A colorimetric method to measure in vitro nitrogenase functionality for engineering nitrogen fixation.

Biological nitrogen fixation (BNF) is the reduction of N2 into NH3 in a group of prokaryotes by an extremely O2-sensitive protein complex called nitrogenase. Transfer of the BNF pathway directly into plants, rather than by association with microorganisms, could generate crops that are less dependent on synthetic nitrogen fertilizers and increase agricultural productivity and sustainability. In the laboratory, nitrogenase activity is commonly determined by measuring ethylene produced from the nitrogenase-dependent reduction of acetylene (ARA) using a gas chromatograph. The ARA is not well suited for analysis of large sample sets nor easily adapted to automated robotic determination of nitrogenase activities. Here, we show that a reduced sulfonated viologen derivative (S2Vred) assay can replace the ARA for simultaneous analysis of isolated nitrogenase proteins using a microplate reader. We used the S2Vred to screen a library of NifH nitrogenase components targeted to mitochondria in yeast. Two NifH proteins presented properties of great interest for engineering of nitrogen fixation in plants, namely NifM independency, to reduce the number of genes to be transferred to the eukaryotic host; and O2 resistance, to expand the half-life of NifH iron-sulfur cluster in a eukaryotic cell. This study established that NifH from Dehalococcoides ethenogenes did not require NifM for solubility, [Fe-S] cluster occupancy or functionality, and that NifH from Geobacter sulfurreducens was more resistant to O2 exposure than the other NifH proteins tested. It demonstrates that nitrogenase components with specific biochemical properties such as a wider range of O2 tolerance exist in Nature, and that their identification should be an area of focus for the engineering of nitrogen-fixing crops.

Environment and coordination of FeMo-co in the nitrogenase metallochaperone NafY.

In nitrogenase biosynthesis, the iron-molybdenum cofactor (FeMo-co) is externally assembled at scaffold proteins and delivered to the NifDK nitrogenase component by the NafY metallochaperone. Here we have used nuclear magnetic resonance, molecular dynamics, and functional analysis to elucidate the environment and coordination of FeMo-co in NafY. H121 stands as the key FeMo-co ligand. Regions near FeMo-co diverge from H121 and include the η1, α1, α2 helical lobe and a narrow path between H121 and C196.

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

Engineering nitrogen fixation in eukaryotes requires high expression of functional nitrogenase structural proteins, a goal that has not yet been achieved. Here we build a knowledge-based library containing 32 nitrogenase nifH sequences from prokaryotes of diverse ecological niches and metabolic features and combine with rapid screening in tobacco to identify superior NifH variants for plant mitochondria expression. Three NifH variants outperform in tobacco mitochondria and are further tested in yeast. Hydrogenobacter thermophilus (Aquificae) NifH is isolated in large quantities from yeast mitochondria and fulfills NifH protein requirements for efficient N2 fixation, including electron transfer for substrate reduction, P-cluster maturation, and FeMo-co biosynthesis. H. thermophilus NifH expressed in tobacco leaves shows lower nitrogenase activity than that from yeast. However, transfer of [Fe4S4] clusters from NifU to NifH in vitro increases 10-fold the activity of the tobacco-isolated NifH, revealing that plant mitochondria [Fe-S] cluster availability constitutes a bottleneck to engineer plant nitrogenases.

Improvement of the Activity of a Fungal Versatile-Lipase Toward Triglycerides: An in silico Mechanistic Description.

Some enzymes that belong to the Candida rugosa-like lipase family (abH03. 01) combine the activities of lipases and sterol esterases. Thus, they can act on water-insoluble carboxylic esters releasing long-chain fatty acids but also on sterol esters, although with different activity and affinity. The differences in the catalytic properties among the proteins of this family are explained by small changes in the hydrophobicity of some regions. One of such versatile enzymes is the sterol esterase/lipase from Ophiostoma piceae (OPE) that acts very efficiently on the two types of substrates. Structurally, OPE is characterized by the presence of a lid formed by a α-helix and two 310-helices rich in hydrophobic amino acids. In this study, the ope gene was modified by directed mutagenesis in order to change specific amino acids in the lid region to modify its structure with the aim of increasing its hydrophobicity. Several recombinant forms of OPE were heterologously produced in Pichia pastoris. In silico molecular dynamics simulations have been used to decipher the mechanistic principles behind the improvements in substrate catalysis. The analyses suggested that the enhanced activity toward hydrophobic substrates such as triglycerides could be due to a better stabilization of the substrate in the lid region as a result of an increased hydrophobicity and an improved topology. These results indicate that in silico simulations can be useful for the optimization of the activity of lipases from the C. rugose-like family for different biotechnological applications.

Non-canonical regulation of glutathione and trehalose biosynthesis characterizes non-Saccharomyces wine yeasts with poor performance in active dry yeast production.

Several yeast species, belonging to Saccharomyces and non-Saccharomyces genera, play fundamental roles during spontaneous must grape fermentation, and recent studies have shown that mixed fermentations, co-inoculated with S. cerevisiae and non-Saccharomyces strains, can improve wine organoleptic properties. During active dry yeast (ADY) production, antioxidant systems play an essential role in yeast survival and vitality as both biomass propagation and dehydration cause cellular oxidative stress and negatively affect technological performance. Mechanisms for adaptation and resistance to desiccation have been described for S. cerevisiae, but no data are available on the physiology and oxidative stress response of non-Saccharomyces wine yeasts and their potential impact on ADY production. In this study we analyzed the oxidative stress response in several non-Saccharomyces yeast species by measuring the activity of reactive oxygen species (ROS) scavenging enzymes, e.g., catalase and glutathione reductase, accumulation of protective metabolites, e.g., trehalose and reduced glutathione (GSH), and lipid and protein oxidation levels. Our data suggest that non-canonical regulation of glutathione and trehalose biosynthesis could cause poor fermentative performance after ADY production, as it corroborates the corrective effect of antioxidant treatments, during biomass propagation, with both pure chemicals and food-grade argan oil.