Yeast cell vacuum infusion into fungal pellets as a novel cell encapsulation methodology.

Immobilized yeast cells are used industrially in winemaking processes such as sparkling wine and Sherry wine production. Here, a novel approach has been explored for the infusion and immobilization of yeast cells into filamentous fungal pellets, which serve as a porous natural material. This was accomplished through vacuum application to force the yeast cells towards the core of the fungal pellets followed by culture in YPD medium to promote their growth from the interior. This method represents an improved variation of a previous approach for the assembly of "yeast biocapsules," which entailed the co-culture of both fungal and yeast cells in the same medium. A comparison was made between both techniques in terms of biocapsule productivity, cell retention capacity, and cell biological activity through an alcoholic fermentation of a grape must. The results indicated a substantial increase in biocapsule productivity (37.40-fold), higher cell retention within the biocapsules (threefold), and reduction in cell leakage during fermentation (twofold). Although the majority of the chemical and sensory variables measured in the produced wine did not exhibit notable differences from those produced utilizing suspended yeast cells (conventional method), some differences (such as herbaceous and toasted smells, acidity, bitterness, and persistence) were perceived and wines positively evaluated by the sensory panel. As the immobilized cells remain functional and the encapsulation technique can be expanded to other microorganisms, it creates potential for additional industrial uses like biofuel, health applications, microbe encapsulation and delivery, bioremediation, and pharmacy. KEY POINTS: • New approach improves biocapsule productivity and cell retention. • Immobilized yeast remains functional in fermentation. • Wine made with immobilized yeast had positive sensory differences.

Flor yeast immobilization in microbial biocapsules for Sherry wine production: microvinification approach.

Sherry wine is a pale-yellowish dry wine produced in Southern-Spain which features are mainly due to biological aging when the metabolism of biofilm-forming yeasts (flor yeasts) consumes ethanol (and other non-fermentable carbon sources) from a previous alcoholic fermentation, and produces volatile compounds such as acetaldehyde. To start aging and maintain the wine stability, a high alcohol content is required, which is achieved by the previous fermentation or by adding ethanol (fortification). Here, an alternative method is proposed which aims to produce a more economic, distinctive Sherry wine without fortification. For this, a flor yeast has been pre-acclimatized to glycerol consumption against ethanol, and later confined in a fungal-based immobilization system known as "microbial biocapsules", to facilitate its inoculum. Once aged, the wines produced using biocapsules and free yeasts (the conventional method) exhibited chemical differences in terms of acidity and volatile concentrations. These differences were evaluated positively by a sensory panel. Pre-acclimatization of flor yeasts to glycerol consumption was not successful but when cells were immobilized in fungal pellets, ethanol consumption was lower. We believe that immobilization of flor yeasts in microbial biocapsules is an economic technique that can be used to produce high quality differentiated Sherry wines.

Use of yeast biocapsules as a fungal-based immobilized cell technology for Indian Pale Ale-type beer brewing.

Immobilized cell technologies (ICT) have been used in wort fermentation, beer maturation, or production of alcohol-free or low-alcohol beer. The purpose of ICT is to restrict intact cells to a specific location while allowing biological function. It improves cell stability, operational flexibility, and control in brewing, as well as ease in executing continuous operations. We investigated the use of yeast biocapsules for Indian Pale Ale (IPA) type beer wort fermentation, a novel ICT in brewing. Yeast biocapsules are a spherical yeast immobilization system in which yeast cells are encapsulated and connected to the hyphae of an inactivated hollow filamentous fungus pellet. Fermentations with yeast encapsulated in alginate beads, as the standard immobilization practice, and in free (non-immobilized) forms were carried out in parallel. We found that yeast biocapsules are a better option for cell reutilization than alginate beads, but worse for beer must clarity. Beer brewed with yeast biocapsules differed in concentration for five volatile compounds (acetaldehyde, diacetyl, ethyl acetate, 1,1-diethoxyethane, and isoamyl alcohol) and three sensory characters (persistency of the foam, malt, and yeast character). KEY POINTS: • Yeast biocapsules were investigated for beer wort fermentation • Biocapsules improve cell reutilization but are limited for beer clarification • Beer brewed with biocapsules is chemically different than conventional beer • Most sensory features did not differ between biocapsule and control beer.

New insights on yeast and filamentous fungus adhesion in a natural co-immobilization system: proposed advances and applications in wine industry.

Fungi possess extraordinary strength in attachment to biotic and abiotic surfaces. This review focuses on adhesion mechanisms of yeast and filamentous fungi and the proposed combination of the adhesive forces of both organisms in an immobilization system called yeast biocapsules, whereby Saccharomyces cerevisiae cells are attached to the hyphae of Penicillium chrysogenum. The natural adherent properties of each organism, one multicellular and another unicellular, allow yeast to be fixated securely on the filamentous fungi and complete alcoholic fermentation. Following alcoholic fermentation, the hyphae become an inert support for yeast cells while maintaining shape and integrity. Biocapsules have been used successfully in both wine and bioethanol production. Investigation of the potential genes involved in fungal-yeast fusion suggests that natural hydrophobic interactions of both organisms play a major role. Analysis of the possible mechanisms involved in fungus and yeast adhesion, future perspectives on improving yeast immobilization, and proposed applications of the biocapsules are explored.

Comparative analysis of intracellular metabolites, proteins and their molecular functions in a flor yeast strain under two enological conditions.

Flor yeasts confer a wide range of organoleptic properties to Sherry-type wines during a process called "biological aging" that takes place after alcoholic fermentation. These kinds of yeasts adapt to a biological aging condition by forming a biofilm known as "flor velum" and by changing from fermentative to oxidative metabolism. It has been reported that some functions such as increase of cell surface hydrophobicity or changes to lipid metabolism are enhanced when yeasts switch to biofilm lifestyle. Here, we attempt to reveal intracellular metabolites and protein molecular functions not documented before that are relevant in biofilm formation and in fermentation by an endometabolome and proteome screening. We report that at early stages of biofilm formation, flor yeasts accumulate mannose, trehalose, glycerol, oleic and stearic acids and synthesize high amounts of GTPases, glycosylases and lipoproteins. On the other hand, in early fermentation, flor yeasts rapidly consume glucose and phosphoric acid; and produce abundant proteins related to chromatin binding, transcription factors and methyl transferases.

Interaction of poly(N-isopropylacrylamide) with sodium dodecyl sulfate below the critical aggregation concentration.

Interaction between the thermoresponsive polymer poly(N-isopropylacrylamide) (P-NIP) and sodium dodecyl sulfate (SDS) both above and below its phase transition temperature was examined under dilute conditions. Above the lower critical solution temperature (LCST) of P-NIP (32 °C), 0.01 wt % P-NIP specifically interacted with 1.0 × 10(-5) mol/L SDS to form a precipitate. However, when SDS was added at concentrations above or below 1.0 × 10(-5) mol/L, the P-NIP solution remained clear above the LCST. A fluorometric probe, N-phenyl-naphthalene, indicated that the hydrophobicity of the aggregates composed of P-NIP and SDS changed at an SDS concentration of 1.0 × 10(-5) mol/L. Although the hydrophobicity of the precipitate was similar to that of P-NIP alone at less than 1.0 × 10(-5) mol/L, it approached that of SDS homomicelles as the SDS concentration increased above 1.0 × 10(-5) mol/L. Dynamic light scattering and turbidimetry studies showed no P-NIP phase transition above an SDS concentration of 1.0 × 10(-5) mol/L, which is much lower than the reported critical association concentration (CAC) of SDS with P-NIP. This indicates that P-NIP interacted with SDS above the LSCT at much lower SDS concentration than the reported CAC.

Filamentous fungal pellets as a novel and sustainable encapsulation matrix for exogenous bioactive compounds.

Edible filamentous fungi (FF) are considered sustainable food materials given their rich nutrient profile and low carbon and water footprints during production. The current study evaluated FF biomass as a natural encapsulation system for exogenous bioactive compounds and as a model system to investigate the complex food matrix-micronutrient interactions during in vitro digestion. Our objective was to compare the fungal pellet, as a multicellular encapsulation system, with single yeast cell-based carriers in terms of loading and release of curcumin, a model compound. The results suggest that the curcumin encapsulation efficiency was similar in single yeast cells and fungal hyphal cells. A vacuum treatment used to facilitate the infusion of curcumin into yeast or fungal cells also enabled rapid internalization of yeast cells into the fungal pellet matrix. Compared to the single-cell encapsulation system, the multicellular systems modified the release kinetics of curcumin during in vitro digestion by eliminating the initial rapid release and reducing the overall release rate of curcumin in the small intestinal phase. These results provide a deeper understanding of the effect of natural edible structures on the bioaccessibility of micronutrients, and demonstrate the potential of using FF biomass as functional food materials.

Edible mycelium as proliferation and differentiation support for anchorage-dependent animal cells in cultivated meat production.

Cultivated meat production requires bioprocess optimization to achieve cell densities that are multiple orders of magnitude higher compared to conventional cell culture techniques. These processes must maximize resource efficiency and cost-effectiveness by attaining high cell growth productivity per unit of medium. Microcarriers, or carriers, are compatible with large-scale bioreactor use, and offer a large surface-area-to-volume ratio for the adhesion and proliferation of anchorage-dependent animal cells. An ongoing challenge persists in the efficient retrieval of cells from the carriers, with conflicting reports on the effectiveness of trypsinization and the need for additional optimization measures such as carrier sieving. To surmount this issue, edible carriers have been proposed, offering the advantage of integration into the final food product while providing opportunities for texture, flavor, and nutritional incorporation. Recently, a proof of concept (POC) utilizing inactivated mycelium biomass derived from edible filamentous fungus demonstrated its potential as a support structure for myoblasts. However, this POC relied on a model mammalian cell line combination with a single mycelium species, limiting realistic applicability to cultivated meat production. This study aims to advance the POC. We found that the species of fungi composing the carriers impacts C2C12 myoblast cell attachment-with carriers derived from Aspergillus oryzae promoting the best proliferation. C2C12 myoblasts effectively differentiated on mycelium carriers when induced in myogenic differentiation media. Mycelium carriers also supported proliferation and differentiation of bovine satellite cells. These findings demonstrate the potential of edible mycelium carrier technology to be readily adapted in product development within the cultivated meat industry.

Specific vulnerability of iPSC-derived motor neurons with TDP-43 gene mutation to oxidative stress.

Amyotrophic lateral sclerosis (ALS) is a disease that affects motor neurons and has a poor prognosis. We focused on TAR DNA-binding protein 43 kDa (TDP-43), which is a common component of neuronal inclusions in many ALS patients. To analyze the contribution of TDP-43 mutations to ALS in human cells, we first introduced TDP-43 mutations into healthy human iPSCs using CRISPR/Cas9 gene editing technology, induced the differentiation of these cells into motor and sensory neurons, and analyzed factors that are assumed to be altered in or associated with ALS (cell morphology, TDP-43 localization and aggregate formation, cell death, TDP-43 splicing function, etc.). We aimed to clarify the pathological alterations caused solely by TDP-43 mutation, i.e., the changes in human iPSC-derived neurons with TDP-43 mutation compared with those with the same genetic background except TDP-43 mutation. Oxidative stress induced by hydrogen peroxide administration caused the death of TDP-43 mutant-expressing motor neurons but not in sensory neurons, indicating the specific vulnerability of human iPSC-derived motor neurons with TDP-43 mutation to oxidative stress. In our model, we observed aggregate formation in a small fraction of TDP-43 mutant-expressing motor neurons, suggesting that aggregate formation seems to be related to ALS pathology but not the direct cause of cell death. This study provides basic knowledge for elucidating the pathogenesis of ALS and developing treatments for the disease.

Assessing Edible Filamentous Fungal Carriers as Cell Supports for Growth of Yeast and Cultivated Meat.

The growth and activity of adherent cells can be enabled or enhanced through attachment to a solid surface. For food and beverage production processes, these solid supports should be food-grade, low-cost, and biocompatible with the cell of interest. Solid supports that are edible can be a part of the final product, thus simplifying downstream operations in the production of fermented beverages and lab grown meat. We provide proof of concept that edible filamentous fungal pellets can function as a solid support by assessing the attachment and growth of two model cell types: yeast, and myoblast cells. The filamentous fungus Aspergillus oryzae was cultured to produce pellets with 0.9 mm diameter. These fugal pellets were inactivated by heat or chemical methods and characterized physicochemically. Chemically inactivated pellets had the lowest dry mass and were the most hydrophobic. Scanning electron microscope images showed that both yeast and myoblast cells naturally adhered to the fungal pellets. Over 48 h of incubation, immobilized yeast increased five-fold on active pellets and six-fold on heat-inactivated pellets. Myoblast cells proliferated best on heat-treated pellets, where viable cell activity increased almost two-fold, whereas on chemically inactivated pellets myoblasts did not increase in the cell mass. These results support the use of filamentous fungi as a novel cell immobilization biomaterial for food technology applications.

Generation and characterization of motor neuron progenitors and motor neurons using metachromatic leukodystrophy-induced pluripotent stem cells.

The pathological consequences leading to primary storage, autophagy impairment, impaired mitochondrial dynamics, and endoplasmic reticulum (ER) stress on neural cell dysfunction and apoptosis in metachromatic leukodystrophy (MLD) have been poorly elucidated. In the present study, we generated 2 cell lines of patient-specific-induced pluripotent stem cells (iPSCs) and modeled the progression of pathological events during the differentiation of iPSCs to motor neuron progenitors (MNPs) and mature motor neurons (MNs). The iPS cells were generated from two late-infantile MLD patient-derived skin fibroblasts using electroporation or the Sendai virus. Olig2+ MNPs were generated from both iPSC lines using a combination of small molecules in a chemically defined neural medium. Furthermore, the MNPs could be differentiated into mature MNs, which was confirmed by RT-PCR and MN markers, including SMI32 and ChAT. The population of MNs was approximately 50% under the culture conditions. Pathological observation of MLD patient-derived iPSCs revealed lysosomal accumulation and impaired autophagy. In addition, both MNPs and MNs derived from MLD-iPSCs showed increased lysosomal accumulation, dysfunctional autophagy, impaired mitophagy, endoplasmic reticulum (ER) stress or unfolded protein response (UPR) activation, and premature cellular death.

Generation of four iPSC lines from a family harboring a 1p36-35 haplotype linked with bipolar disorder and recurrent depressive disorder: Three-generation patients and a healthy sibling.

Induced pluripotent stem cells (iPSCs) obtained from genetically characterized patients benefit the biological study of bipolar disorder (BD). Here, we present iPSC lines from three-generation patients with BD and recurrent depressive disorder (RDD) and a healthy control sibling in a family. All patients shared the specified haplotype in the 1p36-35, previously reported as the susceptibility locus of mood disorders. iPSCs were generated with the reprogramming factors OTC3/4, l-MYC, LIN28, SOX2, KLF4, and p53 shRNA through non-integrated episomal vectors. All iPSC lines strongly expressed pluripotency markers and proved the ability to differentiate into three germ lineages in vitro.

Characterization of the upstream and intron promoters of the gene encoding TAR DNA-binding protein.

TAR DNA-binding protein (TDP-43, encoded by TARDBP) is a multifunctional protein that regulates transcription and RNA metabolism by binding DNA or RNA. TDP-43 has been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS) because abnormal accumulation of cleaved and phosphorylated C-terminal fragments of TDP-43 in motor neurons is a pathological hallmark of ALS. Here, we cloned and analyzed the promoter region of the TARDBP gene. TARDBP upstream sequences and/or intron/luciferase constructs were generated, and their promoter activity was experimentally assessed. The upstream region predictably exhibited promoter activity and identified putative cis-acting elements, including the i-motif, was relevant for the regulation of TDP-43 expression. The cellular abundance of TDP-43 is strictly controlled, and its constancy is critically important for motor neuron survival. A machinery serving to maintain a constant level of TDP-43 is autoregulation via control of mRNA stability, a negative feedback system involving binding to the 3' untranslated region of its own pre-mRNA. However, whether transcriptional mechanisms contribute to TDP-43 autoregulation is unclear. We further showed that TDP-43 negatively regulates the TARDBP promoter and, surprisingly, that disease-causing TDP-43 mutants lacked this regulatory activity. These results allowed the elucidation of a novel transcriptional autoregulatory mechanism of TDP-43.

Metabolic Changes by Wine Flor-Yeasts with Gluconic Acid as the Sole Carbon Source.

Gluconic acid consumption under controlled conditions by a Saccharomyces cerevisiae flor yeast was studied in artificial media. Gluconic acid was the sole carbon source and the compounds derived from this metabolism were tracked by endo-metabolomic analysis using a Gas Chromatography-Mass Spectrometry (GC-MSD) coupled methodology. After 6 days, about 30% of gluconic acid (1.5 g/L) had been consumed and 34 endo-metabolites were identified. Metabolomic pathway analysis showed the TCA cycle, glyoxylate-dicarboxylate, glycine-serine-threonine, and glycerolipid metabolic pathway were significantly affected. These results contribute to the knowledge of intracellular metabolomic fluctuations in flor yeasts during gluconic acid uptake, opening possibilities for future experiments to improve their applications to control gluconic acid contents during the production of fermented beverages.

Mapping the intracellular metabolome of yeast biocapsules - Spherical structures of yeast attached to fungal pellets.

Co-culture conditions are beneficial for study due to the advances which arise from symbiotic interactions and which cannot be replicated under pure culture conditions. Here, the focus is on the connection between two fungi - a yeast, Saccharomyces cerevisiae, and a filamentous fungus, Penicillium chrysogenum - in a yeast immobilization system termed' yeast biocapsules', where the yeast and filamentous fungus are strongly attached to one another, forming spherical structures. This co-culture condition hinders filamentous fungal biomass growth, while immobilization of yeast cells continues to increase. The effect of the co-culture condition on endometabolites or intracellular metabolites were tracked during the beginning and end of the yeast biocapsule formation period, and metabolites analyzed by Gas Chromatography-Mass Spectrometry Detector (GC-MSD). Distinct metabolite profiles were found between single culture conditions, involving each organism separately, and with the co-culture condition, where there were differences in 54 endometabolites. Specifically, co-culture condition compounds such as fructose, glycolic acid and glyceric acid were present in higher concentrations at the end of biocapsule formation. These results shed light on the mechanisms and biochemical impact of the interaction between the yeast and filamentous fungus and serve as a basis to apply and further develop yeast biocapsules as a new biotechnological tool with benefits for industry.

FLO1, FLO5 and FLO11 Flocculation Gene Expression Impacts Saccharomyces cerevisiae Attachment to Penicillium chrysogenum in a Co-immobilization Technique.

A reoccurring flaw of most yeast immobilization systems that limits the potential of the technique is leakage of the cells from the matrix. Leakage may be due to weakly adherent cells, deterioration of the matrix, or to new growth and loss of non-adherent daughter cells. Yeast biocapsules are a spontaneous, cost effective system of immobilization whereby Saccharomyces cerevisiae cells are attached to the hyphae of Penicillium chrysogenum, creating hollow spheres that allow recovery and reutilization. This attachment is based on naturally occurring adherent properties of the yeast cell surface. We hypothesized that proteins associated with flocculation might play a role in adherence to fungal hyphae. To test this hypothesis, yeast strains with overexpressed and deleted flocculation genes (FLO1, FLO5, and FLO11) were evaluated for biocapsule formation to observe the impact of gene expression on biocapsule diameter, number, volume, dry mass, and percent immobilized versus non-immobilized cells. Overexpression of all three genes enhanced immobilization and resulted in larger diameter biocapsules. In particular, overexpression of FLO11 resulted in a five fold increase of absorbed cells versus the wild type isogenic strain. In addition, deletion of FLO1 and FLO11 significantly decreased the number of immobilized yeast cells compared to the wild type BY4742. These results confirm the role of natural adherent properties of yeast cells in attachment to fungal hyphae and offer the potential to create strongly adherent cells that will produce adherent progeny thereby reducing the potential for cell leakage from the matrix.

Loss of DDHD2, whose mutation causes spastic paraplegia, promotes reactive oxygen species generation and apoptosis.

DDHD2/KIAA0725p is a mammalian intracellular phospholipase A1 that exhibits phospholipase and lipase activities. Mutation of the DDHD2 gene causes hereditary spastic paraplegia (SPG54), an inherited neurological disorder characterized by lower limb spasticity and weakness. Although previous studies demonstrated lipid droplet accumulation in the brains of SPG54 patients and DDHD2 knockout mice, the cause of SPG54 remains elusive. Here, we show that ablation of DDHD2 in mice induces age-dependent apoptosis of motor neurons in the spinal cord. In vitro, motor neurons and embryonic fibroblasts from DDHD2 knockout mice fail to survive and are susceptible to apoptotic stimuli. Chemical and probe-based analysis revealed a substantial decrease in cardiolipin content and an increase in reactive oxygen species generation in DDHD2 knockout cells. Reactive oxygen species production in DDHD2 knockout cells was reversed by the expression of wild-type DDHD2, but not by an active-site DDHD2 mutant, DDHD2 mutants related to hereditary spastic paraplegia, or DDHD1, another member of the intracellular phospholipase A1 family whose mutation also causes spastic paraplegia (SPG28). Our results demonstrate the protective role of DDHD2 for mitochondrial integrity and provide a clue to the pathogenic mechanism of SPG54.