acetyl-CoA Synthetase Research Articles

Engineering a Phosphoketolase Pathway to Supplement Cytosolic Acetyl-CoA in Aspergillus niger Enables a Significant Increase in Citric Acid Production

Citric acid is widely used in the food, chemical and pharmaceutical industries. Aspergillus niger is the workhorse used for citric acid production in industry. A canonical citrate biosynthesis that occurred in mitochondria was well established; however, some research suggested that the cytosolic citrate biosynthesis pathway may play a role in this chemical production. Here, the roles of cytosolic phosphoketolase (PK), acetate kinase (ACK) and acetyl-CoA synthetase (ACS) in citrate biosynthesis were investigated by gene deletion and complementation in A. niger. The results indicated that PK, ACK and ACS were important for cytosolic acetyl-CoA accumulation and had significant effects on citric acid biosynthesis. Subsequently, the functions of variant PKs and phosphotransacetylase (PTA) were evaluated, and their efficiencies were determined. Finally, an efficient PK-PTA pathway was reconstructed in A. niger S469 with Ca-PK from Clostridium acetobutylicum and Ts-PTA from Thermoanaerobacterium saccharolyticum. The resultant strain showed an increase of 96.4% and 88% in the citrate titer and yield, respectively, compared with the parent strain in the bioreactor fermentation. These findings indicate that the cytosolic citrate biosynthesis pathway is important for citric acid biosynthesis, and increasing the cytosolic acetyl-CoA level can significantly enhance citric acid production.

Abstract 6048: OGT/CDK5/ACSS2 axis regulates breast cancer brain metastatic growth

Abstract Brain metastases (BMs) in breast cancer patients is considered an end-stage event, with no effective drug treatment and a median survival after diagnosis measured in months. Currently, there are no effective drug treatment for BM patients, thus there is an urgent need to develop novel treatment strategies. Breast cancers that metastasize to the brain must adapt to lack of lipid availability in the brain environment and are highly dependent on fatty acid synthesis for growth and survival. However, the signaling pathways that regulate precursors of fatty acid synthesis and lipid metabolism in breast cancer brain metastatic (BCBM) tumors are not known. Here, we show that BCBM cells can generate acetyl-CoA, a major metabolite feeding lipid biosynthesis, via phosphorylation of acetyl-CoA synthetase 2 (ACSS2) by the cyclin-dependent kinse-5 (CDK5) regulated by the nutrient sensor O-GlcNAc transferase (OGT). Breast cancer cells selected to metastasize to the brain contain higher levels of O-GlcNAcylation, OGT. and ACSS2-Ser267 phosphorylation compared to parental cells, and we show that human breast cancer brain metastatic patient samples contain elevated ACSS2-Ser-267 levels. Additionally, overexpression of the OGT, CDK5, or ACSS2-S267D phospho-mimetic mutant confers a growth advantage of breast cancer cells in the brain in vivo. Importantly, we show that ACSS2 is required for breast cancer growth in the brain but not in the mammary fat pad. Pharmacologically targeting the CDK5 or ACSS2 with respective small molecule inhibitors reduces tumor growth in orthotopic ex vivo brain slice model. These results suggest a crucial role for OGT/CDK5/ACSS2 signaling axis to regulate lipid metabolism in BCBM cells and identify CDK5 and ACSS2 as novel therapeutic targets for treatment of breast cancer brain metastatic growth. Citation Format: Emily Esquea, Lorela Ciraku, Giang Le Ming, Mauricio Reginato. OGT/CDK5/ACSS2 axis regulates breast cancer brain metastatic growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6048.

Improving the Biodiesel Production in Saccharomyces Cerevisiae: Metabolic Engineering and Enzyme Engineering

The yeast Saccharomyces cerevisiae can produce fuels such as biodiesel using the biomass of lignocellulose. S. cerevisiae metabolize monosaccharide of lignocellulosic hydrolysates to supply fuels/energy globally without competition issue with food supply. Ethanol and free fatty acids (FFAs) are the precursors of biodiesel. S. cerevisiae is able to produce these precursors. S. cerevisiae synthesizes biodiesel from ethanol and fatty acyl-coenzyme A thioesters using wax ester acyltransferase (WS). Here, we review some recent strategies applied to metabolic engineering and enzyme engineering of S. cerevisiae for improving the yield of biodiesel. On the metabolic engineering strategy can be done by directing the carbon flux towards the fatty acids biosynthesis pathway through engineering some genes in the central carbon metabolism. By combined overexpression genes of Acetyl-CoA synthetase (ACS), Acetyl-CoA carboxylase (ACC1), and Fatty Acid Synthase (FAS) will improve biodiesel synthesis. In enzyme engineering, we can remove the allosteric inhibition control of Snf1 to Acetyl-CoA carboxylase ((ACC1), an enzyme that convert Acetyl Co-A to malonyl Co-A) by mutation of two phosphorylation site of ACC1 via site-directed mutagenesis which mutates serine1157,659 to alanine1157,629. This result in enhanced carbon flux to fatty acid synthesis and contribute to a higher yield of biodiesel.

Mechanism of ferroptosis in a rat model of premature ovarian insufficiency induced by cisplatin

Ferroptosis is widely present in fibrosis-related diseases. The basic pathology of premature ovarian insufficiency (POI) involves ovarian tissue fibrosis, and there are currently fewer relevant studies addressing the association between ferroptosis and POI. This study aimed to demonstrate that ferroptosis induced by cisplatin (CDDP) caused ovarian tissue fibrosis, leading to POI. Vitamin E (VE), a ferroptosis inhibitor, could repair damaged ovarian function. CDDP was used to establish a rat model of POI, and VE was administered to reverse the reproductive toxicity of CDDP. Ovarian function was assessed by histological section staining, follicle counts, sex hormone levels, as well as fertility assays. The extent of ferroptosis was assessed by transmission electron microscopy (TEM), malondialdehyde (MDA), Perls staining. CCK-8, Ethynyl-2-Deoxyuridine (EdU), and scratch assays were used to determine the effect of CDDP and VE on ovarian granulosa cell (GC) viability. Western blot, quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry were performed to evaluate ferroptosis-related molecular changes. Our results showed that CDDP caused follicle development disorders and ovarian tissue fibrosis, the levels of sex hormones suggested impaired ovarian function, and VE could reverse the reproductive toxicity of CDDP. The results of TEM, MDA and Perls staining suggested that the typical mitochondrial signature of ferroptosis was altered in ovarian GCs from the CDDP group, with significantly higher levels of lipid peroxidation and significant iron deposition in ovarian tissue, whereas VE mitigated the extent of ferroptosis. Molecular experiments then confirmed that the ferroptosis-related molecules acetyl CoA synthetase long chain family member 4 (ACSl4), 15-lipoxygenase-1 (ALOX15), solute carrier family 7 member 11 (SLC7A11), and glutathione peroxidase 4 (GPX4) were differentially expressed in each group. In summary, our study preliminarily demonstrated that CDDP may promote GCs to undergo ferroptosis, cause follicle development disorders, ovarian tissue fibrosis, and induce POI by regulating the expression of ACSl4, ALOX15, SLC7A11, and GPX4, while VE improved impaired ovarian function.

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/HIF-2 signaling facilitates colon cancer growth and metastasis.

The microenvironment of solid tumors is characterized by oxygen and glucose deprivation. Acss2/HIF-2 signaling coordinates essential genetic regulators including acetate-dependent acetyl CoA synthetase 2 (Acss2), Creb binding protein (Cbp), Sirtuin 1 (Sirt1), and Hypoxia Inducible Factor 2α (HIF-2α). We previously shown in mice that exogenous acetate augments growth and metastasis of flank tumors derived from fibrosarcoma-derived HT1080 cells in an Acss2/HIF-2 dependent manner. Colonic epithelial cells are exposed to the highest acetate levels in the body. We reasoned that colon cancer cells, like fibrosarcoma cells, may respond to acetate in a pro-growth manner. In this study, we examine the role of Acss2/HIF-2 signaling in colon cancer. We find that Acss2/HIF-2 signaling is activated by oxygen or glucose deprivation in two human colon cancer-derived cell lines, HCT116 and HT29, and is crucial for colony formation, migration, and invasion in cell culture studies. Flank tumors derived from HCT116 and HT29 cells exhibit augmented growth in mice when supplemented with exogenous acetate in an Acss2/HIF-2 dependent manner. Finally, Acss2 in human colon cancer samples is most frequently localized in the nucleus, consistent with it having a signaling role. Targeted inhibition of Acss2/HIF-2 signaling may have synergistic effects for some colon cancer patients.

Cancer Metabolism: Fasting Reset, the Keto-Paradox and Drugs for Undoing

In tumor cells, ketolysis "via" succinyl-CoA: 3-oxoacid-CoAtransferase (SCOT) and acetyl-CoA acetyltransferase 1 (ACAT1) is a major source of mitochondrial acetyl-CoA. Active ACAT1 tetramers stabilize by tyrosine phosphorylation, which facilitates the SCOT reaction and ketolysis. Tyrosine phosphorylation of pyruvate kinase PK M2 has the opposite effect, stabilizing inactive dimers, while pyruvate dehydrogenase (PDH), which is already inhibited by phosphorylation, is acetylated by ACAT1 and is doubly locked. This closes the glycolytic supply of acetyl-CoA. In addition, since tumor cells must synthesize fatty acids to create new membranes, they automatically turn off the degradation of fatty acids into acetyl-CoA ("via" the malonyl-CoA brake for the fatty acid carnityl transporter). Thus, inhibiting SCOT the specific ketolytic enzyme and ACAT1 should hold back tumor progression. However, tumor cells are still able to take up external acetate and convert it into acetyl-CoA in their cytosol "via" an acetyl-CoA synthetase, which feeds the lipogenic pathway; additionally, inhibiting this enzyme would make it difficult for tumor cells to form new lipid membrane and survive.

Exploring a High-Efficiency Genetic Transformation System for Engineering Saccharopolyspora pogona ASAGF58 To Improve Butenyl-Spinosyn Production

Butenyl-spinosyn is a potent insecticide potentially useful as a broad-spectrum pesticide. Because it is relatively nontoxic to mammals and does not damage the environment, there has been considerable interest in the use of butenyl-spinosyn for increasing agricultural production. However, genetically engineering Saccharopolyspora pogona ASAGF58 to increase its relatively low butenyl-spinosyn content remains challenging because it cannot be transformed efficiently. In this study, genes encoding novel methyltransferases (04455 and 28970) were identified in the Sa. pogona ASAGF58 genome through a bioinformatic-based analysis. Additionally, the transformation efficiency increased by 5.8- and 16.4-fold when foreign DNA was pre-methylated in ET-28970 and ET-04455, respectively, through bypassing of the restriction–modification system. A comparative proteomic analysis of Sa. pogona and Saccharopolyspora spinosa revealed that acetyl-CoA synthetase may be useful for improving butenyl-spinosyn production. The fermentation results indicated that compared with the wild-type butenyl-spinosyn content, overexpressing the acetyl-CoA synthetase gene increased butenyl-spinosyn production by 2-fold. The findings presented herein suggest that the strategy employed in this study may be applicable for the genetic engineering of other nonmodel Saccharopolyspora strains and increase target product yields.

Abstract A023: OGT/CDK5/ACSS2 axis regulates breast cancer brain metastatic growth

Abstract Brain metastases (BMs) in breast cancer patients is considered an end-stage event, with no effective drug treatment and a median survival after diagnosis measured in months. Currently, there are no effective drug treatment for BM patients, thus there is an urgent need to develop novel treatment strategies. Breast cancers that metastasize to the brain must adapt to lack of lipid availability in the brain environment and are highly dependent on fatty acid synthesis for growth and survival. However, the signaling pathways that regulate precursors of fatty acid synthesis and lipid metabolism in breast cancer brain metastatic (BCBM) tumors are not known. Here, we show that BCBM cells can generate acetyl-CoA, a major metabolite feeding lipid biosynthesis, via phosphorylation of acetyl-CoA synthetase 2 (ACSS2) by the cyclin-dependent kinse-5 (CDK5) regulated by the nutrient sensor O-GlcNAc transferase (OGT). Breast cancer cells selected to metastasize to the brain contain higher levels of O-GlcNAcylation, OGT. and ACSS2-Ser267 phosphorylation compared to parental cells, and we show that human breast cancer brain metastatic patient samples contain elevated ACSS2-Ser-267 levels. Additionally, overexpression of the OGT, CDK5, or ACSS2-S267D phospho-mimetic mutant confers a growth advantage of breast cancer cells in the brain in vivo. Importantly, we show that ACSS2 is required for breast cancer growth in the brain but not in the mammary fat pad. Transcriptome analysis shows that ACSS2 regulates E2F-regulated genes and E2F1 protein levels in breast cancer cells. Pharmacologically targeting the CDK5 or ACSS2 with respective small molecule inhibitors reduces tumor growth in orthotopic ex vivo brain slice model. These results suggest a crucial role for OGT/CDK5/ACSS2 signaling axis to regulate lipid metabolism in BCBM cells and identify CDK5 and ACSS2 as novel therapeutic targets for treatment of breast cancer brain metastatic growth. Citation Format: Emily Esquea, Lorela Ciraku, Giang Le Minh, Mauricio Reginato. OGT/CDK5/ACSS2 axis regulates breast cancer brain metastatic growth [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A023.

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.

Engineering the precursor supply pathway in Streptomyces gilvosporeus for overproduction of natamycin

Natamycin is a safe and efficient antimycotics which is widely used in food and medicine industry. The polyene macrolide compound, produced by several bacterial species of the genus Streptomyces, is synthesized by type Ⅰ polyketide synthases using acetyl-CoA, malonyl-CoA, and methylmalonyl-CoA as substrates. In this study, four pathways potentially responsible for the supply of the three precursors were evaluated to identify the effective precursor supply pathway which can support the overproduction of natamycin in Streptomyces gilvosporeus, a natamycin-producing wild-type strain. The results showed that over-expressing acetyl-CoA synthetase and methylmalonyl-CoA mutase increased the yield of natamycin by 44.19% and 20.51%, respectively, compared with the wild type strain under shake flask fermentation. Moreover, the yield of natamycin was increased by 66.29% compared with the wild-type strain by co-overexpression of acetyl-CoA synthetase and methylmalonyl-CoA mutase. The above findings will facilitate natamycin strain improvement as well as development of strains for producing other polyketide compounds.

Adenosine triphosphate can act as a determinant of lysine acetylation of non-native and native substrates

The protein lysine acetylation includes acetyl-CoA (AcCoA) or acetyl phosphate (AcP)-mediated nonenzymatic acetylation, and enzymatic acetylation. It is widespread in the proteomes but the acetylation levels of most sites are very low. A thorough understanding of the determinants of low acetylation levels is highly important for elucidating the physiological relevance of lysine acetylation. In this study, we constructed a non-native substrate library containing 24 synthesized polypeptides, and we showed that ATP could inhibit the AcCoA-mediated nonenzymatic acetylation of these polypeptides through LC-MS/MS analysis. The acetyltransferase PatZ could acetylated these non-native substrates, and the PatZ-catalyzed acetylation of the polypeptides was also inhibited by ATP. Furthermore, the Western blot showed that ATP also inhibited the nonenzymatic (AcCoA or AcP-mediated) and enzymatic (PatZ-catalyzed) acetylation of acetyl-CoA synthetase Acs, which is a native substrate for acetylation. ATP can also inhibit the autoacetylation of acetyltransferase PatZ. Besides, both ADP and AMP could enhance the AcP-mediated acetylation of Acs, but ADP slightly inhibited the AcCoA-mediated acetylation of Acs. However, both ADP and AMP had no evident inhibition on the PatZ-catalyzed acetylation of Acs. Based on these results, we proposed that ATP can act as an inhibitor of acetylation, and it may regulate the function of PatZ by inhibiting its autoacetylation and compensate for the function of deacetylase CobB.

ROS downregulate TCA activity to modulate energy metabolism via the HIF/miR-34/ACS-PK pathway for lifespan extension in Helicoverpa armigera

Previous studies have shown that high levels of reactive oxygen species (ROS) and low tricarboxylic acid (TCA) activity in the brain promote pupal diapause, which is characterized by metabolic depression and lifespan extension. However, it is unclear whether ROS are associated with TCA activity. In this study, we demonstrate that ROS downregulate TCA activity and acetyl-CoA and pyruvate levels in the brains of diapause-destined pupae in the moth Helicoverpa armigera, as well as the protein levels of acetyl-CoA synthetase (ACS) and pyruvate kinase (PK), two proteins involved in the biosynthesis of acetyl-CoA and pyruvate, respectively. Interestingly, miR-34, which is highly expressed in the brains of diapause-destined pupae, can respond to ROS signaling. Furthermore, we show that miR-34 can reduce the expression of ACS and PK by directly targeting their mRNAs. Additionally, hypoxia-inducible factor (HIF), a transcription factor, can be activated by ROS and then promotes miR-34 transcription by binding a cis-element in its promoter. Moreover, we observed delayed pupal development after treatment with a ROS activator paraquat and a HIF activator dimethyloxallyl glycine. Taken together, these results suggest that a novel pathway ROS/HIF/miR-34/ACS-PK was found to negatively regulate TCA activity to promote insect diapause for lifespan extension.

Effect of low expression level of acetyl coenzyme A synthetase gene on secondary metabolite in Monascus

Acetyl-coenzyme A (CoA) is a key metabolite produced by the acetyl-CoA synthetase (ACS) gene in energy metabolism and biosynthetic pathways. ACS is speculated to be the branching site of monacolin K (MK) and citrinin production and related to the metabolite production of Monascus. In this study, the ACS expression was inhibited by ribonucleic acid interference (RNAi). T7 was selected for a follow-up analysis of the lowest ACS expression, which was 0.401 times higher than that of the wild-type strain. The effects on the colony morphology of Monascus were determined. The morphological characteristics of mycelia and spores were observed under a scanning electron microscope. The contents of secondary metabolites, namely, MK and citrinin, were determined through high performance liquid chromatography (HPLC). Colour values were measured with a spectrophotometer. Results showed that the low ACS expression could inhibit the growth of Monascus colonies and the hypha and affect the formation and morphology of Monascus M1 spores. It could also inhibit the production of the main secondary metabolites, namely, MK, citrinin, and pigment.

Glyceryl triacetate feeding in mice increases plasma acetate levels but has no anticonvulsant effects in acute electrical seizure models

IntroductionAcetate has been shown to have neuroprotective and anti-inflammatory effects. It is oxidized by astrocytes and can thus provide auxiliary energy to the brain in addition to glucose. Therefore, we hypothesized that it may protect against seizures, which is investigated here by feeding glyceryl triacetate (GTA), to provide high amounts of acetate without raising sodium or acid levels. MethodCD1 male mice were fed controlled diets with or without GTA for up to three weeks. Body weights, blood glucose levels, plasma short-chain fatty acid levels, and other hematological parameters were monitored. Seizure thresholds were determined in 6 Hz and maximal electroshock seizure threshold (MEST) tests. Antioxidant capacities were evaluated in the cerebral cortex and plasma using a ferric reducing antioxidant power (FRAP) assay and Trolox equivalent antioxidant capacity assay. ResultsBody weight gain was similar with both diets with and without GTA in two experiments. Glyceryl triacetate-fed groups showed 2–3- and 1.6-fold increased acetate and propionate levels in plasma, respectively. Glucose levels were unaltered in blood collected from the tail tip but increased in trunk blood. No differences were found in the activity of cerebral cortex acetyl-CoA synthetase. In the 6 Hz threshold test, seizure thresholds were lower by 3 mA and 2.4 mA after 8 and 14 days, respectively, in the GTA compared to the control diet-fed group, but showed no difference on day 16, showing that GTA has small, but inconsistent proconvulsant effects in this model. In MEST tests, a slightly increased seizure threshold (1 mA) was found on day 19 in the GTA-fed group, but not in another experiment on day 21. There were no differences in antioxidant capacity in plasma or cortex between the two groups. ConclusionGlyceryl triacetate feeding showed no antioxidant effects nor beneficial changes in acute electrical seizure threshold mouse models, despite its ability to increase plasma acetate levels.

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.

A CobB like sirtuin in Oryza sativa indica regulates the mitochondrial machinery under stress conditions

Sirtuins are ubiquitous in nature and are known to play an important role as metabolic regulators. In plants, these NAD+ dependent deacetylases are not well characterized. In this study, we are reporting a new member of sirtuin in Oryza sativa indica. OsCobB shares approx. 89% sequence identity with bacterial sirtuin, a class III sirtuin member. This protein is mostly present in mitochondria with trace amounts in the nucleus. It can physically interact with histones H3 and H4 and can specifically deacetylate histone H3 at Lys9 and Lys18 positions. In mitochondria, acetyl coA synthetase (ACS) and isocitrate dehydrogenase 2 (IDH2) are the targets for its deacetylation. This removal of acetyl group is the mode of regulation under certain stress conditions. Thus, this is the first mitochondrial CobB targeting important plant machinery under changing growth environment. The OsCobB deacetylase activity is not majorly affected by its products, nicotinamide and ADP ribose but are sensitive to certain metal ions like Fe2+ and Mg2+. In contrast to some class III members, it does not have any ADP ribosyl transferase activity. In response to abiotic stress conditions like dehydration and low temperature, this enzyme can also mobilize to the nucleus to regulate the plant metabolism.

Development of a 2-pyrrolidone biosynthetic pathway in Corynebacterium glutamicum by engineering an acetyl-CoA balance route.

2-Pyrrolidone is widely used in the textile and pharmaceutical industries. Here, we established a 2-pyrrolidone biosynthesis pathway in Corynebacterium glutamicum, by expressing glutamate decarboxylase (Gad) mutant and β-alanine CoA transferase (Act) which activates spontaneous dehydration cyclization of GABA to form 2-pyrrolidone. Also, the 5' untranslated regions (UTR) strategy was used to increase the expression of protein. Furthermore, considering the importance of acetyl-CoA in the 2-pyrrolidone synthesis pathway, the acetyl-CoA synthetase (acsA) gene was introduced to convert acetate into acetyl-CoA thus achieving the recyclability of the economy. Finally, the fed-batch fermentation of the final strain in a 5L bioreactor produced 10.5g/L 2-pyrrolidone within 78h, which increased by 42.5% by altering the level of gene expression. This is the first time to build the basic chemical 2-pyrrolidone from glucose in one step in C. glutamicum.

Dual-tracer positron emission tomography/computed tomography as an imaging probe of de novo lipogenesis in preclinical models of hepatocellular carcinoma.

BackgroundDe novo lipogenesis is upregulated in many cancers, and targeting it represents a metabolic approach to cancer treatment. However, the treatment response is unpredictable because lipogenic activity varies greatly among individual tumors, thereby necessitating the assessment of lipogenic activity before treatment. Here, we proposed an imaging probe, positron emission tomography/computed tomography (PET/CT) with dual tracers combining 11C-acetate and 18F-fluorodeoxyglucose (18F-FDG), to assess the lipogenic activity of hepatocellular carcinoma (HCC) and predict the response to lipogenesis-targeted therapy.MethodsWe investigated the association between 11C-acetate/18F-FDG uptake and de novo lipogenesis in three HCC cell lines (from well-differentiated to poorly differentiated: HepG2, Hep3B, and SkHep1) by examining the expression of lipogenic enzymes: acetyl-CoA synthetase 2 (ACSS2), fatty acid synthase (FASN), and ATP citrate lyase (ACLY). The glycolysis level was determined through glycolytic enzymes: pyruvate dehydrogenase expression (PDH). On the basis of the findings of dual-tracer PET/CT, we evaluated the treatment response to a lipase inhibitor (orlistat) in cell culture experiments and xenograft mice.ResultsDual-tracer PET/CT revealed the lipogenic activity of various HCC cells, which was positively associated with 11C-acetate uptake and negatively associated with 18F-FDG uptake. This finding represents the negative association between 11C-acetate and 18F-FDG uptake. Because these two tracers revealed the lipogenic and glycolytic activity, respectively, which implies an antagonism between lipogenic metabolism and glucose metabolism in HCC. In addition, dual-tracer PET/CT not only revealed the lipogenic activity but also predicted the treatment response to lipogenesis-targeted therapy. For example, HepG2 xenografts with high 11C-acetate but low 18F-FDG uptake exhibited high lipogenic activity and responded well to orlistat treatment, whereas SkHep1 xenografts with low 11C-acetate but high 18F-FDG uptake exhibited lower lipogenic activity and poor response to orlistat.ConclusionThe proposed non-invasive dual-tracer PET/CT imaging can reveal the lipogenesis and glycolysis status of HCC, thus providing an ideal imaging probe for predicting the therapeutic response of HCC to lipogenesis-targeted therapy.

Effect of Metschnikowia pulcherrima on Saccharomyces cerevisiae PDH By-Pass in MixedFermentation with Varied Sugar Concentrations of Synthetic Grape Juice and Inoculation Ratios

The effects of Metschnikowia pulcherrima and high glucose osmolality on S. cerevisiae pyruvate dehydrogenase pathway (PDH) by-pass were examined by varying the starting sugar concentration of synthetic grape juice and the inoculation ratio of S. cerevisiae to M. pulcherrima. The findings revealed that M. pulcherrima and osmolarity impacted S. cerevisiae’s PDH by-pass. The inoculation concentration of M. pulcherrima significantly affected pyruvate decarboxylase (PDC) activity and acs2 expression when the initial sugar concentration was 200 g L−1 and 290 g L−1. The osmolarity caused by the initial sugar (380 g L−1) significantly influenced the enzymatic activity of S. cerevisiae, which decreased PDC and acetaldehyde dehydrogenase (ALD) activities while increasing Acetyl-CoA synthetase (ACS) activity. The reduction in acetic acid in the wine was caused by M. pulcherrima altering the initial sugar concentration faced by S. cerevisiae, which in turn affected enzymatic activity. The alteration of enzyme activity and accumulation of primary metabolites revealed why mixed fermentation could reduce the acetic acid content in wine by altering the enzymatic activity and affecting the expression of several key genes. The M. pulcherrima inoculation levels had no significant effect on the acetic acid and glycerol concentration in the same fermentation medium.