Nif variants

Abstract

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

Description

[0001] NIF VARIANTS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates, in part, to modified NifH polypeptides and NifH fusion polypeptides with improved solubility.

[0004] BACKGROUND OF THE INVENTION

[0005] 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.

[0006] 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.

[0007] 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.

[0008] SUMMARY OF THE INVENTION

[0009] The present inventors have identified NifH variants, such as AnfH variants, with improved solubility in mitochondria of a plant cell.

[0010] 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.

[0011] 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.

[0012] 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.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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,

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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,

[0053] 137M, 138P, MOR, 142G, 143K, 144A, 146E, 148Y, 150V, 151A, 152S, 153G, 154E,

[0054] 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,

[0055] 198E, 199F, 204G, 212P, 213R, 215N, 217V, 218Q, 220A, 221E, 222F, 227V, 236Q,

[0056] 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.

[0057] 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.

[0058] 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.

[0059] In an embodiment of the above aspects, the modified NifH polypeptide is a modified AnfH polypeptide.

[0060] 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.

[0061] In an embodiment of the above aspects, the modified NifH polypeptide, preferably a modified AnfH polypeptide, comprises an Fe-S cluster.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] In a preferred embodiment of the above aspects and embodiments, the modified NifH polypeptide is a modified AnfH polypeptide.

[0070] 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.

[0071] In an embodiment, the plant cell further comprises an exogenous polynucleotide encoding a NifM polypeptide.

[0072] 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).

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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 .

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] Furthermore provided is a modified NifH polypeptide as defined herein.

[0082] Also provided is a NifH fusion polypeptide as defined herein.

[0083] In addition, provided is an encoded polypeptide as defined herein.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] In an embodiment, the modified NifH polypeptide is a modified AnfH polypeptide.

[0088] In an embodiment, the NifH fusion polypeptide is an AnfH fusion polypeptide.

[0089] In an embodiment, the encoded polypeptide is an AnfH polypeptide.

[0090] In an embodiment, the exogenous polynucleotide is integrated into the genome of the cell, preferably into the nuclear genome of a plant cell.

[0091] In an embodiment, the protein coding region of the polynucleotide has been codon-modified for expression in a plant cell.

[0092] In a further aspect, the present invention provides a vector comprising the exogenous polynucleotide of the invention.

[0093] 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.

[0094] 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.

[0095] In an embodiment, the part is a transgenic seed.

[0096] 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.

[0097] 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.

[0098] 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).

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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).

[0109] 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.

[0110] 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.

[0111] 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.

[0112] 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.

[0113] 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.

[0114] Any embodiment herein shall be taken to apply mutatis mutandis to any other embodiment unless specifically stated otherwise.

[0115] 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.

[0116] 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.

[0117] 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

[0118] Figure 1. AlphaFold prediction of the tertiary structure of wild-type vinelandii NifU protein, showing the N-terminal, central and C-terminal domains.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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).

[0125] 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)).

[0126] 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.

[0127] Figure 11. Delta-15N of triplicate plant samples as determined by IRMS. A. Increasing amounts of non-purified15N2 gas were added to plants in sealed containers at the indicated percentages and sampled after 24 hr in the dark. B. Non-purified or purified15N2 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.

[0128] 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.

[0129] 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.

[0130] 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.

[0131] 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::

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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).

[0136] 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 in15N2 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.

[0137] 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.

[0138] 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.

[0139] KEY TO THE SEQUENCE LISTING

[0140] SEQ ID NO:1 Amino acid sequence of NifH polypeptide from K. oxyloca. 293 aa.

[0141] SEQ ID NO:2 Amino acid sequence of wild-type NifD polypeptide from K. oxytoca, according to Accession No. X13303.1; 483aa.

[0142] SEQ ID NO:3 Amino acid sequence of NifK polypeptide from K. oxytoca, according to Temme et al. (2012); 520aa.

[0143] SEQ ID NO:4 Amino acid sequence ofNifB polypeptide firom X. oxyloca. 468aa.

[0144] SEQ ID NO:5 Amino acid sequence of NifE polypeptide from K. oxyloca. 457aa.

[0145] SEQ ID NO:6 Amino acid sequence ofNifF polypeptide from ". oxyloca. 176 aa; NCBI Accession No. X03214.

[0146] SEQ ID NO:7 Amino acid sequence of NifI polypeptide from K. oxytoca, 1171 aa; NCBI Accession No. WP_064371580, Cannon et al., (1988).

[0147] SEQ ID NO:8 Amino acid sequence of NifM polypeptide from K. oxyloca. 266 aa; NCBI Accession No. X05887; Paul and Merrick (1987).

[0148] SEQ ID NO:9 Amino acid sequence of NifN polypeptide from K. oxyloca. NCBI Accession No. P08738; 461aa; (Arnold et al., 1988).

[0149] 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.

[0150] SEQ ID NO: 12 Amino acid sequence of NifU polypeptide from K. oxytoca, 274aa. NCBI Accession No. P05343.2 (Arnold et al., 1988).

[0151] SEQ ID NO: 13 Amino acid sequence of NifV polypeptide from K. oxytoca, 381aa. NCBI Accession No. CAA31119.1 (Arnold et al., 1988).

[0152] SEQ ID NO: 14 Amino acid sequence of NifX polypeptide from K. oxytoca, 156aa (Accession No. P09136).

[0153] SEQ ID NO: 15 Amino acid sequence of NifY polypeptide from K. oxytoca, 220aa; NCBI Accession No. CAA31670 (Arnold et al., 1988).

[0154] SEQ ID NO: 16 Amino acid sequence of NifZ polypeptide from K. oxytoca, 148aa; NCBI Accession No. P0A3U2 (Arnold et al., 1988).

[0155] SEQ ID NO: 17. Amino acid sequence ofNifW polypeptide from T. oxytoca.

[0156] SEQ ID NO: 18. Amino acid sequence of wild-type K. oxytoca NifD according to Temme et al. (2012)

[0157] SEQ ID NO: 19. Amino acid sequence of wild-type K. oxytoca according to Temme et al. (2012).

[0158] 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.

[0159] 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.

[0160] SEQ ID NO:23. Peptide sequence.

[0161] 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.

[0162] SEQ ID NO:25. Amino acid sequence of the NifV polypeptide from A. vinelandii (AvNifV; Accession no WP_012698855).

[0163] 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.

[0164] 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.

[0165] 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.

[0166] 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.

[0167] 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.

[0168] 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.

[0169] 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).

[0170] 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).

[0171] SEQ ID NO:35. Amino acid sequence of AnfD from A. vinelandii (Accession No. WP_012703361); 518aa.

[0172] 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.

[0173] SEQ ID NO:38. Amino acid sequence of AnfG from A. vinelandii (Accession No. WP_012703360); 132aa.

[0174] SEQ ID NO:39 Amino acid sequence of the NifH polypeptide from A. vinelandii (AvNifH; Accession No. WP_012698831); 290aa.

[0175] SEQ ID NO:40. Peptide sequence, AnfH motif I, where X represents any amino acid.

[0176] SEQ ID NO:41. Peptide sequence, AnfH motif II.

[0177] SEQ ID NO:42. Peptide sequence, AnfH motif III.

[0178] SEQ ID NO:43. Peptide sequence, AnfH motif IV.

[0179] SEQ ID NO:44. Peptide sequence, AnfH motif V, where X represents any amino acid.

[0180] SEQ ID NO:45. Peptide sequence, AnfH motif VI.

[0181] SEQ ID NO:46. Peptide sequence, AnfH motif VII, where X represents any amino acid.

[0182] SEQ ID NO:47 Amino acid sequence of the FdxN protein of A. vinelandii,' Accession No. WP_012703542; 92aa.

[0183] SEQ ID NO:48 Amino acid sequence of the NafY polypeptide from A. vinelandii (AvNafY; Accession No. AGK13761).

[0184] SEQ ID NO:49-53. C-terminal amino acid sequences of NifK polypeptides.

[0185] SEQ ID NO:54-58. C-terminal amino acid sequences of AnfK polypeptides.

[0186] 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.

[0187] SEQ ID NO:61. Peptide sequence, NifH motif I, where X represents any amino acid.

[0188] SEQ ID NO:62. Peptide sequence, NifH motif II, where X represents any amino acid.

[0189] SEQ ID NO:63. Peptide sequence, NifH motif III, where X represents any amino acid.

[0190] SEQ ID NO:64. Peptide sequence, NifH motif V, where X represents any amino acid.

[0191] SEQ ID NO:65. Peptide sequence, NifH motif VII, where X represents any amino acid.

[0192] 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.

[0193] 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.

[0194] 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.

[0195] 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.

[0196] 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.

[0197] 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.

[0198] 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.

[0199] 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.

[0200] SEQ ID NO :75. Amino acid sequence of variant V4 of AnfH from A. vinelandii, with

[0201] 10 amino acid substitutions compared to wild-type AnAnfH (Table 4); 275aa.

[0202] SEQ ID NO :76. Amino acid sequence of variant V5 of AnfH from A. vinelandii, with

[0203] 11 amino acid substitutions compared to wild-type AnAnfH (Table 4); 275aa.

[0204] 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.

[0205] 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.

[0206] 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.

[0207] 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.

[0208] 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.

[0209] 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.

[0210] 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.

[0211] 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.

[0212] 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.

[0213] 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.

[0214] 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.

[0215] 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.

[0216] 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.

[0217] 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.

[0218] 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.

[0219] 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.

[0220] 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.

[0221] 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.

[0222] 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.

[0223] 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.

[0224] 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.

[0225] 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.

[0226] 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.

[0227] 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.

[0228] 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.

[0229] 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.

[0230] 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.

[0231] 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.

[0232] 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.

[0233] 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.

[0234] 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.

[0235] 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.

[0236] 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.

[0237] 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.

[0238] 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.

[0239] 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.

[0240] 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.

[0241] 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.

[0242] 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.

[0243] 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.

[0244] 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.

[0245] 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.

[0246] 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).

[0247] 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).

[0248] 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).

[0249] SEQ ID NO: 127-150. Peptide sequences.

[0250] 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.

[0251] 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.

[0252] 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.

[0253] 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.

[0254] 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.

[0255] 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.

[0256] DETAILED DESCRIPTION OF THE INVENTION

[0257] General Techniques and Definitions

[0258] 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).

[0259] 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).

[0260] 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.

[0261] 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.

[0262] 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.

[0263] Nitrogenase

[0264] 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.

[0265] 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).

[0266] 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.

[0267] Thermostability and its Relationship with Solubility

[0268] 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.

[0269] 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.

[0270] 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).

[0271] 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

[0272] 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.

[0273] 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.

[0274] 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).

[0275] 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.

[0276] 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.

[0277] 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.

[0278] 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.

[0279] As used herein, the phrase “with reference to SEQ ID NO” refers to the same amino acid position as the defined polypeptide.

[0280] 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.

[0281] 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.

[0282] 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

[0283] (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).

[0284] 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.

[0285] 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.

[0286] 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.

[0287] 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).

[0288] 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.

[0289] 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).

[0290] 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.

[0291] 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.

[0292] 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.

[0293] 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).

[0294] 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.

[0295] 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).

[0296] 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.

[0297] 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.

[0298] 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.

[0299] 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.

[0300] 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).

[0301] 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.

[0302] 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.

[0303] 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.

[0304] 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).

[0305] 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

[0306] (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).

[0307] 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.

[0308] Functional AnfG polypeptides are capable of functioning as the 5 protein structural component of the 012P262 heterohexameric nitrogenase.

[0309] 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).

[0310] 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).

[0311] 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).

[0312] 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

[0313] (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).

[0314] 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

[0315] (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).

[0316] 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

[0317] (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.

[0318] 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.

[0319] 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).

[0320] 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).

[0321] 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).

[0322] 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

[0323] (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.

[0324] 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).

[0325] A. vinelandii NifY and NafY bind to apo-NifD-NifK, at different stages of NifD- NifK holoenzyme maturation, to either a-Cys275or a-His442of 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 His121and 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.

[0326] 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).

[0327] 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).

[0328] 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).

[0329] 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.

[0330] 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

[0331] (WP_069124666.1, 70.65%), Uliginosibacterium sp. TH139 (WP_101942980, 64.47%), Klebsiella michiganensis (WP 049076934. I , 44.26%), Escherichia coli

[0332] (WP_072048756.1, 44.26%), Rhizobium leguminosarum (WPJ30674512.1, 43.86%) and Flavobacterium alvei (WP_103805005.1, 28.57%).

[0333] Sequence Identity and Substitutions

[0334] 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.

[0335] 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).

[0336] 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.

[0337] 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.

[0338] 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.

[0339] 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.

[0340] 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.

[0341] 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.

[0342] 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.

[0343] 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.

[0344] 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.

[0345] 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.

[0346] 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.

[0347] 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.

[0348] 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.

[0349] 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.

[0350] 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.

[0351] 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.

[0352] 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.

[0353] 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.

[0354] 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.

[0355] 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.

[0356] 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.

[0357] 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.

[0358] 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.

[0359] 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.

[0360] 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.

[0361] 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.

[0362] 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.

[0363] 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.

[0364] 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.

[0365] 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.

[0366] 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.

[0367] 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.

[0368] 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.

[0369] 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

[0370] 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.

[0371] 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.

[0372] 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.

[0373] 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,

[0374] 131V, 132C, 133G, 134G, 135F, 136A, 137M, 138P, MOR, 142G, 143K, 144A, 146E,

[0375] 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,

[0376] 185N, 186S, 187R, 189V, 190D, 192E, 198E, 199F, 204G, 212P, 213R, 215N, 217V,

[0377] 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.

[0378] 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.

[0379] 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.

[0380] In an embodiment, the modified NifH polypeptide is a modified AnfH polypeptide.

[0381] 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.

[0382] 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.

[0383] 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.

[0384] 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.

[0385] Mitochondrial Protein Import in Plants

[0386] 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).

[0387] 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).

[0388] 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.

[0389] 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).

[0390] 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.

[0391] 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).

[0392] 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).

[0393] Mitochondrial targeting peptide

[0394] 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.

[0395] 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.

[0396] 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.

[0397] 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.

[0398] 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.

[0399] 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.

[0400] In an embodiment, the MTP is not cleaved, preferably it is cleaved.

[0401] 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.

[0402] 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.

[0403] The skilled person will appreciate that software exists for predicting mitochondrial proteins and their targeting sequence, for example, MitoProtll, PSORT, TargetP and NNPSL.

[0404] 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.

[0405] 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.

[0406] 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.

[0407] 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.

[0408] 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.

[0409] 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

[0410] 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.

[0411] 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.

[0412] 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.

[0413] 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.

[0414] 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.

[0415] 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.

[0416] 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.

[0417] 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.

[0418] Linkers

[0419] 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.

[0420] 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.

[0421] 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.

[0422] 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.

[0423] 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.

[0424] 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.

[0425] In an embodiment, the linker does not comprise a cysteine residue.

[0426] 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.

[0427] 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.

[0428] The use of oligopeptide linkers in modifying polypeptides is reviewed in Chen et al. (2013) and Zhang et al. (2009).

[0429] Tags

[0430] 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.

[0431] 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.

[0432] 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.

[0433] 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.

[0434] 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.

[0435] 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.

[0436] 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).

[0437] Polynucleotides

[0438] 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.

[0439] 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.

[0440] 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.

[0441] 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.

[0442] 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.

[0443] 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 3rdor 4thgeneration 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.

[0444] 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.

[0445] 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.

[0446] 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.

[0447] 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.

[0448] 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).

[0449] 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.

[0450] Nucleic Acid Constructs

[0451] 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.

[0452] 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.

[0453] "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.

[0454] "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.

[0455] 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.

[0456] 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.

[0457] 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.

[0458] Examples of stem-specific promoters include, but are not limited to, those described in US 5,625,136.

[0459] 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.

[0460] 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.

[0461] 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.

[0462] 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).

[0463] Vectors

[0464] 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.

[0465] 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.

[0466] 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.

[0467] 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.

[0468] 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.

[0469] 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.

[0470] 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.

[0471] 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.

[0472] 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.

[0473] Recombinant vectors of the invention comprise fusion sequences which lead to the expression of nucleic acid molecules as fusion proteins.

[0474] 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.

[0475] Recombinant Cells

[0476] 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.

[0477] 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.

[0478] 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.

[0479] 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.

[0480] 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.

[0481] 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.

[0482] Plants

[0483] 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.

[0484] 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.

[0485] 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".

[0486] 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.

[0487] 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.

[0488] 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).

[0489] 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.

[0490] 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.

[0491] 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.

[0492] 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.

[0493] 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

[0494] 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).

[0495] 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.

[0496] 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.

[0497] 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.

[0498] 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.

[0499] 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).

[0500] 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).

[0501] 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.

[0502] 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.

[0503] 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.

[0504] 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).

[0505] 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.

[0506] 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.

[0507] 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.

[0508] 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.

[0509] 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

[0510] 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.

[0511] 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).

[0512] 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.

[0513] 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.

[0514] 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.

[0515] 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.

[0516] 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.

[0517] 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 mo...

Claims

CLAIMS1. 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 acidpositions: 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, 3to 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 / ord) 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 atthe 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.

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