-
Biology Aug 2022Pyruvate decarboxylase (PDC) is a key enzyme involved in ethanol fermentation, and it catalyzes the decarboxylation of pyruvate to acetaldehyde and CO. Bifunctional...
Pyruvate decarboxylase (PDC) is a key enzyme involved in ethanol fermentation, and it catalyzes the decarboxylation of pyruvate to acetaldehyde and CO. Bifunctional PORs/PDCs that also have additional pyruvate:ferredoxin oxidoreductase (POR) activity are found in hyperthermophiles, and they are mostly oxygen-sensitive and CoA-dependent. Thermostable and oxygen-stable PDC activity is highly desirable for biotechnological applications. The enzymes from the thermoacidophiles (formerly ) (Ss, T = 80 °C) and (Sa, T = 80 °C) were purified and characterized, and their biophysical and biochemical properties were determined comparatively. Both enzymes were shown to be heterodimeric, and their two subunits were determined by SDS-PAGE to be 37 ± 3 kDa and 65 ± 2 kDa, respectively. The purified enzymes from and showed both PDC and POR activities which were CoA-dependent, and they were thermostable with half-life times of 2.9 ± 1 and 1.1 ± 1 h at 80 °C, respectively. There was no loss of activity in the presence of oxygen. Optimal pH values for their PDC and POR activity were determined to be 7.9 and 8.6, respectively. In conclusion, both thermostable SsPOR/PDC and SaPOR/PDC catalyze the CoA-dependent production of acetaldehyde from pyruvate in the presence of oxygen.
PubMed: 36009875
DOI: 10.3390/biology11081247 -
Antioxidants (Basel, Switzerland) Aug 2022We have previously shown in a murine model of Non-alcoholic Fatty Liver Disease (NAFLD) that chronic, low-dose exposure to the Harmful Algal Bloom cyanotoxin...
Antioxidant Therapy Significantly Attenuates Hepatotoxicity following Low Dose Exposure to Microcystin-LR in a Murine Model of Diet-Induced Non-Alcoholic Fatty Liver Disease.
We have previously shown in a murine model of Non-alcoholic Fatty Liver Disease (NAFLD) that chronic, low-dose exposure to the Harmful Algal Bloom cyanotoxin microcystin-LR (MC-LR), resulted in significant hepatotoxicity including micro-vesicular lipid accumulation, impaired toxin metabolism as well as dysregulation of the key signaling pathways involved in inflammation, immune response and oxidative stress. On this background we hypothesized that augmentation of hepatic drug metabolism pathways with targeted antioxidant therapies would improve MC-LR metabolism and reduce hepatic injury in NAFLD mice exposed to MC-LR. We chose N-acetylcysteine (NAC, 40 mM), a known antioxidant that augments the glutathione detoxification pathway and a novel peptide (pNaKtide, 25 mg/kg) which is targeted to interrupting a specific Src-kinase mediated pro-oxidant amplification mechanism. Histological analysis showed significant increase in hepatic inflammation in NAFLD mice exposed to MC-LR which was attenuated on treatment with both NAC and pNaKtide (both ≤ 0.05). Oxidative stress, as measured by 8-OHDG levels in urine and protein carbonylation in liver sections, was also significantly downregulated upon treatment with both antioxidants after MC-LR exposure. Genetic analysis of key drug transporters including , Phase I enzyme- and Phase II metabolic enzymes- (Pyruvate kinase, muscle), (Pyruvate kinase, liver, and red blood cell) and (Glutamic acid decarboxylase) was significantly altered by MC-LR exposure as compared to the non-exposed control group (all ≤ 0.05). These changes were significantly attenuated with both pNaKtide and NAC treatment. These results suggest that MC-LR metabolism and detoxification is significantly impaired in the setting of NAFLD, and that these pathways can potentially be reversed with targeted antioxidant treatment.
PubMed: 36009344
DOI: 10.3390/antiox11081625 -
Frontiers in Bioengineering and... 2022Production of 3-hydroxypropionic acid (3-HP) in () the malonyl-CoA pathway has been recently demonstrated using glycerol as a carbon source, but the reported metrics...
Production of 3-hydroxypropionic acid (3-HP) in () the malonyl-CoA pathway has been recently demonstrated using glycerol as a carbon source, but the reported metrics were not commercially relevant. The flux through the heterologous pathway from malonyl-CoA to 3-HP was hypothesized as the main bottleneck. In the present study, different metabolic engineering approaches have been combined to improve the productivity of the original 3-HP producing strains. To do so, an additional copy of the gene encoding for the potential rate-limiting step of the pathway, i.e., the C-terminal domain of the malonyl-CoA reductase, was introduced. In addition, a variant of the endogenous acetyl-CoA carboxylase ( ) was overexpressed with the aim to increase the delivery of malonyl-CoA. Furthermore, the genes encoding for the pyruvate decarboxylase, aldehyde dehydrogenase and acetyl-CoA synthase, respectively, were overexpressed to enhance conversion of pyruvate into cytosolic acetyl-CoA, and the main gene responsible for the production of the by-product D-arabitol was deleted. Three different screening conditions were used to classify the performance of the different strains: 24-deep-well plates batch cultures, small-scale cultures in falcon tubes using FeedBeads® (i.e., slow release of glycerol over time), and mini bioreactor batch cultures. The best two strains from the FeedBeads® screening, PpHP8 and PpHP18, were tested in bioreactor fed-batch cultures using a pre-fixed exponentially increasing feeding rate. The strain PpHP18 produced up to 37.05 g L of 3-HP at 0.712 g L h with a final product yield on glycerol of 0.194 Cmol in fed-batch cultures. Remarkably, PpHP18 did not rank among the 2-top producer strains in small scale batch cultivations in deep-well plates and mini bioreactors, highlighting the importance of multiplexed screening conditions for adequate assessment of metabolic engineering strategies. These results represent a 50% increase in the product yield and final concentration, as well as over 30% increase in volumetric productivity compared to the previously obtained metrics for . Overall, the combination of glycerol as carbon source and a metabolically engineered strain resulted in the highest 3-HP concentration and productivity reported so far in yeast.
PubMed: 35935509
DOI: 10.3389/fbioe.2022.942304 -
The Journal of Biological Chemistry Sep 2022Proximity-dependent protein labeling provides a powerful in vivo strategy to characterize the interactomes of specific proteins. We previously optimized a proximity...
Proximity-dependent protein labeling provides a powerful in vivo strategy to characterize the interactomes of specific proteins. We previously optimized a proximity labeling protocol for Caenorhabditis elegans using the highly active biotin ligase TurboID. A significant constraint on the sensitivity of TurboID is the presence of abundant endogenously biotinylated proteins that take up bandwidth in the mass spectrometer, notably carboxylases that use biotin as a cofactor. In C. elegans, these comprise POD-2/acetyl-CoA carboxylase alpha, PCCA-1/propionyl-CoA carboxylase alpha, PYC-1/pyruvate carboxylase, and MCCC-1/methylcrotonyl-CoA carboxylase alpha. Here, we developed ways to remove these carboxylases prior to streptavidin purification and mass spectrometry by engineering their corresponding genes to add a C-terminal His tag. This allows us to deplete them from C. elegans lysates using immobilized metal affinity chromatography. To demonstrate the method's efficacy, we use it to expand the interactome map of the presynaptic active zone protein ELKS-1. We identify many known active zone proteins, including UNC-10/RIM, SYD-2/liprin-alpha, SAD-1/BRSK1, CLA-1/CLArinet, C16E9.2/Sentryn, as well as previously uncharacterized potentially synaptic proteins such as the ortholog of human angiomotin, F59C12.3 and the uncharacterized protein R148.3. Our approach provides a quick and inexpensive solution to a common contaminant problem in biotin-dependent proximity labeling. The approach may be applicable to other model organisms and will enable deeper and more complete analysis of interactors for proteins of interest.
Topics: Acetyl-CoA Carboxylase; Animals; Biotinylation; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Carboxy-Lyases; Carrier Proteins; Intercellular Signaling Peptides and Proteins; Methylmalonyl-CoA Decarboxylase; Pyruvate Carboxylase; Streptavidin
PubMed: 35933017
DOI: 10.1016/j.jbc.2022.102343 -
MSystems Aug 2022Traditional fermentation processes are driven by complex fungal microbiomes. However, the exact means by which fungal diversity affects fermentation remains unclear. In...
Traditional fermentation processes are driven by complex fungal microbiomes. However, the exact means by which fungal diversity affects fermentation remains unclear. In this study, we systematically investigated the diversity of a fungal community and its functions during the multibatch fermentation process. Metabolomics analysis showed that the metabolic profiles of the were enhanced with an increase in the fermentation time, as determined from the characteristic volatile flavors. High-throughput sequencing technology revealed that the major fungal species involved in sauce-flavor fermentation are sp. (41.75%, average relative abundance), sp. (13.07%), thermophilic species (9.16%), sp. (6.80%), Aspergillus sp. (4.69%), sp. (3.76%), sp. (3.74%), and Zygosaccharomyces sp. (1.41%). In addition, the fungal diversity increased as the number of fermentation batches increased. Moreover, the increased fungal diversity contributed to the modularity of the fungal communities, wherein sp., sp., and sp. maintained the stability of the fungal community. In addition, metatranscriptomics sequencing technologies were used to reconstruct the key metabolic pathways during fermentation, and it was found that the increased microbial diversity significantly promoted glucose-mediated carbon metabolism. Finally, functional gene analysis showed that functional microorganisms, such as Zygosaccharomyces and , can enhance fermentation as a result of the high expression of pyruvate decarboxylase and propanol-preferring alcohol dehydrogenase during the metabolism of pyruvate. These results indicate that fungal biodiversity can be exploited to enhance fermentation-based processes via network interactions and metabolism during multiple-batch fermentation. Biodiversity and network interactions act simultaneously on the microbial community structure in the fermentation process, thereby rendering the microbiome dynamics challenging to manage and predict. Understanding the complex fermentation community and its relationship to community functions is therefore important in the context of developing improved fermentation biotechnology systems. Our work demonstrates that multiple-batch fermentation steps increase microbial diversity and promote community stability. Crucially, the enhanced modularity in the microbial network increases the metabolism of flavor compounds and ethanol. This study highlights the power of biodiversity and network interactions in regulating the function of the microbiome in food fermentation ecosystems.
Topics: Fermentation; Microbiota; Carbohydrate Metabolism; Mycobiome; Pichia; Saccharomyces
PubMed: 35862822
DOI: 10.1128/msystems.00401-22 -
Biotech (Basel (Switzerland)) Mar 2022Drought is one of the most important threats to plants and agriculture. Here, the effects of four drought levels (90%, 55%, 40%, and 25% field capacity) on the relative...
Drought is one of the most important threats to plants and agriculture. Here, the effects of four drought levels (90%, 55%, 40%, and 25% field capacity) on the relative water content (RWC), chlorophyll and carotenoids levels, and mRNA gene expression of metabolic enzymes in (as sensitive to drought) and (as a drought-tolerant species) were evaluated. The physiological results showed that the treatment predominantly affected the RWC, chlorophyll, and carotenoids content. The gene expression analysis demonstrated that moderate and severe drought stress had greater effects on the expression of histone deacetylase-6 (HDA-6) and acetyl-CoA synthetase in both species. Pyruvate decarboxylase-1 (PDC-1) was upregulated in at high drought levels. Finally, succinyl CoA ligase was not affected by drought stress in either species. Data confirmed water stress is able to alter the gene expression of specific enzymes. Furthermore, our results suggest that PDC-1 expression is independent from HDA-6 and the increased expression of ACS can be due to the activation of new pathways involved in carbohydrate production.
PubMed: 35822781
DOI: 10.3390/biotech11020008 -
International Journal of Molecular... Jun 2022The transgenic tobacco ( L.) plants with the modified levels of alternative oxidase (AOX) were used to evaluate the physiological roles of AOX in regulating...
The Role of Alternative Oxidase in the Interplay between Nitric Oxide, Reactive Oxygen Species, and Ethylene in Tobacco ( L.) Plants Incubated under Normoxic and Hypoxic Conditions.
The transgenic tobacco ( L.) plants with the modified levels of alternative oxidase (AOX) were used to evaluate the physiological roles of AOX in regulating nitro-oxidative stress and metabolic changes after exposing plants to hypoxia for 6 h. Under normoxia, AOX expression resulted in the decrease of nitric oxide (NO) levels and of the rate of protein -nitrosylation, while under hypoxia, AOX overexpressors exhibited higher NO and -nitrosylation levels than knockdowns. AOX expression was essential in avoiding hypoxia-induced superoxide and HO levels, and this was achieved via higher activities of catalase and glutathione reductase and the reduced expression of respiratory burst oxidase homolog (Rboh) in overexpressors as compared to knockdowns. The AOX overexpressing lines accumulated less pyruvate and exhibited the increased transcript and activity levels of pyruvate decarboxylase and alcohol dehydrogenase under hypoxia. This suggests that AOX contributes to the energy state of hypoxic tissues by stimulating the increase of pyruvate flow into fermentation pathways. Ethylene biosynthesis genes encoding 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, ACC oxidase, and ethylene-responsive factors (ERFs) were induced during hypoxia and correlated with AOX and NO levels. We conclude that AOX controls the interaction of NO, reactive oxygen species, and ethylene, triggering a coordinated downstream defensive response against hypoxia.
Topics: Ethylenes; Hydrogen Peroxide; Hypoxia; Mitochondrial Proteins; Nitric Oxide; Oxidoreductases; Plant Proteins; Plants, Genetically Modified; Pyruvates; Reactive Oxygen Species; Nicotiana
PubMed: 35806157
DOI: 10.3390/ijms23137153 -
Toxics May 2022Hypoxic environments have an adverse effect on the growth and development of , and this is accompanied by the production of reducing substances such as Fe and Mn. In...
Hypoxic environments have an adverse effect on the growth and development of , and this is accompanied by the production of reducing substances such as Fe and Mn. In this study, the effect of hypoxic stress and Mn concentrations on leaf chlorophyll contents, root morphology, root activity, element absorption, antioxidant enzymes, and respiratory enzyme system of were evaluated in a hydroponics environment. The results revealed that application of Mn during hypoxic stress enhanced leaf chlorophyll contents and boosted up the indexes of the root system. The root activity of was reduced with stresses of hypoxia. The treatment of Mn initially improved and then decreased the root activity of , and attained its maximum with application of 300 μmol/L Mn compared with control. The indexes of antioxidant enzymes of were higher than that of 8 mg/L oxygen concentrations except for variable superoxide dismutase (SOD) in the treatment of 300 μmol/L Mn with hypoxia stress. The application of Mn had inhibited the absorption of mineral elements in . The activities of pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH), and lactic dehydrogenase (LDH) were initially improved and then diminished with hypoxia stress. It is concluded that hypoxia is a key factor affecting the growth and degradation of , while combining it with the increase of Mn concentration enhances the damage to . Our research is helpful for the sustainable management and scientific fertilization management of .
PubMed: 35736899
DOI: 10.3390/toxics10060290 -
International Journal of Molecular... Apr 2022Sucrose nonfermenting-1-related protein kinase 1 (SnRK1) is a central integrator of plant stress and energy starvation signalling pathways. We found that the...
Sucrose nonfermenting-1-related protein kinase 1 (SnRK1) is a central integrator of plant stress and energy starvation signalling pathways. We found that the -overexpression (OE) roots had a higher respiratory rate and tolerance to waterlogging than the -RNAi roots, suggesting that plays a positive role in the regulation of anaerobic respiration under waterlogging. upregulated the activity of anaerobic respiration-related enzymes including hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK), pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH) and lactate dehydrogenase (LDH). also enhanced the ability to quench reactive oxygen species (ROS) by increasing antioxidant enzyme activities. We sequenced the transcriptomes of the roots of both wild-type (WT) and -RNAi plants, and the differentially expressed genes (DEGs) were clearly enriched in the defence response, response to biotic stimuli, and cellular carbohydrate metabolic process. In addition, 42 genes involved in glycolysis and 30 genes involved in pyruvate metabolism were significantly regulated in -RNAi roots. We analysed the transcript levels of two anoxia-related genes and three , and the results showed that , , and were upregulated in response to , indicating that may be involved in the ethylene signalling pathway to improve waterlogging tolerance. In conclusion, increases the expression of and further activates anoxia response genes, thereby enhancing anaerobic respiration metabolism in response to low-oxygen conditions during waterlogging.
Topics: Anaerobiosis; Fragaria; Gene Expression Regulation, Plant; Hypoxia; Plant Roots; Respiratory Rate
PubMed: 35563305
DOI: 10.3390/ijms23094914 -
Cell Metabolism May 2022The folic acid cycle mediates the transfer of one-carbon (1C) units to support nucleotide biosynthesis. While the importance of serine as a mitochondrial and cytosolic...
The folic acid cycle mediates the transfer of one-carbon (1C) units to support nucleotide biosynthesis. While the importance of serine as a mitochondrial and cytosolic donor of folate-mediated 1C units in cancer cells has been thoroughly investigated, a potential role of glycine oxidation remains unclear. We developed an approach for quantifying mitochondrial glycine cleavage system (GCS) flux by combining stable and radioactive isotope tracing with computational flux decomposition. We find high GCS flux in hepatocellular carcinoma (HCC), supporting nucleotide biosynthesis. Surprisingly, other than supplying 1C units, we found that GCS is important for maintaining protein lipoylation and mitochondrial activity. Genetic silencing of glycine decarboxylase inhibits the lipoylation and activity of pyruvate dehydrogenase and impairs tumor growth, suggesting a novel drug target for HCC. Considering the physiological role of liver glycine cleavage, our results support the notion that tissue of origin plays an important role in tumor-specific metabolic rewiring.
Topics: Carcinoma, Hepatocellular; Folic Acid; Glycine; Glycine Dehydrogenase (Decarboxylating); Humans; Lipoylation; Liver Neoplasms; Mitochondrial Proteins; Nucleotides
PubMed: 35508111
DOI: 10.1016/j.cmet.2022.04.006