Sexually dimorphic acetyl-CoA biosynthesis and utilization in response to drought and exogenous acetic acid.

Female willows exhibit greater drought tolerance and benefit more from exogenous acetic acid (AA)-improved drought tolerance than males. However, the potential mechanisms driving these sex-specific responses remain unclear. To comprehensively investigate the sexually dimorphic responsive mechanisms of willows to drought and exogenous AA, here, we performed physiological, proteomic, Lys-acetylproteomic, and transgenic analyses in female and male Salix myrtillacea exposed to drought and AA-applicated drought treatments, focusing on protein abundance and lysine acetylation (LysAc) changes. Drought-tolerant females suffered less drought-induced photosynthetic and oxidative damage, did not activate AA and acetyl-CoA biosynthesis, TCA cycle, fatty acid metabolism, and jasmonic acid signaling as strongly as drought-sensitive males. Exogenous AA caused overaccumulation of endogenous AA and inhibition of acetyl-CoA biosynthesis and utilization in males. However, exogenous AA greatly enhanced acetyl-CoA biosynthesis and utilization and further enhanced drought performance of females, possibly determining that AA improved drought tolerance more in females than in males. Interestingly, overexpression of acetyl-CoA synthetase (ACS) could reprogram fatty acids, increase LysAc levels, and improve drought tolerance, highlighting the involvement of ACS-derived acetyl-CoA in drought responses. In addition, drought and exogenous AA induced sexually dimorphic LysAc associated with histones, transcription factors, and metabolic enzymes in willows. Especially, exogenous AA may greatly improve the photosynthetic capacity of S. myrtillacea males by decreasing LysAc levels and increasing the abundances of photosynthetic proteins. While hyperacetylation in glycolysis, TCA cycle, and fatty acid biosynthesis potentially possibly serve as negative feedback to acclimate acetyl-CoA biosynthesis and utilization in drought-stressed males and AA-applicated females. Thus, acetyl-CoA biosynthesis and utilization determine the sexually dimorphic responses of S. myrtillacea to drought and exogenous AA.

N-glycosylation of SCAP exacerbates hepatocellular inflammation and lipid accumulation via ACSS2-mediated histone H3K27 acetylation.

Sterol regulatory element binding protein (SREBP) cleavage-activating protein (SCAP) is a widely expressed membrane glycoprotein that acts as an important modulator of lipid metabolism and inflammatory stress. N-glycosylation of SCAP has been suggested to modulate cancer development, but its role in nonalcoholic steatohepatitis (NASH) is poorly understood. In this study, the N-glycosylation of SCAP was analyzed by using sequential trypsin proteolysis and glycosidase treatment. The liver cell lines expressing wild-type and N-glycosylation sites mutated SCAP were constructed to investigate the N-glycosylation role of SCAP in regulating inflammation and lipid accumulation as well as the underlying mechanisms. The hepatic SCAP protein levels were significantly increased in C57BL/6J mice fed with Western diet and sugar water (WD + SW) and diabetic db/db mice, which exhibited typical liver steatosis and inflammation accompanied with hyperglycemia. In vitro, the enhanced N-glycosylation by high glucose increased the protein stability of SCAP and hence increased its total protein levels, whereas the ablation of N-glycosylation significantly decreased SCAP protein stability and alleviated lipid accumulation and inflammation in hepatic cell lines. Mechanistically, SCAP N-glycosylation increased not only the SREBP-1-mediated acetyl-CoA synthetase 2 (ACSS2) transcription but also the AMPK-mediated S659 phosphorylation of ACCS2 protein, causing the enhanced ACSS2 levels in nucleus and hence increasing the histone H3K27 acetylation (H3K27ac), which is a key epigenetic modification associated with NASH. Modulating ACSS2 expression or its location in the nuclear abolished the effects of SCAP N-glycosylation on H3K27ac and lipid accumulation and inflammation. In conclusion, SCAP N-glycosylation aggravates inflammation and lipid accumulation through enhancing ACSS2-mediated H3K27ac in hepatocytes.NEW & NOTEWORTHY N-glycosylation of SCAP exacerbates inflammation and lipid accumulation in hepatocytes through ACSS2-mediated H3K27ac. Our data suggest that SCAP N-glycosylation plays a key role in regulating histone H3K27 acetylation and targeting SCAP N-glycosylation may be a new strategy for treating nonalcoholic steatohepatitis (NASH).

Acetyl-CoA synthetase 2 induces pyroptosis and inflammation of renal epithelial tubular cells in sepsis-induced acute kidney injury by upregulating the KLF5/NF-κB pathway.

BACKGROUND: Pyroptosis of the renal tubular epithelial cells (RTECs) and interstitial inflammation are central pathological characteristics of acute kidney injury (AKI). Pyroptosis acts as a pro-inflammatory form of programmed cell death and is mainly dependent on activation of the NLRP3 inflammasome. Previous studies revealed that acetyl-CoA synthetase 2 (ACSS2) promotes inflammation during metabolic stress suggesting that ACSS2 might regulate pyroptosis and inflammatory responses of RTECs in AKI. METHODS AND RESULTS: The expression of ACSS2 was found to be significantly increased in the renal epithelial cells of mice with lipopolysaccharide (LPS)-induced AKI. Pharmacological and genetic strategies demonstrated that ACSS2 regulated NLRP3-mediated caspase-1 activation and pyroptosis through the stimulation of the KLF5/NF-κB pathway in RTECs. The deletion of ACSS2 attenuated renal tubular pathological injury and inflammatory cell infiltration in an LPS-induced mouse model, and ACSS2-deficient mice displayed impaired NLRP3 activation-mediated pyroptosis and decreased IL-1ß production in response to the LPS challenge. In HK-2 cells, ACSS2 deficiency suppressed NLRP3-mediated caspase-1 activation and pyroptosis through the downregulation of the KLF5/NF-κB pathway. The KLF5 inhibitor ML264 suppressed NF-κB activity and NLRP3-mediated caspase-1 activation, thus protecting HK-2 cells from LPS-induced pyroptosis. CONCLUSION: Our results suggested that ACSS2 regulates activation of the NLRP3 inflammasome and pyroptosis by inducing the KLF5/NF-κB pathway in RTECs. These results identified ACSS2 as a potential therapeutic target in AKI.

Acetyl-CoA synthetase 2 promotes diabetic renal tubular injury in mice by rewiring fatty acid metabolism through SIRT1/ChREBP pathway.

Diabetic nephropathy (DN) is characterized by chronic low-grade renal inflammatory responses, which greatly contribute to disease progression. Abnormal glucose metabolism disrupts renal lipid metabolism, leading to lipid accumulation, nephrotoxicity, and subsequent aseptic renal interstitial inflammation. In this study, we investigated the mechanisms underlying the renal inflammation in diabetes, driven by glucose-lipid metabolic rearrangement with a focus on the role of acetyl-CoA synthetase 2 (ACSS2) in lipid accumulation and renal tubular injury. Diabetic models were established in mice by the injection of streptozotocin and in human renal tubular epithelial HK-2 cells cultured under a high glucose (HG, 30 mmol/L) condition. We showed that the expression levels of ACSS2 were significantly increased in renal tubular epithelial cells (RTECs) from the diabetic mice and human diabetic kidney biopsy samples, and ACSS2 was co-localized with the pro-inflammatory cytokine IL-1ß in RTECs. Diabetic ACSS2-deficient mice exhibited reduced renal tubular injury and inflammatory responses. Similarly, ACSS2 knockdown or inhibition of ACSS2 by ACSS2i (10 µmol/L) in HK-2 cells significantly ameliorated HG-induced inflammation, mitochondrial stress, and fatty acid synthesis. Molecular docking revealed that ACSS2 interacted with Sirtuin 1 (SIRT1). In HG-treated HK-2 cells, we demonstrated that ACSS2 suppressed SIRT1 expression and activated fatty acid synthesis by modulating SIRT1-carbohydrate responsive element binding protein (ChREBP) activity, leading to mitochondrial oxidative stress and inflammation. We conclude that ACSS2 promotes mitochondrial oxidative stress and renal tubular inflammation in DN by regulating the SIRT1-ChREBP pathway. This highlights the potential therapeutic value of pharmacological inhibition of ACSS2 for alleviating renal inflammation and dysregulation of fatty acid metabolic homeostasis in DN. Metabolic inflammation in the renal region, driven by lipid metabolism disorder, is a key factor in renal injury in diabetic nephropathy (DN). Acetyl-CoA synthetase 2 (ACSS2) is abundantly expressed in renal tubular epithelial cells (RTECs) and highly upregulated in diabetic kidneys. Deleting ACSS2 reduces renal fatty acid accumulation and markers of renal tubular injury in diabetic mice. We demonstrate that ACSS2 deletion inhibits ChREBP-mediated fatty acid lipogenesis, mitochondrial oxidative stress, and inflammatory response in RTECs, which play a major role in the progression of diabetic renal tubular injury in the kidney. These findings support the potential use of ACSS2 inhibitors in treating patients with DN.

The Roles of Coenzyme A Binding Pocket Residues in Short and Medium Chain Acyl-CoA Synthetases.

Short- and medium-chain acyl-CoA synthetases catalyze similar two-step reactions in which acyl substrate and ATP bind to form an enzyme-bound acyl-adenylate, then CoA binds for formation of the acyl-CoA product. We investigated the roles of active site residues in CoA binding in acetyl-CoA synthetase (Acs) and a medium-chain acyl-CoA synthetase (Macs) that uses 2-methylbutyryl-CoA. Three highly conserved residues, Arg193, Arg528, and Arg586 of Methanothermobacter thermautotrophicus Acs (AcsMt), are predicted to form important interactions with the 5'- and 3'-phosphate groups of CoA. Kinetic characterization of AcsMt variants altered at each of these positions indicates these Arg residues play a critical role in CoA binding and catalysis. The predicted CoA binding site of Methanosarcina acetivorans Macs (MacsMa) is structurally more closely related to that of 4-chlorobenzoate:coenzyme A ligase (CBAL) than Acs. Alteration of MacsMa residues Tyr460, Arg490, Tyr525, and Tyr527, which correspond to CoA binding pocket residues in CBAL, strongly affected CoA binding and catalysis without substantially affecting acyl-adenylate formation. Both enzymes discriminate between 3'-dephospho-CoA and CoA, indicating interaction between the enzyme and the 3'-phosphate group is important. Alteration of MacsMa residues Lys461 and Lys519, located at positions equivalent to AcsMt Arg528 and Arg586, respectively, had only a moderate effect on CoA binding and catalysis. Overall, our results indicate the active site architecture in AcsMt and MacsMa differs even though these enzymes catalyze mechanistically similar reactions. The significance of this study is that we have delineated the active site architecture with respect to CoA binding and catalysis in this important enzyme superfamily.

Acetate and Acetyl-CoA Metabolism of ANME-2 Anaerobic Archaeal Methanotrophs.

Acetyl-CoA synthetase (ACS) and acetate ligase (ACD) are widespread among microorganisms, including archaea, and play an important role in their carbon metabolism, although only a few of these enzymes have been characterized. Anaerobic methanotrophs (ANMEs) have been reported to convert methane anaerobically into CO2, polyhydroxyalkanoate, and acetate. Furthermore, it has been suggested that they might be able to use acetate for anabolism or aceticlastic methanogenesis. To better understand the potential acetate metabolism of ANMEs, we characterized an ACS from ANME-2a as well as an ACS and an ACD from ANME-2d. The conversion of acetate into acetyl-CoA (Vmax of 8.4 µmol mg-1 min-1 and Km of 0.7 mM acetate) by the monomeric 73.8-kDa ACS enzyme from ANME-2a was more favorable than the formation of acetate from acetyl-CoA (Vmax of 0.4 µmol mg-1 min-1 and Km of 0.2 mM acetyl-CoA). The monomeric 73.4-kDa ACS enzyme from ANME-2d had similar Vmax values for both directions (Vmax,acetate of 0.9 µmol mg-1 min-1 versus Vmax,acetyl-CoA of 0.3 µmol mg-1 min-1). The heterotetrameric ACD enzyme from ANME-2d was active solely in the acetate-producing direction. Batch incubations of an enrichment culture dominated by ANME-2d fed with 13C2-labeled acetate produced 3 µmol of [13C]methane in 7 days, suggesting that this anaerobic methanotroph might have the potential to reverse its metabolism and perform aceticlastic methanogenesis using ACS to activate acetate albeit at low rates (2 nmol g [dry weight]-1 min-1). Together, these results show that ANMEs may have the potential to use acetate for assimilation as well as to use part of the surplus acetate for methane production. IMPORTANCE Acetyl-CoA plays a key role in carbon metabolism and is found at the junction of many anabolic and catabolic reactions. This work describes the biochemical properties of ACS and ACD enzymes from ANME-2 archaea. This adds to our knowledge of archaeal ACS and ACD enzymes, only a few of which have been characterized to date. Furthermore, we validated the in situ activity of ACS in ANME-2d, showing the conversion of acetate into methane by an enrichment culture dominated by ANME-2d.

Modulation of plant acetyl-CoA synthetase activity by post-translational lysine acetylation.

Acetyl-CoA synthetase (ACS) is one of several enzymes that generate the key metabolic intermediate, acetyl-CoA. In microbes and mammals ACS activity is regulated by the post-translational acetylation of a key lysine residue. ACS in plant cells is part of a two-enzyme system that maintains acetate homeostasis, but its post-translational regulation is unknown. This study demonstrates that the plant ACS activity can be regulated by the acetylation of a specific lysine residue that is positioned in a homologous position as the microbial and mammalian ACS sequences that regulates ACS activity, occurring in the middle of a conserved motif, near the carboxyl-end of the protein. The inhibitory effect of the acetylation of residue Lys-622 of the Arabidopsis ACS was demonstrated by site-directed mutagenesis of this residue, including its genetic substitution with the non-canonical N-ε-acetyl-lysine residue. This latter modification lowered the catalytic efficiency of the enzyme by a factor of more than 500-fold. Michaelis-Menten kinetic analysis of the mutant enzyme indicates that this acetylation affects the first half-reaction of the ACS catalyzed reaction, namely, the formation of the acetyl adenylate enzyme intermediate. The post-translational acetylation of the plant ACS could affect acetate flux in the plastids and overall acetate homeostasis.

Acss2 Deletion Reveals Functional Versatility via Tissue-Specific Roles in Transcriptional Regulation.

The coordination of cellular biological processes is regulated in part via metabolic enzymes acting to match cellular metabolism to current conditions. The acetate activating enzyme, acyl-coenzyme A synthetase short-chain family member 2 (Acss2), has long been considered to have a predominantly lipogenic function. More recent evidence suggests that this enzyme has regulatory functions in addition to its role in providing acetyl-CoA for lipid synthesis. We used Acss2 knockout mice (Acss2-/-) to further investigate the roles this enzyme plays in three physiologically distinct organ systems that make extensive use of lipid synthesis and storage, including the liver, brain, and adipose tissue. We examined the resulting transcriptomic changes resulting from Acss2 deletion and assessed these changes in relation to fatty acid constitution. We find that loss of Acss2 leads to dysregulation of numerous canonical signaling pathways, upstream transcriptional regulatory molecules, cellular processes, and biological functions, which were distinct in the liver, brain, and mesenteric adipose tissues. The detected organ-specific transcriptional regulatory patterns reflect the complementary functional roles of these organ systems within the context of systemic physiology. While alterations in transcriptional states were evident, the loss of Acss2 resulted in few changes in fatty acid constitution in all three organ systems. Overall, we demonstrate that Acss2 loss institutes organ-specific transcriptional regulatory patterns reflecting the complementary functional roles of these organ systems. Collectively, these findings provide further confirmation that Acss2 regulates key transcription factors and pathways under well-fed, non-stressed conditions and acts as a transcriptional regulatory enzyme.

Development of a new quantification method of Sarcocystis cruzi through detection of the acetyl-CoA synthetase gene.

Sarcocystis cruzi is a member of the genus Sarcocystis, infecting bovine animals such as cattle and bison as intermediate hosts, and canids such as dogs and raccoon dogs as definitive hosts. Acute sarcocystosis of S. cruzi causes occasional symptoms in cattle, including weight loss, reduced milk production, abortions, and death, and similar to other Sarcocystis species can potentially cause food poisoning in humans when raw or undercooked infected cattle meat is consumed. Despite these issues, genetic information on S. cruzi is scarce, and there is no specific quantitative method for the detection and quantification of the parasite in infected cattle. In this study, we aimed to develop a method based on high-throughput sequencing of S. cruzi genome and transcriptome that specifically and quantitatively detects the S. cruzi acetyl-CoA synthetase gene (ScACS). Cardiac muscles were collected from slaughterhouses in Saitama Prefecture to obtain sarcocysts from which DNA and RNA were extracted for the high-throughput sequencing. Using the sequences, we developed a specific quantitative PCR assay which could distinguish S. cruzi ACS from that of Toxoplasma gondii by taking advantage of the differences in their exon/intron organizations and validated the assay with the microscopic counting of the S. cruzi bradyzoites. Thus, this assay will be useful for future studies of S. cruzi pathogenesis in cattle and for the surveillance of infected animals, thereby easing public health concerns.

Emerging insights into keratin 7 roles in tumor progression and metastasis of cancers.

Keratin 7 (KRT7), also known as cytokeratin-7 (CK-7) or K7, constitutes the principal constituent of the intermediate filament cytoskeleton and is primarily expressed in the simple epithelia lining the cavities of the internal organs, glandular ducts, and blood vessels. Various pathological conditions, including cancer, have been linked to the abnormal expression of KRT7. KRT7 overexpression promotes tumor progression and metastasis in different human cancers, although the mechanisms of these processes caused by KRT7 have yet to be established. Studies have indicated that the suppression of KRT7 leads to rapid regression of tumors, highlighting the potential of KRT7 as a novel candidate for therapeutic interventions. This review aims to delineate the various roles played by KRT7 in the progression and metastasis of different human malignancies and to investigate its prognostic significance in cancer treatment. Finally, the differential diagnosis of cancers based on the KRT7 is emphasized.

Succinyl-CoA:acetate CoA-transferase functioning in the oxidative tricarboxylic acid cycle in Desulfurella acetivorans.

Desulfurella acetivorans is a strictly anaerobic sulfur-reducing deltaproteobacterium that possesses a very dynamic metabolism with the ability to revert the citrate synthase version of the tricarboxylic acid (TCA) cycle for autotrophic growth (reversed oxidative TCA cycle) or to use it for acetate oxidation (oxidative TCA cycle). Here we show that for heterotrophic growth on acetate D. acetivorans uses a modified oxidative TCA cycle that was first discovered in acetate-oxidizing sulfate reducers in which a succinyl-CoA:acetate CoA-transferase catalyzes the conversion of succinyl-CoA to succinate, coupled with the activation of acetate to acetyl-CoA. We identified the corresponding enzyme in this bacterium as the AHF96498 gene product and characterized it biochemically. Our phylogenetic analysis of CoA-transferases revealed that the CoA-transferase variant of the oxidative TCA cycle has convergently evolved several times in different bacteria. Its functioning is especially important for anaerobes, as it helps to increase the energetic efficiency of the pathway by using one enzyme for two enzymatic reactions and by allowing to spend just one ATP equivalent for acetate activation.

Negative regulation of the acsA1 gene encoding the major acetyl-CoA synthetase by cAMP receptor protein in Mycobacterium smegmatis.

Acetyl-CoA synthetase (ACS) is the enzyme that irreversibly catalyzes the synthesis of acetyl-CoA from acetate, CoA-SH, and ATP via acetyl-AMP as an intermediate. In this study, we demonstrated that AcsA1 (MSMEG_6179) is the predominantly expressed ACS among four ACSs (MSMEG_6179, MSMEG_0718, MSMEG_3986, and MSMEG_5650) found in Mycobacterium smegmatis and that a deletion mutation of acsA1 in M. smegmatis led to its compromised growth on acetate as the sole carbon source. Expression of acsA1 was demonstrated to be induced during growth on acetate as the sole carbon source. The acsA1 gene was shown to be negatively regulated by Crp1 (MSMEG_6189) that is the major cAMP receptor protein (CRP) in M. smegmatis. Using DNase I footprinting analysis and site-directed mutagenesis, a CRP-binding site (GGTGA-N6-TCACA) was identified in the upstream regulatory region of acsA1, which is important for repression of acsA1 expression. We also demonstrated that inhibition of the respiratory electron transport chain by inactivation of the major terminal oxidase, aa3 cytochrome c oxidase, led to a decrease in acsA1 expression probably through the activation of CRP. In conclusion, AcsA1 is the major ACS in M. smegmatis and its gene is under the negative regulation of Crp1, which contributes to some extent to the induction of acsA1 expression under acetate conditions. The growth of M. smegmatis is severely impaired on acetate as the sole carbon source under respiration-inhibitory conditions.

Coordinated expression of acetyl CoA synthetase and the ace operon enzymes in Escherichia coli in preparation for adaptation to acetate.

Successful adaptation of Escherichia coli to constant environmental challenges demands the operation of a wide range of regulatory control mechanisms, some of which are global, while others are specific. Here, we show that the ability of acetate-negative phenotype strains of E. coli devoid of acetate kinase (AK) and phosphotransacetylase (PTA) to assimilate acetate when challenged at the end of growth on acetogenic substrates is explicable by the co-expression of acetyl CoA-synthetase (AcCoA-S) and acetate permease (AP). Furthermore, mRNA transcript measurements for acs and aceA, together with the enzymatic activities of their corresponding enzymes, acetyl CoA synthetase (AcCoA-S) and isocitrate lyase (ICL), clearly demonstrate that the expression of the two enzymes is inextricably linked and triggered in response to growth rate threshold signal (0.4 h-1± 0.03: n4). Interestingly, further restriction of carbon supply to the level of starvation led to the repression of acs (AcCoA-S), ackA (AK) and pta (PTA). Further, we provide evidence that the reaction sequence catalysed by PTA, AK and AcCoA-S is not in operation at low growth rates and that the reaction catalysed by AcCoA-S is not merely an ATP-dissipating reaction but rather advantageous, as it elevates the available free energy (ΔG°) in central metabolism. Moreover, the transcriptomic data reinforce the view that the expression of PEP carboxykinase is essential in gluconeogenic phenotypes.

Physiological and Molecular Aspects of Two Thymus Species Differently Sensitive to Drought Stress.

Drought is one of the most important threats to plants and agriculture. Here, the effects of four drought levels (90%, 55%, 40%, and 25% field capacity) on the relative water content (RWC), chlorophyll and carotenoids levels, and mRNA gene expression of metabolic enzymes in Thymus vulgaris (as sensitive to drought) and Thymus kotschyanus (as a drought-tolerant species) were evaluated. The physiological results showed that the treatment predominantly affected the RWC, chlorophyll, and carotenoids content. The gene expression analysis demonstrated that moderate and severe drought stress had greater effects on the expression of histone deacetylase-6 (HDA-6) and acetyl-CoA synthetase in both Thymus species. Pyruvate decarboxylase-1 (PDC-1) was upregulated in Thymus vulgaris at high drought levels. Finally, succinyl CoA ligase was not affected by drought stress in either species. Data confirmed water stress is able to alter the gene expression of specific enzymes. Furthermore, our results suggest that PDC-1 expression is independent from HDA-6 and the increased expression of ACS can be due to the activation of new pathways involved in carbohydrate production.

Chemogenomics identifies acetyl-coenzyme A synthetase as a target for malaria treatment and prevention.

We identify the Plasmodium falciparum acetyl-coenzyme A synthetase (PfAcAS) as a druggable target, using genetic and chemical validation. In vitro evolution of resistance with two antiplasmodial drug-like compounds (MMV019721 and MMV084978) selects for mutations in PfAcAS. Metabolic profiling of compound-treated parasites reveals changes in acetyl-CoA levels for both compounds. Genome editing confirms that mutations in PfAcAS are sufficient to confer resistance. Knockdown studies demonstrate that PfAcAS is essential for asexual growth, and partial knockdown induces hypersensitivity to both compounds. In vitro biochemical assays using recombinantly expressed PfAcAS validates that MMV019721 and MMV084978 directly inhibit the enzyme by preventing CoA and acetate binding, respectively. Immunolocalization studies reveal that PfAcAS is primarily localized to the nucleus. Functional studies demonstrate inhibition of histone acetylation in compound-treated wild-type, but not in resistant parasites. Our findings identify and validate PfAcAS as an essential, druggable target involved in the epigenetic regulation of gene expression.

Plasmodium falciparum Acetyl-CoA Synthetase Is Essential for Parasite Intraerythrocytic Development and Chromatin Modification.

The malaria parasite Plasmodium falciparum possesses a unique Acetyl-CoA Synthetase (PfACS), which provides acetyl moieties for different metabolic and regulatory cellular pathways. We characterized PfACS and studied its role focusing on epigenetic modifications using the var gene family as reporter genes. For this, mutant lines to modulate plasmodial ACS expression by degron-mediated protein degradation and ribozyme-induced transcript decay were created. Additionally, an inhibitor of the human Acetyl-CoA Synthetase 2 was tested for its effectiveness in interfering with PfACS. The knockdown of PfACS or its inhibition resulted in impaired parasite growth. Decreased levels of PfACS also led to differential histone acetylation patterns, altered variant gene expression, and concomitantly decreased cytoadherence of infected red blood cells containing knocked-down parasites. Further, ChIP analysis revealed the presence of PfACS in many loci in ring stage parasites, underscoring its involvement in the regulation of chromatin. Due to its central function in the plasmodial metabolism and significant differences to human ACS, PfACS is an interesting target for drug development.

Construction of acetyl-CoA and DBAT hybrid metabolic pathway for acetylation of 10-deacetylbaccatin III to baccatin III.

10-Deacetylbaccatin III (10-DAB) C10 acetylation is an indispensable procedure for Taxol semi-synthesis, which often requires harsh conditions. 10-Deacetylbaccatin III-10-ß-O-acetyltransferase (DBAT) catalyzes the acetylation but acetyl-CoA supply remains a key limiting factor. Here we refactored the innate biosynthetic pathway of acetyl-CoA in Escherichia coli and obtained a chassis with acetyl-CoA productivity over three times higher than that of the host cell. Then, we constructed a microbial cell factory by introducing DBAT gene into this chassis for efficiently converting 10-DAB into baccatin III. We found that baccatin III could be efficiently deacetylated into 10-DAB by DBAT with CoASH and K+ under alkaline condition. Thus, we fed acetic acid to the engineered strain both for serving as a substrate of acetyl-CoA biosynthesis and for alleviating the deacetylation of baccatin III. The fermentation conditions were optimized and the baccatin III titers reached 2, 3 and 4.6 g/L, respectively, in a 3-L bioreactor culture when 2, 3 and 6 g/L of 10-DAB were supplied. Our study provides an environment-friendly approach for the large scale 10-DAB acetylation without addition of acetyl-CoA in the industrial Taxol semi-synthesis. The finding of DBAT deacetylase activity may broaden its application in the structural modification of pharmaceutically important lead compounds.

Increasing the heterologous production of spinosad in Streptomyces albus J1074 by regulating biosynthesis of its polyketide skeleton.

Spinosyns are natural broad-spectrum biological insecticides with a double glycosylated polyketide structure that are produced by aerobic fermentation of the actinomycete, Saccharopolyspora spinosa. However, their large-scale overproduction is hindered by poorly understood bottlenecks in optimizing the original strain, and poor adaptability of the heterologous strain to the production of spinosyn. In this study, we genetically engineered heterologous spinosyn-producer Streptomyces albus J1074 and optimized the fermentation to improve the production of spinosad (spinosyn A and spinosyn D) based on our previous work. We systematically investigated the result of overexpressing polyketide synthase genes (spnA, B, C, D, E) using a constitutive promoter on the spinosad titer in S. albus J1074. The supply of polyketide synthase precursors was then increased to further improve spinosad production. Finally, increasing or replacing the carbon source of the culture medium resulted in a final spinosad titer of ∼70 mg/L, which is the highest titer of spinosad achieved in heterologous Streptomyces species. This research provides useful strategies for efficient heterologous production of natural products.

Mammalian acetate-dependent acetyl CoA synthetase 2 contains multiple protein destabilization and masking elements.

Besides contributing to anabolism, cellular metabolites serve as substrates or cofactors for enzymes and may also have signaling functions. Given these roles, multiple control mechanisms likely ensure fidelity of metabolite-generating enzymes. Acetate-dependent acetyl CoA synthetases (ACS) are de novo sources of acetyl CoA, a building block for fatty acids and a substrate for acetyltransferases. Eukaryotic acetate-dependent acetyl CoA synthetase 2 (Acss2) is predominantly cytosolic, but is also found in the nucleus following oxygen or glucose deprivation, or upon acetate exposure. Acss2-generated acetyl CoA is used in acetylation of Hypoxia-Inducible Factor 2 (HIF-2), a stress-responsive transcription factor. Mutation of a putative nuclear localization signal in endogenous Acss2 abrogates HIF-2 acetylation and signaling, but surprisingly also results in reduced Acss2 protein levels due to unmasking of two protein destabilization elements (PDE) in the Acss2 hinge region. In the current study, we identify up to four additional PDE in the Acss2 hinge region and determine that a previously identified PDE, the ABC domain, consists of two functional PDE. We show that the ABC domain and other PDE are likely masked by intramolecular interactions with other domains in the Acss2 hinge region. We also characterize mice with a prematurely truncated Acss2 that exposes a putative ABC domain PDE, which exhibits reduced Acss2 protein stability and impaired HIF-2 signaling. Finally, using primary mouse embryonic fibroblasts, we demonstrate that the reduced stability of select Acss2 mutant proteins is due to a shortened half-life, which is a result of enhanced degradation via a nonproteasome, nonautophagy pathway.

Sirtuin-Dependent Reversible Lysine Acetylation Controls the Activity of Acetyl Coenzyme A Synthetase in Campylobacter jejuni.

Posttranslational modifications are mechanisms for rapid control of protein function used by cells from all domains of life. Acetylation of the epsilon amino group (Nε) of an active-site lysine of the AMP-forming acetyl coenzyme A (acetyl-CoA) synthetase (Acs) enzyme is the paradigm for the posttranslational control of the activity of metabolic enzymes. In bacteria, this active-site lysine of Acs enzymes can be modified by a number of different GCN5-type N-acetyltransferases (GNATs). Acs activity is lost as a result of acetylation and is restored by deacetylation. Using a heterologous host, we show that Campylobacter jejuni NCTC11168 synthesizes enzymes that control Acs function by reversible lysine acetylation (RLA). This work validates the function of gene products encoded by the cj1537c, cj1715, and cj1050c loci, namely, the AMP-forming acetate-CoA ligase (CjAcs), a type IV GCN5-type lysine acetyltransferase (GNAT [CjLatA]), and a NAD+-dependent (class III) sirtuin deacylase (CjCobB), respectively. To our knowledge, these are the first in vivo and in vitro data on C. jejuni enzymes that control the activity of CjAcs. IMPORTANCE This work provides the experimental evidence needed to support the assignment of function to three key enzymes, two of which control the reversible posttranslational modification of an active-site lysyl residue of the central metabolic enzyme acetyl-CoA synthetase (CjAcs). We can now generate Campylobacter jejuni mutant strains defective in these functions, so we can establish the conditions in which this mode of regulation of CjAcs is triggered in this bacterium. Such knowledge may provide new therapeutic strategies for the control of this pathogen.