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Biochemistry and Biophysics Reports Sep 2016Acetohydroxyacid synthase (AHAS) catalyzes the production of acetolactate from pyruvate. The enzyme from the hyperthermophilic bacterium has been purified and...
Acetohydroxyacid synthase (AHAS) catalyzes the production of acetolactate from pyruvate. The enzyme from the hyperthermophilic bacterium has been purified and characterized ( ~100 s). It was found that the same enzyme also had the ability to catalyze the production of acetaldehyde and CO from pyruvate, an activity of pyruvate decarboxylase (PDC) at a rate approximately 10% of its AHAS activity. Compared to the catalytic subunit, reconstitution of the individually expressed and purified catalytic and regulatory subunits of the AHAS stimulated both activities of PDC and AHAS. Both activities had similar pH and temperature profiles with an optimal pH of 7.0 and temperature of 85 °C. The enzyme kinetic parameters were determined, however, it showed a non-Michaelis-Menten kinetics for pyruvate only. This is the first report on the PDC activity of an AHAS and the second bifunctional enzyme that might be involved in the production of ethanol from pyruvate in hyperthermophilic microorganisms.
PubMed: 28955930
DOI: 10.1016/j.bbrep.2016.07.008 -
Scientific Reports Jul 2021Bioethanol produced by fermentative microorganisms is regarded as an alternative to fossil fuel. Bioethanol to be used as a viable energy source must be produced...
Bioethanol produced by fermentative microorganisms is regarded as an alternative to fossil fuel. Bioethanol to be used as a viable energy source must be produced cost-effectively by removing expense-intensive steps such as the enzymatic hydrolysis of substrate. Consolidated bioprocessing (CBP) is believed to be a practical solution combining saccharification and fermentation in a single step catalyzed by a microorganism. Bacillus subtills with innate ability to grow on a diversity of carbohydrates seems promising for affordable CBP bioethanol production using renewable plant biomass and wastes. In this study, the genes encoding alcohol dehydrogenase from Z. mobilis (adh) and S. cerevisiae (adh) were each used with Z. mobilis pyruvate decarboxylase gene (pdc) to create ethanologenic operons in a lactate-deficient (Δldh) B. subtilis resulting in NZ and NZS strains, respectively. The S. cerevisiae adh caused significantly more ethanol production by NZS and therefore was used to make two other operons including one with double copies of both pdc and adh and the other with a single pdc but double adh genes expressed in N(ZS)2 and NZS2 strains, respectively. In addition, two fusion genes were constructed with pdc and adh in alternate orientations and used for ethanol production by the harboring strains namely NZ:S and NS:Z, respectively. While the increase of gene dosage was not associated with elevated carbon flow for ethanol production, the fusion gene adh:pdc resulted in a more than two times increase of productivity by strain NS:Z as compared with NZS during 48 h fermentation. The CBP ethanol production by NZS and NS:Z using potatoes resulted in 16.3 g/L and 21.5 g/L ethanol during 96 h fermentation, respectively. For the first time in this study, B. subtilis was successfully used for CBP ethanol production with S. cerevisiae alcohol dehydrogenase. The results of the study provide insights on the potentials of B. subtilis for affordable bioethanol production from inexpensive plant biomass and wastes. However, the potentials need to be improved by metabolic and process engineering for higher yields of ethanol production and plant biomass utilization.
Topics: Alcohol Dehydrogenase; Bacillus subtilis; Biomass; Ethanol; Fermentation; Hydrolysis; Lactic Acid; Metabolic Engineering; Pyruvate Decarboxylase; Saccharomyces cerevisiae; Zymomonas
PubMed: 34215768
DOI: 10.1038/s41598-021-92627-9 -
Journal of Plant Research Mar 2022Flooding negatively influences the growth and development of several plant species. Here, we show that the flood tolerance of young Handroanthus chrysotrichus plants...
Flooding negatively influences the growth and development of several plant species. Here, we show that the flood tolerance of young Handroanthus chrysotrichus plants involves growth deficit, carbon assimilation reductions, starch remobilization, and energy regulation. The effect of hypoxia was evaluated in a controlled experiment consisting of plants subjected to normoxia and water-logging, with later recovery. We measured morphological changes, gas exchange, photosynthetic pigments, soluble carbohydrates and starch contents, the activity of the enzymes alcohol dehydrogenase (ADH), and pyruvate decarboxylase (PDC), and ATP and ADP levels. While control plants showed normal appearance and growth, flooded plants exhibited a drastic decrease in growth, necrosis of some root tips, hypertrophic lenticels on the stems, and foliar chlorosis. Oxygen deprivation in root cells led to a significant decrease in stomatal conductance. The low A rates caused a decline in foliar soluble sugar content at 20 days and a subsequent increase in the leaves and roots, coinciding with starch degradation at 40 days. We also observed increases of 220.5% in ADH and 292% in PDC activities in the roots at 20 and 40 days of flooding. The activation of anaerobic metabolism in stressed plants was an essential mechanism for ATP regulation in both tissues used to maintain a minimal metabolism to cope with hypoxia to the detriment of growth. The post-stress recovery process in H. chrysotrichus occurred slowly, with gas exchange gradually resumed and anaerobic metabolism and sugar content maintained to improve energy regulation.
Topics: Carbohydrate Metabolism; Floods; Photosynthesis; Plant Leaves; Plant Roots
PubMed: 35050423
DOI: 10.1007/s10265-022-01370-3 -
Metabolic Engineering Jan 2017The metabolism of Clostridium thermocellum is notable in that it assimilates sugar via the EMP pathway but does not possess a pyruvate kinase enzyme. In the wild type...
The metabolism of Clostridium thermocellum is notable in that it assimilates sugar via the EMP pathway but does not possess a pyruvate kinase enzyme. In the wild type organism, there are three proposed pathways for conversion of phosphoenolpyruvate (PEP) to pyruvate, which differ in their cofactor usage. One path uses pyruvate phosphate dikinase (PPDK), another pathway uses the combined activities of PEP carboxykinase (PEPCK) and oxaloacetate decarboxylase (ODC). Yet another pathway, the malate shunt, uses the combined activities of PEPCK, malate dehydrogenase and malic enzyme. First we showed that there is no flux through the ODC pathway by enzyme assay. Flux through the remaining two pathways (PPDK and malate shunt) was determined by dynamic C labeling. In the wild-type strain, the malate shunt accounts for about 33±2% of the flux to pyruvate, with the remainder via the PPDK pathway. Deletion of the ppdk gene resulted in a redirection of all pyruvate flux through the malate shunt. This provides the first direct evidence of the in-vivo function of the malate shunt.
Topics: Biosynthetic Pathways; Carbon-13 Magnetic Resonance Spectroscopy; Clostridium thermocellum; Glucose; Glycolysis; Malates; Metabolic Flux Analysis; Metabolic Networks and Pathways; Models, Biological; Phosphoenolpyruvate; Pyruvate Kinase; Pyruvic Acid
PubMed: 27914869
DOI: 10.1016/j.ymben.2016.11.011 -
Acta Crystallographica. Section F,... Sep 2016Pyruvate decarboxylase (PDC; EC 4.1.1.1) is a thiamine pyrophosphate- and Mg(2+) ion-dependent enzyme that catalyses the non-oxidative decarboxylation of pyruvate to...
Pyruvate decarboxylase (PDC; EC 4.1.1.1) is a thiamine pyrophosphate- and Mg(2+) ion-dependent enzyme that catalyses the non-oxidative decarboxylation of pyruvate to acetaldehyde and carbon dioxide. It is rare in bacteria, but is a key enzyme in homofermentative metabolism, where ethanol is the major product. Here, the previously unreported crystal structure of the bacterial pyruvate decarboxylase from Zymobacter palmae is presented. The crystals were shown to diffract to 2.15 Å resolution. They belonged to space group P21, with unit-cell parameters a = 204.56, b = 177.39, c = 244.55 Å and Rr.i.m. = 0.175 (0.714 in the highest resolution bin). The structure was solved by molecular replacement using PDB entry 2vbi as a model and the final R values were Rwork = 0.186 (0.271 in the highest resolution bin) and Rfree = 0.220 (0.300 in the highest resolution bin). Each of the six tetramers is a dimer of dimers, with each monomer sharing its thiamine pyrophosphate across the dimer interface, and some contain ethylene glycol mimicking the substrate pyruvate in the active site. Comparison with other bacterial PDCs shows a correlation of higher thermostability with greater tetramer interface area and number of interactions.
Topics: Amino Acid Sequence; Bacterial Proteins; Catalytic Domain; Cations, Divalent; Cloning, Molecular; Crystallography, X-Ray; Escherichia coli; Ethylene Glycol; Gene Expression; Halomonadaceae; Kinetics; Magnesium; Models, Molecular; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Protein Multimerization; Pyruvate Decarboxylase; Pyruvic Acid; Recombinant Proteins; Substrate Specificity; Thiamine Pyrophosphate
PubMed: 27599861
DOI: 10.1107/S2053230X16012012 -
Journal of the Science of Food and... Jun 2021Lactobacillus plantarum, a common species of lactic acid bacteria, is used to improve the flavor of traditional fermented food. Under salt stress, different strains of... (Comparative Study)
Comparative Study
BACKGROUND
Lactobacillus plantarum, a common species of lactic acid bacteria, is used to improve the flavor of traditional fermented food. Under salt stress, different strains of L. plantarum can respond differently. In this work, proteomics and bioinformatics analysis of L. plantarum strains (ATCC14917, FS5-5, and 208) grown under salt stress (240 g L sodium chloride (NaCl)) were investigated based on the isobaric tags for relative and absolute quantitation method.
RESULTS
Although 171 differentially expressed proteins (DEPs) were observed, only 44, 57, and 112 DEPs were identified in the strains ATCC14917, FS5-5, and 208 respectively. There were 33, 191, and 179 specific DEPs in ATCC14917 versus FS5-5, in 208 versus FS5-5, and in strain 208 versus ATCC14917 in 240 g L NaCl. These DEPs indicate that the three strains, from pickles, fermented soybean paste, and fermented milk, may have different salt stress responses. Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes analysis showed that most DEPs observed were involved in protein biosynthesis, nucleotide metabolism, and sugar metabolism. Twenty-six significantly different DEPs that were possibly associated with salt response were selected and further analyzed for gene expression level and pattern by quantitative reverse transcription polymerase chain reaction. Pyruvate kinase and cysteine desulfurase had similar expression patterns in all three strains; glutamate decarboxylase expression was upregulated in FS5-5 and significantly upregulated in strain 208; RNA polymerase subunit alpha was downregulated in FS5-5 but upregulated in strain 208.
CONCLUSIONS
These results also showed that the salt stress response of strain 208 may involve higher numbers of genes than the other strains. This research provides a theoretical basis for improvement of salt tolerance of L. plantarum in industrial production. © 2020 Society of Chemical Industry.
Topics: Bacterial Proteins; Computational Biology; Lactobacillus plantarum; Proteomics; Salt Stress; Sodium Chloride; Stress, Physiological
PubMed: 33270231
DOI: 10.1002/jsfa.10976 -
World Journal of Microbiology &... Mar 2022Klebsiella pneumoniae is a 2,3-butanediol producing bacterium. Nevertheless, a design and construction of L-valine production strain was studied in this paper. The first...
Klebsiella pneumoniae is a 2,3-butanediol producing bacterium. Nevertheless, a design and construction of L-valine production strain was studied in this paper. The first step of 2,3-butanediol synthesis and branched-chain amino acid synthesis pathways share the same step of α-acetolactate synthesis from pyruvate. However, the two pathways are existing in parallel and do not interfere with each other in the wild-type strain. A knockout of budA blocked the 2,3-butanediol synthesis pathway and resulted in the L-valine production. The budA coded an α-acetolactate decarboxylase and catalyzed the acetoin formation from α-acetolactate. Furthermore, blocking the lactic acid synthesis by knocking out of ldhA, which is encoding a lactate dehydrogenase, improved the L-valine synthesis. 2-Ketoisovalerate is the precursor of L-valine, it is also an intermediate of the isobutanol synthesis pathway, while indole-3-pyruvate decarboxylase (ipdC) is responsible for isobutyraldehyde formation from 2-ketoisovalerate. Production of L-valine has been improved by knocking out of ipdC. On the other side, the ilvE, encoding a transaminase B, reversibly transfers one amino group from glutamate to α-ketoisovalerate. Overexpression of ilvE exhibited a distinct improvement of L-valine production. The brnQ encodes a branched-chain amino acid transporter, and L-valine production was further improved by disrupting brnQ. It is also revealed that weak acidic and aerobic conditions favor L-valine production. Based on these findings, L-valine production by metabolically engineered K. pneumonia was examined. In fed-batch fermentation, 22.4 g/L of L-valine was produced by the engineered K. pneumoniae ΔbudA-ΔldhA-ΔipdC-ΔbrnQ-ilvE after 55 h of cultivation, with a substrate conversion ratio of 0.27 mol/mol glucose.
Topics: Biosynthetic Pathways; Butylene Glycols; Klebsiella pneumoniae; Valine
PubMed: 35348886
DOI: 10.1007/s11274-022-03266-9 -
Applied Microbiology and Biotechnology Aug 2023Saccharomyces cerevisiae is the workhorse of fermentation industry. Upon engineering for D-lactate production by a series of gene deletions, this yeast had deficiencies...
Saccharomyces cerevisiae is the workhorse of fermentation industry. Upon engineering for D-lactate production by a series of gene deletions, this yeast had deficiencies in cell growth and D-lactate production at high substrate concentrations. Complex nutrients or high cell density were thus required to support growth and D-lactate production with a potential to increase medium and process cost of industrial-scale D-lactate production. As an alternative microbial biocatalyst, a Crabtree-negative and thermotolerant yeast Kluyveromyces marxianus was engineered in this study to produce high titer and yield of D-lactate at a lower pH without growth defects. Only pyruvate decarboxylase 1 (PDC1) gene was replaced by a codon-optimized bacterial D-lactate dehydrogenase (ldhA). Ethanol, glycerol, or acetic acid was not produced by the resulting strain, KMΔpdc1::ldhA. Aeration rate at 1.5 vvm and culture pH 5.0 at 30 °C provided the highest D-lactate titer of 42.97 ± 0.48 g/L from glucose. Yield and productivity of D-lactate, and glucose-consumption rate were 0.85 ± 0.01 g/g, 0.90 ± 0.01 g/(L·h), and 1.06 ± 0.00 g/(L·h), respectively. Surprisingly, D-lactate titer, productivity, and glucose-consumption rate of 52.29 ± 0.68 g/L, 1.38 ± 0.05 g/(L·h), and 1.22 ± 0.00 g/(L·h), respectively, were higher at 42 °C compared to 30 °C. Sugarcane molasses, a low-value carbon, led to the highest D-lactate titer and yield of 66.26 ± 0.81 g/L and 0.91 ± 0.01 g/g, respectively, in a medium without additional nutrients. This study is a pioneer work of engineering K. marxianus to produce D-lactate at the yield approaching theoretical maximum using simple batch process. Our results support the potential of an engineered K. marxianus for D-lactate production on an industrial scale. KEY POINTS: • K. marxianus was engineered by deleting PDC1 and expressing codon-optimized D-ldhA. • The strain allowed high D-lactate titer and yield under pH ranging from 3.5 to 5.0. • The strain produced 66 g/L D-lactate at 30 °C from molasses without any additional nutrients.
Topics: Lactic Acid; Saccharomyces cerevisiae; Kluyveromyces; L-Lactate Dehydrogenase; Glucose; Pyruvate Decarboxylase; Hydrogen-Ion Concentration; Fermentation
PubMed: 37405435
DOI: 10.1007/s00253-023-12658-2 -
Photosynthesis Research Feb 2021C-like plants represent the penultimate stage of evolution from C to C plants. Although Coleataenia prionitis (formerly Panicum prionitis) has been described as a C...
C-like plants represent the penultimate stage of evolution from C to C plants. Although Coleataenia prionitis (formerly Panicum prionitis) has been described as a C plant, its leaf anatomy and gas exchange traits suggest that it may be a C-like plant. Here, we reexamined the leaf structure and biochemical and physiological traits of photosynthesis in this grass. The large vascular bundles were surrounded by two layers of bundle sheath (BS): a colorless outer BS and a chloroplast-rich inner BS. Small vascular bundles, which generally had a single BS layer with various vascular structures, also occurred throughout the mesophyll together with BS cells not associated with vascular tissue. The mesophyll cells did not show a radial arrangement typical of Kranz anatomy. These features suggest that the leaf anatomy of C. prionitis is on the evolutionary pathway to a complete C Kranz type. Phosphoenolpyruvate carboxylase (PEPC) and pyruvate, Pi dikinase occurred in the mesophyll and outer BS. Glycine decarboxylase was confined to the inner BS. Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) accumulated in the mesophyll and both BSs. C. prionitis had biochemical traits of NADP-malic enzyme type, whereas its gas exchange traits were close to those of C-like intermediate plants rather than C plants. A gas exchange study with a PEPC inhibitor suggested that Rubisco in the mesophyll could fix atmospheric CO. These data demonstrate that C. prionitis is not a true C plant but should be considered as a C-like plant.
Topics: Carbon Dioxide; Chloroplasts; Glycine Dehydrogenase (Decarboxylating); Malate Dehydrogenase; Mesophyll Cells; Phenotype; Phosphoenolpyruvate Carboxylase; Photosynthesis; Plant Leaves; Plant Proteins; Poaceae; Ribulose-Bisphosphate Carboxylase
PubMed: 33393063
DOI: 10.1007/s11120-020-00808-w -
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