Plant expression of NifD protein variants resistant to mitochondrial degradation

Abstract

To engineer Mo-dependent nitrogenase function in plants, expression of the structural proteins NifD and NifK will be an absolute requirement. Although mitochondria have been established as a suitable eukaryotic environment for biosynthesis of oxygen-sensitive enzymes such as NifH, expression of NifD in this organelle has proven difficult due to cryptic NifD degradation. Here, we describe a solution to this problem. Using molecular and proteomic methods, we found NifD degradation to be a consequence of mitochondrial endoprotease activity at a specific motif within NifD. Focusing on this functionally sensitive region, we designed NifD variants comprising between one and three amino acid substitutions and distinguished several that were resistant to degradation when expressed in both plant and yeast mitochondria. Nitrogenase activity assays of these resistant variants in Escherichia coli identified a subset that retained function, including a single amino acid variant (Y100Q). We found that other naturally occurring NifD proteins containing alternate amino acids at the Y100 position were also less susceptible to degradation. The Y100Q variant also enabled expression of a NifD(Y100Q)–linker–NifK translational polyprotein in plant mitochondria, confirmed by identification of the polyprotein in the soluble fraction of plant extracts. The NifD(Y100Q)–linker–NifK retained function in bacterial nitrogenase assays, demonstrating that this polyprotein permits expression of NifD and NifK in a defined stoichiometry supportive of activity. Our results exemplify how protein design can overcome impediments encountered when expressing synthetic proteins in novel environments. Specifically, these findings outline our progress toward the assembly of the catalytic unit of nitrogenase within mitochondria.

Exploiting genetic diversity and gene synthesis to identify superior nitrogenase NifH protein variants to engineer N2-fixation in plants

“…To date, functional NifH, NifU, and NifB have been purified from mitochondria of aerobically cultured Saccharomyces cerevisiae cells 10 , 11 , while active NifU and NifH were isolated from chloroplasts of Nicotiana benthamiana at the end of the dark period 12 . Also, the reported low stability of the NifD protein 13 has now been improved in two recent studies that identified key residues in the NifD sequence as susceptible to cleavage upon mitochondria import 14 , 15 . Notwithstanding these achievements, detailed analysis of yeast mitochondria-targeted Azotobacter vinelandii NifH has been hampered by low protein solubility resulting in suboptimal yields 10 .…”

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

“…Coexpression of either Nif combination did not improve the solubility of any of the Nif protein counterparts (Supplementary Figure 5). Results on coexpression of NifD and NifK targeted to N. benthamiana leaf mitochondria have been reported in a separate publication (Allen et al, 2020).…”

“…NifK was the only protein for which a different construct was made, where the HAtag was included at the N-terminus between pFAg51 and NifK, since any C-terminal fusions render NifK non-functional (Yang et al, 2018). Expression of pFAg51::NifD::HA has been reported as part of a separate study (Allen et al, 2020) and therefore was not included in this study. We constructed control expression plasmids to mimic the processed protein size for all Nif proteins by replacing pFAg51 with 6×His.…”

“…Functional A. vinelandii Nif proteins have been successfully coexpressed within yeast mitochondria (Loṕez-Torrejoń et al, 2016;Bureń et al, 2017b;Bureń et al, 2019), and having these protein combinations present in the one organelle may overcome problems that we report here. Finally, physically linking Nif proteins into larger multi-domain polyproteins (Yang et al, 2018;Allen et al, 2020) could assist in protein assembly and may overcome problems associated with relative abundance and solubility. The function of nitrogenase may be impacted by scar residues originating from the MPP cleavage of the MTP.…”

Analysis of Nitrogenase Fe Protein Activity in Transplastomic Tobacco

“…These select bacteria and archaea reduce N 2 to NH 3 using nitrogenase, a metalloenzyme that in its most prevalent form is composed of the Fe protein (encoded by nifH) and the MoFe protein (encoded by nifD and nifK) [for recent reviews see (Burén et al, 2020;Seefeldt et al, 2020)]. Progress has already been achieved in the path to engineer N 2 fixation by transfer of nif genes to unicellular eukaryotes (Cheng et al, 2005;López-Torrejón et al, 2016;Burén et al, 2017Burén et al, , 2019Burén and Rubio, 2018) and to higher plants (Ivleva et al, 2016;Allen et al, 2020;Eseverri et al, 2020;Jiang et al, 2021).…”