-
International Journal of Biological... Jun 2024Small single-chain variable fragments (scFv) are promising biomolecules to inhibit and neutralize toxins and to act as antivenoms. In this work, we aimed to produce a...
Small single-chain variable fragments (scFv) are promising biomolecules to inhibit and neutralize toxins and to act as antivenoms. In this work, we aimed to produce a functional scFv-6009FV in the yeast Pichia pastoris, which inhibits the pure Cn2 neurotoxin and the whole venom of Centruroides noxius. We were able to achieve yields of up to 31.6 ± 2 mg/L in flasks. Furthermore, the protein showed a structure of 6.1 % α-helix, 49.1 % β-sheet, and 44.8 % of random coil by CD. Mass spectrometry confirmed the amino acid sequence and showed no glycosylation profile for this molecule. Purified scFv-6009FV allowed us to develop anti-scFvs in rabbits, which were then used in affinity columns to purify other scFvs. Determination of its half-maximal inhibitory concentration value (IC) was 40 % better than the scFvs produced by E. coli as a control. Finally, we found that scFv-6009FV was able to inhibit ex vivo the pure Cn2 toxin and the whole venom from C. noxius in murine rescue experiments. These results demonstrated that under the conditions assayed here, P. pastoris is suited to produce scFv-6009FV that, compared to scFvs produced by E. coli, maintains the characteristics of an antibody and neutralizes the Cn2 toxin more effectively.
PubMed: 38945343
DOI: 10.1016/j.ijbiomac.2024.133461 -
Frontiers in Bioscience (Elite Edition) Jun 2024(), also called biotech yeast, is a yeast species with many applications in the biotechnology and pharmaceutical industries. This methylotrophic yeast has garnered... (Review)
Review
(), also called biotech yeast, is a yeast species with many applications in the biotechnology and pharmaceutical industries. This methylotrophic yeast has garnered significant interest as a platform for the production of recombinant proteins. Numerous benefits include effective secretory expression that facilitates the easy purification of heterologous proteins, high cell density with rapid growth, post-translational changes, and stable gene expression with integration into the genome. In the last thirty years, has also been refined as an adaptable cell factory that can produce hundreds of biomolecules in a laboratory setting and on an industrial scale. Indeed, over 5000 recombinant proteins have been generated so far using the expression method, which makes up 30% of the total cell protein or 80% of the total released protein. has been used to manufacture more than 70 commercial products in addition to over 300 industrial processes that have been granted licenses. Among these are useful enzymes for industrial biotechnology, including xylanase, mannanase, lipase, and phytase. The others are biopharmaceuticals, which include human serum albumin, insulin, hepatitis B surface antigen, and epidermal growth factor. Compared to other expression systems, this yeast is also considered a special host for synthesizing subunit vaccines, which have recently been supplanted by alternative vaccination types, such as inactivated/killed and live attenuated vaccines. Moreover, efficient production of recombinant proteins is achieved through multi-level optimization methods, such as codon bias, gene dosage, promoters, signal peptides, and environmental factors. Therefore, although expression systems are efficient and simple with clearly established process procedures, it is still necessary to determine the ideal conditions since these vary depending on the target protein to ensure the highest recombinant protein generation. This review addresses the expression system, its importance in industrial and biopharmaceutical protein production, and some bioprocessing and genetic modification strategies for efficient protein production. will eventually continue contributing as a potent expression system in research areas and industrial applications.
Topics: Saccharomycetales; Recombinant Proteins; Biological Products; Biotechnology; Pichia
PubMed: 38939917
DOI: 10.31083/j.fbe1602019 -
Medical Mycology Jun 2024In response to the growing global threat of fungal infections, in 2020 the World Health Organisation (WHO) established an Expert Group to identify priority fungi and...
In response to the growing global threat of fungal infections, in 2020 the World Health Organisation (WHO) established an Expert Group to identify priority fungi and develop the first WHO fungal priority pathogen list (FPPL). The aim of this systematic review was to evaluate the features and global impact of invasive infections caused by Pichia kudriavzevii (formerly known as Candida krusei). PubMed and Web of Science were used to identify studies published between 1 January 2011 and 18 February 2021 reporting on the criteria of mortality, morbidity (defined as hospitalisation and length of stay), drug resistance, preventability, yearly incidence, and distribution/emergence. Overall, 33 studies were evaluated. Mortality rates of up to 67% in adults were reported. Despite the intrinsic resistance of P. kudriavzevii to fluconazole with decreased susceptibility to amphotericin B, resistance (or non-wild-type rate) to other azoles and echinocandins was low, ranging between 0 and 5%. Risk factors for developing P. kudriavzevii infections included low birth weight, prior use of antibiotics/antifungals, and an underlying diagnosis of gastrointestinal disease or cancer. The incidence of infections caused by P. kudriavzevii is generally low (∼5% of all Candida-like blood isolates) and stable over the 10-year timeframe, although additional surveillance data are needed. Strategies targeting the identified risk factors for developing P. kudriavzevii infections should be developed and tested for effectiveness and feasibility of implementation. Studies presenting data on epidemiology and susceptibility of P. kudriavzevii were scarce, especially in low- and middle-income countries (LMICs). Thus, global surveillance systems are required to monitor the incidence, susceptibility, and morbidity of P. kudriavzevii invasive infections to inform diagnosis and treatment. Timely species-level identification and susceptibility testing should be conducted to reduce the high mortality and limit the spread of P. kudriavzevii in healthcare facilities.
Topics: Humans; Drug Resistance, Fungal; Antifungal Agents; World Health Organization; Pichia; Incidence; Risk Factors; Candidiasis
PubMed: 38935911
DOI: 10.1093/mmy/myad132 -
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 -
Foods (Basel, Switzerland) Jun 2024There is a growing market for craft beverages with unique flavors. This study aimed to obtain a palate-pleasing mead derived from 4A as a monoculture. Different culture...
There is a growing market for craft beverages with unique flavors. This study aimed to obtain a palate-pleasing mead derived from 4A as a monoculture. Different culture media were evaluated to compare the fermentation kinetics and final products. The crucial factors in the medium were ~200 mg L of yeast assimilable nitrogen and a pH of 3.5-5.0. A panel of judges favored the mead derived from 4A (fermented in a medium with honey initially at 23 °Bx) over a commercial sample produced from , considering its appearance, fruity and floral flavors (provided by esters, aldehydes, and higher alcohols), and balance between sweetness (given by the 82.91 g L of residual sugars) and alcohol. The present mead had an 8.57% / ethanol concentration, was elaborated in 28 days, and reached a maximum biomass growth (2.40 g L) on the same fermentation day (6) that the minimum level of pH was reached. The biomass growth yield peaked at 24 and 48 h (~0.049 g g), while the ethanol yield peaked at 24 h (1.525 ± 0.332 g g), in both cases declining thereafter. The Gompertz model adequately describes the kinetics of sugar consumption and the generation of yeast biomass and ethanol. Pathogenic microorganisms, methanol, lead, and arsenic were absent in the mead. Thus, 4A produced a safe and quality mead with probable consumer acceptance.
PubMed: 38928890
DOI: 10.3390/foods13121948 -
International Journal of Molecular... Jun 2024Aflatoxin B (AFB) contamination is a serious threat to nutritional safety and public health. The CotA-laccase from ANSB821 previously reported by our laboratory showed...
Aflatoxin B (AFB) contamination is a serious threat to nutritional safety and public health. The CotA-laccase from ANSB821 previously reported by our laboratory showed great potential to degrade AFB without redox mediators. However, the use of this CotA-laccase to remove AFB in animal feed is limited because of its low catalytic efficiency and low expression level. In order to make better use of this excellent enzyme to effectively degrade AFB, twelve mutants of CotA-laccase were constructed by site-directed mutagenesis. Among these mutants, E186A and E186R showed the best degradation ability of AFB, with degradation ratios of 82.2% and 91.8% within 12 h, which were 1.6- and 1.8-times higher than those of the wild-type CotA-laccase, respectively. The catalytic efficiencies (/K) of E186A and E186R were found to be 1.8- and 3.2-times higher, respectively, than those of the wild-type CotA-laccase. Then the expression vectors pPICZαA-N-E186A and pPICZαA-N-E186R with an optimized signal peptide were constructed and transformed into GS115. The optimized signal peptide improved the secretory expressions of E186A and E186R in GS115. Collectively, the current study provided ideal candidate CotA-laccase mutants for AFB detoxification in food and animal feed and a feasible protocol, which was desperately needed for the industrial production of CotA-laccases.
Topics: Aflatoxin B1; Bacillus licheniformis; Bacterial Proteins; Laccase; Mutagenesis, Site-Directed; Recombinant Proteins; Saccharomycetales
PubMed: 38928160
DOI: 10.3390/ijms25126455 -
Journal of Food Science Jun 2024In vegetable fermentation, pellicle is a common quality deterioration phenomenon. This study investigates the characteristics of glucose, organic acids, amino acids, and...
In vegetable fermentation, pellicle is a common quality deterioration phenomenon. This study investigates the characteristics of glucose, organic acids, amino acids, and biogenic amines during the pellicle occurrence and disappearance of paocai. The results revealed a slight increase in pH of the fermentation system after pellicle occurred, and glucose was the main carbohydrate that microbial activity primary relied on. The microorganisms responsible for pellicle formation consumed organic acids in brine, but the lactic acid in paocai gradually increased and exceeded 25 mg/g. The appearance of pellicle caused a decrease in total free amino acids from 200.390 mg/100 g to 172.079 when pellicle occurred, whereas its impact on biogenic amines was not apparent. Through Kyoto Encyclopedia of Genes and Genomes pathway enrichment of metagenomics sequencing data, screening, and sorting of the key enzymes involved in organic acid metabolism, it was observed that the composition and species of the key microorganisms capable of metabolizing organic acids were more abundant before the appearance of pellicle. When pellicle occurred, lactic acid may be metabolized by Lactobacillus plantarum; in contrast, Lactobacillus and Pichia were associated with citric acid metabolism, and Lactobacillus, Pichia, Saccharomycodes, and Kazachstania were linked to malic acid metabolism. Moreover, Prevotella, Kazachstania, Lactobacillus, Vibrio, and Siphonobacter were implicated in succinic acid metabolism. Additionally, the production of tartaric acid and oxalic acid in paocai and brine resulted from abiotic effects. This knowledge offers a theoretical basis for precise control of paocai fermentation process. PRACTICAL APPLICATION: Our study revealed the specific situation of the metabolites produced by the microorganisms during the pollution and recovery process of pellicle in paocai fermentation, especially the effect of pellicle on the key process of organic acid metabolism. These research results provided theoretical basis for precise control of paocai fermentation.
PubMed: 38922911
DOI: 10.1111/1750-3841.17178 -
Applied Biochemistry and Biotechnology Jun 2024Microbial proteins represent a promising solution to address the escalating global demand for protein, particularly in regions with limited arable land. Yeasts, such as...
Microbial proteins represent a promising solution to address the escalating global demand for protein, particularly in regions with limited arable land. Yeasts, such as Saccharomyces cerevisiae, are robust and safe protein-producing strains. However, the utilization of non-conventional yeast strains for microbial protein production has been hindered, partly due to a lack of comprehensive understanding of protein production traits. In this study, we conducted experimental analyses focusing on the growth, protein content, and amino acid composition of nine yeast strains, including one S. cerevisiae strain, three Yarrowia lipolytica strains, and five Pichia spp. strains. We identified that, though Y. lipolytica and Pichia spp. strains consumed glucose at a slower rate compared to S. cerevisiae, Pichia spp. strains showed a higher cellular protein content, and Y. lipolytica strains showed a higher glucose-to-biomass/protein yield and methionine content. We further applied computational approaches to explain that metabolism economy was the main underlying factor for the limited amount of scarce/carbon-inefficient amino acids (such as methionine) within yeast cell proteins. We additionally verified that the specialized metabolism was a key reason for the high methionine content in Y. lipolytica strains, and proposed Y. lipolytica strain as a potential producer of high-quality single-cell protein rich in scarce amino acids. Through experimental evaluation, we identified Pichia jadinii CICC 1258 as a potential strain for high-quality protein production under unfavorable pH/temperature conditions. Our work suggests a promising avenue for optimizing microbial protein production, identifying the factors influencing amino acid composition, and paving the way for the use of unconventional yeast strains to meet the growing protein demands.
PubMed: 38922492
DOI: 10.1007/s12010-024-04995-3 -
Metabolites May 2024Wild yeast suitable for kiwifruit wine fermentation was isolated and purified, and the fermentation process was optimized to increase the alcohol content of the...
Wild yeast suitable for kiwifruit wine fermentation was isolated and purified, and the fermentation process was optimized to increase the alcohol content of the kiwifruit wine. was isolated from kiwifruit pulp by lineation separating, screened by morphological characteristics in Wallerstein Laboratory Nutrient Agar (WL) medium and microscope observation, and further identified by 26S rDNA D1/D2 domain sequence analysis. Taking alcohol content and sensory evaluation as two indexes, the fermentation condition for kiwifruit wine was optimized by single factor and response surface experiment. The optimal fermentation conditions were optimized as follows: the fermentation temperature was at 24 °C, the initial pH was 3.8, the sugar dosage in second step was 8% (/), and the inoculating quantity of and was 0.15 g/L at equal proportion. Under these optimal conditions, the maximum estimated alcohol content was 15.6 vol%, and the kiwifruit wine was light green in color with strong kiwifruit aroma and mellow taste.
PubMed: 38921445
DOI: 10.3390/metabo14060310 -
Journal of Fungi (Basel, Switzerland) Jun 2024(formerly ) is a methylotrophic yeast widely used in laboratories around the world to produce recombinant proteins. Given its advantageous features, it has also gained... (Review)
Review
(formerly ) is a methylotrophic yeast widely used in laboratories around the world to produce recombinant proteins. Given its advantageous features, it has also gained much interest in the context of modern biotechnology. In this review, we present the utilization of as a platform to produce several products of economic interest such as biopharmaceuticals, renewable chemicals, fuels, biomaterials, and food/feed products. Finally, we present synthetic biology approaches currently used for strain engineering, aiming at the production of new bioproducts.
PubMed: 38921397
DOI: 10.3390/jof10060411