{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,28]],"date-time":"2026-07-28T01:26:46Z","timestamp":1785202006676,"version":"3.55.0"},"reference-count":48,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2020,9,10]],"date-time":"2020-09-10T00:00:00Z","timestamp":1599696000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100000943","name":"Commonwealth Scientific and Industrial Research Organisation","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100000943","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Plant Sci."],"DOI":"10.3389\/fpls.2020.552160","type":"journal-article","created":{"date-parts":[[2020,9,10]],"date-time":"2020-09-10T05:01:23Z","timestamp":1599714083000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":17,"title":["A Synthetic Biology Workflow Reveals Variation in Processing and Solubility of Nitrogenase Proteins Targeted to Plant Mitochondria, and Differing Tolerance of Targeting Sequences in a Bacterial Nitrogenase Assay"],"prefix":"10.3389","volume":"11","author":[{"given":"Shoko","family":"Okada","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christina M.","family":"Gregg","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Robert Silas","family":"Allen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amratha","family":"Menon","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dawar","family":"Hussain","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vanessa","family":"Gillespie","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ema","family":"Johnston","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Keren","family":"Byrne","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michelle Lisa","family":"Colgrave","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Craig C.","family":"Wood","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2020,9,10]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","DOI":"10.3389\/fpls.2017.00287","article-title":"Expression of 16 Nitrogenase Proteins within the Plant Mitochondrial Matrix","volume":"8","author":"Allen","year":"2017","journal-title":"Front. Plant Sci."},{"key":"B2","doi-asserted-by":"publisher","first-page":"202002365","DOI":"10.1073\/pnas.2002365117","article-title":"Plant expression of NifD protein variants resistant to mitochondrial degradation","author":"Allen","year":"2020","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"B3","doi-asserted-by":"publisher","DOI":"10.3389\/fpls.2017.01567","article-title":"Purification and In Vitro Activity of Mitochondria Targeted Nitrogenase Cofactor Maturase NifB","volume":"8","author":"Bur\u00e9n","year":"2017","journal-title":"Front. Plant Sci."},{"key":"B4","doi-asserted-by":"publisher","first-page":"1043","DOI":"10.1021\/acssynbio.6b00371","article-title":"Formation of Nitrogenase NifDK Tetramers in the Mitochondria of Saccharomyces cerevisiae","volume":"6","author":"Bur\u00e9n","year":"2017","journal-title":"ACS Synth. Biol."},{"key":"B5","doi-asserted-by":"publisher","first-page":"25078","DOI":"10.1073\/pnas.1904903116","article-title":"Biosynthesis of the nitrogenase active-site cofactor precursor NifB-co in Saccharomyces cerevisiae","volume":"116","author":"Bur\u00e9n","year":"2019","journal-title":"Proc. Natl. Acad. Sci."},{"key":"B6","doi-asserted-by":"publisher","first-page":"4921","DOI":"10.1021\/acs.chemrev.9b00489","article-title":"Biosynthesis of Nitrogenase Cofactors","volume":"120","author":"Bur\u00e9n","year":"2020","journal-title":"Chem. Rev"},{"key":"B7","doi-asserted-by":"publisher","first-page":"130","DOI":"10.1016\/j.plantsci.2014.06.003","article-title":"Challenges to develop nitrogen-fixing cereals by direct nif-gene transfer","volume":"225","author":"Curatti","year":"2014","journal-title":"Plant Sci."},{"key":"B8","doi-asserted-by":"publisher","first-page":"97","DOI":"10.1021\/sb4001992","article-title":"Rapid and reliable DNA assembly via ligase cycling reaction","volume":"3","author":"de Kok","year":"2014","journal-title":"ACS Synth. Biol."},{"key":"B9","doi-asserted-by":"publisher","first-page":"285","DOI":"10.1016\/0304-4165(66)90383-7","article-title":"Acetylene reduction by nitrogen-fixing preparations from Clostridium pasteurianum","volume":"127","author":"Dilworth","year":"1966","journal-title":"Biochim. Biophys. Acta"},{"key":"B10","doi-asserted-by":"publisher","first-page":"1653","DOI":"10.1126\/science.1529353","article-title":"Crystallographic structure of the nitrogenase iron protein from Azotobacter vinelandii","volume":"257","author":"Georgiadis","year":"1992","journal-title":"Science"},{"key":"B11","doi-asserted-by":"publisher","first-page":"2191","DOI":"10.1042\/BCJ20170521","article-title":"Plant mitochondrial protein import: the ins and outs","volume":"475","author":"Ghifari","year":"2018","journal-title":"Biochem. J."},{"key":"B12","doi-asserted-by":"publisher","DOI":"10.1088\/1748-9326\/9\/10\/105001","article-title":"The Haber Bosch-harmful algal bloom (HB-HAB) link","volume":"9","author":"Glibert","year":"2014","journal-title":"Environ. Res. Lett."},{"key":"B13","doi-asserted-by":"crossref","first-page":"15909","DOI":"10.1016\/S0021-9258(18)55485-5","article-title":"Genes required for formation of the apoMoFe protein of Klebsiella pneumoniae nitrogenase in Escherichia coli","volume":"265","author":"Harris","year":"1990","journal-title":"J. Biol. Chem."},{"key":"B14","doi-asserted-by":"crossref","first-page":"772","DOI":"10.1016\/S0021-9258(17)36161-6","article-title":"Klebsiella pneumoniae nifM gene product is required for stabilization and activation of nitrogenase iron protein in Escherichia coli","volume":"261","author":"Howard","year":"1986","journal-title":"J. Biol. Chem."},{"key":"B15","doi-asserted-by":"publisher","first-page":"1112","DOI":"10.1016\/j.bbabio.2012.12.001","article-title":"Nitrogenase assembly","volume":"1827","author":"Hu","year":"2013","journal-title":"Biochim. Biophys. Acta Bioenerg."},{"key":"B16","doi-asserted-by":"publisher","first-page":"1272","DOI":"10.1104\/pp.109.137885","article-title":"Refining the Definition of Plant Mitochondrial Presequences through Analysis of Sorting Signals, N-Terminal Modifications, and Cleavage Motifs","volume":"150","author":"Huang","year":"2009","journal-title":"Plant Physiol."},{"key":"B17","doi-asserted-by":"publisher","first-page":"1603","DOI":"10.1016\/j.febslet.2014.02.053","article-title":"Modifying the determinants of \u03b1-ketoacid substrate selectivity in Mycobacterium tuberculosis \u03b1-isopropylmalate synthase","volume":"588","author":"Hunter","year":"2014","journal-title":"FEBS Lett."},{"key":"B18","doi-asserted-by":"publisher","first-page":"553","DOI":"10.1038\/360553a0","article-title":"Crystallographic structure and functional implications of the nitrogenase molybdenum-iron protein from Azotobacter vinelandii","volume":"360","author":"Kim","year":"1992","journal-title":"Nature"},{"key":"B19","doi-asserted-by":"publisher","first-page":"974","DOI":"10.1126\/science.1206445","article-title":"X-ray Emission Spectroscopy Evidences a Central Carbon in the Nitrogenase Iron-Molybdenum Cofactor","volume":"334","author":"Lancaster","year":"2011","journal-title":"Science"},{"key":"B20","doi-asserted-by":"publisher","first-page":"5037","DOI":"10.1105\/tpc.112.105361","article-title":"Mitochondrial Targeting of the Arabidopsis F1-ATPase \u03b3-Subunit via Multiple Compensatory and Synergistic Presequence Motifs","volume":"24","author":"Lee","year":"2012","journal-title":"Plant Cell"},{"key":"B21","doi-asserted-by":"publisher","first-page":"186","DOI":"10.1006\/bbrc.1999.1507","article-title":"Regulated expression of the nifM of Azotobacter vinelandii in response to molybdenum and vanadium supplements in Burk\u2019s nitrogen-free growth medium","volume":"264","author":"Lei","year":"1999","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"B22","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms11426","article-title":"Expression of a functional oxygen-labile nitrogenase component in the mitochondrial matrix of aerobically grown yeast","volume":"7","author":"L\u00f3pez-Torrej\u00f3n","year":"2016","journal-title":"Nat. Commun."},{"key":"B23","doi-asserted-by":"publisher","first-page":"11","DOI":"10.1007\/s11816-018-0502-y","article-title":"Gene delivery into the plant mitochondria via organelle-specific peptides","volume":"13","author":"MacMillan","year":"2019","journal-title":"Plant Biotechnol. Rep."},{"key":"B24","doi-asserted-by":"publisher","first-page":"601","DOI":"10.1128\/JB.186.3.601","article-title":"The NifL-NifA System: a Multidomain Transcriptional Regulatory Complex That Integrates Environmental Signals","volume":"186","author":"Martinez-Argudo","year":"2004","journal-title":"J. Bacteriol."},{"key":"B25","doi-asserted-by":"publisher","first-page":"444","DOI":"10.1128\/mmbr.05024-11","article-title":"Biochemistry and Evolution of Anaerobic Energy Metabolism in Eukaryotes","volume":"76","author":"Muller","year":"2012","journal-title":"Microbiol. Mol. Biol. Rev."},{"key":"B26","doi-asserted-by":"publisher","first-page":"250","DOI":"10.1016\/j.freeradbiomed.2019.01.050","article-title":"Geobiological feedbacks, oxygen, and the evolution of nitrogenase","volume":"140","author":"Mus","year":"2019","journal-title":"Free Radic. Biol. Med."},{"key":"B27","doi-asserted-by":"publisher","DOI":"10.1101\/2019.12.23.887703","article-title":"An experimental workflow identifies nitrogenase proteins ready for expression in plant mitochondria","author":"Okada","year":"2019","journal-title":"bioRXiv"},{"key":"B28","doi-asserted-by":"publisher","first-page":"19","DOI":"10.1016\/j.copbio.2013.08.006","article-title":"Biotechnological solutions to the nitrogen problem","volume":"26","author":"Oldroyd","year":"2014","journal-title":"Curr. Opin. Biotechnol."},{"key":"B29","doi-asserted-by":"publisher","first-page":"1181","DOI":"10.1021\/bi9626665","article-title":"Redox-dependent structural changes in the nitrogenase P-cluster","volume":"36","author":"Peters","year":"1997","journal-title":"Biochemistry"},{"key":"B30","doi-asserted-by":"publisher","DOI":"10.1101\/cshperspect.a015990","article-title":"Eukaryotic origins: How and when was the mitochondrion acquired","volume":"6","author":"Poole","year":"2014","journal-title":"Cold Spring Harb. Perspect. Biol."},{"key":"B31","doi-asserted-by":"publisher","first-page":"595","DOI":"10.1128\/JB.11942-13","article-title":"Kinetics of nif Gene Expression in a Nitrogen-Fixing Bacterium","volume":"196","author":"Poza-Carri\u00f3n","year":"2014","journal-title":"J. Bacteriol."},{"key":"B32","doi-asserted-by":"publisher","first-page":"4063","DOI":"10.1021\/cr400463x","article-title":"Biosynthesis of nitrogenase metalloclusters","volume":"114","author":"Ribbe","year":"2014","journal-title":"Chem. Rev."},{"key":"B33","doi-asserted-by":"publisher","first-page":"267","DOI":"10.1128\/JB.136.1.267-279.1978","article-title":"Regulation and characterization of protein products coded by the nif (nitrogen fixation) genes of Klebsiella pneumoniae","volume":"136","author":"Roberts","year":"1978","journal-title":"J. Bacteriol."},{"key":"B34","doi-asserted-by":"publisher","first-page":"743","DOI":"10.1093\/aob\/mct048","article-title":"Biological nitrogen fixation in non-legume plants","volume":"111","author":"Santi","year":"2013","journal-title":"Ann. Bot."},{"key":"B35","doi-asserted-by":"publisher","first-page":"54","DOI":"10.1016\/j.pep.2013.08.021","article-title":"Development of the Twin-Strep-tag\u00ae and its application for purification of recombinant proteins from cell culture supernatants","volume":"92","author":"Schmidt","year":"2013","journal-title":"Protein Expr. Purif."},{"key":"B36","doi-asserted-by":"publisher","first-page":"1241","DOI":"10.1038\/nbt.3063","article-title":"Functional optimization of gene clusters by combinatorial design and assembly","volume":"32","author":"Smanski","year":"2014","journal-title":"Nat. Biotechnol."},{"key":"B37","doi-asserted-by":"publisher","first-page":"415","DOI":"10.1038\/22672","article-title":"Detonator of the population explosion","volume":"400","author":"Smil","year":"1999","journal-title":"Nature"},{"key":"B38","doi-asserted-by":"publisher","first-page":"940","DOI":"10.1126\/science.1214025","article-title":"Evidence for Interstitial Carbon in Nitrogenase FeMo Cofactor","volume":"334","author":"Spatzal","year":"2011","journal-title":"Science"},{"key":"B39","doi-asserted-by":"publisher","first-page":"7085","DOI":"10.1073\/pnas.1120788109","article-title":"Refactoring the nitrogen fixation gene cluster from Klebsiella oxytoca","volume":"109","author":"Temme","year":"2012","journal-title":"Proc. Natl. Acad. Sci."},{"key":"B40","doi-asserted-by":"publisher","first-page":"737","DOI":"10.1890\/1051-0761(1997)007[0737:HAOTGN]2.0.CO;2","article-title":"Human alteration of the global nitrogen cycle: Source and consequences","volume":"7","author":"Vitousek","year":"1997","journal-title":"Ecol. Appl."},{"key":"B41","doi-asserted-by":"publisher","first-page":"119","DOI":"10.1083\/jcb.123.1.119","article-title":"Presequence and mature part of preproteins strongly influence the dependence of mitochondrial protein import on heat shock protein 70 in the matrix","volume":"123","author":"Voos","year":"1993","journal-title":"J. Cell Biol."},{"key":"B42","doi-asserted-by":"publisher","first-page":"2668","DOI":"10.1002\/j.1460-2075.1996.tb00627.x","article-title":"Differential requirement for the mitochondrial Hsp70-Tim44 complex in unfolding and translocation of preproteins","volume":"15","author":"Voos","year":"1996","journal-title":"EMBO J."},{"key":"B43","doi-asserted-by":"publisher","first-page":"e16765","DOI":"10.1371\/journal.pone.0016765","article-title":"A modular cloning system for standardized assembly of multigene constructs","volume":"6","author":"Weber","year":"2011","journal-title":"PloS One"},{"key":"B44","doi-asserted-by":"publisher","first-page":"15435","DOI":"10.1073\/pnas.0507324102","article-title":"Effect of protein structure on mitochondrial import","volume":"102","author":"Wilcox","year":"2005","journal-title":"Proc. Natl. Acad. Sci."},{"key":"B45","doi-asserted-by":"publisher","first-page":"307","DOI":"10.1016\/j.ab.2010.12.004","article-title":"Comparison of ultrafiltration units for proteomic and N-glycoproteomic analysis by the filter-aided sample preparation method","volume":"410","author":"Wi\u015bniewski","year":"2011","journal-title":"Anal. Biochem."},{"key":"B46","doi-asserted-by":"publisher","first-page":"E3718","DOI":"10.1073\/pnas.1411185111","article-title":"Reconstruction and minimal gene requirements for the alternative iron-only nitrogenase in Escherichia coli","volume":"111","author":"Yang","year":"2014","journal-title":"Proc. Nat. Acad. Sci. U.S.A."},{"key":"B47","doi-asserted-by":"publisher","first-page":"E8509","DOI":"10.1073\/pnas.1804992115","article-title":"Polyprotein strategy for stoichiometric assembly of nitrogen fixation components for synthetic biology","volume":"115","author":"Yang","year":"2018","journal-title":"Proc. Natl. Acad. Sci."},{"key":"B48","doi-asserted-by":"publisher","first-page":"1582","DOI":"10.1021\/acs.biomac.8b00323","article-title":"Selective Gene Delivery for Integrating Exogenous DNA into Plastid and Mitochondrial Genomes Using Peptide-DNA Complexes","volume":"19","author":"Yoshizumi","year":"2018","journal-title":"Biomacromolecules"}],"container-title":["Frontiers in Plant Science"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/article\/10.3389\/fpls.2020.552160\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,3,31]],"date-time":"2021-03-31T15:28:53Z","timestamp":1617204533000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/article\/10.3389\/fpls.2020.552160\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,9,10]]},"references-count":48,"alternative-id":["10.3389\/fpls.2020.552160"],"URL":"https:\/\/doi.org\/10.3389\/fpls.2020.552160","relation":{},"ISSN":["1664-462X"],"issn-type":[{"value":"1664-462X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,9,10]]},"article-number":"552160"}}