acetyl-CoA Synthesis Research Articles

Diacerein as a Promising Acyl Donor in Biosynthetic Acetyl-CoA and Glycosyl Esters Mediated by a Multifunctional Maltose O-Acetyltransferase from Escherichia coli.

Acetyl-coenzyme A (acetyl-CoA) is an important donor for acetylation modifications of nutritional supplements. The existing enzymatic methods for acetyl-CoA synthesis suffer from cofactor dependence, donor inaccessibility, and biocatalyst instability, leading to its high cost. Hence, a promising alternative is highly desired. Herein, a maltose O-acetyltransferase (MAT) with cofactor independence had been identified as a stable acetyl-CoA-synthesizing biocatalyst in a screen of the Escherichia coli genome. Under the action of MAT, an anthraquinone medicine containing two acetyl groups, diacerein, was screened as an acetyl donor. Saturation mutagenesis at Glu125 was performed to increase the acetyl-CoA-synthesizing capacity of MAT, while decreasing the accompanying hydrolase activities. A mutant MAT-E125F was thus generated and could convert diacerein and CoA into the highest yield of 3892.70 mg/L acetyl-CoA. Moreover, MAT could synthesize puerarin 6″-O-acetate and other glycosyl esters through acetyl-CoA-dependent acetylation or diacerein-based transesterification reaction. To most of the tested glycosides, the transesterification efficiency was higher than that of acetylation. The mutant MAT-E125V acquired the highest conversion of 94.0% to puerarin 6″-O-acetate through transesterification, while MAT-E125N yielded the highest conversion of 68.5% through acetylation. Taking together, the multifunctional MAT displayed a potent acetyl-CoA- and glycosyl ester-synthesizing capacity using diacerein as an acetyl donor.

Global Transcriptome Analyses Provide Into Several Fatty Acid Biosynthesis-related Genes in Peanut (Arachis hypogaea L.)

Peanut (Arachis hypogaea L.) is one of the important oil crops worldwide. Revealing the molecular basis of seed oil synthesis and analyzing key candidate genes may have important implications for enhancing peanut production. In this study, we investigated the dynamic changes in the oil accumulation rate and gene expression levels in developing peanut seeds. A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that the up-regulated differentially expressed genes (DEGs) among three varieties at 25–35 days after flowering (DAF) were involved in fatty acid synthesis, carbon metabolism, and pyruvate metabolism. Between 25 and 35 DAF, the up-regulated DEGs in the high-oil varieties, but not in the low-oil variety, were related to acetyl-CoA synthesis. We examined the differences in the varieties at the same stages. We analyzed the lipid metabolism-related genes by protein–protein interaction (PPI), and finally identified 57 DEGs that may influence the peanut oil content. These candidate genes included three genes encoding acetyl-CoA carboxylase (ACCase), one gene encoding ketoacyl-ACP synthase III (KASIII), one gene encoding hydroxyacyl-ACP dehydrase (HAD), and one gene encoding enoyl-ACP reductase (EAR), which form the Type II fatty acid synthase complex. Additionally, we also found that 11 transcription factors may affect oil content. These identified candidate genes may help to further clarify the molecular basis of the oil accumulation in peanut seeds.

Synthesis of nucleocytosolic acetyl-CoA regulates mitochondrial respiration and ATP synthesis in budding yeast

• Reduced nucleocytosolic acetyl-CoA synthesis downregulates histone transcription • Decreased acetyl-CoA levels affect PIC assembly at histone promoters • Decreased acetyl-CoA reduces histone acetylation at histone promoters • Decreased histone levels induce respiration and ATP synthesis • Decreased acetyl-CoA synthesis is compensated for by elevated ATP synthesis

Progress in studies on production of chemicals from xylose by Saccharomyces cerevisiae

Effective utilization of xylose is a basis for economic production of biofuels or chemicals from lignocellulose biomass. Over the past 30 years, through metabolic engineering, evolutionary engineering and other strategies, the metabolic capacity of xylose of the traditional ethanol-producing microorganism Saccharomyces cerevisiae has been significantly improved. In recent years, the reported results showed that the transcriptome and metabolome profiles between xylose and glucose metabolism existed significant difference in recombinant yeast strains. Compared with glucose, the overall process of xylose metabolism exhibits Crabtree-negative characteristics, including the limited glycolytic pathway activity, which reduces the metabolic flux of pyruvate to ethanol, and the enhanced cytosolic acetyl-CoA synthesis and respiratory energy metabolism. These traits are helpful to achieve efficient synthesis of downstream products using pyruvate or acetyl-CoA as precursors. This review provides a detailed overview on the modification and optimization of xylose metabolic pathways in S. cerevisiae, the characteristics of xylose metabolism, and the construction of cell factories for production of chemicals using xylose as a carbon source. Meanwhile, the existed difficulties and challenges, and future studies on biosynthesis of bulk chemicals using xylose as an important carbon source are proposed.

ITRAQ-based proteomic analysis of Paracoccidioides brasiliensis in response to hypoxia

Aerobic organisms require oxygen for energy. In the course of the infection, adaptation to hypoxia is crucial for survival of human pathogenic fungi. Members of the Paracoccidioides complex face decreased oxygen tensions during the life cycle stages. In Paracoccidioides brasiliensis proteomic responses to hypoxia have not been investigated and the regulation of the adaptive process is still unknown, and this approach allowed the identification of 216 differentially expressed proteins in hypoxia using iTRAQ-labelling. Data suggest that P. brasiliensis reprograms its metabolism when submitted to hypoxia. The fungus reduces its basal metabolism and general transport proteins. Energy and general metabolism were more representative and up regulated. Glucose is apparently directed towards glycolysis or the production of cell wall polymers. Plasma membrane/cell wall are modulated by increasing ergosterol and glucan, respectively. In addition, molecules such as ethanol and acetate are produced by this fungus indicating that alternative carbon sources probably are activated to obtain energy. Also, detoxification mechanisms are activated. The results were compared with label free proteomics data from Paracoccidioides lutzii. Biochemical pathways involved with acetyl-CoA, pyruvate and ergosterol synthesis were up-regulated in both fungi. On the other hand, proteins from TCA, transcription, protein fate/degradation, cellular transport, signal transduction and cell defense/virulence processes presented different profiles between species. Particularly, proteins related to methylcitrate cycle and those involved with acetate and ethanol synthesis were increased in P. brasiliensis proteome, whereas GABA shunt were accumulated only in P. lutzii. The results emphasize metabolic adaptation processes for distinct Paracoccidioides species.

Abstract PS17-50: Nuclear expression of acetyl-CoA producing enzymes and their roles in epigenetic reprogramming in breast cancer cells

Abstract Metabolic and epigenetic reprogramming are two key hallmarks of cancer. Rapidly proliferating cancer cells often exhibit a higher and differentially reprogrammed transcriptional activity than normal cells of the same tissue. To support transcriptional reprogramming and increased transcriptional load, sustained chromatin acetylation is required to loosen the chromatin and enable RNA transcription. Histone acetylation also requires a constant supply of acetyl-CoA, a labile, membrane impermeable metabolite that is the obligatory donor of the acetyl groups for histone acetylation. However, the pathways responsible for increased synthesis of acetyl-CoA inside the nucleus remain largely elusive. Using estrogen-responsive (MCF7) and their matched endocrine therapy resistant breast cancer cells (LCC9 and MCF7:5C), we show that blocking p300-mediated acetyltransferase activity is crucial for proliferation of endocrine therapy-resistant breast cancer cells. Furthermore, we found nuclear expression of two acetyl-CoA producing enzymes, i.e., pyruvate dehydrogenase complex (PDC), and ATP citrate lyase (ACLY), which implies their functional role inside the nucleus. PDC is a multimeric protein complex enzyme made up of five different subunits. Canonically, PDC is found in mitochondria and is responsible for the conversion of pyruvate to acetyl-CoA, linking glycolysis to the tricarboxylic acid (TCA) cycle. Our results show that multiple PDC enzyme subunits, e.g., pyruvate dehydrogenase (PDH) E1 alpha (E1 alpha), dihydrolipoal dehydrogenase (DLD), and PDHX (also known as E3BP) are expressed in the nucleus of MCF7, LCC9 and MCF7:5C cells. Moreover, expression of nuclear DLD and PDHX subunit is higher in endocrine resistant cells (LCC9 and MCF7:5C) than in their parental MCF7 cells, strongly suggesting a possible role in endocrine resistance. Notably, high expression of PDHX is correlated with poor relapse-free survival in estrogen-receptor positive breast cancer patients in four independent publicly available datasets. ACLY is a tetramer of identical subunits catalyzes conversion of citric acid and co-enzyme A to acetyl-CoA. ACLY is also overexpressed in the nucleus of endocrine-resistant breast cancer cells (LCC9 and MCF7:5C) when compared with their endocrine sensitive MCF7 controls. This study investigates the role of nuclear PDC and ACLY mediated acetyl-CoA synthesis and its impact on histone acetylation enabling reprogrammed transcriptional activity supporting proliferation of endocrine therapy-resistant breast cancer cells. Citation Format: Surojeet Sengupta, Shuait Nair, Lu Jin, Catherine M Sevigny, Brandon Jones, Robert Clarke. Nuclear expression of acetyl-CoA producing enzymes and their roles in epigenetic reprogramming in breast cancer cells [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-50.

Characterization of two enzymes from Psychrobacter cryohalolentis that are required for the biosynthesis of an unusual diacetamido-d-sugar

Psychrobacter cryohalolentis strain K5T is a Gram-negative organism first isolated in 2006. It has a complex O-antigen that contains, in addition to l-rhamnose and d-galactose, two diacetamido- and a triacetamido-sugar. The biochemical pathways for the production of these unusual sugars are presently unknown. Utilizing the published genome sequence of the organism, we hypothesized that the genes 0612, 0638, and 0637 encode for a 4,6-dehydratase, an aminotransferase, and an N-acetyltransferase, respectively, which would be required for the biosynthesis of one of the diacetamido-sugars, 2,4-diacetamido-2,4,6-trideoxy-d-glucose, starting from UDP-N-acetylglucosamine. Here we present functional and structural data on the proteins encoded by the 0638 and 0637 genes. The kinetic properties of these enzymes were investigated by a discontinuous HPLC assay. An X-ray crystallographic structure of 0638, determined in its external aldimine form to 1.3 Å resolution, demonstrated the manner in which the UDP ligand is positioned into the active site. It is strikingly different from that previously observed for PglE from Campylobacter jejuni, which functions on the same substrate. Four X-ray crystallographic structures were also determined for 0637 in various complexed states at resolutions between 1.3 and 1.55 Å. Remarkably, a tetrahedral intermediate mimicking the presumed transition state was trapped in one of the complexes. The data presented herein confirm the hypothesized functions of these enzymes and provide new insight into an unusual sugar biosynthetic pathway in Gram-negative bacteria. We also describe an efficient method for acetyl-CoA synthesis that allowed us to overcome its prohibitive cost for this analysis.

Abstract 15978: Acetyl-coa Catalysis by Atp-citrate Lyase is Essential for Myofibroblast Differentiation and Persistence

A feature of heart failure (HF) is excessive extracellular matrix deposition and cardiac remodeling by a differentiated fibroblast population known as myofibroblasts. Identifying mechanisms of myofibroblast differentiation in cardiac fibrosis could yield novel therapeutic targets to delay or reverse HF. Recent evidence suggests that myofibroblast differentiation requires metabolic reprogramming for transcriptional activation of the myofibroblast gene program by chromatin-dependent mechanisms. We previously reported that inhibition of histone demethylation blocks myofibroblast formation, however, whether histone acetylation (e.g., H3K27ac, a prominent mark associated with gene transcription) is involved in fibroblast reprogramming remains unclear. ATP-citrate lyase (ACLY) synthesizes acetyl-CoA and therein supplies acetyl-CoA to the nucleus, where it is used as a substrate by histone acetyltransferases (HATs). To define the role of acetyl-CoA metabolism in myofibroblast differentiation, we stimulated differentiation in mouse embryonic fibroblasts (MEFs) and adult mouse cardiac fibroblasts (ACFs) with the pro-fibrotic agonist transforming growth factor β (TGFβ) and treated cells with a pharmacological inhibitor of ACLY. ACLY inhibition decreased myofibroblast gene expression in ACF and MEFs in TGFβ-stimulated myofibroblast differentiation, in addition to decreasing the population of αSMA positive MEFs. Genetic deletion of ACLY in MEFs recapitulated the results observed with pharmacological inhibition. Encouragingly, the ACLY inhibitor was sufficient to revert fully differentiated myofibroblasts under continuous TGFβ stimulation to a quiescent, non-fibrotic phenotype. Altogether, our data indicate that ACLY activity is necessary for myofibroblast differentiation and persistence. We hypothesize that ACLY-dependent acetyl-CoA synthesis is necessary for histone acetylation and transcriptional activation of the myofibroblast gene program. Currently, we are examining mechanisms of ACLY-dependent chromatin remodeling in fibroblasts and the in vivo relevance of this mechanism in mutant mice. In summary, ACLY is a potential target to reverse cardiac fibrosis and lessen HF.

Molecular characterization of the ACSS2 gene involved in adaptation to hypoxia in high-altitude cattle breeds

Abstract Under extreme environmental conditions such as hypoxia, insufficient nutrition, and glucose deficiency, the acetyl-CoA synthetase 2 (ACSS2)-mediated acetyl-CoA synthesis pathway plays an alternative role to ensure the normal operation of metabolic activities. To investigate the potential effect of the ACSS2 gene on hypoxic adaptation and its regulatory mechanism of gene expression in high-altitude cattle breeds, we analyzed the genetic variations of the ACSS2 gene in five Bos taurus taurus, Bos taurus indicus, hybrid Bos taurus taurus × Bos taurus indicus Chinese cattle breeds, and two Bos grunniens (yak) breeds distributed at different altitudes (95-3850 m). A total of 58 SNPs was detected in seven populations, and abundant genetic variation was found in high-altitude breeds. We identified the bovine ACSS2 core promoter region between g.-682 and g.-264 by using the luciferase assay in FFB and HepG2 cells. We also identified that the high-altitude hypoxia-specific haplotype (CAGTCT) was composed of six highly linked SNPs. The tagSNP g.-473 T>C (rs23) is located in the core promoter of ACSS2 in the Bos taurus taurus and yak breeds. The recombinant plasmid containing rs23 and analyses of luciferase activity of different genotypes showed that the activity of ACSS2 promoter increased significantly when T was mutated to C. We also found a yak-specific SNP rs20 that consists of 12 base insertions (g.-562 ins GAAAGGACCCTA) in the promoter of yak breeds. Luciferase activity analysis showed that the insertion mutant significantly decreased the promoter activity of ACSS2. Hence, ACSS2 may play an important role in the adaptation to high-altitude hypoxia by generating adaptive alleles to influence gene transcription in cattle. These results signify that different genetic variants and haplotypes affect the activity of the core promoter to regulate ACSS2 gene expression and subsequently overcome and adapt to a high-altitude environment within different cattle breeds. Our findings may have important implications for understanding the mechanism of adaptation to high altitude and for application of molecular breeding in Bos species.

Deltamethrin transformation by Bacillus thuringiensis and the associated metabolic pathways

The biological toxicity of deltamethrin at molecular level has been investigated, whereas, the proteome responsive mechanisms of cells under deltamethrin stress at the phylogenetic level are not clear. The proteome expression, transformation-related pathway and regulatory network of Bacillus thuringiensis during the process of deltamethrin transformation were explored using proteomics and metabolomics approaches in the present study. The results showed that deltamethrin was effectively removed by B. thuringiensis within 48 h. The stress responses of B. thuringiensis were activated to resist deltamethrin stress, with significant differential expression of proteins that were primarily involved in the synthesis of DNA and shock proteins, endospore formation, carbon metabolism. The expression patterns of ribosomal proteins confirmed that the transcription and translation of DNA, and biosynthesis of heat shock proteins were inhibited as deltamethrin transformation. The synthesis of oxaloacetate and acetyl-CoA were also hindered, resulting in downregulated expression of carbohydrate metabolism, TCA cycle and energy metabolism. Meanwhile, endospore formation and germination were promoted to resist oxidative stress induced by deltamethrin. These findings imparted novel insight to elucidate underlying stress response mechanisms of the organism under target contaminants stress, and the interaction between deltamethrin transformation and cellular metabolism at the pathway and network levels.

Cholesterol Homeostasis: An In Silico Investigation into How Aging Disrupts Its Key Hepatic Regulatory Mechanisms.

Simple SummaryCardiovascular disease is the leading cause of death worldwide. High blood cholesterol levels are associated with an increased risk of this condition. How high levels of blood cholesterol result in cardiovascular disease during aging is a difficult question to answer. Computational models have been used over the years to help understand this problem, as they are capable of representing complex systems such as this. The aim of this work was to use a computational model of cholesterol metabolism to better understand why, in certain studies involving the oldest old (persons ≥ 85 years), high blood cholesterol levels have been associated with a decreased risk of death. Using our computational model, we found that key age-associated changes to how cholesterol is processed in the liver could be responsible for this observation. The findings from our work contribute to our understanding of cholesterol metabolism in older people and how treatments could be developed in the future to promote healthy aging.The dysregulation of intracellular cholesterol homeostasis is associated with several age-related diseases, most notably cardiovascular disease (CVD). Research in this area has benefitted from using computational modelling to study the inherent complexity associated with the regulation of this system. In addition to facilitating hypothesis exploration, the utility of modelling lies in its ability to represent an array of rate limiting enzymatic reactions, together with multiple feedback loops, which collectively define the dynamics of cholesterol homeostasis. However, to date no model has specifically investigated the effects aging has on this system. This work addresses this shortcoming by explicitly focusing on the impact of aging on hepatic intracellular cholesterol homeostasis. The model was used to investigate the experimental findings that reactive oxygen species induce the total activation of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGCR). Moreover, the model explored the impact of an age-related decrease in hepatic acetyl-CoA acetyltransferase 2 (ACAT2). The model suggested that an increase in the activity of HMGCR does not have as significant an impact on cholesterol homeostasis as a decrease in hepatic ACAT2 activity. According to the model, a decrease in the activity of hepatic ACAT2 raises free cholesterol (FC) and decreases low-density lipoprotein cholesterol (LDL-C) levels. Increased acetyl CoA synthesis resulted in a reduction in the number of hepatic low-density lipoprotein receptors, and increased LDL-C, FC, and cholesterol esters. The rise in LDL-C was restricted by elevated hepatic FC accumulation. Taken together these findings have important implications for healthspan. This is because emerging clinical data suggest hepatic FC accumulation is relevant to the pathogenesis of non-alcoholic fatty liver disease (NAFLD), which is associated with an increased risk of CVD. These pathophysiological changes could, in part, help to explain the phenomenon of increased mortality associated with low levels of LDL-C which have been observed in certain studies involving the oldest old (≥85 years).

Metabolic engineering of <italic>Saccharomyces cerevisiae</italic> chassis

Saccharomyces cerevisiae acts as a bio-foundry for producing natural products. S. cerevisiae ’s intrinsic pathways highlight microbial cell factories to produce pharmaceutics, food additive, and fine chemicals. For high-titer, yield, and conversion of target compounds, the critical point is that fine-tuning and optimizing intracellular metabolic flux in the yeast cell. Acetyl-CoA is the fundamental precursor for central metabolism and heterologous pathway. In comparison, acetyl-CoA compartmentalizes in mitochondria, nucleus, peroxisome subcellular, and cytosol. Most of the pathway enzymes are expressed in S. cerevisiae cytosol. So, we summarized the fine-tuning strategies of acetyl-CoA synthesis in cytosol matrix. The new acetyl-CoA pathway (phosphoketolase, phosphotransacetylase, and acetaldehyde dehydrogenase, PK/PTA-ADA) shows a high conversion ratio from the carbon source. The PK/PTA-ADA pathway presents improved redox balance, limited ATP requirement, and reduced carbon loss to CO2. Moreover, the heterologous ATP-dependent citrate lyase precisely converts mitochondria metabolism into acetyl-CoA. Knock-out acetyl-CoA depletion pathways retain high acetyl-CoA titer in the cytosol. The continuous metabolic engineering on PK/PTA-ADA pathway is expected for higher acetyl-CoA titer. The Mevalonate pathway has been engineered to produce terpenoids in S. cerevisiae . The exogenous HMG-CoA synthase and HMG-CoA reductase boost mevalonate pathway from acetyl-CoA. Further, the diversified terpenoids are generated from C5 unite Isopentenyl diphosphate and its’ isomer dimethylallyl diphosphate. Intrinsic farnesyl diphosphate synthase (Erg20) shows both dimethylallyltransferase (geranyl diphosphate, C10 product) and geranyltransferase (farnesyl diphosphate, C15 product) activity. The F96W/N127W mutations in Erg20 caused steric hindrance for geranyl diphosphate substrate. So, geranyl diphosphate is accumulated to produce C10 terpenoids or meroterpenoids. Moreover, geranylgeranyl diphosphate synthase (CrtE) convert farnesyl diphosphate (C15) into geranylgeranyl diphosphate (C20) for carotene, lycopene, astaxanthin, and taxol, etc. Interestingly, the novel isopentenol utilization pathway has been constructed in E. coli and Y. lipolytica to supply IPP and IMAPP precursors from isopentanol. This alternative pathway could relieve the acetyl-CoA supply burden in the mevalonate pathway. Fatty acid synthase (FAS) produces fatty acid with specific carbon length and derivatives from acetyl-CoA and malonyl-CoA. Acetyl-CoA carboxylase (ACC1) catalyzes acetyl-CoA to produce malonyl-CoA, which is utilized as the carbon unite in fatty acid pathway. Overexpression of ACC1 and mitochondria located isoenzyme Hfa1 significantly improves fatty acid production. FAS in S. cerevisiae is an α6β6 heterodimer protein and these catalytic domains are engineered to produce specific fatty acids. Directed mutations in ketoacyl synthase (KS), acetyltransferase (AT), and malonyl/palmitoyl transferase (MPT) domains engage FAS to generate high-yield short/medium fatty acid (C6 and C8). Furthermore, fatty acid reductase and acyl-ACP reductase convert fatty acid or fatty acyl-ACP to fatty aldehydes, respectively. Aldehyde deformylating oxygenase produces alkanes from fatty aldehydes substrate. Significantly, fatty acid-derived bio hydrocarbons showed closets properties to petroleum fuel. We reviewed the metabolic strategies of acetyl-CoA synthesis, the novel mevalonate pathway and fatty acid synthesis. These strategies may be valuable for producing high-yield terpene and fatty acid derivatives in Saccharomyces cerevisiae .

Ammonia stress decreased biomarker genes of acetoclastic methanogenesis and second peak of production rates during anaerobic digestion of swine manure

Research shows that anaerobic digestion could acclimate to ammonia stress; however, the acclimation remained unaddressed. In this study, evolution of microbial community, functional gene, and pathway was linked with apparent kinetic and performance in unacclimated inoculum under ammonia stress, to deepen understanding of the acclimation. The second peak in production rate demonstrated crucial kinetic changes under ammonia stress. The methane loss was mainly protein in residual COD. Metagenomic showed initial inhibition in all methane metabolism pathways under ammonia stress, and recovery in acetate uptake was the key to ammonia acclimation. The acclimation was found in alternative pathway of Acetyl-CoA (CH3CO-S-CoA) synthesis from acetate, accompanying by syntrophic methanogenesis. Ammonia inhibited acetoclastic methanogenesis by competing CH3-CO-Pi with pta and formed speculative sediment CH3-CO-PO4[NH4]2. Biomarker of methanogenesis kinetic was suggested as mcr, hdr, and mch. The biomarker could indicate acclimation stages to ammonia, empowering anaerobic digestion by early warning of methane loss.

Crystallographic Characterization of the Carbonylated A-Cluster in Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase.

The Wood–Ljungdahl pathway allows for autotrophic bacterial growth on carbon dioxide, with the last step in acetyl-CoA synthesis catalyzed by the bifunctional enzyme carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS). ACS uses a complex Ni–Fe–S metallocluster termed the A-cluster to assemble acetyl-CoA from carbon monoxide, a methyl moiety and coenzyme A. Here, we report the crystal structure of CODH/ACS from Moorella thermoacetica with substrate carbon monoxide bound at the A-cluster, a state previously uncharacterized by crystallography. Direct structural characterization of this state highlights the role of second sphere residues and conformational dynamics in acetyl-CoA assembly, the biological equivalent of the Monsanto process.

Comprehensive transcriptomic and proteomic analyses of antroquinonol biosynthetic genes and enzymes in Antrodia camphorata

Antroquinonol (AQ) has several remarkable bioactivities in acute myeloid leukaemia and pancreatic cancer, but difficulties in the mass production of AQ hamper its applications. Currently, molecular biotechnology methods, such as gene overexpression, have been widely used to increase the production of metabolites. However, AQ biosynthetic genes and enzymes are poorly understood. In this study, an integrated study coupling RNA-Seq and isobaric tags for relative and absolute quantitation (iTRAQ) were used to identify AQ synthesis-related genes and enzymes in Antrodia camphorata during coenzyme Q0-induced fermentation (FM). The upregulated genes related to acetyl-CoA synthesis indicated that acetyl-CoA enters the mevalonate pathway to form the farnesyl tail precursor of AQ. The metE gene for an enzyme with methyl transfer activity provided sufficient methyl groups for AQ structure formation. The CoQ2 and ubiA genes encode p-hydroxybenzoate polyprenyl transferase, linking coenzyme Q0 and the polyisoprene side chain to form coenzyme Q3. NADH is transformed into NAD+ and releases two electrons, which may be beneficial for the conversion of coenzyme Q3 to AQ. Understanding the biosynthetic genes and enzymes of AQ is important for improving its production by genetic means in the future.

Comparison of Glucose, Acetate and Ethanol as Carbon Resource for Production of Poly(3-Hydroxybutyrate) and Other Acetyl-CoA Derivatives.

Poly(3-hydroxybutyrate) (PHB) is a biodegradable and biocompatible thermoplastic, and synthesized from the central metabolite acetyl-CoA. The acetyl-CoA synthesis from glucose presents low atomic economy due to the release of CO2 in pyruvate decarboxylation. As ethanol and acetate can be converted into acetyl-CoA directly, they were used as carbon source for PHB production in this study. The reductase mutant AdhE A267T/E568K was introduced into Escherichia coli to enable growth on ethanol, and acetate utilization was improved by overexpression of acetyl-CoA synthetase ACS. Comparison of the PHB production using glucose, ethanol or acetate as sole carbon source showed that the production and yield from ethanol was much higher than those from glucose and acetate, and metabolome analysis revealed the differences in metabolism of glucose, ethanol and acetate. Furthermore, other acetyl-CoA derived chemicals including 3-hydroxypropionate and phloroglucinol were produced from those three feedstocks, and similar results were achieved, suggesting that ethanol could be a suitable carbon source for the production of acetyl-CoA derivatives.

SenX3-RegX3, an Important Two-Component System, Regulates Strain Growth and Butenyl-spinosyn Biosynthesis in Saccharopolyspora pogona.

SummaryButenyl-spinosyn produced by Saccharopolyspora pogona exhibits strong insecticidal activity and a broad pesticidal spectrum. Currently, important functional genes involved in butenyl-spinosyn biosynthesis remain unknown, which leads to difficulty in efficient understanding of its regulatory mechanism and improving its production by metabolic engineering. Here, we present data supporting a role of the SenX3-RegX3 system in regulating the butenyl-spinosyn biosynthesis. EMSAs and qRT-PCR demonstrated that RegX3 positively controls butenyl-spinosyn production in an indirect way. Integrated proteomic and metabolomic analysis, regX3 deletion not only strengthens the basal metabolic ability of S. pogona in the mid-growth phase but also promotes the flow of the acetyl-CoA produced via key metabolic pathways into the TCA cycle rather than the butenyl-spinosyn biosynthetic pathway, which ultimately leads to continued growth but reduced butenyl-spinosyn production. The strategy demonstrated here may be valuable for revealing the regulatory role of the SenX3-RegX3 system in the biosynthesis of other natural products.

Peroxisomal oxidation of erucic acid suppresses mitochondrial fatty acid oxidation by stimulating malonyl-CoA formation in the rat liver

Feeding of rapeseed (canola) oil with a high erucic acid concentration is known to cause hepatic steatosis in animals. Mitochondrial fatty acid oxidation plays a central role in liver lipid homeostasis, so it is possible that hepatic metabolism of erucic acid might decrease mitochondrial fatty acid oxidation. However, the precise mechanistic relationship between erucic acid levels and mitochondrial fatty acid oxidation is unclear. Using male Sprague-Dawley rats, along with biochemical and molecular biology approaches, we report here that peroxisomal β-oxidation of erucic acid stimulates malonyl-CoA formation in the liver and thereby suppresses mitochondrial fatty acid oxidation. Excessive hepatic uptake and peroxisomal β-oxidation of erucic acid resulted in appreciable peroxisomal release of free acetate, which was then used in the synthesis of cytosolic acetyl-CoA. Peroxisomal metabolism of erucic acid also remarkably increased the cytosolic NADH/NAD+ ratio, suppressed sirtuin 1 (SIRT1) activity, and thereby activated acetyl-CoA carboxylase, which stimulated malonyl-CoA biosynthesis from acetyl-CoA. Chronic feeding of a diet including high-erucic-acid rapeseed oil diminished mitochondrial fatty acid oxidation and caused hepatic steatosis and insulin resistance in the rats. Of note, administration of a specific peroxisomal β-oxidation inhibitor attenuated these effects. Our findings establish a cross-talk between peroxisomal and mitochondrial fatty acid oxidation. They suggest that peroxisomal oxidation of long-chain fatty acids suppresses mitochondrial fatty acid oxidation by stimulating malonyl-CoA formation, which might play a role in fatty acid-induced hepatic steatosis and related metabolic disorders.

Glutamine Metabolism Controls Chondrocyte Identity and Function.

Correct functioning of chondrocytes is crucial for long bone growth and fracture repair. These cells are highly anabolic but survive and function in an avascular environment, implying specific metabolic requirements that are, however, poorly characterized. Here, we show that chondrocyte identity and function are closely linked with glutamine metabolism in a feedforward process. The master chondrogenic transcription factor SOX9 stimulates glutamine metabolism by increasing glutamine consumption and levels of glutaminase 1 (GLS1), a rate-controlling enzyme in this pathway. Consecutively, GLS1 action is critical for chondrocyte properties and function via a tripartite mechanism. First, glutamine controls chondrogenic gene expression epigenetically through glutamate dehydrogenase-dependent acetyl-CoA synthesis, necessary for histone acetylation. Second, transaminase-mediated aspartate synthesis supports chondrocyte proliferation and matrix synthesis. Third, glutamine-derived glutathione synthesis avoids harmful reactive oxygen species accumulation and allows chondrocyte survival in the avascular growth plate. Collectively, our study identifies glutamine as a metabolic regulator of cartilage fitness during bone development.

RETRACTED ARTICLE: Acetyl-CoA synthetase 3 promotes bladder cancer cell growth under metabolic stress

Cancer cells adapt to nutrient-deprived tumor microenvironment during progression via regulating the level and function of metabolic enzymes. Acetyl-coenzyme A (AcCoA) is a key metabolic intermediate that is crucial for cancer cell metabolism, especially under metabolic stress. It is of special significance to decipher the role acetyl-CoA synthetase short chain family (ACSS) in cancer cells confronting metabolic stress. Here we analyzed the generation of lipogenic AcCoA in bladder cancer cells under metabolic stress and found that in bladder urothelial carcinoma (BLCA) cells, the proportion of lipogenic AcCoA generated from glucose were largely reduced under metabolic stress. Our results revealed that ACSS3 was responsible for lipogenic AcCoA synthesis in BLCA cells under metabolic stress. Interestingly, we found that ACSS3 was required for acetate utilization and histone acetylation. Moreover, our data illustrated that ACSS3 promoted BLCA cell growth. In addition, through analyzing clinical samples, we found that both mRNA and protein levels of ACSS3 were dramatically upregulated in BLCA samples in comparison with adjacent controls and BLCA patients with lower ACSS3 expression were entitled with longer overall survival. Our data revealed an oncogenic role of ACSS3 via regulating AcCoA generation in BLCA and provided a promising target in metabolic pathway for BLCA treatment.