Discovery, Expression, and In Silico Safety Evaluation of Honey Truffle Sweetener, a Sweet Protein Derived from Mattirolomyces terfezioides and Produced by Heterologous Expression in Komagataella phaffii.

Honey truffle sweetener (HTS), a 121 amino acid protein is identified as a high-intensity sweetener found naturally occurring in the Hungarian Sweet Truffle Mattirolomyces terfezioides, an edible mushroom used in regional diets. The protein is intensely sweet, but the truffle is difficult to cultivate; therefore, the protein was systematically characterized, and the gene coding for the protein was expressed in a commonly used host yeast Komagataella phaffii. The heterologously expressed protein maintained the structural characteristics and sweet taste of the truffle. Preliminary safety evaluations for use as a food ingredient were performed on the protein including digestibility and in silico approaches for predicting the allergenicity and toxicity of the protein. HTS is predicted to be nonallergenic, nontoxic, and readily digestible. This protein is readily produced by precision fermentation of the host yeast, making it a potential replacement for both added sugars and small molecule high-intensity sweeteners in food.

Concentration of docosahexaenoic acid from tuna oil via a two lipase-catalyzed reaction.

Docosahexaenoic acid (DHA) was concentrated successfully in the glyceride fractions from tuna oil via a two-step enzyme reaction involving hydrolysis and ethanolysis. In the first step, Candida rugosa lipase-catalyzed hydrolysis was carried out to concentrate DHA in the glyceride fractions. The DHA content in the glyceride fraction after hydrolysis increased from 30% in the initial tuna oil to 46%. In the second step, Lipozyme RM IM-catalyzed ethanolysis was conducted with the reaction mixture from the first step to further concentrate DHA in the glyceride fraction. In this step, the reaction mixture obtained from the first step was employed directly in Lipozyme RM IM-catalyzed ethanolysis without additional steps needed to remove free fatty acid. Finally, DHA was concentrated from an initial content of 30% in the tuna oil to 68.4% in the glyceride fractions via a novel two-step enzyme reaction strategy.

Characterization of recombinant human lactoferrin expressed in Komagataella phaffii.

This work presents a thorough characterization of Helaina recombinant human lactoferrin (rhLF, Effera™) expressed in a yeast system at an industrial scale for the first time. Proteomic analysis confirmed that its amino acid sequence is identical to that of native human LF. N-linked glycans were detected at three known glycosylation sites, namely, Asparagines-156, -497, and -642 and they were predominantly oligomannose structures having five to nine mannoses. Helaina rhLF's protein secondary structure was nearly identical to that of human milk lactoferrin (hmLF), as revealed by microfluidic modulation spectroscopy. Results of small-angle X-ray scattering (SAXS) and analytical ultracentrifugation analyses confirmed that, like hmLF, Helaina rhLF displayed well-folded globular structures in solution. Reconstructed solvent envelopes of Helaina rhLF, obtained through the SAXS analysis, demonstrated a remarkable fit with the reported crystalline structure of iron-bound native hmLF. Differential scanning calorimetry investigations into the thermal stability of Helaina rhLF revealed two distinct denaturation temperatures at 68.7 ± 0.9 °C and 91.9 ± 0.5 °C, consistently mirroring denaturation temperatures observed for apo- and holo-hmLF. Overall, Helaina rhLF differed from hmLF in the N-glycans they possessed; nevertheless, the characterization results affirmed that Helaina rhLF was of high purity and exhibited globular structures closely akin to that of hmLF.

Effects of Yeast Species and Processing on Intestinal Health and Transcriptomic Profiles of Atlantic Salmon (Salmo salar) Fed Soybean Meal-Based Diets in Seawater.

The objective of the current study was to examine the effects of yeasts on intestinal health and transcriptomic profiles from the distal intestine and spleen tissue of Atlantic salmon fed SBM-based diets in seawater. Cyberlindnera jadinii (CJ) and Wickerhamomyces anomalus (WA) yeasts were heat-inactivated with spray-drying (ICJ and IWA) or autolyzed at 50 °C for 16 h (ACJ and AWA), followed by spray-drying. Six diets were formulated, one based on fishmeal (FM), a challenging diet with 30% soybean meal (SBM) and four other diets containing 30% SBM and 10% of each of the four yeast fractions (i.e., ICJ, ACJ, IWA and AWA). The inclusion of CJ yeasts reduced the loss of enterocyte supranuclear vacuolization and reduced the population of CD8α labeled cells present in the lamina propria of fish fed the SBM diet. The CJ yeasts controlled the inflammatory responses of fish fed SBM through up-regulation of pathways related to wound healing and taurine metabolism. The WA yeasts dampened the inflammatory profile of fish fed SBM through down-regulation of pathways related to toll-like receptor signaling, C-lectin receptor, cytokine receptor and signal transduction. This study suggests that the yeast species, Cyberlindnera jadinii and Wickerhamomyces anomalus are novel high-quality protein sources with health-beneficial effects in terms of reducing inflammation associated with feeding plant-based diets to Atlantic salmon.

Antimicrobial activity of sophorolipids produced by Starmerella bombicola against phytopathogens from cherry tomato.

BACKGROUND: Phytopathogenic microorganisms are the main cause of plant diseases, generating significant economic losses for the agricultural and food supply chain. Cherry tomatoes (Solanum lycopersicum var. cerasiforme) are very perishable plants and highly demanding in the use of pesticides; therefore, alternative solutions such as biosurfactants have aroused as a potent substituent. The main objective of the present study was to investigate the antimicrobial activity of sophorolipids against the phytopathogens Botrytis cinerea, Sclerotium rolfsii, Rhizoctonia solani and Pythium ultimum. RESULTS: The biosurfactant inhibited the mycelial growth in vitro with a minimum concentration of 2 mg mL-1 . The application of sophorolipids at 1, 2 and 4 mg mL-1 in detached leaves of tomato before the inoculation of the fungus B. cinerea was the best treatment, reducing leaf necrosis by up to 76.90%. The use of sophorolipids for washing tomato fruits before the inoculation of B. cinerea was able to inhibit the development of gray mold by up to 96.27%. CONCLUSION: The results for tomato leaves and fruits revealed that the biosurfactant acts more effectively when used preventively. Sophorolipids are stable molecules that show promising action for the potential replacement of pesticides in the field and the post-harvest process against the main tomato phytopathogens. © 2021 Society of Chemical Industry.

Formulation of the biological control yeast Meyerozyma caribbica by electrospraying process: effect on postharvest control of anthracnose in mango (Mangifera indica L.) and papaya (Carica papaya L.).

BACKGROUND: Microorganism for biological control of fruit diseases is an eco-friendly alternative to the use of chemical fungicides. RESULTS: This is the first study evaluating the electrospraying process to encapsulate the biocontrol yeast Meyerozyma caribbica. The effect of encapsulating material [Wey protein concentrate (WPC), Fibersol® and Trehalose], its concentration and storage temperature on the cell viability of M. caribbica, and in vitro and in vivo control of Colletotrichum gloeosporioides was evaluated. The processing with commercial resistant maltodextrin (Fibersol®) 30% (w/v) as encapsulating material showed the highest initial cell viability (95.97 ± 1.01%). The storage at 4 ± 1 °C showed lower losses of viability compared to 25 ± 1 °C. Finally, the encapsulated yeast with Fibersol 30% w/v showed inhibitory activity against anthracnose in the in vitro and in vivo tests, similar to yeast fresh cells. CONCLUSION: Electrospraying was a highly efficient process due to the high cell viability, and consequently, a low quantity of capsules is required for the postharvest treatment of fruits. Additionally, the yeast retained its antagonistic power during storage. © 2021 Society of Chemical Industry.

Sucrose gradient chromatin enrichment for quantitative proteomics analysis in budding yeast.

Here, we describe a fractionation protocol optimized to quantify changes in relative abundance of the chromatin-bound proteome (chromatome) by tandem mass tag multiplexing-based tandem mass spectrometry. It has been applied to yeast cells before and after exposure to DNA-damaging drugs to characterize changes in chromatin composition induced by the DNA damage response. We detail steps for stringent chromatin fractionation, sample preparation for mass spectrometry, and its evaluation. For complete details on the use and execution of this protocol, please refer to Challa et al. (2021).

Wickerhamomyces Yeast Killer Toxins' Medical Applications.

Possible implications and applications of the yeast killer phenomenon in the fight against infectious diseases are reviewed, with particular reference to some wide-spectrum killer toxins (KTs) produced by Wickerhamomyces anomalus and other related species. A perspective on the applications of these KTs in the medical field is provided considering (1) a direct use of killer strains, in particular in the symbiotic control of arthropod-borne diseases; (2) a direct use of KTs as experimental therapeutic agents; (3) the production, through the idiotypic network, of immunological derivatives of KTs and their use as potential anti-infective therapeutics. Studies on immunological derivatives of KTs in the context of vaccine development are also described.

Micro-Aqueous Organic System: A Neglected Model in Computational Lipase Design?

Water content is an important factor in lipase-catalyzed reactions in organic media but is frequently ignored in the study of lipases by molecular dynamics (MD) simulation. In this study, Candida antarctica lipase B, Candida rugosa lipase and Rhizopus chinensis lipase were used as research models to explore the mechanisms of lipase in micro-aqueous organic solvent (MAOS) media. MD simulations indicated that lipases in MAOS systems showed unique conformations distinguished from those seen in non-aqueous organic solvent systems. The position of water molecules aggregated on the protein surface in MAOS media is the major determinant of the unique conformations of lipases and particularly impacts the distribution of hydrophilic and hydrophobic amino acids on the lipase surface. Additionally, two maxima were observed in the water-lipase radial distribution function in MAOS systems, implying the formation of two water shells around lipase in these systems. The energy landscapes of lipases along solvent accessible areas of catalytic residues and the minimum energy path indicated the dynamic open states of lipases in MAOS systems differ from those in other solvent environments. This study confirmed the necessity of considering the influence of the microenvironment on MD simulations of lipase-catalyzed reactions in organic media.

Aquaporins - Expression, purification and characterization.

Aquaporin water channels facilitate the bi-directional flow of water and small, neutral solutes down an osmotic gradient in all kingdoms of life. Over the last two decades, the availability of high-quality protein has underpinned progress in the structural and functional characterization of these water channels. In particular, recombinant protein technology has guaranteed the supply of aquaporin samples that were of sufficient quality and quantity for further study. Here we review the features of successful expression, purification and characterization strategies that have underpinned these successes and that will drive further breakthroughs in the field. Overall, Escherichia coli is a suitable host for prokaryotic isoforms, while Pichia pastoris is the most commonly-used recombinant host for eukaryotic variants. Generally, a two-step purification procedure is suitable after solubilization in glucopyranosides and most structures are determined by X-ray following crystallization.

The distribution of phosphatidylinositol 4,5-bisphosphate in the budding yeast plasma membrane.

Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) is generated through phosphorylation of phosphatidylinositol 4-phosphate (PtdIns(4)P) by Mss4p, the only PtdIns phosphate 5-kinase in yeast cells. PtdIns(4,5)P2 is involved in various kinds of yeast functions. PtdIns(4)P is not only the immediate precursor of PtdIns(4,5)P2, but also an essential signaling molecule in the plasma membrane, Golgi, and endosomal system. To analyze the distribution of PtdIns(4,5)P2 and PtdIns(4)P in the yeast plasma membrane at a nanoscale level, we employed a freeze-fracture electron microscopy (EM) method that physically immobilizes lipid molecules in situ. It has been reported that the plasma membrane of budding yeast can be divided into three distinct areas: furrowed, hexagonal, and undifferentiated flat. Previously, using the freeze-fracture EM method, we determined that PtdIns(4)P is localized in the undifferentiated flat area, avoiding the furrowed and hexagonal areas of the plasma membrane. In the present study, we found that PtdIns(4,5)P2 was localized in the cytoplasmic leaflet of the plasma membrane, and concentrated in the furrowed area. There are three types of PtdIns 4-kinases which are encoded by stt4, pik1, and lsb6. The labeling density of PtdIns(4)P in the plasma membrane significantly decreased in both pik1ts and stt4ts mutants. However, the labeling densities of PtdIns(4,5)P2 in the plasma membrane of both the pik1ts and stt4ts mutants were comparable to that of the wild type yeast. These results suggest that PtdIns(4)P produced by either Pik1p or Stt4p is immediately phosphorylated by Mss4p and converted to PtdIns(4,5)P2 at the plasma membrane.

Seeking faster, alternative methods for glycolipid biosurfactant characterization and purification.

Biosurfactants have been investigated as potential alternatives for synthetic surfactants in several areas, for example, in environmental and pharmaceutical fields. In that regard, extensive research has been carried out with sophorolipids and rhamnolipids that also present various biological properties with therapeutic significance. These biosurfactants are obtained as complex mixtures of slightly different molecules, and thus when studying these microbial glycolipids, the ability to identify and purify the produced compounds is of extreme importance. This study aimed to develop improved methodologies for the identification, separation, and purification of sophorolipids and rhamnolipids. Therefore, an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was modified to ensure faster characterization of both sophorolipids and rhamnolipids, enabling the identification and fragmentation pattern description of 10 and 13 congeners, respectively. The separation and purification of these biosurfactants was achieved with novel reversed-phase solid-phase extraction methods guaranteeing the isolation of different glycolipids, including those considered for their significant biological activity (e.g. antimicrobial, anticancer). It was possible to isolate sophorolipids and rhamnolipids with purity of 94% and 99%, respectively. The methods presented herein can be easily implemented and are expected to make purification of these biosurfactants easier, facilitating the study of their individual properties in further works.

Protocol for measuring sphingolipid metabolism in budding yeast.

Sphingolipid biosynthesis occurs in both the endoplasmic reticulum (ER) and the Golgi apparatus. Ceramide synthesized in the ER is transported to the Golgi and incorporated into complex sphingolipids. Here, we present a step-by-step protocol to analyze sphingolipid metabolism in budding yeast. Ceramide and inositolphosphorylceramide (IPC) are classes of sphingolipids present in yeast and are metabolically labeled with radioactive precursors. This protocol for metabolic labeling can be used to investigate ceramide transport in an in vivo environment. For complete details on the use and execution of this protocol, please refer to Ikeda et al. (2020).

Sourdough yeast-bacteria interactions can change ferulic acid metabolism during fermentation.

The metabolism of ferulic acid (FA) was studied during fermentation with different species and strains of lactic acid bacteria (LAB) and yeasts, in synthetic sourdough medium. Yeast strains of Kazachstania humilis, Kazachstania bulderi, and Saccharomyces cerevisiae, as well as lactic acid bacteria strains of Fructilactobacillus sanfranciscensis, Lactiplantibacillus plantarum, Lactiplantibacillus xiangfangensis, Levilactobacillus hammesii, Latilactobacillus curvatus and Latilactobacillus sakei were selected from French natural sourdoughs. Fermentation in presence or absence of FA was carried out in LAB and yeasts monocultures, as well as in LAB/yeast co-cultures. Our results indicated that FA was mainly metabolized into 4-vinylguaiacol (4-VG) by S. cerevisiae strains, and into dihydroferulic acid (DHFA) and 4-VG in the case of LAB. Interactions of LAB and yeasts led to the modification of FA metabolism, with a major formation of DHFA, even by the strains that do not produce it in monoculture. Interestingly, FA was almost completely consumed by the F. sanfranciscensis bFs17 and K. humilis yKh17 pair and converted into DHFA in 89.5 ± 19.6% yield, while neither bFs17, nor yKh17 strains assimilated FA in monoculture.

Co-culture with Tetragenococcus halophilus improved the ethanol tolerance of Zygosaccharomyces rouxii by maintaining cell surface properties.

The accumulation of ethanol has a negative effect on the viability and fermentation performance of microorganisms during the production of fermented foods because of its toxicity. In this study, we investigated the effect of co-culture with Tetragenococcus halophilus on ethanol stress resistance of Zygosaccharomyces rouxii. The result showed that co-culture with T. halophilus promoted cell survival of Z. rouxii under ethanol stress, and the tolerance improved with increasing co-culture time when ethanol content was 8%. Physiological analysis showed that the co-cultured Z. rouxii cells maintained higher intracellular content of trehalose and amino acids including tyrosine, tryptophan, arginine and proline after 8% ethanol stress for 90 min. The membrane integrity analysis and biophysical analysis of the cell surface indicated that the presence of ethanol resulted in cell membrane damage and changes of Young's modulus value and roughness of cell surface. While the co-cultured Z. rouxii cells exhibited better membrane integrity, stiffer and smoother cell surface than single-cultured cells under ethanol stress. As for transcriptomic analyses, the genes involved in unsaturated fatty acid biosynthesis, trehalose biosynthesis, various types of N-glycan biosynthesis, inositol phosphate metabolism, MAPK signaling pathway and tight junction had higher expression in co-cultured Z. rouxii cells with down-regulation of majority of gene expression after stress. And these genes may function in the improvement of ethanol tolerance of Z. rouxii in co-culture.

Molecular characterization of Hsf1 as a master regulator of heat shock response in the thermotolerant methylotrophic yeast Ogataea parapolymorpha.

Ogataea parapolymorpha (Hansenula polymorpha DL-1) is a thermotolerant methylotrophic yeast with biotechnological applications. Here, O. parapolymorpha genes whose expression is induced in response to heat shock were identified by transcriptome analysis and shown to possess heat shock elements (HSEs) in their promoters. The function of O. parapolymorpha HSF1 encoding a putative heat shock transcription factor 1 (OpHsf1) was characterized in the context of heat stress response. Despite exhibiting low sequence identity (26%) to its Saccharomyces cerevisiae homolog, OpHsf1 harbors conserved domains including a DNA binding domain (DBD), domains involved in trimerization (TRI), transcriptional activation (AR1, AR2), transcriptional repression (CE2), and a C-terminal modulator (CTM) domain. OpHSF1 could complement the temperature sensitive (Ts) phenotype of a S. cerevisiae hsf1 mutant. An O. parapolymorpha strain with an H221R mutation in the DBD domain of OpHsf1 exhibited significantly retarded growth and a Ts phenotype. Intriguingly, the expression of heat-shock-protein-coding genes harboring HSEs was significantly decreased in the H221R mutant strain, even under non-stress conditions, indicating the importance of the DBD for the basal growth of O. parapolymorpha. Notably, even though the deletion of C-terminal domains (ΔCE2, ΔAR2, ΔCTM) of OpHsf1 destroyed complementation of the growth defect of the S. cerevisiae hsf1 strain, the C-terminal domains were shown to be dispensable in O. parapolymorpha. Overexpression of OpHsf1 in S. cerevisiae increased resistance to transient heat shock, supporting the idea that OpHsf1 could be useful in the development of heat-shock-resistant yeast host strains.

Secretome analysis as a tool to elucidate bacterial contamination influence during second-generation ethanol production in a Melle-Boinot process.

Melle-boinot fermentation process can be used to increase the ethanol productivity in second-generation ethanol process (2G). However, bacterial contamination can result in decreased ethanol production and sugars consumption. The available literature on microbial contamination in the 2G at the secretome level, microbial interactions and their impacts on ethanol production are scarce. In this context, the cultivation of Spathaspora passalidarum was studied in pure and co-culture with Lactobacillus fermentum under conditions that mimic the Melle-boinot process. Glucose consumption and ethanol production by S. passalidarum were not affected by bacterial contamination. Xylose consumption was higher in pure culture (11.54 ± 2.62, 16.23 ± 1.76 and 6.50 ± 1.68 g) than in co-culture fermentation (11.89 ± 0.38, 7.29 ± 0.49 and 5.54 ± 2.63 g) in cycle 2. The protein profile of the fermented broth was similar in pure and co-culture fermentation. The low effect of L. fermentum on fermentation and protein profile may be associated with the inhibition of the bacteria by the low nutrient fermentation broth, with centrifugation and/or with sulfuric acid washing. Thereby, considering that research on microbial contamination in the 2G fermentation process is very limited, particularly at the omics level, these findings may contribute to the lignocellulosic biomass fermentation industry.

Identification of proteins responsive to heterologous protein production in thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656.

The thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656 is a potential host for heterologous protein production. However, overproduction of heterologous protein can induce cellular stress and limit the level of its secretion. To improve the secretion of heterologous protein, we identified the candidate proteins with altered production during production of heterologous protein in O. thermomethanolica by using a label-free comparative proteomic approach. Four hundred sixty-four proteins with various biological functions showed differential abundance between O. thermomethanolica expressing fungal xylanase (OT + Xyl) and a control strain. The induction of proteins in transport and proteasomal proteolysis was prominently observed. Eight candidate proteins involved in cell wall biosynthesis (Chs3, Gas4), chaperone (Sgt2, Pex19), glycan metabolism (Csf1), protein transport (Ypt35), and vacuole and protein sorting (Cof1, Npr2) were mutated by a CRISPR/Cas9 approach. An Sgt2 mutant showed higher phytase and xylanase activity compared with the control strain (13%-20%), whereas mutants of other genes including Cof1, Pex19, Gas4, and Ypt35 showed lower xylanase activity compared with the control strain (15%-25%). In addition, an Npr2 mutant showed defective growth, while overproduction of Npr2 enhanced xylanase activity. These results reveal genes that can be mutated to modulate heterologous protein production and growth of O. thermomethanolica TBRC656.

Applications of gellan natural polymer microspheres in recombinant catechol-O-methyltransferase direct capture from a Komagataella pastoris lysate.

The present work shows the application of nickel- and magnesium-crosslinked gellan microspheres in ionic and affinity capture strategies to directly extract hSCOMT from the complex Komagataella pastoris lysate through a simple batch method. Both formulations present similar morphology, but nickel-crosslinked microspheres present higher crosslinker content and smaller diameters. Four different capture strategies were established, by manipulating the ionic strength, pH, temperature and competing agents' presence. The most promising results for hSCOMT capture and clarification were obtained employing an ionic strategy with nickel-crosslinked microspheres and an affinity strategy with magnesium-crosslinked microspheres at 4 °C. The bioactivity results (200%) and purification degree (70%) of hSCOMT captured by the ionic strategy were more satisfactory probably due to the soft ionic conditions used (100 mM NaCl). For the first time, the gellan polysaccharide versatility was demonstrated in the microsphere application for the direct capture of hSCOMT from a complex lysate, simplifying isolation biotechnological procedures.

Optimization, Purification and Antitumor Activity of Kodamaea ohmeri ANOMY L-Asparaginase Isolated from Banana Peel.

OBJECTIVE: L-Asparaginase is an important enzyme that converts L-asparagine to L-aspartate and ammonia. Microbial L-asparaginase has important applications as anticancer and food processing agents. METHODS: This study reported the isolation, screening of a local yeast isolate from banana peel for L-asparaginase production using submerged fermentation, optimization of the production, purification, and anticancer assay of L-asparaginase. The yeast isolate was identified as Kodamaea ohmeri ANOMY based on the analysis of nuclear large subunit (26S) rDNA partial sequences. It was a promising L-asparaginase producer with a specific activity of 3059±193 U/mg in a non-optimized medium. The classical one-variable-at-a-time method was used to optimize the production medium components, and it was found that the elimination of K2HPO4 from the medium increased L-asparaginase specific activity (3100.90±180 U/mg). RESULTS: Statistical optimization of L-asparaginase production was done using Plackett-Burman and Box-Behnken designs. The production medium for the maximum L-asparaginase specific activity (8500±578U/mg) was as follows (g/L): L-asparagine (7.50), NaNO3 (0.50), MgSO4.7H2O (0.80), KCl (0.80) associated with an incubation period of 5 days, inoculum size of 5.60 %, and pH (7.0). The optimization process increased L-asparaginase production by 2.78-fold compared to the non-optimized medium. L-Asparaginase was purified using ammonium sulphate precipitation followed by gel filtration on a Sephadex G-100 column. Its molecular weight was 66 KDa by SDS-PAGE analysis. CONCLUSION: The cell morphology technique was used to evaluate the anticancer activity of L-asparaginase against three different cell lines. L-Asparaginase inhibited the growth of HepG-2, MCF-7, and HCT-116 cells at a concentration of 20, 50, and 60 µL, respectively.