Multi-omics view of recombinant Yarrowia lipolytica: Enhanced ketogenic amino acid catabolism increases polyketide-synthase-driven docosahexaenoic production to high selectivity at the gram scale.

DHA is a marine PUFA of commercial value, given its multiple health benefits. The worldwide emerging shortage in DHA supply has increased interest in microbial cell factories that can provide the compound de novo. In this regard, the present work aimed to improve DHA production in the oleaginous yeast strain Y. lipolytica Af4, which synthetized the PUFA via a heterologous myxobacterial polyketide synthase (PKS)-like gene cluster. As starting point, we used transcriptomics, metabolomics, and 13C-based metabolic pathway profiling to study the cellular dynamics of Y. lipolytica Af4. The shift from the growth to the stationary DHA-production phase was associated with fundamental changes in carbon core metabolism, including a strong upregulation of the PUFA gene cluster, as well as an increase in citrate and fatty acid degradation. At the same time, the intracellular levels of the two DHA precursors acetyl-CoA and malonyl-CoA dropped by up to 98% into the picomolar range. Interestingly, the degradation pathways for the ketogenic amino acids l-lysine, l-leucine, and l-isoleucine were transcriptionally activated, presumably to provide extra acetyl-CoA. Supplementation with small amounts of these amino acids at the beginning of the DHA production phase beneficially increased the intracellular CoA-ester pools and boosted the DHA titer by almost 40%. Isotopic 13C-tracer studies revealed that the supplements were efficiently directed toward intracellular CoA-esters and DHA. Hereby, l-lysine was found to be most efficient, as it enabled long-term activation, due to storage within the vacuole and continuous breakdown. The novel strategy enabled DHA production in Y. lipolytica at the gram scale for the first time. DHA was produced at a high selectivity (27% of total fatty acids) and free of the structurally similar PUFA DPA, which facilitates purification for high-value medical applications that require API-grade DHA. The assembled multi-omics picture of the central metabolism of Y. lipolytica provides valuable insights into this important yeast. Beyond our work, the enhanced catabolism of ketogenic amino acids seems promising for the overproduction of other compounds in Y. lipolytica, whose synthesis is limited by the availability of CoA ester precursors.

Genetic inactivation of the Carnitine/Acetyl-Carnitine mitochondrial carrier of Yarrowia lipolytica leads to enhanced odd-chain fatty acid production.

BACKGROUND: Mitochondrial carriers (MCs) can deeply affect the intracellular flux distribution of metabolic pathways. The manipulation of their expression level, to redirect the flux toward the production of a molecule of interest, is an attractive target for the metabolic engineering of eukaryotic microorganisms. The non-conventional yeast Yarrowia lipolytica is able to use a wide range of substrates. As oleaginous yeast, it directs most of the acetyl-CoA therefrom generated towards the synthesis of lipids, which occurs in the cytoplasm. Among them, the odd-chain fatty acids (OCFAs) are promising microbial-based compounds with several applications in the medical, cosmetic, chemical and agricultural industries. RESULTS: In this study, we have identified the MC involved in the Carnitine/Acetyl-Carnitine shuttle in Y. lipolytica, YlCrc1. The Y. lipolytica Ylcrc1 knock-out strain failed to grow on ethanol, acetate and oleic acid, demonstrating the fundamental role of this MC in the transport of acetyl-CoA from peroxisomes and cytoplasm into mitochondria. A metabolic engineering strategy involving the deletion of YlCRC1, and the recombinant expression of propionyl-CoA transferase from Ralstonia eutropha (RePCT), improved propionate utilization and its conversion into OCFAs. These genetic modifications and a lipogenic medium supplemented with glucose and propionate as the sole carbon sources, led to enhanced accumulation of OCFAs in Y. lipolytica. CONCLUSIONS: The Carnitine/Acetyl-Carnitine shuttle of Y. lipolytica involving YlCrc1, is the sole pathway for transporting peroxisomal or cytosolic acetyl-CoA to mitochondria. Manipulation of this carrier can be a promising target for metabolic engineering approaches involving cytosolic acetyl-CoA, as demonstrated by the effect of YlCRC1 deletion on OCFAs synthesis.

Enhanced supply of acetyl-CoA by exogenous pantothenate kinase promotes synthesis of poly(3-hydroxybutyrate).

BACKGROUND: Coenzyme A (CoA) is a carrier of acyl groups. This cofactor is synthesized from pantothenic acid in five steps. The phosphorylation of pantothenate is catalyzed by pantothenate kinase (CoaA), which is a key step in the CoA biosynthetic pathway. To determine whether the enhancement of the CoA biosynthetic pathway is effective for producing useful substances, the effect of elevated acetyl-CoA levels resulting from the introduction of the exogenous coaA gene on poly(3-hydroxybutyrate) [P(3HB)] synthesis was determined in Escherichia coli, which express the genes necessary for cyanobacterial polyhydroxyalkanoate synthesis (phaABEC). RESULTS: E. coli containing the coaA gene in addition to the pha genes accumulated more P(3HB) compared with the transformant containing the pha genes alone. P(3HB) production was enhanced by precursor addition, with P(3HB) content increasing from 18.4% (w/w) to 29.0% in the presence of 0.5 mM pantothenate and 16.3%-28.2% by adding 0.5 mM ß-alanine. Strains expressing the exogenous coaA in the presence of precursors contained acetyl-CoA in excess of 1 nmol/mg of dry cell wt, which promoted the reaction toward P(3HB) formation. The amount of acetate exported into the medium was three times lower in the cells carrying exogenous coaA and pha genes than in the cells carrying pha genes alone. This was attributed to significantly enlarging the intracellular pool size of CoA, which is the recipient of acetic acid and is advantageous for microbial production of value-added materials. CONCLUSIONS: Enhancing the CoA biosynthetic pathway with exogenous CoaA was effective at increasing P(3HB) production. Supplementing the medium with pantothenate facilitated the accumulation of P(3HB). ß-Alanine was able to replace the efficacy of adding pantothenate.

A Pseudomonas taiwanensis malonyl-CoA platform strain for polyketide synthesis.

Malonyl-CoA is a central precursor for biosynthesis of a wide range of complex secondary metabolites. The development of platform strains with increased malonyl-CoA supply can contribute to the efficient production of secondary metabolites, especially if such strains exhibit high tolerance towards these chemicals. In this study, Pseudomonas taiwanensis VLB120 was engineered for increased malonyl-CoA availability to produce bacterial and plant-derived polyketides. A multi-target metabolic engineering strategy focusing on decreasing the malonyl-CoA drain and increasing malonyl-CoA precursor availability, led to an increased production of various malonyl-CoA-derived products, including pinosylvin, resveratrol and flaviolin. The production of flaviolin, a molecule deriving from five malonyl-CoA molecules, was doubled compared to the parental strain by this malonyl-CoA increasing strategy. Additionally, the engineered platform strain enabled production of up to 84 mg L-1 resveratrol from supplemented p-coumarate. One key finding of this study was that acetyl-CoA carboxylase overexpression majorly contributed to an increased malonyl-CoA availability for polyketide production in dependence on the used strain-background and whether downstream fatty acid synthesis was impaired, reflecting its complexity in metabolism. Hence, malonyl-CoA availability is primarily determined by competition of the production pathway with downstream fatty acid synthesis, while supply reactions are of secondary importance for compounds that derive directly from malonyl-CoA in Pseudomonas.

How to Build a Metalloenzyme: Lessons from a Protein-Based Model of Acetyl Coenzyme A Synthase.

"What I cannot create, I do not understand"─Richard Feynman. This sentiment motivates the entire field of artificial metalloenzymes. Naturally occurring enzymes catalyze reactions with efficiencies, rates, and selectivity that generally cannot be achieved in synthetic systems. Many of these processes represent vital building blocks for a sustainable society, including CO2 conversion, nitrogen fixation, water oxidation, and liquid fuel synthesis. Our inability as chemists to fully reproduce the functionality of naturally occurring enzymes implicates yet-unknown contributors to reactivity. To identify these properties, it is necessary to consider all of the components of naturally occurring metalloenzymes, from the active site metal(s) to large-scale dynamics. In this Account, we describe the holistic development of a metalloprotein-based model that functionally reproduces the acetyl coenzyme A synthase (ACS) enzyme.ACS catalyzes the synthesis of a thioester, acetyl coenzyme A, from gaseous carbon monoxide, a methyl group donated by a cobalt corrinoid protein, and coenzyme A. The active site of ACS contains a bimetallic nickel site coupled to a [4Fe-4S] cluster. This reaction mimics Monsanto's acetic acid synthesis and represents an ancient process for incorporating inorganic carbon into cellular biomass through the primordial Wood-Ljungdahl metabolic pathway. From a sustainability standpoint, the reversible conversion of C1 substrates into an acetyl group and selective downstream transfer to a thiolate nucleophile offer opportunities to expand this reactivity to the anthropogenic synthesis of liquid fuels. However, substantial gaps in our understanding of the ACS catalytic mechanism coupled with the enzyme's oxygen sensitivity and general instability have limited these applications. It is our hope that development of an artificial metalloenzyme that carries out ACS-like reactions will advance our mechanistic understanding and enable synthesis of robust compounds with the capacity for similar reactivity.To construct this model, we first focused on the catalytic proximal nickel (NiP) site, which has a single metal center bound by three bridging cysteine residues in a "Y"-shaped arrangement. With an initial emphasis on reproducing the general structure of a low-coordinate metal binding site, the type I cupredoxin, azurin, was selected as the protein scaffold, and a nickel center was incorporated into the mononuclear site. Using numerous spectroscopic and computational techniques, including electron paramagnetic resonance (EPR) spectroscopy, nickel-substituted azurin was shown to have similar electronic and geometric structures to the NiP center in ACS. A substrate access channel was installed, and both carbon monoxide and a methyl group were shown to bind individually to the reduced NiI center. The elusive EPR-active S = 1/2 Ni-CH3 species, which has never been detected in native ACS, was observed in the azurin-based model, establishing the capacity of a biological NiI species to support two-electron organometallic reactions. Pulsed EPR studies on the S = 1/2 Ni-CH3 species in azurin suggested a noncanonical electronic structure with an inverted ligand field, which was proposed to prevent irreversible site degradation. This model azurin protein was ultimately shown to perform carbon-carbon and carbon-sulfur bond formation using sequential, ordered substrate addition for selective, stoichiometric thioester synthesis. X-ray spectroscopic methods were used to provide characterization of the remaining catalytic intermediates, resolving some debate over key mechanistic details.The overall approach and strategies that we employed for the successful construction of a functional protein-based model of ACS are described in this Account. We anticipate that these principles can be adapted across diverse metalloenzyme classes, providing essential mechanistic details and guiding the development of next-generation, functional artificial metalloenzymes.

Effects of Marginal Zn Excess and Thiamine Deficiency on Microglial N9 Cell Metabolism and Their Interactions with Septal SN56 Cholinergic Cells.

Mild thiamine deficiency aggravates Zn accumulation in cholinergic neurons. It leads to the augmentation of Zn toxicity by its interaction with the enzymes of energy metabolism. Within this study, we tested the effect of Zn on microglial cells cultivated in a thiamine-deficient medium, containing 0.003 mmol/L of thiamine vs. 0.009 mmol/L in a control medium. In such conditions, a subtoxic 0.10 mmol/L Zn concentration caused non-significant alterations in the survival and energy metabolism of N9 microglial cells. Both activities of the tricarboxylic acid cycle and the acetyl-CoA level were not decreased in these culture conditions. Amprolium augmented thiamine pyrophosphate deficits in N9 cells. This led to an increase in the intracellular accumulation of free Zn and partially aggravated its toxicity. There was differential sensitivity of neuronal and glial cells to thiamine-deficiency-Zn-evoked toxicity. The co-culture of neuronal SN56 with microglial N9 cells reduced the thiamine-deficiency-Zn-evoked inhibition of acetyl-CoA metabolism and restored the viability of the former. The differential sensitivity of SN56 and N9 cells to borderline thiamine deficiency combined with marginal Zn excess may result from the strong inhibition of pyruvate dehydrogenase in neuronal cells and no inhibition of this enzyme in the glial ones. Therefore, ThDP supplementation can make any brain cell more resistant to Zn excess.

Active post-transcriptional regulation and ACLY-mediated acetyl-CoA synthesis as a pivotal target of Shuang-Huang-Sheng-Bai formula for lung adenocarcinoma treatment.

BACKGROUND: New therapeutic approaches are required to improve the outcomes of lung cancer (LC), a leading cause of cancer-related deaths worldwide. Chinese herbal medicine formulae widely used in China provide a unique opportunity for improving LC treatment, and the Shuang-Huang-Sheng-Bai (SHSB) formula is a typical example. However, the underlying mechanisms of action remains unclear. PURPOSE: This study aimed to confirm the efficacy of SHSB against lung adenocarcinoma (LUAD), which is a major histological type of LC, unveil the downstream targets of this formula, and assess the clinical relevance and biological roles of the newly identified target. METHODS: An experimental metastasis mouse model and a subcutaneous xenograft mouse model were used to evaluate the anti-cancer activity of SHSB. Multi-omics profiling of subcutaneous tumors and metabolomic profiling of sera were performed to identify downstream targets, especially the metabolic targets of SHSB. A clinical trial was conducted to verify the newly identified metabolic targets in patients. Next, the metabolites and enzymes engaged in the metabolic pathway targeted by SHSB were measured in clinical samples. Finally, routine molecular experiments were performed to decipher the biological functions of the metabolic pathways targeted by SHSB. RESULTS: Oral SHSB administration showed overt anti-LUAD efficacy as revealed by the extended overall survival of the metastasis model and impaired growth of implanted tumors in the subcutaneous xenograft model. Mechanistically, SHSB administration altered protein expression in the post-transcriptional layer and modified the metabolome of LUAD xenografts. Integrative analysis demonstrated that SHSB markedly inhibited acetyl-CoA synthesis in tumors by post-transcriptionally downregulating ATP-citrate lyase (ACLY). Consistently, our clinical trial showed that oral SHSB administration declined serum acetyl-CoA levels of patients with LC. Moreover, acetyl-CoA synthesis and ACLY expression were both augmented in clinical LUAD tissues of patients, and high intratumoral ACLY expression predicted a detrimental prognosis. Finally, we showed that ACLY-mediated acetyl-CoA synthesis is essential for LUAD cell growth by promoting G1/S transition and DNA replication. CONCLUSION: Limited downstream targets of SHSB for LC treatment have been reported in previous hypothesis-driven studies. In this study, we conducted a comprehensive multi-omics investigation and demonstrated that SHSB exerted its anti-LUAD efficacy by actively and post-transcriptionally modulating protein expression and particularly restraining ACLY-mediated acetyl-CoA synthesis.

Mitochondrial pyruvate carrier influences ganoderic acid biosynthesis in Ganoderma lucidum.

Mitochondrial pyruvate carriers (MPCs), located in the inner membrane of mitochondria, are essential carriers for pyruvate to enter mitochondria. MPCs regulate a wide range of intracellular metabolic processes, such as glycolysis, the tricarboxylic acid cycle (TCA cycle), fatty acid metabolism, and amino acid metabolism. However, the metabolic regulation of MPCs in macrofungi is poorly studied. We studied the role of MPCs in Ganoderma lucidum (GlMPC) on ganoderic acid (GA) biosynthesis regulation in G. lucidum. In this study, we found that the mitochondrial/cytoplasmic ratio of pyruvate was downregulated about 75% in GlMPC1- and GlMPC2-silenced transformants compared with wild type (WT). In addition, the GA content was 17.72 mg/g and increased by approximately 50% in GlMPC1- and GlMPC2-silenced transformants compared with WT. By assaying the expression levels of three key enzymes and the enzyme activities of isocitrate dehydrogenase (IDH) and α-ketoglutarate dehydrogenase (α-KGDH) of the TCA cycle in GlMPC1- and GlMPC2-silenced transformants, it was found that the decrease in GlMPCs activity did not significantly downregulate the TCA cycle rate, and the enzyme activity of IDH increased by 44% compared with WT. We then verified that fatty acid ß-oxidation (FAO) supplements the TCA cycle by detecting the expression levels of key enzymes involved in FAO. The results showed that compared with WT, the GA content was 1.14 mg/g and reduced by approximately 40% in co-silenced transformants. KEY POINTS: • GlMPCs affects the distribution of pyruvate between mitochondria and the cytoplasm. • Acetyl-CoA produced by FAO maintains the TCA cycle. • Acetyl-CoA produced by FAO promotes the accumulation of GA.

Carnitine octanoyltransferase is important for the assimilation of exogenous acetyl-L-carnitine into acetyl-CoA in mammalian cells.

In eukaryotes, carnitine is best known for its ability to shuttle esterified fatty acids across mitochondrial membranes for ß-oxidation. It also returns to the cytoplasm, in the form of acetyl-L-carnitine (LAC), some of the resulting acetyl groups for posttranslational protein modification and lipid biosynthesis. While dietary LAC supplementation has been clinically investigated, its effects on cellular metabolism are not well understood. To explain how exogenous LAC influences mammalian cell metabolism, we synthesized isotope-labeled forms of LAC and its analogs. In cultures of glucose-limited U87MG glioma cells, exogenous LAC contributed more robustly to intracellular acetyl-CoA pools than did ß-hydroxybutyrate, the predominant circulating ketone body in mammals. The fact that most LAC-derived acetyl-CoA is cytosolic is evident from strong labeling of fatty acids in U87MG cells by exogenous 13C2-acetyl-L-carnitine. We found that the addition of d3-acetyl-L-carnitine increases the supply of acetyl-CoA for cytosolic posttranslational modifications due to its strong kinetic isotope effect on acetyl-CoA carboxylase, the first committed step in fatty acid biosynthesis. Surprisingly, whereas cytosolic carnitine acetyltransferase is believed to catalyze acetyl group transfer from LAC to coenzyme A, CRAT-/- U87MG cells were unimpaired in their ability to assimilate exogenous LAC into acetyl-CoA. We identified carnitine octanoyltransferase as the key enzyme in this process, implicating a role for peroxisomes in efficient LAC utilization. Our work has opened the door to further biochemical investigations of a new pathway for supplying acetyl-CoA to certain glucose-starved cells.

Eating your own fat to stay fit: lipophagy sustains lymphangiogenesis.

Lymphatic endothelial cells (LECs) exploit fatty acid oxidation (FAO) to grow and to maintain lymphatic vessel identity through the epigenetic regulation of the essential transcription factor PROX1. In our recent study, we found that LEC-specific loss of ATG5 prevents injury-induced lymphangiogenesis in vivo. Inadequate degradation of lipid droplets (LDs) caused by genetic ablation of ATG5 in LECs disturbs mitochondrial fitness, and reduces mitochondrial FAO and acetyl-CoA levels, ultimately affecting PROX1-mediated epigenetic regulation of CPT1A and key lymphatic markers, most importantly FLT4/VEGFR3. Supplementing the fatty acid precursor acetate rescues defective inflammation-driven lymphangiogenesis in LEC-specific atg5 knockout mice. Thus, efficient macroautophagy/autophagy-mediated LD breakdown is critical to maintain mitochondrial metabolism and acetyl-CoA levels, which sustain a PROX1-mediated lymphatic gene program required for LEC identity and inflammation-driven lymphangiogenesis.

Disturbance of Fatty Acid Metabolism Promoted Vascular Endothelial Cell Senescence via Acetyl-CoA-Induced Protein Acetylation Modification.

Endothelial cell senescence is the main risk factor contributing to vascular dysfunction and the progression of aging-related cardiovascular diseases. However, the relationship between endothelial cell metabolism and endothelial senescence remains unclear. The present study provides novel insight into fatty acid metabolism in the regulation of endothelial senescence. In the replicative senescence model and H2O2-induced premature senescence model of primary cultured human umbilical vein endothelial cells (HUVECs), fatty acid oxidation (FAO) was suppressed and fatty acid profile was disturbed, accompanied by downregulation of proteins associated with fatty acid uptake and mitochondrial entry, in particular the FAO rate-limiting enzyme carnitine palmitoyl transferase 1A (CPT1A). Impairment of fatty acid metabolism by silencing CPT1A or CPT1A inhibitor etomoxir facilitated the development of endothelial senescence, as implied by the increase of p53, p21, and senescence-associated ß-galactosidase, as well as the decrease of EdU-positive proliferating cells. In the contrary, rescue of FAO by overexpression of CPT1A or supplement of short chain fatty acids (SCFAs) acetate and propionate ameliorated endothelial senescence. In vivo, treatment of acetate for 4 weeks lowered the blood pressure and alleviated the senescence-related phenotypes in aortas of Ang II-infused mice. Mechanistically, fatty acid metabolism regulates endothelial senescence via acetyl-coenzyme A (acetyl-CoA), as implied by the observations that suppression of acetyl-CoA production using the inhibitor of ATP citrate lyase NDI-091143 accelerated senescence of HUVECs and that supplementation of acetyl-CoA prevented H2O2-induced endothelial senescence. Deficiency of acetyl-CoA resulted in alteration of acetylated protein profiles which are associated with cell metabolism and cell cycle. These findings thus suggest that improvement of fatty acid metabolism might ameliorate endothelial senescence-associated cardiovascular diseases.

Evaluating anti-obesity potential, active components, and antioxidant mechanisms of Moringa peregrina seeds extract on high-fat diet-induced obesity.

There are no medical drugs that provide an acceptable weight loss with minimal adverse effects. This study evaluated the Moringa peregrina (MP) seed extract's anti-obesity effect. Twenty-four (6/each group) male Sprague Dawley rats were divided into group Ι (control), group ΙΙ (high-fat diet [HFD]), group ΙΙΙ (HFD+ MP [250 mg/kg b.wt]), and group ΙV (HFD+ MP [500 mg/kg b.wt]). MP administration significantly ameliorated body weight gains and HFD induced elevation in cholesterol, triglycerides, LDL, and reduced HDL. Moreover, MP seed oil showed high free radical-scavenging activity, delayed ß-carotene bleaching and inhibited lipoprotein and pancreatic lipase enzymes. High-performance liquid chromatography (HPLC) revealed three major active components: crypto-chlorogenic acid, isoquercetin, and astragalin. Both quantitative Real-time PCR (RT-PCR) and western blotting revealed that MP seeds oil significantly decreased the expression of lipogenesis-associated genes such as peroxisome proliferator-activated receptors gamma (PPARγ) and fatty acid synthase (FAS) and significantly elevated the expression of lipolysis-associated genes (acetyl-CoA carboxylase1, ACCl). The oil also enhanced phosphorylation of AMP-activated protein kinase alpha (AMPK-α) and suppressed CCAAT/enhancer-binding protein ß (C/EBPß). In conclusion, administration of M. peregrina seeds oil has anti-obesity potential in HFD-induced obesity in rats. PRACTICAL APPLICATIONS: M. peregrina seeds oil had a potential anti-obesity activity that may be attributed to different mechanisms. These included decreasing body weight, and body mass index and improving lipid levels by decreasing total cholesterol, triglycerides and LDL-C, and increasing HDL-C. Also, M. peregrina seeds oil regulated adipogenesis-associated genes, such as downregulating the expression of (PPARγ, C/EBPα, and FAS) and improving and upregulating the expression and phosphorylation of AMPKα and ACCl. Despite that M. peregrina extract has reported clear anti-obesity potential through animal and laboratory studies, the available evidence-based on human clinical trials are very limited. Therefore, further studies are needed that could focus on clinical trials investigating anti-obesity potential different mechanisms of M. peregrina extract in humans.

Overproduction, Purification, and Stability of the Functionally Active Human Carnitine Acetyl Transferase.

Human Carnitine Acetyl Transferase (hCAT) reversibly catalyzes the transfer of the acetyl-moiety from acetyl-CoA to L-carnitine, modulating the acetyl-CoA/CoA ratio in mitochondria. Derangement of acetyl-CoA/CoA ratio leads to metabolic alterations that could result in the onset or worsening of pathological states. Due to the importance of CAT as a pharmacological target and to the European directive for reducing animal experimentation, we have pointed out a procedure to produce a recombinant, pure, and functional hCAT using the E. coli expression system. The cDNA encoding for the hCAT was cloned into the pH6EX3 vector. This construct was used to transform the E. coli Rosetta strain. The optimal conditions for the overexpression of the fully active hCAT include induction with a low concentration of IPTG (0.01 mM) and a low growth temperature (25 °C). The recombinant protein was purified from bacterial homogenate by affinity chromatography. The pure hCAT is very stable in an aqueous solution, retaining full activity for at least two months if stored at - 20 °C. These results could be helpful for a broad set of functional studies on hCAT, including drug-design applications.

L-Arginine enhanced perylenequinone production in the endophytic fungus Shiraia sp. Slf14(w) via NO signaling pathway.

Perylenequinones (PQ) are natural polyketides used as anti-microbial, -cancers, and -viral photodynamic therapy agents. Herein, the effects of L-arginine (Arg) on PQ biosynthesis of Shiraia sp. Slf14(w) and the underlying molecular mechanism were investigated. The total content of PQ reached 817.64 ± 72.53 mg/L under optimal conditions of Arg addition, indicating a 30.52-fold improvement over controls. Comparative transcriptome analysis demonstrated that Arg supplement promoted PQ precursors biosynthesis of Slf14(w) by upregulating the expression of critical genes associated with the glycolysis pathway, and acetyl-CoA and malonyl-CoA synthesis. By downregulating the expression of genes related to the glyoxylate cycle pathway and succinate dehydrogenase, more acetyl-CoA flow into the formation of PQ. Arg supplement upregulated the putative biosynthetic gene clusters for PQ and activated the transporter proteins (MFS and ABC) for exudation of PQ. Further studies showed that Arg increased the gene transcription levels of nitric oxide synthase (NOS) and nitrate reductase (NR), and activated NOS and NR, thus promoting the formation of nitric oxide (NO). A supplement of NO donor sodium nitroprusside (SNP) also confirmed that NO triggered promoted biosynthesis and efflux of PQ. PQ production stimulated by Arg or/and SNP can be significantly inhibited upon the addition of NO scavenger carboxy-PTIO, NOS inhibitor Nω-nitro-L-arginine, or soluble guanylate cyclase inhibitor NS-2028. These results showed that Arg-derived NO, as a signaling molecule, is involved in the biosynthesis and regulation of PQ in Slf14(W) through the NO-cGMP-PKG signaling pathway. Our results provide a valuable strategy for large-scale PQ production and contribute to further understanding of NO signaling in the fungal metabolite biosynthesis. KEY POINTS: • PQ production of Shiraia sp. Slf14(w) was significantly improved by L-arginine addition. • Arginine-derived NO was firstly reported to be involved in the biosynthesis and regulation of PQ. • The NO-cGMP-PKG signaling pathway was proposed for the first time to participate in PQ biosynthesis.

NaCT/SLC13A5 facilitates citrate import and metabolism under nutrient-limited conditions.

Citrate lies at a critical node of metabolism, linking tricarboxylic acid metabolism and lipogenesis via acetyl-coenzyme A. Recent studies have observed that deficiency of the sodium-dependent citrate transporter (NaCT), encoded by SLC13A5, dysregulates hepatic metabolism and drives pediatric epilepsy. To examine how NaCT contributes to citrate metabolism in cells relevant to the pathophysiology of these diseases, we apply 13C isotope tracing to SLC13A5-deficient hepatocellular carcinoma (HCC) cells and primary rat cortical neurons. Exogenous citrate appreciably contributes to intermediary metabolism only under hypoxic conditions. In the absence of glutamine, citrate supplementation increases de novo lipogenesis and growth of HCC cells. Knockout of SLC13A5 in Huh7 cells compromises citrate uptake and catabolism. Citrate supplementation rescues Huh7 cell viability in response to glutamine deprivation or Zn2+ treatment, and NaCT deficiency mitigates these effects. Collectively, these findings demonstrate that NaCT-mediated citrate uptake is metabolically important under nutrient-limited conditions and may facilitate resistance to metal toxicity.

Didymin switches M1-like toward M2-like macrophage to ameliorate ulcerative colitis via fatty acid oxidation.

Inflammatory response by different polarized macrophages has a critical role in a variety of immunological pathophysiology, such as ulcerative colitis (UC). Herein, targeting the paradigm of macrophage phenotypes by small molecular modulators may influence the disease status. In the present study, we firstly demonstrated that didymin, one of the most abundant flavonoid constituents present in the citrus fruits such as oranges and lemons, remarkably attenuated the clinical symptoms of acute and chronic colitis in mice. Mechanistic studies showed that didymin converted pro-inflammatory M1-like to anti-inflammatory M2-like macrophage phenotype, but did not alter the polarization of M2-like macrophages. Metabolic tracing studies revealed that didymin strengthened fatty acid oxidation rather than glycolysis by inducing Hadhb expression. More importantly, in vivo studies verified that promotion of Hadhb expression resulted in the conversion of M1- toward M2-like macrophages and eventually alleviated colitis. Our data highlights the potential of macrophage paradigm in UC inflammation and put forth the stage for considering didymin as a metabolism regulator in reprogramming macrophage polarization, which may serve as a promising therapeutic approach for treatment of inflammation-associated disorders.

Catechin-Rich Green Tea Extract and the Loss-of-TLR4 Signaling Differentially Alter the Hepatic Metabolome in Mice with Nonalcoholic Steatohepatitis.

SCOPE: Catechin-rich green tea extract (GTE) limits inflammation in nonalcoholic steatohepatitis (NASH) consistent with a Toll-like receptor 4 (TLR4)-dependent mechanism. It is hypothesized that GTE supplementation during NASH will shift the hepatic metabolome similar to that attributed to the loss-of-TLR4 signaling. METHODS AND RESULTS: Wild-type (WT) and loss-of-function TLR4-mutant (TLR4mut ) mice are fed a high-fat diet containing 0% or 2% GTE for 8 weeks prior to performing untargeted mass spectrometry-based metabolomics on liver tissue. The loss-of-TLR4 signaling and GTE shift the hepatic metabolome away from that of WT mice. However, relatively few metabolites are altered by GTE in WT mice to the same extent as the loss-of-TLR4 signaling in TLR4mut mice. GTE increases acetyl-coenzyme A precursors and spermidine to a greater extent than the loss-of-TLR4 signaling. Select metabolites associated with thiol metabolism are similarly affected by GTE and the loss-of-TLR4 signaling. Glycerophospholipid catabolites are decreased by GTE, but are unaffected in TLR4mut mice. Conversely, the loss-of-TLR4 signaling but not GTE increases several bile acid metabolites. CONCLUSION: GTE limitedly alters the hepatic metabolome consistent with a TLR4-dependent mechanism. This suggests that the anti-inflammatory activities of GTE and loss-of-TLR4 signaling that regulate hepatic metabolism to abrogate NASH are likely due to distinct mechanisms.

Chronic pantothenic acid supplementation does not affect muscle coenzyme A content or cycling performance.

This study determined if supplementation with pantothenic acid (PA) for 16 weeks could increase skeletal muscle coenzyme A (CoASH) content and exercise performance. Trained male cyclists (n = 14) were matched into control or PA (6 g·day-1) groups. At 0, 4, 8, and 16 weeks, subjects performed an incremental time to exhaustion cycle with muscle biopsies taken prior to and following exercise. Prolonged PA supplementation did not change skeletal muscle CoASH and acetyl-CoA contents or exercise performance. Novelty: Supplementation with pantothenic acid for 16 weeks had no effect on skeletal muscle CoASH and acetyl-CoA content or exercise performance in trained male cyclists.

Tumour Microenvironment: Roles of the Aryl Hydrocarbon Receptor, O-GlcNAcylation, Acetyl-CoA and Melatonergic Pathway in Regulating Dynamic Metabolic Interactions across Cell Types-Tumour Microenvironment and Metabolism.

This article reviews the dynamic interactions of the tumour microenvironment, highlighting the roles of acetyl-CoA and melatonergic pathway regulation in determining the interactions between oxidative phosphorylation (OXPHOS) and glycolysis across the array of cells forming the tumour microenvironment. Many of the factors associated with tumour progression and immune resistance, such as yin yang (YY)1 and glycogen synthase kinase (GSK)3ß, regulate acetyl-CoA and the melatonergic pathway, thereby having significant impacts on the dynamic interactions of the different types of cells present in the tumour microenvironment. The association of the aryl hydrocarbon receptor (AhR) with immune suppression in the tumour microenvironment may be mediated by the AhR-induced cytochrome P450 (CYP)1b1-driven 'backward' conversion of melatonin to its immediate precursor N-acetylserotonin (NAS). NAS within tumours and released from tumour microenvironment cells activates the brain-derived neurotrophic factor (BDNF) receptor, TrkB, thereby increasing the survival and proliferation of cancer stem-like cells. Acetyl-CoA is a crucial co-substrate for initiation of the melatonergic pathway, as well as co-ordinating the interactions of OXPHOS and glycolysis in all cells of the tumour microenvironment. This provides a model of the tumour microenvironment that emphasises the roles of acetyl-CoA and the melatonergic pathway in shaping the dynamic intercellular metabolic interactions of the various cells within the tumour microenvironment. The potentiation of YY1 and GSK3ß by O-GlcNAcylation will drive changes in metabolism in tumours and tumour microenvironment cells in association with their regulation of the melatonergic pathway. The emphasis on metabolic interactions across cell types in the tumour microenvironment provides novel future research and treatment directions.

Effects of propionate concentration on short-term metabolism in liver explants from dairy cows in the postpartum period.

Our objective was to determine the temporal effects of increasing supply of propionate on propionate metabolism in liver tissue of dairy cows in the postpartum (PP) period. A total of 6 dairy cows [primiparous: n = 3, 9.00 ± 1.00 d PP (mean ± SD) and multiparous: n = 3; 4.67 ± 1.15 d PP] were biopsied for liver explants in a block-design experiment. Explants were treated with 3 concentrations of [13C3]sodium propionate of 1, 2, or 4 mM. Explants were incubated in 2 mL of Medium 199 supplemented with 1% BSA, 0.6 mM oleic acid, 2 mM sodium l-lactate, 0.2 mM sodium pyruvate, and 0.5 mMl-glutamine at 38°C and sampled at 0.5, 15, and 60 min. Increasing the concentration of [13C3]propionate increased total 13C% enrichment of propionyl coenzyme A (CoA), succinate, fumarate, malate, and citrate with time. Concentration of propionate did not affect total 13C% enrichment of hepatic glucose or acetyl CoA, but total 13C% enrichment increased with time for hepatic glucose. The 13C labeling from propionate was incorporated into acetyl CoA, but increased concentrations of propionate did not result in greater labeling of acetyl CoA. However, increases in 13C% enrichment of [M+4]citrate and [M+5]citrate concentrations of [13C3]propionate indicate propionate conversion to acetyl CoA and subsequent entry of acetyl CoA into the tricarboxylic acid cycle in dairy cows in the PP period. This research presents evidence that despite an increase in hepatic acetyl CoA concentration and general consensus on the upregulation of gluconeogenesis of dairy cows during the PP period, carbon derived from propionate contributes to the pool of acetyl CoA, which increases as concentration of propionate increases, in addition to stimulating oxidation of acetyl CoA from other sources. Because of the hypophagic effects of propionate, but importance of propionate as a glucose precursor, a balance of propionate supply to dairy cows could lead to improvements in dry matter intake, and subsequently, health and production in dairy cows.