The Ca^(2+)/CaN/ACC and cAMP/PKA/HK signal pathways are required for PBAN-mediated sex pheromone biosynthesis in Conogethes punctiferalis

Conogethes punctiferalis is a crop and fruit pest that has caused serious economic losses to agricultural production.This pest relies heavily on its sex pheromone to ensure sexual encounters and subsequent mating success.However,the molecular mechanism underlying sex pheromone biosynthesis in this species remains elusive.The present study investigated the detailed mechanism underlying PBAN-regulated sex pheromone biosynthesis in C.punctiferalis by transcriptome sequencing of the C.punctiferalis pheromone glands(PGs)and subsequent functional identification of the target genes.The results showed that female mating started from the first scotophase,and peaked at the second to fifth scotophases in accordance with the release of sex pheromones.PBAN regulated sex pheromone biosynthesis by employing Ca^(2+)and cAMP as secondary messengers,as demonstrated by RNA interference(RNAi),pharmacological inhibitors,and behavioral assays.Further investigation revealed that calcineurin(CaN)and acetyl-CoA carboxylase(ACC)were activated by PBAN/Ca^(2+)signaling,and the RNAimediated knockdown of CaN and ACC transcripts significantly reduced sex pheromone production,ultimately leading to a significantly reduced ability of females to attract males.Importantly,hexokinase(HK)was found to regulate sex pheromone biosynthesis in response to the PBAN/cAMP/PKA signaling pathway,as demonstrated by RNAi,enzyme activity,and pharmacological inhibitor assays.Furthermore,Far2 and Desaturase1 were found to participate in PBAN-regulated sex pheromone biosynthesis.Altogether,our findings revealed that PBAN regulates sex pheromone biosynthesis through the PBANR/Ca^(2+)/CaN/ACC and PBANR/cAMP/PKA/HK pathways in C.punctiferalis,which enriches our comprehension of the details of sex pheromone biosynthesis in moths.

高效液相色谱法(HPLC)测定保加利亚乳杆菌胞内Acetyl-CoA

建立了一种快速测定保加利亚乳杆菌胞内Acetyl—CoA含量的HPLC分析方法。以C18（HypersilODS25μm）色谱柱为固定相，以缓冲液A（0．2mol／麟酸钠，pH=5）和缓冲液B（800mL0．25m01／I磷酸钠，pH=5和200mL乙腈的混合物）为流动相，梯度洗脱，流速为1mE]min，柱温25℃，采用紫外检测器（254nm）进行检测。结果表明：该方法可以在17rain内实现EAcetyl—CoA与其他物质完全分离和定量，Acetyl—CoA在0．011-0．359μmol／mL范围内的线性关系良好，回归方程线性相关系数R2=0．9995，检出限（以信噪比（S／N）为3计）为0．28mg／L，定量限（r2S／N为10计）为0．32mg／L。该方法用于保加利亚乳杆菌胞内Acetyl—CoA含量的测定，相对标准偏差（n=6）为0．75％，重现性良好，三个水平的加标回收率为94．8％～103．3％，该方法适用于保加利亚乳杆菌胞内Acetyl—CoA快速、高效分离和定量。

Acetyl-CoA carboxylase inhibitors in non-alcoholic steatohepatitis: Is there a benefit?

De novo lipogenesis(DNL)plays an important role in the pathogenesis of hepatic steatosis and also appears to be implicated in hepatic inflammation and fibrosis.Accordingly,the inhibition of acetyl-CoA carboxylase,which catalyzes the ratelimiting step of DNL,might represent a useful approach in the management of patients with nonalcoholic fatty liver disease(NAFLD).Animal studies and preliminary data in patients with NAFLD consistently showed an improvement in steatosis with the use of these agents.However,effects on fibrosis were variable and an increase in plasma triglyceride levels was observed.Therefore,more longterm studies are needed to clarify the role of these agents in NAFLD and to determine their risk/benefit profile.

Entamoeba histolytica acetyl-CoA synthetase:biomarker of acute amoebic liver abscess

Objective:To characterize the Entamoeba histolytica(E.histolytica)antigen(s)recognized by moribound amoebic liver abscess hamsters.Methods:Crude soluble antigen of E.histolytica was probed with sera of moribund hamsters in 1D-and 2D-Westem blot analyses.The antigenic protein was then sent for tandem mass spectrometry analysis.The corresponding gene was cloned and expressed in Escherichia coli BL21-AI to produce the recombinant E.histolytica ADP-forming acetyl-CoA synthetase(EhACS)protein.A customised ELISA was developed to evaluate the sensitivity and specificity of the recombinant protein.Results:A^75 kDa protein band with a pl value of 5.91-6.5 was found to be antigenic;and not detected by sera of hamsters in the control group.Tandem mass spectrometry analysis revealed the protein to be the 77 kDa E.histolytica ADP-forming acetyl-CoA synthetase(EhACS).The customised ELISA results revealed 100%sensitivity and 100%specificity when tested against infected(n=31)and control group hamsters(n=5)serum samples,respectively.Conclusions:This rinding suggested the significant role of EhACS as a biomarker for moribund hamsters with acute amoebic liver abscess(ALA)infection.It is deemed pertinent that future studies explore the potential roles of EhACS in better understanding the pathogenesis of ALA;and in the development of vaccine and diagnostic tests to control ALA in human populations.

Cloning, Expression and Purification of Wheat Acetyl-CoA Carboxylases CT Domain in E. coil

The entire gene of carboxyltransferase（CT） domain of acetyl-CoA carboxylase（ACCase） from Chinese Spring wheat（CSW） plastid was cloned firstly, and the 2.3 kb gene was inserted into PET28a^＋ vector and expressed in E. coil in a soluble state. The （His）6 fusion protein was identified by SDS-PAGE and Western blot. The recombinant protein was purified by affinity chromatography, and the calculated molecular mass（Mr） was 88000. The results of the sequence analysis indicate that the cloned gene（GeneBank accession No. EU124675） was a supplement and revision of the reported ACCase CT partial cDNA from Chinese Spring wheat plastid. The recombinant protein will be significant for us to investigate the recognizing mechanism between ACCase and herbicides, and further to screen new herbicides.

Detection of Acetyl-CoA Carboxylase （ACCase） Inhibitor Herbicides Resistance in Sterile Wild Oat （Avena sterilis L.） Using Agar Quick Test

This study was conducted using seeds in Petri dish containing agar medium in order to determine acetyl-CoA carboxylase （ACCase） herbicides resistance （R） in Avena sterilis that was grown in wheat fields at Adana province, Turkey. Seeds were collected from one large suspected field, where clodinafop-propargyl （Aryloxyphenoxypropionate “FOPs”） and pinoxaden （Phenylpyrazoline “DEN”） have been applied for many years. Susceptible （S） population was collected from the road side on the same region. Agar media of concentration 14 g/L was prepared and it was melted in microwave. Then the amount of 20 mL agar media was added into each Petri dish. Five seeds were placed on agar mediums containing discriminating dose of clodinafop and pinoxaden. Petri dishes were placed in growth incubator operating at 10 °C. After 15 d, both radicle and hypocotyl length were measured. The percentage of germinated seed and dose-response curves were determined. At these different concentration levels, there were more than 50% of R and less than 40% of S seed germinated for pinoxaden. However, for clodinafop, more than 60% of R and less than 50% of S seeds were germinated. At higher concentration levels, the populations of resistant and susceptible were not germinated for both herbicides. The resistance value of R population was then compared with that of the S biotype. From the resistance index （RI）, the population was more resistant to pinoxaden （7.43 for radicle and 2.47 for hypocotyl） than the clodinafop-propagyl （1.39 for radicle and 3.77 for hypocotyl）. The method provided a simple, quick and cost effective way to identify ACCase herbicides resistance in most grass weeds.

Roles of vitamin A in the regulation of fatty acid synthesis

Dietary macronutrients and micronutrients play important roles in human health.On the other hand,the excessive energy derived from food is stored in the form of triacylglycerol.A variety of dietary and hormonal factors affect this process through the regulation of the activities and expression levels of those key player enzymes involved in fatty acid biosynthesis such as acetyl-CoA carboxylase,fatty acid synthase,fatty acid elongases,and desaturases.As a micronutrient,vitamin A is essential for the health of humans.Recently,vitamin A has been shown to play a role in the regulation of glucose and lipid metabolism.This review summarizes recent research progresses about the roles of vitamin A in fatty acid synthesis.It focuses on the effects of vitamin A on the activities and expression levels of mRNA and proteins of key enzymes for fatty acid synthesis in vitro and in vivo.It appears that vitamin A status and its signaling pathway regulate the expression levels of enzymes involved in fatty acid synthesis.Future research directions are also discussed.

Gaining access to acetyl-CoA by peroxisomal surface display

Synthetic biology is constantly making progress for producing compounds on demand.Recently,Yocum and collaborators have developed an outstanding approach based on the anchoring of biosynthetic enzymes to the peroxisomal membrane.This allowed access to an untapped resource of acetyl-CoA and stimulated the synthesis of a valuable polyketide.

Research progress on unusual acetyl triacylglycerol

Acetyl triacylglycerol (acetyl-TAG) containing acetyl at sn-3 position is rare in nature. It is optically active asymmetric TAG with low viscosity, having improved cold temperature property and low calorific content compared to regular TAGs. It gains increasingly attentions due to its potential applications. Metabolic engineering of plant or microorganism for production of acetyl-TAG is currently attractive. Here we summarize the researches on acetyl-TAG, with emphasis on gene discovery, protein analysis and its potential applications. Comprehensive understanding of current development in acetyl-TAG biosynthesis pathway may contribute to the increase of production and more applications.

Acetyl coenzyme A carboxylase modulates lipogenesis and sugar homeostasis in Blattella germanica

Lipid and sugar homeostasis is critical for insect development and survival.In this study,we characterized an acetyl coenzyme A carboxylase gene in Blattella germanica(BgACC)that is involved in both lipogenesis and sugar homeostasis.We found that BgACC was dominantly expressed in the fat body and integument,and was significantly upregulated after molting.Knockdown of BgACC in 5th-instar nymphs did not affect their normal molting to the next nymphal stage,but it caused a lethal phenotype during adult emergence.BgACC-RNA interference(RNAi)significantly downregulated total free fatty acid(FFA)and triacylglycerol(TAG)levels,and also caused a significant decrease of cuticular hydrocarbons(CHCs).Repression of BgACC in adult females affected the development of oocytes and resulted in sterile females,but BgACC-RNAi did not affect the reproductive ability of males.Interestingly,knockdown of BgACC also changed the expression of insulin-like peptide genes(BglLPs),which mimicked a physiological state of high sugar uptake.In addition,BgACC was upregulated when B.germanica were fed on a high sucrose diet,and repression of BgACC upregulated the expression of the glycogen synthase gene(BgGlyS).Moreover,BgACC-RNAi increased the circulating sugar levels and glycogen storage,and a longevity assay suggested that BgACC was important for the survival of B.germanica under conditions of high sucrose uptake.Our results confirm that BgACC is involved in multiple lipid biogenesis and sugar homeostasis processes,which further modulates insect reproduction and sugar tolerance.This study benefits our understanding of the crosstalk between lipid and sugar metabolism.

Metalloproteins/metalloenzymes for the synthesis of acetyl-CoA in the Wood-Ljungdahl pathway

This paper focuses on the group of metalloproteins/metalloenzymes in the acetyl-coenzyme A synthesis pathway of anaerobic microbes called Wood-Ljungdahl pathway,including formate dehydrogenase (FDH),corrinoid iron sulfur protein (CoFeSP),acetyl-CoA synthase (ACS) and CO dehydrogenase (CODH). FDH,a key metalloenzyme involved in the conversion of carbon dioxide to methyltetrahydrofolate,catalyzes the reversible oxidation of formate to carbon dioxide. CoFeSP,as a methyl group transformer,accepts the methyl group from CH3-H4 folate and then transfers it to ACS. CODH reversibly catalyzes the reduction of CO2 to CO and ACS functions for acetyl-coenzyme A synthesis through condensation of the methyl group,CO and coenzyme A,to finish the whole pathway. This paper introduces the structure,function and reaction mechanisms of these enzymes.

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

10-DeacetylbaccatinⅢ(10-DAB)C10 acetylation is an indispensable procedure for Taxol semi-synthesis,which often requires harsh conditions.10-DeacetylbaccatinⅢ-10-β-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 baccatinⅢ.We found that baccatinⅢcould 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 baccatinⅢ.The fermentation conditions were optimized and the baccatinⅢtiters 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 environmentfriendly 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.

Metabolism along the life journey of T cells

T cells are one of few cell types in adult mammals that can proliferate extensively and differentiate diversely upon stimulation,which serves as an excellent example to dissect the metabolic basis of cell fate decisions.During the last decade,there has been an explosion of research into the metabolic control of T-cell responses.The roles of common metabolic pathways,including glycolysis,lipid metabolism,and mitochondrial oxidative phosphorylation,in T-cell responses have been well characterized,and their mechanisms of action are starting to emerge.In this review,we present several considerations for T-cell metabolism-focused research,while providing an overview of the metabolic control of T-cell fate decisions during their life journey.We try to synthesize principles that explain the causal relationship between cellular metabolism and T-cell fate decision.We also discuss key unresolved questions and challenges in targeting T-cell metabolism to treat disease.

Determination of carbon-fixing potential of Bathyarchaeota in marine sediment by DNA stable isotope probing analysis

Bathyarchaeota is believed to play a crucial role in the global carbon cycle due to its vast biomass,broad distribution,and diverse habitat.However,its physiological and metabolic features are hard to determine without pure culture.While metagenomic analyses have shown that Bathyarchaeota has a complete inorganic carbon fixation(Wood-Ljungdahl,WL)pathway,no direct functional confirmation has been reported.To explore the inorganic carbon fixation ability of Bathyarchaeota,we used lignin and sodium bicarbonate-^(13)C(NaH^(13)CO_(3))in the long-term incubation of marine sediment samples.We found that Bathyarchaeota grew continuously in the cultivation system with lignin,and its abundance increased up to 15.3 times after10 months,increasing its fraction of all archaea from 30%to 80%.We monitored theδ^(13)C of total organic carbon to identify microbial carbon fixation in the cultivation systems,finding that it increased in the first month while NaH^(13)CO_(3)was present but only increased continuously afterward when lignin was also present.Furthermore,ultracentrifugation was performed on DNA extracted from samples at different cultivation stages to separate DNA of different buoyant densities,and bathyarchaeotal and bacterial 16S ribosomal RNA(r RNA)gene abundance were quantified using qPCR.Compared to bacteria,bathyarchaeotal 16S rRNA tended to be concentrated in heavy layers after 4 months of incubation with lignin and NaH^(13)CO_(3),indicating that Bathyarchaeota DNA contained^(13)C through proliferation based on lignin utilization and NaH^(13)CO_(3)assimilation,proving the carbon fixation capacity of Bathyarchaeota.

ACAT1 deficiency in myeloid cells promotes glioblastoma progression by enhancing the accumulation of myeloid-derived suppressor cells

Glioblastoma(GBM)is a highly aggressive and lethal brain tumor with an immunosuppressive tumor microenvironment(TME).In this environment,myeloid cells,such as myeloid-derived suppressor cells(MDSCs),play a pivotal role in suppressing antitumor immunity.Lipometabolism is closely related to the function of myeloid cells.Here,our study reports that acetyl-CoA acetyltransferase 1(ACAT1),the key enzyme of fatty acid oxidation(FAO)and ketogenesis,is significantly downregulated in the MDSCs infiltrated in GBM patients.To investigate the effects of ACAT1 on myeloid cells,we generated mice with myeloid-specific(LyzM-cre)depletion of ACAT1.The results show that these mice exhibited a remarkable accumulation of MDSCs and increased tumor progression both ectopically and orthotopically.The mechanism behind this effect is elevated secretion of C-X-C motif ligand 1(CXCLI)of macrophages(Mo).Overall,our findings demonstrate that ACAT1 could serve as a promising drug target for GBM by regulating the function of MDSCs in the TME.

Differential expression of the TwHMGS gene and its effect on triptolide biosynthesis in Tripterygium wilfordii

3-Hydroxy-3-methylglutaryl-CoA synthase(HMGS) is the first committed enzyme in the MVA pathway and involved in the biosynthesis of terpenes in Tripterygium wilfordii. The full-length cDNA and a 515 bp RNAi target fragment of TwHMGS were ligated into the p H7 WG2 D and p K7 GWIWG2 D vectors to respectively overexpress and silence, Tw HMGS was overexpressed and silenced in T. wilfordii suspension cells using biolistic-gun mediated transformation, which resulted in 2-fold increase and a drop to70% in the expression level compared to cells with empty vector controls. During Tw HMGS overexpression, the expression of TwHMGR, TwDXR and TwTPS7 v2 was significantly upregulated to the control. In the RNAi group, the expression of Tw HMGR,TwDXS, TwDXR and TwMCT visibly displayed downregulation to the control. The cells with TwHMGS overexpressed produced twice higher than the control value. These results proved that differential expression of Tw HMGS determined the production of triptolide in T.wilfordii and laterally caused different trends of relative gene expression in the terpene biosynthetic pathway. Finally, the substrate acetyl-Co A was docked into the active site of TwHMGS, suggesting the key residues including His247, Lys256 and Arg296 undergo electrostatic or H-bond interactions with acetyl-CoA.

Modulation of Guard Cell Turgor and Drought Folerance by a Peroxisomal Acetate-Malate Shunt

In plants, stomatal movements are tightly controlled by changes in cellular turgor pressure. Carbohydrates produced by glycolysis and the tricarboxylic acid cycle play an important role in regulating turgor pressure. Here, we describe anArabidopsis mutant, bzul, isolated in a screen for elevated leaf temperature in response to drought stress, which displays smaller stomatal pores and higher drought resistance than wild-type plants. BZU1 encodes a known acetyl-coenzyme A synthetase, ACN1, which acts in the first step of a metabolic pathway converting acetate to malate in peroxisomes. We showed that BZUl/ACNl-mediated acetate-to-malate conversion provides a shunt that plays an important role in osmoregulation of stomatal turgor. We found that the smaller stomatal pores in the bzul mutant are a consequence of reduced accumu- lation of malate, which acts as an osmoticum and/or a signaling molecule in the control of turgor pressure within guard cells, and these results provided new genetic evidence for malate-regulated stomatal movement. Collectively, our results indicate that a peroxisomal BZUl/ACNl-mediated acetate--malate shunt regulates drought resistance by controlling the turgor pressure of guard cells in Arabidopsis.

Discovery of Novel Long-Chain Alkenyl Diacid Derivatives as ACLY Inhibitors

ATP citrate lyase(ACLY)synthesizes cytosolic acetyl coenzyme A(acetyl-CoA),an essential biosynthetic precursor for lipid synthesis and the acetyl donor required for protein acetylation.The aberrant expression and activity of ACLY has been documented in multiple human cancers.ETC-1002 is an indirect ACLY inhibitor,and it has recently been approved by the FDA as an additional therapeutic option in high-risk hypercholesterolemia patients unable to meet goals with standard therapy.In this work,we identified a series of novel long-chain alkenyl diacids as potent direct ACLY inhibitors,and comprehensive structure-activity relationship analysis showed that compound 18f was the most potent ACLY inhibitor with an IC50 value of 1.5μmol/L.Subsequent ester formation of 18f gave a new series of compounds such as 25f that maintained ACLY inhibitory activity and improved antitumor cell proliferation effects.

Unlocking a new target for streptomycetes strain improvement

Bacteria of the Streptomyces genus are well-known producers of secondary metabolites of high medical value.They contributed nearly 60%of current antibiotics(i.e.,vancomycin,daptomycin and tetracycline),as well as antifungals,antiparasitics(avermectins),anticancer drugs(doxorubicin),immunosuppressants and others[1].Therefore,in the past several decades,Streptomyces bacteria or the therein involved biosynthetic pathways have long been the central topic of strain improvement,metabolic engineering,and bioengineering.Much of this research has focused on regulatory elements;however,it has been a challenge due to the complex regulatory network that controls Streptomyces secondary metabolism and its complex fungus-like morphological differentiation[2].On agar plates,production of secondary metabolites coincides with the switch from vegetative growth(substrate mycelium)to aerial mycelium hyphae and subsequent spore formation,whereas in a liquid culture,early stationary phase when cells stop growing marks the initiation of secondary metabolism.

Reconciling host-microbiota metabolic incompatibility safeguards male fertility

The symbiotic relationship between the host and microbiota is widely acknowledged as mutually beneficial.However,due to significant differences in metabolic substrates and products between prokaryotic bacteria and mammalian cells,mechanisms must exist to reconcile the metabolic incompatibility between the host and microbiota.We report that host enzymes are required to detoxify gut microbiota-derived acetate to maintain male fertility in mice.The combined deletion of acetyl-CoA synthetase short-chain family member 1 and 2(ACSS1 and ACSS2),two enzymes consuming acetate in mammals,leads to excessive accumulation of acetate in circulation.This accumulation causes metabolic acidosis,blocking spermatogenesis and rendering male mice infertile.ACSS1/2-deficient germ cells exhibit comprehensive metabolic alterations with nicotinamide adenine dinucleotide(NAD+)deficiency that impairs betaine production.Supplementation with betaine restores spermatogenesis and fertility in ACSS1/2-deficient mice.Thus,the inevitable production of acetate by gut bacteria and its reproductive toxicity to the host represents an unappreciated metabolic incompatibility between the host and microbiota,which is reconciled by ACSS1/2.