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.

While industrial nitrogen fertilizer is intrinsic to modern agriculture, it is expensive and environmentally harmful. One approach to reduce fertilizer usage is to engineer the bacterial nitrogenase enzyme complex within plant mitochondria, a location that may support enzyme function. Our current strategy involves fusing a mitochondrial targeting peptide (MTP) to nitrogenase (Nif) proteins, enabling their import to the mitochondrial matrix. However, the process of import modifies the N-terminus of each Nif protein and may impact nitrogenase assembly and function. Here we present our workflow assessing the mitochondrial processing, solubility and relative abundance of 16 Klebsiella oxytoca Nif proteins targeted to the mitochondrial matrix in Nicotiana benthamiana leaf. We found that processing and abundance of MTP::Nif proteins varied considerably, despite using the same constitutive promoter and MTP across all Nif proteins tested. Assessment of the solubility for all MTP::Nif proteins when targeted to plant mitochondria found NifF, M, N, S, U, W, X, Y, and Z were soluble, while NifB, E, H, J, K, Q, and V were mostly insoluble. The functional consequence of the N-terminal modifications required for mitochondrial targeting of Nif proteins was tested using a bacterial nitrogenase assay. With the exception of NifM, the Nif proteins generally tolerated the N-terminal extension. Proteomic analysis of Nif proteins expressed in bacteria found that the relative abundance of NifM with an N-terminal extension was increased ~50-fold, while that of the other Nif proteins was not influenced by the N-terminal extension. Based on the solubility, processing and functional assessments, our workflow identified that K. oxytoca NifF, N, S, U, W, Y, and Z successfully met these criteria. For the remaining Nif proteins, their limitations will need to be addressed before proceeding towards assembly of a complete set of plant-ready Nif proteins for reconstituting nitrogenase in plant mitochondria.

Plant expression of NifD protein variants resistant to mitochondrial degradation.

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.

Seed-specific RNAi in safflower generates a superhigh oleic oil with extended oxidative stability.

Vegetable oils extracted from oilseeds are an important component of foods, but are also used in a range of high value oleochemical applications. Despite being biodegradable, nontoxic and renewable current plant oils suffer from the presence of residual polyunsaturated fatty acids that are prone to free radical formation that limit their oxidative stability, and consequently shelf life and functionality. Many decades of plant breeding have been successful in raising the oleic content to ~90%, but have come at the expense of overall field performance, including poor yields. Here, we engineer superhigh oleic (SHO) safflower producing a seed oil with 93% oleic generated from seed produced in multisite field trials spanning five generations. SHO safflower oil is the result of seed-specific hairpin-based RNA interference of two safflower lipid biosynthetic genes, FAD2.2 and FATB, producing seed oil containing less than 1.5% polyunsaturates and only 4% saturates but with no impact on lipid profiles of leaves and roots. Transgenic SHO events were compared to non-GM safflower in multisite trial plots with a wide range of growing season conditions, which showed no evidence of impact on seed yield. The oxidative stability of the field-grown SHO oil produced from various sites was 50 h at 110°C compared to 13 h for conventional ~80% oleic safflower oils. SHO safflower produces a uniquely stable vegetable oil across different field conditions that can provide the scale of production that is required for meeting the global demands for high stability oils in food and the oleochemical industry.