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International Journal of Molecular... Dec 2023A cell population characterized by the release of glucose repression and known as [] emerges spontaneously in the yeast . This study revealed that the [] variants...
A cell population characterized by the release of glucose repression and known as [] emerges spontaneously in the yeast . This study revealed that the [] variants exhibit retarded alcoholic fermentation when glucose is the sole carbon source. To identify the key to the altered glucose response, the gene expression profile of [] cells was examined. Based on RNA-seq data, the [] status was linked to impaired function of the Cyc8p-Tup1p complex. Loss of Cyc8p led to a decrease in the initial rate of alcoholic fermentation under glucose-rich conditions via the inactivation of pyruvate decarboxylase, an enzyme unique to alcoholic fermentation. These results suggest that Cyc8p can become inactive to attenuate alcoholic fermentation. These findings may contribute to the elucidation of the mechanism of non-genetic heterogeneity in yeast alcoholic fermentation.
Topics: Carbon; Fermentation; Glucose; Pyruvate Decarboxylase; Saccharomyces cerevisiae
PubMed: 38203474
DOI: 10.3390/ijms25010304 -
Environmental Science & Technology Dec 2023The EU low-carbon economy aims to reduce the level of CO emission in the EU to 80% by 2050. High efforts are required to achieve this goal, where successful CCU (Carbon...
The EU low-carbon economy aims to reduce the level of CO emission in the EU to 80% by 2050. High efforts are required to achieve this goal, where successful CCU (Carbon Capture and Utilization) technologies will have a high impact. Biocatalysts offer a greener alternative to chemical catalysts for the development of CCU strategies since biocatalysis conforms 10 of the 12 principles of green chemistry. In this study, a multienzymatic system, based on alcohol dehydrogenase (ADH), pyruvate decarboxylase (PDC), and lactate dehydrogenase (LDH), that converts CO and ethanol into lactic acid leading to a 100% atom economy was studied. The system allows cofactor regeneration, thus reducing the process cost. Through reaction media engineering and enzyme ratio study, the performance of the system was able to produce up to 250 μM of lactic acid under the best conditions using 100% CO, corresponding to the highest concentration of lactic acid obtained up to date using this multienzymatic approach. For the first time, the feasibility of the system to be applied under a real industrial environment has been tested using synthetic gas mimicking real blast furnace off-gases composition from the iron and steel industry. Under these conditions, the system was also capable of producing lactic acid, reaching 62 μM.
Topics: Carbon Dioxide; Lactic Acid; Carbon; Biocatalysis; Environment
PubMed: 38078668
DOI: 10.1021/acs.est.3c05455 -
Journal of Bacteriology Jan 2024YisK is an uncharacterized protein in previously shown to interact genetically with the elongasome protein Mbl. YisK overexpression leads to cell widening and lysis,...
YisK is an uncharacterized protein in previously shown to interact genetically with the elongasome protein Mbl. YisK overexpression leads to cell widening and lysis, phenotypes that are dependent on and suppressed by mutations. In the present work, we characterize YisK's localization, structure, and enzymatic activity. We show that YisK localizes as puncta that depend on Mbl. YisK belongs to the fumarylacetoacetate hydrolase (FAH) superfamily, and crystal structures revealed close structural similarity to two oxaloacetate (OAA) decarboxylases: human mitochondrial FAHD1 and Cg1458. We demonstrate that YisK can also catalyze the decarboxylation of OAA ( = 134 µM, = 31 min). A catalytic dead variant (YisK E148A, E150A) retains wild-type localization and still widens cells following overexpression, indicating these activities are not dependent on YisK catalysis. Conversely, a non-localizing variant (YisK E30A) retains wild-type enzymatic activity but localizes diffusely and no longer widens cells following overexpression. Together, these results suggest that YisK may be subject to spatial regulation that depends on the cell envelope synthesis machinery. IMPORTANCE The elongasome is a multiprotein complex that guides lengthwise growth in some bacteria. We previously showed that, in , overexpression of an uncharacterized putative enzyme (YisK) perturbed function of the actin-like elongasome protein Mbl. Here, we show that YisK exhibits Mbl-dependent localization. Through biochemical and structural characterization, we demonstrate that, like its mitochondrial homolog FAHD1, YisK can catalyze the decarboxylation of the oxaloacetate to pyruvate and CO. YisK is the first example of an enzyme implicated in central carbon metabolism with subcellular localization that depends on Mbl.
Topics: Humans; Bacillus subtilis; Carboxy-Lyases; Pyruvic Acid; Oxaloacetates; Hydrolases
PubMed: 38047707
DOI: 10.1128/jb.00202-23 -
Bioresource Technology Feb 2024Klebsiella oxytoca KC004 (ΔadhEΔpta-ackAΔldhAΔbudABΔpflB) was engineered to enhance succinate production. The strain exhibited poor growth without succinate...
Klebsiella oxytoca KC004 (ΔadhEΔpta-ackAΔldhAΔbudABΔpflB) was engineered to enhance succinate production. The strain exhibited poor growth without succinate production due to its deficiencies in ATP production and NADH reoxidation. To overcome obstacles, evolutionary adaptation with over 6,000 generations of growth-based selection was conducted. Under anaerobic conditions, enhanced productions of ATP for growth and succinate for NADH reoxidation by the evolved KC004-TF160 strain were coupled to an increased transcript of PEP carboxykinase (pck) while those of genes in the oxidative branch of TCA cycle (gltA, acnAB, and icd), and pyruvate and acetate metabolisms (pykA, acs, poxB and tdcD) were alleviated. The expression of pyruvate dehydrogenase repressor (pdhR) decreased whereas threonine decarboxylase (tdcE) increased. KC004-TF160 produced succinate at 84 g/L (0.84 g/g, 79 % theoretical maximum). KC004-TF160 produced succinate at 0.87 g/g non-pretreated sugarcane molasses without addition of nutrients and buffers. KC004-TF160 may be a microbial platform for commercial production of bio-succinate.
Topics: Succinic Acid; Metabolic Engineering; Escherichia coli; Klebsiella oxytoca; NAD; Pyruvic Acid; Adenosine Triphosphate
PubMed: 38006983
DOI: 10.1016/j.biortech.2023.130045 -
Journal of the Science of Food and... Feb 2024During postharvest dehydration, grapes are subject to metabolic changes including ethanol anabolism and catabolism. These changes affect the quality of the final product...
BACKGROUND
During postharvest dehydration, grapes are subject to metabolic changes including ethanol anabolism and catabolism. These changes affect the quality of the final product and ethanol production is a key step. Ethanol dissipation has never been measured during postharvest wine grape dehydration. Thus, the present study aimed to: (i) monitor ethanol dissipation and (ii) investigate chemical-biochemical changes in berries during dehydration.
RESULTS
Ethanol dissipation from Raboso grapes, under controlled postharvest dehydration, was found to comprise up to 36% of weight loss (w.l.). Moreover, the activity of enzymes involved in the anaerobic metabolism of grapes was investigated. Ethanol dissipation was highly correlated with grape weight loss (r = 0.989). Alcohol dehydrogenase activity, responsible for the reduction of ethanol to acetaldehyde, declined significantly with w.l. Similarly, pyruvate decarboxylase and lactate dehydrogenase reduced their activity. High lipoxygenase activity was measured at 27% w.l., whereas polyphenol oxidation was constant and declined in the last sampling.
CONCLUSION
Ethanol dissipation during postharvest dehydration allows for reducing anaerobic metabolism and promotes oxidative metabolism. The sensor used can be a useful commercial tool for monitoring berry metabolism. © 2023 Society of Chemical Industry.
Topics: Vitis; Wine; Ethanol; Dehydration; Weight Loss; Fruit
PubMed: 37819862
DOI: 10.1002/jsfa.13042 -
Frontiers in Plant Science 2023Alfalfa, a globally cultivated forage crop, faces significant challenges due to its vulnerability to salt stress. Jasmonates (JAs) play a pivotal role in modulating both...
INTRODUCTION
Alfalfa, a globally cultivated forage crop, faces significant challenges due to its vulnerability to salt stress. Jasmonates (JAs) play a pivotal role in modulating both plant growth and response to stressors.
METHODS
In this study, alfalfa plants were subjected to 150 mM NaCl with or without methyl jasmonate (MeJA). The physiological parameters were detected and a transcriptomic analysis was performed to elucidate the mechanisms underlying MeJA-mediated salt tolerance in alfalfa.
RESULTS
Results showed that exogenous MeJA regulated alfalfa seed germination and primary root growth in a dose-dependent manner, with 5µM MeJA exerting the most efficient in enhancing salt tolerance. MeJA at this concentration elavated the salt tolerance of young alfalfa seedlings by refining plant growth, enhancing antioxidant capacity and ameliorating Na+ overaccumulation. Subsequent transcriptomic analysis identified genes differentially regulated by MeJA+NaCl treatment and NaCl alone. PageMan analysis revealed several significantly enriched categories altered by MeJA+NaCl treatment, compared with NaCl treatment alone, including genes involved in secondary metabolism, glutathione-based redox regulation, cell cycle, transcription factors (TFs), and other signal transductions (such as calcium and ROS). Further weighted gene co-expression network analysis (WGCNA) uncovered that turquoise and yellow gene modules were tightly linked to antioxidant enzymes activity and ion content, respectively. Pyruvate decar-boxylase (PDC) and RNA demethylase (ALKBH10B) were identified as the most central hub genes in these two modules. Also, some TFs-hub genes were identified by WGCNA in these two modules highly positive-related to antioxidant enzymes activity and ion content.
DISCUSSION
MeJA triggered a large-scale transcriptomic remodeling, which might be mediated by transcriptional regulation through TFs or post-transcriptional regulation through demethylation. Our findings contributed new perspectives for understanding the underneath mechanisms by which JA-mediated salt tolerance in alfalfa.
PubMed: 37780521
DOI: 10.3389/fpls.2023.1258498 -
Journal of Fungi (Basel, Switzerland) Sep 2023Phenylacetylcarbinol (PAC) is a precursor for the synthesis of several pharmaceuticals, including ephedrine, pseudoephedrine, and norephedrine. PAC is commonly produced...
Phenylacetylcarbinol (PAC) is a precursor for the synthesis of several pharmaceuticals, including ephedrine, pseudoephedrine, and norephedrine. PAC is commonly produced through biotransformation using microbial pyruvate decarboxylase (PDC) in the form of frozen-thawed whole cells. However, the lack of microorganisms capable of high PDC activity is the main factor in the production of PAC. In addition, researchers are also looking for ways to utilize agro-industrial residues as an inexpensive carbon source through an integrated biorefinery approach in which sugars can be utilized for bioethanol production and frozen-thawed whole cells for PAC synthesis. In the present study, , , and the co-culture of both strains were compared for their biomass and ethanol concentrations, as well as for their volumetric and specific PDC activities when cultivated in a sugarcane bagasse (SCB) hydrolysate medium (SCBHM). The co-culture that resulted in a higher level of PAC (8.65 ± 0.08 mM) with 26.4 ± 0.9 g L ethanol production was chosen for further experiments. Biomass production was scaled up to 100 L and the kinetic parameters were studied. The biomass harvested from the bioreactor was utilized as frozen-thawed whole cells for the selection of an initial pyruvate (Pyr)-to-benzaldehyde (Bz) concentration ([Pyr]/[Bz]) ratio suitable for the PAC biotransformation in a single-phase emulsion system. The initial [Pyr]/[Bz] at 100/120 mM resulted in higher PAC levels with 10.5 ± 0.2 mM when compared to 200/240 mM (8.60 ± 0.01 mM). A subsequent two-phase emulsion system with Pyr in the aqueous phase, Bz in the organic phase, and frozen-thawed whole cells of the co-culture as the biocatalyst produced a 1.46-fold higher PAC level when compared to a single-phase emulsion system. In addition, the cost analysis strategy indicated preliminary costs of USD 0.82 and 1.01/kg PAC for the single-phase and two-phase emulsion systems, respectively. The results of the present study suggested that the co-culture of and can effectively produce bioethanol and PAC from SCB and would decrease the overall production cost on an industrial scale utilizing the two-phase emulsion system with the proposed multiple-pass strategy.
PubMed: 37755036
DOI: 10.3390/jof9090928 -
Food Chemistry Feb 2024Internal browning (IB) is a physiological disorder without external symptoms of postharvest pineapples, but whether and how IB influences pineapple volatiles remain...
Internal browning (IB) is a physiological disorder without external symptoms of postharvest pineapples, but whether and how IB influences pineapple volatiles remain unknown. We examined eigenvalues of volatiles in 'Comte de Paris' pineapples with or without IB using electronic nose (E-nose) and gas chromatography-mass spectrometry (GC-MS). Correlation coefficients between the responses of E-nose sensors S7 and S9 and IB were 0.836 and 0.804, respectively. The multilayer perceptron neural network and radial basis function neural network models classified IB degree with accuracy of 94.77% and 91.67%. GC-MS analysis revealed 30 volatile substances upregulated in pineapple with IB compared to those without, of which 15 were esters. IB regulated volatile compound synthesis through the lipoxygenase pathway which involved lipoxygenase, pyruvate decarboxylase 1, alcohol dehydrogenases, acyl-CoA oxidase 1, and alcohol acyltransferase genes and their related enzymes. These results suggested that volatiles regulated by IB could be used to discriminate IB severity in pineapples.
PubMed: 37688818
DOI: 10.1016/j.foodchem.2023.137358 -
Plants (Basel, Switzerland) Aug 2023Kiwifruit ( spp.) is susceptible to waterlogging stress. Although abundant wild germplasm resources exist among plants for improving the waterlogging tolerance of...
Kiwifruit ( spp.) is susceptible to waterlogging stress. Although abundant wild germplasm resources exist among plants for improving the waterlogging tolerance of kiwifruit cultivars, the underlying mechanisms remain largely unknown. Here, a comparative study was undertaken using one wild germplasm, Maorenshen ( Dunn, MRS), and one cultivar, Miliang-1 ( var. (A.Chev.) A.Chev. cv. Miliang-1, ML). Under stress, the ML plantlets were seriously damaged with wilted chlorotic leaves and blackened rotten roots, whereas the symptoms of injury in the MRS plantlets were much fewer, along with higher photosynthetic rates, chlorophyll fluorescence characteristics and root activity under stress conditions. However, neither aerenchyma in the root nor adventitious roots appeared in both germplasms upon stress exposure. The activities of pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH), as well as their transcript levels, were constitutively higher in MRS than those in ML under both normal and stress conditions. Waterlogging stress significantly enhanced the PDC and ADH enzyme activities in both germplasms, which were 60.8% and 22.4% higher in the MRS roots than those in the ML roots under waterlogging stress, respectively. Moreover, MRS displayed higher activities of antioxidant enzymes, including SOD, CAT, and APX, as well as DPPH-radical scavenging ability, and decreased HO and MDA accumulation under both normal and stress conditions. Our findings suggest that the waterlogging tolerance of the wild germplasm was associated with high PDC and ADH, as well as antioxidant ability.
PubMed: 37571025
DOI: 10.3390/plants12152872 -
Microbial Cell Factories Jul 2023L-arginine is an important amino acid with applications in diverse industrial and pharmaceutical fields. N-acetylglutamate, synthesized from L-glutamate and acetyl-CoA,...
BACKGROUND
L-arginine is an important amino acid with applications in diverse industrial and pharmaceutical fields. N-acetylglutamate, synthesized from L-glutamate and acetyl-CoA, is a precursor of the L-arginine biosynthetic branch in microorganisms. The enzyme that produces N-acetylglutamate, N-acetylglutamate synthase, is allosterically inhibited by L-arginine. L-glutamate, as a central metabolite, provides carbon backbone for diverse biological compounds besides L-arginine. When glucose is the sole carbon source, the theoretical maximum carbon yield towards L-arginine is 96.7%, but the experimental highest yield was 51%. The gap of L-arginine yield indicates the regulation complexity of carbon flux and energy during the L-arginine biosynthesis. Besides endogenous biosynthesis, N-acetylglutamate, the key precursor of L-arginine, can be obtained by chemical acylation of L-glutamate with a high yield of 98%. To achieve high-yield production of L-arginine, we demonstrated a novel approach by directly feeding precursor N-acetylglutamate to engineered Escherichia coli.
RESULTS
We reported a new approach for the high yield of L-arginine production in E. coli. Gene argA encoding N-acetylglutamate synthase was deleted to disable endogenous biosynthesis of N-acetylglutamate. The feasibility of external N-acetylglutamate towards L-arginine was verified via growth assay in argA strain. To improve L-arginine production, astA encoding arginine N-succinyltransferase, speF encoding ornithine decarboxylase, speB encoding agmatinase, and argR encoding an arginine responsive repressor protein were disrupted. Based on overexpression of argDGI, argCBH operons, encoding enzymes of the L-arginine biosynthetic pathway, ~ 4 g/L L-arginine was produced in shake flask fermentation, resulting in a yield of 0.99 mol L-arginine/mol N-acetylglutamate. This strain was further engineered for the co-production of L-arginine and pyruvate by removing genes adhE, ldhA, poxB, pflB, and aceE, encoding enzymes involved in the conversion and degradation of pyruvate. The resulting strain was shown to produce 4 g/L L-arginine and 11.3 g/L pyruvate in shake flask fermentation.
CONCLUSIONS
Here, we developed a novel approach to avoid the strict regulation of L-arginine on ArgA and overcome the metabolism complexity in the L-arginine biosynthesis pathway. We achieve a high yield of L-arginine production from N-acetylglutamate in E. coli. Co-production pyruvate and L-arginine was used as an example to increase the utilization of input carbon sources.
Topics: Escherichia coli; Amino-Acid N-Acetyltransferase; Glutamic Acid; Arginine; Pyruvates; Carbon; Metabolic Engineering
PubMed: 37495979
DOI: 10.1186/s12934-023-02145-8