Integrated genome-transcriptome analysis unveiled the mechanism of Debaryomyces hansenii-mediated arsenic stress amelioration in rice.

Globally, rice is becoming more vulnerable to arsenic (As) pollution, posing a serious threat to public food safety. Previously Debaryomyces hansenii was found to reduce grain As content of rice. To better understand the underlying mechanism, we performed a genome analysis to identify the key genes in D. hansenii responsible for As tolerance and plant growth promotion. Notably, genes related to As resistance (ARR, Ycf1, and Yap) were observed in the genome of D. hansenii. The presence of auxin pathway and glutathione metabolism-related genes may explain the plant growth-promoting potential and As tolerance mechanism of this novel yeast strain. The genome annotation of D. hansenii indicated that it contains a repertoire of genes encoding antioxidants, well corroborated with the in vitro studies of GST, GR, and glutathione content. In addition, the effect of D. hansenii on gene expression profiling of rice plants under As stress was also examined. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database revealed 307 genes, annotated in D. hansenii-treated rice, related to metabolic pathways (184), photosynthesis (12), glutathione (10), tryptophan (4), and biosynthesis of secondary metabolite (117). Higher expression of regulatory elements like AUX/IAA and WRKY transcription factors (TFs), and defense-responsive genes dismutases, catalases, peroxiredoxin, and glutaredoxins during D. hansenii+As exposure was also observed. Combined analysis revealed that D. hansenii genes are contributing to stress mitigation in rice by supporting plant growth and As-tolerance. The study lays the foundation to develop yeast as a beneficial biofertilizer for As-prone areas.

Open (non-sterile) cultivations of Debaryomyces hansenii for recombinant protein production combining industrial side-streams with high salt content.

The halotolerant non-conventional yeast Debaryomyces hansenii can grow in media containing high concentrations of salt (up to 4 M), metabolize alternative carbon sources than glucose, such as lactose or glycerol, and withstand a wide range of temperatures and pH. These inherent capabilities allow this yeast to grow in harsh environments and use alternative feedstock than traditional commercial media. For example, D. hansenii could be a potential cell factory for revalorizing industrial salty by-products, using them as a substrate for producing new valuable bioproducts, boosting a circular economy. In this work, three different salty by-products derived from the dairy and biopharmaceutical industry have been tested as a possible feedstock for D. hansenii's growth. The yeast was not only able to grow efficiently in all of them but also to produce a recombinant protein (Yellow Fluorescent Protein, used as a model) without altering its performance. Moreover, open cultivations at different laboratory scales (1.5 mL and 1 L) were performed under non-sterile conditions and without adding fresh water or any nutritional supplement to the cultivation, making the process cheaper and more sustainable.

Putative 3-nitrotyrosine detoxifying genes identified in the yeast Debaryomyces hansenii: in silico search of regulatory sequences responsive to salt and nitrogen stress

Background: During salt stress, the yeast Debaryomyces hansenii synthesizes tyrosine as a strategy to avoid the oxidation of proteins. Tyrosine reacts with nitrogen radicals to form 3-nitrotyrosine. 3-nitrotyrosine prevents the effects of associated oxidative stress and thus contributes to the high halotolerace of the yeast. However, the mechanism of how D. hansenii counteracts the presence of this toxic compound is unclear. In this work, we evaluated D. hansenii's capacity to assimilate 3-nitrotyrosine as a unique nitrogen source and measured its denitrase activity under salt stress. To identify putative genes related to the assimilation of 3-nitrotyrosine, we performed an in silico search in the promoter regions of D. hansenii genome. Results: We identified 15 genes whose promoters had binding site sequences for transcriptional factors of sodium, nitrogen, and oxidative stress with oxidoreductase and monooxygenase GO annotations. Two of these genes, DEHA2E24178g and DEHA2C00286g, coding for putative denitrases and having GATA sequences, were evaluated by RT-PCR and showed high expression under salt and nitrogen stress. Conclusions: D. hansenii can grow in the presence of 3-nitrotyrosine as the only nitrogen source and has a high specific denitrase activity to degrade 3-nitrotyrosine in 1 and 2 M NaCl stress conditions. The results suggest that given the lack of information on transcriptional factors in D. hansenii, the genes identified in our in silico analysis may help explain 3-nitrotyrosine assimilation mechanisms.

Growth and metabolic activity of conventional and non-conventional yeasts immobilized in foamed alginate.

The aim of this research was to study how the cell immobilization technique of forming foamed alginate gels influences the growth, vitality and metabolic activity of different yeasts. Two distinct strains were used, namely conventional yeast (exemplified by Saccharomyces cerevisiae) and a non-conventional strain (exemplified by Debaryomyces occidentalis). The encapsulation of the yeast cells was performed by the traditional process of droplet formation, but from a foamed alginate solution. The activities of two key enzymes, succinate dehydrogenase and pyruvate decarboxylase, together with the ATP content were measured in both the free and immobilized cells. This novel method of yeast cell entrapment had some notable effects. The number of living immobilized cells reached the level of 10(6)-10(7) per single bead, and was stable during the fermentation process. Reductions in both enzyme activity and ATP content were observed in all immobilized yeasts. However, S. cerevisiae showed higher levels of ATP and enzymatic activity than D. occidentalis. Fermentation trials with immobilized repitching cells showed that the tested yeasts adapted to the specific conditions. Nevertheless, the mechanical endurance of the carriers and the internal structure of the gel need to be improved to enable broad applications of alginate gels in industrial fermentation processes, especially with conventional yeasts. This is one of the few papers and patents that describe the technique of cell immobilization in foamed alginate and shows the fermentative capacities and activities of key enzymes in immobilized yeast cells.

Mitochondria from the salt-tolerant yeast Debaryomyces hansenii (halophilic organelles?).

The yeast Debaryomyces hansenii is considered a marine organism. Sea water contains 0.6 M Na(+) and 10 mM K(+); these cations permeate into the cytoplasm of D. hansenii where proteins and organelles have to adapt to high salt concentrations. The effect of high concentrations of monovalent and divalent cations on isolated mitochondria from D. hansenii was explored. As in S. cerevisiae, these mitochondria underwent a phosphate-sensitive permeability transition (PT) which was inhibited by Ca(2+) or Mg(2+). However, D. hansenii mitochondria require higher phosphate concentrations to inhibit PT. In regard to K(+) and Na(+), and at variance with mitochondria from all other sources known, these monovalent cations promoted closure of the putative mitochondrial unspecific channel. This was evidenced by the K(+)/Na(+)-promoted increase in: respiratory control, transmembrane potential and synthesis of ATP. PT was equally sensitive to either Na(+) or K(+). In the presence of propyl-gallate PT was still observed while in the presence of cyanide the alternative pathway was not active enough to generate a Delta Psi due to a low AOX activity. In D. hansenii mitochondria K(+) and Na(+) optimize oxidative phosphorylation, providing an explanation for the higher growth efficiency in saline environments exhibited by this yeast.

The crystal structures of yeast Get3 suggest a mechanism for tail-anchored protein membrane insertion.

Tail-anchored (TA) proteins represent a unique class of membrane proteins that contain a single C-terminal transmembrane helix. The post-translational insertion of the yeast TA proteins into the ER membrane requires the Golgi ER trafficking (GET) complex which contains Get1, Get2 and Get3. Get3 is an ATPase that recognizes and binds the C-terminal transmembrane domain (TMD) of the TA proteins. We have determined the crystal structures of Get3 from two yeast species, S. cerevisiae and D. hansenii, respectively. These high resolution crystal structures show that Get3 contains a nucleotide-binding domain and a "finger" domain for binding the TA protein TMD. A large hydrophobic groove on the finger domain of S. cerevisiae Get3 structure might represent the binding site for TMD of TA proteins. A hydrophobic helix from a symmetry-related Get3 molecule sits in the TMD-binding groove and mimics the TA binding scenario. Interestingly, the crystal structures of the Get3 dimers from S. cerevisiae and D. hansenii exhibit distinct conformations. The S. cerevisiae Get3 dimer structure does not contain nucleotides and maintains an "open" conformation, while the D. hansenii Get3 dimer structure binds ADP and stays in a "closed" conformation. We propose that the conformational changes to switch the Get3 between the open and closed conformations may facilitate the membrane insertions for TA proteins.