-
PLoS Genetics Jun 2024Lager yeasts are limited to a few strains worldwide, imposing restrictions on flavour and aroma diversity and hindering our understanding of the complex evolutionary...
Lager yeasts are limited to a few strains worldwide, imposing restrictions on flavour and aroma diversity and hindering our understanding of the complex evolutionary mechanisms during yeast domestication. The recent finding of diverse S. eubayanus lineages from Patagonia offers potential for generating new lager yeasts with different flavour profiles. Here, we leverage the natural genetic diversity of S. eubayanus and expand the lager yeast repertoire by including three distinct Patagonian S. eubayanus lineages. We used experimental evolution and selection on desirable traits to enhance the fermentation profiles of novel S. cerevisiae x S. eubayanus hybrids. Our analyses reveal an intricate interplay of pre-existing diversity, selection on species-specific mitochondria, de-novo mutations, and gene copy variations in sugar metabolism genes, resulting in high ethanol production and unique aroma profiles. Hybrids with S. eubayanus mitochondria exhibited greater evolutionary potential and superior fitness post-evolution, analogous to commercial lager hybrids. Using genome-wide screens of the parental subgenomes, we identified genetic changes in IRA2, IMA1, and MALX genes that influence maltose metabolism, and increase glycolytic flux and sugar consumption in the evolved hybrids. Functional validation and transcriptome analyses confirmed increased maltose-related gene expression, influencing greater maltotriose consumption in evolved hybrids. This study demonstrates the potential for generating industrially viable lager yeast hybrids from wild Patagonian strains. Our hybridization, evolution, and mitochondrial selection approach produced hybrids with high fermentation capacity and expands lager beer brewing options.
Topics: Beer; Fermentation; Saccharomyces cerevisiae; Hybridization, Genetic; Saccharomyces; Ethanol; Mitochondria; Genome, Fungal; Evolution, Molecular; Genetic Variation; Maltose; Mutation
PubMed: 38900713
DOI: 10.1371/journal.pgen.1011154 -
Fungal Genetics and Biology : FG & B Jun 2024In the filamentous fungus Aspergillus oryzae, large amounts of amylolytic enzymes are inducibly produced by isomaltose, which is converted from maltose incorporated via...
Glucose-induced endocytic degradation of the maltose transporter MalP is mediated through ubiquitination by the HECT-ubiquitin ligase HulA and its adaptor CreD in Aspergillus oryzae.
In the filamentous fungus Aspergillus oryzae, large amounts of amylolytic enzymes are inducibly produced by isomaltose, which is converted from maltose incorporated via the maltose transporter MalP. In contrast, the preferred sugar glucose strongly represses the expression of both amylolytic and malP genes through carbon catabolite repression. Simultaneously, the addition of glucose triggers the endocytic degradation of MalP on the plasma membrane. In budding yeast, the signal-dependent ubiquitin modification of plasma membrane transporters leads to selective endocytosis into the vacuole for degradation. In addition, during glucose-induced MalP degradation, the homologous of E6AP C-terminus-type E3 ubiquitin ligase (HulA) is responsible for the ubiquitin modification of MalP, and the arrestin-like protein CreD is required for HulA targeting. Although CreD-mediated MalP internalization occurs in response to glucose, the mechanism by which CreD regulates HulA-dependent MalP ubiquitination remains unclear. In this study, we demonstrated that three (P/L)PxY motifs present in the CreD protein are essential for functioning as HulA adaptors so that HulA can recognize MalP in response to glucose stimulation, enabling MalP internalization. Furthermore, four lysine residues (three highly conserved among Aspergillus species and yeast and one conserved among Aspergillus species) of CreD were found to be necessary for its ubiquitination, resulting in efficient glucose-induced MalP endocytosis. The results of this study pave the way for elucidating the regulatory mechanism of MalP endocytic degradation through ubiquitination by the HulA-CreD complex at the molecular level.
PubMed: 38885923
DOI: 10.1016/j.fgb.2024.103909 -
PLoS Computational Biology Jun 2024Periplasmic binding proteins (PBPs) are bacterial proteins commonly used as scaffolds for substrate-detecting biosensors. In these biosensors, effector proteins (for...
Periplasmic binding proteins (PBPs) are bacterial proteins commonly used as scaffolds for substrate-detecting biosensors. In these biosensors, effector proteins (for example fluorescent proteins) are inserted into a PBP such that the effector protein's output changes upon PBP-substate binding. The insertion site is often determined by comparison of PBP apo/holo crystal structures, but random insertion libraries have shown that this can miss the best sites. Here, we present a PBP biosensor design method based on residue contact analysis from molecular dynamics. This computational method identifies the best previously known insertion sites in the maltose binding PBP, and suggests further previously unknown sites. We experimentally characterise fluorescent protein insertions at these new sites, finding they too give functional biosensors. Furthermore, our method is sufficiently flexible to both suggest insertion sites compatible with a variety of effector proteins, and be applied to binding proteins beyond PBPs.
PubMed: 38885277
DOI: 10.1371/journal.pcbi.1012212 -
Frontiers in Bioengineering and... 2024Calcitonin gene-related peptide (CGRP) is involved in trigeminal neuralgia and migraine, and measuring the CGRP concentration in the serum is crucial for the early...
Calcitonin gene-related peptide (CGRP) is involved in trigeminal neuralgia and migraine, and measuring the CGRP concentration in the serum is crucial for the early prediction of these conditions. Current methods for CGRP detection are primarily radioimmunoassay, which needs radioactive substances and enzyme-linked immunosorbent assays (ELISAs) which need long detection time and some have a narrow detection range. The genes of anti-CGRP antibody variable regions were cloned into pDong1 vector to obtain pDong1/Fab-CGRP, with which phage-Fab was prepared, and the concentration of CGRP was detected by competitive ELISA. The pDong1/Fab-CGRP was modified to obtain pDong1/OS-CGRP, with which the co-expression solution containing phage-displayed heavy chain variable fragments (phage-V) and light chain was obtained. CGRP was detected by OS-ELISA based on phage-V, antibody light chain, and anti-light chain antibody. The V gene was cloned into the pMAL vector to obtain pMAL-V (CGRP), with which maltose binding protein fused with V (MBP-V) was prepared. CGRP was detected by OS-ELISA employing MBP-V and phage-V. OS-ELISAs that measure the CGRP concentration by quantifying the interaction between variable regions were investigated. OS-ELISA using phage-V and secreted light chains in the same culture system exhibited a limit of detection (LOD) of 0.05 nM, offering higher sensitivity than competitive assay with an LOD of 0.75 nM, whereas using phage-V and separately prepared MBP-V exhibited an LOD of 0.15 nM and a broader detection range of 0.15-500 nM than competitive ELISA, whose detection range was 0.75-10 nM. The combination of the two OS assays achieved high sensitivity and a broad detection range for CGRP, which may have significance in clinical applications.
PubMed: 38882635
DOI: 10.3389/fbioe.2024.1395330 -
AMB Express Jun 2024α-Amylase plays a crucial role in the industrial degradation of starch. The genus Jeotgalibacillus of the underexplored marine bacteria family Caryophanaceae has not...
α-Amylase plays a crucial role in the industrial degradation of starch. The genus Jeotgalibacillus of the underexplored marine bacteria family Caryophanaceae has not been investigated in terms of α-amylase production. Herein, we report the comprehensive analysis of an α-amylase (AmyJM) from Jeotgalibacillus malaysiensis D5 (= DSM28777 = KCTC33550). Protein phylogenetic analysis indicated that AmyJM belongs to glycoside hydrolase family 13 subfamily 5 (GH13_5) and exhibits low sequence identity with known α-amylases, with its closest counterpart being the GH13_5 α-amylase from Bacillus sp. KSM-K38 (51.05% identity). Purified AmyJM (molecular mass of 70 kDa) is stable at a pH range of 5.5-9.0 and optimally active at pH 7.5. The optimum temperature for AmyJM is 40 °C, where the enzyme is reasonably stable at this temperature. Similar to other α-amylases, the presence of CaCl enhanced both the activity and stability of AmyJM. AmyJM exhibited activity toward raw and gelatinized forms of starches and related α-glucans, generating a mixture of reducing sugars, such as glucose, maltose, maltotriose, maltotetraose, and maltopentaose. In raw starch hydrolysis, AmyJM exhibited its highest efficiency (51.10% degradation) in hydrolyzing raw wheat starch after 3-h incubation at 40 °C. Under the same conditions, AmyJM also hydrolyzed tapioca, sago, potato, rice, and corn raw starches, yielding 16.01-30.05%. These findings highlight the potential of AmyJM as a biocatalyst for the saccharification of raw starches, particularly those derived from wheat.
PubMed: 38874807
DOI: 10.1186/s13568-024-01722-3 -
Food Science & Nutrition Jun 2024Diguo ( Bur.), an ancient wild fruit, is widely spread in southwest China. However, there is little information on the phenotypic traits, quality characteristics, and...
Diguo ( Bur.), an ancient wild fruit, is widely spread in southwest China. However, there is little information on the phenotypic traits, quality characteristics, and aroma compounds available to diguo fruit. The present study is an investigation into the effects of geographical origin on the phenotypic traits and quality characteristics of wild diguo fruit collected from southwest China. The volatile compounds in the mixed fruit samples were also investigated using gas chromatography-mass spectrometry. Our results indicated that significant variation existed among the sampling materials in all the phenotypic parameters. Fruit fresh weight ranged between 2.06 and 4.59 g. Moreover, significant variation existed among the selected materials in all macronutrients (dry matter, total soluble solids, crude protein, crude fat, and ash) and some nutritional parameters (glutamate, arginine, total soluble solids, maltose, and mannose, etc.). Regardless of their geographical origin, diguo fruit is relatively low in fat and fructose and high in fiber and glutamate. A total of 95 volatile constituents were identified in the frozen diguo fruit. In conclusion, diguo fruit with rich nutritional attributes has a promising future for commercial-scale production. The variability of the observed morphological and nutritional features of diguo fruit provides important characteristics for improving the breeding of diguo as a modern fruit crop.
PubMed: 38873439
DOI: 10.1002/fsn3.4106 -
Current Developments in Nutrition Jun 2024Iron supplementation, especially in female athletes, is 1 of the influential factors in aerobic capacity, and its deficiency can lead to significant problems related to...
BACKGROUND
Iron supplementation, especially in female athletes, is 1 of the influential factors in aerobic capacity, and its deficiency can lead to significant problems related to reduced aerobic capacity.
OBJECTIVES
This study aimed to investigate the effect of 3 wk of iron supplementation on the aerobic capacity of female handball players.
METHODS
In this randomized double-blinded, and placebo control trial, 14 elite handball players (age: 21.6 ± 5.68 y; height: 169.5 ± 4.9 cm; weight: 62.2 ± 9.25 kg; body mass index (in kg/m): 21.5 ± 2.9) randomly divided into 2 supplement groups (receiving a 100 mg/d of poly-maltose tri hydroxide iron complex in the form of tablets) and the placebo group (receiving a tablet containing 100 mg/d starch which is the same color and shape as iron tablets). The supplementation protocol was performed for 3 wk during the off-season. Maximal oxygen consumption (VO), amounts of carbon dioxide at the first ventilatory threshold, amounts of carbon dioxide at the second ventilatory threshold, time to exhaustion (TTE), pulmonary ventilation (VE), ventilatory equivalents for oxygen, amounts of oxygen at the first ventilatory threshold, amounts of oxygen at the second ventilatory threshold, time to reach first ventilatory threshold, end-tidal partial pressure of oxygen at the first ventilatory threshold, end-tidal partial pressure of carbon dioxide at the first ventilatory threshold and ventilatory equivalents for carbon dioxide were measured using the Bruce test and gas analyzer in 2 pretest and posttest stages.
RESULTS
There were significant improvements in oxygen at the first ventilatory threshold, time to reach first ventilatory threshold, and end-tidal partial pressure of carbon dioxide at the first ventilatory threshold and a significant decrease in end-tidal partial pressure of oxygen at the first ventilatory threshold ( < 0.05). Also, no significant changes were found in VO, carbon dioxide at the first ventilatory threshold, carbon dioxide at the second ventilatory threshold, oxygen at the second ventilatory threshold, TTE, VE, ventilatory equivalents for oxygen, and ventilatory equivalents for carbon dioxide after 3 wk of iron supplementation ( > 0.05).
CONCLUSIONS
The study found that 3 wk of off-season iron supplementation positively impacted female handball players' aerobic capacity; however, it did not significantly improve their VO.
PubMed: 38868615
DOI: 10.1016/j.cdnut.2024.103767 -
Scientific Reports Jun 2024Xenocoumacin 1 (Xcn 1), antibiotic discovered from secondary metabolites of Xenorhabdus nematophila, had the potential to develop into a new pesticide due to its...
Xenocoumacin 1 (Xcn 1), antibiotic discovered from secondary metabolites of Xenorhabdus nematophila, had the potential to develop into a new pesticide due to its excellent activity against bacteria, oomycetes and fungi. However, the current low yield of Xcn1 limits its development and utilization. To improve the yield of Xcn1, response surface methodology was used to determine the optimal composition of fermentation medium and one factor at a time approach was utilized to optimize the fermentation process. The optimal medium composed of in g/L: proteose peptone 20.8; maltose 12.74; KHPO 3.77. The optimal fermentation conditions were that 25 °C, initial pH 7.0, inoculum size 10%, culture medium 75 mL in a 250 mL shake flask with an agitation rate of 150 rpm for 48 h. Xenorhabdus nematophila YL001 was produced the highest Xcn1 yield (173.99 mg/L) when arginine was added to the broth with 3 mmol/L at the 12th h. Compared with Tryptic Soy Broth medium, the optimized fermentation process resulted in a 243.38% increase in Xcn1 production. The obtained results confirmed that optimizing fermentation technology led to an increase in Xcn1 yield. This work would be helpful for efficient Xcn1 production and lay a foundation for its industrial production.
Topics: Xenorhabdus; Fermentation; Culture Media; Hydrogen-Ion Concentration; Anti-Bacterial Agents; Benzopyrans
PubMed: 38866882
DOI: 10.1038/s41598-024-63794-2 -
Virus Research Aug 2024African swine fever virus (ASFV) is a large double-stranded DNA virus with a complex structural architecture and encodes more than 150 proteins, where many are with...
African swine fever virus (ASFV) is a large double-stranded DNA virus with a complex structural architecture and encodes more than 150 proteins, where many are with unknown functions. E184L has been reported as one of the immunogenic ASFV proteins that may contribute to ASFV pathogenesis and immune evasion. However, the antigenic epitopes of E184L are not yet characterized. In this study, recombinant E184L protein was expressed in prokaryotic expression system and four monoclonal antibodies (mAbs), designated as 1A10, 2D2, 3H6, and 4C10 were generated. All four mAbs reacted specifically with ASFV infected cells. To identify the epitopes of the mAbs, a series of overlapped peptides of E184L were designed and expressed as maltose binding fusion proteins. Accordingly, the expressed fusion proteins were probed with each E184L mAb separately by using Western blot. Following a fine mapping, the minimal linear epitope recognized by mAb 1A10 was identified as IQRQGFL, and mAbs 2D2, 3H6, and 4C10 recognized a region located between DPTEFF. Alignment of amino acids of E184L revealed that the two linear epitopes are highly conserved among different ASFV isolates. Furthermore, the potential application of the two epitopes in ASFV diagnosis was assessed through epitope-based ELISA using 24 ASFV positive and 18 negative pig serum and the method were able to distinguish positive and negative samples, indicating the two epitopes are dominant antigenic sites. To our knowledge, this is the first study to characterize the B cell epitopes of the antigenic E184L protein of ASFV, offering valuable tools for future research, as well as laying a foundation for serological diagnosis and epitope-based marker vaccine development.
Topics: African Swine Fever Virus; Antibodies, Monoclonal; Epitopes, B-Lymphocyte; Animals; Epitope Mapping; Antibodies, Viral; Swine; African Swine Fever; Mice; Viral Proteins; Antigens, Viral; Mice, Inbred BALB C
PubMed: 38838820
DOI: 10.1016/j.virusres.2024.199412 -
The Journal of International Medical... May 2024To investigate the hepatic effects of high-dose intravenous (IV) iron, including those on liver function and the degree of fibrosis, in a rat model of cirrhosis.
OBJECTIVE
To investigate the hepatic effects of high-dose intravenous (IV) iron, including those on liver function and the degree of fibrosis, in a rat model of cirrhosis.
METHODS
We evenly allocated 25 Sprague-Dawley rats into five groups: normal rats (control group), cirrhotic rats receiving IV normal saline (liver cirrhosis [LC] group), and cirrhotic rats receiving 20, 40, or 80 mg/kg IV ferric carboxymaltose (LC-iron20, LC-iron40, and LC-iron80 group, respectively). Biochemical parameters were compared at 0, 7, 14, 21, and 28 days. The degrees of hepatic fibrosis and iron deposition were evaluated. Inflammatory and oxidative stress markers were also compared.
RESULTS
There were no significant differences in the 28-day serum alanine aminotransferase levels among the LC-iron20, LC-iron40, and LC-iron80 groups (69 ± 7, 1003 ± 127, 1064 ± 309, 919 ± 346, and 820 ± 195 IU/L in the control, LC, LC-iron20, LC-iron40, and LC-iron80 groups, respectively). Hepatic iron accumulation increased in a dose-dependent manner, but the degree of hepatic fibrosis was comparable among the groups. The inflammatory and oxidative stress marker levels did not differ significantly according to the IV iron dose.
CONCLUSIONS
Administration of IV iron at various high doses appears safe in our rat model of cirrhosis.
Topics: Animals; Rats, Sprague-Dawley; Liver; Oxidative Stress; Male; Liver Cirrhosis; Disease Models, Animal; Rats; Ferric Compounds; Iron; Injections, Intravenous; Alanine Transaminase; Maltose; Biomarkers; Liver Function Tests; Dose-Response Relationship, Drug
PubMed: 38811356
DOI: 10.1177/03000605241253733