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Journal of Biotechnology Aug 2024Ecallantide comprises Kunitz Domain 1 of Tissue Factor Pathway Inhibitor, mutated at seven amino acid positions to inhibit plasma kallikrein (PK). It is used to treat...
Ecallantide comprises Kunitz Domain 1 of Tissue Factor Pathway Inhibitor, mutated at seven amino acid positions to inhibit plasma kallikrein (PK). It is used to treat acute hereditary angioedema (HAE). We appended hexahistidine tags to the N- or C-terminus of recombinant Ecallantide (rEcall) and expressed and purified the resulting proteins, with or without fusion to human serum albumin (HSA), using Pichia pastoris. The inhibitory constant (K) of rEcall-H6 or H6-rEcall for PK was not increased by albumin fusion. When I-labelled rEcall proteins were injected intravenously into mice, the area under the clearance curve (AUC) was significantly increased, 3.4- and 3.6-fold, for fusion proteins H6-rEcall-HSA and HSA-rEcall-H6 versus their unfused counterparts but remained 2- to 3-fold less than that of HSA-H6. The terminal half-life of H6-rEcall-HSA and HSA-H6 did not differ, although that of HSA-rEcall-H6 was significantly shorter than either other protein. Receptor Associated Protein (RAP), a Low-density lipoprotein Receptor-related Protein (LRP1) antagonist, competed H6-rEcall-HSA clearance more effectively than intravenous immunoglobulin (IVIg), a neonatal Fc receptor (FcRn) antagonist. HSA fusion decreases rEcall clearance in vivo, but LRP1-mediated clearance remains more important than FcRn-mediated recycling for rEcall fusion proteins. The properties of H6-rEcall-HSA warrant investigation in a murine model of HAE.
Topics: Animals; Recombinant Fusion Proteins; Mice; Humans; Half-Life; Plasma Kallikrein; Serum Albumin, Human; Saccharomycetales; Receptors, Fc; Histocompatibility Antigens Class I
PubMed: 38844246
DOI: 10.1016/j.jbiotec.2024.06.002 -
Food Microbiology Sep 2024To evaluate the effects of bioaugmentation fermentation inoculated with one ester-producing strain (Wickerhamomyces anomalus ZX-1) and two strains of lactic acid...
To evaluate the effects of bioaugmentation fermentation inoculated with one ester-producing strain (Wickerhamomyces anomalus ZX-1) and two strains of lactic acid bacteria (Lactobacillus plantarum CGMCC 24035 and Lactobacillus acidophilus R2) for improving the flavor of persimmon vinegar, microbial community, flavor compounds and metabolites were analyzed. The results of microbial diversity analysis showed that bioaugmentation fermentation significantly increased the abundance of Lactobacillus, Saccharomyces, Pichia and Wickerhamomyces, while the abundance of Acetobacter, Apiotrichum, Delftia, Komagataeibacter, Kregervanrija and Aspergillus significantly decreased. After bioaugmentation fermentation, the taste was softer, and the sensory irritancy of acetic acid was significantly reduced. The analysis of HS-SPME-GC-MS and untargeted metabolomics based on LC-MS/MS showed that the contents of citric acid, lactic acid, malic acid, ethyl lactate, methyl acetate, isocitrate, acetoin and 2,3-butanediol were significantly increased. By multivariate analysis, 33 differential metabolites were screened out to construct the correlation between the differential metabolites and microorganisms. Pearson correlation analysis showed that methyl acetate, ethyl lactate, betaine, aconitic acid, acetoin, 2,3-butanediol and isocitrate positively associated with Wickerhamomyces and Lactobacillus. The results confirmed that the quality of persimmon vinegar was improved by bioaugmentation fermentation.
Topics: Fermentation; Acetic Acid; Microbiota; Diospyros; Saccharomycetales; Taste; Flavoring Agents; Lactobacillus plantarum; Food Microbiology; Lactobacillus acidophilus; Bacteria
PubMed: 38839213
DOI: 10.1016/j.fm.2024.104565 -
Metabolic Engineering Jun 2024The development of a heme-responsive biosensor for dynamic pathway regulation in eukaryotes has never been reported, posing a challenge for achieving the efficient...
The development of a heme-responsive biosensor for dynamic pathway regulation in eukaryotes has never been reported, posing a challenge for achieving the efficient synthesis of multifunctional hemoproteins and maintaining intracellular heme homeostasis. Herein, a biosensor containing a newly identified heme-responsive promoter, CRISPR/dCas9, and a degradation tag N-degron was designed and optimized to fine-tune heme biosynthesis in the efficient heme-supplying Pichia pastoris P1H9 chassis. After identifying literature-reported promoters insensitive to heme, the endogenous heme-responsive promoters were mined by transcriptomics, and an optimal biosensor was screened from different combinations of regulatory elements. The dynamic regulation pattern of the biosensor was validated by the transcriptional fluctuations of the HEM2 gene involved in heme biosynthesis and the subsequent responsive changes in intracellular heme titers. We demonstrate the efficiency of this regulatory system by improving the production of high-active porcine myoglobin and soy hemoglobin, which can be used to develop artificial meat and artificial metalloenzymes. Moreover, these findings can offer valuable strategies for the synthesis of other hemoproteins.
PubMed: 38839038
DOI: 10.1016/j.ymben.2024.06.002 -
3 Biotech Jun 2024The lignocellulolytic accessory enzyme, Feruloyl esterase C (FE_5DR), encoded in the genome of thermotolerant was successfully cloned and heterologously expressed in ....
UNLABELLED
The lignocellulolytic accessory enzyme, Feruloyl esterase C (FE_5DR), encoded in the genome of thermotolerant was successfully cloned and heterologously expressed in . The expressed FE_5DR was purified using UNOsphere™ Q anion exchange chromatography column, exhibiting a homogeneous band of ~ 39 kDa. Its optimum temperature was determined to be 60 °C, with an optimal pH of 6.0. Additionally, the enzyme activity of FE_5DR was significantly enhanced by preincubation in a buffer containing Mg, Cu and Ca metal ions. Enzyme kinetic parameters, computed from double reciprocal Lineweaver-Burk plots, yielded observed V and K values of 0.758 U/mg and 0.439 mM, respectively. Furthermore, the potential of custom-made cocktails comprising FE_5DR and benchmark cellulase derived from the developed mutant strain of MAN 40, as well as the biorefinery-relevant lignocellulolytic enzyme Cellic CTec 3, resulted in improved saccharification of unwashed acid pretreated (UWAP) rice straw slurry and mild alkali deacetylated (MAD) rice straw when compared to benchmark MAN 40 and Cellic CTec 3.
SUPPLEMENTARY INFORMATION
The online version contains supplementary material available at 10.1007/s13205-024-04013-7.
PubMed: 38828098
DOI: 10.1007/s13205-024-04013-7 -
MLife Mar 2024is a food-safe yeast with great potential for producing heterologous proteins. Improving the yield in remains a challenge and incorporating large-scale functional...
is a food-safe yeast with great potential for producing heterologous proteins. Improving the yield in remains a challenge and incorporating large-scale functional modules poses a technical obstacle in engineering. To address these issues, linear and circular yeast artificial chromosomes of (KmYACs) were constructed and loaded with disulfide bond formation modules from or . These modules contained up to seven genes with a maximum size of 15 kb. KmYACs carried telomeres either from or . KmYACs were transferred successfully into and stably propagated without affecting the normal growth of the host, regardless of the type of telomeres and configurations of KmYACs. KmYACs increased the overall expression levels of disulfide bond formation genes and significantly enhanced the yield of various heterologous proteins. In high-density fermentation, the use of KmYACs resulted in a glucoamylase yield of 16.8 g/l, the highest reported level to date in . Transcriptomic and metabolomic analysis of cells containing KmYACs suggested increased flavin adenine dinucleotide biosynthesis, enhanced flux entering the tricarboxylic acid cycle, and a preferred demand for lysine and arginine as features of cells overexpressing heterologous proteins. Consistently, supplementing lysine or arginine further improved the yield. Therefore, KmYAC provides a powerful platform for manipulating large modules with enormous potential for industrial applications and fundamental research. Transferring the disulfide bond formation module via YACs proves to be an efficient strategy for improving the yield of heterologous proteins, and this strategy may be applied to optimize other microbial cell factories.
PubMed: 38827505
DOI: 10.1002/mlf2.12115 -
Food Research International (Ottawa,... Jul 2024This study investigated the effects of different pickle brines and glycine additions on biogenic amine formation in pickle fermentation. The results showed that the...
This study investigated the effects of different pickle brines and glycine additions on biogenic amine formation in pickle fermentation. The results showed that the brines with higher biogenic amine content led to the production of more biogenic amines in the simulated pickle fermentation system. This was related to the abundance of biogenic amine-producing microorganisms in the microbial communities of the brines. Metagenome analysis of the brines and metatranscriptome analysis of the fermentation systems showed that putrescine was primarily from Lactobacillus, Oenococcus, and Pichia, while histamine and tyramine were primarily from Lactobacillus and Tetragenococcus. Addition of glycine significantly reduced the accumulation of biogenic amines in the simulated pickle fermentation system by as much as 70 %. The addition of glycine had no inhibitory effect on the amine-producing microorganisms, but it down-regulated the transcription levels of the genes for enzymes related to putrescine synthesis in Pichia, Lactobacillus, and Oenococcus, as well as the histidine decarboxylase genes in Lactobacillus and Tetragenococcus. Catalytic reaction assay using crude solutions of amino acid decarboxylase extracted from Lactobacillus brevis showed that the addition of glycine inhibited 45 %-55 % of ornithine decarboxylase and tyrosine decarboxylase activities. This study may provide a reference for the study and control of the mechanism of biogenic amine formation in pickle fermentation.
Topics: Fermentation; Glycine; Biogenic Amines; Salts; Putrescine; Tyramine; Food Microbiology; Lactobacillus; Fermented Foods; Pichia
PubMed: 38823874
DOI: 10.1016/j.foodres.2024.114501 -
World Journal of Microbiology &... May 2024The β-fructofuranosidase enzyme from Aspergillus niger has been extensively used to commercially produce fructooligosaccharides from sucrose. In this study, the native...
Evaluation of different glycerol fed-batch strategies in a lab-scale bioreactor for the improved production of a novel engineered β-fructofuranosidase enzyme in Pichia pastoris.
The β-fructofuranosidase enzyme from Aspergillus niger has been extensively used to commercially produce fructooligosaccharides from sucrose. In this study, the native and an engineered version of the β-fructofuranosidase enzyme were expressed in Pichia pastoris under control of the glyceraldehyde-3-phosphate dehydrogenase promoter, and production was evaluated in bioreactors using either dissolved oxygen (DO-stat) or constant feed fed-batch feeding strategies. The DO-stat cultivations produced lower biomass concentrations but this resulted in higher volumetric activity for both strains. The native enzyme produced the highest volumetric enzyme activity for both feeding strategies (20.8% and 13.5% higher than that achieved by the engineered enzyme, for DO-stat and constant feed, respectively). However, the constant feed cultivations produced higher biomass concentrations and higher volumetric productivity for both the native as well as engineered enzymes due to shorter process time requirements (59 h for constant feed and 155 h for DO-stat feed). Despite the DO-stat feeding strategy achieving a higher maximum enzyme activity, the constant feed strategy would be preferred for production of the β-fructofuranosidase enzyme using glycerol due to the many industrial advantages related to its enhanced volumetric enzyme productivity.
Topics: beta-Fructofuranosidase; Bioreactors; Glycerol; Biomass; Batch Cell Culture Techniques; Fermentation; Aspergillus niger; Saccharomycetales; Oxygen; Promoter Regions, Genetic; Culture Media; Recombinant Proteins; Pichia; Fungal Proteins; Glyceraldehyde-3-Phosphate Dehydrogenases; Oligosaccharides
PubMed: 38819502
DOI: 10.1007/s11274-024-04027-6 -
Food Chemistry: X Jun 2024Kombucha is a traditional beverage produced by a living culture known as SCOBY or "symbiotic culture of bacteria and yeast". Culture-dependent production is essential...
Kombucha is a traditional beverage produced by a living culture known as SCOBY or "symbiotic culture of bacteria and yeast". Culture-dependent production is essential for stable kombucha fermentation. The aim of this study was to design a microbial community and to determine the effect of that community on the flavor and chemical properties of kombucha. The fermentations were carried out using combinations of selected species including and , which were previously isolated from kombucha. The effects of monocultures and cocultures on fermentation were investigated. The highest acetic acid producer was , which has strong antioxidant properties. In the monoculture and coculture fermentations, aldehydes, acids, and esters were generally observed at the end of fermentation. This study confirms that microbiota reconstruction is a viable approach for achieving the production of kombucha with increased bioactive constituents and consumer acceptance.
PubMed: 38808165
DOI: 10.1016/j.fochx.2024.101469 -
Archives of Microbiology May 2024Yeast, which plays a pivotal role in the brewing, food, and medical industries, exhibits a close relationship with human beings. In this study, we isolated and purified...
Yeast, which plays a pivotal role in the brewing, food, and medical industries, exhibits a close relationship with human beings. In this study, we isolated and purified 60 yeast strains from the natural fermentation broth of Sidamo coffee beans to screen for indigenous beneficial yeasts. Among them, 25 strains were obtained through morphological characterization on nutritional agar medium from Wallerstein Laboratory (WL), with molecular biology identifying Saccharomyces cerevisiae strain YBB-47 and the remaining 24 yeast strains identified as Pichia kudriavzevii. We investigated the fermentation performance, alcohol tolerance, SO tolerance, pH tolerance, sugar tolerance, temperature tolerance, ester production capacity, ethanol production capacity, HS production capacity, and other brewing characteristics of YBB-33 and YBB-47. The results demonstrated that both strains could tolerate up to 3% alcohol by volume at a high sucrose mass concentration (400 g/L) under elevated temperature conditions (40 ℃), while also exhibiting a remarkable ability to withstand an SO mass concentration of 300 g/L at pH 3.2. Moreover, S. cerevisiae YBB-47 displayed a rapid gas production rate and strong ethanol productivity. whereas P. kudriavzevii YBB-33 exhibited excellent alcohol tolerance. Furthermore, this systematic classification and characterization of coffee bean yeast strains from the Sidamo region can potentially uncover additional yeasts that offer high-quality resources for industrial-scale coffee bean production.
Topics: Fermentation; Saccharomyces cerevisiae; Pichia; Ethanol; Hydrogen-Ion Concentration; Coffee; Coffea; Temperature; Seeds; Hydrogen Sulfide
PubMed: 38805051
DOI: 10.1007/s00203-024-04017-0 -
3 Biotech Jun 2024In order to search for high specific activity and the resistant xylanases to XIP-I and provide more alternative xylanases for industrial production, a strain of from...
UNLABELLED
In order to search for high specific activity and the resistant xylanases to XIP-I and provide more alternative xylanases for industrial production, a strain of from produced xylanases was isolated and identified. Three xylanase genes from Z-1 were cloned and successfully expressed in and , respectively. The specific activities of xyn1 xyn2 and xyn3 for birchwood xylan were 38.79, 0.85 and 243.83 U/mg in , and 40.11, 0 and 910.37 U/mg in , respectively. xyn3 and xyn3 had the similar optimum pH at 6.0 and pH stability at 5.0-9.0. However, they had different optimum temperature and thermal stability, with 30 °C for xyn3 and 40 °C for xyn3, and 4-35 °C for xyn3 and 4-40 °C for xyn3, respectively. The substrate spectrum and the kinetic parameters showed that the two xylanases also exhibited the highest xylanase activity and catalytic efficiency (/) toward birchwood xylan, with 243.83 U/mg and 61.44 mL/mg/s for xyn3 and 910.37 U/mg and 910.37 mL/mg/s for xyn3, respectively. This study provided a novel mesophilic xylanase with high specific activity and catalytic efficiency, thus making it a promising candidate for extensive applications in animal feed and food industry.
SUPPLEMENTARY INFORMATION
The online version contains supplementary material available at 10.1007/s13205-024-03973-0.
PubMed: 38803445
DOI: 10.1007/s13205-024-03973-0