Using Synthetic Biology to Overcome Barriers to Stable Expression of Nitrogenase in Eukaryotic Organelles

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 2020 Jul 1

PMID 32601191

Citations 15

Authors

Nan Xiang

Chenyue Guo

Jiwei Liu

Hao Xu

Ray Dixon

Jianguo Yang

Yi-Ping Wang

Affiliations

Soon will be listed here.

Abstract

Engineering biological nitrogen fixation in eukaryotic cells by direct introduction of genes requires elegant synthetic biology approaches to ensure that components required for the biosynthesis of active nitrogenase are stable and expressed in the appropriate stoichiometry. Previously, the NifD subunits of nitrogenase MoFe protein from and were found to be unstable in yeast and plant mitochondria, respectively, presenting a bottleneck to the assembly of active MoFe protein in eukaryotic cells. In this study, we have delineated the region and subsequently a key residue, NifD-R98, from that confers susceptibility to protease-mediated degradation in mitochondria. The effect observed is pervasive, as R98 is conserved among all NifD proteins analyzed. NifD proteins from four representative diazotrophs, but not their R98 variants, were observed to be unstable in yeast mitochondria. Furthermore, by reconstituting mitochondrial-processing peptidases (MPPs) from yeast, , , and in , we demonstrated that MPPs are responsible for cleavage of NifD. These results indicate a pervasive effect on the stability of NifD proteins in mitochondria resulting from cleavage by MPPs. NifD-R98 variants that retained high levels of nitrogenase activity were obtained, with the potential to stably target active MoFe protein to mitochondria. This reconstitution approach could help preevaluate the stability of Nif proteins for plant expression and paves the way for engineering active nitrogenase in plant organelles.

Citing Articles

Co-expression of nitrogenase proteins in cotton (Gossypium hirsutum L.).

Shang Y, Guo W, Liu X, Ma L, Liu D, Chen S PLoS One. 2023; 18(8):e0290556.

PMID: 37616286 PMC: 10449186. DOI: 10.1371/journal.pone.0290556.

Organelle-dependent polyprotein designs enable stoichiometric expression of nitrogen fixation components targeted to mitochondria.

Yang J, Xiang N, Liu Y, Guo C, Li C, Li H Proc Natl Acad Sci U S A. 2023; 120(34):e2305142120.

PMID: 37585462 PMC: 10450427. DOI: 10.1073/pnas.2305142120.

Eight Up-Coming Biotech Tools to Combat Climate Crisis.

Fuchs W, Rachbauer L, Rittmann S, Bochmann G, Ribitsch D, Steger F Microorganisms. 2023; 11(6).

PMID: 37375016 PMC: 10301127. DOI: 10.3390/microorganisms11061514.

Dancing to a different tune, can we switch from chemical to biological nitrogen fixation for sustainable food security?.

Jhu M, Oldroyd G PLoS Biol. 2023; 21(3):e3001982.

PMID: 36917569 PMC: 10013914. DOI: 10.1371/journal.pbio.3001982.

Molecular Mechanism and Agricultural Application of the NifA-NifL System for Nitrogen Fixation.

Zhang W, Chen Y, Huang K, Wang F, Mei Z Int J Mol Sci. 2023; 24(2).

PMID: 36674420 PMC: 9866876. DOI: 10.3390/ijms24020907.

References

Gietz R, Schiestl R . High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc. 2007; 2(1):31-4. DOI: 10.1038/nprot.2007.13. View

Eseverri A, Lopez-Torrejon G, Jiang X, Buren S, Rubio L, Caro E . Use of synthetic biology tools to optimize the production of active nitrogenase Fe protein in chloroplasts of tobacco leaf cells. Plant Biotechnol J. 2020; 18(9):1882-1896. PMC: 7415783. DOI: 10.1111/pbi.13347. View

Yang M, Jensen R, Yaffe M, Oppliger W, Schatz G . Import of proteins into yeast mitochondria: the purified matrix processing protease contains two subunits which are encoded by the nuclear MAS1 and MAS2 genes. EMBO J. 1988; 7(12):3857-62. PMC: 454964. DOI: 10.1002/j.1460-2075.1988.tb03271.x. View

Jimenez-Vicente E, Yang Z, Martin Del Campo J, Cash V, Seefeldt L, Dean D . The NifZ accessory protein has an equivalent function in maturation of both nitrogenase MoFe protein P-clusters. J Biol Chem. 2019; 294(16):6204-6213. PMC: 6484116. DOI: 10.1074/jbc.RA119.007905. View

Naamati A, Regev-Rudzki N, Galperin S, Lill R, Pines O . Dual targeting of Nfs1 and discovery of its novel processing enzyme, Icp55. J Biol Chem. 2009; 284(44):30200-8. PMC: 2781575. DOI: 10.1074/jbc.M109.034694. View

Rubio L, Ludden P . Maturation of nitrogenase: a biochemical puzzle. J Bacteriol. 2005; 187(2):405-14. PMC: 543557. DOI: 10.1128/JB.187.2.405-414.2005. View

Howard K, McLean P, Hansen F, Lemley P, Koblan K, Orme-Johnson W . Klebsiella pneumoniae nifM gene product is required for stabilization and activation of nitrogenase iron protein in Escherichia coli. J Biol Chem. 1986; 261(2):772-8. View

Curatti L, Rubio L . Challenges to develop nitrogen-fixing cereals by direct nif-gene transfer. Plant Sci. 2014; 225:130-7. DOI: 10.1016/j.plantsci.2014.06.003. View

Beatty P, Good A . Plant science. Future prospects for cereals that fix nitrogen. Science. 2011; 333(6041):416-7. DOI: 10.1126/science.1209467. View

10.

Quiros P, Langer T, Lopez-Otin C . New roles for mitochondrial proteases in health, ageing and disease. Nat Rev Mol Cell Biol. 2015; 16(6):345-59. DOI: 10.1038/nrm3984. View

11.

Dixon R, POSTGATE J . Genetic transfer of nitrogen fixation from Klebsiella pneumoniae to Escherichia coli. Nature. 1972; 237(5350):102-3. DOI: 10.1038/237102a0. View

12.

Dos Santos P, Fang Z, Mason S, Setubal J, Dixon R . Distribution of nitrogen fixation and nitrogenase-like sequences amongst microbial genomes. BMC Genomics. 2012; 13:162. PMC: 3464626. DOI: 10.1186/1471-2164-13-162. View

13.

Yang J, Xie X, Xiang N, Tian Z, Dixon R, Wang Y . Polyprotein strategy for stoichiometric assembly of nitrogen fixation components for synthetic biology. Proc Natl Acad Sci U S A. 2018; 115(36):E8509-E8517. PMC: 6130400. DOI: 10.1073/pnas.1804992115. View

14.

Rubio L, Ludden P . Biosynthesis of the iron-molybdenum cofactor of nitrogenase. Annu Rev Microbiol. 2008; 62:93-111. DOI: 10.1146/annurev.micro.62.081307.162737. View

15.

Yang J, Xie X, Yang M, Dixon R, Wang Y . Modular electron-transport chains from eukaryotic organelles function to support nitrogenase activity. Proc Natl Acad Sci U S A. 2017; 114(12):E2460-E2465. PMC: 5373397. DOI: 10.1073/pnas.1620058114. View

16.

Witte C, Jensen R, Yaffe M, Schatz G . MAS1, a gene essential for yeast mitochondrial assembly, encodes a subunit of the mitochondrial processing protease. EMBO J. 1988; 7(5):1439-47. PMC: 458394. DOI: 10.1002/j.1460-2075.1988.tb02961.x. View

17.

Dos Santos P, Smith A, Frazzon J, Cash V, Johnson M, Dean D . Iron-sulfur cluster assembly: NifU-directed activation of the nitrogenase Fe protein. J Biol Chem. 2004; 279(19):19705-11. DOI: 10.1074/jbc.M400278200. View

18.

Hawlitschek G, Schneider H, Schmidt B, Tropschug M, Hartl F, Neupert W . Mitochondrial protein import: identification of processing peptidase and of PEP, a processing enhancing protein. Cell. 1988; 53(5):795-806. DOI: 10.1016/0092-8674(88)90096-7. View

19.

Voos W . Chaperone-protease networks in mitochondrial protein homeostasis. Biochim Biophys Acta. 2012; 1833(2):388-99. DOI: 10.1016/j.bbamcr.2012.06.005. View

20.

Eriksson A, Sjoling S, Glaser E . Characterization of the bifunctional mitochondrial processing peptidase (MPP)/bc1 complex in Spinacia oleracea. J Bioenerg Biomembr. 1996; 28(3):285-92. DOI: 10.1007/BF02110702. View