47 results on '"Phosphotransacetylase"'
Search Results
2. Unravelling the role of anaerobic metabolism (pta-ackA) and virulence (misL and ssa) genes in Salmonella Heidelberg shedding using chicken infection model
- Author
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Monte, Daniel F. M., Saraiva, Mauro M. S., Cabrera, Julia Memrava, de Almeida, Adriana Maria, de Freitas Neto, Oliveiro Caetano, Barrow, Paul A., and Junior, Angelo Berchieri
- Published
- 2024
- Full Text
- View/download PDF
3. Increasing lipid yield in Yarrowia lipolytica through phosphoketolase and phosphotransacetylase expression in a phosphofructokinase deletion strain
- Author
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Annapurna Kamineni, Andrew L. Consiglio, Kyle MacEwen, Shuyan Chen, Gamuchirai Chifamba, A. Joe Shaw, and Vasiliki Tsakraklides
- Subjects
Phosphotransacetylase ,Phosphoketolase ,Lipid yield ,Cell-specific lipid productivity ,Yarrowia lipolytica ,Central carbon metabolism ,Fuel ,TP315-360 ,Biotechnology ,TP248.13-248.65 - Abstract
Abstract Background Lipids are important precursors in the biofuel and oleochemical industries. Yarrowia lipolytica is among the most extensively studied oleaginous microorganisms and has been a focus of metabolic engineering to improve lipid production. Yield improvement, through rewiring of the central carbon metabolism of Y. lipolytica from glucose to the lipid precursor acetyl-CoA, is a key strategy for achieving commercial success in this organism. Results Building on YB-392, a Y. lipolytica isolate known for stable non-hyphal growth and low citrate production with demonstrated potential for high lipid accumulation, we assembled a heterologous pathway that redirects carbon flux from glucose through the pentose phosphate pathway (PPP) to acetyl-CoA. We used phosphofructokinase (Pfk) deletion to block glycolysis and expressed two non-native enzymes, phosphoketolase (Xpk) and phosphotransacetylase (Pta), to convert PPP-produced xylulose-5-P to acetyl-CoA. Introduction of the pathway in a pfk deletion strain that is unable to grow and accumulate lipid from glucose in defined media ensured maximal redirection of carbon flux through Xpk/Pta. Expression of Xpk and Pta restored growth and lipid production from glucose. In 1-L bioreactors, the engineered strains recorded improved lipid yield and cell-specific productivity by up to 19 and 78%, respectively. Conclusions Yields and cell-specific productivities are important bioprocess parameters for large-scale lipid fermentations. Improving these parameters by engineering the Xpk/Pta pathway is an important step towards developing Y. lipolytica as an industrially preferred microbial biocatalyst for lipid production.
- Published
- 2021
- Full Text
- View/download PDF
4. Acetate metabolism in Geobacillus thermoglucosidasius and strain engineering for enhanced bioethanol production
- Author
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Hills, Christopher and Danson, Michael
- Subjects
662 ,Geobacillus thermoglucosidasius ,metabolic engineering ,acetate ,phosphotransacetylase ,acetate kinase - Abstract
Social, economic and political pressures have driven the development of renewable alternatives to fossil fuels. Biofuels, such as bioethanol, have proved to be successful alternatives. Mature technologies are crop-based, but this has brought criticism due to the conflicting use of land for fuel versus food production. Therefore, bioethanol production technologies have shifted to utilising the sugars that derive from the degradation of lignocellulosic biomass. The thermophilic, Gram-positive bacterium, Geobacillus thermoglucosidasius, can naturally utilise a large fraction of these sugars, and metabolic engineering has been used to create a strain that produces ethanol as the major product of fermentation. This strain, G. thermoglucosidasius TM242 (Δldh, Δpfl, pdhup), does however, produce small but significant quantities of acetate, an undesirable by-product of fermentation. Therefore, acetate metabolism in the G. thermoglucosidasius TM242 strain was the focus of this study. During fermentation, ethanol is generated from the central metabolite acetyl-CoA through the activities of a bifunctional enzyme: aldehyde dehydrogenase/alcohol dehydrogenase (ADHE). On the other hand, acetate is generated from acetyl-CoA through catalysis by phosphotransacetylase (PTA) and acetate kinase (AK). Acetate metabolism in G. thermoglucosidasius TM242 was studied in this project by investigating the enzyme activities governing flux from acetyl-CoA, and the feasibility of reduced acetate production was investigated by a pta-deletion strategy. This thesis reports the characterisation of PTA and AK, by studying activities from both native cell lysates and recombinantly expressed proteins. The results indicate that the activities of PTA and AK are greater than those of ADHE, suggesting that the potential metabolic flux is greater towards acetate production than to ethanol. However, the ethanol yield from G. thermoglucosidasius TM242 fermentations is greater than that of acetate, suggesting the existence of a regulatory mechanism controlling acetyl-CoA flux. Several possible regulatory mechanisms were studied in this project and are reported here. The viability of creating a strain that reduces acetate accumulation, and potentially increases ethanol yields, was investigated and reported in this thesis. The gene encoding PTA was deleted from G. thermoglucosidasius TM242, and the resulting strain was characterised. The Δpta strain had approximately 5% of the PTA activity measured in TM242, but acetate was still generated from pentose and hexose fermentations. Additional phosphotransacylase (PTAC) enzymes were discovered in G. thermoglucosidasius TM242 that could catalyse the conversion of acetyl-CoA and orthophosphate to acetyl-phosphate and CoA. A series of PTAC null strains were created and analysed, the results of which indicated that phosphotransbutyrylase (PTB) could be involved in acetate production in vivo. It was discovered that the cell lysates of G. thermoglucosidasius strains carrying deletions to both pta and ptb could no longer catalyse the conversion of acetyl-CoA and orthophosphate to acetyl-phosphate and CoA. However, these strains still accumulated acetate, suggesting the presence of alternative acetate-producing pathways in this organism. In addition, G. thermoglucosidasius strains carrying deletions to both pta and ptb could ferment glucose but not xylose, suggesting that the production of ATP by the PTA-AK pathway is crucial for micro-aerobic growth on pentose sugars.
- Published
- 2015
5. Production of octanoic acid in Saccharomyces cerevisiae: Investigation of new precursor supply engineering strategies and intrinsic limitations.
- Author
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Wernig, Florian, Baumann, Leonie, Boles, Eckhard, and Oreb, Mislav
- Abstract
The eight‐carbon fatty acid octanoic acid (OA) is an important platform chemical and precursor of many industrially relevant products. Its microbial biosynthesis is regarded as a promising alternative to current unsustainable production methods. In Saccharomyces cerevisiae, the production of OA had been previously achieved by rational engineering of the fatty acid synthase. For the supply of the precursor molecule acetyl‐CoA and of the redox cofactor NADPH, the native pyruvate dehydrogenase bypass had been harnessed, or the cells had been additionally provided with a pathway involving a heterologous ATP‐citrate lyase. Here, we redirected the flux of glucose towards the oxidative branch of the pentose phosphate pathway and overexpressed a heterologous phosphoketolase/phosphotransacetylase shunt to improve the supply of NADPH and acetyl‐CoA in a strain background with abolished OA degradation. We show that these modifications lead to an increased yield of OA during the consumption of glucose by more than 60% compared to the parental strain. Furthermore, we investigated different genetic engineering targets to identify potential factors that limit the OA production in yeast. Toxicity assays performed with the engineered strains suggest that the inhibitory effects of OA on cell growth likely impose an upper limit to attainable OA yields. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
6. Engineering cytoplasmic acetyl-CoA synthesis decouples lipid production from nitrogen starvation in the oleaginous yeast Rhodosporidium azoricum
- Author
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Silvia Donzella, Daniela Cucchetti, Claudia Capusoni, Aurora Rizzi, Silvia Galafassi, Gambaro Chiara, and Concetta Compagno
- Subjects
Rhodosporidium azoricum ,Lipid production ,Oleaginous yeasts ,Phosphoketolases ,Phosphotransacetylase ,Lignocellulosic hydrolysates ,Microbiology ,QR1-502 - Abstract
Abstract Background Oleaginous yeasts are able to accumulate very high levels of neutral lipids especially under condition of excess of carbon and nitrogen limitation (medium with high C/N ratio). This makes necessary the use of two-steps processes in order to achieve high level of biomass and lipid. To simplify the process, the decoupling of lipid synthesis from nitrogen starvation, by establishing a cytosolic acetyl-CoA formation pathway alternative to the one catalysed by ATP-citrate lyase, can be useful. Results In this work, we introduced a new cytoplasmic route for acetyl-CoA (AcCoA) formation in Rhodosporidium azoricum by overexpressing genes encoding for homologous phosphoketolase (Xfpk) and heterologous phosphotransacetylase (Pta). The engineered strain PTAPK4 exhibits higher lipid content and produces higher lipid concentration than the wild type strain when it was cultivated in media containing different C/N ratios. In a bioreactor process performed on glucose/xylose mixture, to simulate an industrial process for lipid production from lignocellulosic materials, we obtained an increase of 89% in final lipid concentration by the engineered strain in comparison to the wild type. This indicates that the transformed strain can produce higher cellular biomass with a high lipid content than the wild type. The transformed strain furthermore evidenced the advantage over the wild type in performing this process, being the lipid yields 0.13 and 0.05, respectively. Conclusion Our results show that the overexpression of homologous Xfpk and heterologous Pta activities in R. azoricum creates a new cytosolic AcCoA supply that decouples lipid production from nitrogen starvation. This metabolic modification allows improving lipid production in cultural conditions that can be suitable for the development of industrial bioprocesses using lignocellulosic hydrolysates.
- Published
- 2019
- Full Text
- View/download PDF
7. Increasing lipid yield in Yarrowia lipolytica through phosphoketolase and phosphotransacetylase expression in a phosphofructokinase deletion strain.
- Author
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Kamineni, Annapurna, Consiglio, Andrew L., MacEwen, Kyle, Chen, Shuyan, Chifamba, Gamuchirai, Shaw, A. Joe, and Tsakraklides, Vasiliki
- Subjects
ACETYLCOENZYME A ,PENTOSE phosphate pathway ,MICROBIAL lipids ,LIPIDS ,CARBON metabolism - Abstract
Background: Lipids are important precursors in the biofuel and oleochemical industries. Yarrowia lipolytica is among the most extensively studied oleaginous microorganisms and has been a focus of metabolic engineering to improve lipid production. Yield improvement, through rewiring of the central carbon metabolism of Y. lipolytica from glucose to the lipid precursor acetyl-CoA, is a key strategy for achieving commercial success in this organism. Results: Building on YB-392, a Y. lipolytica isolate known for stable non-hyphal growth and low citrate production with demonstrated potential for high lipid accumulation, we assembled a heterologous pathway that redirects carbon flux from glucose through the pentose phosphate pathway (PPP) to acetyl-CoA. We used phosphofructokinase (Pfk) deletion to block glycolysis and expressed two non-native enzymes, phosphoketolase (Xpk) and phosphotransacetylase (Pta), to convert PPP-produced xylulose-5-P to acetyl-CoA. Introduction of the pathway in a pfk deletion strain that is unable to grow and accumulate lipid from glucose in defined media ensured maximal redirection of carbon flux through Xpk/Pta. Expression of Xpk and Pta restored growth and lipid production from glucose. In 1-L bioreactors, the engineered strains recorded improved lipid yield and cell-specific productivity by up to 19 and 78%, respectively. Conclusions: Yields and cell-specific productivities are important bioprocess parameters for large-scale lipid fermentations. Improving these parameters by engineering the Xpk/Pta pathway is an important step towards developing Y. lipolytica as an industrially preferred microbial biocatalyst for lipid production. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
8. Establishing an innovative carbohydrate metabolic pathway for efficient production of 2-keto-l-gulonic acid in Ketogulonicigenium robustum initiated by intronic promoters
- Author
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Cai-Yun Wang, Ye Li, Zi-Wei Gao, Li-Cheng Liu, Meng-Yue Zhang, Tian-Yuan Zhang, Chun-Fu Wu, and Yi-Xuan Zhang
- Subjects
Ketogulonicigenium ,Metabolic pathway ,Genome analysis ,Promoter ,Phosphoketolase ,Phosphotransacetylase ,Microbiology ,QR1-502 - Abstract
Abstract Background 2-Keto-l-gulonic acid (2-KGA), the precursor of vitamin C, is currently produced by two-step fermentation. In the second step, l-sorbose is transformed into 2-KGA by the symbiosis system composed of Ketogulonicigenium vulgare and Bacillus megaterium. Due to the different nutrient requirements and the uncertain ratio of the two strains, the symbiosis system significantly limits strain improvement and fermentation optimization. Results In this study, Ketogulonicigenium robustum SPU_B003 was reported for its capability to grow well independently and to produce more 2-KGA than that of K. vulgare in a mono-culture system. The complete genome of K. robustum SPU_B003 was sequenced, and the metabolic characteristics were analyzed. Compared to the four reported K. vulgare genomes, K. robustum SPU_B003 contained more tRNAs, rRNAs, NAD and NADP biosynthetic genes, as well as regulation- and cell signaling-related genes. Moreover, the amino acid biosynthesis pathways were more complete. Two species-specific internal promoters, P1 (orf_01408 promoter) and P2 (orf_02221 promoter), were predicted and validated by detecting their initiation activity. To efficiently produce 2-KGA with decreased CO2 release, an innovative acetyl-CoA biosynthetic pathway (XFP-PTA pathway) was introduced into K. robustum SPU_B003 by expressing heterologous phosphoketolase (xfp) and phosphotransacetylase (pta) initiated by internal promoters. After gene optimization, the recombinant strain K. robustum/pBBR-P1_xfp2502-P2_pta2145 enhanced acetyl-CoA approximately 2.4-fold and increased 2-KGA production by 22.27% compared to the control strain K. robustum/pBBR1MCS-2. Accordingly, the transcriptional level of the 6-phosphogluconate dehydrogenase (pgd) and pyruvate dehydrogenase genes (pdh) decreased by 24.33 ± 6.67 and 8.67 ± 5.51%, respectively. The key genes responsible for 2-KGA biosynthesis, sorbose dehydrogenase gene (sdh) and sorbosone dehydrogenase gene (sndh), were up-regulated to different degrees in the recombinant strain. Conclusions The genome-based functional analysis of K. robustum SPU_B003 provided a new understanding of the specific metabolic characteristics. The new XFP-PTA pathway was an efficient route to enhance acetyl-CoA levels and to therefore promote 2-KGA production.
- Published
- 2018
- Full Text
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9. Characterization of the phosphotransacetylase-acetate kinase pathway for ATP production in Porphyromonas gingivalis.
- Author
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Yoshida, Yasuo, Sato, Mitsunari, Nonaka, Takamasa, Hasegawa, Yoshiaki, and Kezuka, Yuichiro
- Subjects
- *
PORPHYROMONAS gingivalis , *OPERONS , *ACETYLCOENZYME A - Abstract
Acetyl phosphate (AcP) is generally produced from acetyl coenzyme A by phosphotransacetylase (Pta), and subsequent reaction with ADP, catalyzed by acetate kinase (Ack), produces ATP. The mechanism of ATP production in Porphyromonas gingivalis is poorly understood. The aim of this study was to explore the molecular basis of the Pta-Ack pathway in this microorganism. Pta and Ack from P. gingivalis ATCC 33277 were enzymatically and structurally characterized. Structural and mutational analyses suggest that Pta is a dimer with two substrate-binding sites in each subunit. Ack is also dimeric, with a catalytic cleft in each subunit, and structural analysis indicates a dramatic domain motion that opens and closes the cleft during catalysis. ATP formation by Ack proceeds via a sequential mechanism. Reverse transcription-PCR analysis demonstrated that the pta (PGN_1179) and ack (PGN_1178) genes, tandemly located in the genome, are cotranscribed as an operon. Inactivation of pta or ack in P. gingivalis by homologous recombination was successful only when the inactivated gene was expressed in trans. Therefore, both pta and ack genes are essential for this microorganism. Insights into the Pta-Ack pathway reported herein would be helpful to understand the energy acquisition in P. gingivalis. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
10. Genetic engineering of Bacillus sp. and fermentation process optimizing for diacetyl production.
- Author
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Wang, Yuepeng, Sun, Wenhui, Zheng, Shihao, Zhang, Yue, and Bao, Yongming
- Subjects
- *
FERMENTATION , *BACILLUS (Bacteria) , *GENETIC engineering , *BACILLUS subtilis , *BACK propagation , *DIACETYL - Abstract
• Deletion of pta gene leading to diacetyl increase markedly in Bacillus sp. DL01-Δ alsD. • Diacetyl biosynthetic flux amplified by overexpressing ALS from Bacillus subtilis 168. • Ferment parameters optimized by BP neural network for increasing diacetyl production. Diacetyl, an important flavor extensively used in the food industry, can be produced from the non-enzymatic oxidative decarboxylation of α-acetolactate in bacteria fermentation. In previous work, we obtained a strain of Bacillus sp. DL01-Δ alsD with low diacetyl accumulation. The strain was engineered and optimized for improving the production of diacetyl in this study. First, deletion of the gene encoding phosphotransacetylase (pta), by homologous recombination with high temperature sensitive shuttle plasmid vector pKS1, led to a reduction of acetate and 130% increase of diacetyl production in B. sp. DL01-Δ alsD- Δ pta. Then overexpression of α-acetolactate synthase (ALS) from B. subtilis 168 in B. sp. DL01-Δ alsD- Δ pta resulted in efficient diacetyl production with a titer of 5.43 g/L. To further increase diacetyl production, single factor and orthogonal experimental data were used to predict the optimal fermentation conditions by Back Propagation neural network. Optimal value of K L a (Dissolved oxygen volume coefficient) was 12.4 h−1 with fermentation parameters of aeration rate 0.66 vvm, agitation speed 179 rpm and temperature 35.7 ℃. A titer of 11.18 g/L diacetyl, the highest reported diacetyl production, was achieved by fed-batch fermentation at the optimal condition using the metabolic engineered strain of B. sp. DL01-Δ alsD- Δ pta-als 168. These results are of great importance as a new way for the efficient production of diacetyl by food-safety bacteria. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
11. Increasing lipid yield in Yarrowia lipolytica through phosphoketolase and phosphotransacetylase expression in a phosphofructokinase deletion strain
- Author
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A. Joe Shaw, Annapurna Kamineni, Gamuchirai Chifamba, Vasiliki Tsakraklides, Kyle MacEwen, Andrew L. Consiglio, and Shuyan Chen
- Subjects
0106 biological sciences ,Yarrowia lipolytica ,Cell-specific lipid productivity ,Phosphoketolase ,Management, Monitoring, Policy and Law ,Pentose phosphate pathway ,01 natural sciences ,Applied Microbiology and Biotechnology ,Metabolic engineering ,03 medical and health sciences ,Central carbon metabolism ,TP315-360 ,010608 biotechnology ,Phosphotransacetylase ,Glycolysis ,Bioprocess ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,biology ,Renewable Energy, Sustainability and the Environment ,Research ,Yarrowia ,biology.organism_classification ,Fuel ,General Energy ,Enzyme ,chemistry ,Biochemistry ,Lipid yield ,TP248.13-248.65 ,Phosphofructokinase ,Biotechnology - Abstract
Background Lipids are important precursors in the biofuel and oleochemical industries. Yarrowia lipolytica is among the most extensively studied oleaginous microorganisms and has been a focus of metabolic engineering to improve lipid production. Yield improvement, through rewiring of the central carbon metabolism of Y. lipolytica from glucose to the lipid precursor acetyl-CoA, is a key strategy for achieving commercial success in this organism. Results Building on YB-392, a Y. lipolytica isolate known for stable non-hyphal growth and low citrate production with demonstrated potential for high lipid accumulation, we assembled a heterologous pathway that redirects carbon flux from glucose through the pentose phosphate pathway (PPP) to acetyl-CoA. We used phosphofructokinase (Pfk) deletion to block glycolysis and expressed two non-native enzymes, phosphoketolase (Xpk) and phosphotransacetylase (Pta), to convert PPP-produced xylulose-5-P to acetyl-CoA. Introduction of the pathway in a pfk deletion strain that is unable to grow and accumulate lipid from glucose in defined media ensured maximal redirection of carbon flux through Xpk/Pta. Expression of Xpk and Pta restored growth and lipid production from glucose. In 1-L bioreactors, the engineered strains recorded improved lipid yield and cell-specific productivity by up to 19 and 78%, respectively. Conclusions Yields and cell-specific productivities are important bioprocess parameters for large-scale lipid fermentations. Improving these parameters by engineering the Xpk/Pta pathway is an important step towards developing Y. lipolytica as an industrially preferred microbial biocatalyst for lipid production.
- Published
- 2021
12. Genetic engineering of Synechocystis sp. PCC6803 for poly-β-hydroxybutyrate overproduction.
- Author
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Carpine, Roberta, Du, Wei, Olivieri, Giuseppe, Pollio, Antonino, Hellingwerf, Klaas J., Marzocchella, Antonio, and Branco dos Santos, Filipe
- Abstract
The biosynthesis of poly-β-hydroxybutyrate (PHB) directly from carbon dioxide is a sustainable alternative for non-renewable, petroleum-based polymer production. Synechocystis sp. PCC6803 can naturally accumulate PHB using CO 2 as the sole carbon source, particularly when major nutrients such as nitrogen become limiting. Many previous studies have tried to genetically engineer PHB overproduction; mostly by increasing the expression of enzymes directly involved in its biosynthesis pathway. Here, we have instead concentrated on engineering the central carbon metabolism of Synechocystis such that ( i ) the PHB synthesis pathway becomes deregulated, and/or ( ii ) the levels of its substrate, acetyl-CoA, were increased. Seven different mutants were constructed harboring, separately or in combination, three different genetic modifications to Synechocystis ' metabolic network. These were the deletions of phosphotransacetylase (Pta) and acetyl-CoA hydrolase (Ach), and the expression of a heterologous phosphoketolase (XfpK) from Bifidobacterium breve . The wild type Synechocystis and the derivative strains were compared in terms of biomass and the PHB production capability during photoautotrophic growth. This was performed in a photobioreactor exposed to a diel light/dark rhythm and using standard BG 11 as the growth medium. We found that the strain that combined all three genetic modifications, i.e. xfpk overexpression in a double pta and ach deletion background, showed the highest levels of PHB production from all the strains tested here. Encouragingly, the production levels obtained: 232 mg L − 1 , ~ 12% (w/w) of the dry biomass weight, and a productivity of 7.3 mg L − 1 d − 1 ; are to the best of our knowledge, the highest ever reported for PHB production directly from CO 2 . [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
13. Phosphotransacetylase as a Key Factor in Biological Production of Polyhydroxybutyrate
- Author
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Miyake, Masato, Miyamoto, Chisako, Schnackenberg, Joerg, Kurane, Ryuichiro, Asada, Yasuo, Finkelstein, Mark, editor, and Davison, Brian H., editor
- Published
- 2000
- Full Text
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14. Metabolic engineering of Klebsiella pneumoniae and in silico investigation for enhanced 2,3-butanediol production.
- Author
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Rathnasingh, Chelladurai, Park, Jong, Kim, Duk-ki, Song, Hyohak, and Chang, Yong
- Subjects
KLEBSIELLA pneumoniae ,BUTANEDIOL ,LACTATE dehydrogenase ,ACETATE kinase ,ALCOHOL dehydrogenase - Abstract
Objectives: To improve the production of 2,3-butanediol (2,3-BD) in Klebsiella pneumoniae, the genes related to the formation of lactic acid, ethanol, and acetic acid were eliminated. Results: Although the cell growth and 2,3-BD production rates of the K. pneumoniae ΔldhA ΔadhE Δpta- ackA strain were lower than those of the wild-type strain, the mutant produced a higher titer of 2,3-BD and a higher yield in batch fermentation: 91 g 2,3-BD/l with a yield of 0.45 g per g glucose and a productivity of 1.62 g/l.h in fed-batch fermentation. The metabolic characteristics of the mutants were consistent with the results of in silico simulation. Conclusions: K. pneumoniae knockout mutants developed with an aid of in silico investigation could produce higher amounts of 2,3-BD with increased titer, yield, and productivity. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
15. Acetate formation in the photoheterotrophic bacterium Chloroflexus aurantiacus involves an archaeal type ADP-forming acetyl- Co A synthetase isoenzyme I.
- Author
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Schmidt, Marcel and Schönheit, Peter
- Subjects
- *
ACETATES , *HETEROTROPHIC bacteria , *CHLOROFLEXUS aurantiacus , *ARCHAEBACTERIA , *ACETYL-CoA synthetase , *ISOENZYMES , *PHOTOSYNTHETIC bacteria - Abstract
The bacterium Chloroflexus aurantiacus excreted significant amounts of acetate during photohetero trophic growth on glucose and in resting cell suspensions. Up to 1.5 mol acetate per mol glucose were formed. In acetate-forming cells, the activities of phosphotransacetylase and acetate kinase, usually involved in acetate formation in Bacteria, could not be detected; instead, the cells contained an acetyl- Co A synthetase ( ADP-forming) ( ACD) (acetyl- Co A + ADP + Pi → acetate + ATP + Co A), an enzyme so far reported in prokaryotes to be specific for acetate-forming Archaea. ACD, which was induced 10-fold during growth on glucose, was purified and the encoding gene was identified as Caur_3920. The recombinant enzyme, a homotetrameric 300-kDa protein composed of 75-kDa subunits, was characterized as functional ACD. Substrate specificities and kinetic constants for acetyl- Co A/acetate and other acyl- Co A esters/acids were determined, showing similarity of the C. aurantiacus ACD to archaeal ACD I isoenzymes, which are involved in acetate formation from sugars. This is the first report of a functional ACD involved in acetate formation in the domain of Bacteria. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
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16. A phosphotransacetylase from uropathogenic Staphylococcus saprophyticus: Characterization, kinetic analysis and its allosteric inhibition by a-ketoglutarate.
- Author
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Yıldız, Hamdi, Aksoy, İlknur Yurtsever, and Türkan, Ali
- Subjects
- *
ACETYLTRANSFERASES , *STAPHYLOCOCCUS , *BACTERIAL enzymes , *ENZYME kinetics , *MOLECULAR cloning , *BACTERIAL genomes , *HYDROGEN-ion concentration , *ADENOSINE triphosphate - Abstract
Objectives: The genome of Staphylococcus saprophyticus, an uropathogen, contains SSP2124 gene encoding a putative phosphotransacetylase (Pta). Pta plays significant role in energy metabolism as well as colonization of uropathogens. In this study, cloning, expression, characterization and kinetic analysis of S. saprophyticus Pta have been undertaken. Methods: The SSP2124 gene was amplified by PCR from the genome of S. saprophyticus and subcloned into pET28a(+) vector and expressed in E. coli BL21. The recombinant Pta was affinity purified by Ni-NTA column. Its characterization as well as its steady-state kinetic analysis has been studied by standard procedures. Results: The recombinant Pta forms dimers as determined by gel filtration. The activity of the enzyme was maximal at a temperature range of 30-35 °C and at the pH range of 7.5-8.5. It was greatly stimulated by K+ and NH4+ ions, but inhibited by Na+ ion. Kinetic analysis and product inhibition studies suggest that there is a kinetic mechanism that proceeds through random addition of both substrates to the enzyme before any product is released. The enzyme was inhibited in both acetyl-CoA-forming (forward) and acetyl phosphate-forming (reverse) directions by adenosine triphosphate (ATP) and α-ketoglutarate but not affected by pyruvate and reduced nicotinamide adenine dinucleotide. ATP was competitive with respect to CoA and noncompetitive with respect to acetyl phosphate. The inhibition by a-ketoglutarate, on the other hand, was allosteric with halfsaturations at 6.5 ± 0.7 mM and Hill coefficients of 2.6 ± 0.8 and 3.3 ± 0.2, in forward and reverse directions, respectively. Conclusions: S. saprophyticus Pta belongs to the Pta I family and show similar biochemical features as well as kinetic mechanism with other members. And the most importantly, this study presents the first example of allosteric regulation of a member of class I Ptas. [ABSTRACT FROM AUTHOR]
- Published
- 2012
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17. Characterization of Escherichia coli EutD: a phosphotransacetylase of the ethanolamine operon.
- Author
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Bologna, Federico, Campos-Bermudez, Valeria, Saavedra, Damián, Andreo, Carlos, and Drincovich, María
- Abstract
The Escherichia coli genes pta and eutD encode proteins containing the phosphate-acetyltransferase domain. EutD is composed only by this domain and belongs to the ethanolamine operon. This enzyme has not been characterized yet, and its relationship to the multimodular E. coli phosphotransacetylase (Pta) remains unclear. In the present work, a detailed characterization of EutD from E. coli (EcEutD) was performed. The enzyme is a more efficient phosphotransacetylase than E. coli Pta (EcPta) in catalyzing its reaction in either direction and assembles as a dimer, being differentially modulated by EcPta effectors. When comparing EutD and Pta, both from E. coli, certain divergent regions of the primary structure responsible for their unique properties can be found. The growth on acetate of the E. coli pta acs double-mutant strain, was complemented by either introducing EcEutD or by inducing the eut operon with ethanolamine. In this case, the expression of a phosphotransacetylase different from Pta was confirmed by activity assays. Overall, the results indicate that EcEutD and Pta, although able to catalyse the same reaction, display differential efficiency and regulation, and also differ in the induction of their expression. However, under certain growth conditions, they can fulfil equal roles in E. coli metabolism. [ABSTRACT FROM AUTHOR]
- Published
- 2010
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18. Functional dissection of Escherichia coli phosphotransacetylase structural domains and analysis of key compounds involved in activity regulation.
- Author
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Campos-Bermudez, Valeria Alina, Bologna, Federico Pablo, Andreo, Carlos Santiago, and Drincovich, María Fabiana
- Subjects
- *
ESCHERICHIA coli , *ACETYLCOENZYME A , *METABOLISM , *CELLULAR signal transduction , *PYRUVATES - Abstract
Escherichia coli phosphotransacetylase (Pta) catalyzes the reversible interconversion of acetyl-CoA and acetyl phosphate. Both compounds are critical in E. coli metabolism, and acetyl phosphate is also involved in the regulation of certain signal transduction pathways. Along with acetate kinase, Pta plays an important role in acetate production when E. coli grows on rich medium; alternatively, it is involved in acetate utilization at high acetate concentrations. E. coli Pta is composed of three different domains, but only the C-terminal one, called PTA_PTB, is specific for all Ptas. In the present work, the characterization of E. coli Pta and deletions from the N-terminal region were performed. E. coli Pta acetyl phosphate-forming and acetyl phosphate-consuming reactions display different maximum activities, and are differentially regulated by pyruvate and phosphoenolpyruvate. These compounds activate acetyl phosphate production, but inhibit acetyl-CoA production, thus playing a critical role in defining the rates of the two Pta reactions. The characterization of three truncated Ptas, which all display Pta activity, indicates that the substrate-binding site is located at the C-terminal PTA_PTB domain. However, the N-terminal P-loop NTPase domain is involved in expression of the maximal catalytic activity, stabilization of the hexameric native state, and Pta activity regulation by NADH, ATP, phosphoenolpyruvate, and pyruvate. The truncated protein Pta-F3 was able to complement the growth on acetate of an E. coli mutant defective in acetyl-CoA synthetase and Pta, indicating that, although not regulated by metabolites, the Pta C-terminal domain is active in vivo. [ABSTRACT FROM AUTHOR]
- Published
- 2010
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19. Enhancement of riboflavin production by overexpression of acetolactate synthase in a pta mutant of Bacillus subtilis.
- Author
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Yingbo Zhu, Xun Chen, Tao Chen, and Xueming Zhao
- Subjects
- *
VITAMIN B2 , *CARBON , *BIOSYNTHESIS , *ACIDS , *BACILLUS subtilis , *GLUCOSE - Abstract
Genetic alterations of carbon flux into the acetoin biosynthesis pathway as a possible means to reduce acid accumulation were investigated in the riboflavin-producing Bacillus subtilis during growth on glucose. The lower rates of cell growth and riboflavin production were found in the pta-disrupted mutant while the rate of acetate formation was reduced. In contrast, acid accumulation was significantly reduced, to one-fifth that of the parental strain RH33::[pRB63]n, and a 50% increase in the riboflavin yield was obtained when the expression of the gene encoding acetolactate synthase was increased in the pta-disrupted mutant. Metabolic analysis, together with enzyme activity assays, indicated that the tricarboxylic acid cycle fluxes are significantly increased in response to acetolactate synthase overexpression in pta-disrupted mutant. Moreover, the intracellular ATP-to-ADP ratio also increased 5.8-fold. The high concentration of ATP could explain the increased riboflavin production. [ABSTRACT FROM AUTHOR]
- Published
- 2007
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20. Genetic engineering of Synechocystis sp. PCC6803 for poly-β-hydroxybutyrate overproduction
- Author
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Wei Du, Klaas J. Hellingwerf, Filipe Branco dos Santos, Antonino Pollio, Giuseppe Olivieri, Roberta Carpine, Antonio Marzocchella, Carpine, Roberta, Du, Wei, Olivieri, Giuseppe, Pollio, Antonino, Hellingwerf, Klaas J., Marzocchella, Antonio, Branco Dos Santos, Filipe, SILS Other Research (FNWI), Systems Biology, and SILS (FNWI)
- Subjects
0106 biological sciences ,0301 basic medicine ,Mutant ,Phosphoketolase ,Photobioreactor ,Biology ,Cyanobacteria ,01 natural sciences ,03 medical and health sciences ,chemistry.chemical_compound ,Biosynthesis ,010608 biotechnology ,Phosphotransacetylase ,Overproduction ,Growth medium ,Acetyl-CoA hydrolase ,Synechocystis ,Wild type ,biology.organism_classification ,030104 developmental biology ,Poly-β-hydroxybutyrate ,chemistry ,Biochemistry ,Genetic engineering ,Agronomy and Crop Science - Abstract
The biosynthesis of poly-β-hydroxybutyrate (PHB) directly from carbon dioxide is a sustainable alternative for non-renewable, petroleum-based polymer production. Synechocystis sp. PCC6803 can naturally accumulate PHB using CO2 as the sole carbon source, particularly when major nutrients such as nitrogen become limiting. Many previous studies have tried to genetically engineer PHB overproduction; mostly by increasing the expression of enzymes directly involved in its biosynthesis pathway. Here, we have instead concentrated on engineering the central carbon metabolism of Synechocystis such that (i) the PHB synthesis pathway becomes deregulated, and/or (ii) the levels of its substrate, acetyl-CoA, were increased. Seven different mutants were constructed harboring, separately or in combination, three different genetic modifications to Synechocystis' metabolic network. These were the deletions of phosphotransacetylase (Pta) and acetyl-CoA hydrolase (Ach), and the expression of a heterologous phosphoketolase (XfpK) from Bifidobacterium breve. The wild type Synechocystis and the derivative strains were compared in terms of biomass and the PHB production capability during photoautotrophic growth. This was performed in a photobioreactor exposed to a diel light/dark rhythm and using standard BG11 as the growth medium. We found that the strain that combined all three genetic modifications, i.e. xfpk overexpression in a double pta and ach deletion background, showed the highest levels of PHB production from all the strains tested here. Encouragingly, the production levels obtained: 232 mg L− 1, ~ 12% (w/w) of the dry biomass weight, and a productivity of 7.3 mg L− 1 d− 1; are to the best of our knowledge, the highest ever reported for PHB production directly from CO2.
- Published
- 2017
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21. Butyric acid and hydrogen production by Clostridium tyrobutyricum ATCC 25755 and mutants
- Author
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Liu, Xiaoguang, Zhu, Ying, and Yang, Shang-Tian
- Subjects
- *
BUTYRIC acid , *BIOCHEMICAL engineering , *GLUCOSE , *FERMENTATION , *INDUSTRIAL microbiology - Abstract
Abstract: Clostridium tyrobutyricum produces butyric acid, acetic acid, hydrogen and carbon dioxide as its main fermentation products. In this work, mutants with inactivated pta gene, encoding phosphotransacetylase (PTA) and ack gene, encoding acetate kinase (AK), were studied for their potential to improve butyric acid production in the fermentation. PTA and AK are two key enzymes in the acetate-producing pathway. PTA and AK activities in the pta-deleted mutant (PPTA-Em) were reduced by 44% and 91%, respectively, whereas AK activity in the ack-deleted mutant (PAK-Em) decreased by 50%. Meanwhile, the activity of butyrate kinase (BK) in PPTA-Em increased by 44% and hydrogenase activity in PAK-Em increased by 40%. As compared with the wild type, the specific growth rate of the mutants decreased by 32% (from 0.28 to 0.19h−1) because of the impaired PTA-AK pathway. Meanwhile, butyric acid production by these mutants was improved greatly, with higher butyric acid yield (>0.4g/g versus 0.34g/g) and final concentration (43g/L versus 29g/L), which also indicated that the mutants had better tolerance to butyric acid inhibition. However, acetate production in the mutants was not significantly reduced even though more butyrate was produced from glucose, suggesting the existence of additional acetate forming pathway in C. tyrobutyricum. Also, hydrogen production by PAK-Em mutant increased significantly, with higher hydrogen yield (2.61mol/mol glucose versus 1.35mol/mol glucose) and H2/CO2 ratio (1.43 versus 1.08). The SDS-PAGE also showed significantly different expression levels of proteins with molecular mass around 32 and 70kDa. These results suggested that integrational mutagenesis resulted in global metabolic shift and phenotypic changes, which also improved production of butyric acid and hydrogen from glucose in the fermentation. [Copyright &y& Elsevier]
- Published
- 2006
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22. Crystal structures of a phosphotransacetylase from Bacillus subtilis and its complex with acetyl phosphate.
- Author
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Xu, Qian Steven, Jancarik, Jarmila, Lou, Yun, Kuznetsova, Kate, Yakunin, Alexander F., Yokota, Hisao, Adams, Paul, Kim, Rosalind, and Kim, Sung-Hou
- Abstract
Phosphotransacetylase (Pta) [EC 2.3.1.8] plays a major role in acetate metabolism by catalyzing the reversible transfer of the acetyl group between coenzyme A (CoA) and orthophosphate: CH
3 COSCoA+HPO $$_{4}^{2-}\rightleftarrows$$ CH3 COOPO+CoASH. In this study, we report the crystal structures of Pta from Bacillus subtilis at 2.75 Å resolution and its complex with acetyl phosphate, one of its substrates, at 2.85 Å resolution. In addition, the Pta activity of the enzyme has been assayed. The enzyme folds into an α/β architecture with two domains separated by a prominent cleft, very similar to two other known Pta structures. The enzyme–acetyl phosphate complex structure reveals a few potential substrate binding sites. Two of them are located in the middle of the interdomain cleft: each one is surrounded by a region of strictly and highly conserved residues. High structural similarities are found with 4-hydroxythreonine-4-phosphate dehydrogenase (PdxA), and isocitrate and isopropylmalate dehydrogenases, all of which utilize NADP+ as their cofactor, which binds in the interdomain cleft. Their substrate binding sites are close to the acetyl phosphate binding sites of Pta in the cleft as well. These results suggest that the CoA is likely to bind to the interdomain cleft of Pta in a similar way as NADP+ binds to the other three enzymes. [ABSTRACT FROM AUTHOR]- Published
- 2005
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23. Free CoA-mediated regulation of intermediary and central metabolism: An hypothesis which accounts for the excretion of α-ketoglutarate during aerobic growth of Escherichia coli on acetate
- Author
-
El-Mansi, Mansi
- Subjects
- *
ESCHERICHIA coli , *DEHYDROGENASES , *PYRUVATES , *ESCHERICHIA - Abstract
Abstract: During growth of Escherichia coli on acetate, phosphotransacetylase and α-ketoglutarate dehydrogenase are in direct competition for their common co-factor, HS-CoA. Such competition is resolved in favour of phosphotransacetylase, thus rendering α-ketoglutarate dehydrogenase rate-limiting (controlling) and, in turn, creating a bottleneck at the level of α-ketoglutarate in the Krebs cycle. Accumulation of α-ketoglutarate is then balanced by its excretion. Addition of pyruvate, glucose or any glycolytic intermediate to acetate-grown culture relieves such a bottleneck by reversing carbon flow through phosphotransacetylase to give acetyl phosphate and much-needed HS-CoA. The urgent need for HS-CoA by the primordial organism might therefore have provided the selective pressure that led to the co-evolution of phosphotransacetylase and the two-malate synthase isoenzymes. [Copyright &y& Elsevier]
- Published
- 2005
- Full Text
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24. The amrG1 gene is involved in the activation of acetate in Corynebacterium glutamicum.
- Author
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Ruan, Hong, Gerstmeir, R., Schnicke, S., and Eikmanns, B.
- Abstract
During growth of Corynebacterium glutamicum on acetate as its carbon and energy source, the expression of the ptaack operon is induced, coding for the acetate-activating enzymes, which are phosphotransacetylase (PTA) and acetate kinase (AK). By transposon rescue, we identified the two genes amrG1 and amrG2 found in the deregulated transposon mutant C. glutamicum G25. The amrG1 gene (NCBI-accession: AF532964) has a size of 732 bp, encoding a polypeptide of 243 amino acids and apparently is partially responsible for the regulation of acetate metabolism in C. glutamicum. We constructed an in-frame deletion mutant and an overexpressing strain of amrG1 in the C. glutamicum ATCC13032 wildtype. The strains were then analyzed with respect to their enzyme activities of PTA and AK during growth on glucose, acetate and glucose or acetate alone as carbon sources. Compared to the parental strain, the amrG1 deletion mutant showed higher specific AK and PTA activities during growth on glucose but showed the same high specific activities of AK and PTA on medium containing acetate plus glucose and on medium containing acetate. In contrast to the gene deletion, overexpression of the amrG1 gene in C. glutamicum 13032 had the adverse regulatory effect. These results indicate that the amrG1 gene encodes a repressor or co-repressor of the ptaack operon. [ABSTRACT FROM AUTHOR]
- Published
- 2005
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- View/download PDF
25. Flux to acetate and lactate excretions in industrial fermentations: physiological and biochemical implications.
- Author
-
El-Mansi, Mansi
- Subjects
- *
ACETATES , *LACTATES , *EXCRETION , *FERMENTATION , *PHYSIOLOGY , *BIOMASS - Abstract
The efficiency of carbon conversion to biomass and desirable end products in industrial fermentations is diminished by the diversion of carbon to acetate and lactate excretions. In this study, the use of prototrophic and mutant strains of Escherichia coli, as well as enzyme active site directed inhibitors, revealed that flux to acetate excretion is physiologically advantageous to the organism as it facilitates a faster growth rate (μ) and permits growth to high cell densities. Moreover, the abolition of flux to acetate excretion was balanced by the excretion of lactate as well as 2-oxoglutarate, isocitrate and citrate, suggesting a ‘bottle-neck’ effect at the level of 2-oxoglutarate in the Krebs cycle. It is proposed that the acetate excreting enzymes, phosphotransacetylase and acetate kinase, constitute an anaplerotic loop or by-pass, the primary function of which is to replenish the Krebs cycle with reduced CoA, thus relieving the bottle-neck effect at the level of 2-oxoglutarate dehydrogenase. Furthermore, flux to lactate excretion plays a central role in regenerating proton gradient and maintaining the redox balance within the cell. The long-held view that flux to acetate and lactate excretions is merely a function of an ‘over-flow’ in central metabolism should, therefore, be re-evaluated. [ABSTRACT FROM AUTHOR]
- Published
- 2004
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- View/download PDF
26. Characterization of the phosphotransacetylase-acetate kinase pathway for ATP production in Porphyromonas gingivalis
- Author
-
Yuichiro Kezuka, Yasuo Yoshida, Mitsunari Sato, Yoshiaki Hasegawa, and Takamasa Nonaka
- Subjects
0301 basic medicine ,Microbiology (medical) ,crystal structure ,lcsh:QR1-502 ,lcsh:Microbiology ,lcsh:Infectious and parasitic diseases ,acetate kinase ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Transferase ,essential genes ,Dentistry (miscellaneous) ,Atp production ,lcsh:RC109-216 ,Porphyromonas gingivalis ,Acetate kinase ,biology ,phosphotransacetylase ,030206 dentistry ,Phosphate ,biology.organism_classification ,ATP ,030104 developmental biology ,Infectious Diseases ,chemistry ,Biochemistry ,Acetyl coenzyme ,Original Article - Abstract
Acetyl phosphate (AcP) is generally produced from acetyl coenzyme A by phosphotransacetylase (Pta), and subsequent reaction with ADP, catalyzed by acetate kinase (Ack), produces ATP. The mechanism of ATP production in Porphyromonas gingivalis is poorly understood. The aim of this study was to explore the molecular basis of the Pta-Ack pathway in this microorganism. Pta and Ack from P. gingivalis ATCC 33277 were enzymatically and structurally characterized. Structural and mutational analyses suggest that Pta is a dimer with two substrate-binding sites in each subunit. Ack is also dimeric, with a catalytic cleft in each subunit, and structural analysis indicates a dramatic domain motion that opens and closes the cleft during catalysis. ATP formation by Ack proceeds via a sequential mechanism. Reverse transcription-PCR analysis demonstrated that the pta (PGN_1179) and ack (PGN_1178) genes, tandemly located in the genome, are cotranscribed as an operon. Inactivation of pta or ack in P. gingivalis by homologous recombination was successful only when the inactivated gene was expressed in trans. Therefore, both pta and ack genes are essential for this microorganism. Insights into the Pta-Ack pathway reported herein would be helpful to understand the energy acquisition in P. gingivalis.
- Published
- 2019
27. Regulation of the Bacillus subtilis Phosphotransacetylase Gene1.
- Author
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Shin, Byung-Sik, Choi, Soo-Keun, and Park, Seung-Hwan
- Subjects
ACETYLTRANSFERASES ,ACYLTRANSFERASES ,NUCLEOTIDES ,NUCLEIC acids ,HEREDITY - Abstract
The enzyme, phosphotransacetylase (Pta), catalyzes the conversion of acetyl coenzyme A to acetyl phosphate. The putative pta gene of Bacillus subtilis, which had been sequenced as part of the Genome Project, was cloned and overexpressed in Escherichia coli. We confirmed that the gene encodes Pta by measuring the enzymatic activity of the purified protein. Insertional mutagenesis of the pta gene resulted in complete loss of the Pta activity, indicating that B. subtilis contains only one kind of pta gene. Expression of a pta-lacZ fusion was induced in the presence of excess glucose in the growth medium, and the intact ccpA gene was required for this activation. The transcriptional start site of the pta gene was located at 37 nucleotides upstream of the pta start codon, and a cre (catabolite responsive element) sequence, a cis-acting element that is responsible for the catabolite repression of a number of carbon utilization genes in B. subtilis, was identified upstream of the tentative promoter site. Experiments involving oligonucleotide-directed mutagenesis showed that the cre sequence is involved in glucose-mediated transcriptional activation. [ABSTRACT FROM AUTHOR]
- Published
- 1999
- Full Text
- View/download PDF
28. Regulation of acetate metabolism in Corynebacterium glutamicum: transcriptional control of the isocitrate lyase and malate synthase genes.
- Author
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Wendisch, Volker F., Spies, Marion, Reinscheid, D. J., Schnicke, Stephanie, Sahm, Hermann, and Eikmanns, B. J.
- Abstract
In the amino-acid-producing microorganism Corynebacterium glutamicum, the specific activities of the acetate-activating enzymes acetate kinase and phosphotransacetylase and those of the glyoxylate cycle enzymes isocitrate lyase and malate synthase were found to be high when the cells were grown on acetate (0.8, 2.9, 2.1, and 1.8 U/mg protein, respectively). When the cells were grown on glucose or on other carbon sources such as lactate, succinate, or glutamate, the specific activities were two- to fourfold (acetate kinase and phosphotransacetylase) and 45- to 100-fold (isocitrate lyase and malate synthase) lower, indicating that the synthesis of the four enzymes is regulated by acetate in the growth medium. A comparative Northern (RNA) analysis of the C. glutamicum isocitrate lyase and malate synthase genes ( aceA and aceB) and transcriptional cat fusion experiments revealed that aceA and aceB are transcribed as 1.6- and 2.7-kb monocistronic messages, respectively, and that the regulation of isocitrate lyase and malate synthase synthesis is exerted at the level of transcription from the respective promoters. Surprisingly, C. glutamicum mutants defective in either acetate kinase or phosphotransacetylase showed low specific activities of the other three enzymes (phosphotransacetylase, isocitrate lyase, and malate synthase or acetate kinase, isocitrate lyase, and malate synthase, respectively) irrespective of the presence or absence of acetate in the medium. This result and a correlation of a high intracellular acetyl coenzyme A concentration with high specific activities of isocitrate lyase, malate synthase, acetate kinase, and phosphotransacetylase suggest that acetyl coenzyme A or a derivative thereof may be a physiological trigger for the genetic regulation of enzymes involved in acetate metabolism of C. glutamicum. [ABSTRACT FROM AUTHOR]
- Published
- 1997
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- View/download PDF
29. Acetate thiokinase and the assimilation of acetate in Methanobacterium thermoautotrophicum.
- Author
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Oberlies, Gerhard, Fuchs, Georg, and Thauer, Rudolf
- Abstract
Methanobacterium thermoautotrophicum growing on H plus CO as sole carbon and energy source was found to contain acetate thiokinase (Acetyl CoA synthetase; EC 6.2.1.1): Acetate+ATP+CoA → Acetyl CoA+AMP+PPi. The apparent K value for acetate was 40 μM. Acetate kinase (EC 2.7.2.1) and phosphotransacetylase (EC 2.3.1.8) could not be detected. The specific activity of acetate thiokinase was high in cells grown with limited H and CO supply (approximately 100nmol/min · mg protein), it was low in exponentially grown cells (2 nmol/min·mg protein). This corresponded with the finding that cells growing linearly in the presence of acetate assimilated the monocarboxylic acid in high amounts (>10% of the cell carbon was derived from acetate), whereas exponentially growing cells did not (<1% of cell carbon was derived from acetate). These latter observations indicated that acetate thiokinase and free acetate are not involved in autotrophic CO fixation in M. thermoautotrophicum. The presence and some kinetic properties of succinate thiokinase (EC 6.2.1.5), adenylate kinase (EC 2.7.4.3), and inorganic pyrophosphatase (EC 3.6.1.1.) are also described. [ABSTRACT FROM AUTHOR]
- Published
- 1980
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- View/download PDF
30. The energy-yielding reactions of Peptococcus prévotii, their behaviour on starvation and the role and regulation of threonine dehydratase.
- Author
-
Bentley, Carolyn and Dawes, Edwin
- Abstract
The principal energy-yielding reactions of the strict anaerobe Peptococcus prévotii comprised the fermentation of l-serine and l-threonine via the enzymes threonine dehydratase, thioclastic enzyme, phosphotransacetylase and acetate kinase. Threonine dehydratase was purified 700-fold and shown to require pyridoxal 5′-phosphate as co-enzyme, and a reducing agent for optimum activity. The ratio of threonine and serine dehydratase activities was unaltered during purification. The optimum pH was 8.5 to 9.5 and isoleucine did not inhibit. Lineweaver-Burk plots were linear at l-threonine concentrations above 1.35 mM and the Kfor threonine was 2.5 mM and for serine 29 mM. Below this concentration co-operativity occurred which was not nullified by individual adenine nucleotides: Hill plots were biphasic. However, the enzyme was controlled by the adenylate energy charge in a novel manner; only at very low threonine concentrations (<1 mM) was control manifest, when a high energy charge inhibited and a low energy charge stimulated activity. During starvation for 33 hrs in phosphate buffer, pH 6.8, viability fell to zero but, of the enzymes of the energy-generating sequence, only the total units and specific activity of threonine dehydratase decreased (by 35%), which was insufficient to explain the loss of ability to generate ATP. [ABSTRACT FROM AUTHOR]
- Published
- 1974
- Full Text
- View/download PDF
31. The NADP-dependent malic enzyme MaeB is a central metabolic hub controlled by the acetyl-CoA to CoASH ratio.
- Author
-
Huergo, Luciano F., Araújo, Gillize A.T., Santos, Adrian S.R., Gerhardt, Edileusa C.M., Pedrosa, Fabio O., Souza, Emanuel M., and Forchhammer, Karl
- Subjects
- *
METABOLIC regulation , *ACETYLCOENZYME A , *ENZYMES , *AZOSPIRILLUM brasilense , *NITROGEN-fixing bacteria - Abstract
Malic enzymes participate in key metabolic processes, the MaeB-like malic enzymes carry a catalytic inactive phosphotransacetylase domain whose function remains elusive. Here we show that acetyl-CoA directly binds and inhibits MaeB-like enzymes with a saturable profile under physiological relevant acetyl-CoA concentrations. A MaeB-like enzyme from the nitrogen-fixing bacterium Azospirillum brasilense , namely AbMaeB1, binds both acetyl-CoA and unesterified CoASH in a way that inhibition of AbMaeB1 by acetyl-CoA is relieved by increasing CoASH concentrations. Hence, AbMaeB1 senses the acetyl-CoA/CoASH ratio. We revisited E. coli MaeB regulation to determine the inhibitory constant for acetyl-CoA. Our data support that the phosphotransacetylase domain of MaeB-like enzymes senses acetyl-CoA to dictate the fate of carbon distribution at the phosphoenol-pyruvate / pyruvate / oxaloacetate metabolic node. • Acetyl-CoA directly binds and inhibits MaeB-like enzymes. • Acetyl-CoA binding augments the MaeB Km for L-malate. • The A. brasilense AbMaeB1 is regulated by the acetyl-CoA/CoASH ratio. • MaeB regulation by acetyl-CoA coordinate carbon distribution in Bacteria. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
32. Engineering cytoplasmic acetyl-CoA synthesis decouples lipid production from nitrogen starvation in the oleaginous yeast Rhodosporidium azoricum.
- Author
-
Donzella, Silvia, Cucchetti, Daniela, Capusoni, Claudia, Rizzi, Aurora, Galafassi, Silvia, Chiara, Gambaro, and Compagno, Concetta
- Subjects
ACETYLCOENZYME A ,LIPID synthesis ,STARVATION ,MANUFACTURING processes ,YEAST ,NITROGEN - Abstract
Background: Oleaginous yeasts are able to accumulate very high levels of neutral lipids especially under condition of excess of carbon and nitrogen limitation (medium with high C/N ratio). This makes necessary the use of two-steps processes in order to achieve high level of biomass and lipid. To simplify the process, the decoupling of lipid synthesis from nitrogen starvation, by establishing a cytosolic acetyl-CoA formation pathway alternative to the one catalysed by ATP-citrate lyase, can be useful. Results: In this work, we introduced a new cytoplasmic route for acetyl-CoA (AcCoA) formation in Rhodosporidium azoricum by overexpressing genes encoding for homologous phosphoketolase (Xfpk) and heterologous phosphotransacetylase (Pta). The engineered strain PTAPK4 exhibits higher lipid content and produces higher lipid concentration than the wild type strain when it was cultivated in media containing different C/N ratios. In a bioreactor process performed on glucose/xylose mixture, to simulate an industrial process for lipid production from lignocellulosic materials, we obtained an increase of 89% in final lipid concentration by the engineered strain in comparison to the wild type. This indicates that the transformed strain can produce higher cellular biomass with a high lipid content than the wild type. The transformed strain furthermore evidenced the advantage over the wild type in performing this process, being the lipid yields 0.13 and 0.05, respectively. Conclusion: Our results show that the overexpression of homologous Xfpk and heterologous Pta activities in R. azoricum creates a new cytosolic AcCoA supply that decouples lipid production from nitrogen starvation. This metabolic modification allows improving lipid production in cultural conditions that can be suitable for the development of industrial bioprocesses using lignocellulosic hydrolysates. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
33. TheamrG1 gene is involved in the activation of acetate inCorynebacterium glutamicum
- Author
-
Ruan, Hong, Gerstmeir, R., Schnicke, S., and Eikmanns, B. J.
- Published
- 2005
- Full Text
- View/download PDF
34. Acetate metabolism in Geobacillus thermoglucosidasius and strain engineering for enhanced bioethanol production
- Author
-
Hills, Christopher
- Subjects
phosphotransacetylase ,Geobacillus thermoglucosidasius ,acetate ,metabolic engineering ,acetate kinase - Abstract
Social, economic and political pressures have driven the development of renewable alternatives to fossil fuels. Biofuels, such as bioethanol, have proved to be successful alternatives. Mature technologies are crop-based, but this has brought criticism due to the conflicting use of land for fuel versus food production. Therefore, bioethanol production technologies have shifted to utilising the sugars that derive from the degradation of lignocellulosic biomass. The thermophilic, Gram-positive bacterium, Geobacillus thermoglucosidasius, can naturally utilise a large fraction of these sugars, and metabolic engineering has been used to create a strain that produces ethanol as the major product of fermentation. This strain, G. thermoglucosidasius TM242 (Δldh, Δpfl, pdhup), does however, produce small but significant quantities of acetate, an undesirable by-product of fermentation. Therefore, acetate metabolism in the G. thermoglucosidasius TM242 strain was the focus of this study.During fermentation, ethanol is generated from the central metabolite acetyl-CoA through the activities of a bifunctional enzyme: aldehyde dehydrogenase/alcohol dehydrogenase (ADHE). On the other hand, acetate is generated from acetyl-CoA through catalysis by phosphotransacetylase (PTA) and acetate kinase (AK). Acetate metabolism in G. thermoglucosidasius TM242 was studied in this project by investigating the enzyme activities governing flux from acetyl-CoA, and the feasibility of reduced acetate production was investigated by a pta-deletion strategy.This thesis reports the characterisation of PTA and AK, by studying activities from both native cell lysates and recombinantly expressed proteins. The results indicate that the activities of PTA and AK are greater than those of ADHE, suggesting that the potential metabolic flux is greater towards acetate production than to ethanol. However, the ethanol yield from G. thermoglucosidasius TM242 fermentations is greater than that of acetate, suggesting the existence of a regulatory mechanism controlling acetyl-CoA flux. Several possible regulatory mechanisms were studied in this project and are reported here.The viability of creating a strain that reduces acetate accumulation, and potentially increases ethanol yields, was investigated and reported in this thesis. The gene encoding PTA was deleted from G. thermoglucosidasius TM242, and the resulting strain was characterised. The Δpta strain had approximately 5% of the PTA activity measured in TM242, but acetate was still generated from pentose and hexose fermentations. Additional phosphotransacylase (PTAC) enzymes were discovered in G. thermoglucosidasius TM242 that could catalyse the conversion of acetyl-CoA and orthophosphate to acetyl-phosphate and CoA. A series of PTAC null strains were created and analysed, the results of which indicated that phosphotransbutyrylase (PTB) could be involved in acetate production in vivo. It was discovered that the cell lysates of G. thermoglucosidasius strains carrying deletions to both pta and ptb could no longer catalyse the conversion of acetyl-CoA and orthophosphate to acetyl-phosphate and CoA. However, these strains still accumulated acetate, suggesting the presence of alternative acetate-producing pathways in this organism. In addition, G. thermoglucosidasius strains carrying deletions to both pta and ptb could ferment glucose but not xylose, suggesting that the production of ATP by the PTA-AK pathway is crucial for micro-aerobic growth on pentose sugars.
- Published
- 2014
35. Superiority of the PCR-based approach for cloning the acetate kinase gene of Clostridium thermocellum
- Author
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Arnold L. Demain, JH Kim, Lee R. Lynd, E Ozcengiz, Gülay Özcengiz, WR Lin, David J. Westenberg, and OpenMETU
- Subjects
Acetate kinase ,Hybridization probe ,Thermophilic bacteria ,Bioengineering ,Methanosarcina ,Biology ,Molecular cloning ,biology.organism_classification ,medicine.disease_cause ,Applied Microbiology and Biotechnology ,Molecular biology ,Clostridium thermocellum ,Complementation ,PCR ,Gene cloning ,Restriction map ,Biochemistry ,medicine ,Phosphotransacetylase ,Escherichia coli ,Biotechnology - Abstract
Cloning of Clostridium thermocellum acetate kinase (ack) and/or phosphotransacetylase (pta) genes in Escherichia coli by functional complementation of ack and/or pta mutants was complicated by an alternative acetate assimilation pathway involving acetyl-CoA synthetase (ACS). In addition to the problems encountered with the complementation approach, cloning of these genes was not readily achieved using heterologous probing with corresponding genes from Escherichia coli and Methanosarcina thermophila due to the lack of sufficient homology. The use of a PCR-based approach, on the other hand, yielded a specific C. thermocellum gene fragment which showed significant sequence identity to the ack gene for which primers were designed. The subcloned ack fragment was then successfully used as a probe for the isolation of the corresponding gene and restriction analysis of that region.
- Published
- 1998
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36. Establishing an innovative carbohydrate metabolic pathway for efficient production of 2-keto-l-gulonic acid in <italic>Ketogulonicigenium robustum</italic> initiated by intronic promoters.
- Author
-
Wang, Cai-Yun, Li, Ye, Gao, Zi-Wei, Liu, Li-Cheng, Zhang, Meng-Yue, Zhang, Tian-Yuan, Wu, Chun-Fu, and Zhang, Yi-Xuan
- Subjects
CARBOHYDRATE metabolism ,VITAMIN C ,BACILLUS megaterium ,RIBOSOMAL RNA ,GENE expression ,CELL communication - Abstract
Background: 2-Keto-l-gulonic acid (2-KGA), the precursor of vitamin C, is currently produced by two-step fermentation. In the second step, l-sorbose is transformed into 2-KGA by the symbiosis system composed of
Ketogulonicigenium vulgare andBacillus megaterium . Due to the different nutrient requirements and the uncertain ratio of the two strains, the symbiosis system significantly limits strain improvement and fermentation optimization. Results: In this study,Ketogulonicigenium robustum SPU_B003 was reported for its capability to grow well independently and to produce more 2-KGA than that ofK. vulgare in a mono-culture system. The complete genome ofK. robustum SPU_B003 was sequenced, and the metabolic characteristics were analyzed. Compared to the four reportedK. vulgare genomes,K. robustum SPU_B003 contained more tRNAs, rRNAs, NAD and NADP biosynthetic genes, as well as regulation- and cell signaling-related genes. Moreover, the amino acid biosynthesis pathways were more complete. Two species-specific internal promoters, P1 (orf_01408 promoter) and P2 (orf_02221 promoter), were predicted and validated by detecting their initiation activity. To efficiently produce 2-KGA with decreased CO2 release, an innovative acetyl-CoA biosynthetic pathway (XFP-PTA pathway) was introduced intoK. robustum SPU_B003 by expressing heterologous phosphoketolase (xfp ) and phosphotransacetylase (pta ) initiated by internal promoters. After gene optimization, the recombinant strainK. robustum /pBBR-P1_xfp2502 -P2_pta2145 enhanced acetyl-CoA approximately 2.4-fold and increased 2-KGA production by 22.27% compared to the control strainK. robustum /pBBR1MCS-2. Accordingly, the transcriptional level of the 6-phosphogluconate dehydrogenase (pgd ) and pyruvate dehydrogenase genes (pdh ) decreased by 24.33 ± 6.67 and 8.67 ± 5.51%, respectively. The key genes responsible for 2-KGA biosynthesis, sorbose dehydrogenase gene (sdh ) and sorbosone dehydrogenase gene (sndh ), were up-regulated to different degrees in the recombinant strain. Conclusions: The genome-based functional analysis ofK. robustum SPU_B003 provided a new understanding of the specific metabolic characteristics. The new XFP-PTA pathway was an efficient route to enhance acetyl-CoA levels and to therefore promote 2-KGA production. [ABSTRACT FROM AUTHOR]- Published
- 2018
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37. Effect of the inactivation of lactate dehydrogenase, ethanol dehydrogenase, and phosphotransacetylase on 2,3-butanediol production in Klebsiella pneumoniae strain
- Author
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Cuiying Zhang, Chaoqun Li, Dongguang Xiao, Yefu Chen, Yazhou Wang, Chun-Hong Cao, Huadong Pei, Xuewu Guo, and Mingyue Wu
- Subjects
biology ,Renewable Energy, Sustainability and the Environment ,Klebsiella pneumoniae ,Acetoin ,Research ,Mutant ,phosphotransacetylase ,Aldehyde dehydrogenase ,lactate dehydrogenase ,2,3-butanediol ,Management, Monitoring, Policy and Law ,biology.organism_classification ,Applied Microbiology and Biotechnology ,chemistry.chemical_compound ,General Energy ,Biochemistry ,chemistry ,Butanediol ,Lactate dehydrogenase ,2,3-Butanediol ,biology.protein ,Fermentation ,ethanol dehydrogenase ,Biotechnology - Abstract
Background 2,3-Butanediol (2,3-BD) is a high-value chemical usually produced petrochemically but which can also be synthesized by some bacteria. To date, Klebsiella pneumoniae is the most powerful 2,3-BD producer which can utilize a wide range of substrates. However, many by-products are also produced by K. pneumoniae, such as ethanol, lactate, and acetate, which negatively regulate the 2,3-BD yield and increase the costs of downstream separation and purification. Results In this study, we constructed K. pneumoniae mutants with lactate dehydrogenase (LDH), acetaldehyde dehydrogenase (ADH), and phosphotransacetylase (PTA) deletion individually by suicide vector conjugation. These mutants showed different behavior of production formation. Knock out of ldhA had little influence on the yield of 2,3-BD, whereas knock out of adhE or pta significantly improved the formation of 2,3-BD. The accumulation of the intermediate of 2,3-BD biosynthesis, acetoin, was decreased in all the mutants. The mutants were then tested in five different carbon sources and increased 2,3-BD was observed. Also a double mutant strain with deletion of adhE and ldhA was constructed which resulted in accelerated fermentation and higher 2,3-BD production. In fed-batch culture this strain achieved more than 100 g/L 2,3-BD from glucose with a relatively high yield of 0.49 g/g. Conclusion 2,3-BD production was dramatically improved with the inactivation of adhE and pta. The inactivation of ldhA could advance faster cell growth and shorter fermentation time. The double mutant strain with deletion of adhE and ldhA resulted in accelerated fermentation and higher 2,3-BD production. These results provide new insights for industrial production of 2,3-BD by K. pneumoniae.
- Published
- 2013
38. TaqMan RT-qPCR targeting the pta gene for Clostridium tyrobutyricum quantification in animal feed, faeces, milk and cheese
- Author
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Bassi, Daniela
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Real time ,cheese ,milk ,phosphotransacetylase ,Settore AGR/16 - MICROBIOLOGIA AGRARIA ,Clostridium tyrobutyricum ,Late blowing - Published
- 2013
39. Fermentative Acetatproduktion durch Homoacetat-Gärung bzw. Acetacetatbildung
- Author
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Straub, Melanie
- Subjects
CO2/H2 ,Acetatkinase ,Acetate ,Acetogene Bakterien ,2,3-Butandiol ,Acetates ,Formation ,Clostridium aceticum ,Wood-Ljungdahl-Weg ,DDC 570 / Life sciences ,Clostridium ljungdahlii ,ddc:570 ,Fermentation ,Phosphotransacetylase ,Acetobacterium woodii ,Clostridium acetobutylicum - Abstract
Für die gentechnische Veränderung von acetogenen Bakterien, wie Clostridium ljungdahlii, Acetobakterium woodii oder Clostridium aceticum, wurde eine Transformationsmethode entwickelt. Durch Überexpression der Gene, welche für die Acetat-Kinase und die Phosphotransacetylase, bzw. der Gene, welche für die Formyl-THF-Synthetase, die Methenyl-THF-Cyclohydrolase, die Methylen-THF-Dehydrogenase und die Methylen-THF-Reduktase des Wood-Ljungdahl-Wegs kodieren, konnte die Acetatproduktion dieser Organismen bei Wachstum auf CO2/H2 gesteigert werden. Durch Inaktivierung der Gene hbd (3-Hydroxybutyryl-CoA-Dehydrogenase) oder crt (Crotonase) in C. acetobutylicum wurde eine erhöhte Ethanolproduktion nachgewiesen. Durch die Inaktivierung des Gens hbd konnte eine Steigerung in der Acetatproduktion beobachtet werden. Eine Überexpression der Gene thlA (Thiolase A) und ctfA/B (Acetacetyl-CoA: Acetat/Butyrat-CoA-Transferase) in der hbd-Integrationsmutante führte ebenfalls zu einer Steigerung in der Acetatproduktion. Die Expression des Gens für die Acetoinreduktase aus C. beijerinkcii in C. acetobutylicum WT bzw. der hbd-Integrationsmutante führte zur Produktion von 2,3-Butandiol.
- Published
- 2012
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40. Analysis of Redox Responses During TNT Transformation by Clostridium acetobutylicum ATCC 824 and Mutants Exhibiting Altered Metabolism
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RICE UNIV HOUSTON TX DEPT OF BIOCHEMISTRY AND CELL BIOLOGY, Cai, Xianpeng, Servinsky, Matthew, Kiel, James, Sund, Christian, Bennett, George N, RICE UNIV HOUSTON TX DEPT OF BIOCHEMISTRY AND CELL BIOLOGY, Cai, Xianpeng, Servinsky, Matthew, Kiel, James, Sund, Christian, and Bennett, George N
- Abstract
The transformation of trinitrotoluene (TNT) by several mutant strains of Clostridium acetobutylicum has been examined to analyze the maximal rate of initial transformation, determine the effects of metabolic mutations of the host on transformation rate and to assess the cell metabolic changes brought about during TNT transformation. Little difference in the maximal rate of TNT degradation in early acid phase cultures was found between the parental ATCC824 strain and strains altered in the acid forming pathways (phosphotransacetylase, or butyrate kinase) or in a high solvent producing strain (mutant B). This result is in agreement with the previous findings of a similar degradation rate in a degenerate strain, (M5) that had lost the ability to produce solvent. A series of antisense constructs were made that reduced the expression of hydA, encoding the Fe-hydrogenase, or hydE and hydF, genes encoding hydrogenase maturating proteins. While the antisense hydA strain had only 30% of the activity of wild type, the antisense hydE strain exhibited a TNT degradation rate around 70% that of the parent. Over expression of hydA modestly increased the TNT degradation rate in acid phase cells, suggesting the amount of reductant flowing into hydrogenase rather than the hydrogenase level itself was a limiting factor in many situations. The redox potential, hydrogen evolution and organic acid metabolites produced during rapid TNT transformation in early log phase cultures were measured. The redox potential of the acid producing culture decreased from -370 to -200 mV immediately after addition of TNT and the hydrogen evolution rate decreased, lowering the hydrogen to carbon dioxide ratio from 1.4 to around 1.1 for 15 minutes. During the time of TNT transformation, the treated acidogenic cells produced less acetate and more butyrate., Published in the Journal of Applied Microbiology and Biotechnology, p1-15, June 2012. The original document contains color images.
- Published
- 2012
41. Pyruvate formate-lyase and a novel route of eukaryotic ATP-synthesis in Chlamydomonas mitochondria
- Author
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Ariane Atteia, Jérôme Garin, Jacques Joyard, Norbert Rolland, Annie Adrait, Robert van Lis, Gabriel Gelius-Dietrich, William Martin, Martin-Laffon, Jacqueline, Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institute of Botany, Heinrich Heine Universität Düsseldorf = Heinrich Heine University [Düsseldorf], Développement de la protéomique comme outil d'investigation fonctionelle et d'annotation des génomes, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA), Heinrich-Heine-Universität Düsseldorf [Düsseldorf], Institut de biologie et chimie des protéines [Lyon] (IBCP), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Université Joseph Fourier - Grenoble 1 (UJF), Laboratoire d'étude de la dynamique des protéomes (LEDyP), Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Institut d’Électronique, de Microélectronique et de Nanotechnologie (IEMN) - UMR 8520 (IEMN), and Centre National de la Recherche Scientifique (CNRS)-Université de Lille-Université Polytechnique Hauts-de-France (UPHF)-Ecole Centrale de Lille-Université Polytechnique Hauts-de-France (UPHF)-Institut supérieur de l'électronique et du numérique (ISEN)
- Subjects
Chloroplasts ,Pyruvate Synthase ,Chlamydomonas reinhardtii ,Pyruvate dehydrogenase phosphatase ,Biochemistry ,Adenosine Triphosphate ,darkness ,carbon energy metabolism ,eukaryote ,bifunctional aldehyde/alcohol dehydrogenase ,Phylogeny ,ComputingMilieux_MISCELLANEOUS ,mass spectrometry ,Expressed Sequence Tags ,algae ,0303 health sciences ,Acetate kinase ,intracellular localization ,biology ,Pyruvate dehydrogenase complex ,Chloroplast ,mitochondria ,immunoblotting ,pyruvate formate lyase ,Pyruvate decarboxylation ,DNA, Complementary ,Pyruvate dehydrogenase kinase ,Molecular Sequence Data ,Pyruvate Dehydrogenase Complex ,Biosynthesis ,Models, Biological ,acetate kinase ,03 medical and health sciences ,proteomics ,Acetyltransferases ,[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Animals ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Amino Acid Sequence ,RNA, Messenger ,Molecular Biology ,Gene Library ,030304 developmental biology ,Sequence Homology, Amino Acid ,030306 microbiology ,Chlamydomonas ,phosphotransacetylase ,anaerobiosis ,Cell Biology ,metabolic pathway ,biology.organism_classification ,Formate ,ATP ,Models, Chemical ,Enzyme ,RNA ,Acetylesterase ,Peptides - Abstract
Pyruvate formate-lyase (PFL) catalyzes the non-oxidative conversion of pyruvate to formate and acetyl-CoA. PFL and its activating enzyme (PFL-AE) are common among strict anaerobic and microaerophilic prokaryotes but are very rare among eukaryotes. In a proteome survey of isolated Chlamydomonas reinhardtii mitochondria, we found several PFL-specific peptides leading to the identification of cDNAs for PFL and PFL-AE, establishing the existence of a PFL system in this photosynthetic algae. Anaerobiosis and darkness led to increased PFL transcripts but had little effect on protein levels, as determined with antiserum raised against C. reinhardtii PFL. Protein blots revealed the occurrence of PFL in both chloroplast and mitochondria purified from aerobically grown cells. Mass spectrometry sequencing of C. reinhardtii mitochondrial proteins, furthermore, identified peptides for phosphotransacetylase and acetate kinase. The phosphotransacetylase-acetate kinase pathway is a common route of ATP synthesis or acetate assimilation among prokaryotes but is novel among eukaryotes. In addition to PFL and pyruvate dehydrogenase, the algae also expresses pyruvate:ferredoxin oxidoreductase and bifunctional aldehyde/alcohol dehydrogenase. Among eukaryotes, the oxygen producer C. reinhardtii has the broadest repertoire of pyruvate-, ethanol-, and acetate-metabolizing enzymes described to date, many of which were previously viewed as specific to anaerobic eukaryotic lineages.
- Published
- 2006
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42. PHOSPHOTRANSACETYLASE AND XYLULOSE 5-PHOSPHATE/FRUCTOSE 6-PHOSPHATE PHOSPHOKETOLASE: TWO EUKARYOTIC PARTNERS OF ACETATE KINASE
- Author
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Taylor, Tonya
- Subjects
- acetate, acetate kinase, Cryptococcus neoformans, phosphoketolase, phosphotransacetylase, Phytophthora ramorum, Biochemistry, Genetics and Genomics, Microbiology
- Abstract
Although acetate is a predominant metabolite produced by many eukaryotic microbes, far less attention has been given to acetate metabolism in eukaryotes than in bacteria and archaea. Acetate kinase (Ack), which catalyzes the reversible phosphorylation of acetate from ATP, is a key enzyme in bacterial acetate metabolism. Ack primarily partners with phosphotransacetylase (Pta), which catalyzes the generation of acetyl phosphate from acetyl-CoA, but can also partner with xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp), which produces acetyl phosphate from either xylulose 5-phosphate or fructose 6-phosphate. The Ack-Pta pathway, found primarily in bacteria, is also present in lower eukaryotes such as the green algae Chlamydomonas reinhardtii and the oomycete, Phytophthora. The Ack-Xfp pathway, which forms a modified pentose phosphoketolase pathway in heterofermentative bacteria, has been found in a number of ascomycete and basidiomycete fungi. Although bacterial and eukaryotic microbes possess these pathways, humans, animals and plants lack these enzymes, making this pathway a potential drug target in eukaryotic pathogens. Two types of Ptas have previously been identified: PtaI and PtaII. PtaII enzymes have an N-terminal regulatory domain that the PtaI enzymes lack. Through sequence analysis, we identified four subtypes, IIa, IIb, IIc, and IId, of the PtaII enzymes based on the presence or absence of two N-terminal subdomains. Here we describe the first biochemical characterization of a eukaryotic Pta, the Phytophthora ramorum Type IIa Pta1 (PrPta1IIa). Although the N-terminus of PrPta1IIa shares only 19% amino acid identity with the N-terminus of the bacterial Escherichia coli and Salmonella enterica PtaIIa enzymes, the effector molecules, ATP, NADH, PEP, and pyruvate, inhibit all three enzymes in the acetyl-CoA-forming direction; whereas, AMP differentially regulates PrPta1IIa compared to SePtaIIa. We hypothesize that Xfp-Ack would function as a modified pentose phosphoketolase pathway to produce acetate and ATP in the opportunistic, fungal pathogen Cryptococcus neoformans, which has two open reading frames, designated as Xfp1 and Xfp2, with sequence identity to Xfp. To investigate the metabolic and physiological role of the Ack-Xfp pathway in C. neoformans, we have generated single XFP1, XFP2 and ACK knockouts, as well as a XFP1/XFP2 double knockout. Our results indicate both Xfp1 and Xfp2 play a role in the survival of C. neoformans within macrophages, and that Ack and Xfp2 most likely partner together under low glucose and possibly low iron environments.
- Published
- 2015
43. Catabolite regulation analysis of Escherichia coli for acetate overflow mechanism and co-consumption of multiple sugars based on systems biology approach using computer simulation.
- Author
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Matsuoka Y and Shimizu K
- Subjects
- Catabolite Repression, Citric Acid Cycle, Escherichia coli genetics, Feedback, Physiological, Fermentation, Fructosediphosphates metabolism, Gene Knockout Techniques, Glycolysis, Models, Biological, Pentose Phosphate Pathway, Phosphoenolpyruvate metabolism, Phosphotransferases genetics, Phosphotransferases metabolism, Pyruvate Kinase genetics, Pyruvate Kinase metabolism, Acetates metabolism, Computer Simulation, Escherichia coli metabolism, Glucose metabolism, Systems Biology methods, Xylose metabolism
- Abstract
It is quite important to understand the basic principle embedded in the main metabolism for the interpretation of the fermentation data. For this, it may be useful to understand the regulation mechanism based on systems biology approach. In the present study, we considered the perturbation analysis together with computer simulation based on the models which include the effects of global regulators on the pathway activation for the main metabolism of Escherichia coli. Main focus is the acetate overflow metabolism and the co-fermentation of multiple carbon sources. The perturbation analysis was first made to understand the nature of the feed-forward loop formed by the activation of Pyk by FDP (F1,6BP), and the feed-back loop formed by the inhibition of Pfk by PEP in the glycolysis. Those together with the effect of transcription factor Cra caused by FDP level affected the glycolysis activity. The PTS (phosphotransferase system) acts as the feed-back system by repressing the glucose uptake rate for the increase in the glucose uptake rate. It was also shown that the increased PTS flux (or glucose consumption rate) causes PEP/PYR ratio to be decreased, and EIIA-P, Cya, cAMP-Crp decreased, where cAMP-Crp in turn repressed TCA cycle and more acetate is formed. This was further verified by the detailed computer simulation. In the case of multiple carbon sources such as glucose and xylose, it was shown that the sequential utilization of carbon sources was observed for wild type, while the co-consumption of multiple carbon sources with slow consumption rates were observed for the ptsG mutant by computer simulation, and this was verified by experiments. Moreover, the effect of a specific gene knockout such as Δpyk on the metabolic characteristics was also investigated based on the computer simulation., (Copyright © 2013 Elsevier B.V. All rights reserved.)
- Published
- 2013
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44. ack::Mu d1-8 (Apr lac) operon fusions of Salmonella typhimurium LT2
- Author
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Kwan, H. S., Chui, H. W., and Wong, K. K.
- Published
- 1988
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45. Allicin, a naturally occurring antibiotic from garlic, specifically inhibits acetyl-CoA synthetase
- Author
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Manfred Focke, Hartmut K. Lichtenthaler, and Andrea Feld
- Subjects
Chloroplasts ,Dithioerythritol ,(Garlic) ,Biophysics ,Acetate-CoA Ligase ,Acetates ,Biology ,Acetyl-CoA synthetase ,Biochemistry ,chemistry.chemical_compound ,Structural Biology ,Yeasts ,Coenzyme A Ligases ,Genetics ,Animals ,Phosphotransacetylase ,Disulfides ,Molecular Biology ,Fatty acid synthesis ,chemistry.chemical_classification ,Acetate kinase ,Allicin ,Bacteria ,Dose-Response Relationship, Drug ,Myocardium ,Fatty Acids ,Cell Biology ,Acetyl—CoA synthetase ,Metabolism ,Sulfinic Acids ,Yeast ,Enzyme ,chemistry ,Cattle - Abstract
Allicin is shown to be a specific inhibitor of the acetyl-CoA synthetases from plants, yeast and mammals. The bacterial acetyl-CoA-forming system, consisting of acetate kinase and phosphotransacetylase, was inhibited too. Non-specific interaction with sulfhydryl-groups could be excluded in experiments with dithioerythritol and P-hydroxymercuribenzoate. Binding of allicin to the enzyme is non-covalent and reversible. [14C]-Acetate incorporation into fatty acids of isolated plastids was inhibited by allicin with an I50-value lower than 10 μM. Other enzymes of the fatty acid synthesis sequence were not affected, as was shown using precursors other than acetate.
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46. Regulation of acetate metabolism in Corynebacterium glutamicum: transcriptional control of the isocitrate lyase and malate synthase genes
- Author
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Bernhard J. Eikmanns, Dieter J. Reinscheid, Volker F. Wendisch, Hermann Sahm, Marion Spies, and Stephanie Schnicke
- Subjects
Transcription, Genetic ,Restriction Mapping ,Succinic Acid ,acetyl phosphate ,Acetates ,Biochemistry ,Corynebacterium glutamicum ,Phosphate Acetyltransferase ,chemistry.chemical_compound ,malate synthase ,sequence-analysis ,Cloning, Molecular ,chemistry.chemical_classification ,Acetate kinase ,glyoxylate ,biology ,Acetyl-CoA ,General Medicine ,Lactates ,clostridium-acetobutylicum ,Plasmids ,acetate metabolism ,Glyoxylate cycle ,Glutamic Acid ,Corynebacterium ,Microbiology ,Gene Expression Regulation, Enzymologic ,acetate kinase ,Transformation, Genetic ,Acetyl Coenzyme A ,Malate synthase ,expression ,Genetics ,biochemical-characterization ,acetyl-phosphate ,Phosphate acetyltransferase ,Molecular Biology ,phosphotransacetylase ,acetyl-coa ,Gene Expression Regulation, Bacterial ,Isocitrate lyase ,Blotting, Northern ,isocitrate lyase ,Molecular biology ,Artificial Gene Fusion ,carbon flux ,enzyme ,Glucose ,Enzyme ,chemistry ,biology.protein ,escherichia-coli ,corynebacterium glutamicum - Abstract
In the amino-acid-producing microorganism Corynebacterium glutamicum, the specific activities of the acetate-activating enzymes acetate kinase and phosphotransacetylase and those of the glyoxylate cycle enzymes isocitrate lyase and malate synthase were found to be high when the cells were grown on acetate (0.8, 2.9, 2.1, and 1.8 U/mg protein, respectively). When the cells were grown on glucose or on other carbon sources such as lactate, succinate, or glutamate, the specific activities were two- to fourfold (acetate kinase and phosphotransacetylase) and 45- to 100-fold (isocitrate lyase and malate synthase) lower, indicating that the synthesis of the four enzymes is regulated by acetate in the growth medium. A comparative Northern (RNA) analysis of the C. glutamicum isocitrate lyase and malate synthase genes (aceA and aceB) and transcriptional cat fusion experiments revealed that aceA and aceB are transcribed as 1.6- and 2.7-kb monocistronic messages, respectively, and that the regulation of isocitrate lyase and malate synthase synthesis is exerted at the level of transcription from the respective promoters. Surprisingly, C. glutamicum mutants defective in either acetate kinase or phosphotransacetylase showed low specific activities of the other three enzymes (phosphotransacetylase, isocitrate lyase, and malate synthase or acetate kinase, isocitrate lyase, and malate synthase, respectively) irrespective of the presence or absence of acetate in the medium. This result and a correlation of a high intracellular acetyl coenzyme A concentration with high specific activities of isocitrate lyase, malate synthase, acetate kinase, and phosphotransacetylase suggest that acetyl coenzyme A or a derivative thereof may be a physiological trigger for the genetic regulation of enzymes involved in acetate metabolism of C. glutamicum.
47. Enzymic Synthesis and Cofactor Activity of 3 ′ -pyrophosphocoenzyme A
- Author
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Mukai, Jun-Ichiro, Sy, Jose, and Lipmann, Fritz
- Published
- 1983
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