ATP Citrate Lyase Gene Research Articles

The prognostic value and its relationship with immune infiltration of ACLY in clear cell renal cell carcinoma

ATP citrate lyase (ACLY) is activated in various cancers, but its role in clear cell renal cell carcinoma (ccRCC) remains poorly understood. Herein, we investigated the prognostic role and potential mechanism of ACLY in ccRCC. The expression profile of ACLY in ccRCC was explored using Gene Expression Profiling Interactive Analysis 2 (GEPIA2), Gene Expression Omnibus (GEO), UALCAN and western blotting assays. The prognosis was investigated using immunohistochemistry (IHC) and Kaplan–Meier plotter assays. The relationship with immune infiltration was further evaluated using Tumor Immune Estimation Resource 2 (TIMER2) and Tumor Immune System Interactions and DrugBank (TISIDB) databases, respectively. Further biological function of ACLY in ccRCC pathogenesis was explored using in vitro experiments. ACLY level was higher in ccRCC than adjacent kidney tissues, and Kaplan–Meier survival analysis showed ACLY mRNA or protein were predictors of poor prognosis in ccRCC patients. Importantly, we reported for the first time that ACLY gene expression was significantly correlated with numerous immune cells and immune inhibitors in ccRCC. ACLY inhibition significantly impaired cell proliferation, induced cell apoptosis, attenuated cell migration, decreased lipid droplets formation, and suppressed epithelial-mesenchymal transition (EMT) of ccRCC. Moreover, these effects might be acted through mammalian target of rapamycin complex 1 (mTORC1) pathway. Collectively, ACLY was not only implicated in ccRCC tumorigenesis and progression, but also potentially interacted with immune infiltration and mTORC1 pathway. Our findings may provide a novel therapeutic strategy by targeting ACLY for ccRCC treatment.

ATP Citrate Lyase Supports Cardiac Function and NAD+/NADH Balance And Is Depressed in Human Heart Failure.

ATP-citrate lyase (ACLY) converts citrate into acetyl-CoA and oxaloacetate in the cytosol. It plays a prominent role in lipogenesis and fat accumulation coupled to excess glucose, and its inhibition is approved for treating hyperlipidemia. In RNAseq analysis of human failing myocardium, we found ACLY gene expression is reduced; however the impact this might have on cardiac function and/or metabolism has not been previously studied. As new ACLY inhibitors are in development for cancer and other disorders, such understanding has added importance. Cardiomyocytes, ex-vivo beating hearts, and in vivo hearts with ACLY inhibited by selective pharmacologic (BMS303141, ACLYi) or genetic suppression, were studied. Regulation of ACLY gene/protein expression, and effects of ACLYi on function, cytotoxicity, tricarboxylic acid (TCA)-cycle metabolism, and redox and NAD+/NADH balance were assessed. Mice with cardiac ACLY knockdown induced by AAV9-acly-shRNA or cardiomyocyte tamoxifen-inducible Acly knockdown were studied. Acly gene expression was reduced more in obese patients with heart failure and preserved EF (HFpEF) than HF with reduced EF. In vivo pressure-overload and in vitro hormonal stress increased ACLY protein expression, whereas it declined upon fatty-acid exposure. Acute ACLYi (1-hr) dose-dependently induced cytotoxicity in adult and neonatal cardiomyocytes, and caused substantial reduction of systolic and diastolic function in myocytes and ex-vivo beating hearts. In the latter, ATP/ADP ratio also fell and lactate increased. U13C-glucose tracing revealed an ACLYdependent TCA-bypass circuit in myocytes, where citrate generated in mitochondria is transported to the cytosol, metabolized by ACLY and then converted to malate to re-enter mitochondria,bypassing several NADH-generating steps. ACLYi lowered NAD+/NADH ratio and restoring this balance ameliorated cardiomyocyte toxicity. Oxidative stress was undetected with ACLYi. Adult hearts following 8-weeks of reduced cardiac and/or cardiomyocyte ACLY downregulation exhibited ventricular dilation and reduced function that was prevented by NAD augmentation. Cardiac dysfunction from ACLY knockdown was worse in hearts subjected to sustained pressureoverload, supporting a role in stress responses. ACLY supports normal cardiac function through maintenance of the NAD+/NADH balance and is upregulated by hemodynamic and hormonal stress, but depressed by lipid excess. ACLY levels are most reduced in human HFpEF with obesity potentially worsening cardio-metabolic reserve.

Fed-batch fermentation of Mucor circinelloides reveals significant improvement in biomass and lipid accumulation through performance evaluation, chemical analysis, and expression profiling

This study aimed to improve the lipid and biomass yields of Mucor circinelloides WJ11 by implementing four different fed-batch fermentation strategies, varied in time and glucose concentration (S1-S4). The S1 fermentation strategy yielded the highest biomass, lipid, and fatty acid content (22 ± 0.7 g/L, 53 ± 1.2 %, and 28 ± 1.6 %) after 120 and 144 h, respectively. The γ-linolenic acid titer of 0.75 ± 0.0 g/L was greatest in S3 after 48 h. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to analyze the transcription of key genes involved in lipid accumulation. The glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and ATP-citrate lyase genes showed increased expression levels. Fourier-transform infrared (FTIR) spectroscopy was used to analyze the biochemical profile during fermentation strategies. Optimal abiotic factors for production efficiency included pH 6.5, 25–26 °C, 15 % (v/v) inoculum, 500 rpm, 20 %-30 % dissolved oxygen, and 120 h fermentation. Glucose co-feeding offers valuable insights to develop effective fermentation strategies for lipid production.

Expression levels of genes involved in lipogenesis and cholesterol synthesis in adenomyosis

Abstract Objectives Adenomyosis is a benign uterine disease that occurs with the invasion of the endometrial gland and stoma into the myometrium. The etiology and molecular pathology of adenomyosis are not yet fully understood. Tissue samples of patients diagnosed with adenomyosis and healthy endometrial tissues were investigated for the lipogenesis and cholesterol synthesis pathways. It was aimed to determine the difference between adenomyosis and healthy endometrial tissues in terms of lipid metabolism and to investigate the mechanism of adenomyosis in this context. Methods Formalin-fixed paraffin-embedded archival tissues were used in the current retrospective study. A total of 76 patient samples and 3 groups were used. Group 1: adenomyotic tissue (n=28), Group 2: eutopic endometrial tissue (n=30), and Control Group (n=18). In these groups, Sterol regulatory element binding protein 1 (SREBP1) molecule, fatty acid synthase (FASN), acetyl-CoA carboxylase (ACACA), ATP-citrate lyase (ACLY), HMG-CoA reductase (HMGCR), and HMG-CoA synthase (HMGCS) markers were evaluated by using RT-PCR method. Results Statistically significant differences (p<0.05) were found between the groups regarding expression levels of HMGCR, HMGCS, ACLY, ACACA, and SREBP1. HMGCR, HMGCS, ACLY, and SREBP1 gene expression levels between Group 1 and Group 2 and HMGCS, ACACA, ACLY, and SREBP1 gene expression levels between Group 1 and Control Group were determined as statistically different. A significant difference was detected only in HMGCR gene expression levels between Group 2 and the Control Group. Conclusions These results show that genes involved in lipid metabolism may be associated with the molecular pathogenesis of adenomyosis.

Herbaceous peony PlACLB2 positively regulates red petal formation by promoting anthocyanin accumulation.

ATP-citrate lyase (ACL) gene catalyzes the formation of acetyl-CoA to provide intermediate precursors for many secondary metabolites, and also plays an important role in anthocyanin biosynthesis of plants. Herbaceous peony (Paeonia lactiflora Pall.) is an international cut flower known for its rich flower colors, however, the function of the ACL gene in flower color regulation is still unclear. Here, double-colored P. lactiflora ‘Hebao Jinlian’ were used to study the molecular mechanism of red petal, and acetyl-CoA and anthocyanin biosynthesis related PlACLB2, PlCHS, PlDFR, PlANS, and PlbHLH1 genes were initially found to highly expressed in the red outer-petals. The expression pattern of PlACLB2 was consistent with the spatial accumulation of anthocyanins. The correlation analysis of PlACLB2 expression pattern, acetyl-CoA content, and anthocyanin accumulation revealed that PlACLB2 was positively correlated with the acetyl-CoA and anthocyanin contents with correlation coefficients of 0.82 and 0.80. Moreover, multiple sequence alignment identified two typical conserved domains in PlACLB2, and phylogenetic analysis clustered PlACLB2 into the ACLB clade. PlACLB2 was localized in the nucleus and cytoplasm. On the one hand, silencing PlACLB2 in P. lactiflora red outer-petal resulted in lighter petal color and decreased acetyl-CoA accumulation, and quantitative analysis detected that PlACLB2-silenced petals lost more anthocyanins than the control groups with a decrease of 31.0%, and the main pigment component cyanidin-3,5-O-diglucoside was reduced by 31.9%. On the other hand, overexpression of PlACLB2 significantly promoted red coloration, acetyl-CoA content, and anthocyanin accumulation in tobacco flowers. These results demonstrated that PlACLB2 promoted anthocyanin accumulation by increasing the abundance of its precursor substrate acetyl-CoA, thereby regulating the formation of the red petals in P. lactiflora.

ACLY Nuclear Translocation in Human Macrophages Drives Proinflammatory Gene Expression by NF-κB Acetylation.

Macrophage stimulation by pathogen-associated molecular patterns (PAMPs) like lipopolysaccharide (LPS) or lipoteichoic acid (LTA) drives a proinflammatory phenotype and induces a metabolic reprogramming to sustain the cell’s function. Nevertheless, the relationship between metabolic shifts and gene expression remains poorly explored. In this context, the metabolic enzyme ATP citrate lyase (ACLY), the producer of citrate-derived acetyl-coenzyme A (CoA), plays a critical role in supporting a proinflammatory response. Through immunocytochemistry and cytosol–nucleus fractionation, we found a short-term ACLY nuclear translocation. Protein immunoprecipitation unveiled the role of nuclear ACLY in NF-κB acetylation and in turn its full activation in human PBMC-derived macrophages. Notably, sepsis in the early hyperinflammatory phase triggers ACLY-mediated NF-κB acetylation. The ACLY/NF-κB axis increases the expression levels of proinflammatory genes, including SLC25A1—which encodes the mitochondrial citrate carrier—and ACLY, thus promoting the existence of a proinflammatory loop involving SLC25A1 and ACLY genes.

Impact of ACLY on type 2 diabetes in UK Biobank: a Mendelian randomisation study

Abstract Background Cholesterol lowering therapies have greatly reduced the burden of cardiovascular disease. Bempedoic acid inhibits ATP citrate lyase (ACLY), a key enzyme in the cholesterol biosynthesis pathway, and has shown promising results in early-phase randomised controlled trials. Targeting this mechanism may therefore provide a further therapeutic option for lowering LDL cholesterol (LDL-C) and cardiovascular risk. In contrast to statin therapy, randomised comparisons suggest that the incidence of new-onset diabetes is lower with bempedoic acid than with placebo. Mendelian randomisation offers the potential to use genetic variants in the ACLY gene to mimic the on-target effects of bempedoic acid. We used this approach to explore the association between genetic inhibition of ACLY and risk of type 2 diabetes (T2DM). Purpose To assess the impact of lifelong genetic inhibition of ACLY associated with lower LDL-C levels on T2DM and glycaemic control (alongside effects on coronary heart disease [CHD]), investigate whether the association varies by background T2DM risk, and explore potential mediating factors. Methods We undertook a 2-sample Mendelian randomisation study of ∼0.3M unrelated White British individuals in UK Biobank, including 26,495 cases of T2DM and 37,272 cases of CHD. Causal effects on these outcomes were estimated using the inverse variance weighted approach, based on 9 independent LDL-C associated ACLY (±500 kb) genetic variants weighted by their LDL-C effects from previously published studies. Results for T2DM and CHD were then meta-analysed with comparable analysis of data from DIAGRAM (n=26,676 T2DM cases) and CARDIoGRAMplusC4D (n=60,801 CHD cases) respectively. Mediation analyses were conducted to explore the relevance of adiposity traits, such as body mass index, on the observed associations. Subgroup analyses considering the effects in individuals at varying risk of T2DM (based on polygenic risk), as well as the effects of ACLY on glycaemic control in those with established diabetes were undertaken. Results Genetic variation in ACLY was associated with a 16% lower risk of T2DM (odds ratio [OR]: 0.84; 95% CI; 0.76–0.92) per 10mg/dL lower ACLY-predicted LDL-C level. The magnitude and direction of this effect was comparable to that observed with CHD (OR: 0.84; 95% CI; 0.77–0.91). There were no differences in the estimated effects of ACLY on T2DM across tertiles of T2DM polygenic risk (p-trend=0.63). Furthermore, the association between ACLY and T2DM was not materially mediated through effects on body mass index. Analyses exploring additional potential mediating factors, the relevance of inflammation, and the effects of ACLY on glycaemic control in individuals with diabetes are also reported. Conclusion This Mendelian randomisation study shows that genetic variants that mimic inhibition of ACLY and are associated with lower LDL-C levels lower the risks of developing T2DM as well as of CHD events. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): British Heart Foundation

Mammalian SIRT6 Represses Invasive Cancer Cell Phenotypes through ATP Citrate Lyase (ACLY)-Dependent Histone Acetylation.

The modulation of dynamic histone acetylation states is key for organizing chromatin structure and modulating gene expression and is regulated by histone acetyltransferase (HAT) and histone deacetylase (HDAC) enzymes. The mammalian SIRT6 protein, a member of the Class III HDAC Sirtuin family of NAD+-dependent enzymes, plays pivotal roles in aging, metabolism, and cancer biology. Through its site-specific histone deacetylation activity, SIRT6 promotes chromatin silencing and transcriptional regulation of aging-associated, metabolic, and tumor suppressive gene expression programs. ATP citrate lyase (ACLY) is a nucleo-cytoplasmic enzyme that produces acetyl coenzyme A (acetyl-CoA), which is the required acetyl donor for lysine acetylation by HATs. In addition to playing a central role in generating cytosolic acetyl-CoA for de novo lipogenesis, a growing body of work indicates that ACLY also functions in the nucleus where it contributes to the nutrient-sensitive regulation of nuclear acetyl-CoA availability for histone acetylation in cancer cells. In this study, we have identified a novel function of SIRT6 in controlling nuclear levels of ACLY and ACLY-dependent tumor suppressive gene regulation. The inactivation of SIRT6 in cancer cells leads to the accumulation of nuclear ACLY protein and increases nuclear acetyl-CoA pools, which in turn drive locus-specific histone acetylation and the expression of cancer cell adhesion and migration genes that promote tumor invasiveness. Our findings uncover a novel mechanism of SIRT6 in suppressing invasive cancer cell phenotypes and identify acetyl-CoA responsive cell migration and adhesion genes as downstream targets of SIRT6.

Biological Behavior and Lipid Metabolism of Colon Cancer Cells are Regulated by a Combination of Sterol Regulatory Element-Binding Protein 1 and ATP Citrate Lyase.

PurposeTo research the effects of ATP citrate lyase (ACLY) and Sterol-regulatory element binding protein 1 (SREBP1) on the biology and lipid metabolism of colorectal cancer cells.MethodsColorectal cancer cells Caco-2 and Lovo were transfected with ACLY or SREBP1 gene knockdown lentiviruses. Four groups were set: ACLY knockdown, SREBP1 knockdown group, empty vector-transfected (negative control), and untreated cells (blank control). Cell proliferation was measured using CCK-8, colony formation, and EdU labeling assays. Apoptosis was detected using Annexin V-APC/7- AAD and JC-1 assay. Transwell migration and wound healing assays analyzed cell migration and invasion. A triglyceride test kit and oil red O stain assessed cell lipid production. Key factors related to lipid metabolism were detected.ResultsACLY and SREBP1 promoted cell proliferation at 48 and 120 h, but there was no significant difference in Caco-2 cells at 24 h, at which point the effect of SREBP1 was more important. ACLY’s effect on cell proliferation was more obvious at 120 h. Colony formation assays in Caco-2 showed similar results to the CCK-8 assay at 120 h, but ACLY knockdown had no effect in Lovo cells. EDU assays showed that ACLY or SREBP1 facilitated DNA reproduction in the two cell lines, in which SREBP1 was more significant. Knockdown of the two genes showed significant differences in Lovo cells. However, ALCY knockdown promoted apoptosis to a greater extent than SREBP1 knockdown in Caco-2 cells. In addition, ACLY and SREBP1 enhanced migration, invasion, and lipid production in both cell lines. Knockdown of ACLY or SREBP1 reduced lipid metabolism pathway gene expression in the two cell lines.ConclusionKnockdown of ACLY and SREBP1 genes inhibit the proliferation, migration, and invasion of colorectal cancer cells, while promoting their apoptosis. Our results identified potential new targets to treat colorectal cancer via lipid synthesis modulation in cancer cells.

Overexpression of SoACLA-1 Gene Confers Drought Tolerance Improvement in Sugarcane

Drought is one of the most severe stresses which limit sugarcane production in China. ATP citrate lyase (ACL) is a major enzyme responsible for the production of acetyl-CoA in cytoplasm and plays an important role in plant metabolism and stress response. In this study, sugarcane ACL gene SoACLA-1 was cloned. The plant overexpression vector of SoACLA-1 was built and transformed into sugarcane calli by Agrobacterium-mediated transformation, and PCR analysis confirmed that SoACLA-1 gene had been stably present in the T0, T1, and T2 generations of the transgenic sugarcane. In order to evaluate the drought resistance of the transgenic lines and verify the function of SoACLA-1 gene in the transgenic sugarcane, T1 generation of the SoACLA-1 transgenic sugarcane lines was used as the material to investigate the physiological and biochemical characteristics at 0 day, 3 days, 6 days, and 9 days after water stress and rewatering for 3 days. Comprehensive evaluation of four indicators (chlorophyll, malondialdehyde, proline, soluble sugar) related to drought resistance was done with membership fuzzy function method. The results showed that the drought resistance of five transgenic sugarcane lines from strong to weak, in turn, was RT2 > RT4 > RT3 > RT1 > WT, and the recovery ability after drought, in turn, was RT1 > RT2 > RT4 > RT3 > WT. The T2 generation of the SoACLA-1 transgenic sugarcane lines was used to analyze the physiological and biochemical changes and the expression of drought-related genes under water stress. The results showed that the transgenic sugarcane lines were more tolerant to drought as compared with the wild-type plants. Our findings indicated that SoACLA-1 gene plays an important role as a positive factor in response to water stress, and overexpression of SoACLA-1 can enhance drought tolerance in transgenic sugarcane plants.

Salidroside simultaneously reduces de novo lipogenesis and cholesterol biosynthesis to attenuate atherosclerosis in mice

Salidroside is a kind of phenylethanoid glycoside and widespread in the plants from Rhodiola and Ligustrum species. Our previous study has reported that salidroside can prevent atherosclerosis progression by ameliorating glyerolipid and glycerophospholipid metabolism in apoE-deficient (apoE−/−) mice. However, its effect on neutral lipids and underlying mechanism remains largely unclear. Here we investigated the molecular mechanism of salidroside action from the perspective of metabolic regulation by integrating metabonomics and transcriptomics pattern. The results showed that salidroside significantly reduced cholesterols, esterified cholesterols, fatty acids, unsaturated fatty acids and triacylclycerols biosynthesis in liver through down-regulating the genes expressions of sterol regulatory element-binding proteins (Srebf1 and Srebf2). The expressions of SREBPs targeted and downstream genes, such as the encoding genes of fatty acid synthase (Fasn), glycerol-3-phosphate acyltransferase (Gpam), stearoyl-CoA desaturase (Scd), 3-hydroxy-3-methylglutaryl-CoA reductase (Hmgcr), and proprotein convertase subtilisin/kexin type 9 (Pcsk9), were also inhibited after salidroside administration. ATP citrate lyase gene (Acly) that encodes an important enzyme producing acetyl-CoA for cholesterol and fatty acid biosynthesis significantly decreased after treatment as well. Moreover, one of ketone body products, 3-hydroxybutyrate, was significantly up-regulated in drug-treated group, indicating that fatty acid degradation was accelerated by salidroside at the same time. Our findings identify salidroside as a regulator of lipid homeostasis in atherosclerotic mice, suggesting its potential to be an alternative medicine for lowering the risks of atherosclerosis-related diseases.

Pistacia lentiscus Hydrosol: Untargeted Metabolomic Analysis and Anti-Inflammatory Activity Mediated by NF-κB and the Citrate Pathway.

Pistacia lentiscus shows a long range of biological activities, and it has been used in traditional medicine for treatment of various kinds of diseases. Moreover, related essential oil keeps important health-promoting properties. However, less is known about P. lentiscus hydrosol, a main by-product of essential oil production, usually used for steam distillation itself or discarded. In this work, by using ultra-high-resolution ESI(+)-FT-ICR mass spectrometry, a direct identification of four main classes of metabolites of P. lentiscus hydrosol (i.e., terpenes, amino acids, peptides, and condensed heterocycles) was obtained. Remarkably, P. lentiscus hydrosol exhibited an anti-inflammatory activity by suppressing the secretion of IL-1β, IL-6, and TNF-α proinflammatory cytokines in lipopolysaccharide- (LPS-) activated primary human monocytes. In LPS-triggered U937 cells, it inhibited NF-κB, a key transcription factor in inflammatory cascade, regulating the expression of both the mitochondrial citrate carrier and the ATP citrate lyase genes. These two main components of the citrate pathway were downregulated by P. lentiscus hydrosol. Therefore, the levels of ROS, NO, and PGE2, the inflammatory mediators downstream the citrate pathway, were reduced. Results shed light on metabolic profile and anti-inflammatory properties of P. lentiscus hydrosol, suggesting its potential as a therapeutic agent.

Cytosolic Acetyl-CoA Generated by ATP-Citrate Lyase Is Essential for Acetylation of Cell Wall Polysaccharides.

Plant cell wall polysaccharides, including xylan, glucomannan, xyloglucan and pectin, are often acetylated. Although a number of acetyltransferases responsible for the acetylation of some of these polysaccharides have been biochemically characterized, little is known about the source of acetyl donors and how acetyl donors are translocated into the Golgi, where these polysaccharides are synthesized. In this report, we investigated roles of ATP-citrate lyase (ACL) that generates cytosolic acetyl-CoA in cell wall polysaccharide acetylation and effects of simultaneous mutations of four Reduced Wall Acetylation (RWA) genes on acetyl-CoA transport into the Golgi in Arabidopsis thaliana. Expression analyses of genes involved in the generation of acetyl-CoA in different subcellular compartments showed that the expression of several ACL genes responsible for cytosolic acetyl-CoA synthesis was elevated in interfascicular fiber cells and induced by secondary wall-associated transcriptional activators. Simultaneous downregulation of the expression of ACL genes was demonstrated to result in a substantial decrease in the degree of xylan acetylation and a severe alteration in secondary wall structure in xylem vessels. In addition, the degree of acetylation of other cell wall polysaccharides, including glucomannan, xyloglucan and pectin, was also reduced. Moreover, Golgi-enriched membrane vesicles isolated from the rwa1/2/3/4 quadruple mutant were found to exhibit a drastic reduction in acetyl-CoA transport activity compared with the wild type. These findings indicate that cytosolic acetyl-CoA generated by ACL is essential for cell wall polysaccharide acetylation and RWAs are required for its transport from the cytosol into the Golgi.

The Role of Methylation of CpG Islands of the csy3 Gene Promotor in the Light Regulation of ATP-Citrate Lyase Activity in Maize Leaves

It was established that the functioning of ATP-citrate lyase in maize (Zea mays L.) leaves is regulated by the light conditions of plants, in particular, irradiation of maize plants with red or blue light caused a sharp increase in enzyme activity compared with the “darkness-” and irradiation with far-red light variants. It was shown that active forms of phytochrome and cryptochrome are involved in increase of ATP-citrate lyase activity by regulating the expression of the csy3 gene. This was probably achieved by interacting with a specific G-site of the csy3 gene promoter. Analysis of the nucleotide sequence of the ATP-citrate lyase gene promoter showed the presence of CpG islands, providing the potential for methylation. The study of the methylation status of individual CG dinucleotides of the csy3 gene promoter revealed a change of this index depending on the conditions of the light regime in plants. A high degree of methylation of CG dinucleotides in the csy3 gene promoter resulted in a decrease in the level of transcripts of this gene, and, as a result, inhibition of ATP-citrate lyase activity in the dark and during irradiation with far-red light.

Metabolic engineering of Saccharomyces cerevisiae by using the CRISPR-Cas9 system for enhanced fatty acid production

Fatty acids produced by the yeast, Saccharomyces cerevisiae, are not only industrially important products, but also highly affect fermentation efficiency. Despite that S. cerevisiae is the potent metabolic platform, traditional plasmid-based genetic manipulations still impose concerns about safety and environmental impact. In this study, using the Cas9 system, S. cerevisiae, was metabolically engineered for increased production of fatty acids by increasing cytosolic acetyl-CoA production via disrupting the isocitrate dehydrogenase gene of the TCA cycle and introducing the cytosolic ATP-citrate lyase gene from Yarrowia lipolytica. By expressing the ATP-citrate lyase gene from Y. lipolytica, total fatty acid production increased by 37.1% compared to that by the wild-type yeast. This total fatty acid production herein increased ∼2 times compared to that by an engineered S. cerevisiae in a previous report, and unsaturated fatty acid levels also increased. Our results suggest that the Cas9 system for S. cerevisiae could be applied to engineer the yeast for industrial production of bio-based products.

Enhanced squalene biosynthesis in Yarrowia lipolytica based on metabolically engineered acetyl-CoA metabolism

As a bioactive triterpenoid, squalene is widely used in the food industry, cosmetics, and pharmacology. Squalene’s major commercial sources are the liver oil of deep-sea sharks and plant oils. In this study, we focused on the enhancement of squalene biosynthesis in Yarrowia lipolytica, with particular attention to the engineering of acetyl-CoA metabolism based on genome-scale metabolic reaction network analysis. Although the overexpression of the rate-limiting endogenous ylHMG1 (3-hydroxy-3-methylglutaryl-CoA reductase gene) could improve squalene synthesis by 3.2-fold over that by the control strain, the availability of the key intracellular precursor, acetyl-CoA, was found to play a more significant role in elevating squalene production. Analysis of metabolic networks with the newly constructed genome-scale metabolic model of Y. lipolytica iYL_2.0 showed that the acetyl-CoA pool size could be increased by redirecting carbon flux of pyruvate dehydrogenation towards the ligation of acetate and CoA or the cleavage of citrate to form oxaloacetate and acetyl-CoA. The overexpression of either acetyl-CoA synthetase gene from Salmonella enterica (acs*) or the endogenous ATP citrate lyase gene (ylACL1) resulted in a more than 50% increase in the cytosolic acetyl-CoA level. Moreover, iterative chromosomal integration of the ylHMG1, asc*, and ylACL1 genes resulted in a significant improvement in squalene production (16.4-fold increase in squalene content over that in the control strain). We also found that supplementation with 10 mM citrate in a flask culture further enhanced squalene production to 10 mg/g DCW. The information obtained in this study demonstrates that rationally engineering acetyl-CoA metabolism to ensure the supply of this key metabolic precursor is an efficient strategy for the enhancement of squalene biosynthesis.

Modes of carbon fixation in an arsenic and CO2-rich shallow hydrothermal ecosystem

The seafloor sediments of Spathi Bay, Milos Island, Greece, are part of the largest arsenic-CO2-rich shallow submarine hydrothermal ecosystem on Earth. Here, white and brown deposits cap chemically distinct sediments with varying hydrothermal influence. All sediments contain abundant genes for autotrophic carbon fixation used in the Calvin-Benson-Bassham (CBB) and reverse tricaboxylic acid (rTCA) cycles. Both forms of RuBisCO, together with ATP citrate lyase genes in the rTCA cycle, increase with distance from the active hydrothermal centres and decrease with sediment depth. Clustering of RuBisCO Form II with a highly prevalent Zetaproteobacteria 16S rRNA gene density infers that iron-oxidizing bacteria contribute significantly to the sediment CBB cycle gene content. Three clusters form from different microbial guilds, each one encompassing one gene involved in CO2 fixation, aside from sulfate reduction. Our study suggests that the microbially mediated CBB cycle drives carbon fixation in the Spathi Bay sediments that are characterized by diffuse hydrothermal activity, high CO2, As emissions and chemically reduced fluids. This study highlights the breadth of conditions influencing the biogeochemistry in shallow CO2-rich hydrothermal systems and the importance of coupling highly specific process indicators to elucidate the complexity of carbon cycling in these ecosystems.

Isoprenoid biosynthesis in dandelion latex is enhanced by the overexpression of three key enzymes involved in the mevalonate pathway

BackgroundLatex from the dandelion species Taraxacum brevicorniculatum contains many high-value isoprenoid end products, e.g. triterpenes and polyisoprenes such as natural rubber. The isopentenyl pyrophosphate units required as precursors for these isoprenoids are provided by the mevalonate (MVA) pathway. The key enzyme in this pathway is 3-hydroxy-methyl-glutaryl-CoA reductase (HMGR) and its activity has been thoroughly characterized in many plant species including dandelion. However, two enzymes acting upstream of HMGR have not been characterized in dandelion latex: ATP citrate lyase (ACL), which provides the acetyl-CoA utilized in the MVA pathway, and acetoacetyl-CoA thiolase (AACT), which catalyzes the first step in the pathway to produce acetoacetyl-CoA. Here we isolated ACL and AACT genes from T. brevicorniculatum latex and characterized their expression profiles. We also overexpressed the well-characterized HMGR, ACL and AACT genes from Arabidopsis thaliana in T. brevicorniculatum to determine their impact on isoprenoid end products in the latex.ResultsThe spatial and temporal expression profiles of T. brevicorniculatum ACL and AACT revealed their pivotal role in the synthesis of precursors necessary for isoprenoid biosynthesis in latex. The overexpression of A. thaliana ACL and AACT and HMGR in T. brevicorniculatum latex resulted in the accumulation of all three enzymes, increased the corresponding enzymatic activities and ultimately increased sterol levels by ~5-fold and pentacyclic triterpene and cis-1,4-isoprene levels by ~2-fold. Remarkably high levels of the triterpene precursor squalene were also detected in the triple-transgenic lines (up to 32 mg/g root dry weight) leading to the formation of numerous lipid droplets which were observed in root cross-sections.ConclusionsWe could show the effective expression of up to three transgenes in T. brevicorniculatum latex which led to increased enzymatic activity and resulted in high level squalene accumulation in the dandelion roots up to an industrially relevant amount. Our data provide insight into the regulation of the MVA pathway in dandelion latex and can be used as a basis for metabolic engineering to enhance the production of isoprenoid end products in this specialized tissue.

ATP-Citrate Lyase Gene (SoACLA-1), a Novel ACLA Gene in Sugarcane, and Its Overexpression Enhance Drought Tolerance of Transgenic Tobacco

ATP-citrate lyase catalyzes cracking citrate to produce acetyl CoA in the cytoplasm and plays an important role in normal growth and development of plants. In this study, SoACLA-1 gene in sugarcane was cloned and characterized. The amino acid sequence of SoACLA-1 showed high homology with other ACLAs families, and they were clustered together in the phylogenetic tree. On transcript level, SoACLA-1 expressed strongly in root and stalk and responded to polyethylene glycol 6000, drought and ABA stresses. The results indicated that SoACLA-1 possibly involves in stress-responsive metabolic process. PCR analysis was performed to confirm the putative transgenic type in T1 tobacco plants. Overexpression of SoACLA-1 enhanced the tolerance of tobacco plants to drought. The sense transgenic tobacco plants were healthier, had longer roots and higher contents of relative water content and chlorophyll and activities of superoxide dismutase, peroxidase and catalase, but lower ion leakage and contents of malondialdehyde (MDA) and H2O2 than the wild-type and antisense transgenic plants under drought stress. In conclusion, SoACLA-1 plays an important role as a positive factor in drought stress response pathways and can be utilized for transformation to improve crop drought tolerance.

Targeting ACLY sensitizes castration-resistant prostate cancer cells to AR antagonism by impinging on an ACLY-AMPK-AR feedback mechanism.

The androgen receptor (AR) plays a central role in prostate tumor growth. Inappropriate reactivation of the AR after androgen deprivation therapy promotes development of incurable castration-resistant prostate cancer (CRPC). In this study, we provide evidence that metabolic features of prostate cancer cells can be exploited to sensitize CRPC cells to AR antagonism. We identify a feedback loop between ATP-citrate lyase (ACLY)-dependent fatty acid synthesis, AMPK, and the AR in prostate cancer cells that could contribute to therapeutic resistance by maintaining AR levels. When combined with an AR antagonist, ACLY inhibition in CRPC cells promotes energetic stress and AMPK activation, resulting in further suppression of AR levels and target gene expression, inhibition of proliferation, and apoptosis. Supplying exogenous fatty acids can restore energetic homeostasis; however, this rescue does not occur through increased β-oxidation to support mitochondrial ATP production. Instead, concurrent inhibition of ACLY and AR may drive excess ATP consumption as cells attempt to cope with endoplasmic reticulum (ER) stress, which is prevented by fatty acid supplementation. Thus, fatty acid metabolism plays a key role in coordinating ER and energetic homeostasis in CRPC cells, thereby sustaining AR action and promoting proliferation. Consistent with a role for fatty acid metabolism in sustaining AR levels in prostate cancer in vivo, AR mRNA levels in human prostate tumors correlate positively with expression of ACLY and other fatty acid synthesis genes. The ACLY-AMPK-AR network can be exploited to sensitize CRPC cells to AR antagonism, suggesting novel therapeutic opportunities for prostate cancer.