Computationally Guided Design of a Soluble, Abundant and Functional AnfH Variant for the Expression of Fe-Only...

Engineering nitrogenase directly into crops is a long-held aspiration in plant biotechnology. Of the three types, the Fe-only nitrogenase is a promising engineering target, as it has a simpler maturation pathway than the MoFe- and VFe-nitrogenases and does not require any heterometals for its cofactor. However, previously, we have reported that the obligate electron donor of the Fe-only nitrogenase from A. vinelandii, AnfH, is mostly insoluble when expressed in plant mitochondria. Here, we employed computational methods, the Protein Repair One-Stop Shop (PROSS) protein optimisation algorithm and Rosetta energy calculations, to design eight variants of AnfH with improved soluble expression. The amino acid substitutions chosen were predicted to lower the free energy of the native state of the protein. All eight AnfH variants, containing between 1 and 11 amino acid substitutions, were more soluble than wild-type AnfH in plant mitochondria. Of these, three variants were isolated from N. benthamiana leaf, of which AnfH variant 6 (AnfH V6, T200A T228V E241H) had the best features, being approx. 90-fold more abundant in the soluble fraction. Importantly, AnfH V6 was also functional in ¹⁵N₂ reduction with A. vinelandii AnfDKG after [Fe₄S₄] cluster reconstitution. These results show that the computational design strategy used here is a powerful approach for engineering nitrogenase into plants and more broadly to plant synthetic biology and recombinant protein production.