Succinate CoA Transferase Research Articles

Contribution of Uncharacterized Target Genes of MxtR/ErdR to Carbon Source Utilization by Pseudomonas putida KT2440.

MxtR/ErdR is a two-component system that has been previously described as a regulator of the utilization of acetate in Vibrio cholerae and in some Pseudomonas species. Regulation is achieved by controlling the expression of the acs gene (acetyl-coenzyme A [CoA] synthetase). However, the physiological significance of other identified target genes is not fully understood. Here, we investigated the role of pp_0154 (scpC) and pp_0354/pp_0353 in the soil bacterium Pseudomonas putida KT2440. To this end, the genes were individually deleted and complemented in trans. Then, the growth of the resulting strains on different carbon sources was analyzed. To obtain information on protein function, a bioinformatic analysis was performed, and ScpC was purified and characterized in vitro. Our results indicated that scpC is important for P. putida KT2440 to cope with high concentrations of acetate. The encoded enzyme catalyzes the transfer of coenzyme A between acetate and succinate. On the contrary, pp_0353 and pp_0354 proved to be unimportant for the growth of the strain on acetate under our conditions. Extending the phenotypic analysis to other carbon sources led to the discovery that mxtR, erdR, and pp_0353 are important for the utilization of pyruvate as a carbon source. Taken together, the findings of this study expand the knowledge about the role of the MxtR/ErdR two-component system in carbon source utilization and about the specific functions of its target genes. IMPORTANCE MxtR/ErdR and homologous two-component systems play important roles in the regulatory networks that control cell metabolism and influence bacterial-host interactions. Using the MxtR/ErdR two-component system of the plant growth-promoting soil bacterium Pseudomonas putida KT2440 as a model, this work elucidates the function of previously uncharacterized target genes of MxtR/ErdR and extends the knowledge of the physiological significance of the two-component system. Our results suggest that the target gene scpC encodes an acetate:succinate CoA transferase that is involved in the detoxification of acetate when it is present in large amounts. Furthermore, it is shown that MxtR/ErdR controls the metabolism of not only acetate but also pyruvate. This control involves the target gene pp_0353 (putative exonuclease). These findings may facilitate the optimization of P. putida KT2440 as a chassis for biotechnological applications and may contribute to a better understanding of the regulatory network of pathogens like Pseudomonas aeruginosa.

Bioproduction of propionic acid using levulinic acid by engineered Pseudomonas putida.

The present study elaborates on the propionic acid (PA) production by the well-known microbial cell factory Pseudomonas putida EM42 and its capacity to utilize biomass-derived levulinic acid (LA). Primarily, the P. putida EM42 strain was engineered to produce PA by deleting the methylcitrate synthase (PrpC) and propionyl-CoA synthase (PrpE) genes. Subsequently, a LA-inducible expression system was employed to express yciA (encoding thioesterase) from Haemophilus influenzae and ygfH (encoding propionyl-CoA: succinate CoA transferase) from Escherichia coli to improve the PA production by up to 10-fold under flask scale cultivation. The engineered P. putida EM42:ΔCE:yciA:ygfH was used to optimize the bioprocess to further improve the PA production titer. Moreover, the fed-batch fermentation performed under optimized conditions in a 5 L bioreactor resulted in the titer, productivity, and molar yield for PA production of 26.8 g/L, 0.3 g/L/h, and 83%, respectively. This study, thus, successfully explored the LA catabolic pathway of P. putida as an alternative route for the sustainable and industrial production of PA from LA.

Coupled synthetic pathways improve the production of 3-hydroxypropionic acid in recombinant Escherichia coli strains

To improve the production of 3-HP with glucose as a substrate, the malonyl-CoA and propionyl-CoA pathways were coupled to regulate NADP+/NADPH regeneration in the recombinant E. coli. The strain Ec-AM that overexpressed the key enzymes of the malonyl-CoA pathway, acetyl CoA carboxylase (ACC) from Ustilago maydis and malonyl CoA reductase (MCR) from Chloroflexus aurantiacus, produced 0.26 g/L of 3-HP in 25-h shake flask cultivation. The strain Ec-P overexpressing the key enzyme of the propionyl-CoA pathway, propionyl-CoA dehydrogenase (PACD) from Candida rugosa, produced 0.11 g/L of 3-HP. However, 3-HP titer of the strain Ec-PAM overexpressing PACD along with ACC and MCR, via two pathways cooperation, was 1.29 g/L. The addition of biotin and bicarbonate improved the 3-HP production of the strain Ec-PAM. 3-HP titer of strain Ec-ΔY-ΔP-PAM with double deletion of ygfH (encoding propionyl-CoA: succinate-CoA transferase) and prpC (encoding methylcitrate synthase) genes reached 1.94 g/L, which was 1.5-fold higher than that of the strain Ec-PAM cultured under the same conditions.

The ASCT/SCS cycle fuels mitochondrial ATP and acetate production in Trypanosoma brucei

Acetate:succinate CoA transferase (ASCT) is a mitochondrial enzyme that catalyzes the production of acetate and succinyl-CoA, which is coupled to ATP production with succinyl-CoA synthetase (SCS) in a process called the ASCT/SCS cycle. This cycle has been studied in Trypanosoma brucei (T. brucei), a pathogen of African sleeping sickness, and is involved in (i) ATP and (ii) acetate production and proceeds independent of oxygen and an electrochemical gradient. Interestingly, knockout of ASCT in procyclic form (PCF) of T. brucei cause oligomycin A-hypersensitivity phenotype indicating that ASCT/SCS cycle complements the deficiency of ATP synthase activity. In bloodstream form (BSF) of T. brucei, ATP synthase works in reverse to maintain the electrochemical gradient by hydrolyzing ATP. However, no information has been available on the source of ATP, although ASCT/SCS cycle could be a potential candidate. Regarding mitochondrial acetate production, which is essential for fatty acid biosynthesis and growth of T. brucei, ASCT or acetyl-CoA hydrolase (ACH) are known to be its source. Despite the importance of this cycle, direct evidence of its function is lacking, and there are no comprehensive biochemical or structural biology studies reported so far. Here, we show that in vitro–reconstituted ASCT/SCS cycle is highly specific towards acetyl-CoA and has a higher kcat than that of yeast and bacterial ATP synthases. Our results provide the first biochemical basis for (i) rescue of ATP synthase-deficient phenotype by ASCT/SCS cycle in PCF and (ii) a potential source of ATP for the reverse reaction of ATP synthase in BSF.

Metabolic engineering of Escherichia coli for the synthesis of polyhydroxyalkanoates using acetate as a main carbon source

BackgroundHigh production cost of bioplastics polyhydroxyalkanoates (PHA) is a major obstacle to replace traditional petro-based plastics. To address the challenges, strategies towards upstream metabolic engineering and downstream fermentation optimizations have been continuously pursued. Given that the feedstocks especially carbon sources account up to a large portion of the production cost, it is of great importance to explore low cost substrates to manufacture PHA economically.ResultsEscherichia coli was metabolically engineered to synthesize poly-3-hydroxybutyrate (P3HB), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4HB), and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) using acetate as a main carbon source. Overexpression of phosphotransacetylase/acetate kinase pathway was shown to be an effective strategy for improving acetate assimilation and biopolymer production. The recombinant strain overexpressing phosphotransacetylase/acetate kinase and P3HB synthesis operon produced 1.27 g/L P3HB when grown on minimal medium supplemented with 10 g/L yeast extract and 5 g/L acetate in shake flask cultures. Further introduction succinate semialdehyde dehydrogenase, 4-hydroxybutyrate dehydrogenase, and CoA transferase lead to the accumulation of P3HB4HB, reaching a titer of 1.71 g/L with a 4-hydroxybutyrate monomer content of 5.79 mol%. When 1 g/L of α-ketoglutarate or citrate was added to the medium, P3HB4HB titer increased to 1.99 and 2.15 g/L, respectively. To achieve PHBV synthesis, acetate and propionate were simultaneously supplied and propionyl-CoA transferase was overexpressed to provide 3-hydroxyvalerate precursor. The resulting strain produced 0.33 g/L PHBV with a 3-hydroxyvalerate monomer content of 6.58 mol%. Further overexpression of propionate permease improved PHBV titer and 3-hydroxyvalerate monomer content to 1.09 g/L and 10.37 mol%, respectively.ConclusionsThe application of acetate as carbon source for microbial fermentation could reduce the consumption of food and agro-based renewable bioresources for biorefineries. Our proposed metabolic engineering strategies illustrate the feasibility for producing polyhydroxyalkanoates using acetate as a main carbon source. Overall, as an abundant and renewable resource, acetate would be developed into a cost-effective feedstock to achieve low cost production of chemicals, materials, and biofuels.

Metabolic engineering of Escherichia coli for the synthesis of the quadripolymer poly(glycolate-co-lactate-co-3-hydroxybutyrate-co-4-hydroxybutyrate) from glucose

Escherichia coli was metabolically engineered to effectively produce a series of biopolymers consisted of four types of monomers including glycolate, lactate, 3-hydroxybutyrate and 4-hydroxybutyrate from glucose as the carbon source. The biosynthetic route of novel quadripolymers was achieved by the overexpression of a range of homologous and heterologous enzymes including isocitrate lyase, isocitrate dehydrogenase kinase/phosphatase, glyoxylate/hydroxypyruvate reductase, propionyl-CoA transferase, β-ketothiolase, acetoacetyl-CoA reductase, succinate semialdehyde dehydrogenase, 4-hydroxybutyrate dehydrogenase, CoA transferase and PHA synthase. In shake flask cultures using Luria-Bertani medium supplemented with glucose, the recombinant E. coli reached 7.10g/l cell dry weight with 52.60wt% biopolymer content. In bioreactor study, the final cell dry weight was 19.61g/l, containing 14.29g/l biopolymer. The structure of the produced polymer was chemically characterized by proton NMR analysis. Assessment of thermal and mechanical properties demonstrated that the quadripolymer possessed decreased crystallinity and improved toughness, in comparison to poly-3-hydroxybutyrate homopolymer. This is the first study reporting efficient microbial production of the quadripolymer poly(glycolate-co-lactate-co-3-hydroxybutyrate-co-4-hydroxybutyrate) from glucose.

Convergent evolution of a modified, acetate-driven TCA cycle in bacteria.

The tricarboxylic acid (TCA) cycle is central to energy production and biosynthetic precursor synthesis in aerobic organisms. There exist few known variations of a complete TCA cycle, with the common notion being that the enzymes involved have already evolved towards optimal performance. Here, we present evidence that an alternative TCA cycle, in which acetate:succinate CoA-transferase (ASCT) replaces the enzymatic step typically performed by succinyl-CoA synthetase (SCS), has arisen in diverse bacterial groups, including microbial symbionts of animals such as humans and insects.

Production of 3-Hydroxypropionic Acid via the Propionyl-CoA Pathway Using Recombinant Escherichia coli Strains.

Our study aimed to produce the commercially promising platform chemical 3-hydroxypropionic acid (3-HP) via the propionyl-CoA pathway in genetically engineered Escherichia coli. Recombinant E. coli Ec-P overexpressing propionyl-CoA dehydrogenase (PACD, encoded by the pacd gene from Candida rugosa) under the T7 promoter produced 1.33 mM of 3-HP in a shake flask culture supplemented with 0.5% propionate. When propionate CoA-transferase (PCT, encoded by the pct gene from Megasphaera elsdenii) and 3-hydroxypropionyl-CoA dehydratase (HPCD, encoded by the hpcd gene from Chloroflexus aurantiacus) were expressed along with PACD, the 3-HP titer of the resulting E. coli Ec-PPH strain was improved by 6-fold. The effect of the cultivation conditions on the 3-HP yield from propionate in the Ec-PPH strain was also investigated. When cultured at 30°C with 1% glucose in addition to propionate, 3-HP production by Ec-PPH increased 2-fold and 12-fold compared to the cultivation at 37°C (4.23 mM) or without glucose (0.68 mM). Deletion of the ygfH gene encoding propionyl-CoA: succinate CoA-transferase from Ec-PPH (resulting in the strain Ec-△Y-PPH) led to increase of 3-HP production in shake flask experiments (15.04 mM), whereas the strain Ec-△Y-PPH with deletion of the prpC gene (encoding methylcitrate synthase in the methylcitrate cycle) produced 17.76 mM of 3-HP. The strain Ec-△Y-△P-PPH with both ygfH and prpC genes deleted produced 24.14 mM of 3-HP, thus showing an 18-fold increase in the 3-HP titer in compare to the strain Ec-P.

Metabolic Capacity of Mitochondrion-related Organelles in the Free-living Anaerobic Stramenopile Cantina marsupialis

Functionally and morphologically degenerate mitochondria, so-called mitochondrion-related organelles (MROs), are frequently found in eukaryotes inhabiting hypoxic or anoxic environments. In the last decade, MROs have been discovered from a phylogenetically broad range of eukaryotic lineages and these organelles have been revealed to possess diverse metabolic capacities. In this study, the biochemical characteristics of an MRO in the free-living anaerobic protist Cantina marsupialis, which represents an independent lineage in stramenopiles, were inferred based on RNA-seq data. We found transcripts for proteins known to function in one form of MROs, the hydrogenosome, such as pyruvate:ferredoxin oxidoreductase, iron-hydrogenase, acetate:succinate CoA-transferase, and succinyl-CoA synthase, along with transcripts for acetyl-CoA synthetase (ADP-forming). These proteins possess putative mitochondrial targeting signals at their N-termini, suggesting dual ATP generation systems through anaerobic pyruvate metabolism in Cantina MROs. In addition, MROs in Cantina were also shown to share several features with canonical mitochondria, including amino acid metabolism and an "incomplete" tricarboxylic acid cycle. Transcripts for all four subunits of complex II (CII) of the electron transport chain were detected, while there was no evidence for the presence of complexes I, III, IV, or F1Fo ATPase. Cantina MRO biochemistry challenges the categories of mitochondrial organelles recently proposed.

Engineering Propionibacterium freudenreichii subsp. shermanii for enhanced propionic acid fermentation: Effects of overexpressing propionyl-CoA:Succinate CoA transferase

Propionibacterium freudenreichii subsp. shermanii naturally forms propionic acid as the main fermentation product with acetate and succinate as two major by-products. In this study, overexpressing the native propionyl-CoA:succinate CoA transferase (CoAT) in P. shermanii was investigated to evaluate its effects on propionic acid fermentation with glucose, glycerol, and their mixtures as carbon source. In general, the mutant produced more propionic acid, with up to 10% increase in yield (0.62 vs. 0.56g/g) and 46% increase in productivity (0.41 vs. 0.28g/Lh), depending on the fermentation conditions. The mutant also produced less acetate and succinate, with the ratios of propionate to acetate (P/A) and succinate (P/S) in the final product increased 50% and 23%, respectively, in the co-fermentation of glucose/glycerol. Metabolic flux analysis elucidated that CoAT overexpression diverted more carbon fluxes toward propionic acid, resulting in higher propionic acid purity and a preference for glycerol over glucose as carbon source.

ATP Synthesis-coupled and -uncoupled Acetate Production from Acetyl-CoA by Mitochondrial Acetate:Succinate CoA-transferase and Acetyl-CoA Thioesterase in Trypanosoma

Insect stage trypanosomes use an "acetate shuttle" to transfer mitochondrial acetyl-CoA to the cytosol for the essential fatty acid biosynthesis. The mitochondrial acetate sources are acetate:succinate CoA-transferase (ASCT) and an unknown enzymatic activity. We have identified a gene encoding acetyl-CoA thioesterase (ACH) activity, which is shown to be the second acetate source. First, RNAi-mediated repression of ASCT in the ACH null background abolishes acetate production from glucose, as opposed to both single ASCT and ACH mutants. Second, incorporation of radiolabeled glucose into fatty acids is also abolished in this ACH/ASCT double mutant. ASCT is involved in ATP production, whereas ACH is not, because the ASCT null mutant is ∼1000 times more sensitive to oligomycin, a specific inhibitor of the mitochondrial F(0)/F(1)-ATP synthase, than wild-type cells or the ACH null mutant. This was confirmed by RNAi repression of the F(0)/F(1)-ATP synthase F(1)β subunit, which is lethal when performed in the ASCT null background but not in the wild-type cells or the ACH null background. We concluded that acetate is produced from both ASCT and ACH; however, only ASCT is responsible, together with the F(0)/F(1)-ATP synthase, for ATP production in the mitochondrion.

Multi‐factorial engineering of heterologous polyketide production in Escherichia coli reveals complex pathway interactions

Polyketides represent a significant fraction of all natural products. Many possess pharmacological activity which makes them attractive drug candidates. The production of the parent macrocyclic aglycones is catalyzed by multi-modular polyketide synthases utilizing short-chain acyl-CoA monomers. When producing polyketides through heterologous hosts, one must not only functionally express the synthase itself, but activate the machinery used to generate the required substrate acyl-CoA's. As a result, metabolic engineering of these pathways is necessary for high-level production of heterologous polyketides. In this study, we over-express three different pathways for provision of the two substrates (propionyl-CoA and (2S)-methylmalonyl-CoA) utilized for the biosynthesis of 6-deoxyerythronolide B (6-dEB; the macrolactone precursor of erythromycin): (1) a propionate → propionyl-CoA → (2S)-methylmalonyl-CoA pathway, (2) a methylmalonate → methylmalonyl-CoA → propionyl-CoA pathway, and (3) a succinate → succinyl-CoA → (2R)-methylmalonyl-CoA → (2S)-methylmalonyl-CoA → propionyl-CoA pathway. The current study revealed that propionate is a necessary component for greater than 5 mg L(-1) titers. Deletion of the propionyl-CoA:succinate CoA transferase (ygfH) or over-expression of the transcriptional activator of short chain fatty acid uptake improved titer to over 100 mg L(-1), while the combination of the two improved titer to over 130 mg L(-1). The addition of exogenous methylmalonate could also improve titer to over 100 mg L(-1). Expression of a Streptomyces coelicolor A3(2) methylmalonyl-CoA epimerase, in conjunction with over-expression of Escherichia coli's native methylmalonyl-CoA mutase, allowed for the incorporation of exogenously fed succinate into the 6-dEB core.

Acetate formation in the energy metabolism of parasitic helminths and protists

Formation and excretion of acetate as a metabolic end product of energy metabolism occurs in many protist and helminth parasites, such as the parasitic helminths Fasciola hepatica, Haemonchus contortus and Ascaris suum, and the protist parasites, Giardia lamblia, Entamoeba histolytica, Trichomonas vaginalis as well as Trypanosoma and Leishmania spp. In all of these parasites acetate is a main end product of their energy metabolism, whereas acetate formation does not occur in their mammalian hosts. Acetate production might therefore harbour novel targets for the development of new anti-parasitic drugs. In parasites, acetate is produced from acetyl-CoA by two different reactions, both involving substrate level phosphorylation, that are catalysed by either a cytosolic acetyl-CoA synthetase (ACS) or an organellar acetate:succinate CoA-transferase (ASCT). The ACS reaction is directly coupled to ATP synthesis, whereas the ASCT reaction yields succinyl-CoA for ATP formation via succinyl-CoA synthetase (SCS). Based on recent work on the ASCTs of F. hepatica, T. vaginalis and Trypanosoma brucei we suggest the existence of three subfamilies of enzymes within the CoA-transferase family I. Enzymes of these three subfamilies catalyse the ASCT reaction in eukaryotes via the same mechanism, but the subfamilies share little sequence homology. The CoA-transferases of the three subfamilies are all present inside ATP-producing organelles of parasites, those of subfamily IA in the mitochondria of trypanosomatids, subfamily IB in the mitochondria of parasitic worms and subfamily IC in hydrogenosome-bearing parasites. Together with the recent characterisation among non-parasitic protists of yet a third route of acetate formation involving acetate kinase (ACK) and phosphotransacetylase (PTA) that was previously unknown among eukaryotes, these recent developments provide a good opportunity to have a closer look at eukaryotic acetate formation.

Investigating the role of native propionyl-CoA and methylmalonyl-CoA metabolism on heterologous polyketide production in Escherichia coli.

6-Deoxyerythronolide B (6dEB) is the macrocyclic aglycone precursor of the antibiotic natural product erythromycin. Heterologous production of 6dEB in Escherichia coli was accomplished, in part, by designed over-expression of a native prpE gene (encoding a propionyl-CoA synthetase) and heterologous pcc genes (encoding a propionyl-CoA carboxylase) to supply the needed propionyl-CoA and (2S)-methylmalonyl-CoA biosynthetic substrates. Separate E. coli metabolism includes three enzymes, Sbm (a methylmalonyl-CoA mutase), YgfG (a methylmalonyl-CoA decarboxylase), and YgfH (a propionyl-CoA:succinate CoA transferase), also involved in propionyl-CoA and methylmalonyl-CoA metabolism. In this study, the sbm, ygfG, and ygfH genes were individually deleted and over-expressed to investigate their effect on heterologous 6dEB production. Our results indicate that the deletion and over-expression of sbm did not influence 6dEB production; ygfG over-expression reduced 6dEB production by fourfold while ygfH deletion increased 6dEB titers from 65 to 129 mg/L in shake flask experiments. It was also found that native E. coli metabolism could support 6dEB biosynthesis in the absence of exogenous propionate and the substrate provision pcc genes. Lastly, the effect of the ygfH deletion was tested in batch bioreactor cultures in which 6dEB titers improved from 206 to 527 mg/L.

Acetate:succinate CoA-transferase in the anaerobic mitochondria of Fasciola hepatica

Fasciola hepatica contains anaerobically functioning mitochondria that produce acetate and propionate, the main endproducts excreted by this parasite. The final reactions in the pathways leading to these endproducts are performed by acetate:succinate CoA-transferase (ASCT) and propionate:succinate CoA-transferase (PSCT), respectively. The enzymes catalysing these essential reactions in anaerobic mitochondria are still not characterized, nor are the corresponding genes identified. Here we describe the identification of the gene that codes for the F. hepatica ASCT. The F. hepatica gene was heterologously expressed and studies on the corresponding enzyme activity showed that the enzyme is indeed a transferase and uses a ping-pong bi-bi reaction mechanism, like most other known CoA-transferases. This F. hepatica CoA-transferase was shown to be a true transferase and not a hydrolase, as it needs an acceptor for optimal activity. Our studies demonstrated that the F. hepatica ASCT can use other CoA-acceptors than succinate, such as propionate, acetate and butyrate, and is in fact a short-chain acyl-CoA-transferase. We further showed that this F. hepatica CoA-transferase can also catalyze the PSCT reaction, which is responsible for the production of propionate. Analysis of the amino acid sequence of F. hepatica clearly indicated the presence of a mitochondrial targeting sequence, and in CHO cells the enzyme is indeed present in the mitochondrial fraction. F. hepatica ASCT is the first ASCT identified in anaerobic mitochondria. It is homologous to the hydrogenosomal ASCT we earlier identified in Trichomonas vaginalis, but not to the ASCT present in the aerobic mitochondria of Trypanosoma brucei.

Biochemical characterization of a mitochondrial-like organelle from Blastocystis sp. subtype 7

A mitochondrion-like organelle (MLO) was isolated from isotonic homogenates of Blastocystis. The organelle sedimented at 5000 g for 10 min, and had an isopycnic density in sucrose of 1.2 g ml(-1). Biochemical characterization enabled the demonstration of several key enzymes that allowed the construction of a metabolic pathway consisting of an incomplete Krebs cycle linked to the oxygen-sensitive enzymes pyruvate : NADP(+) oxidoreductase (PNO), acetate : succinate CoA transferase (ASCT) and succinate thiokinase (STK), which cumulatively are responsible for recycling CoA and generating ATP. The organelle differs from typical aerobic mitochondria in possessing an oxygen-sensitive PNO that can use FAD(+) or FMN(+) as electron acceptor but is inactive with NAD(+), Spinacia oleracea ferredoxin or Clostridium pasteurianum ferredoxin. A gene with 77 % sequence similarity to the PNO mitochondrion precursor cluster from Euglena gracilis sp[Q941N5] was identified in the Blastocystis genome database. A second cluster with 56 % sequence similarity to the pyruvate : ferredoxin oxidoreductase (PFOR) from Trichomonas vaginalis was also identified, which is in agreement with the concept that the PNO gene arose through the fusion of a eubacterial gene for PFOR with the gene for NADPH : cytochrome p450 reductase. Hydrogenase activity was not detected under the conditions used in this study. The Blastocystis oranelle therefore demonstrates significant biochemical differences from traditional mitochondria and hydrogenosomes, but possesses features of both. Based upon the results of this study, the Blastocystis organelle falls into the category of a MLO.

Glucose-induced Remodeling of Intermediary and Energy Metabolism in Procyclic Trypanosoma brucei

The procyclic form of Trypanosoma brucei is a parasitic protozoan that normally dwells in the midgut of its insect vector. In vitro, this parasite prefers d-glucose to l -proline as a carbon source, although this amino acid is the main carbon source available in its natural habitat. Here, we investigated how l -proline is metabolized in glucose-rich and glucose-depleted conditions. Analysis of the excreted end products of (13)C-enriched l -proline metabolism showed that the amino acid is converted into succinate or l -alanine depending on the presence or absence of d-glucose, respectively. The fact that the pathway of l -proline metabolism was truncated in glucose-rich conditions was confirmed by the analysis of 13 separate RNA interference-harboring or knock-out cell lines affecting different steps of this pathway. For instance, RNA interference studies revealed the loss of succinate dehydrogenase activity to be conditionally lethal only in the absence of d-glucose, confirming that in glucose-depleted conditions, l -proline needs to be converted beyond succinate. In addition, depletion of the F(0)/F(1)-ATP synthase activity by RNA interference led to cell death in glucose-depleted medium, but not in glucose-rich medium. This implies that, in the presence of d-glucose, the importance of the F(0)/F(1)-ATP synthase is diminished and ATP is produced by substrate level phosphorylation. We conclude that trypanosomes develop an elaborate adaptation of their energy production pathways in response to carbon source availability.

Acetate:Succinate CoA-transferase in the Hydrogenosomes of Trichomonas vaginalis: IDENTIFICATION AND CHARACTERIZATION

Acetate:succinate CoA-transferases (ASCT) are acetate-producing enzymes in hydrogenosomes, anaerobically functioning mitochondria and in the aerobically functioning mitochondria of trypanosomatids. Although acetate is produced in the hydrogenosomes of a number of anaerobic microbial eukaryotes such as Trichomonas vaginalis, no acetate producing enzyme has ever been identified in these organelles. Acetate production is the last unidentified enzymatic reaction of hydrogenosomal carbohydrate metabolism. We identified a gene encoding an enzyme for acetate production in the genome of the hydrogenosome-containing protozoan parasite T. vaginalis. This gene shows high similarity to Saccharomyces cerevisiae acetyl-CoA hydrolase and Clostridium kluyveri succinyl-CoA:CoA-transferase. Here we demonstrate that this protein is expressed and is present in the hydrogenosomes where it functions as the T. vaginalis acetate:succinate CoA-transferase (TvASCT). Heterologous expression of TvASCT in CHO cells resulted in the expression of an active ASCT. Furthermore, homologous overexpression of the TvASCT gene in T. vaginalis resulted in an equivalent increase in ASCT activity. It was shown that the CoA transferase activity is succinate-dependent. These results demonstrate that this acetyl-CoA hydrolase/transferase homolog functions as the hydrogenosomal ASCT of T. vaginalis. This is the first hydrogenosomal acetate-producing enzyme to be identified. Interestingly, TvASCT does not share any similarity with the mitochondrial ASCT from Trypanosoma brucei, the only other eukaryotic succinate-dependent acetyl-CoA-transferase identified so far. The trichomonad enzyme clearly belongs to a distinct class of acetate:succinate CoA-transferases. Apparently, two completely different enzymes for succinate-dependent acetate production have evolved independently in ATP-generating organelles.

We-P12:284 Oestrogen and females ischemical heart disease

Propionibacterium freudenreichii subsp. shermanii naturally forms propionic acid as the main fermentation product with acetate and succinate as two major by-products. In this study, overexpressing the native propionyl-CoA:succinate CoA transferase (CoAT) in P. shermanii was investigated to evaluate its effects on propionic acid fermentation with glucose, glycerol, and their mixtures as carbon source. In general, the mutant produced more propionic acid, with up to 10% increase in yield (0.62 vs. 0.56 g/g) and 46% increase in productivity (0.41 vs. 0.28 g/L h), depending on the fermentation conditions. The mutant also produced less acetate and succinate, with the ratios of propionate to acetate (P/A) and succinate (P/S) in the final product increased 50% and 23%, respectively, in the co-fermentation of glucose/glycerol. Metabolic flux analysis elucidated that CoAT overexpression diverted more carbon fluxes toward propionic acid, resulting in higher propionic acid purity and a preference for glycerol over glucose as carbon source.

Acetyl:Succinate CoA-transferase in Procyclic Trypanosoma brucei: GENE IDENTIFICATION AND ROLE IN CARBOHYDRATE METABOLISM

Acetyl:succinate CoA-transferase (ASCT) is an acetate-producing enzyme shared by hydrogenosomes, mitochondria of trypanosomatids, and anaerobically functioning mitochondria. The gene encoding ASCT in the protozoan parasite Trypanosoma brucei was identified as a new member of the CoA transferase family. Its assignment to ASCT activity was confirmed by 1) a quantitative correlation of protein expression and activity upon RNA interference-mediated repression, 2) the absence of activity in homozygous Deltaasct/Deltaasct knock out cells, 3) mitochondrial colocalization of protein and activity, 4) increased activity and acetate excretion upon transgenic overexpression, and 5) depletion of ASCT activity from lysates upon immunoprecipitation. Genetic ablation of ASCT produced a severe growth phenotype, increased glucose consumption, and excretion of beta-hydroxybutyrate and pyruvate, indicating accumulation of acetyl-CoA. Analysis of the excreted end products of (13)C-enriched and (14)C-labeled glucose metabolism showed that acetate excretion was only slightly reduced. Adaptation to ASCT deficiency, however, was an infrequent event at the population level, indicating the importance of this enzyme. These studies show that ASCT is indeed involved in acetate production, but is not essential, as apparently it is not the only enzyme that produces acetate in T. brucei.