Intracellular acetyl-CoA Concentrations Research Articles

Small RNA SmsR1 modulates acidogenicity and cariogenic virulence by affecting protein acetylation in Streptococcus mutans.

Post-transcriptional regulation by small RNAs and post-translational modifications (PTM) such as lysine acetylation play fundamental roles in physiological circuits, offering rapid responses to environmental signals with low energy consumption. Yet, the interplay between these regulatory systems remains underexplored. Here, we unveil the cross-talk between sRNAs and lysine acetylation in Streptococcus mutans, a primary cariogenic pathogen known for its potent acidogenic virulence. Through systematic overexpression of sRNAs in S. mutans, we identified sRNA SmsR1 as a critical player in modulating acidogenicity, a key cariogenic virulence feature in S. mutans. Furthermore, combined with the analysis of predicted target mRNA and transcriptome results, potential target genes were identified and experimentally verified. A direct interaction between SmsR1 and 5'-UTR region of pdhC gene was determined by in vitro binding assays. Importantly, we found that overexpression of SmsR1 reduced the expression of pdhC mRNA and increased the intracellular concentration of acetyl-CoA, resulting in global changes in protein acetylation levels. This was verified by acetyl-proteomics in S. mutans, along with an increase in acetylation level and decreased activity of LDH. Our study unravels a novel regulatory paradigm where sRNA bridges post-transcriptional regulation with post-translational modification, underscoring bacterial adeptness in fine-tuning responses to environmental stress.

Engineering a Novel Acetyl-CoA Pathway for Efficient Biosynthesis of Acetyl-CoA-Derived Compounds.

Acetyl-CoA is an essential central metabolite in living organisms and a key precursor for various value-added products as well. However, the intracellular availability of acetyl-CoA limits the efficient production of these target products due to complex and strict regulation. Here, we proposed a new acetyl-CoA pathway, relying on two enzymes, threonine aldolase and acetaldehyde dehydrogenase (acetylating), which can convert one l-threonine into one acetyl-CoA, one glycine, and generate one NADH, without carbon loss. Introducing the acetyl-CoA pathway could increase the intracellular concentration of acetyl-CoA by 8.6-fold compared with the wild-type strain. To develop a cost-competitive and genetically stable acetyl-CoA platform strain, the new acetyl-CoA pathway, driven by the constitutive strong promoter, was integrated into the chromosome of Escherichia coli. We demonstrated the practical application of this new acetyl-CoA pathway by high titer production of β-alanine, mevalonate, and N-acetylglucosamine. At the same time, this pathway achieved a high-yield production of glycine, a value-added commodity chemical for the synthesis of glyphosate and thiamphenicol. This work shows the potential of this new acetyl-CoA pathway for the industrial production of acetyl-CoA-derived compounds.

Calcium-Phosphate Combination Enhances Spinosad Production in Saccharopolyspora spinosa via Regulation of Fatty Acid Metabolism.

Phosphate concentration above 10mM reduces the production of many secondary metabolites; however, the phenomenon is not mechanistically understood yet. Specifically, the problem of phosphorus limitation in antibiotic production remains unresolved. This study investigates the phosphorus inhibition effect on spinosad production and alleviates it by calcium and phosphate supplementation to fermentation media. Furthermore, we examined the mechanism of fatty acids-induced increase in polyketides production. Four phosphates that were supplemented into the fermentation media include NaH2PO4, Na2HPO4, KH2PO4, and K2HPO4 and NaH2PO4 was found to be the most effective phosphate. Under the optimal phosphate condition of supplementing 20mM NaH2PO4 on the fourth day and 5g/L CaCO3, the maximal spinosad production reached 520mg/L, showing a 1.65-fold increase over the control treatment. In the NaH2PO4-CaCO3 system, the de novo fatty acid biosynthesis was significantly downregulated while spinosad biosynthesis and β-oxidation were upregulated. The coordination of de novo fatty acid biosynthesis and β-oxidation promoted intracellular acetyl-CoA concentration. The results demonstrate that NaH2PO4-CaCO3 combined addition is a simple and effective strategy to alleviate phosphorus inhibition effect through the regulation of fatty acid metabolism and accumulation of immediate precursors. This information improves our understanding of phosphates' influence on the large-scale production of polyketides.

Engineering a Central Carbon Metabolism Pathway to Increase the Intracellular Acetyl-CoA Pool in Synechocystis sp. PCC 6803 Grown under Photomixotrophic Conditions.

In cyanobacteria, photomixotrophic growth is considered as a promising strategy to achieve both high cell density and product accumulation. However, the conversion of glucose to acetyl coenzyme A (acetyl-CoA) in the native glycolytic pathway is insufficient, which decreases the carbon utilization and productivity of engineered cyanobacteria under photomixotrophic conditions. To increase the carbon flux from glucose to key intracellular precursor acetyl-CoA in Synechocystis sp. PCC 6803 (hereafter, Synechocystis 6803) under photomixotrophic conditions, a synthetic nonoxidative cyclic glycolysis (NOG) pathway was introduced into the wild type strain, which successfully increased the intracellular pool of acetyl-CoA by approximately 1-fold. To minimize the competition for glucose, the native Embden-Meyerhof-Parnas (EMP) and Entner-Doudoroff (ED) pathways were knocked out, respectively. Notably, eliminating the native ED pathway in the engineered strain carrying the NOG pathway further increased the intracellular pool of acetyl-CoA up to 2.8-fold. Another carbon consuming pathway in Synechocystis 6803, the glycogen biosynthesis pathway, was additionally knocked out in the above-mentioned engineered strain, which enabled an increase of the intracellular acetyl-CoA pool by up to 3.5-fold when compared with the wild type strain. Finally, the content of intracellular lipids was analyzed as an index of the productive capacity of the engineered Synechocystis 6803 cell factory under photomixotrophic conditions. The results showed the total lipids yield increased about 26% compared to the wild type (from 15.71% to 34.12%, g/g glucose), demonstrating that this integrated approach could represent a general strategy not only for the improvement of the intracellular concentration of acetyl-CoA, but also for the production of value-added chemicals that require acetyl-CoA as a key precursor in cyanobacteria.

Acetylation-mediated remodeling of the nucleolus regulates cellular acetyl-CoA responses.

The metabolite acetyl-coenzyme A (acetyl-CoA) serves as an essential element for a wide range of cellular functions including adenosine triphosphate (ATP) production, lipid synthesis, and protein acetylation. Intracellular acetyl-CoA concentrations are associated with nutrient availability, but the mechanisms by which a cell responds to fluctuations in acetyl-CoA levels remain elusive. Here, we generate a cell system to selectively manipulate the nucleo-cytoplasmic levels of acetyl-CoA using clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene editing and acetate supplementation of the culture media. Using this system and quantitative omics analyses, we demonstrate that acetyl-CoA depletion alters the integrity of the nucleolus, impairing ribosomal RNA synthesis and evoking the ribosomal protein-dependent activation of p53. This nucleolar remodeling appears to be mediated through the class IIa histone deacetylases (HDACs). Our findings highlight acetylation-mediated control of the nucleolus as an important hub linking acetyl-CoA fluctuations to cellular stress responses.

Phosphoketolase overexpression increases biomass and lipid yield from methane in an obligate methanotrophic biocatalyst

Microbial conversion of methane to high-value bio-based fuels, chemicals, and materials offers a path to mitigate GHG emissions and valorize this abundant-yet -underutilized carbon source. In addition to fermentation optimization strategies, rational methanotrophic bacterial strain engineering offers a means to reach industrially relevant titers, carbon yields, and productivities of target products. The phosphoketolase pathway functions in heterofermentative bacteria where carbon flux through two sugar catabolic pathways to mixed acids (lactic acid and acetic acid) increases cellular ATP production. Importantly, this pathway also serves as an alternative route to produce acetyl-CoA that bypasses the CO2 lost through pyruvate decarboxylation in the Embden-Meyerhof-Parnas pathway. Thus, the phosphoketolase pathway can be leveraged for carbon efficient biocatalysis to acetyl-CoA-derived intermediates and products. Here, we show that the industrially promising methane biocatalyst, Methylomicrobium buryatense, encodes two phosphoketolase isoforms that are expressed in methanol- and methane-grown cells. Overexpression of the PktB isoform led to a 2-fold increase in intracellular acetyl-CoA concentration, and a 2.6-fold yield enhancement from methane to microbial biomass and lipids compared to wild-type, increasing the potential for methanotroph lipid-based fuel production. Off-gas analysis and metabolite profiling indicated that global metabolic rearrangements, including significant increases in post-translational protein acetylation and gene expression of the tetrahydromethanopterin-linked pathway, along with decreases in several excreted products, coincided with the superior biomass and lipid yield observed in the engineered strain. Further, these data suggest that phosphoketolase may play a key regulatory role in methanotrophic bacterial metabolism. Given that acetyl-CoA is a key intermediate in several biosynthetic pathways, phosphoketolase overexpression offers a viable strategy to enhance the economics of an array of biological methane conversion processes.

Engineering of Serine-Deamination pathway, Entner-Doudoroff pathway and pyruvate dehydrogenase complex to improve poly(3-hydroxybutyrate) production in Escherichia coli.

BackgroundPoly(3-hydroxybutyrate) (PHB), a biodegradable bio-plastic, is one of the most common homopolymer of polyhydroxyalkanoates (PHAs). PHB is synthesized by a variety of microorganisms as intracellular carbon and energy storage compounds in response to environmental stresses. Bio-based production of PHB from renewable feedstock is a promising and sustainable alternative to the petroleum-based chemical synthesis of plastics. In this study, a novel strategy was applied to improve the PHB biosynthesis from different carbon sources.ResultsIn this research, we have constructed E. coli strains to produce PHB by engineering the Serine-Deamination (SD) pathway, the Entner-Doudoroff (ED) pathway, and the pyruvate dehydrogenase (PDH) complex. Firstly, co-overexpression of sdaA (encodes L-serine deaminase), L-serine biosynthesis genes and pgk (encodes phosphoglycerate kinase) activated the SD Pathway, and the resulting strain SD02 (pBHR68), harboring the PHB biosynthesis genes from Ralstonia eutropha, produced 4.86 g/L PHB using glucose as the sole carbon source, representing a 2.34-fold increase compared to the reference strain. In addition, activating the ED pathway together with overexpressing the PDH complex further increased the PHB production to 5.54 g/L with content of 81.1% CDW. The intracellular acetyl-CoA concentration and the [NADPH]/[NADP+] ratio were enhanced after the modification of SD pathway, ED pathway and the PDH complex. Meanwhile, these engineering strains also had a significant increase in PHB concentration and content when xylose or glycerol was used as carbon source.ConclusionsSignificant levels of PHB biosynthesis from different kinds of carbon sources can be achieved by engineering the Serine-Deamination pathway, Entner-Doudoroff pathway and pyruvate dehydrogenase complex in E. coli JM109 harboring the PHB biosynthesis genes from Ralstonia eutropha. This work demonstrates a novel strategy for improving PHB production in E. coli. The strategy reported here should be useful for the bio-based production of PHB from renewable resources.

Acyl-CoA sensing by FasR to adjust fatty acid synthesis in Corynebacterium glutamicum

Corynebacterium glutamicum, like Mycobacterium tuberculosis, is a member of the Corynebacteriales, which have linear fatty acids and as branched fatty acids the mycolic acids. We identified accD1 and fasA as key genes of fatty acid synthesis, encoding the β-subunit of the acetyl-CoA carboxylase and a type-I fatty acid synthase, respectively, and observed their repression during growth on minimal medium with acetate. We also identified the transcriptional regulator FasR and its binding sites in the 5′ upstream regions of accD1 and fasA. In the present work we establish by co-isolation and gel-mobility shifts oleoyl-CoA and palmitoyl-CoA as effectors of FasR, and show by DNA microarray analysis that in presence of exogeneous fatty acids accD1 and fasA are repressed. These results are evidence that acyl-CoA derivatives derived from extracellular fatty acids interact with FasR to repress the genes of fatty acid synthesis. This model also explains the observed repression of accD1 and fasA during growth on acetate, where apparently the known high intracellular acetyl-CoA concentration during growth on this substrate requires reduced accD1 and fasA expression for fine control of de novo fatty acid synthesis. Consequently, this mechanism ensures that membrane lipid homeostasis is maintained when specific nutrients are available resulting in increased acetyl-CoA concentration, as is the case with acetate, or when fatty acids are directly available from the extracellular environment. However, the genes specific to mycolic acid synthesis, which are in part shared with linear fatty acid synthesis, are not controlled by FasR, which is in agreement with the fact that they can not be supplied from the extracellular environment but that their synthesis fully depends on a constant supply of linear fatty acid chains.

Photosynthetic accumulation of poly-(hydroxybutyrate) by cyanobacteria—the metabolism and potential for CO 2 recycling

Regulatory mechanism in PHB [poly-(hydroxybutyrate)] accumulation by cyanobacteria, especially by a thermophilic isolate, Synechococcus MA19 was reviewed in comparison with a genetically engineered strain. The strain, MA19 accumulates PHB under nitrogen starved and photoautotrophic conditions (MA19-N). Little PHB synthase activity was detected in crude extracts from the cells grown in nitrogen sufficient conditions (MA19+N). The activity was detected exclusively in membrane fractions from MA19+N. The change of the enzyme activity was insensitive to chloramphenicol, which suggests post-translational activation. In vitro, acetyl phosphate activated PHB synthase in membrane fractions from MA19+N, and the extent of activation depended on the concentration of acetyl phosphate. Phosphotransacetylase which catalyzes the conversion of acetyl-CoA to acetyl phosphate was detected in crude extracts from MA19-N but not in those from MA19+N. These results suggested that intracellular acetyl phosphate concentration could be controlled, depending on C–N balance and intracellular acetyl-CoA concentration. On the contrary, in genetically-engineered cyanobacterium (transformant with PHB synthesizing genes from Ralstonia eutropha), it did not seem to be PHB synthase but acetyl-CoA flux that limits PHB synthesis. The closer association of PHB granules with thylakoid membranes in MA19 is suggested than that in the genetically-engineered cyanobacterium, which may reflect the difference of distribution of PHB synthase. Transposon-mutagenesis was used to acquire mutants of its altered PHB regulatory mechanism. PHA production by cyanobacteria was considered from the aspects of photobioreactors.

Control of poly-beta-hydroxybutyrate synthase mediated by acetyl phosphate in cyanobacteria.

Poly-beta-hydroxybutyrate (PHB) synthesis in a cyanobacterium, Synechococcus sp. strain MA19, is controlled at the enzyme level and is dependent on the C/N balance in the culture medium. The control involves at least two enzymes. The first enzyme is PHB synthase. Little PHB synthase activity was detected in crude extracts from cells grown under nitrogen-sufficient conditions (MA19(+N)). The activity was detected exclusively in membrane fractions from nitrogen-deprived cells (MA19(-N)) under light but not dark conditions. The shift in the enzyme activity was insensitive to chloramphenicol, which suggests posttranslational activation. Acetyl phosphate activated PHB synthase in membrane fractions from MA19(+N). In vitro, the activation level of PHB synthase changed, depending on the concentration of acetyl phosphate. The second enzyme was phosphotransacetylase (EC 2.3.1.8), which catalyzes the conversion of acetyl coenzyme A (acetyl-CoA) to acetyl phosphate. The activity was detected in crude extracts from MA19(-N) but not in those from MA19(+N). The results suggested that intracellular acetyl phosphate concentration could be controlled, depending on C/N balance and intracellular acetyl-CoA concentration. Acetyl phosphate probably acts as a signal of C/N balance affecting PHB metabolism in MA19.

Regulation of acetyl phosphate synthesis and degradation, and the control of flagellar expression in Escherichia coli

We investigated the relationship between Escherichia coli flagellar expression and the regulation of acetyl phosphate synthesis and degradation. Using cells either wild type for acetyl phosphate metabolism or defective for phosphotransacetylase or acetate kinase, or both, we measured flagellar expression and the intracellular concentration of acetyl phosphate relative to growth phase and temperature. Under the conditions tested, we found that elevated levels of acetyl phosphate corresponded to inhibition of flagellar synthesis. To extend these observations, we measured the intracellular concentration of acetyl-CoA, the level of expression from the pta and ackA promoters, and the activities of phosphotransacetylase and acetate kinase derived from cell lysates. Relative to increasing culture density, acetyl-CoA levels and expression from both the pta and ackA promoters decreased. Relative to increasing temperature, expression from the ackA promoter decreased and phosphotransacetylase activity increased. In contrast, temperature had little or no effect on either acetate kinase activity or expression from the pta promoter. We propose that cells regulate intracellular acetyl phosphate concentrations relative to growth phase and temperature by modulating the availability of acetyl-CoA, the expression of ackA, and the activity of phosphotransacetylase.

Metabolic roles of carnitine expressed through the carnitine acetyltransferase system in Candida pintolopesii

1. 1. The carnitine-responsive mutant yeast, Candida pintolopesii ATCC 26014 and the wild type strain (ATCC 22987) were used to investigate the role of carnitine and the carnitine acetyltransferase system. 2. 2. [ 3H] l-Carnitine, supplied to the cells, was incorporated into acetylcamitine and [ 14C]pantothenate was incorporated into CoA and its derivatives. 3. 3. Both bioautography and quantitative assays indicated that the relative amounts of CoA and acetylCoA were very different in the mutant and wild type cells. 4. 4. The wild type yeast maintained an acetylCoA/CoA ratio of 0.33 ± 0.09 indicating that most of the CoA in the cell is in the free CoA form. Carnitine was not required to establish this ratio nor did its presence lower it further. 5. 5. In contrast, the mutant cells contained a high acetylCoA/CoA ratio (12.8 ± 3.0). 6. 6. In the mutant cells, carnitine lowered the ratio by decreasing the intracellular acetylCoA concentration and releasing free CoA. 7. 7. These data indicated that wild type yeast possess an effective mechanism that is not related to the CAT system for regulating the acetylCoA/CoA ratio. 8. 8. This mechanism appears to be lacking in the mutant. The CAT system decreased the acetylCoA/CoA ratio in the mutant cells but not to the value which is found in the wild type strain. 9. 9. In both stains of Candida pintolopesii, in the presence of carnitine, an acetylcamitine pool can be created whose concentration exceeds that of acetylCoA. 10. 10. The intracellular apparent equilibrium constant ( K app) for carnitine acetyltransferase for wild type Candida pintolopesii ATCC 22987 was 0.73 ± 0.12, close to the established value of 0.6, indicating that the CAT system ran close to equilibrium. 11. 11. The K app for the CAT system of the carnitine-responsive mutant yeast was 7.7 ± 1.7 indicating that this reaction was not at equilibrium.

Regulation of the biosynthesis of platelet‐activating factor in alveolar macrophages

Activities of enzymes which metabolize lysoplatelet-activating factor (lysoPAF) and platelet-activating factor (PAF) were studied in rabbit alveolar macrophage lysates. Substantial acetyltransferase activity was noted in the presence of 100 microM acetyl-coenzyme A (CoA), and this activity was increased in A23187-stimulated cell lysate. On the other hand, in the absence of exogenous acetyl-CoA, lysoPAF was mainly acylated through a transacylation pathway rather than by acetyltransferase in both control and A23187-stimulated cell lysates. We confirmed that the intracellular concentration of acetyl-CoA is relatively low. The observations suggest that the transacylation system may play an equally important role in the regulation of the availability of lysoPAF in intact cells. Intracellular lysoPAF was also maintained at relatively low levels. Interestingly, large amounts of PAF were produced even in unstimulated cells upon addition of an excess of exogenous lysoPAF, suggesting that generation of an adequate amount of lysoPAF within cells may be sufficient to trigger PAF synthesis in this type of cells.

Changes in the intracellular concentration of acetyl-CoA and malonyl-CoA in relation to the carbon and energy metabolism of Escherichia coli K12.

Intracellular concentrations of acetyl-CoA and malonyl-CoA in Escherichia coli K12 were determined by a malonyl-CoA: acetyl-CoA cycling technique. Under aerobic growth conditions with glucose the acetyl-CoA and malonyl-CoA concentrations varied over a range of 0.05-1.5 nmol (mg dry wt)-1 (20-600 microM) and 0.01-0.23 nmol (mg dry wt)-1 (4-90 microM), respectively. The intracellular concentration of acetyl-CoA was highest in exponentially growing cells and it fell rapidly to less than 5% of the maximum level when the organism entered stationary phase after exhaustion of glucose. A linear relationship was observed between the intracellular concentration of total acyl-CoA and the logarithm of the concentration of glucose in the medium. Consequently, the acetyl-CoA/malonyl-CoA ratios also varied drastically, in a range of 0.6-41.7, under different conditions. Of several carbon sources tested, glucose was the most effective for promoting the synthesis of cellular acetyl-CoA. For cells grown on glycerol or acetate the maximum concentrations of total acyl-CoA were significantly lower. In cells incubated with citrate (not used as a carbon source by E. coli), the level was consistent with that in cells starved for exogenous carbon sources.

Glucose metabolism in perfused skeletal muscle. Effects of starvation, diabetes, fatty acids, acetoacetate, insulin and exercise on glucose uptake and disposition.

1. The regulation of glucose uptake and disposition in skeletal muscle was studied in the isolated perfused rat hindquarter. 2. Insulin and exercise, induced by sciatic-nerve stimulation, enhanced glucose uptake about tenfold in fed and starved rats, but were without effect in rats with diabetic ketoacidosis. 3. At rest, the oxidation of lactate (0.44 mumol/min per 30 g muscle in fed rats) was decreased by 75% in both starved and diabetic rats, whereas the release of alanine and lactate (0.41 and 1.35 mumol/min per 30 g respectively in the fed state) was increased. Glycolysis, defined as the sum of lactate+alanine release and lactate oxidation, was not decreased in either starvation or diabetes. 4. In all groups, exercise tripled O2 consumption (from approximately 8 to approximately 25 mumol/min per 30 g of muscle) and increased the release and oxidation of lactate five- to ten-fold. The differences in lactate release between fed, starved and diabetic rats observed at rest were no longer apparent; however, lactate oxidation was still several times greater in the fed group. 5. Perfusion of the hindquarter of a fed rat with palmitate, octanoate or acetoacetate did not alter glucose uptake or lactate release in either resting or exercising muslce; however, lactate oxidation was significantly inhibited by acetoacetate, which also increased the intracellular concentration of acetyl-CoA. 6. The data suggest that neither that neither glycolysis nor the capacity for glucose transport are inhbitied in the perfused hindquarter during starvation or perfusion with fatty acids or ketone bodies. On the other hand, lactate oxidation is inhibited, suggesting diminished activity of pyruvate dehydrogenase. 7. Differences in the regulation of glucose metabolism in heart and skeletal muscle and the role of the glucose/fatty acid cycle in each tissue are discussed.

Characterization and regulation of pyruvate carboxylase of Bacillus licheniformis.

Cell-free extracts of Bacillus licheniformis were found to contain pyruvate carboxylase which catalyzes the reaction between pyruvate and bicarbonate to yield oxalacetate in the presence of adenosine triphosphate (ATP), acetylcoenzyme A (CoA), and manganese. The plot between the reaction velocity of the carboxylation by the partially purified pyruvate carboxylase (25-fold) and the concentration of pyruvate, bicarbonate, manganese, and ATP did not indicate a pronounced deviation from the Michaelis-Menten hyperbola. The enzyme was inhibited by avidin and aspartate. Biotin partially protected the enzyme from avidin inhibition, whereas the amount of inhibition by aspartate was dependent on the concentration of acetyl-CoA present. The intracellular concentration of acetyl-CoA did not vary significantly enough to allow control of the enzyme by this method. Extracts of 4-hr postexponential-phase cells of B. licheniformis were also found to contain phosphoenolpyruvate carboxykinase, which appears to be under catabolite repression control. It is suggested that the endogenous induction of this enzyme is the determining factor allowing the shift to gluconeogenesis from glycolysis during sporulation of glucose-grown cells.