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Heliyon Feb 2024Herbs and spices are food categories known to be at high risk of adulteration. Presence of undeclared foreign plant species has often been reported in oregano and may...
Herbs and spices are food categories known to be at high risk of adulteration. Presence of undeclared foreign plant species has often been reported in oregano and may have a direct impact on its organoleptic quality and potentially the safety of this aromatic herb. A droplet digital PCR approach was developed to assess the purity of oregano by quantifying the DNA copies of oregano versus the total plant DNA copies. Nuclear single-copy genes were selected by targeting the gene from oregano and the plant gene. The reactions were specific to the Origanum genus and plant materials respectively, whereas trueness and precision data confirmed the reliability of the method to quantify oregano. The applicability of the method was further verified on proficiency test samples before being applied on commercial oregano samples.
PubMed: 38420487
DOI: 10.1016/j.heliyon.2024.e25985 -
Microbial Cell Factories Feb 2024Komagataella phaffii (a.k.a. Pichia pastoris) harbors a glutamate utilization pathway in which synthesis of glutamate dehydrogenase 2 and phosphoenolpyruvate...
BACKGROUND
Komagataella phaffii (a.k.a. Pichia pastoris) harbors a glutamate utilization pathway in which synthesis of glutamate dehydrogenase 2 and phosphoenolpyruvate carboxykinase (PEPCK) is induced by glutamate. Glutamate-inducible synthesis of these enzymes is regulated by Rtg1p, a cytosolic, basic helix-loop-helix protein. Here, we report food-grade monosodium glutamate (MSG)-inducible recombinant protein production from K. phaffii PEPCK promoter (P) using green fluorescent protein (GFP) and receptor binding domain of SARS-CoV-2 virus (RBD) as model proteins.
RESULTS
P-RBD/GFP expression cassette was integrated at two different sites in the genome to improve recombinant protein yield from P. The traditional, methanol-inducible alcohol oxidase 1 promoter (P) was used as the benchmark. Initial studies carried out with MSG as the inducer resulted in low recombinant protein yield. A new strategy employing MSG/ethanol mixed feeding improved biomass generation as well as recombinant protein yield. Cell density of 100-120 A units/ml was achieved after 72 h of induction in shake flask cultivations, resulting in recombinant protein yield from P that is comparable or even higher than that from P.
CONCLUSIONS
We have designed an induction medium for recombinant protein production from K. phaffii P in shake flask cultivations. It consists of 1.0% yeast extract, 2.0% peptone, 0.17% yeast nitrogen base with ammonium sulfate, 100 mM potassium phosphate (pH 6.0), 0.4 mg/L biotin, 2.0% MSG, and 2% ethanol. Substitution of ammonium sulphate with 0.5% urea is optional. Carbon source was replenished every 24 h during 72 h induction period. Under these conditions, GFP and RBD yields from P equaled and even surpassed those from P. Compared to the traditional methanol-inducible expression system, the inducers of glutamate-inducible expression system are non-toxic and their metabolism does not generate toxic metabolites such as formaldehyde and hydrogen peroxide. This study sets the stage for MSG-inducible, industrial scale recombinant protein production from K. phaffii P in bioreactors.
Topics: Methanol; Sodium Glutamate; Recombinant Proteins; Glutamates; Saccharomycetales; Ethanol; Pichia
PubMed: 38402195
DOI: 10.1186/s12934-024-02340-1 -
International Journal of Molecular... Feb 2024Phosphoenolpyruvate carboxylase (PEPC) gene family plays a crucial role in both plant growth and response to abiotic stress. Approximately half of the Orchidaceae...
Phosphoenolpyruvate carboxylase (PEPC) gene family plays a crucial role in both plant growth and response to abiotic stress. Approximately half of the Orchidaceae species are estimated to perform CAM pathway, and the availability of sequenced orchid genomes makes them ideal subjects for investigating the gene family in CAM plants. In this study, a total of 33 genes were identified across 15 orchids. Specifically, one gene was found in and ; two in , , , , , , , and ; three in , , , , and . These genes were categorized into four subgroups, namely PEPC-i, PEPC-ii, and PEPC-iii (PTPC), and PEPC-iv (BTPC), supported by the comprehensive analyses of their physicochemical properties, motif, and gene structures. Remarkably, PEPC-iv contained a heretofore unreported orchid gene, identified as . Differences in the number of homolog genes among these species were attributed to segmental duplication, whole-genome duplication (WGD), or gene loss events. -elements identified in promoter regions were predominantly associated with light responsiveness, and circadian-related elements were observed in each PEPC-i and PEPC-ii gene. The expression levels of recruited BTPC, , exhibited a lower expression level than other s in the tested tissues. The expression analyses and RT-qPCR results revealed diverse expression patterns in orchid genes. Duplicated genes exhibited distinct expression patterns, suggesting functional divergence. This study offered a comprehensive analysis to unveil the evolution and function of genes in Orchidaceae.
Topics: Humans; Phosphoenolpyruvate Carboxylase; Plant Proteins; Orchidaceae; Plants; Base Sequence; Phylogeny
PubMed: 38396732
DOI: 10.3390/ijms25042055 -
Frontiers in Microbiology 2024Cellobiose, a β-1,4-linked glucose dimer, is a major cellodextrin resulting from the enzymatic hydrolysis of cellulose. It is a major source of carbon for soil...
Transcriptional regulation of cellobiose utilization by PRD-domain containing Sigma54-dependent transcriptional activator (CelR) and catabolite control protein A (CcpA) in .
Cellobiose, a β-1,4-linked glucose dimer, is a major cellodextrin resulting from the enzymatic hydrolysis of cellulose. It is a major source of carbon for soil bacteria. In bacteria, the phosphoenolpyruvate (PEP): carbohydrate phosphotransferase system (PTS), encoded by the operon, is responsible for the transport and utilization of cellobiose. In this study, we analyzed the transcription and regulation of the operon in (). The operon is composed of five genes forming one transcription unit. β-Galactosidase assays revealed that operon transcription is induced by cellobiose, controlled by Sigma54, and positively regulated by CelR. The HTH-AAA domain of CelR recognized and specifically bound to three possible binding sites in the promoter region. CelR contains two PTS regulation domains (PRD1 and PRD2), which are separated by two PTS-like domains-the mannose transporter enzyme IIA component domain (EIIA) and the galactitol transporter enzyme IIB component domain (EIIB). Mutations of His-546 on the EIIA domain and Cys-682 on the EIIB domain resulted in decreased transcription of the operon, and mutations of His-839 on PRD2 increased transcription of the operon. Glucose repressed the transcription of the operon and catabolite control protein A (CcpA) positively regulated this process by binding the promoter. In the and mutants, PTS activities were decreased, and cellobiose utilization was abolished, suggesting that the operon is essential for cellobiose utilization. has been widely used as a biological pesticide. The metabolic properties of are critical for fermentation. Nutrient utilization is also essential for the environmental adaptation of . Glucose is the preferred energy source for many bacteria, and the presence of the phosphotransferase system allows bacteria to utilize other sugars in addition to glucose. Cellobiose utilization pathways have been of particular interest owing to their potential for developing alternative energy sources for bacteria. The data presented in this study improve our understanding of the transcription patterns of gene clusters. This will further help us to better understand how cellobiose is utilized for bacterial growth.
PubMed: 38357353
DOI: 10.3389/fmicb.2024.1160472 -
Heliyon Feb 2024Diabetes is a dangerous metabolic disorder by increasing incidence in human societies worldwide. Recently, much attention has been focused on the development of...
Diabetes is a dangerous metabolic disorder by increasing incidence in human societies worldwide. Recently, much attention has been focused on the development of hypoglycemic agents, particularly the derivatives of herbal drugs, in the treatment of diabetes. This research aimed to study the anti-diabetic effect of in the diabetic rat models. First, the plant material was extracted from the leaves, and orally administered to the rats. After treating the animals with the aqueous extract of at a dose of 600 mg/kg, animal body weight for 12 weeks, fasting blood glucose, oral glucose tolerance test (OGTT), and body weight changes were examined. To analyze the anti-diabetic function of , we measured the expression of (GLUT4), phosphoenolpyruvate carboxykinase (PEPCK), and glucose 6-phosphatase (G6Pase) genes in healthy and streptozotocin (STZ)-diabetic rats. The expression levels of the genes of interest in muscle and liver tissues were determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC). There were no significant differences in fasting blood glucose and OGTT between normal control (NC) group and the diabetic control (DC) group treated with . In contrast, there was a significant difference with the untreated DC (). The treatment of diabetic rats with significantly increased the expression of GLUT4 in the muscle and decreased the expression levels of PEPCK and G6Pase in the liver compared to the DC group (). These findings clearly show that can improve hyperglycemia by increasing the GLUT4 expression, and inhibiting the gluconeogenesis pathway in the liver. In general, the obtained results provided a new insight into the efficacy of aqueous extract as an anti-diabetic herbal medicine.
PubMed: 38333789
DOI: 10.1016/j.heliyon.2024.e25256 -
Horticulture Research Feb 2024The subfamily Agavoideae comprises crassulacean acid metabolism (CAM), C3, and C4 plants with a young age of speciation and slower mutation accumulation, making it a...
The subfamily Agavoideae comprises crassulacean acid metabolism (CAM), C3, and C4 plants with a young age of speciation and slower mutation accumulation, making it a model crop for studying CAM evolution. However, the genetic mechanism underlying CAM evolution remains unclear because of lacking genomic information. This study assembled the genome of NO.11648, a constitutive CAM plant belonging to subfamily Agavoideae, at the chromosome level using data generated from high-throughput chromosome conformation capture, Nanopore, and Illumina techniques, resulting in 30 pseudo-chromosomes with a size of 4.87 Gb and scaffold N of 186.42 Mb. The genome annotation revealed 58 841 protein-coding genes and 76.91% repetitive sequences, with the dominant repetitive sequences being the I-type repeats (Copia and Gypsy accounting for 18.34% and 13.5% of the genome, respectively). Our findings also provide support for a whole genome duplication event in the lineage leading to , which occurred after its divergence from subfamily Asparagoideae. Moreover, we identified a gene duplication event in the phosphoenolpyruvate carboxylase kinase () gene family and revealed that three genes ( and ) were involved in the CAM pathway. More importantly, we identified transcription factors enriched in the circadian rhythm, MAPK signaling, and plant hormone signal pathway that regulate the expression by analysing the transcriptome and using yeast one-hybrid assays. Our results shed light on CAM evolution and offer an essential resource for the molecular breeding program of spp.
PubMed: 38333731
DOI: 10.1093/hr/uhad269 -
Journal of Industrial Microbiology &... Jan 2024As a key molecular scaffold for various flavonoids, naringenin is a value-added chemical with broad pharmaceutical applicability. For efficient production of naringenin...
UNLABELLED
As a key molecular scaffold for various flavonoids, naringenin is a value-added chemical with broad pharmaceutical applicability. For efficient production of naringenin from acetate, it is crucial to precisely regulate the carbon flux of the oxaloacetate-phosphoenolpyruvate (OAA-PEP) regulatory node through appropriate pckA expression control, as excessive overexpression of pckA can cause extensive loss of OAA and metabolic imbalance. However, considering the critical impact of pckA on naringenin biosynthesis, the conventional strategy of transcriptional regulation of gene expression is limited in its ability to cover the large and balanced solution space. To overcome this hurdle, in this study, pckA expression was fine-tuned at both the transcriptional and translational levels in a combinatorial expression library for the precise exploration of optimal naringenin production from acetate. Additionally, we identified the effects of regulating pckA expression by validating the correlation between phosphoenolpyruvate kinase (PCK) activity and naringenin production. As a result, the flux-optimized strain exhibited a 49.8-fold increase compared with the unoptimized strain, producing 122.12 mg/L of naringenin. Collectively, this study demonstrated the significance of transcriptional and translational flux rebalancing at the key regulatory node, proposing a pivotal metabolic engineering strategy for the biosynthesis of various flavonoids derived from naringenin using acetate.
ONE-SENTENCE SUMMARY
In this study, transcriptional and translational regulation of pckA expression at the crucial regulatory node was conducted to optimize naringenin biosynthesis using acetate in E. coli.
Topics: Escherichia coli; Phosphoenolpyruvate; Flavonoids; Acetates; Flavanones
PubMed: 38285614
DOI: 10.1093/jimb/kuae006 -
Foods (Basel, Switzerland) Nov 2023Malic acid is the predominant organic acid in grape berries, and its content is affected by abiotic factors such as temperature (fruit zone microclimate) and water (vine...
Malic acid is the predominant organic acid in grape berries, and its content is affected by abiotic factors such as temperature (fruit zone microclimate) and water (vine water status). The objectives of this study were to explore the potential mechanisms behind the effects of vine water status on the biosynthesis and degradation of berry malic acid and the potential downstream effects on berry metabolism. This study was conducted over two growing seasons in 2021 and 2022, comprising three watering regimes: no water stress (CK), light water stress (LWS), and moderate water stress (MWS). Compared to CK, a significantly higher level of malic acid was found in berries from the MWS treatment when the berry was still hard and green (E-L 33) in both years. However, water stress reduced the malic acid content at the ripe berry harvest (E-L 38) stage. The activities of NAD-malate dehydrogenase (NAD-MDH) and pyruvate kinase (PK) were enhanced by water stress. Except for the E-L 33 stage, the activity of phosphoenolpyruvate carboxylase (PEPC) was reduced by water stress. The highest phosphoenolpyruvate carboxykinase (PEPCK) activity was observed at the berry veraison (E-L 35) stage and coincided with the onset of a decrease in the malate content. Meanwhile, the expression of was consistent with its enzyme activity. This study showed that water stress changed the content of some free amino acids (GABA, proline, leucine, aspartate, and glutamate), two of which (glutamate and GABA) are primary metabolites of the GABA pathway.
PubMed: 38231685
DOI: 10.3390/foods12234191 -
ACS Omega Jan 2024Conjugated linoleic acid (CLA) holds significant application prospects due to its anticancer, anti-atherosclerosis, lipid-lowering, weight-loss, and growth-promoting...
Conjugated linoleic acid (CLA) holds significant application prospects due to its anticancer, anti-atherosclerosis, lipid-lowering, weight-loss, and growth-promoting functions. The key to its efficient production lies in optimizing the biocatalytic performance of linoleic acid isomerase (LAI). Here, we constructed a mutant library and screened positive mutants with high linoleate isomerase activity. The proteomics and metabolomics were used to explore the mechanism in the regulation of linoleic acid isomerase activity. High-throughput proteomics revealed 104 differentially expressed proteins unique to positive mutant strains of linoleic acid isomerase of which 57 were upregulated and 47 were downregulated. These differentially expressed proteins were primarily involved in galactose metabolism, the phosphotransferase system, starch metabolism, and sucrose metabolism. Differential metabolic pathways were mainly enriched in amino acid biosynthesis, including glutamate metabolism, the Aminoacyl-tRNA biosynthesis pathway, and the ABC transporter pathway. The upregulated metabolites include dl-valine and Acetyl coA, while the downregulated metabolites include Glutamic acid and Phosphoenolpyruvate. Overall, the activity of linoleic acid isomerase in the mutant strain was increased by the regulation of key proteins involved in galactose metabolism, sucrose metabolism, and the phosphotransferase system. This study provides a theoretical basis for the development of high-yield CLA food.
PubMed: 38222669
DOI: 10.1021/acsomega.3c08243 -
PloS One 2024We evaluated the effect of Tucum-do-Cerrado on glucose metabolism homeostasis and its relationship with redox-inflammatory responses in a high-fat (HF) diet-induced...
INTRODUCTION
We evaluated the effect of Tucum-do-Cerrado on glucose metabolism homeostasis and its relationship with redox-inflammatory responses in a high-fat (HF) diet-induced obesity model.
RESULTS
The HF diet increased energy intake, feed efficiency, body weight, muscle and hepatic glycogen, insulin, homeostatic model assessment of insulin resistance (HOMA IR) and beta (β)-cell function, and gut catalase (CAT) activity, and decreased food intake, hepatic glutathione reductase (GR), glutathione peroxidase (GPX), glutathione S-transferase (GST), and superoxide dismutase (SOD) activities, hepatic phosphoenolpyruvate carboxykinase 1 (Pck1), and intestinal solute carrier family 5 member 1 (Slc5a1) mRNA levels compared with the control diet. However, the HF diet with Tucum-do-Cerrado decreased hepatic glycogen, and increased hepatic GR activity, hepatic Slc2a2 mRNA levels and serum Tnfa compared with the HF diet. Tucum-do-Cerrado decreased muscle glycogen, intestinal CAT and GPX activities, muscle PFK-1 and HK activities, and increased hepatic protein (CARB) and intestinal lipid (MDA) oxidation, hepatic GST activity, serum antioxidant potential, hepatic phosphofructokinase-1 (PFK-1) activity, intestinal solute carrier family 2 member 2 (Slc2a2), tumor necrosis factor (Tnf), interleukin-1 beta (Il1b), muscle protein kinase AMP-activated alpha 1 (Prkaa1), solute carrier family 2 member 2 (Slc2a2) mRNA levels, and serum interleukin-6 (IL-6) levels, regardless of diet type.
CONCLUSION
Tucum-do-Cerrado consumption may ameliorate impaired glucose utilization in a HF diet-induced obesity model by increasing liver and muscle glucose uptake and oxidation. These data suggest that Tucum-do-Cerrado consumption improves muscle glucose oxidation in non-obese and obese rats. This response may be related to the improvement in the total antioxidant capacity of rats.
Topics: Rats; Animals; Glucose; Antioxidants; Diet, High-Fat; Liver Glycogen; Obesity; Liver; Arecaceae; RNA, Messenger
PubMed: 38206915
DOI: 10.1371/journal.pone.0293627