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Foods (Basel, Switzerland) Oct 2023Currently, non- yeasts are the subject of interest, among other things, for their contribution to the aromatic complexity of wines. In this study, the characterisation...
Currently, non- yeasts are the subject of interest, among other things, for their contribution to the aromatic complexity of wines. In this study, the characterisation of non- yeasts was addressed by their isolation during spontaneous fermentations of organic Verdejo grapes, obtaining a total of 484 isolates, of which 11% were identified by molecular techniques as non- yeasts. Fermentative isolates belonging to the species , , , , , and were analysed. Significant differences were found in the yeast populations established at the different fermentation stages. Interestingly, stood up as a widely distributed species in vineyards, vintages, and fermentation stages. Several of the strains studied stood out for their biotechnological potential in the production of Verdejo wine, showing the presence of relevant enzymatic activity for the release of varietal aromas and the technological improvement of the winemaking process. Three enzymatic activities were found in an important number of isolates, β-glucosidase, protease, and β-lyase, implicated in the positive aromatic impact on this style of white wine. In that sense, all the isolates of presented those activities. isolates were highlighted for their significant β-lyase activity. In addition, was outlined because of its potential to achieve an elevated fermenting power, as well as the lack of lag phase. The results obtained highlight the importance of maintaining the microbial diversity that contributes to the production of wines with unique and distinctive characteristics of the production region.
PubMed: 37835297
DOI: 10.3390/foods12193644 -
FEMS Microbes 2023The inebriation of wild African elephants from eating the ripened and rotting fruit of the marula tree is a persistent myth in Southern Africa. However, the yeasts...
The inebriation of wild African elephants from eating the ripened and rotting fruit of the marula tree is a persistent myth in Southern Africa. However, the yeasts responsible for alcoholic fermentation to intoxicate the elephants remain poorly documented. In this study, we considered Botswana, a country with the world's largest population of wild elephants, and where the marula tree is indigenous, abundant and protected, to assess the occurrence and biodiversity of yeasts with a potential to ferment and subsequently inebriate the wild elephants. We collected marula fruits from over a stretch of 800 km in Botswana and isolated 106 yeast strains representing 24 yeast species. Over 93% of these isolates, typically known to ferment simple sugars and produce ethanol comprising of high ethanol producers belonging to , and , and intermediate ethanol producers , and . Fermentation of marula juice revealed convincing fermentative and aromatic bouquet credentials to suggest the potential to influence foraging behaviour and inebriate elephants in nature. There is insufficient evidence to refute the aforementioned myth. This work serves as the first work towards understanding the biodiversity marula associated yeasts to debunk the myth or approve the facts.
PubMed: 37854251
DOI: 10.1093/femsmc/xtad018 -
Synthetic and Systems Biotechnology Sep 2023The methylotrophic budding yeast has been utilized to the production of a variety of heterologous recombinant proteins owing to the strong inducible alcohol oxidase...
The methylotrophic budding yeast has been utilized to the production of a variety of heterologous recombinant proteins owing to the strong inducible alcohol oxidase promoter (pAOX1). However, it is difficult to use as the chassis cell factory for high-valuable metabolite biosynthesis due to the low homologous recombination (HR) efficiency and the limitation of handy selective markers, especially in the condition of multistep biosynthetic pathways. Hence, we developed a novel CRISPR/Cas9 system with highly editing efficiencies and recyclable auxotrophic selective marker (HiEE-ReSM) to facilitate cell factory in . Firstly, we improved the HR rates of through knocking out the non-homologous-end-joining gene () and overexpressing HR-related proteins ( and ), resulting in higher positive rate compared to the basal strain, achieved 97%. Then, we used the uracil biosynthetic genes as the reverse screening marker, which can improve the recycling efficiency of marker. Meanwhile, the HR rate is still 100% in uracil auxotrophic yeast. Specially, we improved the growth rate of uracil auxotrophic yeast strains by overexpressing the uracil transporter () to increase the uptake of exogenous uracil from medium. Meanwhile, we explored the optimal concentration of uracil (90 mg/L) for strain growth. In the end, the HiEE-ReSM system has been applied for the inositol production (250 mg/L) derived from methanol in . The systems will contribute to as an attractive cell factory for the complex compound biosynthesis through multistep metabolic pathway engineering and will be a useful tool to improve one carbon (C1) bio-utilization.
PubMed: 37448527
DOI: 10.1016/j.synbio.2023.06.003 -
Heliyon Mar 2024Amylases are enzymes that are known to hydrolyze starch. High efficiency of amylolytic enzymes allows them to compete in the industry with the technology of chemical...
Amylases are enzymes that are known to hydrolyze starch. High efficiency of amylolytic enzymes allows them to compete in the industry with the technology of chemical hydrolysis of starch. A strain with high amylolytic activity was isolated from soil and designated as T5. The gene encoding α-amylase from T5 was successfully expressed in both (rAmyT5-E) and (as rAmyT5-P). According to the study, the recombinant α-amylases rAmyT5-E and rAmyT5-P exhibited the highest activity at pH 6.0 and temperatures of 70 and 80 °C, respectively. Over 80% of the rAmyT5-E enzyme activity was preserved following incubation within the pH range of 5-9; the same was true for rAmyT5-P after incubation at pH 6-9. N-glycosylation reduced the thermal and pH stability of the enzyme. The specific activity and catalytic efficiency of the recombinant AmyT5 α-amylase were also diminished by N-glycosylation.
PubMed: 38515717
DOI: 10.1016/j.heliyon.2024.e28064 -
Nature Communications Aug 2023Saccharomyces cerevisiae is a workhorse of industrial biotechnology owing to the organism's prominence in alcohol fermentation and the suite of sophisticated genetic...
Saccharomyces cerevisiae is a workhorse of industrial biotechnology owing to the organism's prominence in alcohol fermentation and the suite of sophisticated genetic tools available to manipulate its metabolism. However, S. cerevisiae is not suited to overproduce many bulk bioproducts, as toxicity constrains production at high titers. Here, we employ a high-throughput assay to screen 108 publicly accessible yeast strains for tolerance to 20 g L adipic acid (AA), a nylon precursor. We identify 15 tolerant yeasts and select Pichia occidentalis for production of cis,cis-muconic acid (CCM), the precursor to AA. By developing a genome editing toolkit for P. occidentalis, we demonstrate fed-batch production of CCM with a maximum titer (38.8 g L), yield (0.134 g g glucose) and productivity (0.511 g L h) that surpasses all metrics achieved using S. cerevisiae. This work brings us closer to the industrial bioproduction of AA and underscores the importance of host selection in bioprocessing.
Topics: Saccharomyces cerevisiae; Pichia; Sorbic Acid
PubMed: 37652930
DOI: 10.1038/s41467-023-41064-5 -
PloS One 2023In this paper, a novel bifunctional cellulase gene cel1 was cloned from Thermoascus aurantiacus by PCR and heterologously expressed in Pichia pastoris GS115....
In this paper, a novel bifunctional cellulase gene cel1 was cloned from Thermoascus aurantiacus by PCR and heterologously expressed in Pichia pastoris GS115. Bioinformatics and other related tools were used to compare the nucleotide homology of target genes, and analyze the signal peptide, transmembrane domain, hydrophilicity, secondary and tertiary structure of proteins. It was concluded that cel1 has similar endoglucanase nucleotide sequences and falls under the GH5 family. It was also found that cel1 has nucleotide sequences similar to glucosidase, which can infer that cel1 may have the properties of glucosidase, indicating that cel1 is multifunctional. At the same time, a part of the nucleotide sequence of the gene was removed to obtain a new gene cel2, and after highly efficient heterologous expression, its specific activity was found to be 2.1 times higher. Its enhancement is related to the exposure of the protein's hollow three-dimensional structure. This paper provides good material for exploring the relationship between the structure of bifunctional enzymes and their functions, which lays a solid foundation for further research and applications, and provides useful insight for gene mining of other novel enzymes.
Topics: Glycosides; Thermoascus; Cardiac Glycosides; Cellulase; Glucosidases; Cloning, Molecular
PubMed: 37713448
DOI: 10.1371/journal.pone.0285680 -
Archivum Immunologiae Et Therapiae... Aug 2023Fibroblast growth factor 2 (FGF-2) is not only an angiogenic factor, but also a mitogen for epidermal keratinocytes. FGF-2 has been shown to be positively immunoreactive...
Fibroblast growth factor 2 (FGF-2) is not only an angiogenic factor, but also a mitogen for epidermal keratinocytes. FGF-2 has been shown to be positively immunoreactive in the basal layer of psoriatic lesions. In previous work, we used the Escherichia coli (E. coli) expression system to biosynthesize a biologically active anti-FGF-2 nanobody (Nb) screened by phage display technology, but the low yield limited its clinical application. In this study, we aimed to increase the yield of anti-FGF-2 Nb, and evaluate its therapeutic potential for psoriasis by inhibiting FGF-2-mediated mitogenic signaling in psoriatic epidermal keratinocytes. We demonstrated a 16-fold improvement in the yield of anti-FGF-2 Nb produced in the Pichia pastoris (P. pastoris) compared to the E. coli expression system. In vitro, the FGF-2-induced HaCaT cell model (FHCM) was established to mimic the key feature of keratinocyte overproliferation in psoriasis. Anti-FGF-2 Nb was able to effectively inhibit the proliferation and migration of FHCM. In vivo, anti-FGF-2 Nb attenuated the severity of imiquimod (IMQ)-induced psoriatic lesions in mice, and also improved the inflammatory microenvironment by inhibiting the secretion of inflammatory cytokines (IL-1β, IL-6, IL-23, and TNF-α), chemokines (CXCL1 and CCL20), and neutrophil infiltration in skin lesions. These were mainly related to the suppression of FGF-2-mediated mitogenic signaling in psoriatic keratinocytes. In conclusion, we have improved the production of anti-FGF-2 Nb and demonstrated the modality of attenuating the abnormal proliferative behavior of psoriatic keratinocytes by inhibiting FGF-2-mediated mitogenic signaling, which offers the possibility of treating psoriasis.
Topics: Animals; Mice; Escherichia coli; Fibroblast Growth Factor 2; Keratinocytes; Psoriasis; Humans
PubMed: 37632545
DOI: 10.1007/s00005-023-00685-w -
Food Microbiology Aug 2023Contamination of white-brined cheeses (WBCs) with yeasts is of major concern in the dairy industry. This study aimed to identify yeast contaminants and characterize...
Contamination of white-brined cheeses (WBCs) with yeasts is of major concern in the dairy industry. This study aimed to identify yeast contaminants and characterize their succession in white-brined cheese during a shelf-life of 52 weeks. White-brined cheeses added herbs (WBC1) or sundried tomatoes (WBC2) were produced at a Danish dairy and incubated at 5 °C and 10 °C. An increase in yeast counts was observed for both products within the first 12-14 weeks of incubation and stabilized afterwards varying in a range of 4.19-7.08 log CFU/g. Interestingly, higher incubation temperature, especially in WBC2, led to lower yeast counts, concurrently with higher diversity of yeast species. Observed decrease in yeast counts was, most likely, due to negative interactions between yeast species leading to growth inhibition. In total, 469 yeast isolates from WBC1 and WBC2 were genotypically classified using the (GTG)-rep-PCR technique. Out of them, 132 representative isolates were further identified by sequencing the D1/D2 domain of the 26 S rRNA gene. Predominant yeast species in WBCs were Candida zeylanoides and Debaryomyces hansenii, while Candida parapsilosis, Kazachstania bulderi, Kluyveromyces lactis, Pichia fermentans, Pichia kudriavzevii, Rhodotorula mucilaginosa, Torulaspora delbrueckii, and Wickerhamomyces anomalus were found in lower frequency. Heterogeneity of yeast species in WBC2 was generally larger compared to WBC1. This study indicated that, along with contamination levels, taxonomic heterogeneity of yeasts is an important factor influencing yeast cell counts, as well as product quality during storage.
Topics: Cheese; Yeasts; Polymerase Chain Reaction
PubMed: 37098422
DOI: 10.1016/j.fm.2023.104266 -
Turkish Journal of Chemistry 2024yeasts are capable of forming biofilms during vinegar production and causing spoilage in various beverages. In addition, there exists a significant likelihood of...
yeasts are capable of forming biofilms during vinegar production and causing spoilage in various beverages. In addition, there exists a significant likelihood of encountering yeast contamination which can prevent vinegar production. The present study investigates the detection and characterization of the () biofilm on traditionally produced homemade apple vinegar. The unique characteristics of vinegar were analyzed with a focus on the constituent, known as the "mother of vinegar", whose composition is comprised of cellulosic biofilm and acetic acid bacteria, including () Briefly, was isolated from apple vinegar and characterized in terms of the effect of biofilm formation on the surface of the cellulosic film on vinegar production. Microbial identification of vinegar with/without contamination by was analyzed through MALDI-TOF mass spectrometry (MS), and biofilm was characterized by Fourier transform infrared spectroscopy (FT-IR), Scanning electron microscopy (SEM), and crystal violet staining. Accordingly, MS spectrum of isolates was identified as and with a ratio of 2.01 and 1.94, respectively. The FTIR analysis indicated that the peaks within the range of 1150-900 cm revealed a high content of polysaccharide in contaminated biofilm, which is attributed to the stretching vibration of C-C and C-O bonds. The spectral region from 2921.51 to 2853.71 cm exhibited the characteristic of lipids in bacterial cell walls and membranes. SEM images of bacterial biofilms revealed a three-dimensional network composed of ultrafine fibers with a ribbon-like shape; however, the condensed reticulated structure was observed in contaminated biofilms. The presence of two microbial populations was detected regarding the morphological analysis. Crystal violet staining of contaminated-cellulosic biofilms visualized bacterial and yeast colonization. Concisely, this study emphasizes that the proliferation of during apple fermentation has the potential to adversely affect the quality of the homemade vinegar, due to its distinct biofilm characteristics.
PubMed: 38544898
DOI: 10.55730/1300-0527.3640 -
Pharmaceuticals (Basel, Switzerland) May 2024Diabetes mellitus is a heterogeneous metabolic disorder that poses significant health and economic challenges across the globe. Polysaccharides, found abundantly in...
Diabetes mellitus is a heterogeneous metabolic disorder that poses significant health and economic challenges across the globe. Polysaccharides, found abundantly in edible plants, hold promise for managing diabetes by reducing blood glucose levels (BGL) and insulin resistance. However, most of these polysaccharides cannot be digested or absorbed directly by the human body. Here we report the production of antidiabetic oligosaccharides from cress seed mucilage polysaccharides using yeast fermentation. The water-soluble polysaccharides extracted from cress seed mucilage were precipitated using 75% ethanol and fermented with for different time intervals. The digested saccharides were fractionated through gel permeation chromatography using a Bio Gel P-10 column. Structural analysis of the oligosaccharide fractions revealed the presence of galacturonic acid, rhamnose, glucuronic acid, glucose and arabinose. Oligosaccharide fractions exhibited the potential to inhibit α-amylase and α-glucosidase enzymes in a dose-dependent manner in vitro. The fraction DF73 exhibited strong inhibitory activity against α-amylase with IC values of 38.2 ± 1.12 µg/mL, compared to the positive control, acarbose, having an IC value of 29.18 ± 1.76 µg/mL. Similarly, DF72 and DF73 showed the highest inhibition of α-glucosidase, with IC values of 9.26 ± 2.68 and 50.47 ± 5.18 µg/mL, respectively. In in vivo assays in streptozotocin (STZ)-induced diabetic mice, these oligosaccharides significantly reduced BGL and improved lipid profiles compared to the reference drug metformin. Histopathological observations of mouse livers indicated the cytoprotective effects of these sugars. Taken together, our results suggest that oligosaccharides produced through microbial digestion of polysaccharides extracted from cress seed mucilage have the potential to reduce blood glucose levels, possibly through inhibition of carbohydrate-digesting enzymes and regulation of the various signaling pathways.
PubMed: 38931372
DOI: 10.3390/ph17060704