FAy51 ::NifD(Y100Q)::HA fusion polypeptide. Amino acids 1-54 are the MTP-FAy51 which is cleaved by MPP between amino acids 42 and 43, with a C-terminal GG, amino acids 55-482 are the NifD sequence based on the K. oxytoca sequence with the Y100Q substitution, followed by GG, and amino acids 539-547 are a HA epitope; 547aa.
SEQ ID NO:97. Amino acid sequence of the MTP-FAy51 ::HA::NifH fusion polypeptide. Amino acids 1-51 are the MTP-FAy51 which is cleaved by MPP between amino acids 42 and 43, followed by GG at its C-terminus, amino acids 52-64 are the HA epitope tag including a C-terminal GG, amino acids 65-353 are the Geobacter NifH sequence; 353aa.
SEQ ID NO:98. Amino acid sequence of the MTP-FAy51 ::AvNifU::HA fusion polypeptide encoded by SN466, SN499 and SN500. Amino acids 1-54 are the MTP- FAy51 which is cleaved by MPP between amino acids 42 and 43, with a C-terminal GG, amino acids 55-368 are the A. vinelandii NifU sequence followed by GG, and amino acids 369-377 are a HA epitope; 377aa.
SEQ ID NO:99. Amino acid sequence of the MTP-FAy51 : :GFP: :HA fusion polypeptide encoded by SN493. Amino acids 1-54 are the MTP-FAy51 which is cleaved by MPP between amino acids 42 and 43, with a C-terminal GG, amino acids 55-295 are the GFP sequence followed by GG, and amino acids 296-304 are a HA epitope; 304aa.
SEQ ID NO: 100. Amino acid sequence of the MTP-FAy77::GFP fusion polypeptide encoded by pRAl. Amino acids 1-77 are the MTP-FAy77 which is cleaved by MPP between amino acids 42 and 43, with a C-terminal GAP, amino acids 81-319 are the GFP sequence; 319aa.
SEQ ID NO: 101. Amino acid sequence of the MTP-FAy51 ::HA::NifH: linker (TS)::NifH fusion polypeptide encoded by SN655, SN656 and SN657. Amino acids 1- 51 are the MTP-FAy51 which is cleaved by MPP between amino acids 42 and 43, followed by GG at its C-terminus, amino acids 52-64 are the HA epitope tag including a C-terminal GG, amino acids 65-357 and 388-680 are the two T. ox tocaNifH sequences, and amino acids 358-387 are the oligopeptide linker that includes a TwinStrep epitope; 680aa.
SEQ ID NO: 102. Amino acid sequence of the MTP-SU9::KoNifM fusion polypeptide encoded by SN360. Amino acids 1-70 are the MTP-SU9 comprising of the first 68 amino acid residues of the subunit 9 FO-ATPase from Neurospora crassa (Buren et al., 2017a), which is cleaved by MPP between amino acids 66 and 67, with a C-terminal GG, and amino acids 71-336 are the K. ox toca NifM sequence; 336aa.
SEQ ID NO: 103. Amino acid sequence of the MTP-FAy51 ::KoNifH: linker: :NifH::HA fusion polypeptide encoded by SN283. Amino acids 1-51 are the MTP-FAy51 which is cleaved by MPP between amino acids 42 and 43, amino acids 54-345 and 371-663 are the two K. oxytoca NifH sequences, amino acids 346-370 are the oligopeptide linker of 25 amino acids, and amino acids 666-674 are the HA epitope tag; 674aa.
SEQ ID NO:104. Amino acid sequence of the MTP-CoxIV::TS::KoNifH: linker: :NifH fusion polypeptide encoded by SN638, SN649 and SN650. Amino acids 1-29 are the MTP-CoxIV from Saccharomyces cerevisiae cytochrome c oxidase subunit 4 (Jiang et al., 2021) which is cleaved by MPP between amino acids 25 and 26, amino acids 30-61 are the TwinStrep epitope tag including a C-terminal GG, amino acids 62-353 and 379- 671 are the two K. oxytoca NifH sequences, and amino acids 354-378 are the oligopeptide linker; 671aa.
SEQ ID NO:105. Amino acid sequence of the MTP-FAy51::HA::KoNifH::TS::NifH fusion polypeptide encoded by SN656. Amino acids 1-51 are the MTP-FAy51 which is cleaved by MPP between amino acids 42 and 43, followed by GG at its C-terminus, amino acids 52-64 are the HA epitope tag including a C-terminal GG, amino acids 65- 357 and 388-680 are the two K. oxytoca NifH sequences, and amino acids 358-387 are the oligopeptide linker of 30 amino acids that includes a TwinStrep epitope; 680aa.
SEQ ID NO:106. Amino acid sequence of the MTP-FAy51::KoNifD(Y100Q):: HAlinker: :KoNifK fusion polypeptide encoded by SN159. Amino acids 1-51 are the MTP-FAy51 which is cleaved by MPP between amino acids 42 and 43, followed by GG, amino acids 54-535 are the K. oxytoca NifD sequence, amino acids 537-1084 are the K. oxytoca NifK sequence without its start Met and without a C-terminal extension, and amino acids 536-565 are the oligopeptide linker that includes a HA epitope; 1084aa.
SEQ ID NO:107. Amino acid sequence of the MTP-FAy51 ::AvFdxN::HA fusion polypeptide encoded by SN291. Amino acids 1-52 are the MTP-FAy51 which is cleaved by MPP between amino acids 43 and 44, followed by GG, amino acids 55-146 are the vinelandii FdxN sequence, and amino acids 147-157 are a HA epitope; 157aa.
SEQ ID NO: 108. Amino acid sequence of the MTP-FAy51 ::KoNifH::HA fusion polypeptide encoded by SN18. Amino acids 1-51 are the MTP-FAy51 which is cleaved by MPP between amino acids 42 and 43, followed by GG, amino acids 54-346 are the T. ox toca NifH sequence, and amino acids 347-357 are a HA epitope; 357aa.
SEQ ID NO: 109. Amino acid sequence of the MTP-FAy51 : :HA: : AvNifH: linker: :NifH fusion polypeptide encoded by each of SN678, SN679 and SN680. Amino acids 1-51 are the MTP-FAy51 which is cleaved by MPP between amino acids 42 and 43, followed by GG, amino acids 54-62 are the HA epitope followed by GG, amino acids 65-352 and 378-665 are the A. vinelandii NifH sequences (Accession No. AOG20751), and amino acids 353-377 are the oligopeptide linker; 665aa. SEQ ID NO: 110. Amino acid sequence of the MTP-FAy51 ::KoNifH: linker (MPP10)::NifH::HA fusion polypeptide encoded by SN285. Amino acids 1-51 are the MTP-FAy51 which is cleaved by MPP between amino acids 42 and 43, amino acids 54- 345 and 356-648 are the two K. oxytoca NifH sequences, amino acids 346-355 are the oligopeptide linker of 10 amino acids, and amino acids 651-659 are the HA epitope tag; 659aa.
SEQ ID NO:111 Amino acid sequence of the MTP-FAy51 : : AvNifM fusion polypeptide encoded by SN605. Amino acids 1-51 are the MTP-FAy51 which is cleaved by MPP between amino acids 42 and 43, followed by GG, amino acids 54-346 are the A. vinelandii NifM sequence; 346aa.
SEQ ID NO: 112 Amino acid sequence of the MTP-FAy51 : : AvFdxN fusion polypeptide encoded by SN465. Amino acids 1-51 are the MTP-FAy51 which is cleaved by MPP between amino acids 42 and 43, followed by GG, amino acids 54-145 are the A. vinelandii FdxN sequence; 145aa.
SEQ ID NO: 113 Amino acid sequence of the scar: :TS::KoNifH: linker: :NifH fusion polypeptide produced from SN638 and SN650. Amino acids 1-4 are the scar sequence from the MTP-CoxIV, amino acids 5-36 are the TwinStrep epitope tag including a C- terminal GG, amino acids 37-328 and 354-646 are the two K. oxytoca NifH sequences, and amino acids 339-353 are the oligopeptide linker; 646aa.
SEQ ID NO: 114. Amino acid sequence of the MTP-FAy51 ::AvAnfH- V7::linker(TS)::AvAnfH-V7 fusion polypeptide encoded by SN775, SN776 and SN777. Amino acids 1-51 are the MTP-FAy51 which is cleaved by MPP between amino acids 42 and 43, followed by GG at its C-terminus, amino acids 54-328 and 359-633 are the two modified A. vinelandii AnfH-V7 sequences, and amino acids 329-358 are the oligopeptide linker of 30 amino acids that includes a TwinStrep epitope; 633aa.
SEQ ID NO:115 Amino acid sequence of the scar::AvAnfH-V7::linker(TS) ::AvAnfH- V7 fusion polypeptide produced in plant mitochondria from SN775, SN776 and SN777. Amino acids 1-11 are the scar sequence remaining from the C-terminus of the MTP sequence after cleavage by MPP, followed by GG, amino acids 12-286 and 317-591 are the two modified A. vinelandii AnfH-V7 sequences, and amino acids 287-316 are the oligopeptide linker of 30 amino acids that includes a TwinStrep epitope; 591aa. SEQ ID NO: 116. Amino acid sequence of the MTP-FAy51 : : AnfG polypeptide encoded by SN737. Amino acids 1-53 correspond to the MTP-FAy51 sequence including a GG linker at its C-terminus, amino acids 54-185 correspond to the wild-type AnfG sequence from A. vinelandii.
SEQ ID NO: 117. Amino acid sequence of the MTP-FAy51 ::NifB polypeptide encoded by SN598. Amino acids 1-53 correspond to the MTP-FAy51 sequence including a GG linker at its C-terminus, amino acids 54-341 correspond to the wild-type NifB sequence from Methanothermobacter thermautotrophicus.
SEQ ID NO:118. Amino acid sequence of the MTP-FAy51 ::AnfH-V8 polypeptide encoded by SN714. Amino acids 1-53 correspond to the MTP-FAy51 sequence including a GG linker at its C-terminus, amino acids 54-328 correspond to the AnfH-V8 sequence with the substituted amino acids T253A, T281V and G294R, corresponding to substitutions T200A, T228V and T241R of the vinelandii AnfH-V8 sequence.
SEQ ID NO:119. Amino acid sequence of the MTP-FAy51::AnfH-V7 polypeptide encoded by SN754. Amino acids 1-53 correspond to the MTP-FAy51 sequence including a GG linker at its C-terminus, amino acids 54-328 correspond to the AnfH-V7 sequence with the substituted amino acids T253A, T281V and E287H, corresponding to substitutions T200A, T228V and E234H of the vinelandii AnfH-V7 sequence.
SEQ ID NO: 120. Amino acid sequence of the MTP-FAy51 ::AvAnfD::linker(HA) ::AvAnfK fusion polypeptide encoded by SN312, SN734, SN735. Amino acids 1-53 correspond to the MTP-FAy51 with GG at its C-terminus, amino acids 54-570 correspond to wild-type A vinelandii AnfD (AvAnfD) sequence, amino acids 571-596 correspond to the linker including the HA epitope, and amino acids 597-1057 correspond to AnfK (SEQ ID NO: 36) without its N-terminal Met and with its wild-type C-terminus.
SEQ ID NO: 121. Amino acid sequence of the MTP-FAy51 ::NifI fusion polypeptide encoded by SL138 and SN748. Amino acids 1-54 correspond to the MTP-FAy51 with GG, amino acids 55-1225 correspond to T. oxytoca NifI (SEQ ID NO:7). SEQ ID NO: 122. Amino acid sequence of the MTP-FAy51::NifV fusion polypeptide of SN532. Amino acids 1-54 correspond to the MTP-FAy51 sequence with a GG linker, amino acids 55-438 correspond to the NifV sequence from vinelandii.
SEQ ID NO:123. Amino acid sequence of the MTP-FAy51 ::AvFdxN::HA fusion polypeptide encoded by SN736. Amino acids 1-54 are the MTP-FAy51 followed by GG, amino acids 55-148 are the A. vinelandii FdxN sequence without its start methionine, followed by GG, and amino acids 149-157 are a HA epitope; 157aa.
SEQ ID NO: 124. Amino acid sequence of the MTP-FAy51::NifF fusion polypeptide encoded by SL138 and SN747. Amino acids 1-54 correspond to the MTP-FAy51 with GG, amino acids 55-230 correspond to T. oxytoca NifF (SEQ ID NO:6).
SEQ ID NO: 125. Amino acid sequence of the MTP-FAy51 ::AnfD fusion polypeptide encoded by SN733. Amino acids 1-54 correspond to the MTP-FAy51 with GG, amino acids 55-572 correspond to vinelandii AnfD (SEQ ID NO:35).
SEQ ID NO: 126. Amino acid sequence of the MTP-FAy51 ::AnfK fusion polypeptide encoded by SN528. Amino acids 1-54 correspond to the MTP-FAy51 with GG, amino acids 55-572 correspond to A vinelandii AnfK (SEQ ID NO:36).
SEQ ID NO: 127-150. Peptide sequences.
SEQ ID NO: 151. Amino acid sequence of the last four amino acid residues at the C- terminus of the NifK polypeptide from K. oxytoca.
SEQ ID NO: 152. Amino acid sequence of the MTP-FAy51 ::HA::FdxN fusion polypeptide of SL79; 156aa. Amino acids 1-53 correspond to the MTP-FAy51 sequence with a GG linker, amino acids 54-64 correspond to the HA epitope with a GG linker, and amino acids 65-156 correspond to the FdxN sequence without the N-terminal methionine.
SEQ ID NO: 153. Amino acid sequence of the MTP-FAy51::HA::NifV fusion polypeptide of SL48 and SL79; 448aa. Amino acids 1-53 correspond to the MTP- FAy51 sequence with a GG linker, amino acids 54-64 correspond to the HA epitope with a GG linker, and amino acids 65-448 correspond to the NifV sequence from A. vinelandii. SEQ ID NO:154. Amino acid sequence of the MTP-FAy51 ::NifS::HA fusion polypeptide encoded by SL78. Amino acids 1-54 correspond to the MTP-FAy51 with GG at its C-terminus, amino acids 55-454 correspond to K. oxytoca NifS (SEQ ID NO: 19) with its initiator Met, according to Temme et al., (2012), and amino acids 455- 465 include the HA epitope.
SEQ ID NO: 155. Amino acid sequence of the MTP-FAy51 ::NifU::HA fusion polypeptide encoded by SL78. Amino acids 1-54 correspond to the MTP-FAy51 with GG at its C-terminus, amino acids 55-328 correspond to K. oxytoca NifU (SEQ ID NO: 12) with its initiator Met, and amino acids 329-339 include the HA epitope.
SEQ ID NO:156. Amino acid sequence of the MTP-FAy51 ::NifF::HA fusion polypeptide encoded by SL78. Amino acids 1-54 correspond to the MTP-FAy51 with GG, amino acids 55-230 correspond to K. oxytoca NifF (SEQ ID NO:6) and amino acids 231-241 include the HA epitope.
SEQ ID NO: 157. Amino acid sequence of the MTP-FAy51 ::NifI::HA fusion polypeptide encoded by SL78. Amino acids 1-54 correspond to the MTP-FAy51 with GG, amino acids 55-1225 correspond to K. oxytoca NifI (SEQ ID NO:7), and amino acids 1226-1236 include the HA epitope.
DETAILED DESCRIPTION OF THE INVENTION
General Techniques and Definitions
Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in molecular genetics, plant molecular biology, nitrogen fixation, protein chemistry, and biochemistry).
Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).
The term “and/or”, e.g., “X and/or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.
As used herein, the term about, unless stated to the contrary, refers to +/- 10%, or more preferably +/- 5%, even more preferably +/- 1%, of the designated value.
Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
Nitrogenase
Nitrogenase is the enzyme in eubacteria and archaeobacteria that catalyses the reduction of the strong, triple bond of nitrogen (N2) to produce ammonia (NH3). Nitrogenase is found naturally only in bacteria. It is a complex of two enzymes that can be purified separately, namely dinitrogenase and dinitrogenase reductase. Dinitrogenase, also referred to as component I or the molybdenum-iron (MoFe) protein, is a tetramer of two NifD and two NifK polypeptides (0C2B2) that also contains two “P-clusters” and two “FeMo-cof actors” (FeMo-co). Each pair of NifD-NifK subunits contains one P-cluster and one FeMo-co. FeMo-co is a metallocluster composed of a MoFe3-S3 cluster complexed with a homocitrate molecule, which is coordinated to the molybdenum atom, and bridged to a Fe4-S3 cluster by three sulfur ligands. FeMo-co is assembled separately in cells and is then incorporated into apo-MoFe protein. The P-cluster is also a metallocluster and contains 8 Fe atoms and 7 sulfur atoms with a structure similar but different to FeMo-co. The P-clusters are located at the aB subunit interface of dinitrogenase and are coordinated by cysteinyl residues from both subunits. Dinitrogenase reductase, also referred to as component II or the “Fe protein” is a dimer of NifH polypeptides which also contains a single Fe4-S4 cluster at the subunit interface and two Mg- ATP binding sites, one at each subunit. This enzyme is the obligatory electron donor to the dinitrogenase, where the electrons are transferred from the Fe4-S4 cluster to the P-cluster and in turn to the FeMo-co, the site for N2 reduction. Although the Mo-containing nitrogenase is the most commonly found nitrogenase in bacteria, there are two homologous nitrogenases that are genetically distinct but have similar cofactor and subunit compositions, namely the vanadium-containing nitrogenase and the Fe-only nitrogenase, encoded by the Vnf (vanadium nitrogen fixation) and Anf (alternative nitrogen fixation) genes, respectively. Some bacteria in nature possess all three types of nitrogenases, other bacteria contain only the Mo- and V-containing enzymes or only the Mo-containing enzyme, for example, Klebsiella pneumoniae.
A variety of nitrogen fixation (Nif) genes are required for the biosynthesis of FeMo-co and maturation of the nitrogenase components to their catalytically active forms. Roles for the NifB, NifE, NifH, NifN, NifQ, NifV and NifX polypeptides in FeMo-co synthesis have been described (Rubio and Ludden, 2008).
Biological N2 fixation, catalyzed by the prokaryotic enzyme nitrogenase, is an alternative to the use of synthetic N2 fertilizers. The sensitivity of nitrogenase to oxygen is a major barrier to engineering biological nitrogen fixation into plants, for example, into cereal crops, by direct Nif gene transfer.
Thermostability and its Relationship with Solubility
Many proteins misfold and aggregate when taken out of their natural context (Goldenzweig et al., 2016). Protein aggregation may be more likely to occur when nascent peptides misfold and do not form a structurally stable conformational state (Wang and Roberts, 2018). It has also been proposed that naturally occurring proteins are only marginally stable because there is no selective pressure for greater energetic stability within their cellular environments (Magliery, 2015). The compatibility of heterologous protein production and stability in a host is, therefore, difficult to predict and is often impacted by the inherent energetic stability of a protein. It has been reported that the interior of mitochondria functions at a higher temperature than the rest of the organism when the respiratory chain is fully functional (Chretien et al., 2018), at least in endothermic organisms. If this were also true in plant mitochondrial matrix, the relatively higher, localised operating temperature might promote protein aggregation and thereby lessening solubility for proteins such as the wild-type AnfH which are exogenous to plant cells.
Proteins generally exist in cells in a dynamic range of conformations. Each conformation has an associated Gibbs energy that could theoretically be calculated from several parameters, including intermolecular interactions, covalent bond angles and the protein interaction with the solvent (Onuchic et al., 1997). The native fold of a protein is usually in a local energy minimum or energetic ‘well’. This native fold is thought to be stabilised by hydrophobic collapse and intermolecular bonds. Hydrophobic collapse excludes the protein’s hydrophobic residues from the solvent since breaking the hydrogen bonding network of water is energetically unfavourable (Sadqi et al., 2003). Intermolecular interactions involve favourable intermolecular bonds between the protein and the solvent and within the protein (Jaenicke. 2000). For example, a-helices and P- sheets arise as the backbone amino hydrogens and carboxyl oxygen atoms form a hydrogen bonding network. The lower the energy of the native fold of the protein, the more energy is required to change the folded state or ‘unfold’ the protein (Liu et al., 2000). Some proteins have greater thermostability at ambient temperatures, enabling them to retain structure and function at elevated temperatures (Modarres et al., 2016). However, even with our current understanding of protein structure and folding, the stability and functional effect of an amino acid substitution on a protein are challenging to predict (Wilding et al., 2019). Despite this uncertainty, the present inventors used a protein engineering approach to lower the overall energy of AnfH with the aim of improving its stability and solubility.
Analysis of thermophilic proteins suggests that they contain more hydrophobic and charged amino acids compared to less thermophilic proteins (Modarres et al., 2016). These features suggest that thermophilic proteins have better hydrophobic packing and more solvent or salt-bridge interactions. Consensus design has also been used to increase protein stability (Porebski and Buckle, 2016). In this approach, homologous sequences are aligned, and the most frequent amino acids at each position are adopted into a novel protein sequence for expression. It is hypothesised that natural selection removes destabilising amino acids more frequently, such that more common amino acids at the same position across homologous sequences are stabilising (Georgoulis et al., 2020).
In an embodiment, a polypeptide of the invention (e.g., a MTP fusion polypeptide or cleaved product thereof) is at least partially soluble in mitochondria of a plant cell. In this context, the phrase “at least partially soluble” means that the polypeptide is detectable in the soluble fraction of a homogenised sample comprising mitochondria of a plant cell. Suitable methods for detecting solubility of polypeptides are known in the art and include those that are described in Example 1. In an embodiment, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the polypeptide present in mitochondria of the cell is soluble. Nif Polypeptides
As used herein, the terms “Nif polypeptide” and “Nif protein” are used interchangeably and mean a polypeptide which is related in amino acid sequence to naturally occurring polypeptides involved in nitrogenase activity, where the Nif polypeptide of the invention is selected from the group consisting of a NifD polypeptide, a NifH polypeptide, a NifK polypeptide, a NifB polypeptide, a NifE polypeptide, a NifN polypeptide, a NifF polypeptide, a NifI polypeptide, a NifM polypeptide, a NifQ polypeptide, a NifS polypeptide, a NifU polypeptide, a NifV polypeptide, a NifW polypeptide, a NifX polypeptide, a NifY polypeptide and a NifZ polypeptide, each of which as defined herein. In particular, the present invention relates to modified NifH polypeptides and/or NifH fusion polypeptides, preferably modified AnfH polypeptides and/or AnfH fusion polypeptides. Nif polypeptides of the invention include “Nif fusion polypeptides” which, as used herein, means a polypeptide homolog of a naturally occurring Nif polypeptide that has additional amino acid residues joined to the N- terminus or C-terminus, or both, relative to a corresponding naturally occurring Nif polypeptide, and/or which has at least one amino acid substitution when compared to a corresponding wild-type polypeptide. In this context, a “corresponding wild-type polypeptide” means the wild-type polypeptide occurring naturally in bacteria that is the closest in amino acid sequence to the Nif polypeptide of the invention, in terms of the degree of sequence identity, for all of the naturally occurring sequences that were known as of the date of this application. The Nif fusion polypeptide may be lacking the translation initiation Met or the two N-terminal Met residues relative to the corresponding wild-type Nif polypeptide. The amino acid residues of a Nif fusion polypeptide that correspond to the naturally occurring Nif polypeptide, i.e., without the additional amino acid residues joined to the N-terminus or C-terminus or both, are also referred to herein as a Nif polypeptide, abbreviated in this case to “NP”, or as a NifH polypeptide (“NH”) etc. In a preferred embodiment, “additional amino acid residues joined to the N-terminus or C-terminus or both” comprise a mitochondrial targeting peptide (MTP) or the remaining amino acids after protease cleavage of the MTP (processed MTP, or “scar sequence”) joined to the N-terminus of the NP, or an epitope sequence (“tag”) which is N-terminal or C-terminal to the NP or both, or both an MTP or processed MTP and an epitope sequence.
Naturally occurring Nif polypeptides occur only in some bacteria including the nitrogen-fixing bacteria, including free living nitrogen fixing bacteria, associative nitrogen fixing bacteria and symbiotic nitrogen fixing bacteria. Free living nitrogen fixing bacteria are capable of fixing significant levels of nitrogen without the direct interaction with other organisms. Without limitation, said free living nitrogen fixing bacteria include the members of the genera Azotobacter, Beijerinckia, Klebsiella, Cyanobacteria (classified as aerobic organisms) and the members of the genera Clostridium, Desulfovibrio and the named purple sulphur bacteria, purple non-sulphur bacteria and green sulphur bacteria. Associative nitrogen fixing bacteria are those prokaryotic organisms that are able to form close associations with several members of the Poaceae (grasses). These bacteria fix appreciable amounts of nitrogen within the rhizosphere of the host plants. Members of the genera Azospirillum are representative of associative nitrogen fixing bacteria. Symbiotic nitrogen fixation bacteria are those bacteria which fix nitrogen symbiotically by partnering with a host plant. The plant provides sugars from photosynthesis that are utilized by the nitrogen fixing bacteria for the energy it needs for nitrogen fixation. Members of the genera Rhizobia are representative of associative nitrogen fixing bacteria.
The Nif polypeptide or Nif fusion polypeptide useful for the invention include those selected from the group consisting of NifH, NifD, NifK, NifB, NifE, NifN, NifF, Niff, NifM, NifQ, NifS, NifU, NifV, NifW, NifX, NifY and NifZ polypeptides. Function of these polypeptides has been reviewed recently by Buren et al. (2020).
Other polypeptides useful for the invention are considered to be VnfG and AnfG involved in the V-nitrogenase and Fe-nitrogenase, respectively, nitrogenase associated factors (Naf polypeptides) such as, for example, NafY, and ferredoxin polypeptides such as FdxN polypeptides. These polypeptides are preferably encoded and expressed as MTP-fusion polypeptides for mitochondrial targeting.
A polypeptide or class of polypeptides may be defined by the extent of identity (% identity) of its amino acid sequence to a reference amino acid sequence and/or by the presence of certain amino acid motifs or protein family domains, or by having a greater % identity to one reference amino acid sequence than to another. A polypeptide or class of polypeptides may also be defined by having the same biological activity as a naturally occurring Nif polypeptide, in addition to the extent of identity in sequence.
The % identity of a polypeptide is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3, or by Blastp version 2.5 or updated versions thereof (Altschul et al., 1997), where in each case the analysis aligns two sequences including a reference sequence over the entire length of the reference sequence. As used herein, reference sequences include those provided for naturally occurring Nif polypeptides from K. pneumoniae (renamed as K. oxytocaf SEQ ID NOs: l-17, or for AnfH, from Azotobacter vinelandii, SEQ ID NO:37. In the following definitions, the extent of identity of an amino acid sequence to a reference sequence provided as a SEQ ID NO is determined by Blastp, version 2.5 or updated versions thereof (Altschul et al., 1997), using the default parameters except for the maximum number of target sequences which is set at 10,000, and is determined along the full length of the reference amino acid sequence.
As used herein, the phrase “one amino acid substitution” or "an amino acid substitution” refers to the replacement of an amino acid in a wild-type NifH polypeptide with a single, different amino acid. For example, in one embodiment the threonine at position 200 of the NifH polypeptide with an amino acid sequence provided as SEQ ID NO:37 is substituted with an alanine. As used herein, an amino acid substitution excludes insertion or deletion of that amino acid. As a consequence, alignment of the polypeptide having the amino acid substitution, or multiple amino acid substitutions, with the corresponding unmodified polypeptide will not show any gaps due to insertion or deletion i.e. will be the same length. As used herein, the “number of amino acid substitutions” in the NifH polypeptide of the invention is counted along the full length of the alignment of the amino acid sequence of that polypeptide with the amino acid sequence of the corresponding wild-type NifH polypeptide.
As used herein, the phrase “with reference to SEQ ID NO” refers to the same amino acid position as the defined polypeptide.
As used herein, the phrase “at a corresponding amino acid position in the modified NifH polypeptide when its sequence is aligned with SEQ ID NO:37 (or SEQ ID NO:39)” or variations thereof refers to the relative position of the amino acid compared to surrounding amino acids. More specifically, not all wild-type NifH polypeptides have the same length (number of amino acid residues), although any two wild-type NifH polypeptides may have the same length, and hence this embodiment relates to instances where a specific wild-type NifH polypeptide does or does not have the same length as SEQ ID NO:37. The skilled person can nonetheless determine the relevant corresponding amino acid position using protein standard alignment programs, or even by eye.
A NifH polypeptide in naturally occurring bacteria is a structural component of nitrogenase complex and is often termed the iron (Fe) protein. It forms a homodimer, with a Fe4S4 cluster bound between the subunits and two ATP -binding domains. NifH is the obligate electron donor to the nitrogenase protein (NifD/NifK heterotetramer) and therefore functions as the nitrogenase reductase for the molybdenum-type nitrogenase (EC 1.18.6.1). AnfH polypeptides are considered herein as a sub-class of NifH polypeptides and AnfH is the obligate electron donor to the AnfD-AnfH-AnfG nitrogenase protein (AnfDKG, the iron-only nitrogenase) although it can also function with the NifD/NifK heterotetramer. NifH of the molybdenum type is also involved in FeMo-co biosynthesis and apo-MoFe protein maturation (Jasniewski et al., 2018), and AnfH is thought to have the corresponding functions for FeFe-co biosynthesis and apo- FeFe protein maturation. As reviewed in Jasniewski et al., (2018), NifH has three primary recognised functions: (i) involvement in the insertion of Mo and homocitrate in the synthesis of FeMo-co, also involving the NifE-NifN complex, (ii) a reductase function in the formation of P-cluster on NifD-NifK from what is termed P* cluster, which may also involve a small chaperone-like polypeptide NifZ, and (iii) as electron donor to the nitrogenase protein.
As used herein, a “NifH polypeptide” means a polypeptide comprising amino acids whose sequence is at least 41% identical to the amino acid sequence provided as SEQ ID NO: 1 and which comprises one or more of the domains TIGR01287, PRK13236, PRK13233 and cd02040. The TIGR01287 domain is present in each of molybdenumiron nitrogenase reductase (NifH), vanadium-iron nitrogenase reductase (VnfH), and iron-iron nitrogenase reductase (AnfH) but excludes the homologous protein from the light-independent protochlorophyllide reductase. As used herein, NifH polypeptides therefore include the subclass of iron-binding polypeptides which comprise amino acids whose sequence is at least 41% identical to NifH from K. oxytoca (SEQ ID NO: 1), the VnfH iron-binding polypeptides and the AnfH iron-binding polypeptides. A naturally occurring NifH polypeptide typically has a length of between 260 and 300 amino acids and the natural monomer has a molecular weight of about 30 kDa. A great number of NifH polypeptides have been identified and numerous sequences are available in publically available databases. For example, NifH polypeptides have been reported from Klebsiella michiganensis (Accession No. WP 049123239.1, 99% identical to SEQ ID NO: 1), Brenneria goodwinii (WP 048638817.1, 93% identical), Sideroxydans lithotrophicus (WP 013029017.1, 84% identical), Denitrovibrio acetiphilus (WP_013010353.1, 80% identical), Desulfovibrio africanus (WP_014258951.1, 72% identical), Chlorobium phaeobacteroides (WP 011744626.1, 69% identical), Methanosaeta concilii (WP 013718497.1, 64% identical), Rhodobacter
(WP_009565928.1, 61% identical), Methanocaldococcus infernus (WP 013099472.1 , 42% identical) and Desulfosporosinus youngiae (WP 007781874.1, 41% identical). Of particular significance herein and often used as a corresponding wild-type NifH sequence is the NifH from A. vinelandii (SEQ ID NO:39; 290 amino acids) which is 89% identical to SEQ ID NO: 1 (293 amino acids). NifH polypeptides have been described and reviewed in Thiel et al. (1997), Pratte et al. (2006), Boison et al. (2006) and Staples et al. (2007).
As used herein, a functional NifH polypeptide is a NifH polypeptide which is capable of forming a functional nitrogenase protein complex together with the other required subunits, for example, NifD and NifK, or in the case of an AnfH polypeptide, with AnfDKG, and the FeMo-, FeV- or FeFe-cof actor. In this context, the functional nitrogenase complex is able to reduce N2 gas to ammonia and/or acetylene to ethylene, which can be assayed in in vitro reactions as described herein.
As used herein, an “AnfH polypeptide” is a NifH polypeptide which is a member of the nitrogenase conserved superfamily cl25403 (TIGR01287) containing the PRK13233 conserved domain and having at least 69% amino acid sequence identity to the A. vinelandii AnfH polypeptide (SEQ ID NO:37; Accession No. WP_012703362) when measured along the full-length of SEQ ID NO:37. This amino acid sequence is used herein as the reference sequence for AnfH. TIGR01287 AnfH represents the alliron variant of the nitrogenase component II, also known as nitrogenase reductase. As used herein, the AnfH polypeptides are a subset of the NifH polypeptides. AnfH polypeptides do not include the molybdenum type NifH polypeptides and the vanadium type NifH polypeptides (VnfH). The amino acid sequences of AnfH polypeptides in sequence databases were usually annotated as an AnfH polypeptide. As of January 2020, there were 314 specific amino acid sequences in the NCBI protein database in the AnfH set, all of which had amino acid residues specific to AnfH and which were distinct from the molybdenum-type NifH and VnfH, which subsets looked more alike but still distinct. Examples of naturally occurring AnfH polypeptides include AnfH polypeptides from Rhodocyclus tenuis (Accession No. WP_153472986; 92.36% identical), Dickeya paradisiaca (Accession No. WP_015854293; 88.36% identical), Thermodesulfitimonas autotrophica (Accession No. WP_123927773; 78.91% identical), Clostridium kluyveri (Accession No. WP_073538802; 76.36% identical) and Methanophagales archaeon (Accession No. RCV64832; 69.37% identical), each with reference to SEQ ID NO:37.
As described in Example 2 herein, 16 amino acids were identified at defined positions in SEQ ID NO:37 or the corresponding positions in other AnfH sequences that were conserved and characteristic of AnfH polypeptides relative to the molybdenum- type NifH sequences. These 16 amino acid positions can be used to distinguish AnfH polypeptides from other NifH sequences which do not have all 16 amino acids in common. AvNifH (SEQ ID NO:39), KoNifH (SEQ ID NO: 1) and other molybdenum type NifH sequences had motif IV but did not have motifs I, II, III, and V-VII due to one or more amino acid substitutions in each of those, and therefore these motifs (SEQ ID NOs:40-46) could also be used to distinguish the AnfH subset from other NifH polypeptides.
Analogous to other functional NifH polypeptides, functional AnfH polypeptides are capable of functioning as a nitrogenase reductase, being the obligate electron donor to FeFe complex (AnfDKG). Analogous to the molybdenum -type NifH, AnfH is thought to be involved in FeFe-co biosynthesis and maturation of the apo-FeFe complex (AnfDKG).
As used herein, a “NifD polypeptide” means a polypeptide comprising amino acids whose sequence is at least 33% identical to the amino acid sequence provided as SEQ ID NO:2 and which comprises (i) one or both of the domains TIGR01282 and COG2710, both of which are found in the iron-molybdenum binding polypeptides including the polypeptide having the amino acid sequence shown in SEQ ID NO:2, or (ii) the iron-vanadium binding domain TIGR01860 in which case the NifD polypeptide is in the subclass of VnfD polypeptides, or (iii) the iron-iron binding domain TIGR1861 in which case the NifD polypeptide is in the subclass of AnfD polypeptides. The NifD polypeptide may be part of a fusion polypeptide, for example, fused to a MTP and/or NifK, or alternatively may not comprise any N- or C-terminal extensions. In a preferred embodiment, the NifD polypeptide when associated with a NifK polypeptide, binds FeMo-cof actor.
As used herein, NifD polypeptides include the subclass of iron-molybdenum (FeMo-co) binding polypeptides comprising amino acids whose sequence is at least 33% identical to SEQ ID NO:2, the VnfD iron-vanadium polypeptides and the AnfD polypeptides. A naturally occurring NifD polypeptide typically has a length of between 470 and 540 amino acids. A great number of NifD polypeptides have been identified and numerous sequences are available in publically available databases. For example, NifD polypeptides have been reported from Raoultella ornithinolytica (Accession No. WP 044347161.1, 96% identical to SEQ ID NO:2), Kluyvera intermedia (WP_047370273.1, 93% identical), Dickeya dadantii (WP_038902190.1, 89% identical), Tolumonas sp. BRL6-1 (WP 024872642.1, 81% identical), Magnetospirillum gryphiswaldense (WP 024078601.1, 68% identical), Thermoanaerobacterium thermosaccharolyticum (WP_013298320.1, 42% identical), Methanothermobacter thermautotrophicus (WP_010877172.1, 38% identical), Desulfovibrio africanus (WP 014258953.1, 37% identical), Desulfotomaculum sp. LMal (WP 066665786.1, 37% identical), Desulfomicrobium baculatum (WP 015773055.1, 36% identical), the VnfD polypeptide of Fischerella muscicola (WP 016867598.1, 34% identical) and the AnfD polypeptide from Opitutaceae bacterium TAV5 (WP_009512873.1, 33% identical). NifD polypeptides have been described and reviewed in Lawson and Smith (2002), Kim and Rees (1994), Eady (1996), Robson et al. (1989), Dilworth et al. (1988), Dilworth et al. (1993), Miller and Eady (1988), Chiu et al. (2001), Mayer et al. (1999), and Tezcan et al. (2005).
NifD polypeptides of the iron-molybdenum subclass are a key subunit of nitrogenase complexes, being the a subunit of the OC2B2 MoFe protein complex at the core of nitrogenase, and the site of substrate reduction with the FeMo cofactor. As used herein, a functional NifD polypeptide is a NifD polypeptide which is capable of forming a functional nitrogenase protein complex together with the other required subunits, for example, NifH and NifK, and the FeMo or other cofactor.
As used herein a “a NifD polypeptide (ND) which is resistant to protease cleavage” is resistant to cleavage at a defined site or within a defined region, for example within an amino acid sequence corresponding to amino acids 97-100 of SEQ ID NO: 18, when the ND is introduced into plant mitochondria by use of an MTP. As used herein “resistant to protease cleavage” means yielding <10% cleavage when the NifD polypeptide is introduced into plant mitochondria by use of an MTP. In preferred embodiments, less than 5% of the NifD polypeptide is cleaved at the site or within the region, more preferably essentially not cleaved, or cleavage is not detected. The NifD polypeptide may be “relatively resistant to cleavage” compared to a NifD polypeptide comprising the amino acid sequence provided as SEQ ID NO: 18, being cleaved at least 5-fold less often, preferably at least 10-fold less often, as a NifD polypeptide comprising the amino acid sequence provided as SEQ ID NO: 18.
As used herein, an “amino acid sequence other than RRNY (SEQ ID NO: 150) at positions corresponding to amino acids 97-100 of SEQ ID NO: 18” refers to a sequence which comprises four residues at positions corresponding to amino acids 97-100 of SEQ ID NO: 18 and which is not RRNY.
As used herein, an “AnfD polypeptide” is a NifD polypeptide which is specifically a member of the oxidoreductase nitrogenase conserved superfamily cl30843, containing the TIGR01861 conserved domain, and having at least 71% amino acid sequence identity to the Azotobacter vinelandii AnfD polypeptide (SEQ ID NO:35; Accession No. WP_012703361) when measured along the full-length of SEQ ID NO:35. This amino acid sequence is used herein as the reference sequence for AnfD. TIGR01861 AnfD represents the all-iron variant of the nitrogenase component I a-chain. As used herein, an AnfD polypeptide is therefore a subset of the NifD polypeptides. AnfD polypeptides do not include the molybdenum type NifD polypeptides and the vanadium type NifD polypeptides (VnfD) and also do not include protochlorophyllide or chlorophyllide reductase polypeptides (Boyd and Peters, 2013). The amino acid sequences of AnfD polypeptides in the protein sequence database are usually annotated as an AnfD polypeptide. As of January 2020, there were 156 specific amino acid sequences in the NCBI protein database in the AnfD set. Examples of naturally occurring AnfD polypeptides include AnfD polypeptides from Desulfovibrio sp. DV (Accession No. WP_075356167; 87.47% identical), Paenibacillus sp. FSL H7-0357 (Accession No. WP_038590013; 85.52% identical), Rhodobacter capsulatus (Accession No. WP_ 023922817; 80.31% identical), Methanosarcina acetivorans C2A (Accession No. WP_011021232; 77.13% identical) and Bacteroidales bacterium Barb7 (Accession No. OAV73823; 71.25% identical), each with reference to SEQ ID NO:35. Further examples were reported in McRose et al. (2017).
Analogous to other NifD polypeptides which are functional, functional AnfD polypeptides are capable of functioning as the a protein structural component of the 012P282 heterohexameric nitrogenase with the P protein (AnfK) and the 5 protein (AnfG), providing the catalytic complex binding FeFe-co for dinitrogen reduction.
As used herein, a “NifK polypeptide” means a polypeptide comprising amino acids whose sequence is at least 31% identical to the amino acid sequence provided as SEQ ID NO:3 and which comprises one or more of the conserved domains cd01974, TIGR01286, or cd01973 in which case the NifK polypeptide is in the subclass of VnfK polypeptides, or C102775 containing the TIGR02931 conserved domain in which case the NifK polypeptide is in the subclass of AnfK polypeptides. As used herein, NifK polypeptides include the VnfK polypeptides from iron-vanadium nitrogenase and the AnfK iron-binding polypeptides. A naturally occurring NifK polypeptide typically has a length of between 430 and 530 amino acids. A great number of NifK polypeptides have been identified and numerous sequences are available in publically available databases. For example, NifK polypeptides have been reported from Klebsiella michiganensis (Accession No. WP 049080161.1, 99% identical to SEQ ID NO:3), Raoultella ornithinolytica (WP 044347163.1, 96% identical), Klebsiella variicola (SBM87811.1, 94% identical), Kluyvera intermedia (WP_047370272.1, 89% identical), Rahnella aquatilis (WP 014333919.1, 82% identical), Tolumonas auensis (WP_012728880.1, 75% identical), Pseudomonas stutzeri (WP_011912506.1, 68% identical), Vibrio natriegens (WP_065303473.1, 65% identical), Azoarcus toluclasticus (WP_018989051.1, 54% identical), Frankia sp. (prf| |2106319A, 50% identical) and Methanosarcina acetivorans (WP 011021239.1, 31% identical). There are some examples of polypeptides in databases annotated as “NifK” which have less than 31% identity to SEQ ID NO:3 but do not contain any of the domains listed above and are therefore not included as NifK polypeptides herein. NifK polypeptides have been described and reviewed in Kim and Rees (1994), Eady (1996), Robson et al. (1989), Dilworth et al. (1988), Dilworth et al. (1993), Miller and Eady (1988), Igarashi and Seefeldt (2013), Fani et al. (2000) and Rubio and Ludden (2008).
NifK polypeptides of the iron-molybdenum subclass are a key subunit of nitrogenase complexes, being the B subunit of the OC2B2 MoFe protein complex at the core of nitrogenase. As used herein, a functional NifK polypeptide is a NifK polypeptide which is capable of forming a functional nitrogenase protein complex together with the other required subunits, for example, NifD and NifH, and the FeMo or other cofactor. In a preferred embodiment, when aligned with the amino acid sequence SEQ ID NO:3, the amino acid sequence of the NifK polypeptide of the invention has at its C-terminus the amino acids DLVR (SEQ ID NO: 151), the arginine being the C-terminal amino acid. That is, the NifK polypeptide and the NifK fusion polypeptide of the invention preferably has the same C-terminus as the native NifK polypeptides, i.e., it does not have an artificial addition to the C-terminus. Such preferred NifK polypeptides are better able to form a functional nitrogenase complex with NifD and NifH polypeptides.
NifK polypeptides of the iron-molybdenum subclass are a key subunit of nitrogenase complexes, being the B subunit of the OC2B2 MoFe protein complex at the core of nitrogenase. As used herein, a functional NifK polypeptide is a NifK polypeptide which is capable of forming a functional nitrogenase protein complex together with the other required subunits, for example, NifD and NifH, and the FeMo or other cofactor. In a preferred embodiment, when aligned with the amino acid sequence SEQ ID NO:3, the amino acid sequence of the NifK fusion polypeptide and the cleaved NifK polypeptide of the invention have at its C-terminus the amino acids DLVR (SEQ ID NO: 151), the arginine being the C-terminal amino acid. In other preferred embodiments, the amino acid sequence of the NifK fusion polypeptide and the cleaved NifK polypeptide of the invention have at its C-terminus the amino acid sequence DLIR (SEQ ID NO:49), DVVR (SEQ ID NO:50), DIIR (SEQ ID NO:51), DLTR (SEQ ID NO:52) or INVW (SEQ ID NO:53), which are typically not present in native AnfK sequences. The NifK polypeptide and the NifK fusion polypeptide of the invention, and the cleaved NifK polypeptide therefrom, preferably has the same C-terminus as a native NifK polypeptide, i.e., it does not have an artificial addition to the C-terminus, and it does not have any amino acids deleted from the C-terminus when aligned with a native NifK polypeptide. Such preferred NifK polypeptides are better able to form a functional nitrogenase complex with NifD and NifH polypeptides. As used herein, an “AnfK polypeptide” is a polypeptide which is a member of the oxidoreductase nitrogenase conserved superfamily C102775, containing the TIGR02931 conserved domain, and having at least 54% amino acid sequence identity to the A. vinelandii AnfK polypeptide (SEQ ID NO:36; Accession No. WP_012703359) when measured along the full-length of SEQ ID NO:36. This amino acid sequence is used herein as the reference sequence for AnfK. TIGR02931 :AnfK represents the alliron variant of the nitrogenase component I P-chain. As used herein, an AnfK polypeptide may be a NifK polypeptide, having at least 31% amino acid identity to SEQ ID NO:3. Other AnfK polypeptides are less homologous and are only 25-31% identical to SEQ ID NO: 3 but are nevertheless included in AnfK polypeptides of the invention. AnfK polypeptides do not include the molybdenum type NifK polypeptides and the vanadium type NifK polypeptides (VnfK). The AnfK fusion polypeptide and the cleaved AnfK polypeptide of the invention preferably have the same C-terminus as a native AnfK polypeptide, i.e., it does not have an artificial addition to the C-terminus, and it does not have any amino acids deleted from the C-terminus when aligned with a native AnfK polypeptide such as SEQ ID NO:36. In preferred embodiments, the amino acid sequence of the AnfK fusion polypeptide and the cleaved AnfK polypeptide of the invention has at its C-terminus the amino acid sequence LNVW (SEQ ID NO: 54), LNTW (SEQ ID NO:55), LNMW (SEQ ID NO:56), LAMW (SEQ ID NO:57) or LSVW (SEQ ID NO:58). The amino acid sequences of AnfK polypeptides in the protein sequence database are usually annotated as an AnfK polypeptide. As of January 2020, there were 155 specific amino acid sequences in the protein database in the AnfK set, which were distinct from the molybdenum-type NifK and VnfK polypeptide sequences. Examples of naturally occurring AnfK polypeptides include AnfK polypeptides from Azomonas agilis (Accession No. WP_144571040; 91.34% identical), Clostridium sp. BL-8 (Accession No. WP_077859050; 78.35% identical), Lucifera butyrica (Accession No. WP 122630336; 62.34% identical) and Rhodoblastus acidophilus (Accession No. WP_088520366; 54% identical), each with reference to SEQ ID NO:36.
Analogous to other NifK polypeptides which are functional, functional AnfK polypeptides are capable of functioning as the P protein structural component of the 012P282 heterohexameric nitrogenase with the a protein (AnfD) and the 5 protein (AnfG) to form the complex having the active site for dinitrogen reduction on FeFe-co.
A NifB polypeptide in naturally occurring bacteria is a protein which converts [4Fe-4S] clusters into NifB-co, an Fe-S cluster of higher nuclearity with a central C atom that serves as a precursor of FeMo-co, FeV-co and FeFe-co synthesis (Guo et al., 2016). NifB therefore catalyses the first committed step in the FeMo-co, FeV-co and FeFe-co synthesis pathways and is therefore essential for nitrogenase function. The NifB-co product of NifB is able to bind to the NifE-NifN complex and can be shuttled from NifB to NifE-NifN by the metallocluster carrier protein NifX.
As used herein, a “NifB polypeptide” means a polypeptide whose amino acid sequence comprises amino acids whose sequence is at least 27% identical to the amino acid sequence provided as SEQ ID NO:4. Most NifB polypeptides comprise one or more of the conserved domain TIGR01290, the NifB conserved domain cd00852, the NifX- NifB superfamily conserved domain cl00252 and the Radical SAM conserved domain cd01335. As used herein, NifB polypeptides include naturally occurring polypeptides which have been annotated as having NifB function but which do not have one of these domains. NifB polypeptides from Klebsiella, Azolobacler, Rhizobium, Bradyrhizobium and other bacteria have a C-terminal NifX-like extension, whereas most archeal NifB polypeptides lack the NifX-like domain and are referred to as “truncated NifB polypeptides”. A naturally occurring NifB polypeptide typically has a length of between 440 and 500 amino acids and the natural monomer has a molecular weight of about 50 kDa. A great number of NifB polypeptides have been identified and numerous sequences are available in publically available databases. For example, NifB polypeptides have been reported from Raoultella ornithinolytica (Accession No. WP 041145602.1, 91% identical to SEQ ID NO:4), Kosakonia radicincitans (WP 043953592.1, 80% identical), Dickeya chrysanthemi (WP 040003311.1, 76% identical), Pectobacterium atrosepticum (WP_011094468.1, 70% identical), Brenneria goodwinii (WP_048638849.1, 63% identical), Halorhodospira halophila (WP 011813098.1, 59% identical, lacking a NifX domain), Methanosarcina barkeri (WP_048108879.1, 50% identical, lacking a NifX domain), Clostridium purinilyticum (WP 050355163.1, 40% identical, lacking a NifX domain) and Desulfovibrio salexigens (WP 015850328.1, 27% identical). As used herein, a “functional NifB polypeptide” is a NifB polypeptide which is capable of forming NifB-co from [4Fe-4S] clusters. Functional NifB requires S-adenosyl- methionine (SAM) for its function. NifB polypeptides have been described and reviewed in Curatti et al. (2006) and Allen et al. (1995).
Boyd et al. (2011) investigated the phylogenetic relationship of Anf/Vnf/NifDKEN and NifB from 40 taxa and made the following conclusions: (1) Lateral gene transfer of the Nif cluster encoding a NifB lacking a C-terminal NifX domain occurred from a methanogen ancestor in the order Methanosarcinales to an anaerobic Firmicutes ancestor, where the two organisms coexisted in an anaerobic environment and where molybdenum was available, and (2) after this lateral gene transfer event, fusion of NifB and NifX occurred in the Firmicutes, from which the diazotrophic bacterial lineage evolved. The following evidence was provided to support this theory: (1) None of the methanogenic archaea (Methanococcales, Methanosarcinales and Methanobacteriales) have a NifB with a C-terminal NifX domain, (2) NifB sequences from Methanobacteriales and Methanococcales indicate early divergence from those of Methanosarcinales and Bacteria, and (3) some of the anaerobic Firmicutes, Chloroflexi and Proteobacteria that have a NifB without the C- terminal NifX domain diverged early from the Firmicute lineage, supposedly shortly after the Nif lateral gene transfer event.
To determine the presence or absence of a C-terminal NifX domain in NifB polypeptides, a NifB amino acid sequence can be aligned using Constraint-based Multiple Alignment Tool (COBALT, NCBI, with representative NifB sequences such as from Klebsiella michiganensis NifB (Accession No. Pl 0930), Klebsiella michiganensis NifX (KZT46636.1), NifY (KZT46633.1), A. vinelandii NifX (AGK13791.1), NifY (AGK13792.1), NafY (AGK13761.1), and NifX/NifY/NafY/VnfX family protein (AGK14217.1). The ‘dinitrogenase FeMo- cofactor binding site’ (Pfam family PF02579) in each sequence can be identified by PfamScan (EMBL-EBI, www.ebi.ac.uk/Tools/pfa/pfamscan/), using the Pfam-A database with the expectation value set to 10.
The NifEN complex is a scaffold complex that is required for the correct assembly of dinitrogenase, functioning as the scaffold for NifB-co maturation into FeMo- co which process also requires NifH function, and is also structurally similar to the dinitrogenase (Fay et al., 2016). The NifEN complex is comprised of 2 subunits of each of NifE and NifN, respectively, forming a heterotetramer, here termed EN0C2B2. A NifE polypeptide in naturally occurring bacteria is a polypeptide which is the a subunit of the EN0C2B2 tetramer with the NifN polypeptide, and this EN0C2B2 tetramer is required for FeMo-co synthesis and is proposed to function as a scaffold on which FeMo-co is synthesized.
As used herein, a “NifE polypeptide” means a polypeptide comprising amino acids whose sequence is at least 32% identical to the amino acid sequence provided as SEQ ID NO:5 and which comprises one or both of the domains TIGR01283 and PRK14478. Members of TIGR01283 domain protein family are also members of the superfamily cl02775. A naturally occurring NifE polypeptide typically has a length of between 440 and 490 amino acids and the natural monomer has a molecular weight of about 50 kDa. A great number of NifE polypeptides have been identified and numerous sequences are available in publically available databases. For example, NifE polypeptides have been reported from Klebsiella michiganensis (Accession No. WP 049114606.1, 99% identical to SEQ ID NO:5), Klebsiella variicola (SBM87755.1, 92% identical), Dickeya paradisiaca (WP_012764127.1, 89% identical), Tolumonas auensis (WP_012728883.1, 75% identical), Pseudomonas stutzeri (WP_003297989.1, 69% identical), Azotobacter vinelandii (WP_012698965.1, 62% identical), Trichormus azollae (WP_013190624.1, 55% identical), Paenibacillus durus (WP_025698318.1, 50% identical), Sulfuricurvum kujiense (WP_013460149.1, 44% identical), Methanobacterium formicicum (AIS31022.1, 39% identical), Anaeromusa acidaminophila (WP_018701501.1, 35% identical) and Megasphaera cerevisiae (WP_048514099.1, 32% identical). As used herein, a “functional NifE polypeptide” is a NifE polypeptide which is capable of forming a functional tetramer together with NifN such that the complex is capable of synthesizing FeMo-co. This synthesis of FeMo-co involves other polypeptides including NifH and NifB and may involve NifX. NifE polypeptides have been described and reviewed in Fay et al. (2016), Hu et al. (2005), Hu et al. (2006) and Hu et al. (2008).
A NifF polypeptide in naturally occurring diazotrophs is a flavodoxin which is an electron donor to NifH. As used herein, a “NifF polypeptide” means a polypeptide comprising amino acids whose sequence is at least 34% identical to the amino acid sequence provided as SEQ ID NO: 6 and which comprises one or both of the flavodoxin long domain TIGR01752 and the flavodoxin FLDA domain found on Nif proteins from Azobacter and other bacterial genera PRK09267. NifF polypeptides encompass flavodoxins associated with pyruvate formate-lyase activation and cobalamin-dependent methionine synthase activity in non-nitrogen fixing bacteria but exclude other flavodoxins involved in broader functions. A naturally occurring NifF polypeptide typically has a length of between 160 and 200 amino acids and the natural monomer has a molecular weight of about 19 kDa. A great number of NifF polypeptides have been identified and numerous sequences are available in publically available databases. For example, NifF polypeptides have been reported from Klebsiella michiganensis (Accession No. WP_004122417.1, 99% identical to SEQ ID NO:6), Klebsiella variicola (WP_040968713.1, 85% identical), Kosakonia radicincitans (WP_035885760.1, 76% identical), Dickeya chrysanthemi (WP 039999438.1, 72% identical), Brenneria goodwinii (WP 048638838.1, 62% identical), Methylomonas methanica
(WP_064006977.1, 56% identical), Azotobacter vinelandii (WP_012698862.1, 50% identical), Chlorobaculum tepidum (WP 010933399.1, 39% identical), Campylobacter showae (WP_002949173.1, 37% identical) and Azotobacter chromococcum (WP_039801725.1, 34% identical). As used herein, a “functional NifF polypeptide” is a NifF polypeptide which is capable of being an electron donor to a NifH polypeptide. NifF polypeptides have been described and reviewed in Drummond (1985).
As used herein, an “AnfG polypeptide” is a member of the nitrogenase conserved superfamily cl03910 (pfam03139-AnfG), containing the TIGR02929 conserved domain, and having at least 42% amino acid sequence identity to WQ Azotobacter vinelandii AnfG polypeptide (SEQ ID NO:38; Accession No. WP_012703360) when measured along the full-length of SEQ ID NO:38. This amino acid sequence is used herein as the reference sequence for AnfG. TIGR02929 represents the all-iron variant of the nitrogenase component I 5-chain. AnfG polypeptides do not include the vanadium type NifG polypeptides (VnfG). The amino acid sequences of AnfG polypeptides in the protein sequence database are usually annotated as an AnfG polypeptide. As of January 2020, there were 150 specific amino acid sequences in the protein database in the AnfG set. Examples of naturally occurring AnfG polypeptides include AnfG polypeptides from Azomonas agilis (Accession No. WP_144571041; 84.73% identical), Firmicutes bacterium (Accession No. HBE76208; 70.37% identical), Sporomusa termitida (Accession No. WP_144349445; 68.75% identical), Rhodovulum viride (Accession No. WP_1 12317428; 57.14% identical) and Megasphaera cerevisiae (Accession No. WP_048515315; 42.86% identical), each with reference to SEQ ID NO:38.
Functional AnfG polypeptides are capable of functioning as the 5 protein structural component of the 012P262 heterohexameric nitrogenase.
A NifI polypeptide in naturally occurring bacteria is a pyruvate :flavodoxin (ferredoxin) oxidoreductase which is an electron donor to NifH. As used herein, a “NifI polypeptide” means a polypeptide comprising amino acids whose sequence is at least 40% identical to the amino acid sequence provided as SEQ ID NO: 7 and which comprises the conserved domain TIGR02176. A naturally occurring NifI polypeptide typically has a length of between 1100 and 1200 amino acids and the natural monomer has a molecular weight of about 128 kDa. A great number of NifI polypeptides have been identified and numerous sequences are available in publically available databases. For example, NifI polypeptides have been reported from Klebsiella michiganensis (Accession No. WP_024360006.1, 99% identical to SEQ ID NO:7), Raoultella ornithinolytica (WP 044347157.1, 95% identical), Klebsiella quasipneumoniae (WP_050533844.1, 92% identical), Kosakonia oryzae (WP_064566543.1, 82% identical), Dickeya solani (WP 057084649.1, 78% identical), Rahnella aquatilis (WP_014683040.1, 72% identical), Thermoanaerobacter mathranii (WP_013149847.1, 64% identical), Clostridium botulinum (WP 053341220.1, 60% identical), Spirochaeta africana (WP_014454638.1, 52% identical) and Vibrio cholerae (CSA83023.1, 40% identical). As used herein, a “functional NifJ polypeptide” is a Niff polypeptide which is capable of being an electron donor to a NifH polypeptide. NifI polypeptides have been described and reviewed in Schmitz et al. (2001).
A NifM polypeptide in naturally occurring bacteria is a polypeptide required for maturation of some but not all NifH polypeptides. In the absence of NifM, K oxytoca NifH was present at only low levels in E. coli and yeast when expressed heterologously and was not able to donate electrons to NifD-NifK. As used herein, a “NifM polypeptide” means a polypeptide comprising amino acids whose sequence is at least 26% identical to the amino acid sequence provided as SEQ ID NO: 8 and which comprises the domain TIGR02933. NifM polypeptides are homologous to peptidyl- prolyl cis-trans isomerases (PPIase), a group of enzymes that promote protein folding by catalysing the cis-trans isomerisation of proline imidic peptide bonds, having a PpiC- type domain, and appear to be accessory proteins for some NifH polypeptides, including at least some VnfH and AnfH polypeptides. A naturally occurring NifM polypeptide typically has a length of between 240 and 300 amino acids and the natural monomer has a molecular weight of about 30 kDa. A great number of NifM polypeptides have been identified and numerous sequences are available in publically available databases. For example, NifM polypeptides have been reported from Klebsiella oxytoca (Accession No. WP_064342940.1, 99% identical to SEQ ID NO:8), Klebsiella michiganensis (WP_004122413.1, 97% identical), Raoultella ornithinolytica (WP_044347181.1, 85% identical), Klebsiella variicola (WP 063105800.1, 75% identical), Kosakonia radicincitans (WP 035885759.1, 59% identical), Pectobacterium atrosepticum (WP_011094472.1, 42% identical), Brenneria goodwinii (WP_048638837.1, 33% identical), Pseudomonas aeruginosa PAO1 (CAA75544.1, 28% identical), Marinobacterium sp. AK27 (WP_051692859.1, 27% identical) and Teredinibacter turnerae (WP_018415157.1, 26% identical). As used herein, a “functional NifM polypeptide” is a NifM polypeptide which is capable of complexing with a NifH polypeptide for maturation of the NifH polypeptide. NifM polypeptides have been described and reviewed in Petrova et al. (2000).
A NifN polypeptide in naturally occurring bacteria is the B subunit of the EN0C2B2 tetramer with the NifE polypeptide, and the EN0C2B2 tetramer is required for FeMo-co synthesis and is proposed to function as a scaffold on which FeMo-co is synthesized. As used herein, a “NifN polypeptide” means (i) a polypeptide comprising amino acids whose sequence is at least 76% identical to the sequence provided as SEQ ID NO:9 and/or (ii) a polypeptide comprising amino acids whose sequence is at least 34% identical to the sequence provided as SEQ ID NO:9 and which comprises one or more of the conserved domains TIGR01285, cd01966 and PRK14476. NifN is related in structure to the molybdenum-iron protein B chain NifK. Polypeptides comprising the conserved TIGR01285 covers most examples of NifN polypeptides but excludes some NifN polypeptides, such as the putative NifN of Chlorobium tepidum, and therefore the definition of NifN is not limited to polypeptides comprising the conserved TIGR01285 domain. Members of PRK14476 domain protein family are also members of the superfamily cl02775. A naturally occurring NifN polypeptide typically has a length of between 410 and 470 amino acids, although when fused naturally to NifE it may have about 900 amino acid residues, and the natural monomer has a molecular weight of about 50 kDa. A great number of NifN polypeptides have been identified and numerous sequences are available in publically available databases. For example, NifN polypeptides have been reported from Klebsiella oxytoca (Accession No. WP 064391778.1, 97% identical to SEQ ID NO:9), Kluyvera intermedia (WP_047370268.1, 80% identical), Rahnella aquatilis (WP_014683026.1, 70% identical), Brenneria goodwinii (WP 048638830.1, 65% identical), Methylobacter tundripaludum (WP 027147663.1, 46% identical), Calothrix parietina (WP_015195966.1, 41% identical), Zymomonas mobilis (WP_023593609.1, 37% identical), Paenibacillus massiliensis (WP 025677480.1, 35% identical) and Desulfitobacterium hafniense (WP_018306265.1, 34% identical). As used herein, a “functional NifN polypeptide” is a NifN polypeptide which is capable of forming a functional tetramer together with NifE such that the complex is capable of synthesizing FeMo-co. NifN polypeptides have been described and reviewed in Fay et al. (2016), Brigle et al. (1987), Fani et al. (2000), and Hu et al. (2005).
A NifQ polypeptide in naturally occurring bacteria is a polypeptide involved in FeMo-co synthesis, probably in early MoCh2' processing. The conserved C-terminal cysteine residues may be involved in metal binding. As used herein, a “NifQ polypeptide” means a polypeptide comprising amino acids whose sequence is at least 34% identical to the amino acid sequence provided as SEQ ID NO: 10 and which is a member of the CL04826 domain protein family and a member of the pfam04891 domain protein family. A naturally occurring NifQ polypeptide typically has a length of between 160 and 250 amino acids, although they may be as long as 350 amino acid residues, and the natural monomer has a molecular weight of about 20 kDa. A great number of NifQ polypeptides have been identified and numerous sequences are available in publically available databases. For example, NifQ polypeptides have been reported from Klebsiella oxytoca (Accession No. WP_064391765.1, 95% identical to SEQ ID NO: 10), Klebsiella variicola (CTQ06350.1, 75% identical), Kluyvera intermedia (WP_047370257.1, 63% identical), P ectobacterium atrosepticum (WP 043878077.1, 59% identical), Mesorhizobium metallidurans (WP 008878174.1, 46% identical), Rhodopseudomonas palustris (WP_011501504.1, 42% identical), Paraburkholderia sprentiae
(WP 027196569.1, 41% identical), Burkholderia stabilis (GAU06296.1, 39% identical) and Cupriavidus oxalaticus (WP 063239464.1, 34% identical). As used herein, a “functional NifQ polypeptide” is a NifQ polypeptide which is capable of processing MOO42'. NifQ polypeptides have been described and reviewed in Allen et al. (1995) and Siddavattam et al. (1993).
A NifS polypeptide in naturally occurring bacteria is a cysteine desulfurase involved in iron-sulfur (FeS) cluster biosynthesis e.g. which is involved in mobilisation of sulfur for Fe-S cluster synthesis and repair. As used herein, a “NifS polypeptide” means (i) a polypeptide comprising amino acids whose sequence is at least 90% identical to the amino acid sequence provided as SEQ ID NO: 19 and/or (ii) a polypeptide comprising amino acids whose sequence is at least 36% identical to the sequence provided as SEQ ID NO: 19 and which comprises one or both of the conserved domains TIGR03402 and COG1104. The TIGR03402 domain protein family includes a clade nearly always found in extended nitrogen fixation systems plus a second clade more closely related to the first than to IscS and also part of NifS-like/NifU-like systems. The TIGR03402 domain protein family does not extend to a more distant clade found in the epsilon proteobacteria such as Helicobacter pylori, also named NifS in the literature, built instead in TIGR03403. The COG1104 domain protein family includes cysteine sulfinate desulfinase/cysteine desulfurase or related enzymes. Some NifS polypeptides include the asparate aminotransferase domain cl 18945. A naturally occurring NifS polypeptide typically has a length of between 370 and 440 amino acids and the natural monomer has a molecular weight of about 43 kDa. A great number of NifS polypeptides have been identified and numerous sequences are available in publically available databases. For example, NifS polypeptides have been reported from Klebsiella michiganensis (Accession No. WP_004138780.1, 99% identical to SEQ ID NO: 19), Raoultella terrigena (WP 045858151.1, 89% identical), Kluyvera intermedia (WP_047370265.1, 80% identical), Rahnella aquatilis (WP_014333911.1, 73% identical), Agarivorans gilvus (WP_055731597.1, 64% identical), Azospirillum brasilense (WP_014239770.1, 60% identical), Desulfosarcina cetonica
(WP_054691765.1, 55% identical), Clostridium intestinale (WP_021802294.1, 47% identical), Clostridiisalibacter paucivorans (WP 026894054.1, 36% identical) and Bacillus coagulans (WP 061575621.1, 42% identical and which is in COG1104). As used herein, a “functional NifS polypeptide” is a NifS polypeptide which is capable of functioning in iron-sulfur (FeS) cluster biosynthesis and/or repair. NifS polypeptides have been described and reviewed in Clausen et al. (2000), Johnson et al. (2005), Olson et al. (2000) and Yuvaniyama et al. (2000).
A NifU polypeptide in naturally occurring bacteria is a molecular scaffold polypeptide involved in iron-sulfur (FeS) cluster biosynthesis for nitrogenase components. As used herein, a “NifU polypeptide” means a polypeptide comprising amino acids whose sequence is at least 31% identical to the sequence provided as SEQ ID NO: 12 and which comprises the domain TIGR02000. Members of the TIGR02000 domain protein family are specifically involved in nitrogenase maturation. NifU comprises an N-terminal domain (pfam01592) and a C-terminal domain (pfam01106). Three different but partially homologous Fe-S cluster assembly systems have been described: Isc, Suf, and Nif. The Nif system, of which NifU is a part, is associated with donation of an Fe-S cluster to nitrogenase in a number of nitrogen-fixing species. Isc and Suf homologs with an equivalent domain architecture from Helicobacter and Campylobacter are excluded from the definition of NifU herein. NifU, therefore, is specific for NifU polypeptides involved in nitrogenase maturation. Members of the related TIGR01999 domain protein family which are IscU proteins (from for example, Escherichia, coli and Saccharomyces cerevisiae and Homo sapiens) that comprise a homolog of the N-terminal region of NifU are also excluded from the definition of NifU herein. A naturally occurring NifU polypeptide typically has a length of between 260 and 310 amino acids and the natural monomer has a molecular weight of about 29 kDa. A great number of NifU polypeptides have been identified and numerous sequences are available in publically available databases. For example, NifU polypeptides have been reported from Klebsiella michiganensis (Accession No. WP 049136164.1, 97% identical to SEQ ID NO: 12), Klebsiella variicola (WP 050887862.1, 90% identical), Dickeya solani (WP 057084657.1, 80% identical), Brenneria goodwinii
(WP_048638833.1, 73% identical), Tolumonas auensis (WP_012728889.1, 66% identical), Agarivorans gilvus (WP 055731596.1, 58% identical), Desulfocurvus vexinensis (WP 028587630.1, 54% identical), Rhodopseudomonas palustris (WP_044417303.1, 49% identical), Helicobacter pylori (WP_001051984.1, 31% identical) and Sulfurovum sp. PC08-66 (KIM05011.1, 31% identical). As used herein, a “functional NifU polypeptide” is a NifU polypeptide which is capable of functioning as a molecular scaffold polypeptide involved in iron-sulfur (FeS) cluster biosynthesis. NifU polypeptides have been described and reviewed in Hwang et al. (1996), Muhlenhoff et al. (2003) and Ouzounis et al. (1994). NifS is a pyridoxal phosphate (PLP, vitamin B6) dependent cysteine desulfurase which generates the inorganic sulphide required for Fe-S cluster synthesis from cysteine. The reaction produces alanine as a byproduct. The reaction proceeds via a protein-bound cysteine persulfide intermediate that is formed by the nucleophilic attack of a highly conserved cysteine residue (Cys325 in Azotobacter vinelandii) on the cysteine-PLP adduct (Zheng et al., 1994). The sulphide is the provided to NifU for the sequential formation of [Fe2S2] and [Fe4S4] clusters. The NifS enzyme functions in bacteria as a homodimer.
NifU provides a scaffold for [Fe4S4] cluster formation, functioning as a homodimer. The NifU polypeptide contains three domains, namely a N-terminal scaffolding domain, a central domain and a C-terminal scaffolding domain (Smith et al., 2005). The N-terminal domain has a high sequence homology to IscU proteins from bacteria and Isu proteins from eukaryotes, while the C-terminal domain is homologous to Nfu proteins found in mitochondria and chloroplasts. The central domain contains one permanent redox-active [Fe2S2]2+ cluster per NifU subunit which, due to its stability, is thought not to be transferred to other Nif proteins. That cluster is thought to be coordinated by four conserved cysteine residues (Cysl37, 139, 172 and 175 in A. vinelandii NifU) (Fu et al., 1994). In bacteria, NifU forms a homodimer and its N- terminal domain can bind one [Fe2S2] cluster per monomer. The [Fe2S2] clusters in the monomers can be reductively fused to form one [Fe4S4] cluster per NifU dimer. A pair of [Fe4S4] clusters are then delivered from NifU to NifB and processed into an 8Fe core on NifB which is subsequently used for the synthesis of FeMoco. In a divergent pathway for the Fe-S clusters, one [Fe4S4] cluster bound to either the N-terminal or C-terminal scaffolding domain of NifU is transferred to apo-NifH for maturation of nitrogenase reductase, the NifH protein (Smith et al., 2005). It has been proposed that NifU also donates two [Fe4S4] clusters to a NifD-NifK protein complex (designated herein as stage 0 D-K), and that NifH condenses that pair of clusters into a mature P-cluster [Fes-S?] (Dos Santos et al., 2004). These N-terminal clusters are thought to be extremely labile and are not retained during purification (Smith et al., 2005). The C terminal domain can hold one [Fe4S4] cluster per monomer. In contrast to the N-terminal cluster, the assembly of the C terminal [Fe4S4] cluster is rapid and no intermediate [Fe2S2] cluster has been detected (Smith et al., 2005). The C-terminal clusters are more stable than the N-terminal clusters and can be retained during purification. However, upon reduction with dithionite, the C-terminal clusters are rapidly degraded (Smith et al., 2005). Using cysteine to alanine mutations in NifU, Dos Santos and colleagues showed that both the N- and C- terminal clusters can be transferred to apo-NifH. Lopez-Torrejon et al. (2016) reported that a NifH protein capable of donating electrons to holoNifD-NifK can be generated within yeast mitochondria via the expression of both NifH and NifM. These authors found that, in the yeast cells, NifS and NifU were not required for the generation of NifH protein with this function. They concluded that endogenous iron sulphur cluster assembly pathways in the yeast cells, presumably mitochondrial -located Nfsl and Nful proteins which are related proteins in yeast, were capable of donating [Fe4S4] clusters to NifH. It therefore is possible that NifS and NifU will not be required for reconstituting the NifH protein, the Fe-protein or dinitrogenase reductase in yeast, but NifS and NifU may be required for NifB and/or NifD-NifK maturation and function. Whether plant mitochondria have similar endogenous ability for forming sufficient [Fe4S4] clusters for nitrogenase activity is unknown.
A NifV polypeptide in naturally occurring bacteria is a homocitrate synthase (EC 2.3.3.14), producing homocitrate by the transfer of the acetyl group from acetylcoenzyme A (acetyl-CoA) to 2-oxoglutarate. Homocitrate is then used in the synthesis of FeMo-co, FeV-co and FeFe-co. As used herein, a “NifV polypeptide” means a polypeptide comprising amino acids whose sequence is at least 39% identical to the amino acid sequence provided as SEQ ID NO: 13 and which comprises one or both of the domains TIGR02660 and DRE TIM. Members of the TIGR02660 domain protein family are homologous to enzymes that include 2-isopropylmalate synthase, (R)- citramalate synthase, and homocitrate synthase associated with processes other than nitrogen fixation. The cd07939 domain protein family also includes the NifV proteins of Heliobacterium chlorum and Gluconacetobacter diazolrophicus. which appear to be orthologous to FrbC. This family belongs to the DRE-TIM metallolyase superfamily. DRE-TIM metallolyases include 2-isopropylmalate synthase (IPMS), alphaisopropylmalate synthase (LeuA), 3 -hydroxy-3 -methylglutaryl-CoA lyase, homocitrate synthase, citramalate synthase, 4-hydroxy-2-oxovalerate aldolase, re-citrate synthase, transcarboxylase 5S, pyruvate carboxylase, AksA, and FrbC. These members all share a conserved triose-phosphate isomerase (TIM) barrel domain consisting of a core beta(8)-alpha(8) motif with the eight parallel beta strands forming an enclosed barrel surrounded by eight alpha helices. The domain has a catalytic center containing a divalent cation-binding site formed by a cluster of invariant residues that cap the core of the barrel. In addition, the catalytic site includes three invariant residues - an aspartate (D), an arginine (R), and a glutamate (E) - which is the basis for the domain name "DRETIM". A naturally occurring NifV polypeptide typically has a length of between 360 and 390 amino acids, although some members are about 490 amino acid residues in length, and the natural monomer has a molecular weight of about 41 kDa. A great number of NifV polypeptides have been identified and numerous sequences are available in publically available databases. For example, NifV polypeptides have been reported from Klebsiella michiganensis (Accession No. WP 049083341.1, 95% identical to SEQ ID NO: 13), Raoultella ornithinolytica (WP 045858154.1, 86% identical), Kluyvera intermedia (WP_047370264.1, 81% identical), Dickeya dadantii (WP_038912041.1, 70% identical), Brenneria goodwinii (WP_048638835.1, 59% identical), Magnetococcus marinus (WP_011712856.1, 46% identical), Sphingomonaswittichii (WP_037528703.1, 43% identical), Frankia sp. EI5c (OAA29062.1, 41% identical) and Clostridium sp. Maddingley MBC34-26 (EKQ56006.1, 39% identical). As used herein, a “functional NifV polypeptide” is a NifV polypeptide which is capable of functioning as a homocitrate synthase. NifV polypeptides have been described and reviewed in Hu et al. (2008), Lee et al. (2000), Masukawa et al. (2007) and Zheng et al. (1997).
NifX polypeptide in Azotobacter vinelandii binds NifB-co (Fee-S9-C), which is passed on to NifE-NifN for FeMo-co assembly (Hernandez et al., 2007). It has also been shown to exchange VK-clusters (Fes-Ssi-C or Mo-Fe?-S9-C) between NifE-NifN, suggesting its role as a transient reservoir for FeMo-co precursors. Hernandez et al. (2007) reported that NifX may act as a chaperone that stabilises the NifE-NifN or NifD- NifK complexes during transfer of FeMo-co to apo-NifD-NifK, and/or reposition the proteins in a favorable orientation for FeMoco transfer and so act to regulate FeMoco synthesis. Activation of apo-NifD-NifK by exogenous FeMo-co with dinitrogenase complexes extracted from A. vinelandii mutants deficient in different accessory protein combinations of NifY/NafY/NifX indicated that NifX can also assist in FeMo-co insertion of apo-NifD-NifK (Rubio et al., 2002). This additional function of NifX may be responsible for the retention of acetylene reduction activity in the Klebsiella AnifY mutant shown by Homer et al. (1993).
A NifX polypeptide in naturally occurring bacteria is a polypeptide which is involved in FeMo-co synthesis, at least assisting in transferring FeMo-co precursors from NifB to NifE-NifN or FeMo-co to NifD-NifK. As used herein, a “NifX polypeptide” means a polypeptide comprising amino acids whose sequence is at least 29% identical to the amino acid sequence provided as SEQ ID NO: 14 and which comprises one or both of the conserved domains TIGR02663 and cd00853. NifX is included in a larger family of iron-molybdenum cluster-binding proteins that includes some NifB sequences and NifY, in that NifX, NafY and the C-terminal region of some NifB polypeptides all comprise the pfam02579 domain, and each are involved in the synthesis of one or more or all of FeMo-co, FeV-co or FeFe-co. Other NifB polypeptides, specifically from methanogenic archaea and some anaerobic firmicutes, lack a NifX-like domain (Boyd et al., 2011), including NifB from H. halophila. M. barkeri and C. purinilyticum mentioned above. Some NifX polypeptides have been annotated in databases as NifY, and vice versa. A naturally occurring NifX polypeptide, produced on its own rather than as a natural fusion as part of a NifB polypeptide, typically has a length of between 110 and 160 amino acids and the natural monomer has a molecular weight of about 15 kDa. A great number of NifX polypeptides have been identified and numerous sequences are available in publically available databases. For example, NifX polypeptides have been reported from Klebsiella michiganensis (Accession No. WP_049070199.1, 97% identical to SEQ ID NO: 14), Klebsiella oxytoca (WP_064342937.1, 97% identical), Raoultella ornithinolytica (WP 044347173.1, 91% identical), Klebsiella variicola (WP_044612922.1, 83% identical), Kosakonia radicincitans (WP_043953583.1, 75% identical), Dickeya chrysanthemi (WP 039999416.1, 68% identical), Rahnella aquatilis (WP_047608097.1, 58% identical), Azotobacter chroococcum (WP_039800848.1, 34% identical), Beggiatoa leptomitiformis (WP 062149047.1, 33% identical) and Methyloversatilis discipulorum (WP_020165972.1, 29% identical). As used herein, a “functional NifX polypeptide” is a NifX polypeptide which is capable of transferring FeMo-co precursors from NifB to NifE-NifN. NifX polypeptides have been described and reviewed in Allen et al. (1994) and Shah et al. (1999).
A NifY polypeptide in naturally occurring bacteria is a polypeptide which is involved in FeMo-co synthesis, at least assisting in transferring FeMo-co precursors from NifB to NifE-NifN. As used herein, a “NifY polypeptide” means a polypeptide comprising amino acids whose sequence is at least 34% identical to the amino acid sequence provided as SEQ ID NO: 15 and which comprises one or both of the conserved domains TIGR02663 and cd00853. NifY is included in a larger family of ironmolybdenum cluster-binding proteins that includes NifB and NifX, in that NifX, NafY and the C-terminal region of NifB all comprise the pfam02579 domain, and each are involved in the synthesis of FeMo-co. A great number of NifY polypeptides have been identified and numerous sequences are available in publically available databases. For example, NifY polypeptides have been reported from Klebsiella michiganensis (Accession No. WP_049089500.1, 99% identical to SEQ ID NO: 15), Klebsiella oxytoca (WP_064342935.1, 98% identical), Klebsiella quasipneumoniae (WP_044524054.1, 90% identical), Klebsiella variicola (WP_049010739.1, 81% identical), Kluyvera intermedia (WP_047370270.1, 69% identical), Dickeya chrysanthemi
(WP 039999411.1, 62% identical), Serratia sp. ATCC 39006 (WP_037382461.1, 57% identical), Rahnella aquatilis (WP 014683024.1, 47% identical), Pseudomonas putida (AEX25784.1, 37% identical) and Azotobacter vinelandii (WP_012698835.1, 34% identical). As used herein, a “functional NifY polypeptide” is a NifY polypeptide which is capable of transferring FeMo-co precursors from NifB to NifE-NifN.
When isolated from NifB or NifN-NifE mutant strains of either K oxytoca or A. vinelandii, apo-NifD-NifK was associated with an additional polypeptide termed the y protein (Paustian et al, 1990; Homer et al., 1993), forming a heterohexamer with NifD and NifK polypeptides (012P2Y2). In K oxytoca, the third polypeptide was encoded by the NifY gene (Homer et al., 1993) and the addition of purified FeMo-co to purified heterohexamer 012 272 complex was sufficient to yield catalytically active nitrogenase. Addition of FeMo-co resulted in dissociation of NifY from the complex with formation of the holoenzyme (01232). In A. vinelandii, the third polypeptide was encoded by the NafY gene (nitrogenase associated factor Y; Accession No. AGK13761, Rubio et al., 2002) which was different but related to the product of the NifY gene in A. vinelandii (Accession No. AGK13792). The third polypeptide in each case was thought to be involved in assisting in the insertion of FeMo-co to form the active enzyme. This was supported by the ability of NafY and NifY to bind FeMo-co (Homer et al., 1995).
A. vinelandii NifY and NafY bind to apo-NifD-NifK, at different stages of NifD- NifK holoenzyme maturation, to either a-Cys275 or a-His442 of NifD, both amino acid residues of which covalently anchor FeMo-co (Jimenez-Vincente et al., 2018). That is, NifY and NafY do not bind to apo-NifD-NifK simultaneously. The order of binding of NifY and NafY to apo-NifD-NifK is currently unknown. Dissociation of NifY from NifD-NifK upon FeMo-co insertion has been demonstrated for K. oxytoca nitrogenase (Homer et al., 1993) and NafY from NifD-NifK upon FeMo-co insertion for vinelandii (Homer et al., 1995). NafY is also thought to bind FeMo-co through His121 and possibly NifB-co as well, suggesting its role as a FeMo-co or FeMo-co precursor insertase (Rubio et al., 2004). A. vinelandii NifY seems to be functionally redundant based on lack of a phenotype in AnifY mutants (Rubio et al., 2002) and NafY is proposed to be the primary accessory protein to apo-NifD-NifK that supports FeMo-co insertion. On the other hand, Klebsiella species do not have a NafY gene and only have NifY to support FeMo-co insertion into apo-NifD-NifK, although a Klebsiella AnifY mutant still retained 60% of acetylene reduction activity (Homer et al., 1993). This retention of function indicated presence of another accessory protein in Klebsiella that could partially cover NifY function in its absence, such as NifX as described above.
As used herein, a “NafY polypeptide” means a polypeptide comprising amino acids whose sequence is at least 50% identical to the sequence provided as SEQ ID NO:48 (A. vinelandii NafY, Accession No. AGK13761, 243aa) along its full-length and which comprises the conserved domain pfam 16844. This domain of about 91 amino acid residues in length is found by itself in some members and in the amino terminal half of longer NafY proteins. This region is negatively charged and appears to function for recognising and interacting with apo-NifD-NifK. A naturally occurring NafY polypeptide typically has a length of between 230 and 250 amino acids and the natural monomer has a molecular weight of -25-28 kDa. A great number of NafY polypeptides have been identified and numerous sequences are available in publically available databases; some have been annotated as NifX polypeptides because of the relatedness of NafY and NifX sequences. For example, NafY polypeptides have been reported from Azotobacter beijerinckii (WP 090728988, 93% identical to SEQ ID NO:48), Pseudomonas slulzeri. (WP 011912501, 69% identical), Halomonas endophytica (WP_102654474, 68% identical), Pseudomonas linyingensis (WP_090313081, 67% identical), Acidihalobacter prosperus (WP 038093031, 56% identical), Oscillatoriales cyanobacterium (WP 009769409, 50% identical) As used herein, a “functional NafY polypeptide” is a NafY polypeptide which is capable of binding to apo-NifD-NifK and to FeMo-co. The three-dimensional structure of NafY polypeptide from A. vinelandii and a comparison and distinction of NafY and NifY, NifX, VnfX and NifB polypeptide sequences was reported in Dyer et al. (2003).
A NifZ polypeptide in naturally occurring bacteria is a polypeptide which is involved in Fe-S cluster synthesis, specifically functioning in the coupling of a second Fe4S4 pair in the formation of the second P-cluster of the MoFe protein. NifZ is thought to act as a chaperone that induces a conformational change in at least the second half of apo-MoFe protein, allowing for the formation of the second P-cluster together with NifH. Deletion of NifZ in A. vinelandii decreased MoFe protein activity by 66% but had no effect on NifH activity. As used herein, a “NifZ polypeptide” means a polypeptide comprising amino acids whose sequence is at least 28% identical to the sequence provided as SEQ ID NO: 16 and which comprises the conserved domain pfam04319. This domain of about 75 amino acid residues is found in isolation in some members and in the amino terminal half of the longer NifZ proteins. A naturally occurring NifZ polypeptide typically has a length of between 70 and 150 amino acids and the natural monomer has a molecular weight of about 9 to about 16 kDa. A great number of NifZ polypeptides have been identified and numerous sequences are available in publically available databases. For example, NifZ polypeptides have been reported from Klebsiella michiganensis (Accession No. WP_057173223.1, 93% identical to SEQ ID NO: 16), Klebsiella oxytoca (WP 064342939.1, 95% identical), Klebsiella variicola (WP_043875005.1, 77% identical), Kosakonia radicincitans (WP_043953588.1, 67% identical), Kosakonia sacchari (WP 065368553.1, 58% identical), Ferriphaselus amnicola (WP 062627625.1, 47% identical), Paraburkholderia xenovorans (WP_011491838.1, 41% identical), Acidithiobacillus ferrivorans (WP_014029050.1, 35% identical) and Bradyrhizobium oligotrophicum (WP_015665422.1, 28% identical). As used herein, a “functional NifZ polypeptide” is a NifZ polypeptide which is capable of coupling a Fe4S4 cluster in Fe-S cluster synthesis. NifZ polypeptides have been described and reviewed in Cotton (2009) and Hu et al. (2004).
A NifW polypeptide in naturally occurring bacteria is a polypeptide which associates with NifZ polypeptide to form higher order complexes (Lee et al., 1998), and is involved in MoFe protein (NifD-NifK) synthesis or activity. NifW and NifZ appear to be involved in the formation or accumulation of MoFe protein (Paul and Merrick, 1987). As used herein, a “NifW polypeptide” means a polypeptide whose amino acid sequence comprises amino acids whose sequence is at least 28% identical to the amino acid sequence provided as SEQ ID NO: 17 and which comprises the conserved NifW superfamily protein domain, architecture ID number 10505077 and is in Pfamily PF03206. A number of NifW polypeptides have been identified and numerous sequences are available in publically available databases. For example, NifW polypeptides have been reported from Klebsiella oxytoca (Accession No. WP_064342938.1, 98% identical to SEQ ID NO: 17), Klebsiella michiganensis (WP 049080155.1, 94% identical), Enterobacter sp. 10-1 (WP 095103586.1, 90% identical), Klebsiella quasipneumoniae (WP 065877373. I , 81% identical), P ectobacterium polaris (WP_095699971.1, 69% identical), Dickeya paradisiaca (WP 012764136.1, 58% identical), Brenneria goodwinii (WP_053085547.1, 36% identical), Aquaspirillum sp. LM1 (WP_077299824.1, 44% identical), Candidatus Muproteobacteria bacterium RBG 16 64 10 (OGI40729, 34% identical), Azotobacter vinelandii (ACO76430.1, 32% identical) and Methylocaldum marinum (BBA37427.1, 28% identical). As used herein, a “functional NifW polypeptide” is a NifW polypeptide which promotes or enhances one or more of the formation, accumulation or activity of MoFe protein. A functional NifW may interact with NifZ and/or play a role in the oxygen protection of the MoFe-protein (Gavini et al., 1998).
Most organisms including both bacteria and eukaryotes such as plants have numerous ferredoxins. For example, there are 15 or 16 proteins annotated as ferredoxin or ferredoxin-like in the vinelandii DJ and CA genomes, respectively. As used herein, a “ferredoxin polypeptide” is an electron carrier protein having one or two iron-sulfur clusters of the [2Fe-2S], [3Fe-4S] and/or [4Fe-4S] type that form their reactive centers, see review by Matsubara and Saeki (1992). They are involved in a variety of metabolic processes, including ferredoxin polypeptides which are involved in nitrogen fixation, generally of lower molecular weight than those not involved in nitrogenase. Based on the wide diversity of ferredoxins in most cells and the variations observed in several studies on the compatibility or specificity of different ferredoxins in complementing the function of FdxN forNifB-co synthesis (Yates, 1972; Jimenez- Vincente et al., 2014), ferredoxins including ones such as FdxN are best defined based on the presence of the iron-sulfur clusters and their function rather than on amino acid identity to a standard sequence such as A. vinelandii FdxN (SEQ ID NO:47; Accession No. WP_012703542). As used herein, a “FdxN polypeptide” is a ferredoxin or ferredoxin-like polypeptide which functions for donating electrons to mature dinitrogenase reductase NifH and/or for NifB-co synthesis for nitrogenase and/or serves as an intermediate carrier of [4Fe-4S] clusters. FdxN may function by donating electrons to mature dinitrogenase reductase NifH which then transfers the electrons to NifD-NifK heterohexamer (see Yang et al., 2017; Rhizobium japonicum FdxN, Carter et al., 1980; R. meliloti FdxN, Riedel et al., 1995; Rhodobacter capsulatus FdxN, Jouanneau et al., 1995), or donating electrons to NifB polypeptide for NifB-co synthesis (A. vinelandii'. Jimenez- Vincente et al., 2014), or serves as an intermediate carrier of [4Fe-4S] clusters (A. vinelandii'. Buren et al., 2019), or a combination of any of these functions.
Representative examples of FdxN polypeptides include the following, identified by searching the non-redundant protein database using SEQ ID NO:47 as query in BLASTP and showing percentage identity to that sequence: Pseudomonas syringae (WP 065835964.1, 85.87%), Candidatus Thiodiazotropha endolucinida
(WP_069124666.1, 70.65%), Uliginosibacterium sp. TH139 (WP_101942980, 64.47%), Klebsiella michiganensis (WP 049076934. I , 44.26%), Escherichia coli
(WP_072048756.1, 44.26%), Rhizobium leguminosarum (WPJ30674512.1, 43.86%) and Flavobacterium alvei (WP_103805005.1, 28.57%).
Sequence Identity and Substitutions
With regard to a defined polypeptide, it will be appreciated that % identity figures higher than those provided above will encompass preferred embodiments. Thus, where applicable, in light of the minimum % identity figures, it is preferred that the polypeptide comprises an amino acid sequence which is at least 30%, more preferably at least 35%, more preferably at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant nominated SEQ ID NO.
As used herein, the “free energy” or “AG” refers to the Gibbs free energy in protein folding which is directly related to enthalpy and entropy as a polypeptide folds into its active structure. For a negative AG to arise and for protein folding to become thermodynamically favorable, then either enthalpy, entropy, or both terms must be favorable. Methods of determining the free energy of a polypeptide are known in the art and include those described in Leman et al. (2020).
As used herein, “AAG” is a measure of the change in energy between the folded and unfolded states (AG folding) and the change in AG folding when an amino acid substitution is present.
As used herein, a wild-type polypeptide is a polypeptide that exists in nature, examples of which are provided herein. In one instance, the wild-type NifH polypeptide has a sequence of amino acids as provided in SEQ ID NO: 1 or 39.
Amino acid sequence variants of the polypeptides defined herein can be prepared by introducing appropriate nucleotide changes into a nucleic acid defined herein. Such variants include for example, one or more amino acid deletions, insertions, or substitutions. A combination of deletion, insertion and substitution mutations can be made to arrive at the final construct, provided that the final polypeptide product possesses the desired characteristics. Preferred amino acid sequence mutants have only one, two, three, four or less than 10 amino acid changes relative to the reference wild-type polypeptide. Nevertheless, more preferred NifH polypeptides of the invention are NifH polypeptides which have at least one amino acid substitution, for example 2-5 or 3-5 substitutions, and no deletions or insertions relative to a corresponding wild-type NifH polypeptide.
Mutant (altered) or variant polypeptides can be prepared using any technique known in the art, for example, using directed evolution or rational design strategies (see below). Products derived from mutated/altered DNA can readily be screened using techniques described herein to determine if their expression in a plant alters the properties of the mutant or variant polypeptide, for example for its solubility in mitochondria of the plant cells, or the plant phenotype relative to a corresponding wild-type plant, for example, if their expression results in increased yield, biomass, growth rate, vigor, nitrogen gain derived from biological nitrogen fixation, nitrogen use efficiency, abiotic stress tolerance, and/or tolerance to nutrient deficiency relative to the corresponding wild-type plant.
In designing amino acid sequence variants, the location of the variation site and the nature of the variation will depend on characteristic(s) to be modified. The sites for variation can be modified individually or in series for example, by (1) substituting first with conservative amino acid choices and then with more radical selections depending upon the results achieved, (2) deleting the target residue, or (3) inserting other residues adjacent to the located site.
Polypeptides of the invention that have at least one amino acid substitution have at least one amino acid residue in the wild-type polypeptide molecule removed and a different residue inserted in the place of each amino acid removed i.e. a swap or substitution of each of the at least one amino acids. Where multiple amino acids are substituted, each new amino acid is selected independently of the others. Where it is desirable to maintain a certain activity it is preferable to make no, or only conservative substitutions, at amino acid positions which are highly conserved in the relevant protein family. Examples of conservative substitutions are shown in Table 1 under the heading of "exemplary substitutions". Preferred amino acid substitutions, which may not be conservative substitutions, in the NifH polypeptides of the invention, preferably in AnfH polypeptides, are described in Tables 4, 5, 9, 10 and 11 herein. Where relevant, when determining a suitable amino acid substitution if there is a conflict between Table 1 and those Tables, Tables 4, 5, 9, 10 and 11, take precedent.
In an embodiment, the at least one amino acid substitution is at an amino acid position selected from the group consisting of amino acid positions: 2, 5, 7, 19, 23, 24, 26 to 35, 45, 48, 49, 51, 53, 54, 56 to 59, 61, 62, 64 to 74, 76 to 78, 80 to 84, 102, 105, 107, 111 to 114, 116 to 118, 121 to 124, 139, 145, 147, 149, 158, 165, 166, 168, 169, 171, 179, 182, 183, 188, 191, 193 to 197, 200 to 203, 205 to 211, 214, 216, 219, 223 to 226, 228 to 235, 237, 238, 241, 242, 244 to 246, 248, 249, 251 to 253, 257, 259 to 264, 266 to 271 and 273 to 275 with reference to SEQ ID NO: 37, or at a corresponding amino acid position in the modified NifH polypeptide when its sequence is aligned with SEQ ID NO:37. Alternatively, or in addition, in an embodiment, the at least one amino acid substitution is at an amino acid position selected from the group consisting of amino acid positions: 3, 6, 8, 20, 24, 25, 27 to 35, 45, 48, 49, 51, 53, 54, 56 to 59, 61, 62, 64 to 75, 77 to 79, 81 to 85, 103, 106, 108, 112 to 115, 117 to 119, 122 to 125, 140, 146, 148, 150, 159, 166, 167, 169, 170, 172, 180, 183, 184, 189, 192, 194 to 198, 201 to 204, 206 to 212, 215, 217, 220, 224 to 227, 229 to 236, 238, 239, 242, 243, 245 to 247, 249, 250, 252 to 254, 258, 260 to 265, 267 to 272 and 274 to 276 with reference to SEQ ID NO:39, or at a corresponding amino acid position in the modified NifH polypeptide when its sequence is aligned with SEQ ID NO:39.
In an embodiment, the modified NifH polypeptide comprises at least one amino acid substitution, or two or three amino acid substitutions, or four or more amino acid substitutions, selected from the group of amino acid substitutions listed in one or more of Tables 4, 5, 9, 10 or 11, or corresponding amino acid substitutions when the modified NifH polypeptide sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment, the modified NifH polypeptide comprises at least one amino acid substitution, or preferably two or three amino acid substitutions, or four or more amino acid substitutions, at amino acid position(s) selected from the group consisting of amino acid positions 69, 168, 200, 201, 224, 228, 234, 241, 252 and 263 with reference to SEQ ID NO: 37, or at a corresponding amino acid position in the modified NifH polypeptide when its sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment, the at least one amino acid substitution, or preferably two or three amino acid substitutions, or four or more amino acid substitutions, are selected from the group consisting of 69N, 1681, 200A, 20 IK, 224R, either 2281 or 228V, either 234H or 234C, either 241R or 241A, 252M and 263E, wherein the amino acid positions correspond to the amino acid sequence provided as SEQ ID NO: 37, or at a corresponding amino acid position in the modified NifH polypeptide when its sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment, the at least one amino acid substitution, or preferably two or three amino acid substitutions, or four or more amino acid substitutions, are selected from the group consisting of positions corresponding to amino acids 69, 168, 200, 201, 224, 228, 234, 252 and 263 of SEQ ID NO:37, or at a corresponding amino acid position in the modified NifH polypeptide when its sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment, the at least one amino acid substitution, or two or three amino acid substitutions, or four or more amino acid substitutions, are selected from the group consisting of 69N, 1681, 200A, 201K, 224R, either 2281 or 228V, either 234H or 234C, 252M and 263E, wherein the amino acid positions correspond to the amino acid sequence provided as SEQ ID NO: 37, or at a corresponding amino acid position in the modified NifH polypeptide when its sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39. In an embodiment, the modified NifH polypeptide has one, two, three, four, five, six, seven, eighth, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 1 to 20, 1 to 15, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 2 to 20, 2 to 15, 2 to 11, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 or 3, 3 to 20, 3 to 15, 3 to 11, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 or 4, preferably 1 to 3, 1 to 4, or 1 to 5, more preferably 2 to 4 or 2 to 5, most preferably 3 to 5, amino acid substitution(s) when compared to the corresponding wild-type NifH polypeptide.
In an embodiment, the modified NifH polypeptide comprises at least one amino acid substitution which is 228V with reference to SEQ ID NO:37, or the same amino acid substitution at a corresponding amino acid position when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment, the modified NifH polypeptide comprises at least one amino acid substitution which is 2281 with reference to SEQ ID NO:37, or the same amino acid substitution at a corresponding amino acid position when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment, the modified NifH polypeptide comprises at least one amino acid substitution which is 200A with reference to SEQ ID NO:37, or the same amino acid substitution at a corresponding amino acid position when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment, the modified NifH polypeptide comprises at least one amino acid substitution which is 234H with reference to SEQ ID NO:37, or the same amino acid substitution at a corresponding amino acid position when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment, the modified NifH polypeptide comprises at least two amino acid substitutions which are 200A and 228V with reference to SEQ ID NO:37, or the same amino acid substitutions at corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment, the modified NifH polypeptide comprises at least two amino acid substitutions which are 200A and 2281 with reference to SEQ ID NO:37, or the same amino acid substitutions at corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment, the modified NifH polypeptide comprises at least two amino acid substitutions which are 228V and 234H with reference to SEQ ID NO:37, or the same amino acid substitutions at corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39. In an embodiment, the modified NifH polypeptide comprises at least two amino acid substitutions which are 2281 and 234H with reference to SEQ ID NO:37, or the same amino acid substitutions at corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment, the modified NifH polypeptide comprises at least two amino acid substitutions which are 200A, 228V and 234H with reference to SEQ ID NO:37, or the same amino acid substitutions at corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment, the modified NifH polypeptide comprises at least two amino acid substitutions which are 200A, 2281 and 234H with reference to SEQ ID NO:37, or the same amino acid substitutions at corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment, the modified NifH polypeptide comprises at least two amino acid substitutions which are 200A, 228V or 2281, 234H and 241R with reference to SEQ ID NO:37, or the same amino acid substitutions at corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment, the modified NifH polypeptide comprises at least two amino acid substitutions which are 1681, 200A, 2281 or 228V and 234H with reference to SEQ ID NO:37, or the same amino acid substitutions at corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment, the modified NifH polypeptide comprises at least two amino acid substitutions which are 69N, 1681, 200A, 228V or 2281, 234H, 252M and 263E with reference to SEQ ID NO:37, or the same amino acid substitutions at corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment, the modified NifH polypeptide comprises at least two amino acid substitutions which are 69N, 1681, 200A, 20 IK, 228V or 2281, 234H, 252M and 263E with reference to SEQ ID NO:37, or the same amino acid substitutions at corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment, the modified NifH polypeptide comprises at least two amino acid substitutions which are 69N, 1681, 200A, 20 IK, 224R, 2281 or 228V, 234H, 252M and 263E with reference to SEQ ID NO:37, or the same amino acid substitutions at corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39. In an embodiment, the modified NifH polypeptide comprises at least two amino acid substitutions which are 1681, 200A, 2281 or 228V, 234H and 241R with reference to SEQ ID NO:37, or the same amino acid substitutions at corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment, the modified NifH polypeptide comprises at least two amino acid substitutions which are 69N, 1681, 200A, 228V or 2281, 234H, 241R, 252M and 263E with reference to SEQ ID NO:37, or the same amino acid substitutions at corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment, the modified NifH polypeptide comprises at least two amino acid substitutions which are 69N, 1681, 200A, 20 IK, 228V or 2281, 234H, 241R, 252M and 263E with reference to SEQ ID NO:37, or the same amino acid substitutions at corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment, the modified NifH polypeptide comprises at least two amino acid substitutions which are 69N, 1681, 200A, 20 IK, 224R, 2281 or 228V, 234H, 241R, 252M and 263E with reference to SEQ ID NO:37, or the same amino acid substitutions at corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment, the modified NifH polypeptide comprises at least two amino acid substitutions which are 112L, 200A, 228V or 2281, 234H and 241R with reference to SEQ ID NO:37, or the same amino acid substitutions at corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment, the modified NifH polypeptide comprises at least two amino acid substitutions which are 112L, 1681, 200A, 2281 or 228V, 234H and 241R with reference to SEQ ID NO:37, or the same amino acid substitutions at corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment, the modified NifH polypeptide comprises at least two amino acid substitutions which are 69N, 112L, 1681, 200A, 228V or 2281, 234H, 241R, 252M and 263E with reference to SEQ ID NO:37, or the same amino acid substitutions at corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39. In an embodiment, the modified NifH polypeptide comprises at least two amino acid substitutions which are 69N, 112L, 1681, 200A, 201K, 228V or 2281, 234H, 241R, 252M and 263E with reference to SEQ ID NO:37, or the same amino acid substitutions at corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment, the modified NifH polypeptide comprises at least two amino acid substitutions which are 69N, 112L, 1681, 200A, 201K, 224R, 2281 or 228V, 234H, 241R, 252M and 263E with reference to SEQ ID NO:37, or the same amino acid substitutions at corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment, the modified NifH polypeptide comprises amino acids 200A, 228V and 234H with reference to SEQ ID NO:37, or the same amino acids at the corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO: 11539
In an embodiment, the modified NifH polypeptide comprises one or more or all of the following motifs: YGKGGIGKSTTXQN (SEQ ID NO:61), IXGCDPKAD (SEQ ID NO:62), CXESGGPEPGVGCAGRG (SEQ ID NO:63), DVLGDVVCGGFAMP (SEQ ID NO:43), VXSGEMMAXYAANNI (SEQ ID NO:64), and CNSRXXD (motif VII, SEQ ID NO:65), preferably at least DVLGDVVCGGFAMP (SEQ ID NO:43), where each X independently represents any amino acid.
In an embodiment, the modified NifH polypeptide comprises one or more or all of the following motifs: YGKGGIGKSTTXQNT (SEQ ID NO:40), IHGCDPKAD (SEQ ID NO:41), CVESGGPEPGVGCAGRG (SEQ ID NO:42), DVLGDVVCGGFAMP (SEQ ID NO:43), VASGEMMAXYAANNI (SEQ ID NO:44), QSGVR (SEQ ID NO:45) and CNSRXVD (SEQ ID NO:46), preferably at least DVLGDVVCGGFAMP (SEQ ID NO:43), where each X independently represents any amino acid.
In an embodiment, the modified NifH polypeptide has 12, 13, 14, 15 or all of following amino acids: 4K, 22T, 37H, 52G, 60D, 63R, 108L, 109M, 142G, 151A, 174Q, 189V, 198E, 199F, 222F and 2471, with reference to SEQ ID NO:37, or the same amino acids at the corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment, the modified NifH polypeptide has 130, 131, 132, 133, 134, 135, 136 or all 137 of the following amino acids: 3R, 4K, 6A, 8Y, 9G, 10K, 11G, 12G, 131, 14G, 15K, 16S, 17T, 18T, 20Q, 21N, 22T, 25A, 361, 37H, 38G, 39C, 40D, 41P, 42K, 43A, 44D, 46T, 47R, 50L, 52G, 55Q, 60D, 63R, 75V, 79G, 85C, 86V, 87E, 88S, 89G, 90G, 91P, 92E, 93P, 94G, 95V, 96G, 97C, 98A, 99G, 100R, 101G, 1031, 104T, 1061, 108L, 109M, 110E, 115Y, 119L, 120D, 125D, 126 V, 127L, 128G, 129D, 130V,
131V, 132C, 133G, 134G, 135F, 136A, 137M, 138P, MOR, 142G, 143K, 144A, 146E,
148Y, 150V, 151A, 152S, 153G, 154E, 155M, 156M, 157A, 159Y, 160A, 161A, 162N, 163N, 1641, 167G, 170K, 172 A, 174Q, 175S, 176G, 177 V, 178R, 180G, 181G, 184C,
185N, 186S, 187R, 189V, 190D, 192E, 198E, 199F, 204G, 212P, 213R, 215N, 217V,
218Q, 220A, 221E, 222F, 227V, 236Q, 239E, 240Y, 243L, 2471, 250N, 254V, 2551, 256P, 258P, 265E and 272G, with reference to SEQ ID NO:37, or the same amino acids at the corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment, the modified NifH polypeptide has one or both of the following amino acids: 141D and 173K, with reference to SEQ ID NO:37, or the same amino acids at the corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment, the modified NifH polypeptide has an amino acid sequence which is at least 60% identical, preferably at least 70% identical or at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to the amino acid sequence provided as SEQ ID NO:37 and/or SEQ ID NO:39.
In an embodiment, the modified NifH polypeptide is a modified AnfH polypeptide.
In an embodiment, the modified AnfH polypeptide has an amino acid sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to the amino acid sequence provided as SEQ ID NO:37.
In an embodiment, the modified NifH polypeptide comprises at least amino acids 2-275 of the amino acid sequence provided as SEQ ID NO:78, or comprises SEQ ID NO:78.
In an embodiment a modified polypeptide of the invention has one or two or three or four conservative amino acid changes when compared to a naturally occurring (wildtype) polypeptide. Details of conservative amino acid changes are provided in Table 1. In a preferred embodiment, the changes are not in one or more of the motifs or domains which are highly conserved between the different polypeptides of the invention, as described in Examples 2, 8, 9 and 18 herein. As the skilled person would be aware, such minor changes can reasonably be predicted not to alter the activity of the polypeptide when expressed in a recombinant cell. Table 1. Exemplary conservative substitutions.
The primary amino acid sequence of a polypeptide of the invention can be used to design variants/mutants or modified forms thereof based on comparisons with closely related polypeptides. As the skilled person will appreciate, residues highly conserved amongst closely related proteins are less likely to be able to be altered, especially with non-conservative substitutions, and activity maintained than less conserved residues (see above). A more stringent test to identify conserved amino acid residues is to align more distantly related polypeptides of the same function. Highly conserved residues should be maintained in order to retain function, whereas non-conserved residues are more amenable to substitutions or deletion while maintaining function. Also included within the scope of the invention are polypeptides of the present invention which are differentially modified during or after synthesis in a cell, e.g., by glycosylation, acetylation, phosphorylation or proteolytic cleavage.
Mitochondrial Protein Import in Plants
Almost all mitochondrial proteins are nuclear encoded and translated in the cytosol, therefore requiring their translocation into the mitochondria. Signal sequences within the polypeptides direct their import to four different intra-mitochondrial locations: the outer membrane (OM), the intermembrane space (IS), the inner membrane (IM), or the matrix (MM). These signal sequences are distinguished by their biochemical properties and guide trafficking via at least four distinct import pathways which direct the polypeptides to one or more of the four locations (Chacinska et al., 2009). These four pathways are: (1) the general import pathway, also referred to as the “classical” presequence pathway, which directs polypeptides to the MM, the IS or the IM; (2) the carrier import pathway, used for transport to the IM, (3) the mitochondrial intermembrane space (MIA) assembly pathway, and (4) the sorting and assembly machinery (SAM) pathway used for transport of polypeptides to the OM. The general import pathway imports polypeptides having a cleavable pre-sequence, also known as a signal sequence. These polypeptides may also have a hydrophobic sorting signal (HSS). The carrier import pathway imports polypeptides with internal pre-sequence like signals and a hydrophobic region. The MIA pathway imports polypeptides with twin cysteine residues. The SAM pathway imports polypeptides that contain a P signal and a putative TOM20 signal. All of these pathways make use of a translocase of the outer membrane (TOM) and the first and second pathways also use a TIM23 translocase of the intermembrane complex. Only the first pathway uses matrix processing peptidase (matrix processing protease, MPP).
A common characteristic of all mitochondrial targeted polypeptides is the presence of at least one domain within the polypeptide that guides transport to the correct location. The best studied of these is the “classic” N-terminal pre-sequence domain that is cleaved in the matrix by MPP (Murcha et al., 2004). It has been estimated that about 70% of plant and animal mitochondrial proteins have a cleavable pre-sequence but both internal and C-terminal signal sequences have also been found (reviewed in Pfanner and Geissler (2001), Schleiff and Soil (2000)). In Arabidopsis. these pre-sequences range in length from 11 to 109 amino acid residues with an average length of 50 amino acid residues. Although there is no consensus sequence that fully defines a pre-sequence for the first pathway, they tend to contain a high proportion of hydrophobic and positively charged amino acids. A further characteristic is their ability to form an amphiphilic a- helix, usually starting within the first 10 amino acid residues (Roise et al., 1986). These domains are rich in hydrophobic (Ala, Leu, Phe, Vai), hydroxylated (Ser, Thr) and positively charged (Arg, Lys) amino acid residues, and deficient in acidic amino acids. Over a large number of mitochondrial proteins, serine (16-17%) and alanine (12-13%) are greatly over-represented in mitochondrial signal peptides, and arginine is abundant (12%). The MPP cleavage point is defined for most pre-sequences by the presence of a conserved arginine residue, usually at position P2 (-2 aa from the scissile bond), or P3 in most other cases (Huang et al., 2009).
Mitochondrial pre-sequences interact with the Tom20 receptor through hydrophobic residues. Studies have shown that the hydrophobic surface of the a-helix facilitates recognition of the peptide by the TOM20 component of the TOM import complex, whereas the positive charges are recognised by the TOM22 subunit (Abe et al., 2000). Finally, most pre-sequences guide transport of the polypeptide in association with Hsp70, and accordingly nearly all plant pre-sequences contain at least one binding motif for Hsp70 molecular chaperone (Zhang and Glaser, 2002). The chaperone Hsp70 is involved in protein folding, prevents protein aggregations, and functions as a molecular motor, pulling the precursor across the mitochondrial membranes. The electrical membrane potential (Ay) (-100 mV, negative inside) across the inner membrane also drives translocation of the positively charged pre-sequence via an electrophoretic effect.
The majority of proteins with cleavable pre-sequences are destined for the mitochondrial matrix via the general import pathway, which utilises the transporter of the outer membrane (TOM) complex and the transporter of the inner membrane 23 complex (TIM23). However some proteins with cleavable pre-sequences can assemble in the inner membrane (Murcha et al., 2004) or the inter membrane space, if they also contain a hydrophobic sorting signal (HSS) (Glick et al., 1992). There are very few examples of matrix localised proteins that do not have their pre-sequences cleaved. In Arabidopsis, only Glutamate dehydrogenase has been found in the matrix with an unprocessed full length pre-sequence (Huang et al., 2009).
For proteins that are not matrix targeted, a variety of internal non-cleavable localisation signals are employed. These are typically associated with a specific trafficking pathway, and are additionally tailored for the particular class of protein. In plants, no studies thus far have determined what precisely constitutes an internal signal sequence for intermembrane space proteins. However, it appears a motif with twin cysteine residues is associated with transport via the mitochondrial intermembrane space assembly pathway (MIA) (Carrie et al., 2010; Darshi et al., 2012). Finally, non-cleavable internal sequences are also utilised by proteins destined for the inner membrane via the carrier pathway, which utilises the TOM and TIM22 apparatus to insert proteins with multiple transmembrane regions (Kerscher et al., 1997; Sirrenberg et al., 1996). These sequences typically contain a hydrophobic region followed by a pre-sequence like internal sequence, and are thus similar to N-terminal pre-sequences, but distinguished by their internal location within their cognate protein.
In photosynthetic organisms, nuclear encoded mitochondrial proteins have a requirement for differentiation between chloroplast and mitochondrial trafficking, despite many similarities between these two organelles and their proteomes. The a-helix that occurs mostly in mitochondria pre-sequences is usually absent in chloroplast presequences (Zhang and Glaser, 2002), which tend to be more unstructured and show high P sheet domain structure (Bruce, 2001).
In plants, the MPP is anchored to the inner membrane bound Cytbci complex, although the active MPP site is located facing the matrix, and the functions of the two proteins are independent (Glaser and Dessi, 1999).
Mitochondrial targeting peptide
As used herein, the term "mitochondrial targeting peptide” or “MTP” means an amino acid sequence, comprising at least 10 amino acids and preferably between 10 and about 80 amino acid residues in length that directs a target protein to a mitochondrion and which can be used heterologously in an MTP -target protein translational fusion to direct a selected a Nif polypeptide to a mitochondrion.
The MTP typically comprises at its N-terminus a translation initiator methionine of the polypeptide from which it is derived. The MTP is translationally fused to a Nif polypeptide or “target protein” by a peptide bond to the Met residue that corresponds to the initiator Met of the target protein, or that Met residue may be omitted and the peptide bond is directly fused to the amino acid residue that in the wild-type is the second amino acid of the target protein. The MTP is typically rich in basic and hydroxylated amino acids and usually lacks acidic amino acids or extended hydrophobic stretches. The MTP may form amphiphilic helices.
While not wanting to be limited by theory, the MTP typically comprises an uptake-targeting sequence that binds to receptors on the outer membrane of the mitochondrion. Upon binding to the outer membrane, the fusion polypeptide preferably undergoes membrane translocation to transport channel proteins, and passages through the double membrane of the mitochondrion to the mitochondrial matrix (MM). The uptake-targeting sequence is then typically cleaved and the mature fusion protein folded. The MTP may comprise additional signals that subsequently target the protein to different regions of the mitochondria, such as the mitochondrial matrix (MM). In an embodiment, the uptake-targeting sequence is a matrix targeting sequence.
The MTP may be cleavable or non-cleavable when translationally fused to the Nif polypeptide. Thus, in an embodiment, the MTP-Nif fusion polypeptide, such as a MTP-modified NifH polypeptide or MTP -NifH fusion polypeptide of the invention, preferably a MTP-modified AnfH polypeptide or MTP-AnfH fusion polypeptide, is at least partially cleaved in mitochondria of the plant cell. In this regard, the phrase “at least partially cleaved” refers to a detectable amount of cleavage of a MTP-modified NifH polypeptide or MTP-Nif fusion polypeptide when expressed in a plant cell. In an embodiment, at least 50% of the MTP-Nif fusion polypeptide that is produced in the cell is cleaved within the MTP sequence, preferably at least 75% is cleaved, more preferably at least 90% is cleaved. In an alternative, less preferred embodiment, less than 50% of the MTP-Nif fusion polypeptide is cleaved in the cell, for example, the MTP is not cleaved. In an embodiment, the MTP does not comprise a cleavage site for MPP. The MTP preferably comprises a cleavage site for MPP. Upon cleavage, the N-terminal part of the resultant processed product (i.e., the mature NP or “cleavage product”) may comprise one or more C-terminal amino acids of the MTP, also referred to herein as a “scar sequence” or “scar peptide”, or it may not comprise any C-terminal amino acids of the MTP. In the latter case, cleavage by MPP is immediately adjacent to the MTP sequence, for example at the junction between the MTP and NifH sequence. When present, the scar sequence is preferable 1 to 45 amino acids in length, more preferably 1 to 20 amino acids, even more preferably 1 to 12 amino acids, or most preferably 4 to 12 amino acids. The scar sequence may be 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acids in length, not counting the amino acids of the NifH sequence. Alternatively, the cleavage site may be located within the fusion polypeptide such that the entire MTP sequence is cleaved off, for example, the linker may comprise the cleavage sequence.
Native mitochondrial targeting peptides are localized at the N-terminus of the precursor proteins and a N-terminal part are typically cleaved off during or after import into mitochondria. Cleavage is typically catalysed by the general matrix processing protease (MPP), which, in plants, is integrated into the bci complex of the respiratory chain. This protease recognizes the cleavage sites of nearly 1000 precursor proteins that have a wide range of amino acid sequences which show little conservation. In an embodiment, the MTP comprises a protease cleavage site for MPP. In a further embodiment, the processed product is produced by cleavage of the fusion protein within, or immediately after, the MTP by MPP. In this context, the phrase “immediately after” or “immediately adjacent” means that following cleavage by MPP, there are no amino acids remaining from the MTP fused to the Nif polypeptide. Thus, where the fusion polypeptide is cleaved “immediately after” the MTP, the MPP cleavage site is immediately after the C-terminal amino acid of the MTP.
The terms “cleaved product” or “cleavage product”, as used herein in the context of a MTP-modified Nif or MTP -Nif fusion polypeptide, preferably a modified NifH polypeptide or NifH dimer polypeptide as described herein, more preferably a modified AnfH polypeptide or AnfH dimer polypeptide, refer to a polypeptide resulting from protease cleavage either within or immediately after the MTP amino acid sequence. In this regard, the cleaved product of the MTP fusion polypeptide is obtainable by cleavage by MPP. The cleaved product may retain one or more amino acids from the MTP after cleavage (i.e., a scar peptide), or it may not have any amino acids remaining from the MTP after cleavage. In an embodiment, a cleaved product produced from a MTP- modified Nif or MTP -Nif fusion polypeptide of the invention comprises at least 95% or all of the amino acids present in the Nif polypeptide sequence.
In an embodiment, the MTP is not cleaved, preferably it is cleaved.
Suitable MTPs that can be used in the context of the present invention include, without limitation, peptides having the general structure as defined by von Heijne (1986) or by Roise and Schatz (1988). Non limiting examples of MTPs are the mitochondrial targeting peptides defined in Table I of von Heijne (1986) or disclosed herein.
In an embodiment, the MTP is an F 1 -ATPase y-subunit (MTP -F Ay). An example of a suitable FAy MTP is that from A. thaliana (Lee et al., 2012). In a preferred embodiment, the MTP -FAy is less than 77 amino acids in length. For example, the MTP- FAy may be about 51 amino acids in length, the cleavage of which by an MMP leaves 9 MTP residues at the N-terminal end of the fusion polypeptide. The scar sequence may also comprise a GG two-amino acid sequence between the amino acids from the C- terminal end of the MTP and the NifH sequence, the result of GoldenGate cloning methods.
The skilled person will appreciate that software exists for predicting mitochondrial proteins and their targeting sequence, for example, MitoProtll, PSORT, TargetP and NNPSL.
MitoProtll is a program that predicts mitochondrial localization of a sequence based on several physiochemical parameters (e.g., amino acid composition in the N- terminal part, or the highest total hydrophobicity for a 17 residues window). PSORT is a program that predicts subcellular locations based on various sequence-derived features such as the presence of sequence motifs and amino acid compositions. TargetP predicts the subcellular location of eukaryotic proteins based on the predicted presence of any of the N-terminal presequences: chloroplast transit peptide, mitochondrial targeting peptide or secretory pathway signal peptide. TargetP requires the N-terminal sequence as an input into two layers of artificial neural networks (ANN), utilizing the earlier binary predictors, SignalP and ChloroP. For the sequences predicted to contain an N-terminal presequence a potential cleavage site can also be predicted. NNPSL is another ANN- based method using the amino acid composition to assign one of four subcellular localization (cytosolic, extracellular, nuclear and mitochondrial) to a query sequence.
The skilled person would be readily able to determine if the chosen MTP targeted the fusion polypeptide to the mitochondrial matrix based on routine methods and methods disclosed herein.
It may be useful in some embodiments of this invention to use multiple tandem copies of a chosen MTP. The coding sequence for a duplicated or multiplied targeting peptide may be obtained through genetic engineering from an existing MTP. The amount of MTP can be measured by cellular fractionation, followed by, for example, quantitative immunoblot analysis. Thus, in the present invention, the term "mitochondrial targeting peptide” or “MTP” encompasses one or more copies of one amino acid peptide that directs a target Nif protein to the mitochondria. In a preferred embodiment, the MTP comprises two copies of a chosen MTP. In another embodiment, the MTP comprises three copies of a chosen MTP. In another embodiment, the MTP comprises four copies or more of a chosen MTP.
The skilled person will appreciate that the MTP sequence is not limited to native MTP sequences but may comprise amino acid substitutions, deletions and/or insertions, relative to a naturally-occurring MTP, provided that the sequence variant still functions for mitochondrial targeting.
The skilled person will understand that the MTP may be flanked by amino acids at its N- or C-terminal ends as a result of the cloning strategy and may function as a linker. These additional amino acids may be considered to form part of the MTP.
The skilled person will also understand that the MTP may be N- or C-terminally fused to an oligopeptide linker and/or tag such as an epitope tag. In a preferred embodiment, one or more or all of the Nif fusion polypeptides of the invention produced in a plant cell lack added epitope tags relative to a corresponding wild-type Nif polypeptide. Mitochondrial Targeting Peptide (MTP)-Nif Fusion Polypeptides
The present invention includes mitochondrial targeting peptide (MTP)-Nif fusion polypeptides, also referred to herein as the “encoded polypeptides” since they are the immediate translational product encoded by the exogenous polynucleotide of the invention, and their cleaved polypeptide products. In particular, the present invention includes MTP -modified NifH polypeptides and MTP -NifH fusion polypeptides, preferably MTP-modified AnfH polypeptides or MTP-AnfH dimer polypeptides, and their cleaved polypeptide products. When a MTP-modified Nif polypeptide or MTP-Nif fusion polypeptide of the invention (encoded polypeptide) is expressed in a plant cell, either the MTP-modified Nif polypeptide or MTP-Nif fusion polypeptide and/or the cleaved polypeptide product is targeted to the mitochondrial matrix (MM). Preferably, the fusion polypeptides confer nitrogenase reductase and/or nitrogenase activity to the plant cell, or an activity which is the same as that conferred by a corresponding wild-type Nif polypeptide in bacteria.
As used herein, the term "fusion polypeptide" means a polypeptide which comprises two or more polypeptide domains which are covalently joined by a peptide bond, one of which is a Nif sequence, preferably a NifH sequence which has at least one amino acid substitution, or two NifH sequences joined by an oligopeptide linker, more preferably an AnfH sequence which has at least one amino acid substitution, or two AnfH sequences joined by an oligopeptide linker. The fusion polypeptide is encoded as a single polypeptide chain by a chimeric polynucleotide of the invention. In an embodiment, fusion polypeptides of the invention comprise a mitochondrial targeting peptide (MTP) and a Nif polypeptide (NP). In this embodiment, the C-terminal end of the MTP is translationally fused to the N-terminal end of the NP by a peptide bond. In an alternative embodiment, fusion polypeptides of the invention comprise a C-terminal part of an MTP and a NP, where the C-terminal part results from cleavage of the MTP by MPP. Such a C-terminal part of an MTP is referred to herein as a “scar” sequence. In this embodiment, the C-terminal amino acid of the C-terminal part of the MTP is translationally fused to the N-terminal amino acid of the NP by a peptide bond. In these embodiments, the fusion polypeptide may comprise one or more additional amino acids between the MTP and the NP, such as a GlyGly sequence, and/or an added methionine as a translation start amino acid. In an embodiment, a cell, plant or part thereof of the invention comprises a fusion polypeptide comprising two NifH polypeptides, preferably AnfH polypeptides, translationally fused via a linker.
As used herein, the term "translationally fused at the N-terminal end" means that the C-terminal end of the MTP polypeptide or linker polypeptide is covalently joined by a peptide bond to the N-terminal end of a NP, thereby being a fusion polypeptide. In an embodiment, the NP does not comprise its native translation start methionine (Met) residue or its two N-terminal Met residues relative to a corresponding wild-type NP. In an alternative embodiment, the NP comprises the translation start Met or one or both of the two N-terminal Met residues of the wild-type NP polypeptide such as, for example, for NifH.
Such polypeptides are typically produced by expression of a chimeric protein coding region where the translational reading frame of the nucleotides encoding the MTP are joined in-frame with the reading frame of the nucleotides encoding the NP. The skilled person will appreciate that the C-terminal amino acid of the MTP can be translationally fused to the N-terminal amino acid of the NP without a linker or via a linker of one or more amino acid residues, for example of 1-5 amino acid residues. Such a linker can also be considered to be part of the MTP. Expression of the protein coding region may be followed by cleavage of the MTP in the MM of a plant cell, and such cleavage (if it occurs) is included in the concept of production of the fusion polypeptide of the invention.
The fusion polypeptide or the processed Nif polypeptide preferably has functional Nif activity. In a preferred embodiment, the activity is similar to that of the corresponding wild-type Nif polypeptide. The functional activity of the fusion polypeptide or the processed Nif polypeptide may be determined in bacterial and biochemical complementation assays. In a preferred embodiment, the fusion polypeptide or the processed Nif polypeptide has between about 70-100% of the activity of the wild-type Nif activity. Nif polypeptides which do not have Nif function still have utility, for example, as research tools to test for expression levels from genetic constructs or for association with other Nif polypeptides.
A cell, plant or part thereof of the invention may comprise a fusion polypeptide with more than one MTP and/or more than one NP, for example, the fusion polypeptide may comprise a MTP, a NifD polypeptide and a NifK polypeptide. The fusion polypeptide may also comprise an oligopeptide linker, for example, linking two NPs. Preferably, the linker is of sufficient length to allow the two or more functional domains, for example, two NPs such as NifH and NifH (homodimer), NifD and NifK or NifE and NifN, to associate in a functional configuration in a plant cell. In a preferred embodiment, the NifD polypeptide is an AnfD polypeptide and the NifK polypeptide is an AnfK polypeptide. Such a linker may be between 8 and 50 amino acid residues in length, preferably about 25-35 amino acids in length, more preferably about 30 amino acid residues in length or about 26 amino acid residues in length for an AnfD-linker- AnfK fusion polypeptide. A fusion polypeptide may be obtained by conventional means, e.g., by means of gene expression of the polynucleotide sequence encoding for said fusion polypeptide in a suitable cell.
In an embodiment, a polypeptide of the invention is a substantially purified polypeptide. As used herein, a "substantially purified polypeptide" means a polypeptide which is substantially free from components (e.g., lipids, nucleic acids, carbohydrates) that normally associate with the polypeptide, for example, in a cell. Preferably, the substantially purified polypeptide is at least 90% free from said components.
Plant cells, transgenic plants and parts thereof of the invention comprise an exogenous polynucleotide encoding a polypeptide of the invention. Polypeptides of the invention are not naturally occurring in plant cells, in particular not in the mitochondria of plant cells, and therefore the polynucleotide encoding the polypeptide may be referred to herein as an exogenous polynucleotide since it is not naturally occurring in a plant cell but has been introduced into the plant cell or a progenitor cell. The cells, plants and plant parts of the invention which produce a polypeptide of the invention can therefore be said to produce a recombinant polypeptide. The term "recombinant" in the context of a polypeptide refers to the polypeptide encoded by an exogenous polynucleotide when produced by a cell, which polynucleotide has been introduced into the cell or a progenitor cell by recombinant DNA or RNA techniques such as, for example, transformation. Typically, the plant cell, plant or plant part comprises a non-endogenous gene that causes an amount of the polypeptide to be produced, at least at some time in the life-cycle of the plant cell or plant. Preferably the exogenous polynucleotide is integrated into the nuclear genome of the plant cell and/or is transcribed in the nucleus of the cell.
Linkers
As used herein in the context of polypeptides, the term "linker" or “oligopeptide linker” means one or more amino acids that covalently join two or more functional domains, for example, the MTP and the NP, two NPs, a NP and a tag. As used herein, the “linker” does not include the amino acids of the Nif sequence, for example the NifH or AnfH sequence, where those amino acids are also present in the corresponding wildtype Nif sequence. The “linker” also does not include the amino acids of the MTP sequence, if present, where those amino acids are the same as a naturally occurring MTP sequence. The amino acids are covalently joined through peptide bonds, both within the linker and between linker and functional domains. The linker may provide for freedom of movement of one functional domain with respect to the other, without causing a substantial detrimental effect on the function of the two or more domains. The linker may help promote proper folding and functioning of one or both of the functional domains or, as described herein, assist with increasing solubility of the fusion polypeptide. The skilled person will understand that the size of a linker can be determined empirically or can be modelled based on protein folding information.
The linker may comprise a cleavage site for a protease such as MPP. Such a linker can also be considered to be part of an MTP.
The skilled person will appreciate that the C-terminal end of the MTP can be translationally fused to the N-terminal amino acid of the NP without a linker or via a linker of one or more amino acid residues, for example of 1-5 amino acid residues.
In embodiments, the linker comprises at least 1 amino acid, at least 2 amino acids, at least 3 amino acids, at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, at least 10 amino acids, at least 12 amino acids, at least 14 amino acids, at least 16 amino acids, at least 18 amino acids, at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 35 amino acids, at least 40 amino acids, the least 45 amino acids, at least 50 amino acids, at least 60 amino acids, at least 70 amino acids, at least 80 amino acids, at least 90 amino acids, or about 100 amino acids. In embodiments, the maximal size of the linker is 100 amino acids, preferably 60 amino acids, more preferably 40 amino acids.
In some embodiments, the linker will permit the movement of one functional domain with respect to the other in order to increase stability of the fusion polypeptide. If desired, the linker can encompass either: repetitions of poly-glycine or combinations of glycine, proline and alanine residues.
Linkers for joining two Nif polypeptides such as NifH-linker-NifH, preferably AnfH-linker-AnfH, are preferably selected, for the number and sequence of the amino acids in the linker, based on several criteria. These are: a lack of cysteine residues to avoid formation of unwanted disulphide linkages, few or preferably no charged residues (Glu, Asp, Arg, Lys) to reduce the likelihood of unwanted surface salt bridge interactions, few or no hydrophobic residues (Phe, Trp, Tyr, Met, Vai, He, Leu) as such residues may promote a tendency to penetrate the surface of the polypeptide, and lacking amino acids which may be post-translationally modified. In this context “few charged residues” means less than 10% of the amino acid residues in the linker, and “few hydrophobic residues” means less than 15% of the amino acid residues in the linker.
In an embodiment, the linker does not comprise a cysteine residue.
In an embodiment, the linker comprises in increasingly preferred order, four, three, or two, or one, or no charged residues. Preferably, in total the linker comprises four, three, or two, or one, or no glutamic acid, aspartic acid, arginine and lysine residues. In an embodiment, the linker comprises four, three, or two, or one or no hydrophobic residues. Preferably, in total the linker comprises four, three, or two, or one or no phenylalanine, tryptophan, tyrosine, methionine, valine, isoleucine and leucine residues.
In an embodiment, at least 70%, or at least 80%, or at least 90%, of the linker comprises residues selected from threonine, serine, glycine and alanine.
The use of oligopeptide linkers in modifying polypeptides is reviewed in Chen et al. (2013) and Zhang et al. (2009).
Tags
In a particular embodiment, the fusion polypeptide comprises at least one tag adequate for detection or purification of the fusion polypeptide or a processed product thereof. The tag is typically bound to the C-terminal or N-terminal domain of the fusion polypeptide, or preferably as part of a linker in the NifH-linker-NifH fusion polypeptide. In a preferred embodiment, the tag is joined to the N-terminal end of the Nif sequence. The tag is generally a peptide or amino acid sequence capable of binding to one or more ligands, for example, one or more ligands of an affinity matrix such as a chromatography support or bead, or an antibody, with high affinity. The skilled person will understand that the tag should be located in the fusion protein at a location which does not result in the removal of the tag from the NP once the MTP is cleaved off after import into the mitochondria. Further, the tag should not interfere with the mitochondria import machinery. In a preferred embodiment, the polynucleotide of the invention encodes a fusion polypeptide that comprises, in the N- to C-terminal order, a N-terminal MTP, the Nif polypeptide and the detection/purification tag, for example a N-terminal MTP, a NifH polypeptide and the detection/purification tag, optionally followed by a second NifH polypeptide. In an alternate embodiment, the fusion polypeptide comprises, in the N- to C-terminal order, a N-terminal MTP, the detection/purification tag and the Nif polypeptide for example a N-terminal MTP, the detection/purification tag, for example a N-terminal MTP, a NifH polypeptide and the detection/purification tag, and a NifH polypeptide, optionally followed by a linker and a second NifH polypeptide.
Additional illustrative, non-limiting examples of tags useful for detecting, isolating or purifying a fusion polypeptide or a processed product thereof include, human influenza hemagglutinin (HA) tag, histidine tags comprising for example, 6 or 8 histidine residues, fluoresecent tags such as fluorescein, resourfm and derivatives thereof, Argtag, FLAG-tag, Strep-tag, an epitope capable of being recognized by an antibody, such as c-myc-tag (recognized by an anti-c-myc antibody), SBP-tag, S-tag, calmodulin binding peptide, cellulose binding domain, chitin binding domain, glutathione S- transferase-tag, maltose binding protein, NusA, TrxA, DsbA, Avi-tag, etc.
Translational Fusions Involving Nif Polypeptides Translational fusions have been made to several Nif polypeptides as reported in the scientific literature. These are summarised in Table 2 and in the review by Buren and Rubio (2018). Most of them involve the artificial addition of epitopes or binding domains such as Histidine tags or Strep tags to the proteins for detection and purification purposes and only a few have been expressed in plant cells. There are a few reports of naturally occurring fusions between Nif polypeptides, in bacteria. For assays in bacterial hosts, His tags of different lengths (7-10 histidines) were added to NifD (Christiansen et al., 1998), NifE (Goodwin et al., 1998), NifM (Gavini et al., 2006) and both full length and truncated versions of NifB (Fay et al., 2015). In each case, Nif function was retained for the modified Nif polypeptide as demonstrated in bacteria or in in vitro nitrogenase reconstitution assays.
Table 2. Summary of gene fusions of Nif polypeptides as reported in the literature Thiel et al. (1995) identified a naturally occurring deletion of 29 nucleotides and therefore deleting 9 amino acids and the NifE stop codon in the intergenic region between the NifE and NifN genes in the blue-green Anabaena variabilis. The deletion resulted in a NifE-NifN polypeptide fusion which retained at least some nitrogenase function of the NifE and NifN polypeptides. The NifE-NifN fusion polypeptide also had 19 other amino acid substitutions in the region of the fusion junction, which might have affected Nif function but in unknown ways. The fusion gene was expressed but only under strictly anaerobic conditions. It was not reported if there was a reduction in activity relative to the non-fused genes.
Suh et al. (2003) created an artificial junction between the NifE) and NifK genes of the chromosome of A. vinelandii by a deletion including the stop codon of NifD and the translation start codon (ATG) of NifK, forming a vector designated pBG1404. The deletion resulted in a net loss of three amino acids and seven amino acid substitutions in amino acids 2-10 of the NifK polypeptide. The A. vinelandii host cells containing pBG1404 were compromised in their growth in low nitrogen media relative to the corresponding wild-type bacteria.
Wiig at al. (2011) used a naturally occurring translational fusion between NifN and NifB genes found in Clostridium pastuerianum and determined that it is functional for NifN and NifB activity in bacterial and biochemical complementation assays. This fusion was direct without any peptide linker, i.e. the C-terminal end of NifN was directly covalently linked to the N-terminal end of NifB.
In yeast and plant cells, translational fusions have been used to direct proteins encoded in the nucleus to mitochondrial matrix. In yeast expression assays, translational fusions of mitochondrial targeting peptide (MTP) and some Nif polypeptides (NifH, NifM, NifS, and NifU) were shown to be functional when grown under aerobic conditions (Lopez-Torrejon et al., 2016). Epitope fusions (FLAG and HIS) were also shown to be functional when fused to NifH, NifM, NifS and NifU, although these fusions were intended for localisation within the yeast cytoplasm and were only functional when the yeast were grown under anaerobic conditions. Buren et al. (2017b) showed that a mitochondrial -matrix targeted version of a soluble variant of NifB was functional in in vitro complementation assays when re-isolated from the mitochondria of yeast. This version of NifB included a N-terminal MTP, a truncated variant of NifB (without the NifX-like domain) and a C-terminal lOxHis epitope tag. A large number of MTP -Nif fusions were also generated in yeast expression assays. However, this large ensemble of co-expressed proteins failed to show activity in yeast (Buren et al., 2017b). An MTP from a CPN-60 gene was fused to the N-terminal end of NifH, NifM, NifS and NifU and shown to be functional via in vitro complementation assays when the FeProtein was re-isolated from plants grown under reduced oxygen tension at 10% oxygen (US2016/0304842).
Polynucleotides
The terms "polynucleotide" and "nucleic acid" are used interchangeably herein. They mean a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide defined herein may be of genomic, cDNA, semi synthetic, or synthetic origin, single-stranded or preferably doublestranded and by virtue of its origin or manipulation: (1) is not associated with all or a portion of a polynucleotide with which it is associated in nature (e.g., a /polynucleotide that does not comprise a native promoter encoding sequence), (2) is linked to a polynucleotide other than that to which it is linked in nature (e.g., a Nif polynucleotide linked to a MTP encoding nucleotide sequence and/or a non-native promoter encoding sequence), or (3) does not occur in nature (e.g., polynucleotides encoding MTP -Nif fusion polypeptides of the invention). The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, chimeric DNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization such as by conjugation with a labeling component.
An "isolated polynucleotide" is substantially free from components that are normally linked (e.g., regulatory sequences) or associate with the polynucleotide. Thus, an isolated polynucleotide is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Preferably, the isolated polynucleotide is at least 60% free, more preferably at least 75% free, and more preferably at least 90% free from said components.
As used herein, the phrase “exogenous polynucleotide” refers to a polynucleotide that has a sequence originating from outside the cell or organism that the exogenous polynucleotide is present in. The exogenous polynucleotide therefore does not naturally occur in the cell or organism. It may be a chimeric polynucleotide formed from two or more nucleic acid sequences that do not naturally occur joined together. For example, the promoter is heterologous to the Nif protein coding region. Additionally, the Nif protein coding region may have been codon-modified relative to a Nif sequence encoding the protein in a bacterium. When the Nif polypeptide comprises at least one amino acid substitution, the polynucleotide encoding it is necessarily an exogenous polynucleotide.
As used herein, the term "gene" is to be taken in its broadest context and includes the deoxyribonucleotide sequences comprising the transcribed region and, if translated, the protein coding region, of a structural gene and including sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of at least about 2 kb on either end and which are involved in expression of the gene. In this regard, the gene includes control signals such as promoters, enhancers, translation and transcription termination and/or polyadenylation signals that are naturally associated with a given gene, or heterologous control signals, in which case, the gene is referred to as a "chimeric gene". The sequences which are located 5' of the protein coding region and which are present on the mRNA are referred to as 5' non-translated sequences. The sequences which are located 3' or downstream of the protein coding region and which are present on the mRNA are referred to as 3' non-translated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene contains the coding region which may be interrupted with non-coding sequences termed "introns", "intervening regions", or "intervening sequences." Introns are segments of a gene which are transcribed into nuclear RNA (nRNA). Introns may contain regulatory elements such as enhancers. Introns are removed or "spliced out" from the nuclear or primary transcript; introns therefore are absent in the mRNA transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide. The term "gene" includes a synthetic or fusion molecule encoding all or part of the proteins of the invention described herein and a complementary nucleotide sequence to any one of the above.
As used herein, "chimeric DNA", also referred to herein as a "DNA construct", means any DNA molecule that is not naturally found in nature but which artificially joins two DNA parts into a single molecule, each part of which might be found in nature but the whole is not found in nature. For example, a DNA construct encoding a MTP- modified NifH polypeptide or MTP-Nif fusion polypeptide of the invention. Typically, chimeric DNA comprises regulatory and transcribed or protein coding sequences that are not naturally found together in nature (e.g., a Nif polynucleotide linked to a non-native promoter encoding sequence). Accordingly, chimeric DNA may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. The open reading frame may or may not be linked to its natural upstream and downstream regulatory elements. The open reading frame may be incorporated into, for example, the plant genome, in a non-natural location, or in a replicon or vector where it is not naturally found such as a bacterial plasmid or a viral vector. The term "chimeric DNA" is not limited to DNA molecules which are replicable in a host, but includes DNA capable of being ligated into a replicon by, for example, specific adaptor sequences.
A "transgene" is a gene that has been introduced into the genome by a transformation procedure. The term includes a gene in a progeny cell, plant, seed, nonhuman organism or part thereof which was introducing into the genome of a progenitor cell thereof. Such progeny cells etc may be at least a 3rd or 4th generation progeny from the progenitor cell which was the primary transformed cell. Progeny may be produced by sexual reproduction or vegetatively such as, for example, from tubers in potatoes or ratoons in sugarcane. The term "genetically modified", and variations thereof, is a broader term that includes introducing a gene into a cell by transformation or transduction, mutating a gene in a cell and genetically altering or modulating the regulation of a gene in a cell, or the progeny of any cell modified as described above.
A "genomic region" as used herein refers to a position within the genome where a transgene, or group of transgenes (also referred to herein as a cluster), have been inserted into a cell, or predecessor thereof. Such regions only comprise nucleotides that have been incorporated by the intervention of man such as by methods described herein.
A "recombinant polynucleotide" of the invention refers to a nucleic acid molecule which has been constructed or modified by artificial recombinant methods. The recombinant polynucleotide may be present in a cell in an altered amount or expressed at an altered rate (e.g., in the case of mRNA) compared to its native state. In one embodiment, the polynucleotide is introduced into a cell that does not naturally comprise the polynucleotide. Typically an exogenous DNA is used as a template for transcription of mRNA which is then translated into a continuous sequence of amino acid residues coding for a polypeptide of the invention within the transformed cell. In another embodiment, the polynucleotide is endogenous to a bacterial cell and its expression is altered by recombinant means, for example, an exogenous control sequence is introduced upstream of an endogenous gene of interest to enable the transformed cell to express the polypeptide encoded by the gene. A recombinant polynucleotide of the invention includes polynucleotides which have not been separated from other components of the cell-based or cell-free expression system, in which it is present, and polynucleotides produced in said cell-based or cell- free systems which are subsequently purified away from at least some other components. The polynucleotide can be a contiguous stretch of nucleotides existing in nature (e.g., Nif polynucleotide), or comprise two or more contiguous stretches of nucleotides from different sources (naturally occurring and/or synthetic) joined to form a single polynucleotide (e.g., a /polynucleotide linked to a MTP encoding nucleotide sequence and/or a non-native promoter encoding sequence). Typically, such chimeric polynucleotides comprise at least an open reading frame encoding a polypeptide of the invention operably linked to a promoter suitable of driving transcription of the open reading frame in a cell of interest. Reference to “a promoter” herein encompasses a single promoter or multiple promoters.
With regard to the defined polynucleotides, it will be appreciated that % identity figures higher than those provided above will encompass preferred embodiments. Thus, where applicable, in light of the minimum % identity figures, it is preferred that the polynucleotide comprises a polynucleotide sequence which is at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant nominated SEQ ID NO.
A polynucleotide of, or useful for, the present invention may selectively hybridise, under stringent conditions, to a polynucleotide defined herein. As used herein, stringent conditions are those that: (1) employ during hybridisation a denaturing agent such as formamide, for example, 50% (v/v) formamide with 0.1% (w/v) bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42°C; or (2) employ 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt' s solution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS and 10% dextran sulfate at 42°C in 0.2 x SSC and 0.1% SDS, and/or (3) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50°C.
Polynucleotides of the invention may possess, when compared to naturally occurring molecules, one or more mutations which are deletions, insertions, or substitutions of nucleotide residues. Polynucleotides which have mutations relative to a reference sequence can be either naturally occurring (that is to say, isolated from a natural source) or synthetic (for example, by performing site-directed mutagenesis or DNA shuffling on the nucleic acid as described above).
Polynucleotides of the invention may be codon-modified for expression in a plant cell. The skilled person will appreciated that the protein coding region may be codon optimised relative to, for example, the coding region of a naturally occurring polynucleotide in a nitrogen fixing bacterium.
Nucleic Acid Constructs
The present invention includes nucleic acid constructs comprising one or more polynucleotides of the invention, and vectors and host cells containing these, methods of their production and use, and uses thereof. The present invention refers to elements which are operably connected or linked. "Operably connected" or "operably linked" and the like refer to a linkage of polynucleotide elements in a functional relationship. Typically, operably connected nucleic acid sequences are contiguously linked and, where necessary to join two protein coding regions, contiguous and in reading frame. A coding sequence is "operably connected to" another coding sequence when RNA polymerase will transcribe the two coding sequences into a single RNA, which if translated is then translated into a single polypeptide having amino acids derived from both coding sequences. The coding sequences need not be contiguous to one another so long as the expressed sequences are ultimately processed to produce the desired protein.
As used herein, the term "cis-acting sequence", "cis-acting element" or "cis- regulatory region" or "regulatory region" or similar term shall be taken to mean any sequence of nucleotides, which when positioned appropriately and connected relative to an expressible genetic sequence, is capable of regulating, at least in part, the expression of the genetic sequence. Those skilled in the art will be aware that a cis-regulatory region may be capable of activating, silencing, enhancing, repressing or otherwise altering the level of expression and/or cell-type-specificity and/or developmental specificity of a gene sequence at the transcriptional or post-transcriptional level. In preferred embodiments of the present invention, the cis-acting sequence is an activator sequence that enhances or stimulates the expression of an expressible genetic sequence. "Operably connecting" a promoter or enhancer element to a transcribable polynucleotide means placing the transcribable polynucleotide (e.g., protein-encoding polynucleotide or other transcript) under the regulatory control of a promoter, which then controls the transcription of that polynucleotide. In the construction of heterologous promoter/structural gene combinations, it is generally preferred to position a promoter or variant thereof at a distance from the transcription start site of the transcribable polynucleotide which is approximately the same as the distance between that promoter and the protein coding region it controls in its natural setting; i.e., the gene from which the promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of function. Similarly, the preferred positioning of a regulatory sequence element (e.g., an operator, enhancer etc) with respect to a transcribable polynucleotide to be placed under its control is defined by the positioning of the element in its natural setting; i.e., the gene from which it is derived.
"Promoter" or "promoter sequence" as used herein refers to a region of a gene, generally upstream (5') of the RNA encoding region, which controls the initiation and level of transcription in the cell of interest. A "promoter" includes the transcriptional regulatory sequences of a classical genomic gene, such as a TATA box and CCAAT box sequences, as well as additional regulatory elements (i.e., upstream activating sequences, enhancers and silencers) that alter gene expression in response to developmental and/or environmental stimuli, or in a tissue-specific or cell-type-specific manner. A promoter is usually, but not necessarily (for example, some PolIII promoters), positioned upstream of a structural gene, the expression of which it regulates. Furthermore, the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the gene. Promoters may contain additional specific regulatory elements, located more distal to the start site to further enhance expression in a cell, and/or to alter the timing or inducibility of expression of a structural gene to which it is operably connected.
"Constitutive promoter" refers to a promoter that directs expression of an operably linked transcribed sequence in many or all tissues of an organism such as a plant. A preferred constitutive promoter is the CaMV 35S promoter, or CaMV e35S promoter, as used herein. The term “constitutive” as used herein does not necessarily indicate that a gene is expressed at the same level in all cell types, but that the gene is expressed in a wide range of cell types, although some variation in level is often detectable. "Selective expression" as used herein refers to expression almost exclusively in specific organs of, for example, the plant, such as, for example, endosperm, embryo, leaves, fruit, tubers or root. In a preferred embodiment, a promoter is expressed selectively or preferentially in roots, leaves and/or stems of a plant, preferably a cereal plant. Selective expression may therefore be contrasted with constitutive expression, which refers to expression in many or all tissues of a plant under most or all of the conditions experienced by the plant.
Selective expression may also result in compartmentation of the products of gene expression in specific plant tissues, organs or developmental stages. Compartmentation in specific subcellular locations such as the plastid, cytosol, vacuole, or apoplastic space may be achieved by the inclusion in the structure of the gene product of appropriate signals, eg. a signal peptide, for transport to the required cellular compartment, or in the case of the semi-autonomous organelles (plastids and mitochondria) by integration of the transgene with appropriate regulatory sequences directly into the organelle genome.
A "tissue-specific promoter" or "organ-specific promoter" is a promoter that is preferentially expressed in one tissue or organ relative to many other tissues or organs, preferably most if not all other tissues or organs in, for example, a plant. Typically, the promoter is expressed at a level 10-fold higher in the specific tissue or organ than in other tissues or organs.
In an embodiment, the promoter is a stem-specific promoter, a leaf-specific promoter or a promoter which directs gene expression in an aerial part of the plant (at least stems and leaves) (green tissue specific promoter) such as a ribulose- 1,5- bisphosphate carboxylase oxygenase (RUBISCO) promoter.
Examples of stem-specific promoters include, but are not limited to, those described in US 5,625,136.
In an embodiment, the promoter is a root specific promoter. Examples of root specific promoters include, but are not limited to, the promoter for the acid chitinase gene and specific subdomains of the CaMV 35S promoter.
The promoters contemplated by the present invention may be native to the host plant to be transformed or may be derived from an alternative source, where the region is functional in the host plant. Other sources include the Agrobacterium T-DNA genes, such as the promoters of genes for the biosynthesis of nopaline, octapine, mannopine, or other opine promoters, tissue specific promoters (see, e.g., US 5,459,252 and WO 91/13992); promoters from viruses (including host specific viruses), or partially or wholly synthetic promoters. Numerous promoters that are functional in mono- and dicotyledonous plants are well known in the art (see, for example, Salomon et al., 1984; Garfinkel et al., 1983; Barker et al., 1983); including various promoters isolated from plants and viruses such as the cauliflower mosaic virus promoter (CaMV 35S, 19S). Non-limiting methods for assessing promoter activity are disclosed by Medberry et al. (1992, 1993), Sambrook et al. (1989, supra) and US 5,164,316. Alternatively or additionally, the promoter may be an inducible promoter or a developmentally regulated promoter which is capable of driving expression of the introduced polynucleotide at an appropriate developmental stage of the, for example, plant. Other c/.s-acting sequences which may be employed include transcriptional and/or translational enhancers. Enhancer regions are well known to persons skilled in the art, and can include an ATG translational initiation codon and adjacent sequences. When included, the initiation codon should be in phase with the reading frame of the coding sequence relating to the foreign or exogenous polynucleotide to ensure translation of the entire sequence if it is to be translated. Translational initiation regions may be provided from the source of the transcriptional initiation region, or from a foreign or exogenous polynucleotide. The sequence can also be derived from the source of the promoter selected to drive transcription and can be specifically modified so as to increase translation of the mRNA.
The nucleic acid construct of the present invention may comprise a 3' nontranslated sequence from about 50 to 1,000 nucleotide base pairs which may include a transcription termination sequence. A 3' non-translated sequence may contain a transcription termination signal which may or may not include a polyadenylation signal and any other regulatory signals capable of effecting mRNA processing. A polyadenylation signal functions for addition of polyadenylic acid tracts to the 3' end of a mRNA precursor. Polyadenylation signals are commonly recognized by the presence of homology to the canonical form 5' AATAAA-3' although variations are not uncommon. Transcription termination sequences which do not include a polyadenylation signal include terminators for Poll or PolIII RNA polymerase which comprise a run of four or more thymidines. Examples of suitable 3' non-translated sequences are the 3' transcribed non-translated regions containing a polyadenylation signal from an octopine synthase (ocs) gene or nopaline synthase (nos) gene of Agrobacterium tumefaciens (Bevan et al., 1983). Suitable 3' non-translated sequences may also be derived from plant genes such as the ribulose- 1,5 -bisphosphate carboxylase (ssRUBISCO) gene, although other 3' elements known to those of skill in the art can also be employed. A preferred transcription terminator sequence is the TTm terminator described herein, or a functionally equivalent terminator have multiple transcription terminator regions and optionally a MAR region.
As the DNA sequence inserted between the transcription initiation site and the start of the coding sequence, i.e., the untranslated 5’ leader sequence (5’UTR), can influence gene expression if it is translated as well as transcribed, one can also employ a particular leader sequence. Suitable leader sequences include those that comprise sequences selected to direct optimum expression of the foreign or endogenous DNA sequence. For example, such leader sequences include a preferred consensus sequence which can increase or maintain mRNA stability and prevent inappropriate initiation of translation as, for example, described by Joshi (1987).
Vectors
The present invention includes use of vectors for manipulation or transfer of genetic constructs. A vector is a nucleic acid molecule, preferably a DNA molecule, that can be used to artificially carry foreign genetic material; into another cell, where it can be replicated or expressed. A vector containing foreign DNA is referred to as a “recombinant vector”. Examples of vectors include, but are not limited to, plasmids and viral vectors, for example the Gemini virus based replicative vectors described herein. The vector may comprise a transposable element.
A vector preferably is double-stranded DNA and contains one or more unique restriction sites and may be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or capable of integration into the genome, preferably the nuclear genome, of the defined host such that the cloned sequence is reproducible. Accordingly, the vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed circular plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into a cell, is integrated into the genome, preferably the nuclear genome, of the recipient cell and replicated together with the chromosome(s) into which it has been integrated. A vector system may comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the host cell, or a transposon. The choice of the vector will typically depend on the compatibility of the vector with the cell into which the vector is to be introduced. The vector may also include a selection marker such as an antibiotic resistance gene, a herbicide resistance gene or other gene that can be used for selection of suitable transformants. Examples of such genes are well known to those of skill in the art.
The nucleic acid construct of the invention can be introduced into a vector, such as a plasmid. Plasmid vectors typically include additional nucleic acid sequences that provide for easy selection, amplification, and transformation of the expression cassette in prokaryotic and eukaryotic cells, for example, pUC-derived vectors, pSK-derived vectors, pGEM-derived vectors, pSP-derived vectors, pBS-derived vectors, or binary vectors containing one or more T-DNA regions. Additional nucleic acid sequences include origins of replication to provide for autonomous replication of the vector, selectable marker genes, preferably encoding antibiotic or herbicide resistance, unique multiple cloning sites providing for multiple sites to insert nucleic acid sequences or genes encoded in the nucleic acid construct, and sequences that enhance transformation of prokaryotic and eukaryotic (especially plant) cells.
By "marker gene" is meant a gene that imparts a distinct phenotype to cells expressing the marker gene and thus allows such transformed cells to be distinguished from cells that do not have the marker. A selectable marker gene confers a trait for which one can "select" based on resistance to a selective agent (e.g., a herbicide, antibiotic, radiation, heat, or other treatment damaging to untransformed cells). A screenable marker gene (or reporter gene) confers a trait that one can identify through observation or testing, i.e., by "screening" (e.g., P-glucuronidase, luciferase, GFP or other enzyme activity not present in untransformed cells). The marker gene and the nucleotide sequence of interest do not have to be linked.
To facilitate identification of transformants, the nucleic acid construct desirably comprises a selectable or screenable marker gene as, or in addition to, the foreign or exogenous polynucleotide. The actual choice of a marker is not crucial as long as it is functional (i.e., selective) in combination with the host cell, preferably a plant host cell. The marker gene and the foreign or exogenous polynucleotide of interest do not have to be linked, since co-transformation of unlinked genes as, for example, described in US 4,399,216 is also an efficient process in plant transformation.
Examples of bacterial selectable markers are markers that confer antibiotic resistance such as ampicillin, erythromycin, chloramphenicol or tetracycline resistance, preferably kanamycin resistance. Exemplary selectable markers for selection of plant transformants include, but are not limited to, a hyg gene which encodes hygromycin B resistance; a neomycin phosphotransferase (nptll) gene conferring resistance to kanamycin, paromomycin, G418; a glutathione-S-transferase gene from rat liver conferring resistance to glutathione derived herbicides as, for example, described in EP 256223; a glutamine synthetase gene conferring, upon overexpression, resistance to glutamine synthetase inhibitors such as phosphinothricin as, for example, described in WO 87/05327; an acetyltransferase gene from Streptomyces viridochromogenes conferring resistance to the selective agent phosphinothricin as, for example, described in EP 275957; a gene encoding a 5 -enol shikimate-3 -phosphate synthase (EPSPS) conferring tolerance to N-phosphonom ethylglycine as, for example, described by Hinchee et al. (1988); a bar gene conferring resistance against bialaphos as, for example, described in WO91/02071; a nitrilase gene such as bxn from Klebsiella ozaenae which confers resistance to bromoxynil (Stalker et al., 1988); a dihydrofolate reductase (DHFR) gene conferring resistance to methotrexate (Thillet et al., 1988); a mutant acetolactate synthase gene (ALS), which confers resistance to imidazolinone, sulfonylurea or other ALS-inhibiting chemicals (EP 154,204); a mutated anthranilate synthase gene that confers resistance to 5-methyl tryptophan; or a dalapon dehalogenase gene that confers resistance to the herbicide.
Preferred screenable markers include, but are not limited to, a uidA gene encoding a P-glucuronidase (GUS) enzyme for which various chromogenic substrates are known; a P-galactosidase gene encoding an enzyme for which chromogenic substrates are known; an aequorin gene (Prasher et al., 1985), which may be employed in calciumsensitive bioluminescence detection; a green fluorescent protein gene (Niedz et al., 1995) or derivatives thereof; a luciferase (/wc) gene (Ow et al., 1986), which allows for bioluminescence detection, and others known in the art. By "reporter molecule" as used in the present specification is meant a molecule that, by its chemical nature, provides an analytically identifiable signal that facilitates determination of promoter activity by reference to protein product.
Preferably, the nucleic acid construct is stably incorporated into the genome of, for example, the plant, more preferably into the nuclear genome of the plant. The nucleic acid construct may also be integrated into the plastid genome or mitochondria genome of the plant. Accordingly, the nucleic acid comprises appropriate elements which allow the molecule to be incorporated into the genome, or the construct is placed in an appropriate vector which can be incorporated into a chromosome of a plant cell.
One embodiment of the present invention includes a recombinant vector, which comprises at least one polynucleotide defined herein, and is capable of delivering the polynucleotide into a host cell. Such a vector contains heterologous nucleic acid sequences, that is nucleic acid sequences that are not naturally found adjacent to nucleic acid molecules of the present invention and that preferably are derived from a species other than the species from which the nucleic acid molecule(s) are derived. The vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a virus or a plasmid.
Recombinant vectors of the invention comprise fusion sequences which lead to the expression of nucleic acid molecules as fusion proteins.
Recombinant vectors may also include intervening and/or untranslated sequences surrounding and/or within the nucleic acid sequence of a polynucleotide defined herein. Preferably, the recombinant vector is stably incorporated into the genome of a host cell such as a plant cell. Accordingly, the recombinant vector may comprise appropriate elements which allow the vector to be incorporated into the genome, or into a chromosome of the cell.
Recombinant Cells
Another embodiment of the present invention includes a recombinant cell, for example, a recombinant plant cell, which is a host cell transformed with one or more polynucleotides, constructs, or vectors of the present invention, or progeny cells thereof. The term "recombinant cell" is used interchangeably with the term "transgenic cell" herein.
Transformation of a nucleic acid molecule into a cell can be accomplished by any method by which a nucleic acid molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. A recombinant cell may remain unicellular or may grow into a tissue, organ or a multicellular organism. Transformed nucleic acid molecules of the present invention can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed cell in such a manner that their ability to be expressed is retained.
Preferred host cells are plant cells, more preferably cells of a cereal plant, more preferably barley or wheat cells, and even more preferably a wheat cell.
The recombinant cell may be a cell in culture, a cell in vitro, or in an organism such as, for example, a plant, or in an organ such as, for example, a root, leaf or stem. Preferably, the cell is in a plant, more preferably in roots, leaves, and/or stems of a plant.
In an embodiment, expression of active NifH in a plant cell requires expression of NifM, a NifS, a NifU, a NifD and a NifK. In an embodiment, the NifD and NifK are present as a NifD-NifK fusion polypeptide.
In another or further embodiment, expression of active NifH in a plant cell requires expression of NifH and NifM and optionally, NifU and/or NifN.
Plants
The term "plant" as used herein as a noun refers to whole plants and refers to any member of the Kingdom Plantae, but as used as an adjective refers to any substance which is present in, obtained from, derived from, or related to a plant, such as for example, plant organs (e.g. leaves, stems, roots, flowers), single cells (e.g. pollen), seeds, plant cells and the like. Plantlets and germinated seeds from which roots and shoots have emerged are also included within the meaning of "plant". The term "plant parts" as used herein refers to one or more plant tissues or organs which are obtained from a plant and which comprises genomic DNA of the plant. Plant parts include vegetative structures (for example, leaves, stems), roots, floral organs/structures, seed (including embryo, cotyledons, and seed coat), plant tissue (for example, vascular tissue, ground tissue, and the like), cells and progeny of the same. In a preferred embodiment, the plant part is a seed. The term "plant cell" as used herein refers to a cell obtained from a plant or in a plant and includes protoplasts or other cells derived from plants, gamete-producing cells, and cells which regenerate into whole plants. Plant cells may be cells in culture. By "plant tissue" is meant differentiated tissue in a plant or obtained from a plant ("explant") or undifferentiated tissue derived from immature or mature embryos, seeds, roots, shoots, fruits, tubers, pollen, tumor tissue, such as crown galls, and various forms of aggregations of plant cells in culture, such as calli. Exemplary plant tissues in or from seeds are cotyledon, embryo and embryo axis. The invention accordingly includes plants and plant parts and products comprising these.
As used herein, the term "seed" refers to "mature seed" of a plant, which is either ready for harvesting or has been harvested from the plant, such as is typically harvested commercially in the field, or as "developing seed" which occurs in a plant after fertilisation and prior to seed dormancy being established and before harvest.
A "transgenic plant" as used herein refers to a plant that contains a nucleic acid construct not found in a wild-type plant of the same species, variety or cultivar. That is, transgenic plants (transformed plants) contain genetic material (a transgene) that they did not contain prior to the transformation. The transgene may include genetic sequences obtained from or derived from a plant cell, or another plant cell, or a non-plant source, or a synthetic sequence. Typically, the transgene has been introduced into the plant by human manipulation such as, for example, by transformation but any method can be used as one of skill in the art recognizes. The genetic material is preferably stably integrated into the genome of the plant, preferably the nuclear genome. The introduced genetic material may comprise sequences that naturally occur in the same species but in a rearranged order or in a different arrangement of elements, for example an antisense sequence. Plants containing such sequences are included herein in "transgenic plants".
In a preferred embodiment, the transgenic plants are homozygous for each and every gene that has been introduced (transgene) so that their progeny do not segregate for the desired phenotype. The transgenic plants may also be heterozygous for the introduced transgene(s), such as, for example, in Fl progeny which have been grown from hybrid seed. Such plants may provide advantages such as hybrid vigour, well known in the art.
Transgenic plants, as defined in the context of the present invention include progeny of the plants which have been genetically modified using recombinant techniques, wherein the progeny comprise the transgene of interest. Such progeny may be obtained by self-fertilisation of the primary transgenic plant or by crossing such plants with another plant of the same species. This would generally be to modulate the production of at least one protein defined herein in the desired plant or plant organ. Transgenic plant parts include all parts and cells of said plants comprising the transgene such as, for example, cultured tissues, callus and protoplasts.
Transgenic plants can be produced using techniques known in the art, such as those generally described in A. Slater et al., Plant Biotechnology - The Genetic Manipulation of Plants, Oxford University Press (2003), and P. Christou and H. Klee, Handbook of Plant Biotechnology, John Wiley and Sons (2004).
A "non-transgenic plant" is one which has not been genetically modified by the introduction of genetic material by recombinant DNA techniques. As used herein, the term "compared to an isogenic plant", or similar phrases, refers to a plant which is isogenic relative to the transgenic plant but without the transgene of interest. Preferably, the corresponding non-transgenic plant is of the same cultivar or variety as the progenitor of the transgenic plant of interest, or a sibling plant line which lacks the construct, often termed a "segregant", or a plant of the same cultivar or variety transformed with an "empty vector" construct, and may be a non-transgenic plant. "Wild type", as used herein, refers to a cell, tissue or plant that has not been modified according to the invention. Wild-type cells, tissue or plants may be used as controls to compare levels of expression of an exogenous nucleic acid or the extent and nature of trait modification with cells, tissue or plants modified as described herein.
Transgenic plants, as defined in the context of the present invention include progeny of the plants which have been genetically modified using recombinant techniques, wherein the progeny comprise the transgene of interest. Such progeny may be obtained by self-fertilisation of the primary transgenic plant or by crossing such plants with another plant of the same species. Transgenic plant parts include all parts and cells of said plants comprising the transgene such as, for example, cultured tissues, callus and protoplasts.
Plants contemplated for use in the practice of the present invention include both monocotyledons and dicotyledons. Target plants include, but are not limited to, the following: cereals (for example, wheat, barley, rye, oats, rice, maize, sorghum and related crops); grapes; beet (sugar beet and fodder beet); pomes, stone fruit and soft fruit (apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries and black-berries); leguminous plants (beans, lentils, peas, soybeans); oil plants (rape or other Brassicas, mustard, poppy, olives, sunflowers, safflower, flax, coconut, castor oil plants, cocoa beans, groundnuts); cucumber plants (marrows, cucumbers, melons); fibre plants (cotton, flax, hemp, jute); citrus fruit (oranges, lemons, grapefruit, mandarins); vegetables (spinach, lettuce, asparagus, cabbages, carrots, onions, tomatoes, potatoes, paprika); lauraceae (avocados, cinnamon, camphor); or plants such as maize, tobacco, nuts, coffee, sugar cane, tea, vines, hops, turf, bananas and natural rubber plants, as well as ornamentals (flowers, shrubs, broad-leaved trees and evergreens, such as conifers). Preferably, the plant is a cereal plant, more preferably wheat, rice, maize, triticale, oats or barley, even more preferably wheat.
As used herein, the term "wheat" refers to any species of the Genus Triticum, including progenitors thereof, as well as progeny thereof produced by crosses with other species. Wheat includes "hexapioid wheat" which has genome organization of AABBDD, comprised of 42 chromosomes, and "tetrapioid wheat" which has genome organization of AABB, comprised of 28 chromosomes. Hexapioid wheat includes T. aestivum, T. spelta, T. macha. T. compactum, T. sphaerococcum, T. vavilovii, and interspecies cross thereof. A preferred species of hexapioid wheat is T. aestivum ssp aestivum (also termed "breadwheat"). Tetrapioid wheat includes T. durum (also referred to herein as durum wheat or Triticum turgidum ssp. durum), T. dicoccoides, T. dicoccum, T. polonicum, and interspecies cross thereof. In addition, the term "wheat" includes potential progenitors of hexapioid or tetrapioid Triticum sp. such as T. uartu, T. monococcum or T. boeoticum for the A genome, Aegilops speltoides for the B genome, and T. tauschii (also known as Aegilops squarrosa or Aegilops tauschii) for the D genome. Particularly preferred progenitors are those of the A genome, even more preferably the A genome progenitor is T. monococcum. A wheat cultivar for use in the present invention may belong to, but is not limited to, any of the above-listed species. Also encompassed are plants that are produced by conventional techniques using Triticum sp. as a parent in a sexual cross with a non-Triticum species (such as rye [Secale cereale]), including but not limited to Triticale.
As used herein, the term "barley" refers to any species of the Genus Hordeum, including progenitors thereof, as well as progeny thereof produced by crosses with other species. It is preferred that the plant is of a Hordeum species which is commercially cultivated such as, for example, a strain or cultivar or variety of Hordeum vulgare or suitable for commercial production of grain. Methods for producing transgenic plants
Four general methods for direct delivery of a gene into cells have been described: (1) chemical methods (Graham et al., 1973); (2) physical methods such as microinjection (Capecchi, 1980); electroporation (see, for example, WO 87/06614, US 5,472,869, 5,384,253, WO 92/09696 and WO 93/21335); and the gene gun (see, for example, US 4,945,050 and US 5,141,131); (3) viral vectors (Clapp, 1993; Lu et al., 1993; Eglitis et al., 1988); and (4) receptor-mediated mechanisms (Curiel et al., 1992; Wagner et al., 1992).
Acceleration methods that may be used include, for example, microprojectile bombardment and the like. One example of a method for delivering transforming nucleic acid molecules to plant cells is microprojectile bombardment. This method has been reviewed by Yang et al., Particle Bombardment Technology for Gene Transfer, Oxford Press, Oxford, England (1994). Non-biological particles (microprojectiles) that may be coated with nucleic acids and delivered into cells by a propelling force. Exemplary particles include those comprised of tungsten, gold, platinum, and the like. A particular advantage of microprojectile bombardment, in addition to it being an effective means of reproducibly transforming monocots, is that neither the isolation of protoplasts, nor the susceptibility of Agrobacterium infection are required. A particle delivery system suitable for use with the present invention is the helium acceleration PDS-1000/He gun is available from Bio-Rad Laboratories. For the bombardment, immature embryos or derived target cells such as scutella or calli from immature embryos may be arranged on solid culture medium.
In another alternative embodiment, plastids can be stably transformed. Method disclosed for plastid transformation in higher plants include particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination (US 5, 451,513, US 5,545,818, US 5,877,402, US 5,932479, and WO 99/05265.
Agrobacterium-mQ \&iQ transfer is a widely applicable system for introducing genes into plant cells because the DNA can be introduced into whole plant tissues, thereby bypassing the need for regeneration of an intact plant from a protoplast. The use of Agrobacterium-mQ \&iQ plant integrating vectors to introduce DNA into plant cells is well known in the art (see, for example, US 5,177,010, US 5,104,310, US 5,004,863, US 5,159,135). Further, the integration of the T-DNA is a relatively precise process resulting in few rearrangements. The region of DNA to be transferred is defined by the border sequences, and intervening DNA is usually inserted into the plant genome. Agrobacterium transformation vectors are capable of replication in E. coli as well as Agrobacterium, allowing for convenient manipulations as described (Klee et al., Plant DNA Infectious Agents, Hohn and Schell, (editors), Springer-Verlag, New York, (1985): 179-203). Moreover, technological advances in vectors for d zYz/ zcVc vz/zzz-mediated gene transfer have improved the arrangement of genes and restriction sites in the vectors to facilitate construction of vectors capable of expressing various polypeptide coding genes. The vectors described have convenient multi-linker regions flanked by a promoter and a polyadenylation site for direct expression of inserted polypeptide coding genes and are suitable for present purposes. In addition, Agrobacterium containing both armed and disarmed Ti genes can be used for the transformations. In those plant varieties where HgroZ>rzctez7z/zzz-mediated transformation is efficient, it is the method of choice because of the facile and defined nature of the gene transfer.
A transgenic plant formed using Agrobacterium transformation methods typically contains a single genetic locus on one chromosome. Such transgenic plants can be referred to as being hemizygous for the added gene. More preferred is a transgenic plant that is homozygous for the added structural gene; i.e., a transgenic plant that contains two added genes, one gene at the same locus on each chromosome of a chromosome pair. A homozygous transgenic plant can be obtained by sexually mating (selfing) an independent segregant transgenic plant that contains a single added gene, germinating some of the seed produced and analyzing the resulting plants for the gene of interest.
It is also to be understood that two different transgenic plants can also be mated to produce offspring that contain two independently segregating exogenous genes. Selfing of appropriate progeny can produce plants that are homozygous for both exogenous genes. Back-crossing to a parental plant and out-crossing with a non- transgenic plant are also contemplated, as is vegetative propagation. Descriptions of other breeding methods that are commonly used for different traits and crops can be found in Fehr, Breeding Methods for Cultivar Development, J. Wilcox (editor) American Society of Agronomy, Madison Wis. (1987).
Transformation of plant protoplasts can be achieved using methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments. Application of these systems to different plant varieties depends upon the ability to regenerate that particular plant strain from protoplasts. Illustrative methods for the regeneration of cereals from protoplasts are described (Fujimura et al., 1985; Toriyama et al., 1986; Abdullah et al., 1986).
Other methods of cell transformation can also be used and include but are not limited to introduction of DNA into plants by direct DNA transfer into pollen, by direct I l l injection of DNA into reproductive organs of a plant, or by direct injection of DNA into the cells of immature embryos followed by the rehydration of desiccated embryos.
The regeneration, development, and cultivation of plants from single plant protoplast transformants or from various transformed explants is well known in the art (Weissbach et al., Methods for Plant Molecular Biology, Academic Press, San Diego, (1988)). This regeneration and growth process typically includes the steps of selection of transformed cells, culturing those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil.
The development or regeneration of plants containing the foreign, exogenous gene is well known in the art. Preferably, the regenerated plants are self-pollinated to provide homozygous transgenic plants. Otherwise, pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important lines. Conversely, pollen from plants of these important lines is used to pollinate regenerated plants. A transgenic plant of the present invention containing a desired exogenous nucleic acid is cultivated using methods well known to one skilled in the art.
Methods for transforming dicots, primarily by use of Agrobacterium lumefaciens. and obtaining transgenic plants have been published for cotton (US 5,004,863, US 5,159,135, US 5,518,908); soybean (US 5,569,834, US 5,416,011); Brassica (US 5,463,174); peanut (Cheng et al., 1996); and pea (Grant et al., 1995).
Methods for transformation of cereal plants such as wheat and barley for introducing genetic variation into the plant by introduction of an exogenous nucleic acid and for regeneration of plants from protoplasts or immature plant embryos are well known in the art, see for example, CA 2,092,588, AU 61781/94, AU 667939, US 6,100,447, WO 97/048814, US 5,589,617, US 6,541,257, and other methods are set out in WO 99/14314. Preferably, transgenic wheat or barley plants are produced by Agrobacterium tumefaciens mediated transformation procedures. Vectors carrying the desired nucleic acid construct may be introduced into regenerable wheat cells of tissue cultured plants or explants, or suitable plant systems such as protoplasts. The regenerable wheat cells are preferably from the scutellum of immature embryos, mature embryos, callus derived from these, or the meristematic tissue.
To confirm the presence of the transgenes in transgenic cells and plants, a polymerase chain reaction (PCR) amplification or Southern blot analysis can be performed using methods known to those skilled in the art. Expression products of the transgenes can be detected in any of a variety of ways, depending upon the nature of the product, and include Western blot and enzyme assay. One particularly useful way to quantitate protein expression and to detect replication in different plant tissues is to use a reporter gene, such as GUS. Once transgenic plants have been obtained, they may be grown to produce plant tissues or parts having the desired phenotype. The plant tissue or plant parts, may be harvested, and/or the seed collected. The seed may serve as a source for growing additional plants with tissues or parts having the desired characteristics.
The "polymerase chain reaction" ("PCR") is a reaction in which replicate copies are made of a target polynucleotide using a "pair of primers" or "set of primers" consisting of "upstream" and a "downstream" primer, and a catalyst of polymerization, such as a DNA polymerase, and typically a thermally-stable polymerase enzyme. Methods for PCR are known in the art, and are taught, for example, in "PCR" (M.J. McPherson and S.G Moller (editors), BIOS Scientific Publishers Ltd, Oxford, (2000)). PCR can be performed on cDNA obtained from reverse transcribing mRNA isolated from plant cells expressing a polynucleotide of the invention. However, it will generally be easier if PCR is performed on genomic DNA isolated from a plant.
A primer is an oligonucleotide sequence that is capable of hybridising in a sequence specific fashion to the target sequence and being extended during the PCR. Amplicons or PCR products or PCR fragments or amplification products are extension products that comprise the primer and the newly synthesized copies of the target sequences. Multiplex PCR systems contain multiple sets of primers that result in simultaneous production of more than one amplicon. Primers may be perfectly matched to the target sequence or they may contain internal mismatched bases that can result in the introduction of restriction enzyme or catalytic nucleic acid recognition/cleavage sites in specific target sequences. Primers may also contain additional sequences and/or contain modified or labelled nucleotides to facilitate capture or detection of amplicons. Repeated cycles of heat denaturation of the DNA, annealing of primers to their complementary sequences and extension of the annealed primers with polymerase result in exponential amplification of the target sequence. The terms target or target sequence or template refer to nucleic acid sequences which are amplified.
Methods for direct sequencing of nucleotide sequences are well known to those skilled in the art and can be found for example in Ausubel et al. (supra) and Sambrook et al. (supra). Sequencing can be carried out by any suitable method, for example, dideoxy sequencing, chemical sequencing or variations thereof. Direct sequencing has the advantage of determining variation in any base pair of a particular sequence. Plant/Grain Processing
Grain/seed of the invention, preferably cereal grain, or other plant parts of the invention, can be processed to produce a food ingredient, food or non-food product using any technique known in the art.
In one embodiment, the product is whole grain flour such as, for example, an ultrafme-milled whole grain flour, or a flour made from about 100% of the grain. The whole grain flour includes a refined flour constituent (refined flour or refined flour) and a coarse fraction (an ultrafme-milled coarse fraction).
Refined flour may be flour which is prepared, for example, by grinding and bolting cleaned grain such as wheat or barley grain. The particle size of refined flour is described as flour in which not less than 98% passes through a cloth having openings not larger than those of woven wire cloth designated "212 micrometers (U.S. Wire 70)". The coarse fraction includes at least one of: bran and germ. For instance, the germ is an embryonic plant found within the grain kernel. The germ includes lipids, fiber, vitamins, protein, minerals and phytonutrients, such as flavonoids. The bran includes several cell layers and has a significant amount of lipids, fiber, vitamins, protein, minerals and phytonutrients, such as flavonoids. Further, the coarse fraction may include an aleurone layer which also includes lipids, fiber, vitamins, protein, minerals and phytonutrients, such as flavonoids. The aleurone layer, while technically considered part of the endosperm, exhibits many of the same characteristics as the bran and therefore is typically removed with the bran and germ during the milling process. The aleurone layer contains proteins, vitamins and phytonutrients, such as ferulic acid.
Further, the coarse fraction may be blended with the refined flour constituent. The coarse fraction may be mixed with the refined flour constituent to form the whole grain flour, thus providing a whole grain flour with increased nutritional value, fiber content, and antioxidant capacity as compared to refined flour. For example, the coarse fraction or whole grain flour may be used in various amounts to replace refined or whole grain flour in baked goods, snack products, and food products. The whole grain flour of the present invention (i.e. -ultrafme-milled whole grain flour) may also be marketed directly to consumers for use in their homemade baked products. In an exemplary embodiment, a granulation profile of the whole grain flour is such that 98% of particles by weight of the whole grain flour are less than 212 micrometers.
In further embodiments, enzymes found within the bran and germ of the whole grain flour and/or coarse fraction are inactivated in order to stabilize the whole grain flour and/or coarse fraction. Stabilization is a process that uses steam, heat, radiation, or other treatments to inactivate the enzymes found in the bran and germ layer. Flour that has been stabilized retains its cooking characteristics and has a longer shelf life.
In additional embodiments, the whole grain flour, the coarse fraction, or the refined flour may be a component (ingredient) of a food product and may be used to product a food product. For example, the food product may be a bagel, a biscuit, a bread, a bun, a croissant, a dumpling, an English muffin, a muffin, a pita bread, a quickbread, a refrigerated/frozen dough product, dough, baked beans, a burrito, chili, a taco, a tamale, a tortilla, a pot pie, a ready to eat cereal, a ready to eat meal, stuffing, a microwaveable meal, a brownie, a cake, a cheesecake, a coffee cake, a cookie, a dessert, a pastry, a sweet roll, a candy bar, a pie crust, pie filling, baby food, a baking mix, a batter, a breading, a gravy mix, a meat extender, a meat substitute, a seasoning mix, a soup mix, a gravy, a roux, a salad dressing, a soup, sour cream, a noodle, a pasta, ramen noodles, chow mein noodles, lo mein noodles, an ice cream inclusion, an ice cream bar, an ice cream cone, an ice cream sandwich, a cracker, a crouton, a doughnut, an egg roll, an extruded snack, a fruit and grain bar, a microwaveable snack product, a nutritional bar, a pancake, a par- baked bakery product, a pretzel, a pudding, a granola-based product, a snack chip, a snack food, a snack mix, a waffle, a pizza crust, animal food or pet food.
In alternative embodiments, the whole grain flour, refined flour, or coarse fraction may be a component of a nutritional supplement. For instance, the nutritional supplement may be a product that is added to the diet containing one or more additional ingredients, typically including: vitamins, minerals, herbs, amino acids, enzymes, antioxidants, herbs, spices, probiotics, extracts, prebiotics and fiber. The whole grain flour, refined flour or coarse fraction of the present invention includes vitamins, minerals, amino acids, enzymes, and fiber. For instance, the coarse fraction contains a concentrated amount of dietary fiber as well as other essential nutrients, such as B- vitamins, selenium, chromium, manganese, magnesium, and antioxidants, which are essential for a healthy diet. For example 22 grams of the coarse fraction of the present invention delivers 33% of an individual's daily recommend consumption of fiber. The nutritional supplement may include any known nutritional ingredients that will aid in the overall health of an individual, examples include but are not limited to vitamins, minerals, other fiber components, fatty acids, antioxidants, amino acids, peptides, proteins, lutein, ribose, omega-3 fatty acids, and/or other nutritional ingredients. The supplement may be delivered in, but is not limited to the following forms: instant beverage mixes, ready -to-drink beverages, nutritional bars, wafers, cookies, crackers, gel shots, capsules, chews, chewable tablets, and pills. One embodiment delivers the fiber supplement in the form of a flavored shake or malt type beverage, this embodiment may be particularly attractive as a fiber supplement for children.
In an additional embodiment, a milling process may be used to make a multi-grain flour or a multi-grain coarse fraction. For example, bran and germ from one type of grain may be ground and blended with ground endosperm or whole grain cereal flour of another type of cereal. Alternatively bran and germ of one type of grain may be ground and blended with ground endosperm or whole grain flour of another type of grain. It is contemplated that the present invention encompasses mixing any combination of one or more of bran, germ, endosperm, and whole grain flour of one or more grains. This multigrain approach may be used to make custom flour and capitalize on the qualities and nutritional contents of multiple types of cereal grains to make one flour.
It is contemplated that the whole grain flour, coarse fraction and/or grain products of the present invention may be produced by any milling process known in the art. An exemplary embodiment involves grinding grain in a single stream without separating endosperm, bran, and germ of the grain into separate streams. Clean and tempered grain is conveyed to a first passage grinder, such as a hammermill, roller mill, pin mill, impact mill, disc mill, air attrition mill, gap mill, or the like. After grinding, the grain is discharged and conveyed to a sifter. Further, it is contemplated that the whole grain flour, coarse fraction and/or grain products of the present invention may be modified or enhanced by way of numerous other processes such as: fermentation, instantizing, extrusion, encapsulation, toasting, roasting, or the like.
Malting
A malt-based beverage provided by the present invention involves alcohol beverages (including distilled beverages) and non-alcohol beverages that are produced by using malt as a part or whole of their starting material. Examples include beer, happoshu (low-malt beer beverage), whisky, low-alcohol malt-based beverages (e.g., malt-based beverages containing less than 1% of alcohols), and non-alcohol beverages.
Malting is a process of controlled steeping and germination followed by drying of the grain such as barley and wheat grain. This sequence of events is important for the synthesis of numerous enzymes that cause grain modification, a process that principally depolymerizes the dead endosperm cell walls and mobilizes the grain nutrients. In the subsequent drying process, flavour and colour are produced due to chemical browning reactions. Although the primary use of malt is for beverage production, it can also be utilized in other industrial processes, for example as an enzyme source in the baking industry, or as a flavouring and colouring agent in the food industry, for example as malt or as a malt flour, or indirectly as a malt syrup, etc.
In one embodiment, the present invention relates to methods of producing a malt composition. The method preferably comprises the steps of:
(i) providing grain, such as barley or wheat grain, of the invention,
(ii) steeping said grain,
(iii) germinating the steeped grains under predetermined conditions and
(iv) drying said germinated grains.
For example, the malt may be produced by any of the methods described in Hoseney (Principles of Cereal Science and Technology, Second Edition, 1994: American Association of Cereal Chemists, St. Paul, Minn.). However, any other suitable method for producing malt may also be used with the present invention, such as methods for production of speciality malts, including, but limited to, methods of roasting the malt.
Malt is mainly used for brewing beer, but also for the production of distilled spirits. Brewing comprises wort production, main and secondary fermentations and posttreatment. First the malt is milled, stirred into water and heated. During this "mashing", the enzymes activated in the malting degrade the starch of the kernel into fermentable sugars. The produced wort is clarified, yeast is added, the mixture is fermented and a post-treatment is performed.
Detection of Nitrogenase Complex
Detection of the nitrogenase complex can be carried out by any method which allows for the detection of the interaction between the NifDK protein complex and the NifH protein. Methods suitable for detecting the interaction between the NifDK protein complex and the NifH protein include any method known in the art for detecting proteinprotein interaction including co-immunoprecipitation, affinity blotting, pull down, FRET and the like.
Alternatively, the detection of the nitrogenase complex can be carried out by measuring the activity of the resulting nitrogenase complex.
Methods suitable for measuring nitrogenase activity include any method known in the art for detecting the enzymatic reduction of dinitrogen to ammonia wherein electrons are transferred from the NifH protein to the NifDK protein complex. For example, the nitrogen fixation activity can be estimated by the acetylene reduction assay. Briefly, this technique is an indirect method which uses the ability of the nitrogenase complex to reduce triple bounded substrates. The nitrogenase enzyme reduces acetylene (C2H2) to ethylene (C2H4). Both gases can be quantified using gas chromatography. Nitrogen fixation may also be measured by the hydrogen evolution assay. H2 is an obligate by-product of N2 fixation. An indirect measure of nitrogenase activity can therefore be obtained by quantifying the H2 concentration in a gas stream using a flow- through H2 sensor or gas chromatograph.
Detection of N2 fixation
Nitrogen fixation can be estimated by determining a net increase in total N of a plant-soil system (N balance method); 2) separating plant N into the fraction taken up from the soil and the fraction derived from the N2 fixation (N difference, 15N natural abundance, 15N isotype dilution and ureide methods) and 3) measuring the activity of the nitrogenase (acetylene reduction and hydrogen evolution assays). The acetylene reduction and N2 reduction assays can be adapted for in vitro use as described herein.
EXAMPLES
Example 1. Materials and Methods
Growth of Azotobacter vinelandii and induction of the Fe-nitrogenase
A. vinelandii strains DJ2102 and DJ2241 were kindly provided by Dennis Dean, Virginia Polytechnic Institute and State University, USA. DJ2102 cells from stocks were streaked onto plates containing Burke’s medium (Strandberg and Wilson, 1968) supplemented with 13 mM ammonium acetate as nitrogen source (BN medium). The cells were transferred to 100 mL liquid BN medium in 250 mL shake flasks and grown at 30°C and 250 rpm overnight. The next morning, a new culture of 250 mL in a 1 L shake flask was set up with an initial optical density at 600 nm (OD600) of 0.1. In the evening, 10 L medium was inoculated with 250 mL of the pre-culture. The larger culture was grown in a bioreactor (Infers, Labfors-5) with the following parameters: dissolved oxygen level, pCh, was controlled by a cascade with variation in stirring speed and air flow rate. The setpoints were as follows: pCh = 25% (min: 15%, max: 100%), stirring speed = 125 rpm (min: 125 rpm, max: 400 rpm), air flow rate = 10 L/min (min: 1 L/min, max: 10/ L min).
To induce the cells for producing active nitrogenase, the DJ2102 cells were grown in the BN medium until a buffer exchange to Burke’s medium with no nitrogen (B medium) as previously described (Jimenez-Vicente et al., 2018). Approximately 16 h after starting the culture, the culture was centrifuged at 7,187 x g for 20 min and then resuspended in 10 L B medium in the bioreactor. Cells were harvested 3-4 hours after the cells had been re-suspended in B medium by centrifugation at 7,187 x g for 20 min. The cells were frozen under liquid nitrogen and stored at -80°C until further use. Strain DJ2241 was grown in a two-stage process as described (Perez-Gonzalez et al., 2021) with minor modifications. The A. vinelandii cells were cultured in 500 mL aliquots in 2 L flasks in an orbital shaker with aeration. 300 mM ammonium ferric citrate was added to the pre-inoculum medium to increase the Fe-S loading. For an inoculum, strain DJ2241 was streaked on modified Burk’s Nitrogen media plate containing 1.64 mM KH2PO4, 5.2 mM K2HPO4, 10 mM urea, 300 mM ammonium ferric citrate, 20% (v/v) sucrose, 0.2% (v/v) MgSC . FFO, 0.09% (w/v) CaCh 2H2O and 0.2 mM Fe(II)SO4.7H2O (Modified BN medium) solidified with 2% (v/v) agar, and incubated for 2-3 days at 30°C. From the plate, a small loop full of cells was used to inoculate a 200 mL pre-inoculum Modified BN medium and grown for 15 hours at 30°C with shaking at 250 rpm to an ODeoo 1.0. Cells were centrifuged at 4,000 rpm for 10 min and washed with Low NB medium to remove excess urea. Low NB medium was the same as Modified BN medium except that the urea concentration was 1.0 mM rather than 10 mM. The supernatant was removed via centrifugation at 4,000 rpm for 10 min and the pelleted cells gently resuspended in B medium and inoculated in 2 L flasks containing 500 mL of B medium supplemented with 300 mM ammonium ferric citrate and 1.0 mM urea to an initial ODeoo of 0.1. The 2 L flasks were sealed using micropore tape and grown for 21 hours at 30°C with shaking at 250 rpm. After 21 hours of growth, cells were centrifuged at 5,000 rpm for 20 min. The resulting cellular paste was stored at -80°C. Typically, 12 L of growth medium provided between 20-30 gram of cellular paste which formed enough material for a single protein purification of the AnfH and AnfDKG enzymes.
Isolation of AnfDKG and AnfH from A. vinelandii
The bacterial AnfD-AnfK-AnfG (AnfDKG) complex and AnfH component were isolated and purified as described by Perez-Gonzalez et al. (2021) and Harris et al. (2018) with minor modifications. In brief and under anaerobic conditions, the Streptactin XT column and the CaptoQ anionic column were connected and equilibrated with 100 mL strep wash buffer (50 mM Tris-HCl, pH 8.0, 75 mM NaCl, 20% glycerol and 2 mM sodium dithionite). The CaptoQ anionic column with its positively charged resin was used to separate proteins and protein complexes according to differences in charge, enabling the purification of the native AnfH dimer enzyme. The complexes are eluted using an anionic gradient, based on their charge and interaction with the column. The frozen A. vinelandii DJ2241 cell paste was resuspended under anaerobic conditions in lysis buffer containing 50 mM Tris-HCl, pH 8.0, 75 mM NaCl, 20% (v/v) glycerol with added 2 mM sodium dithionite, 10 mM phenylmethyl sulfonyl fluoride (PMSF) and 10 mM leupeptin. Resuspended cells were sonicated in a rosette glass cooling cell on ice, with 3 x 5 min bursts, with the sonicator set to 50% power and 50% cycle to ensure the sample did not overheat. Lysed cells were then centrifuged for 1 hour at 35,000 rpm to remove cellular debris. The supernatant was next passed over a Streptactin XT column (Strep-Tactin XT Superflow IBA Lifesciences 2-4030-010) to bind the strep tagged AnfDKG complex. The flowthrough was further passed over the CaptoQ anionic column to isolate the AnfH protein. Both the Streptactin XT and CaptoQ anionic columns were then washed with 100 mL strep wash buffer (50 mM Tris-HCl, pH 8.0, 75 mM NaCl, 20% (v/v) glycerol with added 2 mM sodium dithionite). Elution buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 20% (v/v) glycerol, 2 mM sodium dithionite and 50 mM biotin was used to elute the bound AnfDKG protein from the Streptactin XT column. Eluted fractions were collected and concentrated by centrifugation using an Amicon (EMD Millipore, Billerica, MA) concentrator with a 30 kDa cut-off.
The CaptoQ anionic column was washed with 100 mL of buffer containing 50 mM Tris-HCl pH 8.0 and 2 mM DTH, and lacking salt. Proteins were separated with a NaCl gradient of 0-50% NaCl (w/v) at 5 mL/min. AnfH eluted off the column as a dark brown fraction at 27-29% NaCl. AnfH fractions were further purified by size exclusion with a Sephacryl S-200 column using a buffer containing 50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 5% (v/v) glycerol and 2 mM sodium dithionite. Pooled fractions containing the AnfH polypeptide were concentrated using an Amicon (EMD Millipore, Billerica, MA) concentrator with a 10 kDa cut-off membrane and stored under liquid nitrogen. Protein concentrations were determined by the Bicinchoninic acid assay using Bovine Serum Albumin (BSA) as a standard (Copper-based total protein quantification- ThermoFisher). A single protein purification typically yielded 1,200 pL of the AnfDKG enzyme preparation and 1,000 pL of the AnfH enzyme preparation.
Isolation of NifDK from 4. vinelandii
NifD-NifK (NifDK) was purified from A. vinelandii strain DJ2102. Cells were lysed and NifDK was purified using the same procedure as described for AnfDKG from strain DJ2241 except that the gradient for the CaptoQ column was different. For the purification of Nif proteins, the gradient consisted of a steep gradient increase to 200 mM NaCl with 50 mL buffer, and then a shallower gradient increase to 500 mM NaCl with 300 mL buffer. The fractions were analysed by SDS-PAGE. NifH eluted at first and then NifDK. NifDK was then purified using a Strep-Tactin®XT column as described above for AnfDKG. Molecular cloning and heterologous expression of A. vinelandii NifU in E. coli
A. vinelandii NifU was co-expressed with A. vinelandii NifS in E. coli in the EcoFlex cloning system (Moore et al., 2016) using the pTU2S-a vector with a pl 5a origin of replication. The construct was named EFL2-30. NifU contained a Twin-Strep-tag® at the N-terminus for purification. For expression, EFL2-30 was transformed into E. coli BL21 (DE3). A single colony was cultured overnight in 100 mL LB medium. The cells were subcultured and grown in 2 L Schott bottles in modified terrific broth (mTB + additional glycerol) medium supplemented with 35 pg mL chloramphenicol and were stirred with a magnetic stirrer at room temperature. When the OD600 reached 0.6 ± 0.1, expression was induced with 0.1 mM isopropyl P-D-thiogalactopyranoside (IPTG), the culture was supplemented with 2 mM (NH4)2Fe(SO4)2 and 0.5 mM cysteine, and the lids of the bottles were closed to transition to anaerobic growth. After 16 hours the culture was harvested by centrifugation at 15,000 x g for 10 min. The pellets were frozen in liquid nitrogen and stored at -80 °C until further use.
Alternatively, the cells were grown in a 10 L bioreactor (Labfors-5). The cells were grown at 37°C until induction and the dissolved oxygen level, pCh, was controlled by a cascade with variation in stirring speed and air flow rate. The setpoints were as follows: pO2 = 25% (min: 15%, max: 100%), stirring speed = 125 rpm (min: 125 rpm, max: 400 rpm), flow rate = 10 L/min (min: 1 L/min, max: 10 L/min). After the OD600 reached 0.6 ± 0.1, the cells were induced with 0.1 mM IPTG, the culture was supplemented with 2 mM (NH4)2Fe(SO4)2 and 0.5 mM cysteine, the temperature was decreased to 18°C and the air flow was turned off. After 16 hours the culture was harvested by centrifugation at 7,187 x g for 20 min, the pellets were frozen in liquid nitrogen and stored at -80 °C until further use.
Purification of NifU from E. coli
The purification of NifU was performed in a COY anaerobic chamber in a 97- 98% N2/2-3% H2 atmosphere with an AKTA pure instrument. The cell pellet was resuspended in 50 mM Tris-HCl pH 7.4, 200 mM NaCl, 5% glycerol, 2 mM TCEP (buffer A) with 10 pM leupeptin, 0.2 mM PMSF and 25 mg/L lysozyme, and sonicated for 3 x 3 min (Hielscher sonicator UP200Ht) at 50% cycle and 50% amplitude. The suspension was centrifuged at 142,400 x g in polycarbonate bottles with cap assembly (Beckman Coulter). The supernatant was loaded onto a Strep-Tactin®XT (IB A) column equilibrated in buffer A, washed with 10 column volumes of buffer A, and then eluted in buffer A with 50 mM biotin. The eluate was then concentrated using an Amicon® Ultra centrifugal filter (30kDa MW cutoff) and frozen in liquid nitrogen until further use. Chemical reconstitution ofFe-S clusters on NifU proteins
NifU fusion polypeptides were chemically reconstituted using a similar procedure as previously described (Lopez-Torrejon et al., 2016; Marengo et al., 2021). The TS::NifU polypeptide was diluted in 25 mM Tris pH 7.4, 150 mM NaCl, 2.5 mM dithiothreitol (DTT) to a concentration in the range of 1 - 5 mg/mL. A solution of 40 mM (NH4)2Fe(SO4)2 was then added to a final concentration of 2.5 mM. After that, 40 mM Na2S was added to a final concentration of 0.5 mM with gentle, constant mixing. A colour change was observed within a few minutes of the addition of Fe2+ and S2'. The reaction was incubated overnight in the anaerobic chamber at approximately 28°C. The next day, the buffer was exchanged to Mg-ATP buffer using a MiniTrap Sephadex G25 column to remove excess Fe2+ and S2'. Mg-ATP buffer was prepared by dissolving 10 mM MgCh and 2 mM ATP in 25 mM Tris pH 7.4, which was followed by a pH adjustment by addition of 1/10 volume 1 M NaOH, resulting in pH 7.8. The protein solution was concentrated using an Amicon®Ultra centrifugal filter (30 kDaMW cutoff).
NifU reconstitution of AnfH
Buffers for the reconstitution were prepared as follows: Prior to use all buffers and solutions used were tested for O2 contamination using electrochemically reduced methyl viologen. Mg-ATP buffer was prepared by dissolving 10 mM MgCh and 5 mM ATP in 25 mM MOPS, pH 7.0, which was followed by pH adjustment by addition of 1/50 volume 1 M NaOH, resulting in pH 7.7. To remove interfering compounds such as DTH and biotin, the elution buffer of AnfH was exchanged to Mg-ATP buffer using a centrifugal filter (Amicon® Ultra, 10 kDa or 30 kDa MW cutoff). This procedure was carried out twice and the minimum dilution achieved was 200-fold. Chemically reconstituted NifU was added to AnfH at a ratio of 2: 1 and incubated overnight at approx. 28°C.
Determination of protein concentration
To determine the protein concentration of purified and concentrated Anf and Nif proteins, a standard curve was developed using a series of Bovine Serum Albumin (BSA) protein standards in the 0 pg/mL to 1,000 pg/mL range. The absorbance of each sample was measured at 562 nm. Protein concentrations of samples were determined using the standard curve from plotted protein standards of known concentration. Sample concentration was determined using the BCA Protein Assay kit. Inductively Coupled Plasma Mass Spectrometry
Inductively Coupled Plasma Mass Spectrometry (ICPMS) analysis to measure the metal content of purified protein samples was conducted by a facility at the Australian National University. Samples were analysed for levels of Mg, V, Mn, Fe, Co, Ni, Cu, Zn and Mo using a ThermoFisher iCap RQ Q-ICP-MS with quartz sample introduction system, including double-pass cyclonic spraychamber and MicroMist® nebuliser. Samples were acidified to 2.0% (v/v) with triple-distilled nitric acid prior to analysis. The Q-ICP-MS was operated using Qtegra™ software in high-sensitivity standard mode with nickel sample and skimmer cones and a high-sensitivity skimmer cone insert. Quantification was achieved using a 7-point external calibration series, and independently prepared quality control samples measured to verify calibration accuracy and long-term instrument performance.
Acetylene reduction assay for FeFe nitrogenase function
Acetylene reduction assays (ARA) were conducted using sealed 3.5 cc reaction vials as previously described (Perez-Gonzalez et al., 2021). In brief, vials contained an assay buffer including a Mg-ATP regeneration system containing 5 mM DTH, 2 mM ATP, 20 mM MgCh, 10 mM phosphocreatine and 0.1 mg/mL phosphocreatine kinase from rabbit muscle in 25 mM Tris pH 8.0 buffer. The reaction mixtures were made anaerobic and the headspace gases in the vials were adjusted to the desired partial pressures containing 0.1 atm acetylene (C2H2). The remaining gas space was filled with argon. For assays with AnfH and its variants, 0.1 mg of purified AnfD-AnfK-AnfG protein (FeFe protein) was added to each assay vial. These reactions were initiated by the addition of bacterial or plant isolated AnfH protein at a molar ratio of 10, 20 or 30 Fe protein per FeFe protein. Reaction mixtures were incubated at 30°C with shaking for 18 hours at 200 rpm. The ethylene (C2H4) product from the acetylene reduction assay was quantified using a gas chromatograph with Flame Ionization Detector (GC-FID) according to published methods with minor modifications (Allen et al., 2020). Ethylene standards were purchased from BOC gas at 100 ppm. Positive controls were conducted with purified AnfH isolated from A. vinelandii.
When used, the A. vinelandii NifU polypeptide loaded with Fe-S was used at a molar ratio of 40 per FeFe protein. The NifU was added where indicated prior to addition of sodium dithionite (DTH) containing buffer. Acetylene reduction assays for NifH and variants other than AnfH
Prior to acetylene reduction assay (ARA), the buffer of purified NifH fusion polypeptides, including for NifH dimer polypeptides, was replaced with 50 mM Tris, pH 7.7, containing 100 mM NaCl using a 50 kDa cut-off centrifugal filter unit. Protein concentration of the buffer-replaced NifH preparations was measured with the BCA protein assay kit (Thermo Fisher Scientific) against known concentrations of bovine serum albumin used to generate the standard curve. Acetylene reduction assay (ARA) was performed according to Jiang et al. (2021) with the following modifications: The final volume of each reaction was 370 pl aqueous medium assembled into a 3.7 ml Exetainer vial, injected with 370 pl of acetylene and incubated at 30°C for 30 minutes. Production of ethylene was measured using the method described in Allen et al. (2020). Positive controls were conducted with NifH isolated from A. vinelandii.
Purification of 15N2 gas for nitrogenase activity assays
To remove the contaminating N compounds in commercially-available 15N2 gas (at least 98% pure and isotopically labelled; Cambridge Isotope Laboratories Inc, NLM- 363-0), a device published by Kennedy and Wood (2001) (the ‘Turret’) was employed. This allowed syringes with luer slip tips to transport the gas through, and mix with, different aqueous solutions. Due to the nature of the suspected contaminants, two solutions were decided upon, a base/oxidant solution and an acid solution. The base/oxidant solution contained 2M hydrogen peroxide and IM sodium hydroxide and was used to remove any nitrites (NCU) by oxidation to nitrates (NOs'), to form sodium nitrate, a stable and highly soluble salt in aqueous solution. The ratio of the two concentrations was important to obtain the oxidant sodium peroxide Na2O2 in low concentrations and minimise the decomposition of hydrogen peroxide to water and oxygen. The second, an acid solution contained 3M sulfuric acid (H2SO4) and 0.344 M potassium sulfate (K2SO4) and was used to convert ammonia (NH3) to ammonium sulfate ((NH4)2SO4). The potassium sulfate served to reduce the amount of nitrogen dissolved into solution during this process.
The gas scrubbing process involved the use of three 25 mL syringes with the Turret. One syringe was filled with the 15N2 gas, one filled with 5 mL of the base/oxidant solution and one with 5 mL of the acid solution. The Turret has three ports, A, B and C. The base/oxidant syringe was connected to port B and the acid syringe to C. The 15N2 syringe was connected to port A. The Turret contained a knob that allowed gas transfer to occur between A and B, B and C but not C to A. Upon connecting the 15N2 syringe, the gas was injected into port B where it was bubbled through the base solution and shaken for 2 min. The gas was then moved from port B to port C, where it was bubbled through the acid solution and shaken for 2 min. After the 2 minutes, the knob was moved into a position that closed off the acid syringe to port B so the base syringe could be removed and replaced with the 15N2 syringe again but connected to port B. The knob was adjusted to allow exchange between ports B and C and the now purified 15N2 then injected back into its original syringe, to be used immediately.
Nitrogenase assays in planta using gas containing 15N
Plants were exposed to purified 15N2 gas, or leaf discs were exposed to the gas, for a 24hr period at a concentration of up to 10% (v/v) of the gas, preferably in the dark. Prior to addition of the gas, the equivalent headspace to be added to the sample flask was evacuated to maintain a 1 atm pressure overall. After 24hr, leaf disks were removed from their suba sealed conical flasks, or whole leaves were excised from plants or disks cut from the leaves using the end of a 50 mL polypropylene tube, and dried overnight in an oven at 65°C in 6-well plates. Samples were then distributed to 2 mL tubes for tissue lysing with bead-beating at 20 kHz for 5 min. Triplicate samples of about 2 mg of the powders were accurately weighed out into tin capsules and subjected to Isotope Ratio Mass Spectrometry (IRMS). The IRMS was tuned using nitrogen gas that originated from liquid nitrogen evaporation and wheat flour was used as the system suitability sample. The delta-15N was calculated for each sample using the formula:
5 = 1000(Rsample — Rstandardj/Rstandard where Rsampie = the quotient 15N/14N for the sample and Rstandard = the quotient 15N/14N for atmospheric nitrogen, taken as 0.336/99.63.
Nitrogen fixation assay in vitro in aqueous mixtures
NifU fusion polypeptides were chemically reconstituted using a similar procedure as previously described (Lopez-Torrejon et al., 2016; Marengo et al., 2021). The TS::NifU polypeptide was diluted in 25 mM Tris pH 7.4, 150 mM NaCl, 2.5 mM dithiothreitol (DTT) to a concentration in the range of 1 - 5 mg/mL. A solution of 40 mM (NH4)2Fe(SO4)2 was then added to a final concentration of 2.5 mM. After that, 40 mM Na2S was added to a final concentration of 0.5 mM with gentle, constant mixing. A colour change was observed within a few minutes of the addition of Fe2+ and S2'. The reaction was incubated overnight in the anaerobic chamber at approximately 28°C. The next day, the buffer was exchanged to Mg-ATP buffer using a MiniTrap Sephadex G25 column to remove excess Fe2+ and S2'. Mg-ATP buffer was prepared by dissolving 10 mM MgCh and 2 mM ATP in 25 mM Tris pH 7.4, which was followed by a pH adjustment by addition of 1/10 volume 1 M NaOH, resulting in pH 7.8. The protein solution was concentrated using an Amicon®Ultra centrifugal filter (30 kDaMW cutoff).
Construction of plasmids for expression of Nif genes in plant cells
Unless otherwise stated, plasmids for transient expression of Nif genes, including Anf genes, in N benthamiana leaves or in stably transform plants were constructed using a modular cloning system with Golden Gate assembly (Peccoud et al., 2011, Weber et al., 2011). DNA components were commercially synthesized (Thermo Fisher Scientific, ENSA or Twist Bioscience) and first assembled as individual plasmids, each containing the CaMV 35S promoter (internal part number EC51288) and a CaMV polyadenylation signal/transcription terminator (EC41414) for expression in the plant cells, unless otherwise stated. All of the Nif genes, including Anf genes, were synthesised using a plant-derived codon bias for optimised expression. Plant codon-optimised components included protein coding regions nifH (EC38011), nifK (EC38015), nifY (EC38019), nifE (EC38016), nifN (EC38024), nif J (EC38022), nifB (EC38017), nifQ (EC38025), nifF (EC38021), nifU (EC38026), nifS (EC38018), nifV (EC38020), nifW (EC38027), nifZ (EC38029), nifM (EC38023) and nifX (EC38028). When desired, duplicate sequences were made to provide for translational fusions at the N- and/or C-terminus of the Nif/ Anf polypeptide to allow for either mitochondrial or cytoplasmic localisation. Plant codon optimized DNA parts including protein coding regions for anfD (EC38103, EC38106), anfK (EC38102), anfH (EC38100) and anfG (EC38101) were used to construct SN and SL plasmids. In many constructs, a nucleotide sequence coding for the first 51 amino acids of the Arabidopsis thaliana Fl-ATPase y subunit (MTP-FAy51) was included at the N-terminus of the Nif polypeptide for localization of the encoded fusion polypeptides to the mitochondrial matrix. MTP-FAy51 includes a well-characterised MPP cleavage site and was followed with or without an epitope tag such as the HA tag (EC38095 or EC38006, respectively) followed by the Nif sequence. In some constructs, a CoxIV mitochondrial targeting peptide including a processable MPP cleavage site followed by the twin-streptactin epitope tag (TwinStrep or TS, EC38091) was used instead of MTP- FAy51-HA. Other MTP sequences were used as described. The expression cassettes were assembled in suitable backbone Ll-acceptor vectors (EC47772, EC47742, EC47761, EC47781, EC47751) that included standard T-DNA based flanking regions using Type IIS restriction cloning with the Bsal enzyme for SN Golden Gate construction. SN constructs with the LI acceptors were then constructed into multi -gene constructs using Type IIS restriction cloning, with the Bbsl/BpiA enzyme for SL Golden Gate construction. The SN constructs were assembled onto a suitable SL acceptor plasmid (EC50505) with a kanamycin resistance gene in position one on the SL constructs as a selectable marker gene for plant transformation. All individual genes retained their own CaMV 35S promoter and CaMV polyadenylation signal/ transcription terminator (EC41414). The construct to express the silencing suppressor protein Pl 9 and other standard constructs were as described previously (Wood et al., 2009).
Expression of genes in plant cells in a transient expression system
Genes were expressed in plant cells using a transient expression system essentially as described by Wood et al. (2009), with various modifications as outlined below. Nicotiana benthamiana plants were grown in a growth chamber at 23 °C under a 16:8 h light: dark cycle with 90 pmol/min light intensity provided by cool white fluorescent lamps. Binary vectors containing the coding region to be expressed in plant cells by a strong, constitutive 35S promoter or the enhanced 35S promoter (e35S; Kay et al., 1987) were introduced into Agrobacterium tumefaciens strain GV3101 (SN or SL constructs). A chimeric binary vector, 35S::pl9, for expression ofthe P19 viral silencing suppressor was separately introduced into A. tumefaciens strain AGL1, as described in WO20 10/057246. This viral silencing suppressor was routinely included in the infiltrations to maintain gene expression of transgenes introduced together with it. The recombinant A. tumefaciens cells were grown to stationary phase at 28°C in LB broth supplemented with 50 mg/L carbenicillin or 50 mg/L kanamycin, according to the selectable marker gene on the vector, and 50 mg/L rifampicin. Acetosyringone was added to the culture to a final concentration of 100 pM and the culture then incubated at 28°C with shaking for another 2.5 hr. The bacteria were then pelleted by centrifugation at 5000 x g for 10 min at room temperature. The supernatant was discarded and the pellet was resuspended in a solution containing 10 mM MES pH 5.7, 10 mM MgCh and 100 pM acetosyringone (infiltration buffer) after which the OD600 was measured. A volume of each culture, including the culture containing the viral suppressor construct 35S::pl9, required to reach a final concentration of OD600 = 0.10 was added to a fresh tube. The final volume was made up with the infiltration buffer. Leaves were then infiltrated with the culture mixture and the plants were typically grown for a further three to five days after infiltration before leaf discs were recovered for analysis, or whole leaves if they were entirely infiltrated for protein purifications. A control infiltration was included in the same batch of plants which had only the viral suppressor construct 35S::pl9. For over-expression of more than one gene of interest in combination, each additional gene was introduced separately into an A. tumefaciens strain and grown as described above. Bacterial suspensions were mixed so that each bacterial strain was at a final concentration of OD600 = 0.10. The bacterial strain containing the gene encoding the P19 viral silencing suppressor was included in all mixtures at the same concentration. For example, to express four genes in a transient leaf assay and including the viral suppressor construct, the final OD600 of the infiltrated mixture was 5 x 0.10 = 0.50 units. The simultaneous over-expression of at least five genes each from separate T-DNA vectors within plant cells in the transient assay format has previously been demonstrated using 7V. benthamiana (Wood et al., 2009).
RNA extraction, cDNA synthesis and analysis
In order to extract RNA from plant leaf samples such as those which have been infiltrated w\'A\ Agrobaclerium , leaf pieces of about 2x2 cm in area are frozen with liquid nitrogen, ground to a powder, and 500 pl of Trizol buffer (Thermo Fisher Scientific) added per sample. Following this, the Trizol supplier’s instructions are followed except with these modifications: the chloroform extraction was repeated and the RNA was dissolved at 37°C. The extracted RNA was treated with RQ1 DNAse (Promega) to remove any extracted DNA. The RNA preparations were then further purified using Plant RNeasy columns (Qiagen). When performed, cDNA synthesis was carried out using Superscript III reverse transcriptase (Thermo Fisher Scientific) according to the supplier’ s protocol with an oligo-dT primer. For RT-PCR analysis of each RNA sample, three separate cDNA synthesis reactions were carried out. The 20 pl cDNA reactions were diluted 20-fold in nuclease free water. qRT-PCR was carried out on a Qiagen rotor gene Q real-time PCR machine. 9.6 pl of each cDNA was added to 10 pl of 2x sensifast no ROX SYBR Taq (Bioline) and 0.4 pl of forward and reverse primers at 10 pmol each, for a final reaction volume of 20 pl. All qPCR reactions, for both reference and specific genes, were carried out in triplicate, for example under the following cycling conditions: 1 cycle of 95°C/5 min, 45 cycles of 95°C/15 sec, 60°C/15 sec and 72°C/20 sec. Fluorescence was measured at the 72°C step. A 55°C to 99°C melting cycle was then carried out. Control amplifications for a constitutively expressed N benthamiana GADPH mRNA were used to normalise gene expression using the comparative quantitation program in the rotor gene software package. The values for each set of three cDNAs, representing the average of triplicate assays, were averaged, allowing for a calculation of the standard error of the mean (SEM). Preparation of anaerobic buffers
All buffers used for anaerobic experiments as described below were filtered through a 0.2 pm Whatman Nucleopore membrane (Sigma-Aldrich). Buffers were prepared as described by Okada et al. (2020). Anaerobic buffers were then stored under anaerobic conditions in a COY anaerobic hood (COY Laboratory products) with oxygen levels maintained at 0-5ppm prior to use. The COY anaerobic hood was filled with a H2/N2 mix of 2-3% / 98-97% and oxygen removed by the use of palladium catalysts.
Protein extraction from leaf tissue and Western blot analysis
To analyse the amount and properties of specific polypeptides produced in plant cells after T-DNA introduction, infiltrated zones from N. benthamiana leaves were harvested, 4 or 5 days after infiltration, as leaf discs with a diameter of 1.5cm. Proteins were extracted and protein expression detected by Western blot as described by Allen et al. (2017). After blotting onto the PVDF membrane, the gels were retained and stained with Coomassie stain (SimplyBlue SafeStain, Thermo Fisher Scientific) overnight, then rinsed in water for visualisation of remaining proteins to confirm that transfer of the polypeptides had occurred. Coomassie staining also provided confirmation of the equal loading of protein amounts per gel lane, using the levels of highly abundant proteins such as Rubisco large and small subunits as an indicator of equal protein loading per lane. The anti -HA antibody (Monoclonal Anti -HA, Sigma) was used at 1 :5000 dilution, the anti- Strep/HRP conjugate antibody (Strep-MAB-conjugate HRP, IBA Lifesciences) was used at 1 : 10,000 in the Western blot detections.
For testing the solubility of plant-expressed proteins, the leaf tissue was ground in ice-cold extraction buffer (100 mM Tris pH 8.0, 150 mM NaCl, 0.25 M mannitol, 5% (v/v) glycerol, 1% (v/v) Tween 20, 1% (w/v) PVP, 2 mM TCEP, 0.2 mM PMSF, 10 pM leupeptin) and transferred to a microfuge tube. The sample was centrifuged at 20,000 x g for 5 min to divide the sample into soluble (supernatant) and insoluble (pellet) fractions. The supernatant was transferred to a fresh microfuge tube and centrifuged again at 20,000 x g for 5 min. The insoluble fraction was washed three times by resuspension of the pellet in 1 mL extraction buffer. The pellet was dispersed by repeated strokes of the pipette and centrifuged at 20,000 x g for 5 min, this cycle was repeated twice more. Laemmli buffer was added to the resulting soluble and insoluble fractions and subjected to SDS-PAGE followed by Western blot analysis (Allen et al., 2017; Okada et al., 2020). Samples and molecular weight markers (BenchMark ladder- Thermo Fisher; Pre-stained PageRuler ladder- Thermo Fisher) were electrophoresed on 4-20% NuPage gels for 60 minutes at 200 V, using 20 pL of sample per lane. For Western blot analysis of anaerobically extracted proteins, the extractions were carried out in an anaerobic chamber (COY Laboratory Products) filled with a H2/N2 atmosphere (2-3%/97-98%). Anaerobic extraction solutions were prepared at a Schlenk line in a bottle equipped with a butyl rubber septum by at least four cycles of evacuating and purging with N2.
Purification from plants of polypeptides fused to a TwinStrep epitope
Unless otherwise stated, N. benthamiana leaves were harvested four or five days after infiltration with A. tumefaciens cells containing the genetic construct of interest, or from stably transformed plant leaves, and treated as follows. Leaf material, typically 10- 20g but up to about 120g fresh weight, was macerated in 100 ml cold extraction buffer (100 mM Tris-HCl pH 8.6, 200 mM NaCl, 10% v/v glycerol, 2 mM DTH, 0.2 mM phenylmethylsuflonyl fluoride (PMSF), 10 pM leupeptin) under anaerobic conditions (<5ppm O2) using a Nutribullet blender, keeping the mixture cold on ice throughout. The homogenised mixture was filtered through four layers of Miracloth (Merck Millipore) and the filtrate (70-80 ml) centrifuged for 1 hr at 20,000 g at 4°C. The resulting supernatant was filtered through a 0.2 pm PVDF membrane filter to further remove fine particulates and loaded onto a 4 ml Streptactin® XT 4Flow® (IBA Life Sciences) at 2.5 ml/min. After loading, the column was washed with 10 column volumes of wash buffer (100 mM Tris-HCl pH 8.6, 200 mM NaCl, 10% v/v glycerol, 2 mM DTH), after which the protein was eluted with wash buffer containing 50 mM biotin and concentrated down to approximately 300 pl with an Amicon 50 kDa cutoff centrifugal filter unit (Merck Millipore). When Fe and S (in the form of cysteine) were exogenously supplied to the lysate supernatant after centrifugation, the lysis buffer contained 2 mM TCEP as the reductant instead of DTH.
The purified protein concentrate was frozen in liquid nitrogen for future analysis. Samples were retained from each step of the purification process for Western blot analysis. Samples and molecular weight markers (BenchMark ladder- Thermo Fisher, Pre-stained Pageruler ladder- Thermo Fisher) were electrophoresed on 4-20% NuPage gels for 60 minutes at 200 V, using 20 pL of sample per lane. Proteins were blotted to PVDF membrane using an iBLOT apparatus and proteins containing an epitope detected by using anti-HA (1 :5,000 dilution), HRP (1 :5,000 dilution) or anti-Strep:HRP conjugate (1 : 10,000 dilution) antibodies.
The protein concentration of samples was determined using the BCA Protein Assay kit from ThermoFisher Scientific. The protein concentration of samples was calculated by reference to a standard curve of absorbances at 562 nm from bovine serum albumin. Densitometry of the Coomassie stain of the SDS-PAGE was used to determine the proportion of the purified polypeptides in the concentrated eluate using the analysis software in the Amersham Imager 600 (GE Healthcare).
Purification of HA epitope tagged proteins using anti -HA agarose
N. benthamiana leaf samples were harvested four-days post infiltration with A. tumefaciens cells containing the genetic SN or SL construct of interest and treated as outlined in Allen et al. (2020). In brief, leaf material of approximately 3.0 g was macerated by hand using a mortar and pestle in 10 mL of Extraction buffer. Soluble and insoluble fractions were separated by centrifugation for 15 min at 5,500 rpm at 4°C. The insoluble fraction was then discarded. 250 pL of anti-HA agarose suspension (Sigma) was gently washed with 1.0 mL of 50 mM Tris, pH 8.0, buffer containing 75 mM NaCl. The anti-HA agarose was centrifuged 2 min at 1,000 rpm at 4°C and the supernatant removed from the agarose suspension. Plant supernatant was then added to the anti-HA agarose and incubated for 1 hour with gentle rotation at 3 rpm at 4°C. Samples were next centrifuged for 2 min at 1,000 rpm and the supernatant separated. Protein bound anti-HA agarose beads were next washed five times with 1.0 mL of 50 mM Tris, pH 8.0, 75 mM NaCl buffer. Samples were next centrifuged for 2 min at 1,000 rpm and the supernatant removed. Anti-HA agarose was next resuspended in 200 pL of 4 x SDS buffer and heated for 5 min at 95°C. Heated samples were then analysed via SDS-PAGE and Western blotting. Protein samples from the HA-agarose purification were further used to develop targeted peptides for proteomic analysis.
Treatment of protein extracts with trypsin prior to LC-MS analyses
A variety of protein sources expressed from single gene and multigene constructs as described herein were used for LC-MS analysis to confirm the identity and quantitate specific polypeptides. Protein samples were prepared either in-solution or as excised gel slices. The in-gel digests were prepared as described previously (Colgrave et al., 2019). The solutions were subjected to the filter-aided sample preparation (FASP), a method used for the on-filter digestion of proteins prior to mass spectrometry-based analyses (Wisniewski et al., 2009). In brief, 100 pL with about 200 pg of protein was diluted in 8 M urea, 100 mM Tris-HCl, pH 8.5 (UA buffer) and applied to a 10 kDa molecular weight cut-off filter (Merck Millipore, Australia). The filters were centrifuged at 20,800 * for 15 min and washed with 200 pL of UA buffer. The total protein extracts were reduced with 50 mM dithiothreitol (DTT) and the mixture incubated at room temperature for 50 min with shaking. The filters were again centrifuged and washed with two 200 pL volumes of UA buffer. For cysteine alkylation, 100 pL of 50 mM iodoacetamide in UA buffer was added and the mixture incubated at room temperature in the dark for 30 min. The filters were again centrifuged to remove excess iodoacetamide and washed with two 200 pL volumes of UA buffer. The buffer was exchanged using 50 mM ammonium bicarbonate (pH 8.5) by two consecutive wash/centrifugation steps. Trypsin (Promega, Alexandria, Australia) solution (200 pL, 20 pg/mL in 50 mM ammonium bicarbonate, 1 mM CaCh) was added and the mixture incubated overnight at 37°C. The 10 kDa filters were transferred to fresh collection tubes and the filtrates, tryptic peptides were again collected by centrifugation. The filters were washed with 200 pL of 50 mM ammonium bicarbonate and filtrates combined and lyophilised. The tryptic peptides were resuspended in 50 pL of 1% formic acid and stored at 4°C until LC-MS/MS analysis.
Global proteomic profiling
The trypsin digested peptides from in-solution digests were analyzed using chromatographic separation (4 pL) on an Ekspert nanoLC415 (Eksigent, Dublin, CA, U.S.A.) directly coupled to a TripleTOF 6600 liquid chromatography tandem mass spectrometer (SCIEX, Redwood City, CA, USA). The peptides were desalted for 5 min on a ChromXP C18 (3 pm, 120 A, 10 mm x 0.3 mm) trap column at a flow rate of 10 pL/min 0.1% formic acid (FA), and separated on a ChromXP C18 (3 pm, 120 A, 150 mm x 0.3 mm) column at a flow rate of 5 pL/min at 30°C. A linear gradient from 3-25% solvent B over 68 min was employed followed by: 5 min from 25% B to 35% B; 2 min 35% B to 80% B; 3 min at 80% B, 80-3% B, 1 min; and 8 min re-equilibration. The solvents were: (A) 5% dimethylsulfoxide (DMSO), 0.1% FA, 94.9% water; (B) 5% DMSO, 0.1% FA, 90% acetonitrile, 4.9% water. The instrument parameters were: ion spray voltage 5500 V, curtain gas 25 psi, GS1 15 psi and GS2 15 psi, heated interface 150°C. Data were acquired in information-dependent acquisition mode comprising a time-of-flight (TOF)-MS survey scan followed by 30 MS/MS, each with a 40 ms accumulation time. First stage MS analysis was performed in positive ion mode, mass range m/z 400-1250 and 0.25 s accumulation time. Tandem mass spectra were acquired on precursor ions >150 counts/s with charge state 2-5 and dynamic exclusion for 15 s with a 100 ppm mass tolerance. Spectra were acquired over the mass range of m/z 100-1500 using the manufacturer’ s rolling collision energy based on the mass and charge of the precursor ion. The in-gel digests were analyzed using chromatographic separation (5 pL) employing a shorter linear gradient time frame from 3-25% solvent B over 38 min followed by: 5 min from 25% B to 33% B; 2 min 35% B to 80% B; 3 min at 80% B, 80- 3% B, 1 min; and 8 min re-equilibration, and a wider mass range m/z 300-1250 for the data acquisition. Protein identification was undertaken using ProteinPilot™ 5.0.2 software (SCIEX) with searches conducted against the Nicotiana benthamiana of the Uniprot-SwissProt database (2018/08) appended with a custom nitrogenase database and a contaminant database (Common Repository of Adventitious Proteins). The total number of proteins in the custom database was 2,622. The resulting proteomics data has been deposited to the CSIRO Data Access Portal (https://doi.org/10.25919/5f0d49f60dl57).
MRM method development
After database searching, tryptic peptides were selected for target proteins such as, for example, AnfD, AnfK, AnfG and AnfH. The selection of peptides was based on the criteria: MS response (high intensity), specific/unique to the target protein and of a size amenable to LC-MS, i.e. 5-20 amino acids in length. The MS/MS data and subsequent database search results were used to select the highest intensity precursor ion (QI m/z) and product ions (Q3 m/z, n = 4) for each peptide, which were subsequently defined as the MRM method.
Targeted proteomics
Targeted liquid chromatography-multiple reaction monitoring-mass spectrometry (LC-MRM-MS) was performed for target protein detection. Reduced and alkylated tryptic peptides (15 pL) were chromatographically separated on a Kinetex Cl 8 column (2.1 mm x 100 mm, Phenomenex) using a linear gradient of 5-45% acetonitrile in 0.1% formic acid, over 10 min at a flow rate of 400 pL/min. The eluent from the Shimadzu Nexera UHPLC was directed to a QTRAP 6500 mass spectrometer (SCIEX) equipped with a TurboV ion source operated in positive ion mode for data acquisition and analysis. The MS parameters were as follows: ion spray voltage, 5500 V; curtain gas, 35; GS1, 35; GS2, 40; source temperature, 500°C; declustering potential, 70 V; and entrance potential, 10 V. Peptides were fragmented in the collision cell with nitrogen gas based on rolling collision energy dependent on the mass and charge of the precursor ion. Protein detection was confirmed using scheduled MRM scanning experiments with a 60 second detection window around the expected retention time (RT) and a 0.3 second cycle time. Data were acquired using Analyst vl .7 software. Peak areas of the MRM transitions were integrated using Skyline (MacLean et al., 2010) wherein all transitions (n=4) were required to co-elute with a signal-to-noise (S/N) > 3 and intensity >1000 counts per second (cps) for detection (Suppl. Figures 8-13). Two synthetic peptides (SpikeTides™ TQL; JPT Peptide Technologies GmbH, Berlin, Germany) were synthesised for AnfG and AnfH. These four peptides were isotopically labelled with stable isotopes at the C- terminal arginine (R; 13C6; 15N4) or lysine (K; 13C6; 15N2), resulting in a mass shift of +10 or +8 Da, respectively. The heavy peptides were used as standards to confirm the detection of the low abundant AnfG and AnfH light peptides.
Affinity bead purification of mitochondria
A. tumefaciens cells containing the genetic construct (SN197) encoding a TwinStrep-mTurquoise2-Metaxin fusion protein, together with A. tumefaciens cells containing other genetic constructs for expression and localisation of polypeptides to the mitochondria if desired, were infiltrated into N. benthamiana leaves along with A. tumefaciens cells containing a separate construct for expression of the Pl 9 silencing suppressor. For example, A. tumefaciens cells containing the multi-gene construct SL26 encoding four different fusion polypeptides AnfD, AnfK, AnfG and AnfH, each fused with a MTP and a HA epitope, was prepared at an OD of 0.1 and mixed with the cells containing SN197 and cells with the construct for expressing P19 to a total OD600 of 0.4. Appropriate control mixtures having SN197 and P19 without SL26 were separately infiltrated into N. benthamiana leaves, as well as a mixture of cells containing separate constructs for expression of the P19 silencing suppressor and GFP (pRAOl). After four days, infiltration zones were excised, providing samples of about 100 mg fresh weight in a 4 cm x 2 cm leaf piece. The following steps were performed at 4°C. Leaf material was ground by hand in a mortar and pestle using 500 pL KPBS buffer (KPBS buffer: 1.36 M KC1, 100 mM KH2PO4, pH 7.25). The slurry was centrifuged at low speed, 1000 g for 5 min, to pellet cell wall debris but leaving most mitochondria in suspension. 300 pL of supernatant was applied to 50 pL of a slurry of magnetic beads coated with streptavidin (DynaBeads MyOne Cl) in a 1.5 ml Eppendorf tube, after the beads had been washed once with KPBS buffer. The magnetic beads in the mixture were collected to the wall of the tube using a magnet and the remainder of the liquid was carefully removed. The magnetic beads were then washed twice with 1 mL KPBS buffer, each time collecting them as before with the magnet, and finally resuspended in 50 pL KPBS. The purification process was analyzed via Western blot assays. To do this, polypeptides bound to the magnetic beads were released and denatured by the addition of 50 pL of Laemmli buffer and heated at 95 °C.
Imaging mitochondria
Streptavidin affinity beads post mitochondrial extraction were immediately mounted on a slide and sequentially imaged with a Leica SP8 laser-scanning confocal microscope (Leica Microsystems) equipped with a 40x water-immersion objective (NA=1.1) and operated via the LasX software. GFP (kex=488 nm; Xem=500-520 nm, gated at 0.30-3.80 ns) and transmitted light (kex=488 nm) were imaged in the same track.
Molecular modelling
A structural model of a heterodecamer complex of AnfD, AnfK, AnfG and AnfH was constructed in Swiss-Model (Waterhouse et al., 2018) using the crystal structure of V- nitrogenase (PDB ID: 5N6Y) (Sippel and Einsle, 2017) as a template, after which the two AnfH homodimers with homology models constructed based upon those in PDB ID: 1N2C (Schindelin et al., 1997) were positioned based upon a superposition of the AnfD and AnfK units of the homology model with those in 1N2C. The AnfH and AnfG units were manually moved away from the AnfDK complex to remove steric clashes that occurred as artefacts of the superposition, then the apo-complex was subjected to a very brief (6.5 ns) MD simulation using Amberl8 (D.A. Case, 2020) (ffl4SB forcefield, 298 K, NVT ensemble, TIP3P truncated octahedral water box with 12 A boundary) to allow the AnfH homodimers and the AnfG subunits to ‘settle’ into a plausible assembly, forming the full AnfDKGH complex.
Example 2. Analysis of AnfH polypeptides
As described herein, an AnfH polypeptide is a NifH polypeptide which is a member of the nitrogenase conserved superfamily cl25403 containing the PRK13233 conserved domain and having at least 69% amino acid sequence identity to the Azotobacter vinelandii AnfH polypeptide (SEQ ID NO: 37) when measured along the full length of 275 amino acid residues of SEQ ID NO:37. The inventors analysed AnfH polypeptide sequences present in databases and aligned these and compared them to a representative molybdenum-type NifH.
Databases were searched for AnfH amino acid sequences. These were identified as having the PRK13233 conserved domain and at least 69% identity to SEQ ID NO:37. This identified 314 such sequences. These were aligned with NCBI COBALT and a consensus sequence developed which had 300 residue positions including gaps. This consensus sequence was 89% identical to SEQ ID NO:37. The aligned AnfH amino acid sequences remarkably had 138 amino acids of the 300 positions that were identical in all of the 314 naturally occurring AnfH polypeptides and many other amino acids that were conserved in many of the AnfH polypeptides. Since the 138 conserved amino acids within the PRK13233 domain spanned most of the AnfH sequences, it was concluded that the PRK13233 domain covered most of the AnfH sequences and that PRK13233 was indicative of a family of sequences, not of one particular sequence. The 138 conserved amino acids included the sequence motifs YGKGGIGKSTTXQNT (motif I, SEQ ID NO:40), H4GCDPKAD (motif II, SEQ ID NO:41), CVESGGPEPGVGCAGRG (motif III, SEQ ID NO:42), DVLGDVVCGGFAMP (motif IV, SEQ ID NO:43), VASGEMMAXYAANNI (motif V, SEQ ID NO:44), QSGVR (motif VI, SEQ ID NO:45) and CNSRXVD (motif VII, SEQ ID NO:46), where X represents any amino acid. All of the motifs I- VII were present in all 314 AnfH sequences analysed.
Many of the NifH sequences other than the AnfH sequences had identical motif IV, but the sequences corresponding to the other motifs often had 1-3 amino substitutions relative to motifs I-III and V-VII. The corresponding motifs I-III, V and VII in many NifH sequences other than AnfH sequences, such as K. oxytoca NifH and A. vinelandii NifH, were YGKGGIGKSTTXQN (SEQ ID NO:61), IXGCDPKAD (SEQ ID NO: 62), CXESGGPEPGVGCAGRG (SEQ ID NO:63), VXSGEMMAXYAANNI (motif V; SEQ ID NO:64), and CNSRXXD (motif VII; SEQ ID NO:65), where X in each case is any amino acid.
The 138 amino acids that were fully conserved were as follows, with the number referring to the amino acid position in SEQ ID NO:37 and the letter to the amino acid at that position: IM, 3R, 4K, 6A, 8Y, 9G, 10K, 11G, 12G, 131, 14G, 15K, 16S, 17T, 18T, 20Q, 21N, 22T, 25A, 361, 37H, 38G, 39C, 40D, 41P, 42K, 43A, 44D, 46T, 47R, 50L, 52G, 55Q, 60D, 63R, 75V, 79G, 85C, 86V, 87E, 88S, 89G, 90G, 91P, 92E, 93P, 94G, 95V, 96G, 97C, 98 A, 99G, 100R, 101G, 1031, 104T, 1061, 108L, 109M, 110E, 115Y, 119L, 120D, 125D, 126V, 127L, 128G, 129D, 130 V, 131V, 132C, 133G, 134G, 135F,
136A, 137M, 138P, 140R, 142G, 143K, 144A, 146E, 148Y, 150V, 151A, 152S, 153G,
154E, 155M, 156M, 157A, 159Y, 160A, 161A, 162N, 163N, 1641, 167G, 170K, 172A, 174Q, 175S, 176G, 177 V, 178R, 180G, 181G, 184C, 185N, 186S, 187R, 189 V, 190D,
192E, 198E, 199F, 204G, 212P, 213R, 215N, 217V, 218Q, 220A, 221E, 222F, 227V,
236Q, 239E, 240Y, 243L, 2471, 250N, 254V, 2551, 256P, 258P, 265E, 272G. When aligned with the vinelandii NifH sequence (AvNifH; SEQ ID NO: 39), 122 of the 138 fully conserved amino acids from AnfH sequences were also present in the corresponding positions of AvNifH. The 16 amino acids that were conserved in all of the AnfH sequences but not at the corresponding positions in AvNifH were: 4K, 22T, 37H, 52G, 60D, 63R, 108L, 109M, 142G, 151A, 174Q, 189V, 198E, 199F, 222F and 2471 with reference to SEQ ID NO:37. These 16 amino acids were therefore characteristic of AnfH polypeptides relative to the molybdenum-type NifH sequence from A. vinelandii and can be used to distinguish AnfH polypeptides from other NifH sequences which do not have all 16 amino acids in common. AvNifH, KoNifH (SEQ ID NO: 1) and other molybdenum type NifH sequences had motifs III and IV but did not have motifs I, II, V-VII, instead have motifs I-III, V and VII with broader sequences (SEQ ID NOs:61-65) and therefore these motifs could also be used to distinguish the AnfH subset from other NifH polypeptides.
Example 3. Expression and solubility of multiple Nif polypeptides expressed in plant cells and targeted to the mitochondria
Introduction
The iron-only nitrogenase system is found in some diazotrophic bacteria, for example in the well-studied bacterium A. vinelandii (Av) which has three nitrogenase systems based on molybdenum (Mo), vanadium (V) and iron-only (Fe), using the cofactors termed FeMoco, FeVco and FeFeco, respectively (Davis et al., 1996; Robson et al., 1986). The latter system is also called the ‘Fe-nitrogenase’ or alternative nitrogenase (Anf) system. All organisms recorded to date that contain one or both of the V- or Fe-nitrogenases also contain the Mo-nitrogenase. Generally, the V- and Fe- nitrogenases are repressed by the expression of the Mo-nitrogenase and are expressed only when the availability of Mo becomes limiting.
The Fe-nitrogenase is the least studied of the three nitrogenases. It has the lowest catalytic activity of the three for reducing dinitrogen but its biogenesis appears to be simpler, requiring less proteins for producing a functional nitrogenase. There are six known Fe-nitrogenase proteins produced in A. vinelandii, namely AnfD, AnfK, AnfH, AnfG, AnfO and AnfR, which are distinct for Fe-nitrogenase. Of these six, the first four are required for the activity of the nitrogenase enzyme. Indeed, AnfD, AnfK and AnfG form the catalytic Fe-nitrogenase complex and AnfH is the obligate electron donor to the complex. The Mo- and V-nitrogenase systems use the corresponding proteins designated as Nif, or Vnf, D, K and H. The V- and Fe-nitrogenases also require the additional structural protein designated VnfG or AnfG, respectively, which is not required by the Mo-nitrogenase. The remaining minimal accessory genes required for activity of the Fe- nitrogenase are common to the Mo-nitrogenase pathway, namely NifS, NifU, NifB, NifV, NifF and NifI (Yang et al., 2014). The iron-only nitrogenase therefore has a minimal set of 4 Anf and 6 accessory Nif polypeptides required for heterologous function within E. coli (Yang et al., 2014). It is not yet known what the minimal polypeptide set is in plant cells.
In the Fe-nitrogenase system, the dinitrogenase enzyme is a heterohexamer made up of two AnfD polypeptides as the a unit, two AnfK polypeptides as the P unit and two AnfG polypeptides as the 5 unit, so in a 012P262 conformation. The dinitrogenase reductase enzyme, the obligate electron donor to the dinitrogenase enzyme, is a homodimer with 2 identical AnfH polypeptides. The dinitrogenase reductase AnfH dimer contains a single [Fe4S4] cluster at the interface of its subunits (Buren et al., 2017b). The AnfH protein is predicted to have two other functions, namely being required for the maturation of the dinitrogenase enzyme in analogous fashion to the NifH and VnfH gene products in the Mo- and V-nitrogenases. All of the key nitrogenase enzymes are extremely oxygen sensitive and quickly become irreversibly inactivated when exposed to oxygen.
W02013/068039 reported experiments to express four synthetic Anf genes in plant cells for mitochondrial localisation of the encoded Anf fusion polypeptides and demonstrated an association of the AnfD, AnfK and AnfG polypeptides in the mitochondria of plant cells after MPP processing. Furthermore, constructs for the four structural Anf genes were co-introduced with constructs to express up to six so-called accessory Nif genes encoding NifV, NifS, NifU, Niff, NifF and NifB fusion polypeptides, again targeted to the mitochondria. The sequences for the Anf polypeptides were based on the bacterium A. vinelandii and, for the other Nif polypeptides, on the bacterium K. oxytoca (see, for example, the vector SL54 described in W02013/068039). Each encoded fusion polypeptide had addition of an N-terminal MTP sequence and an epitope tag for detection by Western blot analysis.
The in planta mitochondrial expression of the individual K. oxytoca Niff, NifF, NifS, NifU, NifB and NifV fusion polypeptides was also reported by Okada et al. (2020). They reported that the Niff, NifV and NifB fusion polypeptides from K. oxytoca, after processing by MPP, were predominantly present within the insoluble protein fraction when targeted to the plant mitochondrial matrix. In contrast, the K. oxytoca NifS, NifU, and NifF fusion polypeptides were shown to be partially soluble when expressed in planta and targeted to the plant mitochondrial matrix (Okada et al., 2020).
Co-expression and solubility of mitochondrially targeted NiffFSUB fusion polypeptides The inventors made a multi-gene construct, SL78, for in planta expression and mitochondrial targeting of fusion polypeptides based on the K. oxytoca Niff, NifF, NifS, NifU and Methanocaldococcus infernus NifB amino acid sequences. Each fusion polypeptide except for the NifB polypeptide had a HA epitope fused at the C-terminus of the Nif sequence. The NifB fusion polypeptide had the HA epitope fused between the MTP and NifB sequences. Table 3 provides the molecular weights of the encoded proteins prior to MPP processing and after processing by cleavage within the MTP sequence. Each protein coding region was expressed from its own 35S promoter with a 3’ polyadenylation region-transcription terminator (Tm) from CaMV. After infiltration into N. benthamiana leaves, total, soluble and insoluble protein fractions were extracted and assessed by Western blotting.
Table 3. Summary of key features of Nif fusion polypeptides expressed from multigene constructs. HA and TS are epitope tags, MTP-pFAy51and MTP-CoxIV are mitochondrial targeting peptides.
The Western blot of the total protein sample showed protein bands migrating to a size consistent with the MPP-processed Niff, the unprocessed MTP-pFAy51 ::NifS and processed form thereof, the processed MTP-pFAy51 ::NifB, the unprocessed NifU and processed form thereof, and the unprocessed NifF protein and processed form thereof. Protein bands migrating to sizes expected for the processed products scar::Niff::HA and scar::NifB::HA, and the unprocessed product MTP-pFAy51 ::NifF::HA were detected within the insoluble protein fraction. Within the soluble protein sample, single protein bands were observed to migrate to sizes consistent with the processed products for scar::Niff::HA, scar::NifF::HA, scar: :NifB::HA and scar::NifU::HA. Two protein bands migrating to a size consistent with the unprocessed and processed version of the NifS: :HA protein were also observed within the soluble fraction. As used herein, the term ‘ scar’ refers to the amino acids from the C-terminal part of the MTP sequence that remain covalently bonded to the N-terminus of the fusion polypeptide after processing by MPP in the plant mitochondria.
LC-MS/MS proteomic assessment by tryptic cleavage and mass spectrometry, performed as described in Example 1, was used to confirm the presence of the Nif proteins within the total and soluble protein fractions, indicating that each Nif polypeptide was present within the soluble protein fraction of the plant mitochondrial matrix when expressed from the multi-gene vector. At least one tryptic peptide from each of the five polypeptides was detected at the 95% confidence level. The NifB protein was detected at the lowest level, consistent with the weak detection of the NifB protein in the Western blot analysis, indicating that the NifB fusion polypeptide was only partially soluble when co-expressed in the plant mitochondria. In summary, each of the Nif polypeptides was expressed abundantly, underwent mitochondrial targeting at least partially, and was at least partially soluble when targeted to the plant mitochondrial matrix when expressed from the multi-gene vector.
Co-expression in plant cells of multiple mitochondrially targeted Anf fusion polypeptides with Nif accessory polypeptides
Two other vectors, SL48 and SL79, were made to express the Anf components for the Fe-nitrogenase in the plant cells and localise them to the mitochondria, including a fused AnfD-linker-AnfK protein (W02013/068039). Each polypeptide had a MTP sequence fused at the N-terminus and were tagged with one or both of the HA and TS epitopes. Relative to SL48, the SL79 vector had an additional gene to express the ferredoxin FdxN.
The SL78 and SL48 vectors were co-infiltrated into N. benthamiana leaves and the expressed polypeptides analysed as before. The Western blot analysis of the total protein extracts showed that the Anf and Nif proteins migrated to the expected size for each polypeptide, being either fully processed or partially processed. The band highest on the blot migrated to a size consistent with the processed NifI polypeptide. The band for the AnfD-linker-AnfK polypeptide migrated to a size slightly smaller than the NifI band on the blot. Single bands were present that migrated to a size consistent with the MPP-processed forms scar::NifS, scar::NifV, scar::NifB, scar::AnfH and scar::AnfG. The NifU and NifF polypeptides were both present as two separate bands, one migrating to a size consistent with the unprocessed polypeptide and the second band migrating to a size consistent with a MPP-processed fusion polypeptide. A number of smaller bands were observed from the total protein fraction that did not migrate to a size predicted for the Anf or Nif proteins and were likely breakdown products of the larger NifI or AnfD- linker-AnfK polypeptides.
The soluble protein fraction showed protein bands migrating to a size consistent with the MPP-processed AnfD-linker-AnfK, NifI, NifV, NifS, NifB, NifU, NifF and AnfH polypeptides and a faintly visible AnfG band. Polypeptide bands migrating to a size consistent with both the processed and unprocessed forms of MTP: :NifF were visible within the soluble protein extract. The remaining polypeptides migrated to a size consistent with the processed version of each expressed protein, including the polypeptides NifI, AnfD-linker-AnfK, NifV, NifS, NifB, NifU, NifF, AnfH and AnfG. The insoluble protein fraction showed visible bands migrating to sizes consistent with the processed version of NifI, AnfD-linker-AnfK and NifB, and two protein bands migrating to sizes consistent with both the unprocessed and processed forms of NifS and NifV. The NifF, NifU, AnfH, and AnfG polypeptides were not detected in the insoluble protein fraction. It was concluded from these results that the Nif proteins including the Anf proteins were expressed and underwent targeting and localisation to the plant mitochondrial matrix when co-expressed in N. benthamiana leaves. In summary, the coexpression of the A. vinelandii AnfD, AnfK, AnfH, AnfG and NifV, with the K. oxytoca NifI, NifF, NifS, NifU and the M. infernus NifB fusion polypeptides within the plant mitochondrial matrix was successful.
Example 4. Characterisation of Anf polypeptides expressed in plant cells
Purification of AnfD-AnfK-AnfG and AnfH from A. vinelandii
The native AnfDKG nitrogenase and AnfH nitrogenase reductase enzymes were purified from vinelandii strain DI2241 cells under anaerobic conditions as described in Example 1. This was done to provide purified, active enzymes for in vitro acetylene reduction assays to allow for testing of the plant-produced polypeptides, for example testing a plant produced AnfH polypeptide with a bacterially produced AnfDKG complex. Since the enzymes were oxygen sensitive, the purifications had to be under anaerobic conditions to provide active enzymes. Strain DI2241 had a genetic modification in the anfD gene whereby the encoded AnfD polypeptide had a streptactin epitope tag fused to its C-terminus, allowing for affinity purification of the AnfD fusion polypeptide. The purification was carried out under conditions so that the AnfK and AnfG polypeptides remained bound to the AnfD polypeptide as an active complex. The same DI2241 cell preparation was used to separately purify the AnfH polypeptide as described in Example 1. The purifications were confirmed by SDS-PAGE, Western blotting and proteomic assessments. The purified AnfD-AnfK-AnfG protein was concentrated and displayed a dark brown colour, indicating that Fe-S clusters were retained on the catalytic enzyme. The SDS-PAGE gel stained with Coomassie showed clear protein bands migrating to a size consistent with that predicted for AnfD, AnfK, and AnfG. The Western blot probed with a streptactin-antibody showed an intense band migrating to a size consistent with the AnfD protein, along with several smaller, less intense bands that were considered likely to be breakdown products of the AnfD protein that may have occurred during the purification or analysis.
Due to the transient interaction of the AnfH protein with the AnfD-AnfK-AnfG complex, the AnfH protein was not likely to co-purify with the AnfD-AnfK-AnfG complex. The AnfH protein was therefore separately purified and concentrated from the DJ2241 cells by native purification techniques using an anionic exchange column and size exclusion chromatography as described in Example 1. The AnfH homodimer was determined to elute from the anionic exchange column at a NaCl concentration of 27- 29%. The purified protein also displayed a dark brown colour, indicating the presence of Fe-S clusters. The Coomassie stained SDS-PAGE gel of the purified protein showed an abundant band migrating at a size consistent with that predicted for AnfH. A Western blot probed with an antibody to the native NifH, that cross reacted with the AnfH peptide due to the homology in the amino acid sequence, showed three bands, one band consistent with the AnfH protein and two bands of smaller size, representing potential breakdown products of the AnfH peptide.
The purified protein preparations were also assessed by LC MS/MS analysis which showed that only minor traces of AnfH were present in the AnfDKG preparation and, conversely, only the slightest traces of AnfD, AnfK an AnfG in the AnfH preparation. Overall, the expression and purification of these enzymes was successful.
Example 5. In vitro loading of FeS onto purified TS::NifU fusion polypeptide
Molecular cloning and heterologous expression of a NifU fusion polypeptide in E. coli
For following experiments aiming to transfer Fe-S clusters to isolated NifH or AnfH fusion polypeptides produced in planta, a TS::NifU fusion polypeptide was produced and purified from E. coli. The purification made use of the TwinStrep epitope for affinity purification. To do this, a gene encoding a NifU fusion polypeptide having a TwinStrep (TS) epitope sequence fused at the N-terminus of the NifU sequence from A. vinelandii was expressed in the EcoFlex cloning system (Moore et al., 2016) using the pTU2S-a vector with a pl 5a origin of replication as the starting vector. The E. coli strain used was BL21(DE3) which provided the T7 RNA polymerase over-expression system. The gene encoding the TS::NifU fusion polypeptide (SEQ ID NO:67) was co-expressed with a gene encoding vinelandii NifS (SEQ ID NO:60). The construct containing both genes was named EFL2-30. The TS: :NifU fusion protein was extracted under anaerobic conditions and purified making use of the TwinStrep tag, as described in Example 1.
Chemical reconstitution of Fe-S clusters on TS::NifU protein
The TS::NifU polypeptide was chemically reconstituted to ensure it was loaded with Fe-S using a similar procedure as previously described (Lopez-Torrejon et al., 2016; Marengo et al., 2021), as described in Example 1.
TS::NifU reconstitution of AnfH or apoNifH
Buffers for the reconstitution were prepared as follows. Prior to use, all buffers and solutions used were tested for O2 contamination using electrochemically reduced methyl viologen. Mg- ATP buffer was prepared by dissolving 10 mM MgCh and 5 mM ATP in 25 mM MOPS pH 7.0, which was followed by pH adjustment by addition of 1/50 volume 1 M NaOH, resulting in pH 7.7.
To remove interfering compounds such as DTH and biotin, the elution buffer of AnfH was exchanged to Mg- ATP buffer using a centrifugal filter (Amicon® Ultra, 10 kDa or 30 kDa MWCO). This procedure was carried out twice and the minimum dilution achieved was 200-fold. Chemically reconstituted NifU was added to AnfH at a ratio of 2: 1 and incubated overnight at approx. 28°C.
AnfH that was purified from plants was reconstituted with TS::NifU purified from E. coli
The TS::NifU was produced in E. coli in the presence of co-expressed NifS and isolated (Example 1). After isolation from E. coli, the Fe content of NifU was only 0.2 Fe/protein monomer as measured by ICP-MS, which showed that it did not contain a complete set of iron-sulfur clusters. To increase cluster loading on the isolated TS::NifU polypeptide, a chemical reconstitution with sulfide and Fe2+ was carried out. Prior to AnfH reconstitution with NifU, the DTH in the AnfH samples was removed as DTH has been shown to destabilise the [Fe4S4] clusters of NifU (Smith et al., 2005b). To reconstitute AnfH with NifU, both proteins were incubated overnight at a ratio of 1 :2 AnfH:NifU in the presence of Mg-ATP. AnfH activity was then determined using the acetylene reduction assay with NifDK. Example 6. Co-expression in plant cells of NifS and NifU with targeting to mitochondria
Introduction
Several nitrogenase components contain metalloclusters that are essential for enzymatic function. These include a [FesS?] complex, referred to as a P-cluster, at each a/p subunit interface of the catalytic tetramer for the Mo-nitrogenase, or hexamer for the V- and Fe-only nitrogenases. The FeMo-, FeV- and Fe-only cofactors within each a subunit are also based on Fe-S clusters. Further, the nitrogenase reductase component which is a homodimer of NifH in case of the Mo-nitrogenase, or specifically homodimers of the VnfH or AnfH polypeptides for the V- and Fe-only nitrogenases, respectively, each contain a subunit-bridging Fe4S4 cluster. These Fe4S4 clusters are essential for transfer of electrons to the nitrogenase for the reduction of N2. These proteins with their metalloclusters are all highly oxygen-sensitive and become irreversibly inactivated upon exposure to oxygen, especially the NifH/VnfH/AnfH proteins. The synthesis and structure of nitrogenase is reviewed in Rubio and Ludden (2005) and the biosynthesis of the nitrogenase cofactors is reviewed thoroughly in Buren et al. (2020).
The first step towards the generation of the metalloclusters for the nitrogenase polypeptides is the formation of Fe2S2 clusters, and from a pair of them, Fe4S4 clusters. These reactions are catalyzed in diazotrophic bacteria by the NifS and NifU proteins. The Fe4S4 clusters are thought to then be transferred to the NifH/VnfH/AnfH polypeptides to form the active nitrogenase reductases, to NifB to eventually form an intermediate called NifB-co as a step toward formation of the FeMo-, FeV- and Fe-only cofactors, to the NifD-NifK tetramer (NifDK) or AnfD-AnfH-AnfG hexamer (AnfDKG) to form the P- clusters, and to NifQ to form a [MoFe3S4] group for FeMo-co synthesis (Barahona et al., 2023). NifS and NifU are therefore not only involved in the assembly of the Modependent nitrogenase, but also in the assembly of FeFe nitrogenase and FeFe nitrogenase reductase for synthesis of their Fe-S metalloclusters (Kennedy and Dean, 1992).
The NifS and NifU activities have been well studied in bacteria. NifS is a pyridoxal phosphate-dependent cysteine desulfurase which generates the inorganic sulphide required for Fe-S cluster synthesis from cysteine. The reaction produces alanine as a byproduct. The sulphide is then provided to NifU which provides a scaffold for Fe- S cluster formation, first for the formation of Fe2S2 clusters and then Fe4S4 clusters. The NifS and NifU enzymes both function in bacteria as homodimers. The N-terminal domain of NifU can bind one Fe2S2 cluster per monomer and the central domain also binds an Fe-S cluster, referred to as the permanent cluster. The Fe2S2 clusters in the monomers can be reductively fused to form one Fe4S4 cluster per NifU dimer. The C- terminal domain of NifU can also hold one Fe4S4 cluster per monomer. NifU then donates a Fe4S4 cluster to the downstream Nif proteins, including to apo-NifH/VnfH/AnfH for maturation of nitrogenase reductase.
It has been reported in W02021/068039 that NifS and NifU polypeptides each fused to MTP sequences can form a complex when co-expressed in plant cells and targeted to the mitochondria, after cleavage of the MTP sequences by MPP in the mitochondrial matrix. The fusion polypeptides in those experiments were based on the K. oxytoca Nif sequences. The encoded polypeptides each had a MTP-FAy51 sequence at the N-terminus of the Nif sequence for mitochondrial targeting and either a HA or TwinStrep epitope for detection by Western blotting or for isolation by affinity chromatography. Most of the MPP-processed NifU polypeptide accumulated in a soluble form in the plant mitochondria, but only some (<50%) of the processed NifS polypeptide accumulated in a soluble form. In the following experiments, the present inventors switched to use of NifS and NifU fusion polypeptides based on the A. vinelandii Nif sequences. The wild-type amino acid sequences for A. vinelandii NifS is provided herein as SEQ ID NO:60 and for NifU as SEQ ID NO:59.
Modelling the structure of NifU from A. vinelandii
The present inventors first modelled the structure of a NifU monomer from A. vinelandii using the AlphaFold program (https://alphafold.ebi.ac.uk/; Varadi et al., 2022; Varadi and Velankar, 2022). The structure that was produced is shown schematically in Figure 1. This revealed three distinct, linked domains in the polypeptide, consistent with the biochemical evidence that NifU has three different functional domains, namely N- and C-terminal domains and a central domain (Smith et al., 2005). The N- and C-terminal domains are thought to bind so-called transient Fe-S clusters that can be donated to apo- NifH, whereas the central domain contains the so-called permanent Fe-S cluster. The N- terminal domain (amino acids 1-131 of SEQ ID NO:59) had about 49% sequence identity to IscU proteins from E. coli and about 45% identity to Isul protein from Arabidopsis. while the C-terminal domain was homologous to ferredoxin proteins found in mitochondria and chloroplasts.
Construction of plasmid encoding a NifU fusion polypeptide with TwinStrep epitope.
A genetic construct SL122 was designed and made with a gene for expression in plant cells of a NifS fusion polypeptide and a separate gene for expression of a NifU fusion polypeptide, each with an N-terminal fused MTP sequence for mitochondrial targeting. One gene encoded a MTP-FAy51 ::NifS fusion polypeptide (SEQ ID NO:68) and the other a MTP-CoxIV : :TwinStrep: :NifU fusion polypeptide (SEQ ID NO:70). The amino acid sequences of the NifS and NifU regions of the fusion polypeptides were based on the amino acid sequences of the A. vinelandii proteins. Previous experiments with a MTP-FAy51 ::NifS fusion polypeptide using the K. oxytoca NifS sequence had shown that MTP-FAy51 was a favourable MTP sequence for expression and processing for NifS, therefore that MTP was used for the A. vinelandii NifS fusion polypeptide. The TwinStrep (TS) epitope was chosen for the NifU polypeptide as it had a high affinity for binding to StrepTactinXT resin under essentially physiological conditions and was thus ideally suited to the purification of the protein comprising the epitope, even at low protein concentrations. The MTP -NifS polypeptide encoded by SL122 did not have an epitope tag. The nucleotide sequences of the protein coding regions were codon optimized for improved expression in plant cells. Each sequence was linked to a e35S CaMV promoter sequence and a 35S polyadenylation region/transcription terminator (Tm) for expression in the plant cells. These constructs were made using the GoldenGate assembly strategy (Weber et al., 2011; Engler et al., 2014), using analogous methods to those described above. The amino acid sequences of the scar::NifS and scar::TS::NifU polypeptides produced after processing with MPP in the mitochondria are provided as SEQ ID NO:69 and SEQ ID NO:71, respectively.
Production of a soluble NifU fusion polypeptide in mitochondria of plant cells
SL122 was introduced into N. benthamiana leaves as described in Example 1 in order to test the production and enable the purification of soluble MPP-processed NifU fusion polypeptide. It was observed that the scar::TS::NifU fusion polypeptide was almost fully soluble when produced in the plant cell mitochondria and could be readily purified by the affinity chromatography method. For an initial isolation, soluble protein was extracted from about 40 g of leaf tissue under non-denaturing, non-aerobic conditions to minimise the presence of oxygen. The extraction buffer contained 100 mM Tris-HCl pH 8.0, 150 mM NaCl, 5% (v/v) glycerol, 2 mM DTH or TCEP as reducing agent, 1% (w/v) PVP (average MW 40 kDa) and 0.1% Tween 20. A 2 ml StrepTactinXT column was washed with buffer containing 100 mM Tris pH 8.0, 150 mM NaCl and 2 mM reducing agent (wash buffer) and then loaded with the protein extract from SL122. After washing the column to remove unbound proteins, the bound proteins were eluted with wash buffer containing 50 mM biotin. Samples containing the eluted protein were concentrated to a volume of 200-500 pl using a 15 mL Amicon Ultra 30 kD MWCO concentrator. The eluate was light brown coloured, likely indicating the presence of some bound Fe-S clusters in the isolated protein. Aliquots of 20 pl were subjected to SDS- PAG electrophoresis and Western blotting using the antibody Streptactin HRP. Duplicate gels were stained with Coomassie blue to stain proteins.
The Western blot showed that the scar::TS::NifU fusion polypeptide had indeed been purified from the SL122 infiltrated tissues through the use of the StrepTactinXT column. It was concluded that the extract from the SL122 infiltrated leaves yielded isolated polypeptide which appeared to be mostly of the MPP-processed form i.e scar::TS::NifU polypeptide. The corresponding gels stained with Coomassie blue confirmed that a high degree of enrichment had occurred in the isolation process.
Gel slices were cut out of the Coomassie stained gel and the polypeptides in those slices subjected to N-terminal amino acid analysis. This confirmed that most of the MTP- CoxIV::TS::NifU fusion polypeptide encoded by SL122 had been cleaved by MPP at the intended site in the MTP sequence, since the purified polypeptide had the N-terminal sequence resulting from the intended processing.
The isolated protein preparation was analysed by ICP-MS to measure the Fe content, which was calculated to be about 0.3 Fe per polypeptide monomer. In a second isolation experiment where Fe and cysteine were added to the plant extract after the centrifugation step but before loading onto the StreptActin column, the Fe content increased to about 1.0 Fe per monomer. These amounts were lower than the theoretical maximum of fully loaded NifU protein at 8 Fe per monomer. Despite this, the isolated protein with about 1.0 Fe per monomer had some activity in reconstituting apoNifH (below).
Eluate containing StreptactinXT column-purified scar::TS::NifU from another purification was applied to a PD10 column (GE Healthcare) equilibrated in 50 mM Tris- HC1 pH 8.0 and 300 mM NaCl to remove biotin and excess Fe2+ and cysteine.
It was concluded from these experiments that the NifU fusion polypeptide targeted to the mitochondria when co-expressed with a NifS fusion polypeptide was processed in the mitochondria in sufficiently soluble form to allow for purification, and that it contained some, albeit low, Fe-S clusters as determined by measurement of the Fe content.
Reconstitution of apoNifH with NifU purified from plant cells
The activity of plant-produced and isolated scar: :TS: :NifU (SEQ ID NO:71) was tested in an apoNifH reconstitution assay (DosSantos et al., 2004b). Briefly, the isolated scar::TS::NifU polypeptide was incubated with apoNifH for 10 min, and then added to purified A. vinelandii NifDK which had been isolated as described in Example 1. Acetylene was added in an assay as described in Example 1 and the reaction analysed for production of ethylene. It was observed that the combination of NifDK and apoNifH that had not been treated with NifU was not active, as expected for that negative control. In contrast, the combination of the apoNifH with the scar::TS::NifU preparation that had been isolated in the presence of added Fe and cysteine in the extraction buffer, was active in the acetylene reduction assay with NifDK, showing that scar::TS::NifU had successfully transferred some Fe4S4 clusters to apoNifH despite the relatively low Fe content of the isolated scar::TS::NifU.
Since the isolated NifU fusion polypeptide seemed to lose most of its Fe-S clusters under the extraction conditions used, it was not clear how well it was loaded with Fe-S when produced in plant mitochondria in the presence of NifS. Experiments were therefore designed to test this by using NifH and AnfH variants, which should receive Fe-S clusters from NifU, as kind of a reporter system in planta. This is described in the following Examples.
Example 7. Modelling the structure of the Anf nitrogenase
Using the VFe nitrogenase crystal structure to develop a homology-based model for the FeFe nitrogenase/nitrogenase reductase complex
Crystal structures have been reported for the MoFe and VFe nitrogenase enzymes (Kim and Rees 1992; Kim et al., 1993; Sippel and Einsle, 2017). At the time this present study was done, there are no reports for a crystal structure of the FeFe nitrogenase. More recently, however, Schmidt et al. (2023) reported a crystal structure for the iron-only nitrogenase. The crystal structure of the nitrogenase reductase AnfH component purified from vinelandii has also been reported recently (Trncik et al., 2022). Even though this AnfH polypeptide is only 60% identical in amino acid sequence to the A. vinelandii NifH and VnfH polypeptides, all three show much the same conformation when purified from the bacterium, even with the different metal ions Mo, V or Fe in the cofactor at the active site.
To address the lack of a crystal structure at the time, the present inventors applied in silica modelling to generate a structural model for the FeFe nitrogenase of A. vinelandii, showing the structure of the nitrogenase formed by the interaction of AnfD, AnfK and AnfG (also referred to as AnfDKG, Component I) with the nitrogenase reductase AnfH (Component II), forming the AnfDKGH complex. The model of AnfDKGH was constructed in Swiss-Model (Waterhouse et al., 2018) using the crystal structure of VFe nitrogenase (PDB ID: 5N6Y, Sippel and Einsle 2017) as a template. The two AnfH homodimers, with structure based on the homology models in PDB ID: 1N2C (Schindelin et al., 1997), were positioned based upon a superposition of the AnfD and AnfK polypeptides of the homology model with those in 1N2C. The AnfH and AnfG units were manually moved away from the AnfD-AnfK complex to remove steric clashes that occurred as artefacts of the superposition. The apo-complex was then subjected to a very brief (6.5 ns) MD simulation using Amberl8 (Case et al., 2020) AMBER 2020, using the settings ffl4SB forcefield, 298 K, NVT ensemble and TIP3P truncated octahedral water box with 12 A boundary, to allow the AnfH homodimers and the AnfG subunits to ‘settle’ into a plausible assembly, forming the full AnfDKGH complex.
The structural model is shown in Figure 2. This model shows the stoichiometric ratio of AnfD:K:G (2:2:2), and the four subunits of AnfH, in two pairs upper and lower, all complexing as a heterodecamer. An interesting feature observed from the model was the apparent association of the C-terminus of AnfG with the AnfH homodimer, evident at both ends of the complex. The contact areas between the AnfG and AnfD monomers, calculated using the PISA webserver (Krissinel and Henri ck 2007), was 1,010 A2 and 1,231 A2, respectively, with free energy AG of -10.7 and -12.7 kcal/mol, respectively, while the AnfH homodimers had interaction surface areas of 1529 A2 and 1267 A2 with their adjacent AnfD and AnfK units, respectively, with free energy AG of -14.8 and -5.7 kcal/mol, respectively. These high contact area to AG ratios suggest quite weak interactions that are possibly transient in nature between the AnfD and AnfH polypeptides.
Since there was no reported crystal structure of the whole AnfH-AnfDKG complex, a structural model of the complex was developed using AlphaFold (Jumper et al., 2021; Varadi et al., 2022) to provide information on amino acids involved in interactions between the polypeptide subunits.
Example 8. Artificial AnfH variants for increased solubility after production in plant cells
Introduction
Previous studies demonstrated that the MPP -processed product of a MTP-AnfH fusion polypeptide was only partially soluble in the mitochondrial matrix when expressed from a transgene in N. benthamiana plant leaves (W02021/068039). The partial solubility was confirmed by the present inventors when AnfH was expressed as an MTP- FAy51 ::HA::AnfH fusion polypeptide from construct SN651 (below), showing that the majority of the polypeptide produced in N benthamiana leaves was insoluble and therefore not available for purification. Densitometric scanning of Coomassie stained gels showed that only about 30% of the scar: : FAy51 : :HA: AnfH polypeptide having the wild-type AnfH sequence was soluble - see Figure 3 for wild-type AnfH. Consequently, yields of the purified AnfH fusion polypeptide were low when using the TwinStrep tag in affinity chromatography experiments. In contrast, when an AnfH fusion polypeptide without a MTP sequence was expressed for localisation in the cytoplasm, the AnfH polypeptide was completely soluble. Attempts were therefore made by the present inventors to increase the solubility of AnfH fusion polypeptides in mitochondria by introducing amino acid substitutions into the A. vinelandii AnfH polypeptide (wild-type: SEQ ID NO:37) while still retaining AnfH activity of the variants. As an initial step, amino acid substitutions that were predicted to increase thermal stability were identified, since there may be an association of thermal stability and solubility. This is described in more detail as follows.
Identifying conserved amino acids from the structural model of the Anf nitrogenase
To identify candidate amino acid substitutions that might increase thermal stability while avoiding amino acid positions that should not be substituted in order to retain Anf nitrogenase reductase function, the present inventors first identified conserved amino acid positions that were likely to be required for that function. The 275 amino acid sequence of the AnfH protein from A. vinelandii (SEQ ID NO: 37) was analysed with use of a crystal structure of AnfH obtained from Oliver Einsle (Albert-Ludwigs-Universitat Freiburg, Freiburg, Germany: PDB:7QQA) (Tmcik et al., 2022) and the model produced by SwissModel (Example 7 herein) to identify amino acid residues that were involved in the interaction of AnfH with AnfD-AnfK-AnfG (AnfDKG). The identified amino acids to be conserved in AnfH included functionally important positions defined by Trncik et al. (2022) for AnfH. These included the nucleotide binding P-loop region at positions 9- 16, the switch I region at positions 39-45, and the switch II region at positions 125-132 with reference to SEQ ID NO:37. Other amino acids that were identified to be retained included some close to the P-loop and switch regions, and amino acids involved in the AnfH to AnfDKG interface, in particular amino acids D 141, KI 73 and QI 74 which were selected from the AlphaFold model and visualised using PyMOL. The cysteines at positions 97 and 132 that coordinate the Fe4S4 metallocluster were considered essential to retain, i.e. C97 and C132. Additionally, amino acids in the vicinity of the metallocluster and those facilitating dimerisation were retained. Notably, the lysine at position 69 (K69) was not included in the list of conserved amino acids even though it was thought to be involved in the AnfH to AnfDKG interface. Altogether, the amino acids selected to be retained (non-substituted) in AnfH were at positions: 1, 3, 4, 6, 8-18, 20-22, 25, 36-44, 46, 47, 50, 52, 55, 60, 63, 75, 79, 85-101, 103, 104, 106, 108-110, 115, 119, 120, 125-138, 140-144, 146, 148, 150-157, 159-164, 167, 170, 172-178, 180, 181, 184-187, 189, 190, 192, 198, 199, 204, 212, 213, 215, 217, 218, 220-222, 227, 236, 239, 240, 243, 247, 250, 254-256, 258, 265 and 272. These positions were in common with the 138 highly conserved amino acids identified in Example 2 herein with the addition of positions 141 and 173.
Identification of candidate amino acid substitution variants of AnfH
The Protein Repair One-Stop Shop (PROSS, http://pross.weizmann.ac.il) is an algorithm that can be used to design artificial variants of a naturally occurring protein with amino acid substitutions. These may increase the thermal stability and possibly the solubility of a protein (Goldenzweig et al., 2016). The algorithm takes a protein structure and its amino acid sequence as inputs. A multiple sequence alignment is generated from the sequence, generating a log-likelihood position-specific substitution matrix. This matrix has the amino acid positions, 275 in the case of A. vinelandii AnfH, along the protein sequence as columns and the twenty canonical amino acids as rows. Each row column value is the log-likelihood of observing that amino acid at that position in nature. Substitutions favoured for stability scoring above zero are assessed for protein energy minimisation by the Rosetta energy function. A change in energy value is calculated by generating the Rosetta energy of the wild-type protein and subtracting the generated Rosetta energy of the substituted protein variant. From these values, Rosetta combinatorial sequence design is used to select substitutions that are more likely to work together optimally.
The PROSS webserver was used to generate candidate sequence variants of the wild-type vinelandii AnfH polypeptide (SEQ ID NO:37), as follows. Firstly, the PDB file of the structure of AnfH of A. vinelandii was uploaded to the web server. The complete sequence of AnfH was pasted into the submission box. Since the crystal structure did not resolve some residues, the translation initiator methionine and the C- terminal aspartic acid were included. Chain identifier A, the first monomer on the PDB file, was selected for substitution. Either monomer could have been chosen due to the homogeneity between monomers of AnfH (homodimer). Since AlphaFol d2 software was not used to generate this structure, the AlphaFold checkbox was set to ‘off . The Fe4S4 metallocluster was assigned to Chain B and named FS4 in the PDB file at position 301. This was designated as a ‘ Small-molecule ligand’ under PROSS, entered as ‘FS4301 B’. Chain B was set as an ‘Interacting chain’ on the PROSS web server, entered as ‘B’.
Specific amino acids were selected to be left unmodified by PROSS, as listed above. The Multiple Sequence Alignment (MSA) option was selected to be automatically generated by PROSS rather than by manual input. The PROSS MSA generation was left at the default settings; minimal sequence identity at 35%, coverage at 75, maximum targets at 4000 and BLASTp E-value at 0.0001. The energy function selected was the Rosetta Energy Function (Park et al., 2016) termed ‘talaris2014’ to calculate the change in energy from wild-type to the substituted variant. The PROSS output included 8 candidate designs having between 5 and 22 amino acid substitutions. In contrast, when no amino acids were selected to be left unmodified, the PROSS output provided an entirely different set of candidate amino acid sets for substitution with up to 33 substitutions.
A subset of variants, designated VI to V5 and having between 5 and 11 substitutions were therefore selected; these are listed in Table 4. The amino acid sequences of these variant AnfH polypeptides are provided as SEQ ID NOs:72-76. Substitutions are listed with the position number and the substitute amino acid, using the single letter code for amino acids. These can also be listed in standard format, for example the variant designated VI has five substitutions, namely N112L, T200A, T228V, E234H and G241R. Variants V2-V5 had an increasing number of substitutions, each incorporating additional substitutions to those of the previous variant, to a maximum of 11 substitutions. Another variant with 15 substitutions was also designed, based on the PROSS output.
Table 4. Amino acid substitutions in candidate AnfH variants. Blanks indicate no change at that position. All other amino acid residues were as for the wild-type AnfH polypeptide.
Testing of candidate AnfH variants for solubility after targeting to plant cell mitochondria
To test the candidate AnfH variants for expression, mitochondrial processing and solubility, expression vectors were designed and made using GoldenGate cloning methods. Each vector contained in order: the e35S promoter, a 5’UTR from TMV for enhancing translation, then a protein coding sequence for a fusion polypeptide which had an MTP for mitochondrial localisation followed by an epitope tag for either HA or TwinStrep (TS) for detection, translationally fused to the coding region for the full-length AnfH polypeptide including its translation initiator Met. The constructs then had a CaMV 3’ polyadenylation region/transcription terminator (Tm) or a hybrid terminator (TTm, Example 13 herein). In some constructs, the encoded MTP was from a. thaliana Fl- ATPase y-subunit protein (MTP-FAy51; W02021/068039), 51 amino acids in length. Alternatively, an MTP-CoxIV was used, most often when the epitope was the TwinStrep tag. The encoded fusion proteins were therefore configured as, for example, MTP- FAy51 ::HA::AnfH or MTP-CoxIV::TS::AnfH polypeptides. The constructs were named as listed in Table 4. The amino acid sequences for the two fusion polypeptides with the wild-type AvAnfH sequence are provided as (SEQ ID NO:28) and (SEQ ID NO:80), respectively. The amino acid sequences for the fusion polypeptides comprising the variant AnfH sequences, VI to V5, were the same as the wild-type AnfH fusion polypeptides except for the amino acid substitutions.
Each of the constructs was transformed into Agrobacterium tumefaciens strain GV3101 by electroporation with selection on plates containing the antibiotics rifampicin, gentamycin and carbenicillin. Cultures of transformed cells were grown and infiltrated into N benthamiana plant leaves as described in Example 1. Five days later, leaf discs were excised from the infiltrated zones and extracted as described in Example 1 for total protein and soluble protein extracts. Samples were run on SDS-PAGE gels and analysed by Western blot using antibodies binding to the HA or TS epitopes as appropriate.
The Western blot showed that, under the SDS-PAGE conditions used, a single band was observed for the AnfH fusion polypeptide with a mobility between those of the 33kDa and 40kDa polypeptide markers (Figure 3). From comparisons with previous experiments (Johnston et al., 2023), and later experiments with variants V7 and V8, it was concluded that the band corresponded to the fusion polypeptides after MPP- mediated cleavage within the MTP peptide, indicating that all of the wild-type and variant polypeptides were efficiently processed upon entry into the mitochondria. That is, the band in each case was for the scar:: HA: AnfH processed polypeptide. It was also seen that the wild-type AnfH fusion polypeptide encoded by SN651, after MPP -mediated cleavage, was only slightly soluble, consistent with previous observations. In contrast, the intensity of the bands from the soluble extracts for the variant fusion polypeptides V1-V5 appeared much the same as the bands from the total protein extracts. These polypeptides were therefore clearly much more soluble in the mitochondria relative to the corresponding wild-type AnfH polypeptide. This result was unexpected to the inventors and could not have been predicted without this experiment having been conducted.
In a further experiment, constructs for the wild-type AnfH fusion polypeptide (SN651) and variant VI fusion polypeptide (SN641) were separately introduced into five zones across three N. benthamiana plants, to provide biological repeats and to look for variation between samples. Leaf discs were processed as before to produce total protein and soluble protein extracts and analysed by Western blot with the anti-HA antibody. The intensity of the band for each sample was measured by optical densitometry and variances analysed by ANOVA. No significant difference was found between the wildtype and variant V 1 for the intensities of the scar: :HA: AnfH bands from the total protein extracts. However, the intensity of the band for the VI variant in the soluble extracts differed significantly from the band for the wild-type (p < 0.05). In addition, a Mann- Whitney U test confirmed a significant difference in means between the wild-type AnfH and the VI variant for the soluble protein samples (p < 0.05). However, the Mann- Whitney U test showed no significant difference between the wild-type and VI polypeptides from the total protein extracts, indicating that the increase in the variants in the soluble fraction was not due to increased production from the constructs or decreased degradation.
Purification of AnfH variants after targeting to plant cell mitochondria
The analogous construct SN676 (Table 4) encoding the MTP-CoxIV::TS::AnfH- VI fusion polypeptide (SEQ ID NO: 81) was introduced into N benthamiana leaves along with constructs SL133 that encoded two fusion polypeptides, namely MTP- CPN60::NifS (SEQ ID NO:89) and MTP-SU9::NifU (SEQ ID NOVO) and the construct SN465 encoding a MTP-FAy51 ::FdxN fusion polypeptide (SEQ ID NOV 1), and the P19 silencing suppressor. A NifM polypeptide was not included since, in contrast to NifH, AnfH does not require NifM for its maturation (Yang et al., 2014). The mixtures were infiltrated into the leaves of at least 20 N. benthamiana plants. After five days, 120 g of infiltrated leaves were harvested and soluble proteins extracted under anaerobic conditions as described in Example 1. The fusion polypeptides containing the TS epitope were purified on a StrepTactin column as described in Example 1. This resulted in isolation of 140 pg of protein at a concentration of 6.3 pg/pl. The yield of isolated protein per leaf was 1.16 pg/g leaf wet weight. Samples taken from the blended leaf material, the supernatant, the pellet, the flow through material and the eluant were electrophoresed on SDS-PAGE gels and subjected to Western blot analysis with an anti-Strep antibody, as before.
A photograph of the Western blot is shown in Figure 4, as well as the Coomassie- stained gel. The eluted protein had high purity for the MPP -processed AnfH variant VI polypeptide, designated scar: :TS:: AnfH- VI (SEQ ID NO:85), as seen from the Coomassie stained gel. No scar::TS::AnfH-Vl polypeptide was detected in the Western blot of the pellet fraction, whereas it was detected in the supernatant fraction, indicating that the scar: :TS: AnfH polypeptide was essentially fully soluble in the mitochondria and extractable. Furthermore, the polypeptide was not detected in the flowthrough material, showing that the protein had bound tightly to the StrepTactinXT column. A single, intense band of the expected mobility was observed for the Western blot of the eluted protein sample, showing that the scar: :TS:: AnfH- VI polypeptide had been successfully purified and was intact. It was concluded that the AnfH variant VI fusion polypeptide was produced in the plant cells at high levels in soluble form, in contrast to the corresponding wild-type AnfH polypeptide which was mostly insoluble, and could be purified from plant leaves at adequate quantities for further analysis. It was also concluded that the MPP-processed, variant polypeptides were much more soluble than the wild-type polypeptide because of the amino acid substitutions.
Inductively coupled plasma mass spectrometry (ICPMS) on the purified scar: :TS: AnfH variant VI polypeptide showed iron and sulfur concentrations at background levels, suggesting a lack of metallocluster loading on this polypeptide or a loss of the metallocluster during the purification procedure. Furthermore, acetylene reductase assays in the presence of A. vinelandii NifD and NifK proteins did not yield production of ethylene, indicating that the protein ‘as isolated’ was not functional for electron donation to NifDK, possibly because the purified polypeptide lacked the essential Fe4S4 metallocluster. Alternately, the lack of acetylene reduction activity might have been due to the presence of the G241R substitution, which later experiments showed was likely deleterious to activity, or to both reasons. Experiments were carried out as described below to test this and to reconstitute functional enzyme with two other variants, V7 and V8, as described in the next two Examples. Example 9. Design of further candidate stabilising substitutions in AnfH Computational investigation of stabilising mutations
Following the demonstration of the inactivity of the purified AnfH variant VI ‘as isolated’ in acetylene reduction assays (Example 8), further computational analysis of the amino acid sequence was conducted. Firstly, the PROSS algorithm was partially recreated with a Rosetta energy amino acid substitution scan to reveal insights into the amino acid substitution selection process used by PROSS, as detailed below. Secondly, a molecular dynamics simulation was run on the wild-type AnfH and AnfH variant VI to assess and rationalise the effects of the amino acid substitutions. These analyses were then used to generate new substituted AnfH variants as candidates for further solubility and functional testing.
Rosetta energy and Positon Specific Substitution analysis
The methodology that PROSS uses to assess and select amino acid substitutions was investigated by coding a python script and accessing the position-specific substitution matrix (PSSM) generated by PROSS. A Rosetta energy amino acid substitution scan was run using the ROSIE web server (Lyskov et al., 2013). This energy scan calculated the difference in Rosetta energy units between the polypeptides for wildtype AnfH and AnfH variant VI with selected single amino acid substitutions, without the scar or epitope sequences at the N-terminus. Positions selected for the scan were regions that were considered non-conserved for function, as in the original PROSS prediction described above i.e. all positions in the sequence were scanned other than the highly conserved positions, with the exception of position 222 which was included in the scan. The output showed a calculated change in free energy (AAG) in Rosetta energy units for each of the 19 amino acid possible substitutions at each of those positions, ranging from -5.81 to 44.18. It was considered that substitutions having a calculated positive AAG would be destabilising and so these were not considered further as candidates for substitution. Substitutions having a AAG of between -0.5 and zero were also set aside as likely having insufficient effect, so focussing more on those having a lower AAG, i.e. more negative than -0.5.
There were numerous amino acid positions where one or more substitutions at that position was calculated to provide the desired change in AAG. These are listed in Table 5, in order of more preferred to less preferred substitutions, using the single letter amino acid code, at each position based on the AAG, showing the candidate substitutions for increasing stability of the polypeptide and thereby its solubility in plant cell mitochondria. For some of the positions, e.g. position 222, as many as 13 of the possible 19 substitutions were calculated to change the AAG negatively by more than 0.5 units, whereas for other positions there were only 1-3 substitutions having AAG of less than - 0.5. Of the amino acid positions that were scanned, the positions where no substitutions were predicted to provide a AAG below -0.5 were: 23, 28, 29, 35, 36, 49, 72, 77, 80, 81, 82, 83, 121, 123, 124, 147, 193, 195, 196, 203, 206, 216, 219, 223, 224, 230, 238, 242, 245, 248 and 263. It is therefore preferred that these positions in AnfH from A. vinelandii. and corresponding positions in other wild-type AnfH polypeptides, be set aside from consideration for modification. The substitutions listed in Table 5 were candidates for testing in combinations of 2, 3 or more substitutions. In particular, one or more substitutions from T200A, T228V, T228I, E234S and E234H are combined with one or more of the other substitutions listed in Table 5.
Table 5. Change in free energy (REU) calculated for candidate amino acid substitutions in AnfH.
All non-conserved positions in the AnfH sequence that were analysed for contributing to a reduction in free energy were also considered for those positions where the wild-type amino acid in A. vinelandii AnfH was not the most prevalent amino acid at that sequence position in the PSSM. This data was combined into a scatter plot, presented in Figure 5. Each point on the scatter plot represented a potential amino acid substitution in AnfH, characterised by the change in Rosetta energy units on the x-axis and the probability of the substitution from the PROSS PSSM on the y-axis. It was considered that, in most instances, the more negative the change in Rosetta energy units and the higher the substitution probability, the more likely the substitution was stabilising. Most substitutions generated by PROSS lay within the plot’s top-left quadrant, i.e. for likely stabilising substitutions. The majority of potential substitutions in Figure 5 lay in the bottom right-hand side quadrant; these were unlikely to be stabilising. For example, it was inferred from the scatter plot that PROSS had chosen the E234H substitution above the E234C substitution due to a lower change in energy score, despite being a substitution that was comparatively less likely to occur in nature. It was also interesting to note the inclusion by PROSS of the N112L substitution, which had a comparatively lower logscale frequency and Rosetta energy value than the other substitutions at that position (Table 5). This analysis revealed that the N112L substitution was only marginally stabilising, i.e., change in free energy = -0.18 REU compared to wildtype AnfH. The T200A, T228V, E234H and G241R substitutions were more stabilising at REU = -4.37, REU = -5.59, REU = -2.8 and REU = -3.88, respectively. It was concluded that the N112L substitution was not as stabilising as T228V, G241R, E234H and T200A, and therefore this substitution was omitted from new candidate variants (below). Given that E234H and G241R were more drastic changes from a structural point of view, these variations were tested separately in new variants V7 and V8 (below) in combination with T200A and T228V.
Molecular dynamics simulation
A molecular dynamics simulation was run to compare the motions of the wildtype AnfH and the AnfH variant VI molecules. The simulation was run on NAMD interfaced by a molecular dynamics script collection developed by Dr M. Kuiper, CSIRO. Simulations were run in triplicate at 100ns per simulation, with a timestep of 2 fsec. Due to the difficulties in generating force-field parameter files for the metallocluster of AnfH, an AnfH without the metallocluster was simulated. The simulation was run at 310 K, and periodic boundary conditions were used. Data was collected every 40 fsec of simulation time. Root mean squared deviation (RMSD) was assessed for the protein conformation every 40 fsec, referenced to the starting protein conformation. In the region of substitutions T228V, E234H and G241R, the RMSF of the residues was lower than that of the wild-type. T200A had an equivalent RMSF. It was observed that the hydrophobic substitutions T228V and N112L completely removed hydrogen bonding on the amino acid side chain. Despite threonine being capable of forming hydrogen bonds, the threonine at position 200 on the wild-type AnfH formed few hydrogen bonds in the simulation. The E234H substitution minimised hydrogen bonding of the side chain. However, the wild-type glutamic acid at position 234 had a hydrogen bond occupancy of mostly water, with an occupancy of 321% with the solvent and occupancy of less than 1% for other amino acids on the protein. This high percentage was considered due to the number of hydrogen bonds glutamic acid can form with the solvent. The histidine substitution formed hydrogen bonds with the backbone of glutamine at position 236, with an occupancy of 31%, and other hydrogen bond occupancies of less than one percent. G241R introduced hydrogen bonds with the solvent.
From these observations, it was concluded that the N112L substitution was likely destabilising due to no change in the RMSF at that position and the removal of hydrogen bonding. T200A also appeared to have little effect on the RMSF energy and therefore needed to be tested empirically for its effect on solubility and function.
Example 10. Testing of further variants having substitutions in AnfH fusion polypeptides as candidates for function
Investigating the effects of permutations of substitutions of AnfH VI on solubility
Following the computational investigation of molecular dynamics and the PROSS algorithm and rationalising that fewer substitutions might be less likely to perturb AnfH function, the present inventors screened new variants having a subset of 3 or 4 of the five substitutions in AnfH variant VI. Initially, two constructs SN798 and SN711 were made encoding MTP-FAy51 ::HA::AnfH-V7 and -V8 polypeptides, each having three amino acid substitutions. The solubility of the processed proteins from these constructs was examined after expression in N. benthamiana leaves, and each was shown to be as soluble as the corresponding AnfH-Vl polypeptide. Three more new constructs designated SN708-710 (Table 4) were therefore made using the TS epitope rather than the HA tag, to provide for isolation of the proteins, and these were tested for solubility and enzyme function as for variant VI. The encoded fusion polypeptides comprising variant AnfH sequences, designated variants V6, V7 and V8, had either 3 or 4 (V6) amino acid substitutions relative to the wild-type sequence from amongst the five in VI (Table 4). The polypeptide (SEQ ID NO:82) encoded by SN708 included the V6 sequence which had the substitutions T200A, T228V, E234H and G241R, the polypeptide (SEQ ID NO:83) encoded by SN709 included the V7 sequence which had the substitutions T200A, T228V and E234H, and the polypeptide (SEQ ID NO:84) encoded by SN710 included the V8 sequence which had the substitutions T200A, T228V and G241R, in each case relative to the wild-type AvAnfH sequence SEQ ID NO:37. The corresponding MPP-processed polypeptides produced after localisation to the mitochondria are provided as SEQ ID NOs:86-88. Each MTP-CoxIV::TS::AnfH fusion polypeptide comprised the TwinStrep epitope to allow for affinity purification of its MPP-processed product from the plant leaves. The constructs were introduced into A. tumefaciens and the transformed bacterial cells infiltrated into N benthamiana leaves as before. Leaves were harvested after growth of the plants in the dark for 8 hr, and total protein and soluble protein extracts prepared. Five biological repeats were conducted on each variant and tested first for solubility. The Western blot was developed using an anti-Strep HRP conjugate antibody. The Western blot results are presented in Figure 6 for two of the biological repeats. The other three repeats gave essentially the same results.
From the intensities of the bands in the Western blots, it was concluded that all three of the processed fusion polypeptides (SEQ ID NOs:86-88) comprising the variant AnfH sequences V6, V7 and V8 had increased solubility compared to the corresponding fusion polypeptide with the wild-type AnfH. The bands were analysed by densitometry across the five biological repeats using the Amersham 600 Imager to process the numeric band densitometry value (Figure 6, lower panel). Analysis by ANOVA indicated that the major difference between the wild-type, VI and V6-8 samples was attributed to the polypeptide variant (p < 0.05). Tukey analysis confirmed what was evident by eye, namely that the AnfH fusion polypeptide band was significantly more intense for all three variants V6-8 than the wild-type AnfH polypeptide, from both the total protein and soluble protein extracts (p < 0.05). Furthermore, variants V7 and V8 in the soluble extracts yielded significantly more intense bands than VI, and V8 from the total extract had a significantly more intense band than VI from the corresponding total extract, suggesting improved production and/or accumulation of the MPP-processed polypeptide, again indicative of increased stability.
In view of these results, another AvAnfH variant was designed and made having a single amino acid substitution, T228V, designated herein as V9. The genetic construct SN778 encoded the MTP-CoxIV::TS::AnfH-V9 fusion polypeptide. When this construct was expressed in N. benthamiana leaves, the MPP-processed scar: :TS: : AnfH- V9 fusion polypeptide was more soluble than the corresponding wild-type polypeptide but less soluble than the processed products comprising the V7 and V8 sequences having three amino acid substitutions. The processed product was isolated by affinity chromatography as for the others, yielding less isolated protein than for the V7 and V8 proteins, but nevertheless it could be isolated. It was concluded that AnfH variants having more than one substitution were likely better for increased solubility than variants with only one substitution.
Other Av AnfH variants were designed with only two amino acid substitutions relative to the wild-type sequence, including one with the T228V and E234H, another with the T228V and T200A substitutions. Other variants were designed having the E234H substitution, present in V7, or the T200A substitution, in each case along with at least one other substitution identified above, excluding the 137 highly conserved amino acid positions. Further variants are designed having one or more substitutions selected from T200A, T228V, T228I, E234S and E234H combined with one or more of the other substitutions listed in Table 5. The increased solubility of these variants relative to the corresponding wild-type AnfH fusion polypeptide is confirmed by testing using the methods as for SN708-710.
Functional testing with bacterial NifD and NifK for acetylene reduction
Given that the position specific substitution matrix used as described in Examples 8 and 9 contained a majority of NifH and VnfH proteins, and that the specific group of wild-type AnfH proteins included in the analysis had certain amino acid positions that were highly conserved (Example 2 herein) in wild-type sequences, it was important to test whether the soluble AnfH variants would function with AnfDKG or NifDK, or both, to reduce the substrates acetylene and N2. To test function of the MPP-processed polypeptides in in vitro ARA and 15N2 reduction assays, the genetic constructs SN709 and SN710 were expressed in N benthamiana plant leaves with co-expression of MTP::NifS and MTP::NifU fusion polypeptides from the construct SL133, as before. In bacteria, NifS and NifU are involved in [Fe4S4] cluster maturation of NifH and are thought to play an analogous role for AnfH maturation (Kennedy and Dean, 1992; DosSantos et al., 2004). NifM was not included in the infiltrated mixtures since, in contrast to NifH, AnfH does not require NifM for its maturation in bacteria (Yang et al., 2014). The co-expression of NifS and NifU from SL133 in the leaf tissue is confirmed by proteomic analysis of the supernatant from the leaf extractions, which detects the presence of diagnostic peptides for these fusion polypeptides.
The MPP-processed AnfH polypeptides were extracted from the leaves and isolated by affinity chromatography, as before. The plant leaves were harvested after a period of 16 hr growth in the dark, seeking to lower the presence of intracellular O2 or reactive oxygen species that are produced during photosynthesis and which might damage [Fe4S4] clusters bound to the AnfH polypeptides. The plants were not supplemented during growth with additional iron. All steps during the isolation process including cell lysis and the affinity chromatography were carried out under anaerobic conditions.
Both MPP-processed AnfH polypeptides were successfully isolated with final purities of approximately 70% as determined by densitometric analysis of the gel stained with Coomassie stain. Aliquots of the extracts at the steps in the purification process were analysed by SDS-PAGE - the Western blots and Coomassie stained gels are shown in Figure 7. The Coomassie staining of the eluted proteins for the V7 and V8 variants showed a main, intense band for the scar: :TS: AnfH polypeptide, indicating a high level of enrichment during the isolation process, and showing that the variants could be readily purified. Multiple attempts were also made to isolate the corresponding scar: :TS: AnfH fusion polypeptide having the wild-type AnfH sequence as a control for the assays, but it proved difficult to isolate sufficient protein to assay. After repeated attempts, some wildtype AnfH fusion polypeptide was isolated but the preparation was relatively impure; its purity was only approximately 13% and the yield was low. It was also observed (Figure 7, panel A) that the wild-type AnfH purification yielded a band smaller in size than the expected scar::TS::AnfH polypeptide as well as the expected band for the full length polypeptide, suggesting cleavage of some of the full length polypeptide during the purification process. The comparison between the wild-type and corresponding variant polypeptides was clear: the variant polypeptides produced in the plant cell mitochondria from SN709 and SN710 were much more soluble and more easily isolated at much higher yield and purity.
The isolated scar::TS::AnfH-V7 polypeptide is analysed by ICP-MS to measure the iron content of the protein ‘as isolated’ which, from the colour of the preparation, appears to be low.
Although AnfH functions to donate electrons to AnfDKG in the native bacterial host, it has been shown that isolated and purified AnfH can perform a similar electron donation to NifD-NifK (NifDK) to reduce acetylene under in vitro assay conditions (Lopez-Torrejon et al., 2021). In preliminary in vitro acetylene reduction assays carried out by the present inventors, more ethylene was produced by the combination of purified, bacterial AnfH with purified, bacterial NifDK than with AnfDKG when using a 20: 1 ratio for AnfH:NifDK proteins. The isolated AnfH variant polypeptides produced from SN709 and SN710 were therefore assayed in vitro with NifDK from A. vinelandii strain DJ2102. The NifDK had been purified as described in Example 1 and had been shown to not be contaminated with bacterial NifH. The ratio of AnfH:NifDK proteins used in the in vitro reactions was from 20: 1 up to 100: 1. However, the isolated AnfH fusion polypeptides did not produce detectable amounts of ethylene when combined with NifDK. In further assays, the in vitro reactions are modified by including purified, bacterial AnfDKG rather than NifDK, isolated from A. vinelandii strain DJ2241 as described in Example 1. In addition, the isolated AnfH protein was suspended in a different buffer thought to be more suitable for activity, exchanging the buffer to 50 mM Tris, pH 8.0, containing 75 mM NaCl and 2 mM DTH. Again, no ethylene was produced. The observed lack of acetylene reduction was considered to be most likely due to the absence of [Fe4S4] clusters bound to the scar::TS::AnfH-V7 and -V8 polypeptides.
The experiment was repeated in the same manner except that the construct SL139, encoding MTP::AnfD, MTP::AnfK and MTP::AnfG polypeptides in separate genes on the one construct, was co-expressed with SL133 and SN709 in the plant leaves. Once again, the isolated MPP -processed AnfH-V7 polypeptide produced from SN709 was not active in reducing acetylene when combined in vitro with the purified NifDK. Coexpression of the AnfDKG proteins with the AnfH polypeptide in the plant leaves had not resulted in activity in the ARA assay of the ‘as isolated’ AnfH polypeptide.
The isolated scar: :TS: : AnfH-V7 polypeptide was also tested in vitro for reduction of N2 gas using the 15N-labelled nitrogen gas as described in Example 1. In that experiment, the polypeptide was isolated from N benthamiana leaves without the coexpression of NifS and NifU from SL133, in view of results described in Example 15 herein. No significant change in the delta-15N value was observed in that experiment, without reloading of the AnfH polypeptide with Fe-S clusters. It was concluded that the isolated variant AnfH polypeptides did not have acetylene or N2 reduction activity ‘as isolated’. As shown below, this was due to a lack of Fe-S clusters on the isolated proteins.
Reconstitution of AnfH polypeptides with Fe-S clusters from bacterially-produced NifU To assess whether AnfH activity could be restored for the V7 and V8 variant AnfH polypeptides by insertion of a [Fe4S4] cluster, the isolated polypeptides were treated with purified, bacterial NifU protein. That protein had a TS epitope at its N- terminus to allow for purification by affinity chromatography as described in Example 1. The treatment involved incubating the isolated AnfH polypeptides in vitro with activated bacterial NifU protein. Although it is not completely understood how NifU donates the iron-sulfur clusters to AnfH in bacteria, it has been shown that apoNifH can be activated in vitro by incubation with NifU (Dos-Santos et al., 2004b), and that reaction was used by the present inventors as a positive control for the NifU treatment.
To obtain the purified NifU polypeptide for this purpose, a gene encoding a TS: :NifU fusion polypeptide was co-expressed with NifS in E. coli and purified from the bacterial cells as described in Examples 1 and 6. After purification, however, the ability of the NifU protein ‘as isolated’ to provide iron-sulfur clusters to apoNifH was low as shown by control reactions in vitro with apoNifH, indicating that the isolated TS::NifU polypeptide from the E. coli did not contain a complete set of iron-sulfur clusters. A chemical treatment of the TS::NifU polypeptide with sulfide and Fe2+ was therefore carried out as described in Example 1. The Fe content of the purified, treated NifU was greatly increased. This confirmed that the ‘as isolated’ TS::NifU did not contain a complete set of iron-sulfur clusters, as well as showing that these could be reloaded onto the TS: :NifU polypeptide by the treatment with sulfide and Fe2+. This reconstituted NifU protein was therefore used with the AnfH polypeptides, as follows.
Since the reducing agent dithionite (DTH) has been shown to destabilise [Fe4S4] clusters on NifU (Smith et al., 2005b), the DTH present in the buffers containing the isolated, variant AnfH fusion polypeptides produced from SN709 and SN710 was first removed by a buffer exchange process using micro-filtration units. The new buffer composition was 50 mM Tris, pH 7.7 containing 5 mM ATP and 10 mM MgCh. The AnfH polypeptide samples were then incubated with the purified, reloaded TS::NifU protein overnight at a ratio of 1 :2 AnfH:NifU in the presence of Mg- ATP at 5mM.
The NifU-treated scar::TS::AnfH fusion polypeptides from SN709 and SN710 were then tested for reduction of acetylene in the in vitro assay with NifDK, and compared to the corresponding fusion polypeptide having the wild-type AnfH sequence. The ratio of AnfH protein to NifDK protein was 20: 1. The amount of AnfH was adjusted based on a densitometric analysis of the SDS-PAGE gel, accounting for contaminating proteins that remained in the AnfH preparations (Figure 7). Positive control reactions were carried out with purified, wild-type AnfH isolated from A. vinelandii, having undergone the same NifU reconstitution procedure and therefore presumed to be fully active. Encouragingly, the scar: :TS:: AnfH variant polypeptide produced from SN709 comprising the V7 sequence was active in producing ethylene after it had been treated with the re-loaded TS: :NifU polypeptide. Over five experiments, including with different batches of isolated AnfH and TS::NifU polypeptides, the level of acetylene reduction varied between 9-110% relative to the positive control reactions. The results for two experiments are shown graphically in Figure 8, normalised to the amount of ethylene produced in the positive control reactions, which was 243 nmol/min/mg NifDK protein in the first experiment and 232 nmol/min/mg NifDK in the second. In contrast to the AnfH-V7 variant, the corresponding AnfH variant polypeptide produced from SN710 comprising the V8 sequence produced a very low amount of ethylene, at about 1% of the amount in the positive control reaction, in one experiment but none in a second experiment. Due to the limited amount of corresponding scar: :TS: AnfH polypeptide having the wild-type AnfH sequence that could be recovered from plants, only a single experiment with the wild-type polypeptide could be carried out. However, probably due to the relatively impure preparation of that polypeptide, the reconstitution procedure with re-loaded NifU did not produce an active polypeptide for reducing acetylene.
Attempts were made to measure the acetylene reduction for the isolated scar::TS::AnfH-V7 in combination with purified, bacterial AnfDKG, but only low levels of ethylene were produced, including for the positive control, and therefore could not be reliably measured.
Activity of the AnfH polypeptides together with AnfDKG to fix N2 gas
The isolated scar: :TS: AnfH fusion polypeptides produced from SN709 (V7) and SN710 (V8) were also tested in vitro for reduction of N2 gas by the method using 15N2 as described in Examples 1 and 12. The isolated fusion polypeptides were first treated with the purified and re-loaded NifU protein as described above and then combined with the purified AnfDKG protein. These proteins were combined in the ratio of 40:20: 1 for the NifU: AnfH variant: AnfDKG proteins. The polypeptide from SN709 reduced the 15N2 gas at a rate equal to approximately 90% in a first experiment, and 78% in a second experiment, normalised to the positive control (Figure 8). Surprisingly in view of the lack of activity for acetylene reduction, the polypeptide from SN710 was able to reduce the 15N2 gas in one experiment at 43% normalised to the positive control (Figure 8).
AnfH variants having other combinations of amino acid substitutions as described in Example 9 that are confirmed to have increased solubility are tested for acetylene reduction and N2 reduction using the same methods. Variants that retain enzymatic function are selected.
In conclusion, the three amino acid substitutions in the AnfH-V7 fusion polypeptide not only greatly increased the solubility of the MPP-processed polypeptide when expressed in plant cells and localised to the mitochondria, but also was active for reducing acetylene to ethylene and N2 gas to ammonia in in vitro reactions, provided that the fusion polypeptide was first loaded with Fe-S clusters from active NifU. In contrast, the corresponding AnfH-V8 polypeptide was essentially inactive for reducing acetylene but was still active for reducing N2, albeit at a modest level. It was concluded that the difference between the two variants was likely due to the presence of the G241R substitution in the latter being deleterious for acetylene reduction activity, even though that variant was highly soluble. Example 11. The development of a colourimetric assay to test the activity of AnfH
Milton et al. (2017) reported that methyl viologen (MV) could be used as a chemical mediator to provide electrons to NifH purified from A. vinelandii. In their system, MV was reduced by an electron provided by an electrode connected to a potentiostat set in chronoamperometry mode and poised at -0.6 V vs NHE which resulted in MV donating the electron to NifH and, from there, to NifD-NifK (NifDK). In the presence of ATP, MgCh, phosphocreatine and creatine phosphokinase, a catalytic current could be measured, indicating catalysis by the NifDK nitrogenase and the production of ammonia. The main technique used in that 2017 study was open circuit potentiometry where the faradaic efficiency was measured for the number of electrons transferred from MV that ended up in the product ammonia. This technical advance was significant as a demonstration of the consumption of electrons as a substrate in the nitrogenase reaction. This technique was therefore referred to as a ‘front-end’ nitrogenase assay rather than the traditional ‘back-end assay’ where the products produced by the enzyme were measured such as ethylene or ammonia from acetylene or N2 gas, respectively. After Milton et al. (2017), Seefeldt and co-workers (Badalyan et al., 2019) recognised that viologens could be utilised to develop a colourimetric assay for nitrogenase activity. This colourimetric assay was based on the change in colour of an electron donor that had two different, defined colours in its oxidised and reduced state and a redox potential that aligned with its electron acceptor, NifH.
MV and other viologen derivatives appear blue after a one electron reduction, to the radical cation, and are colourless upon a one electron oxidation, to the di-cation. Due to the redox potential of MV at -0.44 V vs normal hydrogen electrode (NHE), the gradual change in colour observed is related to the passing of the electron to NifH which has a redox potential of -0.3 V vs NHE when unbound to NifDK. A colour change was not observed as NifH passed the electron to NifDK (redox potential -0.23 V vs NHE) to undertake catalysis on a substrate. In those experiments, a higher concentration of the viologen was required, typically around 0.5 mM to observe absorbance changes. In comparison, cyclic voltammetry used concentrations less than 0.1 mM and still obtained a quantifiable change in current. At concentrations greater than 0.1 mM, the catalytic activity of NifH/NifDK was inhibited, thus creating a less than optimal ‘front end’ assay. Badalyan et al. (2019), hypothesised that the cause of the inhibition was a cation radical dimer which was formed when 2MV molecules are reduced by one electron and H-H stack. To overcome this problem with MV, a derivative of MV was synthesised having charged-alkyl groups which would prevent the formation of radical cation dimers. They showed that a MV-derivative with propylsulfonate groups added to the viologen, (1,1'- bis(3-sulfonatopropyl)-4,4'-bipyridinium (Figure 9), herein abbreviated to S2V, did not exhibit inhibition of nitrogenase at concentrations up to 2 mM and reported an identical redox potential. The S2V sulfonated derivative could be synthesised from 4,4-bipyridine following the method of Debruler et al. (2018).
In setting up the colourimetric assay, the S2V was first reduced using a high surface area electrode in a process known as bulk electrolysis, whereby a potential was applied below the reduction potential of the viologen. The reduction reaction was followed by observing the colour change from colourless to blue and by examining the current curve plotted by the potentiostat vs time. The reduction was complete when the current curve plateaued. Although the usual electron donor in nitrogenase assays was sodium dithionite (DTH), its lack of colour or absorbance in a unique area of the visible spectrum and complicated reaction and decomposition chemistry meant it was not suitable for a colourimetric assay. Therefore, reduced S2V was selected by the present inventors as a suitable electron donor, also referred to as an electrochemical mediator, for nitrogenase assays in place of DTH, whereby the loss of colour indicated the consumption of electrons from the S2V electron donor. The rate of electron consumption can be monitored by measuring the decrease in absorbance at 600 nm, which can allow the catalytic rate of the reaction (kobs) to be calculated.
Paya-Tormo et al. (2022) adapted the assay to measure the activity of NifH expressed in, and isolated from, transgenic yeast. They adapted the assay to work in a 96 well plate format to allow higher throughput to assay NifH variants. In these assays, the change in absorbance at 600 nm was measured using a plate reader in an aerobic environment and loss of colour (lower absorbance) indicated catalytic activity.
The present inventors tested whether the colourimetric assay using either MV or S2V could be used to assay NifH and AnfH polypeptides purified from plant leaves. Initially, MV was tested with nitrogenase components purified from A. vinelandii. It was initially observed that a MV-based colourimetric assay was possible with a combination of AvNifH and AvNifD-NifK at an MV concentration of 0.5 mM. However, no colour change was detected using MV as the mediator with the purified bacterial proteins AvAnfH and AvAnfDKG from A. vinelandii. After synthesising S2V following the method of Debruler et al. (2018), the catalytic activity could be measured with S2V for both combinations of AvNifH/ AvNifDK and Av AnfH/ AvAnfDKG. It was noted, however, that the presence of DTH in the protein samples, used to provide protection against oxygen during purification and storage under liquid nitrogen, was at a high enough concentration that it was able to reduce S2V and essentially complicate the monitoring of the colour change. That is, the DTH could continually reduce S2V until all of the DTH had undergone an irreversible one electron oxidation. This was particularly evident when using more dilute protein samples such as those typically purified from the plants as described herein.
The present inventors therefore developed a procedure to remove DTH from the isolated nitrogenase components by exchanging the buffer solution in the preparations with one that contained no DTH. This was done using Amicon centrifugal units (0.5 mL, 30 kDa for AvNifH or AvAnfH, 50 kDa for AvNifDK or AvAnfDKG) for exchanging the buffer and then concentrating the enzyme components. A degassed 100 mM MOPS, pH 7 buffer was used throughout. The exchange buffer to remove the DTH contained 5 mM ATP and 6.7 mM MgCh.2H2O. Care was taken that the buffer and centrifugal units were thoroughly anaerobic. To achieve this, this procedure was performed in an anaerobic chamber containing 95% N2 and 5% H2. All solutions were degassed using a Schlenk line with typical vacuum/N2 cycles happening every 10 min for at least an hour. Once transferred to the anaerobic chamber, the buffers were stirred for at least 12 hr and the centrifugal units were washed in the anaerobic buffer twice with centrifugation prior to use. A further step taken to avoid oxygen within the assay buffers was to add the oxidised S2V to the ATP regeneration solution containing ATP, MgCh, creatine phosphokinase, phosphocreatine and BSA and carry out bulk electrolysis on this mixture with stirring. In this way, any residual oxygen from the powders added or within the buffer would be reduced by this procedure.
The colourimetric solution contained 5 mM ATP, 6.7 mM MgCh.2H2O, 30 mM creatine phosphate, 1.3 mg/mL BSA, 0.2 mg/mL of creatine phosphokinase and 500 pM S2V, all dissolved in 100 mM MOPS buffer. The addition of BSA was hypothesized to improve the solubility and stability of the proteins in solution and is commonly added to electrochemical nitrogenase assays. The electrochemical cell used to reduce S2V had a working electrode made of carbon paper attached with silver epoxide to a titanium wire and an Ag| AgCl reference electrode. These were placed in the solution and connected to the potentiostat. A separate glass tube with frit, filled with 100 mM MOPS to the same level as the colourimetric solution and containing the platinum counter electrode, was placed in the colourimetric solution. A potential of -0.6 V vs NHE was applied to reduce S2V.
A total reaction volume of 200 pL was found to be optimal for the 96-well plate format. The colourimetric solution without any nitrogenase component added was used as a negative control for all experiments. Due to the redox potential of S2V, any presence of O2 in the anaerobic environment would see the S2V oxidised. For AvNifDK: AvNifH, a ratio of 1 :20 was used and for AvAnfDKG: AvAnfH a ratio of 1 :30. In this Example, the prefix ‘Av’ refers to proteins purified from the bacterium vinelandii as described in Example 1. Different concentrations were examined, with 0.1 pM:2.0 pM and 0.1 pM:3.0 pM, respectively, being the optimal concentration ratios for the proteins to accurately monitor absorbance changes at 600 nm over a 30 - 60 min time period using a Cerillo Stratus plate reader.
Three combinations of components were tested in vitro'. AvNifH/AvNifDK, AvAnfH/AvAnfDKG and a hybrid combination of AvAnfH/AvNifDK. The results averaged from triplicate samples are shown in Figure 10. For AvNifH/AvNifDK and AvAnfDKG:AvAnfH, the reactions were complete within 30 min, with AvNifH/AvNifDK shown to have faster kinetics than AvAnfDKG: AvAnfH. Curiously, only very low S2V-mediated activity was noted when the AvAnfH/AvNifDK combination was tested. This was in contrast to that seen in acetylene reduction assays (ARA) where the AvAnfH/AvNifDK combination was able to reduce acetylene to ethylene at a measurable rate (Examples 10 and 16 herein). The hybrid system has also been observed to reduce nitrogen to ammonia via IRMS. It was concluded that S2V could be used as the electrochemical mediator in a colourimetric assay for nitrogenase activity for Nif-Nif and Anf-Anf systems, provided that the DTH level in the reaction mix was minimised, but not for hybrid Nif-Anf combinations.
Example 12. Development of methods to assess nitrogenase function in planta
The present inventors sought to develop a method to detect and measure nitrogenase activity in planta, either in whole plants or in excised leaf discs, in order to test combinations of mitochondrially-targeted Nif and Anf proteins expressed in plant cells for nitrogenase function. Several methods could potentially be used to detect nitrogenase activity, for example the acetylene reduction assay (ARA, Example 1) which produces ethylene, or detection of H2 production. Differing requirements were considered to influence the choice of assay method, including speed, scalability, sensitivity and robustness of the potential assays. The present inventors chose to test a direct assay using the nitrogen isotope 15N in the form of 15N2 gas as a substrate. The use of 15N assays to detect nitrogenase activity and the assessment of the contribution biologically-fixed nitrogen in plants has been reviewed by Chalk et al. (2017). The 15N abundance of plants usually closely reflects the 15N abundance of their major immediate N sources, i.e. plant-available N in the soil in the case of non-nitrogen fixing plants, and atmospheric N2 as well as plant-available soil N in the case of N2 fixing plants. Typically, the 15N isotope is present at about 0.336% of the total nitrogen content, with a 15N:14N ratio of 0.336/99.63. Positive nitrogenase activity would be detected by incorporation of the 15N into fixed ammonia or other nitrogen-containing compounds produced from ammonia, since no other known biological process besides nitrogenase activity can reduce N2 gas. It was considered that the 15N system might be the most sensitive and reliable for unambiguously demonstrating biological N2 reduction. This was tested as follows.
In a first experiment, whole N. benthamiana plants that had been grown five weeks in vermiculite and irrigated with Hyper Grow Complete hydroponic nutrient mix (Nutriflo, New South Wales, AU) under the conditions of 16 hr of light and 8 hr of dark were placed in 2.5 L airtight jars (Oxoid). In addition, leaf discs from such plants were excised and placed into 25 ml conical flasks and sealed with suba seals. 15N2 gas that had not been further purified was injected to 10% of total volume of each flask. After 24 hours, leaf samples were subject to Isotope Ratio Mass Spectrometry (IRMS) as described in Example 1. Positive delta-15N values were observed for all samples, including negative controls, for both leaf disk and whole plants.
In a second experiment, the same, not further purified 15N2 gas was added to untreated leaf disks cut from aseptically grown tobacco plants in a 25 mL conical flask (35 mL headspace), also containing 3 mL of sterile 5% (w/v) sucrose solution. The plants had been grown in tissue culture medium (Murashige and Skoog, MS) under aseptic conditions and were handled aseptically to ensure there was no possible nitrogen fixation from contaminating bacteria. The 15N2 gas was added at 5%, 10% and 20% of total volume, or omitted as a control, for triplicate samples for each. After 24 hours, leaf samples were subject to IRMS as before. It was observed that the delta-15N values increased with increasing levels of added 15N2 gas, correlating with the added amount (Figure 11). Notably, no increase in delta-15N over background was detected in the leaf discs that had no gas added. It was concluded that the observed positive delta-15N values in these two experiments were likely to have resulted from contaminating 15N compounds present in the gas other than 15N2 such as, for example, nitrites or ammonia. These contaminating compounds needed to be removed for an assay with less background.
In a third experiment, therefore, contaminating N compounds in the 15N2 gas such as nitrites or ammonia were first removed using a device published by Kennedy and Wood (2001), with acid and base/oxidant solutions as described in Example 1. The plant experiment was then repeated with aseptically-grown leaf disks to compare the further purified gas with non-purified gas. Using the further purified gas, no change was observed in delta-15N values above background, whereas the non-purified gas provided a positive delta-15N value as before (Figure 11 (B)). This indicated that the non-purified gas did contain contaminating compounds that were 15N labelled and which became incorporated into the leaf samples, and that the additional purification process removed these. It was concluded that using the purified gas containing 15N2 ensured negligible background levels of 15N incorporation from non-nitrogen fixing sources into the plant material and therefore any true nitrogenase activity above background levels would be detected.
To test the sensitivity of the assay, a mock nitrogenase activity assay was performed by infiltrating live, A. vinelandii cells of strain DJ2102, grown in BN medium and induced for nitrogenase activity with B medium (Example 1), into N. benthamiana leaves. The cells were infiltrated 5 hr after induction at an OD of 5. Leaf disks were then cut and put into sterile 25 mL conical flasks containing a 5% (w/v) sucrose solution. The flasks were sealed with suba seals and 3.5 mL of purified 15N2 added to provide a 10% concentration in the headspace of the flasks. Leaf discs were harvested after keeping the flasks 24 hr in the dark and processed for IRMS as before. This time, the delta-15N values were significantly higher than background for non-infiltrated leaves as a control (Figure 11 (C)). This result demonstrated that nitrogenase activity could be detected with sensitivity and measured in plant leaves using the purified 15N2 gas.
The more detailed methods used for measuring nitrogenase activity in vitro and in planta with the purified 15N2 gas are described in Example 1.
Example 13. Design of vectors for increasing expression of nitrogenase components in plant cells
Introduction
In nitrogen fixing bacteria during the transition to diazotrophy, up to 40% of total cellular protein is comprised of nitrogenase components (Waite et al., 2021). In contrast to the bacteria, some nitrogenase proteins were poorly expressed when targeted to plant mitochondria when produced from conventional expression constructs (Allen et al., 2017; Okada et al., 2020). Furthermore, in vitro activity assays of individual Nif/Anf components produced in plant cells require considerable amounts of purified protein to provide reliable activity measurements, and poor accumulation of the proteins has up till this time hindered this endeavour. Previous experiments (Allen et al., 2017; Okada et al., 2020) used the enhanced 35S (e35S) promoter from CaMV with a 5’ untranslated region providing translational enhancement from Tobacco Mosaic Virus (TMV 5’UTR) and a 35S polyadenylation region/transcription terminator (3’-35S) to drive Nif gene expression in the plant cells. Those experiments used the GoldenGate toolkit for plants (Engler, 2011) to construct the expression constructs. In many cases, but not all, this combination of expression elements provided high levels of protein production in leaves, such as in Nicotiana plants.
The present inventors therefore investigated different methods to further enhance Nif/Anf gene expression. There are several, general known ways to increase protein expression in plants (Egelkrout et al., 2012). One method relies on the use of replicating viral vectors, for example vectors based on gemini virus (GV) genomes. These work by multiplying the copy number of a gene of interest within a cell. The present inventors therefore tested GV vectors for nitrogenase fusion protein production, adapted to a format that was compatible with the GoldenGate cloning system. This would also allow rapid interchange and test of elements such as addition of mitochondrial targeting peptides and antibody epitopes.
Design and construction of replicative expression vectors adapted for GoldenGate cloning
Gemini Virus (GV) replicative expression vectors were designed and made based on the structure of a LSL Bean Yellow Dwarf Virus (BeYDV) plasmid vector (Baltes et al., 2014), as follows. The virus BeYDV has a single-stranded DNA genome and has a relatively wide host range among dicotyledonous plants. Vectors based on its replicative regions are therefore expected to replicate in many dicotyledonous plants, including N. benthamiana used as described herein. The LSL vector includes the large intergenic region (LIR) and short intergenic region (SIR) required for viral replication as well as a gene encoding the trans-acting replication (Rep/RepA) proteins but does not have the genes encoding the viral coat protein and the movement protein required for cell to cell spread of the wild-type virus. Those genes were deleted to provide space for inserting heterologous genes.
To adapt the LSL-BeYDV vector to be suitable for type IIS restriction enzyme cloning in the GoldenGate system for the construction of LI modules, a guanosine at nucleotide position 1,119 with reference to SEQ ID NO: 94, within the Rep/RepA coding region, was replaced with an adenine to remove a Bsal site that otherwise would interfere with subsequent cloning, providing the nucleic acid having SEQ ID NO:94. This substitution did not change the encoded Rep amino acid sequence and, as shown afterward, did not adversely affect Rep function. This vector backbone is referred to herein with the notation “GV”. Further modifications were made where an adenosine at position 1,469 in the Rep 5’UTR sequence, with reference to SEQ ID NO:94, was additionally changed to either a guanosine as per Diamos and Mason (2019) in the GVc vector, or an adenosine in the GVt vector. The complementary nucleotides C or T in the sense strand are less favoured for translation initiation in their context 3 basepairs upstream of the ATG start codon. This was done with the aim of decreasing translation of the Rep protein to reduce cell necrosis that tended to occur with GV vectors. Both vectors were designed to be assembled from /.sal-ended DNA fragments as modules and this strategy thereby provided flexibility and adaptability as for the GoldenGate cloning system, allowing various combinations of modules and switching of any one module for another.
DNA fragments for the modules were synthesized commercially with a Bsal restriction enzyme site close to each end of the sequences and inserted into a standard cloning vector to provide L0 vectors. One DNA designated as “P module” (SEQ ID NO:92) included the LIR region from BeYDV and had an upstream Bsal -ligation site for cloning into a GoldenGate LI acceptor vector and a downstream Asz/I-ligation site for joining with a second module, designated the “U” module (SEQ ID NO:93). In this construction, the U module included a CaMV e35S promoter joined to a 5’ UTR sequence from tobacco mosaic virus (TMV), including an ATG start codon, for efficient transcription and translation of a downstream protein coding region of interest. For this U module (based on Fragment number EC51288), the upstream Bsal -ligation site was compatible with the 3’ Asz/I-ligation site of the P module, and the downstream Bsal- ligation site was compatible with “C”, “SCI”, “Cl” or “SC” modules for cloning polypeptides of interest containing N terminal extensions, C terminal extensions, both N and C terminal extensions, or neither, respectively. To allow incorporation of a 3’ polyadenylation region/transcription terminator and the necessary GV replicative regions downstream of the S module, a module designated as “T” (SEQ ID NO:94) was synthesized that comprised a 35S transcription terminator (35S Tm) followed by the SIR/Rep/RepA/LIR region from the BeYDV genome. The P module (LIR5’) has the Fragment number EN38510, the U module (35S+TMV 5 ’UTR) has EN38509 and the T module (35S Tm SIR/Rep/RepA/LIR) has EN38511 for the GVc and EN38512 for the GVt vectors.
The design of these modules allows for the modular assembly of standardised GoldenGate compatible parts (Figure 12) into a plant cell-replicative vector with the GV replication components and including any “S”, “C”, “SCI”, “Cl” or “SC” modules encoding a polypeptide such as a Nif fusion polypeptide, depending on the presence or absence of an epitope tag sequence. These encoded fusion polypeptides typically comprised an MTP sequence at the N-terminus, followed by an epitope tag or not, then a Nif polypeptide coding sequence and finally with or without a C-terminal epitope tag as desired. Results with replicating vectors
To assess the GVc and GVt vectors for protein expression and accumulation, several constructs were designed and made with S modules encoding any one of three different MTP-Nif fusion polypeptides for mitochondrial targeting and processing as well as a control construct for GFP. These comprised a Klebsiella oxytoca NifD sequence containing the Y100Q mutation (Allen et al., 2020; SEQ ID NO:96), Geobacter NifH (SEQ ID NO: 97), A. vinelandii NifU (SEQ ID NO: 98) and GFP (SEQ ID NO: 99), each with the MTP-FAy51 sequence at its N-terminus, or the MTP-FAy77 sequence in the case of pRAl (SEQ ID NO: 100), and a HA epitope tag at either its C-terminus or between the MTP and NifH sequences in the case of the Geobacter NifH. The genetic constructs were assembled from the P, U, S/C/Cl and T modules by digestion with Bsa\ and ligation into the T-DNA vector EC47742 (Engler et al., 2014). These genetic constructs were introduced into A. tumefaciens and transformed cells used to infiltrate N. benthamiana leaves as per Example 1. Total protein extracts were prepared from infiltrated leaf discs after four days and analysed by gel electrophoresis and Western blotting as above. The accumulation of the polypeptides, either processed by MPP or unprocessed, was assessed from the size and intensity of the protein bands. It was observed that accumulation of the fusion polypeptides was greatly increased using the GV vectors compared to the corresponding non-replicating vector encoding the same fusion polypeptide, for all tested fusion polypeptides (Figure 13). For the GVc and GVt vectors, but not for the nonreplicating control vector, bands at the sizes of the expressed polypeptides were even visible on a total protein gel stained with Coomassie stain, indicating very high levels of accumulation of the proteins from the GV vectors. No noticeable difference was observed in protein accumulation levels between the GVc and GVt vectors. However, necrosis was slightly less pronounced with the GVc vector, although some necrosis was still observed at 5 dpi. These results indicated that the replicative GVc and GVt vectors adapted for the GoldenGate methodology were well suited to substantially increase the expression level of nitrogenase polypeptides, with preference for the GVc vector.
Several other polypeptides were over-expressed in leaf cells using the GVc vector, including a MTP-CoxIV::TS: ifH: linker: ifH polypeptide expressed from SN650 and MTP-FAy51 ::AnfD::linker(HA)::AnfK polypeptide from SN735, both mitochondrially targeted, and compared to the same polypeptide expressed from a nonreplicating vector. In each case, the GVc vector produced much more of the specific protein, to the extent that a protein band of the expected size was visible on a Coomassie- stained SDS-PAGE gel. It was concluded that the replicative vectors were generally useful for accumulating high levels of mitochondrially-localised fusion polypeptides in plant cells, including Nif and Anf polypeptides.
Modifying a non-replicative vector for increased expression levels in plant cells
It has been reported that GV vectors may elicit a hypersensitive response when expressed in some plant cells, leading to necrosis and cell death (Diamos and Mason 2019). Despite efforts to attenuate this by incorporating single nucleotide substitutions into the GVc and GVt vectors as described above, some necrosis still occurred with the GV-based constructs. The present inventors therefore explored further non-replicating vector components where ultra-high protein expression was not required. One fruitful approach has been the use of multiple, chimeric 3’ flanking regions (Diamos and Mason, 2018). Those authors reported the effect of combining multiple transcription terminators and a matrix attachment region (MAR) as downstream elements for gene expression. They reported that expression of a GFP reporter gene was significantly enhanced by combining an intron-less tobacco extensin gene 3’UTR (EU Tm) with a 35S terminator and a MAR associated with a tobacco Rb7 gene. Furthermore, it was found that the MAR region could be reduced in size and still aid production of increased levels of GFP protein when combined with the dual polyadenylation/transcription terminator regions. Accordingly, the present inventors made a “hybrid terminator” (TTm, SEQ ID NO: 95) with three components, namely in order, the EU Tm, the 35S Tm and the reduced MAR region fused at the 3’ end. This combination of three elements is sometimes referred to herein as the “triple terminator”. The smaller size of the MAR region was advantageous to reduce the gene size needed for expression of multiple genes from multi-gene constructs. This TTm terminator region was made GoldenGate compatible as a replacement for the conventional 35S terminator (Tm), with appropriate Bsal sites close to the ends of the sequence.
To test the TTm terminator for its effect on production of mitochondrially- targeted proteins in comparison to the standard Tm and GVc-based vectors, three genetic constructs were made, each encoding a MTP-FAy51 ::NifH::linker(TS)::NifH fusion polypeptide (SEQ ID NO: 101), i.e. a NifH dimer. These were the conventional expression vector (SN655) with a single Tm, a TTm terminator vector (SN656) and a GVc vector (SN657). Each therefore encoded the same MTP-fusion polypeptide but differed in the presence of downstream Tm or TTm regions (SN655 vs SN656) or from a non-replicating or GVc-replicating vector (SN655 and SN656 vs SN657). The MTP sequence was the FAy51 sequence, the two NifH sequences were the K. oxytoca sequence, and the linker incorporated a TwinStrep epitope for detection with antibody in the Western blot. These genetic constructs were introduced from A. tumefaciens cells into N. benthamiana leaves by the method as described in Example 1, with co-infiltration of a construct encoding a MTP::NifM fusion polypeptide (SN360, SEQ ID NO: 102). After culturing the plants for four days post-infiltration, leaf samples were harvested immediately after a 16hr dark period, and total, soluble and insoluble protein extracts were prepared and analysed by SDS-PAGE and Western blotting (Figure 14). It was observed that although the TTm and GVc constructs expressed and accumulated much more scar::NifH::linker(TS)::NifH polypeptide in the total protein extracts than the standard Tm vector SN655, more soluble polypeptide accumulated in the plant cells when expressed from the vector SN656 having the hybrid terminator. Furthermore, most of the MTP::NifH::linker(TS)::NifH fusion polypeptide expressed from the GVc vector was not processed by MPP, while most of the fusion polypeptide encoded by the TTm vector SN656 was processed. This demonstrated that the TTm terminator vector was well suited to increase expression of mitochondrially-targeted Nif proteins to levels beyond those achievable using conventional constructs with a single terminator, even when using the same promoter. More generally, it was concluded that use of different expression systems could provide for increased accumulation levels of desired proteins within plant mitochondria.
Example 14. A synthetic dimer of NifH and its production in plant cells
Introduction
The NifH protein, also known as the Fe protein, that functions as the dinitrogenase reductase enzyme is an essential component of the biological nitrogen fixation pathway in diazotrophic bacteria. The protein is encoded in the bacteria by the nifH gene and functions as a homodimer. In bacteria such as Klebsiella oxytoca and A. vinelandii. activity of the mature protein requires the function of a NifM protein which is thought to be a maturase with isomerase activity, specifically a putative peptidyl-prolyl cis-trans isomerase activity (Gavini et al., 2006). NifM has also been reported to form a physical interaction with NifH as a heterodimer (Petrova et al., 2000 and 2002). Aside from its function as dinitrogenase reductase in transferring electrons from NifF to holo-NifDK during catalysis, NifH is known to have two other functions, namely involvement in maturation of pre-P clusters into P-clusters and insertion of M-clusters into the apo- NifDK heterotetramer (Buren et al., 2020). To date it has been shown that a NifH active for electron donation is also capable of assisting both P- and M-cluster biogenesis (Jiang et al., 2021). Okada et al. (2020) reported that a fusion polypeptide including K. oxytoca NifH, designated herein as KoNifH, was insoluble when targeted to the plant mitochondrial matrix, similar to other canonical NifH proteins, hindering efforts to isolate an active enzyme from the organelle. Jiang et al. (2021) screened 32 MTP-NifH fusion polypeptides for solubility in plant cell mitochondria after co-expression with NifM, NifS and NifU fusion polypeptides and identified only three of the NifH polypeptides that were soluble. That study expressed NifH from the thermophilic, obligate chemolithoautotrophic bacterium Hydrogenobacter thermophilus (Ht), in N. benthamiana and purified the protein from the plant cells. When tested in in vitro acetylene reduction assays, the protein was inactive “as isolated”. However, partial activity of the purified HtNifH protein was observed for electron donation to A. vinelandii NifD-NifK when the host plants were supplied with additional iron during growth and leaves expressing the HtNifH were harvested after a growth period in the dark, pre-dawn, before isolation. This suggested that plants grown under normal conditions were not supportive for functional, wild-type NifH and that oxygen produced by photosynthesis during growth in the light was hampering production of functional NifH. A much larger increase in activity of the plant-isolated and purified HtNifH was reported when the purified HtNifH protein was treated in vitro with purified vinelandii (Av) NifU protein produced in A. coli (Jiang et al., 2021), presumably to reconstitute Fe- S clusters in the HtNifH protein.
In contrast to the insolubility of most NifH polypeptides targeted to the plant mitochondria, cytosolically localised NifH from K. oxytoca was fully soluble and did not require NifM for solubility. This was further confirmed as described below. However, there is no evidence that the monomer NifH polypeptide in the cytosol was correctly folded or functional, despite being soluble.
More controversial is whether NifD and NifK together help stabilise the structure of NifH, an idea based on the interlocking crystal structures of NifH/NifDK (Schlesier et al., 2016) and the increase in activity and solubility of AnfH seen when co-expressed with AnfD and AnfK in yeast (Lopez-Torrejon et al., 2021).
Production of a soluble form of the NifH protein in mitochondria is considered essential to constitute nitrogenase activity when expressed in plant cells with mitochondrial localisation. The observed insolubility of KoNifH (Okada et al., 2020) as confirmed herein therefore precludes the use of that form of NifH in mitochondria for nitrogenase function. Attempts to improve solubility of MTP-NifH fusion polypeptides localised to mitochondria
In initial experiments expressing a genetic construct (SN18) encoding a monomeric MTP-FAy51 ::NifH::HA polypeptide in N. benthamiana leaf cells, the encoded fusion polypeptide (SEQ ID NO:20) and its MPP-processed product were both essentially insoluble when targeted to the mitochondria. The NifH sequence was based on the K. oxytoca NifH and had the MTP-FAy51 at its N-terminus and a HA epitope at its C-terminus. Total protein, soluble protein and insoluble protein extracts were analysed by Western blotting with the anti-HA antibody, as before. It was observed that the scar::KoNifH::HA fusion polypeptide was almost completely insoluble. The soluble protein extract showed only a very faint band for the MPP-processed fusion polypeptide, whereas the corresponding band in the insoluble extract was as intense as the band in the total protein extract. This confirmed the observation of Okada et al. (2020) that the K. oxytoca NifH monomer polypeptide was essentially insoluble in mitochondria.
The present inventors sought to increase scar: :KoNifH solubility in several ways, such as by co-expression with MTP-SU9: :KoNifM (SEQ ID NO: 102) from the construct SN360. The NifM sequence was based on the homologous sequence from K. oxytoca (SEQ ID NO: 8), joined to the MTP sequence from SU9 at the N-terminus. That experiment resulted in only slightly increased solubility of the processed scar::KoNifH::HA polypeptide. When another construct encoding a MTP::GFP fusion polypeptide (pRAl; SEQ ID NO: 100) was co-infiltrated as a control, no increase in solubility of the scar::KoNifH::HA polypeptide was observed, indicating that the slight effect on increasing solubility was caused by the NifM polypeptide that was cointroduced. However, the scar: :KoNifH: :HA polypeptide was still mostly insoluble. Further addition of constructs to co-express fusion polypeptides from SL133 (Table 6), encoding MTP-CPN60::AvNifS (SEQ ID NO:89) and MTP-SU9::AvNifU (SEQ ID NO: 90), both having the A. vinelandii sequences and without epitope tags, did not further enhance the scar: :KoNifH: :HA monomer solubility. Further addition of SN465 encoding MTP-FAy51 ::AvFdxN (SEQ ID NO:91) likewise did not further enhance the scar::KoNifH::HA monomer solubility. In similar fashion, co-expression of MTP- CPN60::AvNifS and MTP-SU9::AvNifU without MTP-SU9::KoNifM did not enhance scar::KoNifH::HA monomer solubility. The improvement in solubility, rather modest, was therefore specific to the presence of NifM, presumably by improved folding of the NifH fusion protein. A synthetic dimer of NifH
The present inventors then conceived of the idea that a synthetic dimer of a NifH protein might have better solubility than the corresponding monomer when expressed in plant cells and localised to the mitochondria. Several genetic constructs were therefore designed and made that encoded translational fusions of dimerised NifH polypeptides, with the two monomer units separated by a 25 amino acid linker region (Lahiri et al., 2008; Yang et al., 2018), thought to be beneficial for correct folding of the two NifH units. The encoded fusion polypeptides, designated MTP::NifH::linker::NifH, with the two NifH sequences based on the K. oxytoca sequence in the first instance, had an MTP sequence at the N-terminus for localisation to the mitochondrial matrix of the plant cells. The polypeptides also included either a HA or TwinStrep (TS) epitope for easy detection and/or purification in the case of the TS version. These constructs were co-infiltrated with one or more constructs to express various combinations of AvNifS and AvNifU or additionally a ferredoxin AvFdxN fusion polypeptide, each based on the A. vinelandii protein sequences. A K. oxytoca NifM fusion polypeptide with MTP was included, as above, and in one instance a MTP-NifD: dinker: :NifK fusion polypeptide based on the K. oxytoca NifD and NifK sequences but having the Y100Q amino acid substitution in the NifD sequence for improved protein stability (Allen et al., 2020). Each of these accessory proteins was translationally fused to a MTP sequence for mitochondrial localisation with the MTP at the N-terminus of the polypeptide. Table 6 lists the constructs that were made and provides a complete notation for the encoded fusion polypeptides with the SEQ ID NOs.
In one experiment, each synthetic gene was expressed using the 35S promoter with a single polyadenylation/transcription terminator sequence (Tm). The Western blot analysis of the protein extracts after infiltration of the construct (SN283) to express a MTP::NifH::linker::NifH::HA fusion polypeptide (SEQ ID NO: 103) yielded a unexpectedly different and surprising increase in solubility of the resultant MPP- processed polypeptide, compared to the monomer construct. This was particularly observed in the presence of co-introduced MTP -NifM from SN360. As shown in Figure 15, lanes 1-3, the scar::NifH::linker::NifH::HA fusion polypeptide was partially soluble on its own, more so than the corresponding scar: :NifH: :HA monomer, but the dimer polypeptide was much more abundant and soluble when co-expressed with the MTP::NifM construct (lanes 7-9). Specifically, the NifH dimer was predominantly (>50%) soluble in the mitochondria when produced in the presence of the NifM fusion polypeptide. Further addition of one or two constructs to co-express fusion polypeptides for MTP:: AvNifS and MTP:: AvNifU from SL133 and MTP:: AvFdxN from SN465, each based on the A. vinelandii sequences, in combination did not further enhance the scar: NifH: linker: NifH fusion polypeptide abundance or solubility. It was later observed that the AvFdxN polypeptide from SN465 was insoluble and likely not functional, so that addition might not be meaningful. Addition of SL133 to co-express fusion polypeptides for MTP::AvNifS and MTP::AvNifU in the absence of MTP::NifM did not appear to enhance the solubility of the scar: NifH: linker: NifH: :HA fusion polypeptide, showing that the effects on increasing the abundance and solubility was primarily due to the presence of the NifM polypeptide. The inventors considered that the likely reason for the observed effect was that the NifM polypeptide was assisting the correct folding of the scar: NifH: linker: NifH polypeptide in the mitochondria and thereby increasing its solubility and probably reducing turnover of the polypeptide. It was also considered that formation of the NifH homodimer was improved by the dimerised translational fusion rather than the two separate subunits binding only by electrostatic interactions as occurs for the NifH monomers.
The solubility of the MPP-processed product from MTP- CoxIV::TS::NifH: linker: :NifH (SEQ ID NOTCH) expressed from a second construct (SN650, Table 6) was also increased by co-expression of K. oxytoca NifM, although it was not as pronounced as for scar: NifH: linker:: NifH:: HA.
These observations were in contrast to the solubility of cytosolically localised NifH from K. oxytoca which was fully soluble and did not require the addition of NifM for solubility. This was further confirmed as described below.
The MTP-NifH dimer was processed by MPP in the plant mitochondria
To demonstrate that the encoded MTP-NifH dimer fusion polypeptides were cleaved by MPP at the expected site within the MTP sequence, the size of the accumulated protein was compared to that of two control polypeptides, namely a nonprocessed analog of the polypeptide having alanine substitutions in the MTP sequence, notated as “alaMTP”, and an analog that did not have the MTP sequence but instead had six histidines at the N-terminus, notated as “6xHis” polypeptides, which approximated the size of the MPP-processed fusion polypeptide. That polypeptide was expected to remain localised in the cytosol, lacking an MTP sequence. The NifH sequences were all based on the ", oxytoca sequence. The control constructs were SN807 encoding the nonprocessed alaMTP-FAy51 : NifH: linker: NifH: :HA, SN303 encoding the 6xHis: NifH: linker: NifH: :HA and SN808 for the non-processed alaMTP- FAy51 ::HA::NifH::TS::NifH. The results (Figure 16) showed that the fusion polypeptides having the functional MTP sequence encoded by SN283 and SN656 yielded polypeptides of the same mobility as the 6xHis polypeptide and therefore were mostly (>50%) fully processed.
Table 6. Genetic constructs for experiments testing NifH dimer fusion polypeptides. These observations for cleavage by MPP at the expected cleavage sites and therefore mitochondrial localisation are confirmed by proteomics to identify the N- terminal tryptic peptide by mass spectrometry and by use of a metaxin-mediated mitochondrial enrichment by the methods described in W02021/068039.
It was concluded that the processed fusion polypeptide products comprising the NifH-linker-NifH dimer were localised to the mitochondria in a soluble form.
Alternative MTP-NifH dimer polypeptides
To test whether the increased accumulation and solubility of a scar: NifH dimer when co-expressed in the presence of MTP::NifM was generally true for different NifH sequences, three genetic constructs (SN678, SN679, SN680) each encoding an analogous MTP: AvNifH: dinker: AvNifH dimer (SEQ ID NO: 109) were designed and made using the NifH sequence from A. vinelandii. The same 25-amino acid linker was used to join the two NifH units as for the previous polypeptides using the K. oxytoca NifH units. Each of these constructs was separately introduced along with SN605 which encoded a homologous MTP-AvNifM polypeptide, also based on the A. vinelandii NifM sequence, and expressed in N benthamiana leaves. The protein products were analysed by SDS- PAGE and Western blotting as before. It was observed from the blot (Figure 17) of the total protein, soluble protein and insoluble extracts that the solubility of the scar::HA: AvNifH: :linker: AvNifH dimer based on the A. vinelandii sequence was also increased relative to the corresponding monomer when the construct was co-expressed with the MTP -NifM polypeptide, but not as much as for the analogous K. oxytoca NifH dimer.
The role of the MTP sequence and the position of the TwinStrep epitope in influencing the solubility of the processed protein product was investigated by comparing the accumulation of processed fusion polypeptides after introduction of SN638, expressing the MTP-CoxIV::TS::NifH::linker::NifH fusion polypeptide (SEQ ID NO: 104), with SN656, encoding the MTP-FAy51 ::HA::NifH::linker(TS)::NifH fusion polypeptide (SEQ ID NO: 105). The linker(TS) was 30 amino acids in length, longer than the 25 amino acids used previously, and with a different amino acid composition an including a TwinStrep tag. These two constructs were transiently expressed in N. benthamiana leaves either with or without co-introduction of the construct SN360 encoding the MTP-NifM polypeptide, and protein extracts analysed as before. This experiment showed that both processed fusion polypeptides were at least partially soluble in the presence of the NifM construct, but the increased solubility was much more marked for SN656. This also showed that the nature of the MTP and the position of the epitope sequence could influence the extent of the increase in solubility of the processed NifH dimer polypeptide. The optimum structure for solubility can be determined empirically by this strategy.
Testing different expression systems for NifH dimer production
Three analogous sets, each of three genetic constructs, were made to test the production, MPP-processing and solubility of NifH dimer fusion polypeptides using three different expression systems, each in N. benthamiana leaves: two using the enhanced CaMV promoter (e35S), of which one had a single polyadenylation/transcription termination sequence while the other had a hybrid terminator design (TTm, see Example 13 herein), and the third with a geminiviral replicon system (Example 13 herein). A first set had SN638, SN649 and SN650, each encoding the MTP-CoxIV::TS::KoNifH::linker::KoNifH polypeptide (SEQ ID NO: 104). The second set had SN655, SN656 and SN657, each encoding the MTP- FAy51 ::HA::KoNifH::linker(TS)::KoNifH polypeptide (SEQ ID NO: 105). The third set had SN678, SN679 and SN680, each encoding the MTP-
FAy51 ::HA::AvNifH: linker: :AvNifH polypeptide (SEQ ID NO: 109). These sets differed in their NifH sequence, their MTP sequence and the position of the epitope, therefore comparing the three expression systems for a range of fusion polypeptides. In two sets, the TS epitope was used to allow for protein purification on a streptactin sepharose column.
The three sets of constructs were expressed in N benthamiana leaves and total and soluble extracts analysed as before. The constructs with the hybrid terminator (TTm) and the replicative GV vectors in each case outperformed the corresponding non- replicative vector with the single terminator in terms of the amount of fusion polypeptide that was produced and processed. For example, on average per 100g of leaf tissue, the geminiviral vector SN650 encoding MTP-CoxIV::TS::KoNifH: linker: :KoNifH, produced approximately six-fold more soluble, processed scar::TS::KoNifH::linker::KoNifH protein compared to that using p35S, at 180 pg purified protein compared to 28 pg for SN638. The hybrid terminator construct SN656 produced, on average, 127 pg of purified soluble protein per 100 g fresh leaf weight, of the processed polypeptide scar::HA::KoNifH::linker(TS)::KoNifH. This amount of purified protein readily allowed for the in vitro testing of function in acetylene reduction assays, as described below. Later experiments showed that the hybrid terminator construct with omission of the HA tag provided at least as processed much polypeptide scar: :KoNifH: linker(TS): :KoNifH. Constructs for modifying the linker length
To test the effectiveness of a shorter linker length in the MTP-NifH-linker-NifH context, a construct (SN285) encoding the MTP::KoNifH: linker: :KoNifH::HA polypeptide (SEQ ID NO: 110) with a 10-amino acid linker (SN285) was co-infiltrated with or without MTP: :NifM in N. benthamiana leaves and the protein products analysed by SDS-PAGE and Western blotting, as before. This protein product was marginally soluble without co-expression of MTP::NifM, but became more soluble (50%) when coexpressed with MTP::NifM, although its increase in abundance and solubility was not as pronounced compared to the polypeptides produced from SN656 and SN283.
Example 15. Enzyme function of NifH dimer polypeptides produced in plant cells
Prior to the experiment described above comparing the solubility of the polypeptides produced from constructs SN638 and SN656, the construct SN638, encoding a MTP-CoxIV::TS::KoNifK::linker::KoNifH fusion polypeptide (SEQ ID NO: 104) expressed from an e35S promoter, was introduced into A. benthamiana leaves along with three other co-infiltrated constructs expressing four additional polypeptides. These were SL133 which encoded MTP-CPN60::AvNifS and MTP-SU9::AvNifU, SN291 or SN465 which encoded MTP-FAy51 : : AvFdxN: :HA or MTP-FAy51 : : AvFdxN, respectively, and SN360 encoding MTP::KoNifM (Table 6). The resultant MPP- processed scar: :TS::KoNifH: linker: :KoNifH polypeptide (SEQ ID NO: 113) was isolated from about 200g of infiltrated leaves four days post-infiltration using the method described in Example 1, purified using streptactin affinity chromatography, and concentrated down to a volume of 300-385 pl. A second, parallel protein production and isolation was done using the geminiviral construct SN650 expressing the same MTP- CoxIV::TS::KoNifH: linker: :KoNifH fusion polypeptide. In that second production, SN650 was co-expressed in the plant cells with the construct SN159 encoding MTP- FAy51::KoNifD(Y100Q)::HAlinker::KoNifK (SEQ ID NO: 106) in addition to the vectors encoding MTP-CPN60::AvNifS, MTP-SU9::AvNifU, MTP- FAy51 ::AvFdxN::HA or MTP-FAy51 ::AvFdxN and MTP::KoNifM. In further parallel isolations, final concentrations of 2 mM Fe and 0.5 mM cysteine were added to the supernatants of the leaf lysates during the isolation process from SN638 and SN650 with the intent to maintain or further enhance Fe-S cluster loading onto the NifH dimer polypeptide via the co-expressed NifU polypeptide. This experiment therefore produced four different NifH dimer preparations. The purified polypeptides were tested in vitro for acetylene reduction activity in reaction mixes together with purified, wild-type NifD-NifK isolated from A. vinelandii strain DJ2102, as described in Examples 1 and 3. The isolated polypeptide produced from the non-replicative construct SN638, having the e35S promoter, and from the replicative geminiviral construct SN650 both reduced acetylene to ethylene when combined with the purified vinelandii NifD-NifK (Figure 18). The amount of ethylene produced was 0.5% and 1.7%, respectively, of the ethylene produced using wild-type NifH isolated and purified from A. vinelandii as the positive control. Addition of the Fe and cysteine during the isolation and purification increased the acetylene reduction for the NifH dimer polypeptide produced from SN638 but not significantly for SN650.
In that experiment, the plants had not been applied with extra iron during growth, and the leaves had been harvested after 2-4 hr of incubation in the light. In a following experiment, the level of acetylene reduction was therefore compared between isolated scar: :TS::KoNifH: linker: :KoNifH polypeptide produced in N. benthamiana leaves harvested after a growth period in the light vs leaves harvested at the end of a 16hr period of darkness. The polypeptides were produced from SN650 co-expressed with vectors encoding MTP-KoNiflVI, MTP-AvNifS, MTP-AvNifU, MTP-AvFdxN: :HA, as before, as well as MTP-KoNifD(Y100Q)::KoNifK (SEQ ID NO: 106) from SN159. The scar: :TS::KoNifH: linker: :KoNifH polypeptide purified from dark-harvested leaves was approximately four-fold more active for acetylene reduction compared to that from light- harvested leaves (Figure 18). This increase was observed even though the light-harvested scar: :KoNifH: linker: :KoNifH had a higher NifH:NifD-NifK ratio (60: 1) in the ARA reaction mix compared to that from dark-harvested scar: :NifH: linker: :NifH (22: 1).
In another experiment, the acetylene reduction assays of three scar: :TS::KoNifH: linker: :KoNifH isolates produced from SN638 or SN650 were compared in in vitro assays in combination with purified vinelandii NifD-NifK. This showed that the assay with scar: :TS::KoNifH: linker: :KoNifH polypeptide from SN638 produced 2 units of ethylene (at a NifH:NifD-NifK ratio of 12: 1) when Fe and cysteine were not added during the isolation, compared to 4 units (at a NifH:NifD-NifK ratio of 15: 1) when the same polypeptide was isolated in the presence of Fe and cysteine. The same polypeptide produced from the geminiviral vector SN650 produced 7 units of ethylene (at a NifH:NifD-NifK ratio of 60: 1). In comparison, the positive control assay using purified A vinelandii NifH with the NifD-NifK (at a NifH:NifD-NifK ratio of 20: 1) produced 400 units of ethylene at 2hr incubation.
In a further experiment testing the three different expression systems with nonreplicating vectors SN655, SN656 and the replicative vector SN657, each encoding the MTP-FAy51::HA::KoNifH:linker(TS)::KoNifH fusion polypeptide (SEQ ID NO:101), plant leaves were harvested after a period of 16hr incubation in the dark. Some of the N. benthamiana plants had been applied with additional iron during growth before infiltration with the genetic constructs in view of a reported benefit for added iron (Jiang et al., 2021 ; Baysal et al., 2022) and to test whether standard growing conditions provided enough iron to the plants. Fe supplementation was applied to the plants at 3, 4 and 5 weeks post-sowing by adding vermiculite drenched with 100 mg/L Fe chelate. The final concentrations of the three isolated scar::HA::KoNifH::linker(TS)::KoNifH protein preparations ranged from 1.4 to 1.9 mg/ml. Coomassie staining of the proteins and densitometric scanning of the gels indicated that from SN655 and SN656, 8-8.5% of the total protein preparation was the processed NifH dimer, whereas for the geminiviral construct SN657, 27% of the total isolated protein was the processed NifH dimer. However, the isolated NifH dimer from the plants supplemented with Fe chelate exhibited a 3-4 fold decreased acetylene reduction activity compared to the same polypeptide isolated from plants that weren't supplemented with Fe chelate. This observation was in contrast to the reports from Jiang et al. (2021) and Baysal et al. (2022), where N. benthamiana and rice plants expressing H. thermophilus NifH were only active ‘as-isolated’ when supplemented with Fe during growth. This difference may be due to the N. benthamiana plants described herein being grown in a synthetic medium already supplemented with a comprehensive nutrient solution that included Fe, and therefore the additional Fe chelate supplementation may have had a negative effect on the overall health of the plants which may have compromised the Fe-S cluster loading onto the scar: NifH: linker: NifH fusion polypeptide.
Testing activity of NifH dimer polypeptides after co-expression with other Nif proteins In order to identify a possible minimal combination of proteins required to obtain an active scar: ifH: linker: ifH ‘as isolated’, the construct SN656 encoding a MTP- FAy5 l ::HA: NifH: linker(TS): NifH fusion polypeptide (SEQ ID NO: 101) was introduced into TV. benthamiana leaves along with SN360 encoding MTP::KoNifM, and either no additional genes (other than pl 9) or one or more genes encoding additional Nif fusion polypeptides. As described above, SN656 produced more soluble, processed NifH dimer protein than SN638 when co-expressed with MTP-NifM and so was the preferred vector for this experiment. Isolation of the processed protein by affinity chromatography under anaerobic conditions yielded on average 1.07 mg isolated protein/kg fresh leaf wt, with an average purity of 27% based on densitometry analysis of the Coomassie stained SDS PAGE gel. The isolated NifH dimer fusion polypeptide reduced acetylene to ethylene when combined in vitro with purified wild-type A. vinelandii NifD and NifK proteins. The amount of ethylene produced was 2.6% of the ethylene produced using wild-type NifH isolated and purified from A. vinelandii, producing ethylene at an average of 37 nmol/min/mg NifD + NifK compared to about 1500-1600 for the positive control using bacterially-produced NifH protein (Figure 19, n=3). Surprisingly to the inventors, the isolated polypeptide had been produced in the plant cells in the absence of NifS and NifU. This result demonstrated that at least some of the scar::HA::NifH::linker::NifH fusion polypeptide was activated, albeit at a relatively low level compared to wild-type bacterial NifH, by an endogenous mitochondrial [Fe4S4] cluster biosynthesis and delivery mechanism that provided the function in the plant mitochondria that otherwise would have been provided together by NifS and NifU. That is, at least some of the NifH dimer polypeptide produced in the plant was active for acetylene reduction ‘as isolated’.
Combinations with additional Nif and FdxN proteins were also tested. Coexpression of the MTP-NifH dimer construct from SN656 together with MTP-AvNifS and MTP-AvNifU from SL133 as well as the MTP-KoNifM did not increase the activity of the processed, dimeric NifH isolated from plant leaf mitochondria compared to when co-expressed with MTP-NifM alone (Figure 19).
The low level of activity of the ‘as isolated” NifH dimer polypeptide might have been due to the loss of Fe-S clusters during the isolation process, perhaps because of the presence of traces of oxygen, or because the NifH dimer polypeptide was only partially loaded with Fe-S clusters in the plant mitochondria in the first place. Attempts were therefore made to reconstitute isolated scar::NifH::linker(TS)::NifH polypeptide with [Fe4S4] clusters using purified, loaded AvNifU that was expressed and isolated from E. coli as described in Example 1. The NifU was purified in an active form as described in Example 1. This attempt did not increase acetylene reduction activity of the dimeric NifH protein in the assays with purified bacterial NifD and NifK (Figure 19). The positive control showed that the purified AvNifU was able to transfer its Fe-S clusters to apoNifH which had been previously stripped of its Fe-S clusters. The lack of increase in acetylene reduction for the isolated dimeric NifH protein was thought to be due to a presence of contaminants in the dimeric NifH preparation from plants that inhibited possible Fe-S cluster loading, or possibly the linker joining the two NifH units inhibited Fe-S cluster loading by the NifU protein.
It was concluded from these experiments that a soluble NifH dimer was expressed from an exogenous polynucleotide in plant cells and localised to the mitochondria, provided NifM polypeptide was co-expressed, to provide a processed fusion polypeptide with at least some acetylene reduction activity ‘as isolated’, i.e. not requiring addition of iron-sulfur clusters in vitro, improving on the results in rice leaf reported by Bay sal et al. (2022). The NifM polypeptide was preferably from the same species as the NifH sequence i.e. homologous to the NifH sequence or closely related thereto.
Metal content of isolated NifH dimer polypeptide
Based on its partial (albeit modest) activity, the scar::NifH::linker(TS)::NifH polypeptide that was isolated from N benthamiana leaves expressing SN656 and MTP: :KoNifM, with or without MTP: : AvNifS and MTP: : AvNifU would be expected to contain at least some Fe-S clusters. A sample of the isolated scar: :NifH: dinker(TS): :NifH is analysed by inductively coupled plasma mass spectrometry (ICPMS), which detects the presence of iron in the preparation.
Testing isolated NifH dimer polypeptide in 15N assays
The isolated NifH dimer protein was also tested in in vitro nitrogenase assays when combined with NifD-NifK using 15N2 gas, as follows. Reaction mixes were set up that contained scar::NifH::linker(TS)::NifH polypeptide isolated from N. benthamiana leaves co-expressing SN656 with MTP::KoNifM, either with or without MTP: AvNifS and MTP::AvNifU, and AvNifDK isolated from A. vinelandii DJ2102. These reactions were set up in the same manner as for the acetylene reduction assay described in Example 1, except that 1 ml of 15N2 gas that had been bubbled through H2O containing sodium dithionite to remove any oxygen was added to the reaction mix instead of acetylene gas. After 30 min with shaking at 30°C, the reactions were terminated by adding 25 pl of 1 M HC1. An aliquot of 100 pl of the resulting reaction mix was dried down in a tin capsule with inert chromosorb for 48 h in a desiccator at room temperature, weighed, and analyzed by using a SerCon hydra 20-20 isotope ratio mass spectrometer to measure the ratio of 15N to 14N. Delta-15N values of scar::NifH::linker(TS)::NifH polypeptide combined with AvNifDK were as low as those of the negative control comprising only AvNifDK at an average of 0.7 and 0.8%, respectively, whereas the positive control of AvNifH isolated from A. vinelandii DJ2102 and AvNifDK had on average 158%. That is, despite the ability to reduce acetylene to ethylene, this dimeric NifH when combined with bacterial AvNifD-NifK was unable to fix 15N2 gas (Figure 19).
The inability of the dimeric NifH to fix 15N gas could be due to the linker hindering effective electron donation to NifDK, as reduction of N2 gas requires more electrons than reduction of acetylene (Seefeldt et al., 2013). Alternately, perhaps more likely, contaminating substances in the isolated scar::NifH::linker(TS)::NifH polypeptide preparations were inhibiting electron donation to NifDK. General discussion
In these experiments, the inventors demonstrated function of a nitrogenase Fe protein in the form of a NifH-linker-NifH dimer ‘as isolated’ from plant mitochondria, in its ability to transfer electrons to NifDK to reduce acetylene. In order to show that function, it was necessary to isolate sufficient amounts of soluble NifH, which was achieved by (1) expressing two NifH molecules translationally fused with a linker in the plant mitochondria, and (2) co-expressed with NifM. These two conditions significantly increased NifH solubility when expressed in N. benthamiana mitochondria, which allowed isolation of sufficient amounts of NifH to conduct the hybrid ARA assay with A. vinelandii NifDK. Assuming that the NifH:NifDK ratio used in the assay at 40: 1 provided the maximum amount of ethylene production at 400 units, it was calculated that approximately 1.7 % of the NifH-linker-NifH dimer protein was loaded with Fe-S clusters compared to the A. vinelandii NifH that was used for the positive control.
It was observed that enhancing the dimerization of NifH translational fusion of the two NifH units significantly increased solubility of the dimer relative to the corresponding NifH monomer when expressed in plant cells and localised to the mitochondria, in the presence of NifM. NifM is a putative peptidyl-prolyl cis-trans isomerase (Gavini et al., 2006) and is reported to form a physical interaction with NifH as a heterodimer (Petrova et al., 2000 and 2002). While it is unlikely that the physical interaction between NifH and NifM assisted in Fe-S cluster insertion into NifH, the physical interaction of NifH with NifM is likely to have changed the conformation of NifH such that the apo-NifH dimer was more stable to be able to receive the Fe-S cluster and become an active holo-NifH. This hypothesis was supported by observations reported by Zou et al. (2000), showing that niJM mutants of Klebsiella were slower at accumulating NifH. They suggested that there was an isomerase other than NifM that might have fulfilled the same role, albeit less efficiently.
Jiang et al. (2021) reported that harvesting leaves expressing NifH after an extended dark period from 8 hrs to 16 hrs along with Fe addition to the soil the plants were growing in resulted in isolation of an active H. thermophilus NifH. The level of activity the present inventors saw as described herein with the KoNifH: linker: :NifH dimer, compared to the positive control, at 1.4% was similar to that observed by Jiang et al. (2021) with H. thermophilus NifH at 1.1%. Example 16. Synthetic dimers of AnfH and their production in plant cells
After the demonstration of the increased solubility and enzymatic function of artificial AnfH variants (Examples 8-10) and synthetic dimers ofNifH (Examples 13 and 14), the present inventors conceived of combining the two beneficial modifications. A series of constructs were designed and made, each to express a MTP::AnfHmod: linker: :AnfHmod fusion polypeptide. In this context, ‘AnfHmod’ refers to an AnfH sequence having one or more artificial amino acid substitutions relative to the wild-type AnfH. The first AnfHmod dimer tested was based on the A. vinelandii wild-type sequence but with the T200A, T228V and E234H substitutions that had been tested and shown active in the monomer AnfH context i.e. variant V7 (Example 8). Three constructs were made using the non-replicating and replicative vector expression systems described in Example 13. These constructs were SN775 having the e35S promoter and single polyadenylation region/transcription terminator (Tm), SN776 having the e35S promoter and the hybrid transcription terminator (TTm) and SN777 which was a replicative GV vector having e35S and Tm. Each construct encoded the same MTP- FAy51 ::Av AnfH- V7::linker(TS)::Av AnfH- V7 polypeptide (SEQ ID NO: 114). The FAy51 sequence was chosen as the MTP sequence since preliminary experiments to express AnfH dimers with the wild-type A. vinelandii sequence had shown production of slightly more soluble MPP-processed protein with MTP- FAy51 than MTP-CoxIV. The encoded polypeptide did not have a HA epitope in order to shorten the N-terminal extension, in particular after cleavage of the MTP by MPP, but it had a TS epitope incorporated into the 30-amino acid linker to allow for isolation and purification of the processed protein from plant cells.
The three vectors were introduced separately into N. benthamiana leaves from A. tumefaciens by infiltration, along with the vector encoding the pl9 silencing suppressor protein, and total protein and soluble protein extracts prepared four days after infiltration. The leaves were harvested 2-3 hr after lights had come on in the growth cabinet, and the extracts were prepared under aerobic conditions. Samples of the total and soluble protein extracts were analysed on SDS-PAGE and Western blotting to detect polypeptides having the TS epitope, as before. It was observed (Figure 20) that all three expression systems produced mostly, if not fully, soluble MPP-processed polypeptide, here designated as scar::AvAnfH-V7::linker(TS)::AvAnfH-V7 fusion polypeptide (SEQ ID NO: 115) based on the intensity of the protein band from the soluble extract being as intense as the band from the total protein extract. It was also clearly observed that the variant V7 dimer polypeptide was much more soluble when produced in the plant mitochondria compared to the corresponding dimer polypeptide having the wild-type AnfH sequence. That is, the three amino acid substitutions relative to the wild-type sequence were effective to increase the solubility in the AnfH dimer context as for the monomer context.
Two other AnfH variant monomers that showed an increase in solubility compared to the wild-type AnfH polypeptide, namely the V6 and V8 variants, were also tested for solubility in the same dimer format as the V7 variant. The genetic constructs for these were SN720 (V8) and SN721 (V6). The MPP-processed polypeptides produced from these constructs were also more soluble compared to the corresponding scar:: Av AnfH: :linker(TS):: Av AnfH polypeptide having the wild-type AnfH sequence produced from SN719 in N. benthamiana leaf. It was concluded that the increased solubility observed in the AnfH monomer context equally extended to the AnfH dimer context.
In a further experiment, the scar::AvAnfH-V7::linker(TS)::AvAnfH-V7 fusion polypeptide produced from SN776 was isolated from N. benthamiana leaves by affinity chromatography using the TS epitope, as before. The leaves had been co-infiltrated with SL133 expressing MTP-NifS and MTP-NifU polypeptides and the leaves were harvested after the last 16 hr of growth in the dark, and the isolation was performed under anaerobic conditions. Approximately 2 mg of isolated protein was obtained from 100 g of leaves (wet weight). The isolated protein exhibited a very intense, concentrated band on SDS- PAGE gels (Figure 21), which was estimated to be approximately 68% pure by densitometric scanning of the Coomassie-stained gel. This showed that plentiful amounts of the processed fusion polypeptide having the AnfH dimer with amino acid substitutions could be readily purified from plant cell mitochondria.
Functional analysis of the isolated AnfH dimer protein
The isolated AnfH-V7 dimer polypeptide was tested in acetylene reduction and 15N2 reduction assays using the methods described in Examples 1, 11 and 12. When combined with purified, bacterial NifD-NifK from A. vinelandii at either a 20: 1 or 40:1 ratio for AnfH:NifDK proteins, no ethylene was produced after 1 hr incubation with acetylene at 30°C (Figure 22). That is, the ‘as isolated’ AnfH-V7 dimer polypeptide was inactive in the acetylene reduction assay. However, when the isolated AnfH-V7 dimer polypeptide was incubated overnight with purified TS: :NifU protein that had been treated with Fe and cysteine, using the methods described in Examples 1 and 6, followed by testing in the acetylene reduction assay, ethylene was produced at approximately 600% and 260% of the amount compared to the positive control reaction at AnfH:NifDK protein ratios of 40: 1 and 20: 1, respectively (Figure 22). Those levels of activities were observed in the assays after 1 hr incubation at 30°C. The positive control reaction in those assays was the purified AnfH protein from A. vinelandii strain DJ2241 combined with the purified NifDK protein isolated from A. vinelandii strain DJ2102, as described in Example 1.
The greater acetylene reduction activity observed in the assay relative to the positive control may have been due to loss or oxidative damage of some of the Fe-S clusters from some of the purified, bacterial AnfH polypeptides. To test whether that is the case, the bacterial, purified AnfH protein is stripped of its Fe-S clusters by treatment with 5 mM (final concentration) of 2,2'-bipyridine as chelator of the Fe-S clusters, followed by passing through a PD-10 column to remove the remaining bipyridine, and then reconstituted with the purified, loaded TS::NifU protein. This re-constituted AnfH protein, which should be fully loaded with Fe-S clusters, is then used in the positive control reactions.
To test for the ability of the isolated AnfH-V7 dimer polypeptide to reduce nitrogen gas, either ‘as isolated’ or after re-loading with Fe-S clusters, the isolated polypeptide from SN776 was combined with purified, bacterial AnfDKG isolated from A. vinelandii strain DJ2241 at either a 20: 1 or 40: 1 ratio for AnfH: AnfDKG proteins. The 15N2 gas was scrubbed of oxygen prior to addition and was added at 33% of the reaction vial space. After incubation for 1 hr at 30°C, there was no 15N2 reduction detected in the reaction mix for the ‘as isolated’ polypeptide. In contrast, when the isolated AnfH-V7 dimer polypeptide was incubated overnight with purified TS: :NifU protein that had been treated with Fe and cysteine, using the methods described in Examples 1 and 6, followed by adding 15N2 gas, 15N2 reduction was detected in the reaction mix at approximately 120% and 250% of the amount compared to the positive control reaction (Figure 22). This was observed at AnfH: AnfDKG protein ratios of 20: 1 and 40: 1, respectively. As before, the positive control reaction in these N2 assays was the purified AnfH protein from A. vinelandii strain DJ2241 combined with the purified AnfDKG protein, also isolated from A. vinelandii strain DJ2241, as described in Examples 1 and 4.
Several conclusions were evident from these experiments: (i) the AnfH-V7 variant dimer polypeptide could be activated by addition of Fe-S clusters to high activity in both acetylene and N2 reduction assays, showing that it was capable of receiving Fe- S clusters from NifU, (ii) the lack of activity of the dimer protein ‘as isolated’ indicated that any Fe-S clusters bound to the polypeptide in the plant mitochondria were likely lost during the isolation process, or Fe-S clusters were not bound to the dimer polypeptide in the first place, (iii) the linker and the scar sequence at the N-terminus did not inhibit the loading of the dimer protein with Fe-S clusters i.e. did not prevent the interaction with the NifU protein, and (iv) the linker and the scar sequence were fully capable of providing electrons to both the NifDK and AnfDKG enzymes for reducing acetylene and N2 gas, respectively, and therefore did not did not prevent the interaction with the NifDK and AnfDKG enzymes.
To test whether the Fe-S clusters were lost from AnfH variant polypeptides during isolation and the purification process, the following experiment was done. An amount of the isolated AnfH variant polypeptide produced from SN709 was loaded with Fe-S clusters using purified NifU protein in vitro as above, making it enzymatically active. The reconstituted polypeptide was added to N. benthamiana leaf lysate from leaves which had not been infiltrated with genetic constructs and the polypeptide was then reisolated by affinity chromatography. This re-isolated AnfH variant polypeptide, which had a brown colour indicating it potentially still had bound Fe-S, was then tested for activity by acetylene reduction, but no detectable ethylene was produced. It was concluded that the isolation conditions were not conducive to retention of the Fe-S clusters on the AnfH polypeptide.
If the re-isolated AnfH variant polypeptide had retained its Fe-S cluster and therefore activity in that experiment, that would have indicated that it was not acquiring Fe-S clusters during its production in planta. However, it was apparent that the polypeptide was not retaining its Fe-S clusters during isolation, so it could not be concluded whether or not the AnfH variant polypeptides were acquiring Fe-S clusters in planta.
Further variants in the AnfH-linker-AnfH context
AnfH variants having other combinations of amino acid substitutions as described in Example 9 that are confirmed to have increased solubility are incorporated into the AnfH-linker-AnfH context, analogous to the construct SN776 (above). These are tested for increased solubility compared to the corresponding dimer having the wild-type AnfH sequence and those that have increased solubility are tested for acetylene reduction and N2 reduction using the same methods as for SN776. Variants that retain enzymatic function are selected.
Example 17. Testing combinations of Nif polypeptides in planta
Measuring the relative abundance of co-expressed soluble Nif/Anf polypeptides
To date, successful transfer of an entire nitrogenase pathway has only been demonstrated between prokaryotes. Thus far the smallest Nif set transferred to a non- diazotroph comprised eight Nif proteins and a HesA protein (Wang et al., 2013). However the nitrogenase activity was very low, although it could be increased by expressing further Nif proteins (Li et al., 2016). In E.coli, expression of eleven genes for expression of components of the iron-only nitrogenase system provided some nitrogenase function (Yang et al., 2014). This so-called “minimal set” of components for the iron-only system comprised AnfD, AnfG, AnfK and AnfH components that were specific for the iron-only system, and NifS, NifU, NifV, NifB, NifF and NifI components which were in common with the Mo- and V-nitrogenases. In view of the goal of assembling an iron-only nitrogenase system in plant mitochondria, the present inventors decided to extend the experiments described in Example 3 herein and express the iron- only nitrogenase components in plant cells with localisation to the mitochondria, making use of the improvements in the solubility and activity of modified AnfH polypeptides as described in Examples 8-10 and 16 herein.
Five different sets of genes (Table 8) were put together, each designed to express the full ‘minimal set’ of iron-only nitrogenase components, in order to compare several expression systems and to test and develop the detection methods using mass spectrometry. Each gene in each set encoded an Anf or Nif polypeptide, except for the NifS and NifU polypeptides, which had an N-terminal MTP-FAy51 sequence for mitochondrial localisation. Specifically, each set of polypeptides differed from the combinations described in Example 3 herein by (i) the AnfH sequence contained three amino acid substitutions T200A, T228V and G241R that were shown (Example 10) to enhance solubility of the polypeptide within plant mitochondria - this modified AnfH was selected before the experiments described in Example 10 had been completed showing the reduced enzyme activity of that polypeptide; and (ii) the NifS and NifU sequences were selected from A. vinelandii as these proteins were thought to be more likely compatible with the AnfH from A. vinelandii and had better solubility in plant mitochondria than for the corresponding K. oxytoca polypeptides (Okada et al., 2020). The NifV from A. vinelandii was used in each set as this protein was both soluble and functional in plant mitochondria for homocitrate production (W02021/068039). Further, the NifB from Methanothermobacter thermautotrophicus (MtNifB) was selected as it has been shown to be active in vitro for FeMo-co synthesis when isolated from plants and reconstituted with Fe-S clusters, molybdate, R-homocitrate and SAM (Buren et al., 2017b). Each set of encoded fusion polypeptides used the same AnfG sequence from vinelandii, a FdxN: :HA polypeptide based on the FdxN sequence from A. vinelandii, and the NifF and NifI sequences from K. oxytoca.
These gene sets were also intended to measure the expression and abundance of separate AnfD and AnfK fusion polypeptides and compare with a translationally linked AnfD: linker: :AnfK polypeptide when expressed in the context of the minimal set of iron-only nitrogenase components. Further, these gene sets were designed to compare the effect of enhanced expression systems, namely the use of the hybrid transcription terminator (TTm) and the replicative expression system (GV) as described in Example 13 herein, for selected genes to increase the abundance of certain Nif components that previously have been difficult to produce at substantial quantities in plant mitochondria and/or were desired to be expressed at high levels for nitrogenase activity, especially the AnfH and AnfD-AnfK components. The different combinations of genes and their encoded fusion polypeptides are listed in Table 8.
All of the genetic constructs were assembled by GoldenGate methods, as before, and mixtures of A. tumefaciens cells transformed with the constructs infiltrated into N. benthamiana leaves, together with the construct expressing the pl9 silencing suppressor, as before. Four days after co-infiltration of these 5 sets of gene combinations, soluble protein fractions were prepared as described above and subjected to Multiple Reaction Monitoring (MRM) analysis as described in Example 1. Briefly, the in-solution proteins were solubilised in UTC buffer (100 mM Tris pH 8.5, 8 M urea, 2 M thiourea, 4% CHAPS) containing 50 mM DTT and shaken for 50 min at room temperature. An 80 pg sample of each extract was applied to a 3 kDa exclusion filter to remove low molecular weight compounds, prior to reduction, alkylation and tryptic digestion using a standard FASP protocol. Sequencing grade porcine trypsin (Promega, Alexandria, Australia) was added to the samples at an amount of 1.6 pg in 200 pL of 50 mM ammonium bicarbonate with 1 mM CaCh and the mixtures incubated for 16 h at 37°C in a thermomixer at 300rpm. The extracted tryptic peptides were dried and resuspended in 50 pL of 0.1% formic acid. Initially, 5 pL of the tryptic peptides from each of the digests were injected into the 6600 TripleTOF mass spectrometer using an Eksigent microHPLC (85 min). Residual tryptic peptides were stored at -20°C for analysis using MRM. For MRM, the MS data quality was observed to be stable. Repeated injections of a standard (sp|B- gal Eco/z digest + iRT (Biognosysis iRT-Kit)) interspersed throughout the sample run showed that the MS was stable across the 17 peptides measured. The diagnostic peptide GDFQFNISR (QI m/z 542.26++, SEQ ID NO: 127) showed stable signal intensity of 2x10e6 across three injections of B-Gal+iRT, one at each of the start, middle and end of the sample batch, meeting specifications for good quality data acquisition.
Firstly, four tryptic peptides unique to pl9 were monitored and the peak areas integrated. The peak area for each of the four peptides measured across the five sets were 26.24, 36.45, 17.21, 19.34, respectively, thereby demonstrating a level consistent enough to allow for proper comparison between the five infiltrated gene sets. Next, specific peptides for the Anf and Nif fusion polypeptides were identified and quantitated by MRM across the different gene sets 1-5. It was observed that, apart from the scar::FdxN::HA polypeptide, all polypeptides were identified and quantitated in the soluble protein extracts for each gene set (Table 8), although abundances varied considerably. Notably, set 3 that included a replicative GV vector for expression of the MTP::AnfD::linker::AnfK fusion polypeptide exhibited a greater amount of peptides for not only AnfD and AnfK but also for AnfG. This may have indicated the potential of the MPP-processed AnfD: dinker: AnfK protein to interact with and stabilise the processed AnfG polypeptide within mitochondria. Set 3 also yielded a substantial level of the peptide SISTQVVR (SEQ ID NO:23) coming from unprocessed fusion polypeptides still having the MTP sequence, which suggested there was a substantial amount of unprocessed MTP: AnfD: : linker: AnfK polypeptide present after expression from a GV vector in set 3. This was consistent with other results for GV vectors shown by Western analysis (Example 13). Nevertheless, the level of the peptide ISTQVVR (SEQ ID NO:22) from set 3 was almost double the amount for the other sets. Since this peptide resulted from MPP-processing of the encoded polypeptides, this indicated that significantly greater amounts of scar: Anf or scar::Nif polypeptides, likely to be the scar: AnfD: dinker: AnfK polypeptide, were accumulating in the plant cell mitochondria for set 3. Interestingly, set 5 that expressed separate MTP-AnfD and MTP-AnfK polypeptides using the hybrid transcription terminator (TTm) expression system also yielded relatively greater amounts of both AnfD and AnfK peptides, although AnfH was less abundant in this set.
As the scar::FdxN::HA polypeptide was not detected in that experiment by the MRM mass spectrometry method, the methods were modified in an attempt to detect this protein. The tryptic peptides GGMALK (SEQ ID NO: 128), CTECDGDYAEK (SEQ ID NO: 129) and LAEAMR (SEQ ID NO: 130) that should have been detected from that polypeptide were investigated by Enhanced Production Ion (EPI) scanning to acquire full scan MS/MS spectra for the target MRM on the 6500 Qtrap. The MRM included scanning for modified forms of the peptides having an oxidised methionine residue or a pyroGlu residue, with a total of 78 transitions. This yielded putative peaks for the modified peptide with an oxidised Met residue (99% confidence) and the unmodified peptide (66% confidence). Scans were therefore made to detect this peptide in the tryptic digests of the plant extracts in an attempt to detect the FdxN polypeptide by this method.
An additional gene set, designated set 6, was prepared with some modifications to the previous gene sets 1-5, again for expression in N. benthamiana leaves. Set 6 included a gene to express a fusion polypeptide having the AnfH-V7 sequence, having the E234H substitution instead of the G241R substitution in variant V8, in view of the activity analyses described in Example 10. Set 6 also used the TTm expression system to express the NifF and NifI fusion polypeptides, in an attempt to increase their accumulation in the plant cell mitochondria. All polypeptides encoded by the genes of set 6 were found in the soluble fraction, indicating the AnfH-V7 variant could be coexpressed in the soluble fractions together with the so-called minimal set of Anf/Nif proteins.
The abundance of these polypeptides was also assessed after the infiltrated plants were enclosed in a IL airtight container for 24 hr, starting 5 days after infiltration. This was to determine if these proteins were still present in an enclosed environment that would be required to assay nitrogenase activity using 15N2 labelled gas. For this, leaf discs were sampled at 4 dpi and at 6 dpi, the latter after 24 hr enclosure in the airtight container which commenced at 5dpi. After protein extraction of the soluble fractions as previously, peptides were detected for all of the Nif/ Anf polypeptides within the sets at both 4 dpi and 6dpi post 24 hour enclosure. Most of the Anf/Nif polypeptides did not appreciably diminish in amount after 24 hr in the airtight container. It was concluded that enclosure of the plants in an airtight container for 24 hr did not prevent accumulation of the Nif and Anf proteins and should allow assays for nitrogenase activity using 15N2 labelled gas.
Another gene set is prepared including a construct to express a MTP::AnfH: linker: :AnfH fusion polypeptide having the three amino acid substitutions in the V7 sequence (Example 16) in both of the AnfH units, instead of SN754 in set 6. Together these results demonstrated that a set of genetic constructs encoding the ‘minimal set’ of Anf and Nif polypeptides could be expressed in plant cells and detected and quantitated within the mitochondria.
Table 7. Gene sets to express Anf and Nif fusion polypeptides.
Table 8. Abundance of specific peptides detected by MRM mass spectrometry after tryptic digestion of soluble protein extracts from N. benthamiana leaves infiltrated with genetic constructs for sets 1-5. The protein from which the peptide sequence was derived, used in the MRM detection, is indicated. Values for set 1 to 6 represent the
MRM integrated peak area/10,000.
Example 18. NifH variants with amino acid substitutions
Examples 8-10 and 17 herein demonstrated the increased solubility and enzymatic function of artificial AnfH variants as either monomers or dimers. The present inventors conceived that artificial amino acid substitutions to NifH proteins other than AnfH proteins might increase the solubility and function of those proteins as well. As a first step, the present inventors considered the 11 positions that were substituted in AnfH-V5 (Table 9) and the corresponding amino acids in wild-type NifHs from A. vinelandii (AvNifH), K. oxytoca (KoNifH) and Methanocaldococcus infernus (MiNifH, Genbank Accession No. WP 013099459). The amino acids at these positions in the wild-type sequences and the amino acid substitutions that correspond to the stabilising mutations in AnfH-V5 are shown in Table 9. It was noted that the three wild-type NifH sequences already had preferred amino acids at 5-7 of the eleven positions, whilst the remainder of the positions did not, pointing to amino acid substitutions that might provide increased stability and solubility. The effect of these candidate amino acid substitutions is assessed with Rosetta, as in Examples 8 and 9 above. Constructs to express AvNifH and KoNifH having one or more amino acid substitutions at these positions, predicted to lower AG and increase solubility, are prepared and expressed in plant leaves with mitochondrial localisation. The polypeptides in the plant mitochondria are assessed for increased solubility.
It was also noted that the MiNifH amino acid sequence was closer at 71% identity to the wild-type Av AnfH sequence than the 63% identity to the AvNifH sequence and so was more like an AnfH protein.
Next, the amino acid substitutions listed in Table 5 which were considered likely to provide some increased solubility to AnfH from A. vinelandii were considered at the corresponding positions in the AvNifH, KoNifH and MiNifH sequences as candidates for increasing solubility. This was based on the alignment of the amino acid sequences along the full length sequence of Av AnfH using the BLASTp program. Table 10 lists the corresponding substitutions in those sequences, also showing with parentheses where the preferred amino acid was already present in the wild-type sequence. These sequences are analysed by PROSS and Rosetta in the same manner as for AnfH (Examples 8 and 9). Table 9. Proposed amino acid substitutions in NifH polypeptides vinelandii (AvNifH), K. oxytoca (KoNifH) and Methanocaldococcus infernus (MiNifH), for the 11 positions modified in AvAnfH-V5. Subsitutions are shown with the wild-type amino acid before the position number and the new amino acid after the position number in AvNifH, KoNifH or MiNifH, using the single letter amino acid code. Amino acids and the position number in parentheses are positions where the AvNifH, KoNifH or MiNifH already has the preferred amino acid for lower AG. Table 10. Proposed amino acid substitutions in NifH polypeptides A. vinelandii (AvNifH), K. oxytoca (KoNifH) and Methanocaldococcus infernus (MiNifH), for the positions corresponding to those shown in Table 5 for AvAnfH. Subsitutions are shown with the wild-type amino acid before the position number and the new amino acid after the position number in AvNifH, KoNifH or MiNifH. Amino acids and the position number in parentheses are positions where the AvNifH, KoNifH or MiNifH already has the preferred amino acid as predicted for lower free energy and thereby increased solubility.
Jiang et al. (2021) tested 32 NifH variants, each based on a wild-type NifH sequence from one of a diverse range of natural diazotrophic prokaryotes, for solubility in N. benthamiana mitochondria when expressed in the plants. Each polypeptide was fused to an N-terminal MTP sequence and a TS epitope and was co-expressed with fusion polypeptides comprising NifM, NifS and NifU. Only 2 of the 32 NifH polypeptides consistently yielded soluble protein in the tobacco mitochondria, with a third NifH at a low level in one experiment, whereas almost all of the other NifH polypeptides that could be detected were essentially insoluble in the plant mitochondria. The two variants that yielded soluble NifH fusion polypeptides were derived from the wild-type sequences in Methanocaldococcus infernus (MiNifH) and Hydrogenobacter thermophilus (HtNifH), and the third one was from Methanothermobacter marburgensis (MmNifH). All three of those organisms were thermophilic organisms and could be expected to have more thermo-stable NifH proteins. In view of this, the present inventors examined the sequence alignment generated as described in Example 2 herein to compare NifH amino acid sequences from thermophilic organisms to NifH sequences from mesophilic or psychrophilic organisms. These NifH sequences were from 273 to 294 amino acids in length. That alignment had 300 amino acid positions, with gaps within sequences to provide the best alignment. In particular, most of the 137 highly conserved amino acid positions listed in Example 2 were excluded from the analysis, but the other less conserved positions were considered to identify preferred amino acids for NifH as candidates for increased thermostability and solubility. Certain positions were identified in the alignment where there was a clear difference between the most common amino acid in NifH sequences from mesophiles, the NifH proteins generally being insoluble in mitochondria when tested, and the preferred amino acid in the thermophilic NifH sequences. Examples of the plots of some segments of the sequences are shown in Figure 23. Clear differences were seen, for example, at positions 71, 75, 125, 153, 200 and 204 of the alignment, with reference to the consensus sequence. Table 11 lists the preferred amino acids at these positions with reference to the wild-type sequences for AvNifH, KoNifH or MiNifH, based on the preferred amino acid in the thermophilic NifHs. It was noted that the MiNifH sequence already had five of the six preferred amino acids at those positions, whereas the AvNifH and KoNifH had non-preferred amino acids at all six positions. The preferred amino acids at these positions were therefore candidate substitutions to be included. For example, the proposed substitutions for KoNifH were at positions 63, 67, 117, 143, 190 and 194 with reference to the wild-type KoNifH sequence (SEQ ID NO: 1), with the preferred substitutions being A63R, S67E, E117T, N143G, T190V and D194R. Constructs encoding mitochondrially targeted NifH fusion polypeptides, either in the NifH monomer or NifH-linker-NifH dimer form, comprising substitutions at these positions are made. These are tested by expression in N. benthamiana leaf mitochondria, extraction and analysis for soluble processed polypeptides, using the methods described above. Variants with increased solubility in the mitochondria are selected.
Table 11. Proposed amino acid substitutions in NifH polypeptides from A. vinelandii (AvNifH), K. oxytoca (KoNifH) and Methanocaldococcus infernus (MiNifH), for six positions identified in an alignment. Subsitutions are shown with the wild-type amino acid before the position number and the new amino acid after the position number in AvNifH, KoNifH or MiNifH, using the single letter amino acid code. Amino acids and the position number in parentheses are positions where the AvNifH, KoNifH or MiNifH already has the preferred amino acid.
Example 19. Optimising extraction conditions for NifH polypeptides from plant cells
As described in Example 16 herein, an AnfH-V7 dimer polypeptide that was isolated from plant cells after expression from the construct SN776 was inactive “as isolated” for reduction of either acetylene or 15N2 gas but could be activated by addition of Fe-S clusters when incubated with purified TS-NifU protein loaded with Fe-S clusters. When a purified, activated TS-AnfH-V7 monomer polypeptide produced from SN709 in plant cells was added to plant extracts and subjected to an extraction process and then reisolated, the resultant purified polypeptide was inactive for producing ethylene from acetylene. It was concluded (Example 16) that the isolation conditions were not conducive for the retention of the Fe-S clusters on the AnfH polypeptide. The present inventors therefore tested modifications of the extraction and isolation procedures in order to identify conditions which would allow retention of the Fe-S clusters on NifH polypeptides, as follows.
In order to optimise the extraction and isolation procedure for retention of the oxygen-sensitive Fe-S clusters, an active NifH polypeptide with a Twin Strep (TS) epitope was desired which could be purified in an active form, then added to leaf tissue (‘spiked in’) and subjected to the purification process. It was considered that the activity changes through this process would provide information on the damage to the Fe-S clusters caused by the purification process. Since NifH polypeptides do not express well in Azotobacter when fused to either Strep or Twin Strep (TS) epitopes (personal communication with Denis Dean), an E.coli expression system was used to produce sufficient quantities of active NifH polypeptide.
The purification process was initially tested using a TS::NifH fusion polypeptide where the NifH component was from Hydrogenobacter thermophilus (HtNifH). This NifH sequence was selected for this experiment since a MTP -HtNifH polypeptide was reported to be expressed in a soluble form in plant mitochondria, in contrast to many other NifH polypeptides that were essentially insoluble in plant cells (Jiang et al., 2021). The present inventors therefore expressed a TS::HtNifH polypeptide in E. coli from an Ecoflex vector (Moore et al., 2016) designated EL2-36, which had genes encoding not only the TS::HtNifH polypeptide but also the maturation proteins NifM, NifU and NifS, all from A. vinelandii. The TS : :HtNifH polypeptide was purified from the induced E. coli cells as described in Example 1 with the exception that 2 mM DTH replaced 2 mM TCEP as the reductant in the extraction buffer. The entire bacterial growth, induction and isolation process was carried out under strict anoxic conditions. The purified polypeptide was tested using the standard in vitro Acetylene Reduction Assay (ARA) when combined with purified, wild-type NifD-NifK from A. vinelandii and found to have a specific activity of approximately 400 nmol ethylene produced per milligram NifD-NifK added per min (nmol C2H4 mg NifDK'1 min'1). In a parallel experiment, the TS::HtNifH polypeptide was also shown to be active when produced in E. coli cells co-expressing NifM from the EcoFlex vector EL2-38, but without the NifS and NifU proteins.
About 1 mg of the TS::HtNifH polypeptide in a volume of 100 pL at a concentration of 10 mg/mL was added to 50 g of wild-type, non-infiltrated N. benthamiana leaf tissue and 75 mL lysis buffer containing 2 mM DTH. The extraction and purification process by affinity chromatography was carried out as described in Example 1. The TS::HtNifH polypeptide was eluted from the Strep-Tactin column with 50 mM biotin. The acetylene reduction activity of the eluted protein was measured to be approximately 100 nmol C2H4 mg NifDK'1 min'1, indicating that only about 25% of the activity was recovered through the purification process.
The inventors considered that the observed loss of activity during the purification process may have been due to exposure of the protein to residual oxygen trapped within the plant leaf tissues. Attempts were therefore made to minimise exposure to oxygen and its effects. Firstly, the concentration of DTH in the extraction buffer was increased from 2 mM to 5 mM. DTH is known to scrub oxygen and the higher concentrations of DTH would provide extra protection to any oxygen within the leaves. Secondly, an initial washing step of the leaf tissue was added whereby the leaf material was fully submerged in about 100 mL of wash buffer (Example 1) with 5 mM DTH and then placed into the COY airlock and subjected to 5 vacuum cycles. It was anticipated that passing the solution through these additional vacuum cycles would remove any oxygen trapped within the leaves. Furthermore, small quantities (~ 5 pL) of a commercial surfactant mixture, Silwet, were added to the wash buffer to reduce surface tension and assist oxygen removal. Following the vacuum cycle, the solution containing wash buffer with 5 mM DTH and Silwet was poured off and replaced with the standard extraction buffer containing 0.2 mM PMSF, 10 pM leupeptin and with DTH increased to 5 mM.
This modified procedure was then tested using TS::HtNifH, where the purified TS::HtNifH polypeptide was added to the final lysis buffer shortly before blending the plant leaf tissue. The extraction and purification process was then performed as described in Example 1. Using the revised conditions, the activity of the protein recovered from the process was found to be approximately 400 nmol C2H4 mg NifDK'1 min'1, indicating that the revised conditions allowed NifH polypeptides to be purified with full retention of the Fe-S clusters on the polypeptide.
The modified extraction and purification process was then applied to the TS::AnfH-V7 monomer polypeptide purified from E. coli after expression using the Ecoflex expression system. The E. coli cells contained an EL2-35 vector with genes encoding the TS::AnfH-V7 polypeptide having the T200A, T228A and E234H amino acid substitutions as well as genes encoding AvNifS and AvNifU polypeptides. Again, the entire bacterial growth, induction and isolation process was carried out under strict anoxic conditions. However, the purified TS::AnfH-V7 monomer polypeptide from this heterologous system yielded a polypeptide which had only 2.5% activity in the in vitro ARA assay compared to the wild type AnfH purified from A. vinelandii. Nevertheless, when ‘spiked in’ to the modified extraction and purification process described above for TS::HtNifH, the TS-AnfH-V7 monomer polypeptide retained its level of specific activity, as had been observed with the TS::HtNifH polypeptide. It was concluded that the modified extraction and purification procedure was suitable for retention of the Fe-S clusters and therefore activity of the NifH polypeptides, including the AnfH polypeptides.
At the time the experiment was done, the reason for this low level of 2.5% activity for the TS::AvAnfH-V7 polypeptide produced in E. coli was not clear.
Example 20. Developing a more rapid ARA assay to screen for active gene combinations
The present inventors considered that adding genes involved with Fe-S cluster maturation in diazotrophic bacteria might be able to assist with loading Fe-S clusters onto a NifH polypeptide expressed in plant cells, including soluble AnfH polypeptides. Given the high number of candidate genes that could be tested, a simpler and more rapid assay was desired to increase the screening throughput.
The activity of NifH polypeptides expressed in plants is typically reported as a specific activity defined as nmol C2H4 mg NifDK'1 min'1 in the ARA assay. In order to obtain maximum specific activities, a large excess of NifH polypeptide, usually 20 times or more, is added to low concentrations of NifD-NifK at about 0.1 - 0.2 pM. Specific activity can be further increased by using short incubation periods with acetylene, normally less than 30 min, before the enzymatic reaction is stopped by the addition of either strong base or acid. It is known from the literature, however, that the absolute amount of ethylene produced by a nitrogenase is not dependent on having a large excess of NifH over NifD-NifK. Tests conducted by the present inventors using NifH and NifD- NifK purified from A. vinelandii confirmed that ethylene production increased when a 1 : 1 ratio of NifH:NifD-NifK at 1 or 2 pM was used and that a similar amount of ethylene was produced when an excess of NifD-NifK was added at a ratio of 1 :2 of NifH:NifD- NifK compared to the 20: 1 ratio. It was concluded that less NifH polypeptide could be used in an ARA assay.
The present inventors therefore developed what is referred to as a ‘crude assay’ for NifH activity, including AnfH activity, wherein the concentration of NifH polypeptide purified from plants was not measured before the assay. However, the hybrid in vitro assay still included a standard amount of purified NifD-NifK, normally between 0.2 pM and 1 pM. Acetylene was added to the sample and the sample was incubated overnight. The following day the quantity of ethylene produced was measured using GC as described in Example 1. This modification had the advantage of allowing the assay to be carried out on the same day as purification of the NifH polypeptide, which avoided any losses caused by freezing and thawing the purified polypeptides and allowed a quick initial screen to be carried out, looking for gene combinations which result in improved ethylene production. It was concluded that this simpler, more rapid assay was effective for testing the activity of NifH polypeptides produced in plant cells.
Example 21. Stably transformed plants producing AnfH polypeptides targeted to mitochondria
Examples 8-10 herein describe the generation of AnfH variants based on the A. vinelandii AnfH sequence but having amino acid substitutions. The solubility of the AnfH variant polypeptides when expressed in plant cells and targeted to the mitochondria was greatly increased compared to the corresponding wild-type AnfH. Following this success, the present inventors next assessed whether the variant polypeptides could be expressed in stably transformed plants, and to what level. This experiment also tested whether co-expression of NifS and NifU fusion polypeptides in the transgenic plants might provide for insertion of Fe-S clusters into the AnfH polypeptides in the mitochondria. Relevant to this, prior publications reported failure to detect accumulation of NifS and NifU fusion polypeptides in transgenic rice, despite accumulation of their mRNAs (Bay sal et al. 2022; He et al. 2022).
To prepare stably transformed plants, two genetic constructs designated SL149 and SL151 were designed and made for stable plant transformation, both being binary vectors forHgro zctera/m-mediated transformation. The T-DNA of SL151 contained a gene encoding a MTP-CoxIV::TS::AnfH-V7 fusion polypeptide (SEQ ID NO:83, Example 8) and the T-DNA of SL149 contained that gene and additionally genes encoding MTP-CPN60::AvNifS (SEQ ID NO:89) and MTP-Su9::AvNifU (SEQ ID NO:90). Both vectors also contained a kanamycin selectable marker gene within the T- DNA region flanked by the LB and RB border sequences, to provide for selection of transgenic cells.
Transgenic N. benthamiana plants were generated using the method described in W02021/068039. Twenty primary transgenic (TO) plants were recovered for each construct. All of the TO plants were morphologically indistinguishable from wild-type plants and produced the same quantity of seed as the wild-type. The TO plants were assayed for expression of the MTP-CoxIV::TS::AnfH-V7 polypeptide and its MPP- processed product scar::TS::AnfH-V7 (SEQ ID NO:87) using western blotting as previously described for the TS epitope (Figure 24). The level of expression in the transgenic leaves was compared with that in transiently infiltrated N benthamiana leaves produced using the same constructs.
A band was observed in the western blots for the AnfH polypeptide that was consistent with the size of an MPP-processed polypeptide resulting from cleavage within the MTP sequence. A range of expression levels was observed in the transgenic leaves, as often happens in a set of transgenic events. Notably, however, similar or higher expression levels were observed in some of the independent transgenic plants compared to the transiently infiltrated leaves in comparable non-transgenic plants. This was confirmed by quantifying diagnostic tryptic peptides from the exogenous AnfH polypeptide in proteomic analysis. For example, leaves of the TO plant SL149-20 accumulated more peptides from the AnfH-V7 polypeptide than the transiently infiltrated control sample, indicating a higher level of MTP-CoxIV::TS::AnfH-V7 expression and accumulation of the scar:: TS::AnfH-V7 polypeptide.
Surprisingly in view of previous reports, the proteomic analysis of leaf samples transformed with the T-DNA of SL149, including the plant SL149-20, also identified high levels of peptides that were diagnostic for the MPP-processed AvNifS and AvNifU peptides. This experiment was therefore the first successful demonstration of NifS and NifU fusion polypeptide expression targeted to mitochondria in stable plants, contrary to the reports of Baysal et al. (2022) and He et al. (2022). The coding regions expressed in the SL149-transformed plants used different codon-optimisation and also used a different MTP sequence than the constructs of Baysal et al. (2022).
To assess stability of expression in these transgenic lines, T1 seed from several selected plants was sown and the resultant T1 plants grown and analysed for expression levels and for transgene copy number using a digital droplet PCR method. This analysis showed that the level of accumulation of the scar::TS::AvAnfH-V7 polypeptide correlated with the transgene copy number, for example plants that were homozygous (2 copies) of the transgene accumulated about twice as much of the processed polypeptide compared to heterozygous plants (1 copy) in the same line.
Finally the level of accumulation of the scar::TS::AvAnfH-V7 polypeptide in transgenic roots was assessed in several plants. Roots were considered by the inventors to be a potentially useful location for nitrogenase assembly given the reduced oxygen availability in the root tissues because of oxygen consumption there, together with the lack of chloroplasts that produce oxygen in the root cells, whilst having mitochondria. Several T1 plants were shown by western blotting to express the MTP- CoxIV::TS::AnfH-V7 polypeptide in the roots, including in transgenic line 149-20.
In conclusion, the production of transgenic plants with the transgene integrated into the nuclear genome that expressed high levels of a soluble AnfH variant was demonstrated. These plants will be assayed for AnfH activity in different tissues and will provide starting material for super-infiltration experiments of candidate nitrogenase and accessory proteins for functional improvement, for example for provision of Fe-S clusters to the NifH polypeptides targeted to mitochondria.
The present application claims priority from AU 2023902807 filed 31 August 2023, the entire contents of which are incorporated herein by reference.
It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
All publications discussed and/or referenced herein are incorporated herein in their entirety. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.
REFERENCES
Abe et al. (2000) Cell 100:551-560.
Abdullah et al. (1986) Biotechnology 4: 1087.
Allen et al. (1994) Crit. Rev. Biotechnol. 14:225-249.
Allen et al. (1995) J. Biol. Chem. 270:26890-26896.
Allen et al. (2017) Front. Plant Sci. 8:287. doi: 10.3389/fpls.2017.00287
Allen et al. (2020) PNAS 117:23165-23173.
Arnold et al. (1988) Tree physiology, 4:291-300.
Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.
Badalyan et al. (2019) Biochemistry 58:4590-4595.
Baltes et al . (2014) The Plant Cell 26 : 151 - 163. doi : 10.1105/tpc.113.119792.
Barahona et al. (2023) BioRxiv preprint doi: https://doi.Org/10. l 101/2021.12.06.471366.
Barker et al. (1983) Plant Molecular Biology, 2(6):335-350.
Baysal et al. (2022). Communications Biology 5: 1006.
Bevan et al. (1983) Nature 304: 184-187.
Boison et al. (2006) Arch. Microbiol. 186:367-376.
Boyd et al. (2011) Geobiology 9:221-232
Boyd and Peters (2013) Front.Microbiol. 4:201. doi: 10.3389/fmicb.2013.00201.
Brigle et al. (1987) J. Bacteriol. 169: 1547-1553.
Bruce (2001) Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 1541 :2- 21.
Buren et al. (2017a) Front Plant Sci 8: 1567.
Buren et al. (2017b) ACS Synthetic Biology 6: 1043-1055.
Buren and Rubio (2018) Fems Microbiol Lett 365:fnx274; doi: 10.1093/femsle/fnx274.
Buren et al. (2019) PNAS 116:25078-25086.
Buren et al. (2020) Chemical Reviews doi.org/10.1021/acs.chemrev.9b00489.
Cannon et al. (1988) Nucleic Acids Res. 16: 11379.
Capecchi (1980) Cell 22:479-488.
Carrie et al. (2010) Journal of Biological Chemistry 285:36138-36148.
Carter et al. (1980) J. Biol. Chem. 255:4213-4223.
Case et al. (2020) AMBER 2020.
Chen et al. (2013) Advanced Drug Delivery Reviews 65: 1357-1369.
Cheng et al. (2005) Biochemical and Biophysical Research Communications 329:966- 975.
Chacinska et al. (2009) Cell 138:628-644. Chalk et al. (2017) Soil Biology & Biochemistry 106:36-50.
Cheng et al. (1996) PNAS 93:3636-3641.
Chiu et al. (2001) Biochemistry 40:641-650.
Chretien et al. (2018) PLOS Biology, e2003992.
Christiansen et al. (1998) Biochemistry-Us 37: 12611-12623.
Clapp (1993) Clinics in Perinatology, 20(1): 155-168.
Clausen et al. (2000) PNAS 97:3856-3861.
Cotton (2009) J. Am. Chem. Soc. 131 :4558-4559.
Colgrave et al. (2019) Food and Chemical Toxicology 126:313-321.
Cui et al. (2013) PNAS 110, 2052-2057.
Curatti et al. (2006) PNAS 103:5297-5301.
Curatti and Rubio (2014) Plant Sci 225: 130-137.
Curiel et al. (1992) Am J Respir Cell Mol Biol, 6: 247-252.
Darshi et al. (2012) Journal of Biological Chemistry 287:39480-39491.
Davis et al. (1996) J. Bacteriol. 178: 1445-1450.
De'ath et al. (2012) PNAS 109: 17995-17999. de Bruijn (2015) In: Biological Nitrogen Fixation pp. 1087-1101. John Wiley & Sons, Inc.
Debruler et al. (2018) ACS Energy Letters 3:663-68.
Dos Santos et al. (2004) Chem Rev. 104:1159-73.
Dos Santos et al. (2012) BMC Genomics 13: 162.
Diamos and Mason (2018) Plant Biotech J. 16: 1971-1982.
Diamos and Mason (2019) Frontiers in Plant Science 9. doi: 10.3389/fpls.2018.01974.
Dilworth et al. (1988) Biochem. J. 249:745-751.
Dilworth et al. (1993) Biochem. J. 289:395-400.
Drummond (1985) Biochem. J. 232:891-896.
Dyer et al. (2003) J. Biol. Chem. 278:32150-32156.
Eady (1996) Chem. Rev. 96:3013-3030.
Egelkrout et al. (2012) Plant Sci 184:83-101. doi: 10.1016/j.plantsci.2011.12.005.
Eglitis et al. (1988) Advances in Experimental Medicine and Biology 241 : 19-27.
Engler et al. (2014) ACS Synthetic Biology 3(11):839-843.
Fani et al. (2000) J. Mol. Evol. 51 : 1-11.
Fay et al. (2015) PNAS 112: 14829-14833.
Fay et al. (2016) PNAS 2016:9504-9508.
Fu et al. (1994) Biochemistry 1994 33: 13455-63.
Fujimura et al. (1985) Plant Tissue Cult Lett. 2:74-75. Garfinkel et al. (1983) Cell 27: 143-153.
Gavini et al. (1998) Biochemical and Biophysical Research Communications. 244:498- 504.
Gavini et al. (2006) Journal of Bacteriology 188:6020-6025.
Geddes et al. (2015) Curr Opin Biotech 32:216-222.
Georgoulis et al. (2020) Extremophiles 24:293-306.
Glibert et al. (2014) Environ Res Lett 9:el05001; doi.org/10.1088/1748- 9326/9/10/105001.
Glaser and Deshi (1999) J Bioenerg Biomembr 31 :259-274.
Glick et al. (1992) Cell 69:809-822.
Goldenzweig et al. (2016) Molecular Cell 63:337-346.
Good and Beatty (2011) PLoS Biol 9, elOOl 124.
Graham et al. (1973) Virology 52 456-467.
Goodwin et al. (1998) Biochemistry-Us 37: 10420-10428.
Grant et al. (1995) J. Agric. Sci. 124: 437-445
Guo et al. (2016) Angewandete Chemie 55: 12764-12767
Harris et al. (2018) Biochemistry 57:701-710.
He et al. (2022). ACS Synth Biol, 11 : 3028-3036.
Hellinga (1997) PNAS 94: 19 10015-10017.
Hernandez et al. (2007) Mol. Microbiol. 63:177-192.
Hinchee et al. (1988) Nature Biotechnology 6:915-922.
Homer et al. (1993) J. Bacteriol. 175:4907-4910.
Homer et al. (1995) J. Biol. Chem. 270:24745-24752.
Hu et al. (2004) J. Biol. Chem. 279:54963-54971.
Hu et al. (2005) PNAS 102:3236-3241.
Hu et al. (2006) PNAS 103: 17119-17124.
Hu et al. (2008) Biochemistry 47:3973-3981.
Hu and Ribbe (2013) Bba-Bioenergetics 1827: 1112-1122.
Huang et al. (2009) Plant Physiology 150(3): 1272-1285.
Hwang et al. (1996) J. Mol. Evol. Nov; 43:536-540.
Igarashi and Seefeldt (2003) Crit. Rev. Biochem. Mol. Biol. 38:351-384.
Jaenicke (2000) Journal of Biotechnology 79: 193-203.
Jiang et al. (2021) Commun Biol 4:4. doi: 10.1038/s42003-020-01536-6
Jimenez- Vicente et al. (2014) FEBS Letters 588:512-516.
Jimenez-vicente et al. (2018) J. Biol. Chem. 293:9812-9823.
Jasniewski et al. (2018) Inorganics 6:25. Johnson et al. (2005) Biochem. Soc. Trans. 33:90-93.
Johnston et al. (2023) Plant Molecular Biology doi.org/10.1007/sl 1103-023-01363-3
Joshi (1987) Nucleic Acids Res. 15:6643-6653.
Jouanneau et al. (1995) Biochim. Biophys. Acta 1232:33-42.
Jumper et al. (2021) Nature 596:583-589.
Kay et al. (1987) Science 236: 1299-1302.
Kennedy and Dean (1992) Mol Gen Genet. 231 :494-498.
Kerscher et al. (1997) The Journal of Cell Biology 139: 1663-1675.
Kim et al. (1993) Biochemistry 32:7104-7115.
Kim and Rees (1992) Nature 360:553-560.
Kim and Rees (1994) Biochemistry 33:389-397.
Krissinel and Henrick (2007) Journal of Molecular Biology 372:774-797.
Lahiri et al. (2008) Biochem Biophys Res Commun 368:311-317.
Lawson and Smith (2002) Met Ions Biol Syst; 39:75-119.
Lee et al. (1998) Biochemical and Biophysical Research Communications 244: 2 498- 504.
Lee et al. (2000) J. Bacteriol. 182:7088-7091.
Lee et al. (2012) Plant Cell 24:5037-5057.
Liu et al. (2000) J. Mol. Biol. 295: 127
Lopez-Torrejon et al. (2016) Nature Communications 7: 11426.
Lopez-Torrejon et al. (2021) Microbial Biotechnology 14: 1073-1083.
Lu et al. (1993) Sci China B. 36: 11 1342-51.
Lyskov et al. (2013) PLoS ONE 8, e63906.
McRose et al. (2017) Frontiers Microbiol. 8:267 doi: 10.3389/fmicb.2017.00267.
MacLean et al. (2010) Bioinformatics 26:966-968.
Magliery (2015) Current opinion in structural biology 33: 161-168.
Marengo et al. (2021) Enzymatic and Chemical In Vitro Reconstitution of Iron- Sulfur Cluster Proteins, pp 79-95, Springer US.
Masukawa et al. (2007) Appl. Environ. Microbiol. 73:7562-7570.
Matsubara and Saeki (1992) Adv. Inorganic Chem. 38:223-280.
Mayer et al. (1999) J. Mol. Biol. 292:871-891.
Medberry et al. (1992) The Plant Cell 4: 2 185-192.
Medberry et al. (1993) Plant J. 1993 3: 4 619-26.
Merrick and Dixon (1984) Trends Biotechnol 2: 162-166.
Miller and Eady (1988) Biochem. J. 256:429-432.
Milton et al. (2017) Angewandte Chemie. 129:2724-2727. Modarres et al. (2016) RSC Advances 6.116. Royal Soc. Chem., pp. 115252- 115270.
Moore et al. (2016) ACS Synthetic Biology 5: 1059-1069.
Mueller et al. (2012) Nature 490:254-257.
Muhlenhoff et al. (2003) EMBO J. 22:4815-4825.
Murcha et al. (2004) J Mol Biol 344:443-454.
Niedz et al. (1995) Plant Cell Reports 14: 7 403-6.
Okada et al. (2020) Front Plant Sci 11 :552160. doi: 10.3389/fpls.2020.552160
Oldroyd and Dixon (2014) Curr Opin Biotechnol 26:19-24.
Olson et al. (2000) Biochemistry; 39: 16213-16219.
Onuchic et al. (1997) Annual review of physical chemistry 48:545-600.
Ouzounis et al. (1994) Trends Biochem. Sci. 19: 199-200.
Ow et al. (1986) Science 234:856-859.
Park et al. (2016) J. Chem. Theory Comput. 2016:6201-621.
Paul and Merrick (1987) Eur. J. Biochem. 170:259-265.
Paustian et al. (1990) Biochemistry 29:3515-3522.
Paya-Tormo et al. (2022) Scientific Reports 12: 10367: 1-12.
Peccoud et al. (2011) PLoS ONE 6:el6765.
Perez-Gonzalez et al. (2021) mBio 12.e01568-21.
Petrova et al. (2000) Biochem. Biophys. Res. Commun. 270:863-867.
Pfanner and Geissler (2001) Nat. Rev. Mol. Cell Biol. 2:339-349.
Porebski and Buckle (2016) Protein Eng Des Sei 29:245-251.
Prasher et al. (1985) Trends in Genetics 11 : 8 320-3.
Pratte et al. (2006) J. Bacteriol. 188:5806-5811.
Riedel et al. (1995) Eur. J. Biochem. 231 :742-746.
Robson and Postgate (1980) Annual Review of Microbiology 34: 183-207.
Robson et al. (1986) Nature, 322:388-390.
Robson et al. (1989) EMBO J. 8: 1217-1224.
Rockstrom et al. (2009) Nature 461 :472-475.
Rodriguez-Quinones et al. (1993) J. Bacteriol. 175:2926-2935.
Roise et al. (1986) The EMBO Journal 5: 1327-1334.
Roise and Schatz (1988) J.Biol.Chem. 263:4509-4511.
Rubio et al. (2002) J. Biol. Chem. 277: 14299-14305.
Rubio et al. (2004) J Biol Chem 279: 19739-19746.
Rubio and Ludden (2005) J. Bacteriol. 187:405-414.
Rubio and Ludden (2008) Annu Rev Microbiol 62:93-111.
Sadqi et al. (2003) PNAS 100: 12117-12122. Salomon et al. (1984) EMBO 3: 1 141 - 146.
Santi et al. (2013) Ann Bot 111 :743-767.
Schindelin et al. (1997) Nature 387:370-376.
Schleiff and Soil (2000) Planta 211 :449-456.
Schlesier et al. (2016) J Am Chem Soc 138:239-247.
Schmidt et al. (2023) BioRxiv preprint doi: https://doi.org/10.1101/2023.05.02.539077
Schmitz et al. (2001) FEMS Microbiol Lett. 195:97-102.
Seefeldt et al. (2013) Biochim Biophys Acta 1827: 1102-1111.
Seefeldt et al. (2009) Annu Rev Biochem 78:701-722.
Shah et al. (1999) J. Bacteriol. 181 :2797-2801.
Siddavattam et al. (1993) Mol. Gen. Genet. 239:435-440.
Sippel and Einsle, (2017) Nature Chemical Biology, 13:956. doi : 10.1038/nchembio.2428
Sirrenberg et al. (1996) Nature 384: 6609 582-5.
Smil (2002) Ambio 31 : 126-131.
Smith et al. (2005) Annu. Rev. Biochem. 74:247-281.
Smith et al. (2005b) Biochemistry 44: 12955-12969.
Stalker et al. (1988) J. Biol. Chem. 263(13):6310-6314.
Staples et al. (2007) J. Bacteriol. 189:7392-7398.
Strandberg and Wilson (1968) J Microbiol 14:25-31.
Suh et al. (2003) Journal of Biological Chemistry 278:5353-5360.
Sutton et al. (2008) Environ Pollut 156:583-604.
Temme et al. (2012) PNAS 109:7085-7090.
Tezcan et al. (2005) Science 309: 1377-1380.
Thiel et al. (1995) PNAS 92: 9358-9362.
Thiel et al. (1997) J. Bacteriol. 179:5222-5225.
Thillet et al. (1988) J. Biol. Chem 263: 12500-12508
Toriyama et al. (1986) Theor. Appl. Genet. 73: 16-19
Tmcik et al. (2022) J Inorganic Biochem. 227: 111690.
Varadi et al. (2022) Nucl Acid Res. 50 (DI), p.D439-D444.
Varadi and Velankar (2022) Proteomics 2022, 2200128. von Heijne (1986) EMBO J. 5: 1335-1342.
Wang et al. (2013) PLoS Genet 9, el003865.
Wagner et al. (1992) PNAS 89:6099-6103
Waite et al. (2021) Front Microbiol. 12:718487.
Wang and Roberts (2018) International journal of pharmaceutics 550:251-268. Waterhouse et al. (2018) Nucleic Acids Res. 46(W1), W296-W303.
Weber et al. (2011) PloS one. 6(2), pp.el6765.
Wiig et al. (2011) PNAS 108: 8623-8627.
Wilding et al. (2019) Biochemical Society Transactions 47:701-711. Wisniewski et al. (2009) Nature Methods 6:359-360.
Wood et al. (2009) Plant Biotechnol J. 7:914-924.
Wood and Kennedy (2001) Analytical Biochem. 296: 147-149.
Yang et al. (2014) PNAS 11 LE3718-E3725.
Yang et al. (2017) PNAS 114:E2460-E2465. Yang et al. (2018) PNAS doi/10.1073/pnas.1804992115
Yates (1972) FEBS Lett 27:63-67.
Yuvaniyama et al. (2000) PNAS 97:599-604.
Zhang et al. (2009) Progress in Natural Science 19: 1197-1200.
Zhang and Glaser (2002) Trends Plant Sci 7: 14-21. Zheng et al. (1994) Biochemistry of Vitamin B6 and PQQ. Advances in Life Sciences. Birkhauser Basel. 211-215.
Zheng et al. (1997) J. Bacteriol. 179:5963-5966.

Claims

1. A plant cell comprising a modified NifH polypeptide, wherein the modified NifH polypeptide comprises at least one amino acid substitution when compared to a corresponding wild-type NifH polypeptide, and wherein the modified NifH polypeptide is more soluble in mitochondria of the cell than the corresponding wild-type NifH polypeptide.
2. The plant cell of claim 1, wherein the at least one amino acid substitution is at an amino acid position selected from (i) the group consisting of amino acid positions: 2, 5, 7, 19, 23, 24, 26 to 35, 45, 48, 49, 51, 53, 54, 56 to 59, 61, 62, 64 to 74, 76 to 78, 80 to 84, 102, 105, 107, 111 to 114, 116 to 118, 121 to 124, 139, 145, 147, 149, 158, 165, 166, 168, 169, 171, 179, 182, 183, 188, 191, 193 to 197, 200 to 203, 205 to 211, 214, 216, 219, 223 to 226, 228 to 235, 237, 238, 241, 242, 244 to 246, 248, 249, 251 to 253, 257, 259 to 264, 266 to 271 and 273 to 275 with reference to SEQ ID NO:37, or at a corresponding amino acid position in the modified NifH polypeptide when its sequence is aligned with SEQ ID NO:37, and/or (ii) the group consisting of amino acid positions:
3. 6, 8, 20, 24, 25, 27 to 35, 45, 48, 49, 51, 53, 54, 56 to 59, 61, 62, 64 to 75, 77 to 79, 81 to 85, 103, 106, 108, 112 to 115, 117 to 119, 122 to 125, 140, 146, 148, 150, 159, 166, 167, 169, 170, 172, 180, 183, 184, 189, 192, 194 to 198, 201 to 204, 206 to 212, 215, 217, 220, 224 to 227, 229 to 236, 238, 239, 242, 243, 245 to 247, 249, 250, 252 to 254, 258, 260 to 265, 267 to 272 and 274 to 276 with reference to SEQ ID NO:39, or at a corresponding amino acid position in the modified NifH polypeptide when its sequence is aligned with SEQ ID NO:39.
3. A plant cell comprising a modified NifH polypeptide, wherein the modified NifH polypeptide comprises at least one amino acid substitution when compared to a corresponding wild-type NifH polypeptide, and wherein the at least one amino acid substitution is at an amino acid position selected from (i) the group consisting of amino acid positions: 2, 5, 7, 19, 23, 24, 26 to 35, 45, 48, 49, 51, 53, 54, 56 to 59, 61, 62, 64 to 74, 76 to 78, 80 to 84, 102, 105, 107, 111 to 114, 116 to 118, 121 to 124, 139, 145, 147, 149, 158, 165, 166, 168, 169, 171, 179, 182, 183, 188, 191, 193 to 197, 200 to 203, 205 to 211, 214, 216, 219, 223 to 226, 228 to 235, 237, 238, 241, 242, 244 to 246, 248, 249, 251 to 253, 257, 259 to 264, 266 to 271 and 273 to 275 with reference to SEQ ID NO:37, or at a corresponding amino acid position in the modified NifH polypeptide when its sequence is aligned with SEQ ID NO:37, and/or (ii) the group consisting of amino acid positions: 3, 6, 8, 20, 24, 25, 27 to 35, 45, 48, 49, 51, 53, 54, 56 to 59, 61, 62, 64 to 75, 77 to 79, 81 to 85, 103, 106, 108, 112 to 115, 117 to 119, 122 to 125, 140, 146, 148, 150, 159, 166, 167, 169, 170, 172, 180, 183, 184, 189, 192, 194 to 198, 201 to 204, 206 to 212, 215, 217, 220, 224 to 227, 229 to 236, 238, 239, 242, 243, 245 to 247, 249, 250, 252 to 254, 258, 260 to 265, 267 to 272 and 274 to 276 with reference to SEQ ID NO:39, or at a corresponding amino acid position in the modified NifH polypeptide when its sequence is aligned with SEQ ID NO:39.
4. The plant cell of claim 3, wherein the modified NifH polypeptide is more soluble in mitochondria of the cell than the corresponding wild-type NifH polypeptide, preferably wherein: a) at least 15%, at least 20%, at least 35%, at least 40%, at least 45%, at least 50%, or 15% to 90%, 15% to 80%, 15% to 70% or 15% to 60%, of the modified NifH polypeptide in mitochondria of the cell is soluble; and/or b) at least 2-fold, preferably at least 3 -fold, at least 4-fold or at least 5-fold, or between 2-fold and 10-fold, more of the NifH polypeptide in mitochondria of the cell is soluble when compared to the corresponding wild-type NifH polypeptide.
5. The plant cell according to any one of claims 1 to 4, wherein: a) the modified NifH polypeptide has a lower free energy than the wild-type NifH polypeptide, and/or wherein the amino acid substitution reduces the free energy of the modified NifH polypeptide relative to the wild-type NifH polypeptide, preferably wherein the modified NifH polypeptide with the substituted amino acid has a free energy which is reduced by at least 0.5, at least 1.0, at least 1.5, at least 2, at least 3, at least 4, at least 5, or between 2 and 6 units relative to a corresponding NifH polypeptide which is identical in amino acid sequence to the modified NifH polypeptide except for the substituted amino acid; and/or b) the modified NifH polypeptide comprises at least one, preferably at least two or at least three, amino acid substitution when compared to the corresponding wild-type NifH polypeptide, wherein each substituted amino acid reduces the free energy of the modified NifH polypeptide by at least 0.5, at least 1.0, at least 1.5, at least 2, at least 3, at least 4, at least 5 units, or between 2 and 6 units, and/or wherein the amino acid substitution(s), in sum, reduce the free energy of the modified NifH polypeptide by at least 4.0, at least 5.0, at least 6.0, at least 7.0, at least 8.0, at least 9.0, at least 10.0, at least 12.0 units, or between 4.0 and 15.0, between 4.0 and 13.0, or between 4.0 and 12.0 units.
6. The plant cell according to any one of claims 1 to 5, wherein: a) the modified NifH polypeptide comprises at least one amino acid substitution, or two or three amino acid substitutions, or four or more amino acid substitutions, selected from the group of amino acid substitutions listed in one or more of Tables 4, 5, 9, 10 or 11, or corresponding amino acid substitutions when the modified NifH polypeptide sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39; and/or b) the modified NifH polypeptide comprises at least one amino acid substitution, or preferably two or three amino acid substitutions, or four or more amino acid substitutions, at amino acid position(s) selected from the group consisting of amino acid positions 69, 168, 200, 201, 224, 228, 234, 241, 252 and 263 with reference to SEQ ID NO: 37, or at a corresponding amino acid position in the modified NifH polypeptide when its sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39, optionally wherein the at least one amino acid substitution, or preferably two or three amino acid substitutions, or four or more amino acid substitutions, are selected from the group consisting of 69N, 1681, 200A, 201K, 224R, either 2281 or 228V, either 234H or 234C, either 241R or 241A, 252M and 263E, wherein the amino acid positions correspond to the amino acid sequence provided as SEQ ID NO: 37, or at a corresponding amino acid position in the modified NifH polypeptide when its sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO: 39; and/or c) the at least one amino acid substitution, or preferably two or three amino acid substitutions, or four or more amino acid substitutions, are selected from the group consisting of positions corresponding to amino acids 69, 168, 200, 201, 224, 228, 234, 252 and 263 of SEQ ID NO: 37, or at a corresponding amino acid position in the modified NifH polypeptide when its sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39, optionally wherein the at least one amino acid substitution, or two or three amino acid substitutions, or four or more amino acid substitutions, are selected from the group consisting of 69N, 1681, 200A, 201K, 224R, either 2281 or 228V, either 234H or 234C, 252M and 263E, wherein the amino acid positions correspond to the amino acid sequence provided as SEQ ID NO: 37, or at a corresponding amino acid position in the modified NifH polypeptide when its sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39; and/or d) the modified NifH polypeptide has one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 1 to 20, 1 to 15, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 2 to 20, 2 to 15, 2 to 11, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 or 3, 3 to 20, 3 to 15, 3 to 11, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 or 4, preferably 1 to 3, 1 to 4, or 1 to 5, more preferably 2 to 4 or 2 to 5, most preferably 3 to 5, amino acid substitution(s) when compared to the corresponding wild-type NifH polypeptide.
7. The plant cell according to any one of claims 1 to 6, wherein the modified NifH polypeptide comprises at least one amino acid substitution which is i) 228V, ii) 2281, iii) 200A, or iv) 234H, or at least two amino acid substitutions which are v) 200A and 228V, vi) 200A and 2281, vii) 228V and 234H, viii) 2281 and 234H, ix) 200A, 228V and 234H, x) 200A, 2281 and 234H, xi) 200A, 228V or 2281, 234H and 241R, xii) 1681, 200A, 2281 or 228V and 234H, xiii) 69N, 1681, 200A, 228V or 2281, 234H, 252M and 263E, xiv) 69N, 1681, 200A, 20 IK, 228V or 2281, 234H, 252M and 263E, xv) 69N, 1681, 200A, 20 IK, 224R, 2281 or 228V, 234H, 252M and 263E, xvi) 1681, 200A, 2281 or 228V, 234H and 241R, xvii) 69N, 1681, 200A, 228V or 2281, 234H, 241R, 252M and 263E, xviii) 69N, 1681, 200A, 20 IK, 228V or 2281, 234H, 241R, 252M and 263E, xix) 69N, 1681, 200A, 20 IK, 224R, 2281 or 228V, 234H, 241R, 252M and 263E, xx) 112L, 200A, 228V or 2281, 234H and 241R, xxi) 112L, 1681, 200A, 2281 or 228V, 234H and 241R, xxii) 69N, 112L, 1681, 200A, 228V or 2281, 234H, 241R, 252M and 263E, xxiii) 69N, 112L, 1681, 200A, 201K, 228V or 2281, 234H, 241R, 252M and 263E, or xxiv) 69N, 112L, 1681, 200A, 201K, 224R, 2281 or 228V, 234H, 241R, 252M and 263E, with reference to SEQ ID NO:37, or the same amino acid substitution(s) at a corresponding amino acid position(s) when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
8. The plant cell of claim 7, wherein the modified NifH polypeptide comprises amino acids 200A, 228V and 234H with reference to SEQ ID NO:37, or the same amino acids at the corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO:39.
9. The plant cell according to any one of claims 1 to 8, wherein: a) the modified NifH polypeptide comprises one or more or all of the following motifs: YGKGGIGKSTTXQN (SEQ ID NO:61), IXGCDPKAD (SEQ ID NO: 62), CXESGGPEPGVGCAGRG (SEQ ID NO:63), DVLGDVVCGGFAMP (SEQ ID NO:43), VXSGEMMAXYAANNI (SEQ ID NO:64), and CNSRXXD (motif VII, SEQ ID NO:65), preferably at least DVLGDVVCGGFAMP (SEQ ID NO:43), where each X independently represents any amino acid; or b) the modified NifH polypeptide comprises one or more or all of the following motifs: YGKGGIGKSTTXQNT (SEQ ID NO:40), IHGCDPKAD (SEQ ID NO:41), CVESGGPEPGVGCAGRG (SEQ ID NO:42), DVLGDVVCGGFAMP (SEQ ID NO:43), VASGEMMAXYAANNI (SEQ ID NO:44), QSGVR (SEQ ID NO:45) and CNSRXVD (SEQ ID NO:46), preferably at least DVLGDVVCGGFAMP (SEQ ID NO:43), where each X independently represents any amino acid.
10. The plant cell according to any one of claims 1 to 9, wherein: a) the modified NifH polypeptide has 12, 13, 14, 15 or all of following amino acids: 4K, 22T, 37H, 52G, 60D, 63R, 108L, 109M, 142G, 151A, 174Q, 189V, 198E, 199F, 222F and 2471, with reference to SEQ ID NO: 37, or the same amino acids at the corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO: 39; and/or b) the modified NifH polypeptide has 130, 131, 132, 133, 134, 135, 136 or all 137 of the following amino acids: 3R, 4K, 6A, 8Y, 9G, 10K, 11G, 12G, 131, 14G, 15K, 16S, 17T, 18T, 20Q, 21N, 22T, 25A, 361, 37H, 38G, 39C, 40D, 41P, 42K, 43A, 44D, 46T, 47R, 50L, 52G, 55Q, 60D, 63R, 75V, 79G, 85C, 86V, 87E, 88S, 89G, 90G, 91P, 92E, 93P, 94G, 95V, 96G, 97C, 98A, 99G, 100R, 101G, 1031, 104T, 1061, 108L, 109M, 110E, 115Y, 119L, 120D, 125D, 126V, 127L, 128G, 129D, 130 V, 131V, 132C, 133G, 134G,
135F, 136A, 137M, 138P, 140R, 142G, 143K, 144A, 146E, 148Y, 150V, 151A, 152S,
153G, 154E, 155M, 156M, 157A, 159Y, 160A, 161A, 162N, 163N, 1641, 167G, 170K, 172A, 174Q, 175S, 176G, 177 V, 178R, 180G, 181G, 184C, 185N, 186S, 187R, 189 V,
190D, 192E, 198E, 199F, 204G, 212P, 213R, 215N, 217V, 218Q, 220A, 221E, 222F, 227V, 236Q, 239E, 240Y, 243L, 2471, 250N, 254V, 2551, 256P, 258P, 265E and 272G, with reference to SEQ ID NO:37, or the same amino acids at the corresponding amino acid positions when the modified NifH sequence is aligned with SEQ ID NO:37 and/or SEQ ID NO: 39; and/or c) the modified NifH polypeptide has an amino acid sequence which is at least 60% identical, preferably at least 70% identical or at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to the amino acid sequence provided as SEQ ID NO:37 and/or SEQ ID NO:39.
11. The plant cell according to any one of claims 1 to 10, wherein the modified NifH polypeptide is a modified AnfH polypeptide, optionally wherein the modified AnfH polypeptide has an amino acid sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to the amino acid sequence provided as SEQ ID NO: 37, preferably wherein the modified NifH polypeptide comprises at least amino acids 2-275 of the amino acid sequence provided as SEQ ID NO:78, or comprises SEQ ID NO:78.
12. The plant cell according to any one of claims 1 to 11, wherein a) the modified NifH polypeptide comprises an Fe-S cluster; and/or b) the modified NifH polypeptide is a cleavage product of a NifH fusion polypeptide which comprises a mitochondrial targeting peptide (MTP) translationally fused to the modified NifH polypeptide, wherein the MTP is preferably translationally fused at the N-terminus of the modified NifH polypeptide, wherein the modified NifH polypeptide is produced in the plant cell by protease cleavage of the NifH fusion polypeptide within or immediately adjacent to the MTP, optionally wherein the NifH fusion polypeptide is cleaved within the MTP by mitochondrial processing protease (MPP) to produce the modified NifH polypeptide, wherein the modified NifH polypeptide comprises either (i) at its N-terminal end, a C-terminal peptide from the MTP (scar peptide), or (ii) does not comprise a C-terminal peptide from the MTP; and/or c) the modified NifH polypeptide is capable, in combination with NifD and NifK polypeptides, or in combination with AnfD, AnfK and AnfG polypeptides, of reducing
(i) N2 gas to produce ammonia and/or (ii) acetylene to ethylene, preferably both (i) and
(ii), preferably wherein the modified NifH polypeptide is a modified AnfH polypeptide; and/or d) the plant cell further comprises exogenous polynucleotides encoding (a) a NifM polypeptide, a NifS polypeptide, a NifU polypeptide and either (i) a NifD and a NifK polypeptide or (ii) a NifD-NifK fusion polypeptide, or (b) a NifS polypeptide, a NifU polypeptide, and AnfG polypeptide and either (iii) a AnfD and a AnfK polypeptide, or (iv) a AnfD-AnfK fusion polypeptide.
13. The plant cell according to any one of claims 1 to 12, wherein the modified NifH polypeptide comprises two NifH polypeptides which are covalently linked by an oligopeptide linker in the N-terminal to C-terminal order NifH: : linker ::NifH, preferably two modified AnfH polypeptides which are covalently linked by an oligopeptide linker in the N-terminal to C-terminal order AnfH: linker: AnfH, optionally further comprising at its N-terminal end, a C-terminal peptide from a mitochondrial targeting peptide (MTP), optionally wherein the linker has a length of 10-50 residues, preferably 16-50 residues in length or 20-35 residues in length, more preferably about 25 or about 30 residues in length, or most preferably is 25 or 30 residues in length.
14. A plant cell comprising a NifH fusion polypeptide which comprises two NifH polypeptides, preferably two AnfH polypeptides, which are covalently linked by an oligopeptide linker in the N-terminal to C-terminal order NifH: : linker ::NifH, preferably in the order AnfH: linker: AnfH, optionally further comprising at its N-terminal end, a C-terminal peptide from a mitochondrial targeting peptide (MTP).
15. The plant cell of claim 14, wherein a) the two NifH polypeptides, preferably the two AnfH polypeptides, are the same or where they differ only by the presence of a methionine at amino acid position 1 of one of the NifH polypeptides; and/or b) the NifH fusion polypeptide, preferably the AnfH fusion polypeptide, is more soluble in mitochondria of the cell than a corresponding NifH or AnfH polypeptide which has only a single NifH or AnfH polypeptide; and/or c) the two NifH polypeptides, preferably the two AnfH polypeptides, are each at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical to an amino acid sequence provided as SEQ ID NO:37 and/or SEQ ID NO: 39; and/or d) the NifH fusion polypeptide is a cleavage product of an encoded polypeptide which comprises a mitochondrial targeting peptide (MTP) translationally fused to the NifH: linker: :NifH polypeptide, wherein the MTP is preferably translationally fused at the N-terminus of a NifH polypeptide, wherein the NifH fusion polypeptide is produced in the plant cell by protease cleavage of the encoded polypeptide within or immediately adjacent to the MTP, wherein NifH fusion polypeptide comprises either (i) at its N- terminal end, a C-terminal peptide from the MTP, or (ii) does not comprise a C-terminal peptide from the MTP.
16. The plant cell according to any one of claims 1 to 15, wherein the modified NifH polypeptide or the NifH fusion polypeptide is a cleavage product of a polypeptide encoded by an exogenous polynucleotide in the plant cell, wherein the encoded polypeptide comprises a mitochondrial targeting peptide (MTP) translationally fused to a modified NifH polypeptide or the NifH: dinker: :NifH polypeptide, wherein the MTP is preferably translationally fused at the N-terminus of the modified NifH polypeptide or the NifH::linker::NifH polypeptide, wherein the modified NifH polypeptide or the NifH fusion polypeptide is produced in the plant cell by protease cleavage of the encoded polypeptide within or immediately adjacent to the MTP, optionally wherein the exogenous polypeptide is integrated into the genome, preferably into the nuclear genome, of the plant cell, and/or wherein the modified NifH polypeptide is a modified AnfH polypeptide and/or the NifH fusion polypeptide is an AnfH fusion polypeptide.
17. A modified NifH polypeptide as defined in any one of claims 1 to 13, a NifH fusion polypeptide as defined in claim 14 or claim 15, or an encoded polypeptide as defined in claim 15 or claim 16.
18. The encoded polypeptide of claim 17, wherein the MTP is translationally fused at the N-terminus of the modified NifH polypeptide or the NifH: linker: :NifH polypeptide.
19. The modified NifH polypeptide, NifH fusion polypeptide or encoded polypeptide of claim 17 or claim 18, which is a modified AnfH polypeptide, an AnfH fusion polypeptide or an encoded polypeptide which comprises one or two AnfH polypeptides, respectively, which is optionally further characterised by one or more of the features as defined in any one of claims 1 to 16.
20. The encoded polypeptide of claim 19 which comprises an AnfH polypeptide and a mitochondrial targeting peptide (MTP) which is translationally fused to the AnfH polypeptide, preferably at the N-terminus of the AnfH polypeptide.
21. An exogenous polynucleotide, comprising a promoter operably linked to a nucleotide sequence which encodes the modified NifH polypeptide as defined in any one of claims 1 to 13, a NifH fusion polypeptide as defined in claim 14 or claim 15, or an encoded polypeptide as defined in claim 15 or claim 16, wherein the promoter directs expression of the nucleotide sequence in a cell, preferably a plant cell.
22. The exogenous polynucleotide of claim 21, wherein a) the modified NifH polypeptide is a modified AnfH polypeptide, the NifH fusion polypeptide is a AnfH fusion polypeptide, or the encoded polypeptide is a AnfH polypeptide; and/or b) the exogenous polynucleotide is integrated into the genome of the cell, preferably into the nuclear genome of a plant cell; and/or c) the protein coding region of the polynucleotide has been codon-modified for expression in a plant cell.
23. A vector comprising the exogenous polynucleotide according to claim 21 or claim 22.
24. A transgenic plant or part thereof, comprising one or more of a plant cell according to any one of claims 1 to 16, a modified NifH polypeptide as defined in any one of claims 1 to 13, a NifH fusion polypeptide as defined in claim 14 or claim 15, an encoded polypeptide as defined claim 15 or claim 16 or an exogenous polynucleotide as defined in claim 21 or claim 22.
25. The transgenic plant or part thereof of claim 24, wherein the modified NifH polypeptide, NifH fusion polypeptide or encoded polypeptide is a modified AnfH polypeptide, an AnfH fusion polypeptide or an encoded polypeptide which comprises one or two AnfH polypeptides, respectively, as defined in any one of claims 1 to 16.
26. The transgenic plant or part thereof of claim 24 or claim 25, wherein the part is a transgenic seed.
27. The transgenic plant or part thereof according to any one of claims 24 to 26, which is a cereal plant, preferably a wheat, rice, maize, triticale, oat or barley plant, or part thereof.
28. A method of selecting a modified NifH polypeptide, preferably a modified AnfH polypeptide, or a NifH fusion polypeptide, preferably an AnfH fusion polypeptide, the method comprising i) expressing an exogenous polynucleotide according to claim 21 or claim 22 in a plant cell, ii) extracting proteins comprising the modified NifH polypeptide or NifH fusion polypeptide from the mitochondria of the plant cell, iii) determining the level of solubility of the modified NifH polypeptide or NifH fusion polypeptide in the extracted proteins, and iv) selecting the modified NifH polypeptide or NifH fusion polypeptide, wherein at least 15%, preferably at least 50%, of the modified NifH polypeptide or NifH fusion polypeptide in the cell is soluble.
29. A method of producing a modified NifH polypeptide, preferably a modified AnfH polypeptide, or a NifH fusion polypeptide, preferably an AnfH fusion polypeptide, the method comprising expressing an exogenous polynucleotide according to claim 21 or claim 22 in a plant cell or a transgenic plant.
30. A method of selecting a plant cell which produces a modified NifH polypeptide, preferably a modified AnfH polypeptide, or a NifH fusion polypeptide, preferably an AnfH fusion polypeptide, the method comprising i) expressing an exogenous polynucleotide according to claim 21 or claim 22 in a plant cell, ii) determining whether the modified NifH polypeptide or NifH fusion polypeptide is produced at a desired level and/or has a desired activity, iii) selecting the plant cell on the basis of the results of step ii), iv) optionally producing a transgenic plant from the selected plant cell, and v) optionally producing transgenic progeny plants and/or transgenic seed from the transgenic plant.
31. A method of selecting a plant which produces a modified NifH polypeptide, preferably a modified AnfH polypeptide, or a NifH fusion polypeptide, preferably an AnfH fusion polypeptide, the method comprising i) expressing an exogenous polynucleotide according to claim 21 or claim 22 in a plant or in multiple plants, ii) determining whether the modified NifH polypeptide or NifH fusion polypeptide is produced at a desired level and/or has a desired activity in the plant or multiple plants, iii) selecting a plant from step ii) which produces the modified NifH polypeptide or NifH fusion polypeptide at a desired level and/or has a desired activity in the plant or a part thereof, and iv) optionally producing transgenic progeny plants and/or transgenic seed from the transgenic plant.
32. The method of any one of claims 28 to 31, wherein a) the modified NifH has one or more of the features of the modified NifH polypeptide as defined in any one of claims 1 to 19, or the NifH fusion polypeptide has one or more of the features of the NifH fusion polypeptide as defined in any one of claims 14 to 19; and/or b) the modified NifH polypeptide has at least one amino acid substitution which confers onto the modified NifH polypeptide a lower free energy when compared to a corresponding NifH polypeptide which lacks the at least one amino acid substitution.
33. Use of an exogenous polynucleotide according to claim 21 or claim 22, and/or a vector of claim 23, for producing a transgenic plant cell.
34. A method of producing a transgenic plant, the method comprising the steps of i) introducing one or more exogenous polynucleotides according to claim 21 or claim 22, and/or a vector of claim 23, into a plant cell, ii) from the cell of step i), regenerating a transgenic plant according to any one of claims 24 to 27, and iii) optionally, producing transgenic seed and/or progeny plants from the transgenic plant regenerated in step ii).
35. A method of producing transgenic seed, comprising i) harvesting seed from the transgenic plant according to any one of claims 24 to 27, and/or ii) harvesting seed from one or more transgenic progeny plants produced by the method of claim 34.
36. A method of producing flour, wholemeal, starch, oil, seedmeal or other product obtained from seed, the method comprising extracting flour, wholemeal, starch, oil or other product, or producing the seedmeal, from the seed of claim 26.
37. A product produced from the transgenic plant or part thereof according to any one of claims 24 to 27, wherein the product comprises one or more or all of a modified NifH polypeptide as defined in any one of claims 1 to 13, a NifH fusion polypeptide as defined in claim 14 or claim 15, an encoded polypeptide as defined in claim 15 or claim 16 and an exogenous polynucleotide according to claim 21 or claim 22.
38. A method of preparing a food product, the method comprising mixing seed of claim 26, or flour, wholemeal, starch, oil or other product from the seed, with another food ingredient.
39. A process of feeding an animal, comprising providing to the animal the plant or part thereof according to any one of claims 24 to 27, or the product of claim 37.
EP24857521.9A 2023-08-31 2024-08-30 Nif variants Pending EP4771133A1 (en)

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