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Cell Oct 2018Acetate is a major nutrient that supports acetyl-coenzyme A (Ac-CoA) metabolism and thus lipogenesis and protein acetylation. However, its source is unclear. Here, we...
Acetate is a major nutrient that supports acetyl-coenzyme A (Ac-CoA) metabolism and thus lipogenesis and protein acetylation. However, its source is unclear. Here, we report that pyruvate, the end product of glycolysis and key node in central carbon metabolism, quantitatively generates acetate in mammals. This phenomenon becomes more pronounced in the context of nutritional excess, such as during hyperactive glucose metabolism. Conversion of pyruvate to acetate occurs through two mechanisms: (1) coupling to reactive oxygen species (ROS) and (2) neomorphic enzyme activity from keto acid dehydrogenases that enable function as pyruvate decarboxylases. Further, we demonstrate that de novo acetate production sustains Ac-CoA pools and cell proliferation in limited metabolic environments, such as during mitochondrial dysfunction or ATP citrate lyase (ACLY) deficiency. By virtue of de novo acetate production being coupled to mitochondrial metabolism, there are numerous possible regulatory mechanisms and links to pathophysiology.
Topics: ATP Citrate (pro-S)-Lyase; Acetates; Acetyl Coenzyme A; Acetylation; Animals; Female; Glucose; Glycolysis; Lipogenesis; Male; Mammals; Mice; Mice, Inbred C57BL; Mitochondria; Oxidoreductases; Pyruvate Decarboxylase; Pyruvic Acid; Reactive Oxygen Species
PubMed: 30245009
DOI: 10.1016/j.cell.2018.08.040 -
BMC Research Notes May 2021Zymomonas mobilis is an alpha-proteobacterium with a rapid ethanologenic pathway, involving Entner-Doudoroff (E-D) glycolysis, pyruvate decarboxylase (Pdc) and two...
OBJECTIVE
Zymomonas mobilis is an alpha-proteobacterium with a rapid ethanologenic pathway, involving Entner-Doudoroff (E-D) glycolysis, pyruvate decarboxylase (Pdc) and two alcohol dehydrogenase (ADH) isoenzymes. Pyruvate is the end-product of the E-D pathway and the substrate for Pdc. Construction and study of Pdc-deficient strains is of key importance for Z. mobilis metabolic engineering, because the pyruvate node represents the central branching point, most novel pathways divert from ethanol synthesis. In the present work, we examined the aerobic metabolism of a strain with partly inactivated Pdc.
RESULTS
Relative to its parent strain the mutant produced more pyruvate. Yet, it also yielded more acetaldehyde, the product of the Pdc reaction and the substrate for ADH, although the bulk ADH activity was similar in both strains, while the Pdc activity in the mutant was reduced by half. Simulations with the kinetic model of Z. mobilis E-D pathway indicated that, for the observed acetaldehyde to ethanol production ratio in the mutant, the ratio between its respiratory NADH oxidase and ADH activities should be significantly higher, than the measured values. Implications of this finding for the directionality of the ADH isoenzyme operation in vivo and interactions between ADH and Pdc are discussed.
Topics: Alcohol Dehydrogenase; Metabolic Engineering; Pyruvate Decarboxylase; Respiration; Zymomonas
PubMed: 34049566
DOI: 10.1186/s13104-021-05625-5 -
Biotechnology For Biofuels and... May 2022Klebsiella pneumoniae contains an endogenous isobutanol synthesis pathway. The ipdC gene annotated as an indole-3-pyruvate decarboxylase (Kp-IpdC), was identified to...
BACKGROUND
Klebsiella pneumoniae contains an endogenous isobutanol synthesis pathway. The ipdC gene annotated as an indole-3-pyruvate decarboxylase (Kp-IpdC), was identified to catalyze the formation of isobutyraldehyde from 2-ketoisovalerate.
RESULTS
Compared with 2-ketoisovalerate decarboxylase from Lactococcus lactis (KivD), a decarboxylase commonly used in artificial isobutanol synthesis pathways, Kp-IpdC has an 2.8-fold lower K for 2-ketoisovalerate, leading to higher isobutanol production without induction. However, expression of ipdC by IPTG induction resulted in a low isobutanol titer. In vitro enzymatic reactions showed that Kp-IpdC exhibits promiscuous pyruvate decarboxylase activity, which adversely consume the available pyruvate precursor for isobutanol synthesis. To address this, we have engineered Kp-IpdC to reduce pyruvate decarboxylase activity. From computational modeling, we identified 10 amino acid residues surrounding the active site for mutagenesis. Ten designs consisting of eight single-point mutants and two double-point mutants were selected for exploration. Mutants L546W and T290L that showed only 5.1% and 22.1% of catalytic efficiency on pyruvate compared to Kp-IpdC, were then expressed in K. pneumoniae for in vivo testing. Isobutanol production by K. pneumoniae T290L was 25% higher than that of the control strain, and a final titer of 5.5 g/L isobutanol was obtained with a substrate conversion ratio of 0.16 mol/mol glucose.
CONCLUSIONS
This research provides a new way to improve the efficiency of the biological route of isobutanol production.
PubMed: 35501883
DOI: 10.1186/s13068-022-02144-8 -
The FEBS Journal Dec 2013Thiamine diphosphate-dependent enzymes are broadly distributed in all organisms, and they catalyse a broad range of C-C bond forming and breaking reactions. Enzymes... (Review)
Review
Thiamine diphosphate-dependent enzymes are broadly distributed in all organisms, and they catalyse a broad range of C-C bond forming and breaking reactions. Enzymes belonging to the structural families of decarboxylases and transketolases have been particularly well investigated concerning their substrate range, mechanism of stereoselective carboligation and carbolyase reaction. Both structurally different enzyme families differ also in stereoselectivity: enzymes from the decarboxylase family are predominantly R-selective, whereas those from the transketolase family are S-selective. In recent years a key focus of our studies has been on stereoselective benzoin condensation-like 1,2-additions. Meanwhile, several S-selective variants of pyruvate decarboxylase, benzoylformate decarboxylase and 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate (SEPHCHC) synthase as well as R-selective transketolase variants were created that allow access to a broad range of enantiocomplementary α-hydroxyketones and α,α'-dihydroxyketones. This review covers recent studies and the mechanistic understanding of stereoselective C-C bond forming thiamine diphosphate-dependent enzymes, which has been guided by structure-function analyses based on mutagenesis studies and from influences of different substrates and organic co-solvents on stereoselectivity.
Topics: Animals; Carboxy-Lyases; Catalysis; Humans; Stereoisomerism; Thiamine Pyrophosphate; Transketolase
PubMed: 24034356
DOI: 10.1111/febs.12496 -
PloS One 2023Understanding metabolism in the pathogen Candida glabrata is key to identifying new targets for antifungals. The thiamine biosynthetic (THI) pathway is partially...
Understanding metabolism in the pathogen Candida glabrata is key to identifying new targets for antifungals. The thiamine biosynthetic (THI) pathway is partially defective in C. glabrata, but the transcription factor CgPdc2 upregulates some thiamine biosynthetic and transport genes. One of these genes encodes a recently evolved thiamine pyrophosphatase (CgPMU3) that is critical for accessing external thiamine. Here, we demonstrate that CgPdc2 primarily regulates THI genes. In Saccharomyces cerevisiae, Pdc2 regulates both THI and pyruvate decarboxylase (PDC) genes, with PDC proteins being a major thiamine sink. Deletion of PDC2 is lethal in S. cerevisiae in standard growth conditions, but not in C. glabrata. We uncover cryptic cis elements in C. glabrata PDC promoters that still allow for regulation by ScPdc2, even when that regulation is not apparent in C. glabrata. C. glabrata lacks Thi2, and it is likely that inclusion of Thi2 into transcriptional regulation in S. cerevisiae allows for a more complex regulation pattern and regulation of THI and PDC genes. We present evidence that Pdc2 functions independent of Thi2 and Thi3 in both species. The C-terminal activation domain of Pdc2 is intrinsically disordered and critical for species differences. Truncation of the disordered domains leads to a gradual loss of activity. Through a series of cross species complementation assays of transcription, we suggest that there are multiple Pdc2-containing complexes, and C. glabrata appears to have the simplest requirement set for THI genes, except for CgPMU3. CgPMU3 has different cis requirements, but still requires Pdc2 and Thi3 to be upregulated by thiamine starvation. We identify the minimal region sufficient for thiamine regulation in CgTHI20, CgPMU3, and ScPDC5 promoters. Defining the cis and trans requirements for THI promoters should lead to an understanding of how to interrupt their upregulation and provide targets in metabolism for antifungals.
Topics: Saccharomyces cerevisiae; Candida glabrata; Transcription Factors; Fungal Proteins; Pyruvate Decarboxylase; Thiamine; Carboxy-Lyases; Promoter Regions, Genetic; Intrinsically Disordered Proteins; Gene Expression Regulation, Fungal
PubMed: 37285346
DOI: 10.1371/journal.pone.0286744 -
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 -
Journal of Applied Microbiology Jul 2015Ethanol production directly from CO2 , utilizing genetically engineered photosynthetic cyanobacteria as a biocatalyst, offers significant potential as a renewable and... (Review)
Review
Ethanol production directly from CO2 , utilizing genetically engineered photosynthetic cyanobacteria as a biocatalyst, offers significant potential as a renewable and sustainable source of biofuel. Despite the current absence of a commercially successful production system, significant resources have been deployed to realize this goal. Utilizing the pyruvate decarboxylase from Zymomonas species, metabolically derived pyruvate can be converted to ethanol. This review of both peer-reviewed and patent literature focuses on the genetic modifications utilized for metabolic engineering and the resultant effect on ethanol yield. Gene dosage, induced expression and cassette optimizat-ion have been analyzed to optimize production, with production rates of 0·1-0·5 g L(-1) day(-1) being achieved. The current 'toolbox' of molecular manipulations and future directions focusing on applicability, addressing the primary challenges facing commercialization of cyanobacterial technologies are discussed.
Topics: Autotrophic Processes; Biofuels; Cyanobacteria; Ethanol; Industrial Microbiology; Photosynthesis
PubMed: 25865951
DOI: 10.1111/jam.12821 -
Current Opinion in Chemical Biology Dec 2020Saccharomyces cerevisiae, Baker's yeast, is the industrial workhorse for producing ethanol and the subject of substantial metabolic engineering research in both industry... (Review)
Review
Saccharomyces cerevisiae, Baker's yeast, is the industrial workhorse for producing ethanol and the subject of substantial metabolic engineering research in both industry and academia. S. cerevisiae has been used to demonstrate production of a wide range of chemical products from glucose. However, in many cases, the demonstrations report titers and yields that fall below thresholds for industrial feasibility. Ethanol synthesis is a central part of S. cerevisiae metabolism, and redirecting flux to other products remains a barrier to industrialize strains for producing other molecules. Removing ethanol producing pathways leads to poor fitness, such as impaired growth on glucose. Here, we review metabolic engineering efforts aimed at restoring growth in non-ethanol producing strains with emphasis on relieving glucose repression associated with the Crabtree effect and rewiring metabolism to provide access to critical cellular building blocks. Substantial progress has been made in the past decade, but many opportunities for improvement remain.
Topics: Acetyl Coenzyme A; Ethanol; Glucose; Industrial Microbiology; Metabolic Engineering; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 33032255
DOI: 10.1016/j.cbpa.2020.08.005 -
Plant Signaling & Behavior 2014This review focuses on the energy metabolism during pollen maturation and tube growth and updates current knowledge. Pollen tube growth is essential for male... (Review)
Review
This review focuses on the energy metabolism during pollen maturation and tube growth and updates current knowledge. Pollen tube growth is essential for male reproductive success and extremely fast. Therefore, pollen development and tube growth are high energy-demanding processes. During the last years, various publications (including research papers and reviews) emphasize the importance of mitochondrial respiration and fermentation during male gametogenesis and pollen tube elongation. These pathways obviously contribute to satisfy the high energy demand, and there are many studies which suggest that respiration and fermentation are the only pathways to generate the needed energy. Here, we review data which show for the first time that in addition plastidial glycolysis and the balancing of the ATP/NAD(P)H ratio (by malate valves and NAD(+) biosynthesis) contribute to satisfy the energy demand during pollen development. Although the importance of energy generation by plastids was discounted during the last years (possibly due to the controversial opinion about their existence in pollen grains and pollen tubes), the available data underline their prime role during pollen maturation and tube growth.
Topics: Energy Metabolism; Fermentation; Mitochondria; Oxidation-Reduction; Plastids; Pollen Tube
PubMed: 25482752
DOI: 10.4161/15592324.2014.977200 -
Archaea (Vancouver, B.C.) 2014The hyperthermophilic archaeon Thermococcus guaymasensis produces ethanol as a metabolic end product, and an alcohol dehydrogenase (ADH) catalyzing the reduction of...
The hyperthermophilic archaeon Thermococcus guaymasensis produces ethanol as a metabolic end product, and an alcohol dehydrogenase (ADH) catalyzing the reduction of acetaldehyde to ethanol has been purified and characterized. However, the enzyme catalyzing the formation of acetaldehyde has not been identified. In this study an enzyme catalyzing the production of acetaldehyde from pyruvate was purified and characterized from T. guaymasensis under strictly anaerobic conditions. The enzyme had both pyruvate decarboxylase (PDC) and pyruvate ferredoxin oxidoreductase (POR) activities. It was oxygen sensitive, and the optimal temperatures were 85°C and >95°C for the PDC and POR activities, respectively. The purified enzyme had activities of 3.8 ± 0.22 U mg(-1) and 20.2 ± 1.8 U mg(-1), with optimal pH-values of 9.5 and 8.4 for each activity, respectively. Coenzyme A was essential for both activities, although it did not serve as a substrate for the former. Enzyme kinetic parameters were determined separately for each activity. The purified enzyme was a heterotetramer. The sequences of the genes encoding the subunits of the bifunctional PDC/POR were determined. It is predicted that all hyperthermophilic β -keto acids ferredoxin oxidoreductases are bifunctional, catalyzing the activities of nonoxidative and oxidative decarboxylation of the corresponding β -keto acids.
Topics: Acetaldehyde; DNA, Archaeal; Enzyme Inhibitors; Enzyme Stability; Ethanol; Hydrogen-Ion Concentration; Kinetics; Molecular Sequence Data; Oxygen; Protein Multimerization; Pyruvate Decarboxylase; Pyruvate Synthase; Pyruvic Acid; Sequence Analysis, DNA; Temperature; Thermococcus
PubMed: 24982594
DOI: 10.1155/2014/349379