Investigating the response of the butyrate production potential to major fibers in dietary intervention studies.

Interventions involving dietary fibers are known to benefit host health. A leading contribution of gut microbiota is commonly recognized with production of short chain fatty acids (SCFA) suspected to play a key role. However, the detailed mechanisms are largely unknown, and apart from a well-described bifidogenic effect of some fibers, results for other bacterial taxa are often incongruent between studies. We performed pooled analyses of 16S rRNA gene data derived from intervention studies (n = 14) based on three fibers, namely, inulin-type fructans (ITF), resistant starch (RS), and arabinoxylan-oligosaccharides (AXOS), harmonizing the bioinformatics workflow to reveal taxa stimulated by those substrates, specifically focusing on the SCFA-production potential. The results showed an increased butyrate production potential after ITF (p < 0.05) and RS (p < 0.1) treatment via an increase in bacteria exhibiting the enzyme butyryl-CoA:acetate CoA-transferase (but) that was governed by Faecalibacterium, Anaerostipes (ITF) and Agathobacter (RS) respectively. AXOS did not promote an increase in butyrate producers, nor were pathways linked to propionate production stimulated by any intervention. A bifidogenic effect was observed for AXOS and ITF, which was only partly associated with the behavior of but-containing bacteria and largely represented a separate response. Low and high Ruminococcus abundances pre-intervention for ITF and RS, respectively, promoted an increase in but-containing taxa (p < 0.05) upon interventions, whereas initial Prevotella abundance was negatively associated with responses of butyrate producers for both fibers. Collectively, our data demonstrate targeted stimulation of specific taxa by individual fibers increasing the potential to synthesize butyrate, where gut microbiota composition pre-intervention strongly controlled outcomes.

Characterization, Structure, and Inhibition of the Human Succinyl-CoA:glutarate-CoA Transferase, a Putative Genetic Modifier of Glutaric Aciduria Type 1.

Glutaric Aciduria Type 1 (GA1) is a serious inborn error of metabolism with no pharmacological treatments. A novel strategy to treat this disease is to divert the toxic biochemical intermediates to less toxic or nontoxic metabolites. Here, we report a putative novel target, succinyl-CoA:glutarate-CoA transferase (SUGCT), which we hypothesize suppresses the GA1 metabolic phenotype through decreasing glutaryl-CoA and the derived 3-hydroxyglutaric acid. SUGCT is a type III CoA transferase that uses succinyl-CoA and glutaric acid as substrates. We report the structure of SUGCT, develop enzyme- and cell-based assays, and identify valsartan and losartan carboxylic acid as inhibitors of the enzyme in a high-throughput screen of FDA-approved compounds. The cocrystal structure of SUGCT with losartan carboxylic acid revealed a novel pocket in the active site and further validated the high-throughput screening approach. These results may form the basis for the future development of new pharmacological intervention to treat GA1.

Complete sequence randomness of lactate-based copolymers (LAHBs) with varied lactate monomer fractions employing a series of propionyl-CoA transferases.

Previously, we biosynthesized an evolved version of a bio-based polylactide (PLA) on microbial platforms using our engineered lactate-polymerizing enzyme (LPE). This lactate (LA)-based copolyester, LAHB, has advantages over PLA, including improved flexibility and biodegradability, and its properties can be regulated through the LA fraction. To expand the LA-incorporation capacity and improve polymer properties, in the state of in vivo LAHB production, propionyl-CoA transferases (PCTs) that exhibited enhanced production of LA-CoA than the conventional PCTs were selected. Here, the present study has demonstrated that the LA fraction of LAHB could be altered using various PCTs. Enhanced PCT performance was achieved by balancing polymer production and cell growth. Both events are governed by the use of acetyl-CoA, a commonly shared key metabolite. This could be attributed to the different reactivities of individual PCTs towards acetyl-CoA, which serves both as a CoA donor and a leading compound in the TCA cycle. Interestingly, we found complete sequence randomness in the LAHB copolymers, independent of the LA fraction. The mechanism of LA fraction-independent sequence randomness is discussed. This new PCT-based strategy synergistically combines with the evolution of LPE to advance the LAHB project, and enables us to perform advanced applications other than LAHB production utilizing CoA-linked substrates.

Targeting OXCT1-mediated ketone metabolism reprograms macrophages to promote antitumor immunity via CD8+ T cells in hepatocellular carcinoma.

BACKGROUND & AIMS: The liver is the main organ of ketogenesis, while ketones are mainly metabolized in peripheral tissues via the critical enzyme 3-oxoacid CoA-transferase 1 (OXCT1). We previously found that ketolysis is reactivated in hepatocellular carcinoma (HCC) cells through OXCT1 expression to promote tumor progression; however, whether OXCT1 regulates antitumor immunity remains unclear. METHODS: To investigate the expression pattern of OXCT1 in HCC in vivo, we conducted multiplex immunohistochemistry experiments on human HCC specimens. To explore the role of OXCT1 in mouse HCC tumor-associated macrophages (TAMs), we generated LysMcreOXCT1f/f (OXCT1 conditional knockout in macrophages) mice. RESULTS: Here, we found that inhibiting OXCT1 expression in tumor-associated macrophages reduced CD8+ T-cell exhaustion through the succinate-H3K4me3-Arg1 axis. Initially, we found that OXCT1 was highly expressed in liver macrophages under steady state and that OXCT expression was further increased in TAMs. OXCT1 deficiency in macrophages suppressed tumor growth by reprogramming TAMs toward an antitumor phenotype, reducing CD8+ T-cell exhaustion and increasing CD8+ T-cell cytotoxicity. Mechanistically, high OXCT1 expression induced the accumulation of succinate, a byproduct of ketolysis, in TAMs, which promoted Arg1 transcription by increasing the H3K4me3 level in the Arg1 promoter. In addition, pimozide, an inhibitor of OXCT1, suppressed Arg1 expression as well as TAM polarization toward the protumor phenotype, leading to decreased CD8+ T-cell exhaustion and slower tumor growth. Finally, high expression of OXCT1 in macrophages was positively associated with poor survival in patients with HCC. CONCLUSIONS: In conclusion, our results demonstrate that OXCT1 epigenetically suppresses antitumor immunity, suggesting that suppressing OXCT1 activity in TAMs could be an effective approach for treating liver cancer. IMPACT AND IMPLICATIONS: The intricate metabolism of liver macrophages plays a critical role in shaping hepatocellular carcinoma progression and immune modulation. Targeting macrophage metabolism to counteract immune suppression presents a promising avenue for hepatocellular carcinoma treatment. Herein, we found that the ketogenesis gene OXCT1 was highly expressed in tumor-associated macrophages (TAMs) and promoted tumor growth by reprogramming TAMs toward a protumor phenotype. Pharmacological targeting or genetic downregulation of OXCT1 in TAMs enhances antitumor immunity and slows tumor growth. Our results suggest that suppressing OXCT1 activity in TAMs could be an effective approach for treating liver cancer.

A Synthetic Pathway for the Production of Benzylsuccinate in Escherichia coli.

(R)-Benzylsuccinate is generated in anaerobic toluene degradation by the radical addition of toluene to fumarate and further degraded to benzoyl-CoA by a ß-oxidation pathway. Using metabolic modules for benzoate transport and activation to benzoyl-CoA and the enzymes of benzylsuccinate ß-oxidation, we established an artificial pathway for benzylsuccinate production in Escherichia coli, which is based on its degradation pathway running in reverse. Benzoate is supplied to the medium but needs to be converted to benzoyl-CoA by an uptake transporter and a benzoate-CoA ligase or CoA-transferase. In contrast, the second substrate succinate is endogenously produced from glucose under anaerobic conditions, and the constructed pathway includes a succinyl-CoA:benzylsuccinate CoA-transferase that activates it to the CoA-thioester. We present first evidence for the feasibility of this pathway and explore product yields under different growth conditions. Compared to aerobic cultures, the product yield increased more than 1000-fold in anaerobic glucose-fermenting cultures and showed further improvement under fumarate-respiring conditions. An important bottleneck to overcome appears to be product excretion, based on much higher recorded intracellular concentrations of benzylsuccinate, compared to those excreted. While no export system is known for benzylsuccinate, we observed an increased product yield after adding an unspecific mechanosensitive channel to the constructed pathway.

AMPKα2 regulates fasting-induced hyperketonemia by suppressing SCOT ubiquitination and degradation.

Ketone bodies serve as an energy source, especially in the absence of carbohydrates or in the extended exercise. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a crucial energy sensor that regulates lipid and glucose metabolism. However, whether AMPK regulates ketone metabolism in whole body is unclear even though AMPK regulates ketogenesis in liver. Prolonged resulted in a significant increase in blood and urine levels of ketone bodies in wild-type (WT) mice. Interestingly, fasting AMPKα2-/- and AMPKα1-/- mice exhibited significantly higher levels of ketone bodies in both blood and urine compared to fasting WT mice. BHB tolerance assays revealed that both AMPKα2-/- and AMPKα1-/- mice exhibited slower ketone consumption compared to WT mice, as indicated by higher blood BHB or urine BHB levels in the AMPKα2-/- and AMPKα1-/- mice even after the peak. Interestingly, fasting AMPKα2-/- and AMPKα1-/- mice exhibited significantly higher levels of ketone bodies in both blood and urine compared to fasting WT mice. . Specifically, AMPKα2ΔMusc mice showed approximately a twofold increase in blood BHB levels, and AMPKα2ΔMyo mice exhibited a 1.5-fold increase compared to their WT littermates after a 48-h fasting. However, blood BHB levels in AMPKα1ΔMusc and AMPKα1ΔMyo mice were as same as in WT mice. Notably, AMPKα2ΔMusc mice demonstrated a slower rate of BHB consumption in the BHB tolerance assay, whereas AMPKα1ΔMusc mice did not show such an effect. Declining rates of body weights and blood glucoses were similar among all the mice. Protein levels of SCOT, the rate-limiting enzyme of ketolysis, decreased in skeletal muscle of AMPKα2-/- mice. Moreover, SCOT protein ubiquitination increased in C2C12 cells either transfected with kinase-dead AMPKα2 or subjected to AMPKα2 inhibition. AMPKα2 physiologically binds and stabilizes SCOT, which is dependent on AMPKα2 activity.

OXCT1 regulates hippocampal neurogenesis and alleviates cognitive impairment via the Akt/GSK-3ß/ß-catenin pathway after subarachnoid hemorrhage.

BACKGROUND: Subarachnoid hemorrhage (SAH) is a life-threatening neurological disease that usually has a poor prognosis. Neurogenesis is a potential therapeutic target for brain injury. Ketone metabolism also plays neuroprotective roles in many neurological disorders. OXCT1 (3-Oxoacid CoA-Transferase 1) is the rate-limiting enzyme of ketone body oxidation. In this study, we explored whether increasing ketone oxidation by upregulating OXCT1 in neurons could promote neurogenesis after SAH, and evaluated the potential mechanism involved in this process. METHODS: The ß-hydroxybutyrate content was measured using an enzymatic colorimetric assay. Adeno-associated virus targeting neurons was injected to overexpress OXCT1, and the expression and localization of proteins were evaluated by western blotting and immunofluorescence staining. Adult hippocampal neurogenesis was evaluated by dual staining with doublecortin and 5-Ethynyl-2'-Deoxyuridine. LY294002 was intracerebroventricularly administered to inhibit Akt activity. The Morris water maze and Y-maze tests were employed to assess cognitive function after SAH. RESULTS: The results showed that OXCT1 expression and hippocampal neurogenesis significantly decreased in the early stage of SAH. Overexpression of OXCT1 successfully increased hippocampal neurogenesis via activation of Akt/GSK-3ß/ß-catenin signaling and improved cognitive function, both of which were reversed by administration of LY294002. CONCLUSIONS: OXCT1 regulated hippocampal ketone body metabolism and increased neurogenesis through mechanisms mediated by the Akt/GSK-3ß/ß-catenin pathway, improving cognitive impairment after SAH.

Detection of oxalyl-CoA decarboxylase (oxc) and formyl-CoA transferase (frc) genes in novel probiotic isolates capable of oxalate degradation in vitro.

Oxalate degradation is one of lactic acid bacteria's desirable activities. It is achieved by two enzymes, formyl coenzyme A transferase (frc) and oxalyl coenzyme A decarboxylase (oxc). The current study aimed to screen 15 locally isolated lactic acid bacteria to select those with the highest oxalate degradation ability. It also aimed to amplify the genes involved in degradation. MRS broth supplemented with 20 mM sodium oxalate was used to culture the tested isolates for 72 h. This was followed by an enzymatic assay to detect remaining oxalate. All isolates showed oxalate degradation activity to variable degrees. Five isolates demonstrated high oxalate degradation, 78 to 88%. To investigate the oxalate-degradation potential of the selected isolates, they have been further tested for the presence of genes that encode for enzymes involved in oxalate catabolism, formyl coenzyme A transferase (frc) and oxalyl coenzyme A decarboxylase (oxc). Three strains showed bands with the specific OXC and FRC forward and reverse primers designated as (SA-5, 9 and 37). Species-level identification revealed Loigolactobacillus bifermentans, Lacticaseibacillus paracasei, and Lactiplantibacillus plantarum. Preliminary results revealed that the tested probiotic strains harbored both oxc and frc whose products are putatively involved in oxalate catabolism. The probiotic potential of the selected strains was evaluated, and they showed high survival rates to both simulated gastric and intestinal fluids and variable degrees of antagonism against the tested Gram-positive and negative pathogens and were sensitive to clarithromycin but resistant to both metronidazole and ceftazidime. Finally, these strains could be exploited as an innovative approach to establish oxalate homeostasis in humans and prevent kidney stone formation.

Identifying Acss1, Mtfp1 and Oxct1 as key regulators and promising biomarkers of sarcopenia in various models.

Mitochondrial dysfunction plays a critical role in muscular homeostasis, but the molecular mechanism underlying mitochondrial dynamics and sarcopenia awaits to be uncovered. We all know that malnutrition, cachexia, and type 2 diabetes are significant contributors to the development of sarcopenia.Therefore, we analyzed a bioinformatic analysis on cathectic differentially expressed genes (cDEGs), fasted differentially genes (fDEGs) and mitochondria-related genes. The overlapping genes identified were then validated by RT-qPCR and Western blotting experiments in various sarcopenia mice models and used to predict aging-related muscle loss in humans. First, the correlation analysis and PPI network indicated 6 overlapping candidates (Bdh1, Gdap1, Acss1, Mtfp1, Idh2, Oxct1) may constitute a regulatory effect in mitochondrial dynamics and muscle wasting. Next, we successfully established fasted, Lewis lung carcinoma (LLC) and Diabetes Mellitus (DM) induced sarcopenia mice models and verified that Acss1, Mtfp1 and Oxct1 shared common and significant variation tendency in these sarcopenia mice models. Further-more, Pearson correlation analysis showed that Acss1 was negatively related to the weight of gastrocnemius while Mtfp1 and Oxct1 displayed a significantly positive correlation with gastrocnemius weight in sarcopenic mice model induced by LLC, fasting and DM. What's more, ROC analysis based on human aging-related datasets indicated Acss1, Mtfp1, Oxct1 had outstanding diagnostic capabilities for sarcopenia. In general, we identified three hub genes (Acss1, Mtfp1 and Oxct1) that are strongly associated with mitochondrial dysfunction in sarcopenia and may provide novel and reliable indicators for screening, diagnosis, and prognosis, as well as potential therapeutic targets for patients with sarcopenia.

[Succinyl CoA:3 oxoacid CoA transferase deficiency: A case report]. / Deficiencia de succinil-CoA acetoacetato transferasa: reporte de un caso.

Background: Succinyl-CoA:3 oxoacid CoA transferase deficiency (SCOTD) is a rare autosomal recessive disease, characterized by altered utilization of ketone bodies, with acute episodes of ketoacidosis. Clinical case: It is presented the case of a patient with SCOTD, with a first atypical episode accompanied by hyperglycemia, with 4 subsequent episodes with classic manifestations of the disease, presenting with a biochemical pattern of permanent ketonuria with marked elevation of ketone bodies (acetoacetate, 3 beta-hydroxybutyrate) in the study of urinary organic acids by gas chromatography and mass spectrometry, together with the clinical picture granting the diagnosis. It was started a maintenance therapy with a characteristic feeding plan; it was shown an adequate response to treatment, and the absence of permanent ketosis was surmised. Conclusion: Being a rare disease, the categorization of these patients as diabetic ketoacidosis is frequent. The clinical and biochemical characteristics with ketosis or persistent ketonuria should be analyzed very carefully, especially in patients presenting with hyperglycemia, which is an atypical manifestation of the disease, in order to make an early diagnosis and treatment, positively impacting the prognosis of patients.

Introducción: la deficiencia de succinil-CoA acetoacetato transferasa (SCOT) es una enfermedad rara, autosómica recesiva, caracterizada por alteración en la utilización de cuerpos cetónicos, con episodios agudos de cetoacidosis. Caso clínico: se presenta el caso de un paciente con deficiencia de SCOT, con un primer episodio atípico acompañado con hiperglucemia, con 4 episodios posteriores con manifestaciones clásicas de la enfermedad, que presentó patrón bioquímico de cetonuria permanente con marcada elevación de cuerpos cetónicos (acetoacetato, 3 beta-hidroxibutirato) en estudio de ácidos orgánicos urinarios por cromatografía de gases y espectrometría de masas, aunado a cuadro clínico que otorgó el diagnóstico. Se inició terapia de mantenimiento con plan de alimentación característico; se demostró una adecuada respuesta al tratamiento, y se infirió una ausencia de cetosis permanente. Conclusiones: al ser una enfermedad rara, la categorización de estos pacientes como cetoacidosis diabética es frecuente. Se deben analizar de forma muy minuciosa las características clínicas y bioquímicas con cetosis o cetonuria persistente, sobre todo en pacientes que se presenten con hiperglucemia, que es una manifestación atípica de la enfermedad, para realizar un diagnóstico y tratamiento temprano que impacte de forma positiva el pronóstico de los pacientes.

Long-term benefit of DAAs on gut dysbiosis and microbial translocation in HCV-infected patients with and without HIV coinfection.

Long-term effect of Direct-acting antivirals (DAAs) on gut microbiota, short-chain fatty acids (SCFAs) and microbial translocation in patients with hepatitis C virus (HCV) infection who achieve sustained virological response (SVR) were limited. A longitudinal study of 50 patients with HCV monoinfection and 19 patients with HCV/HIV coinfection received DAAs were conducted. Fecal specimens collected at baseline and at week 72 after treatment completion (FUw72) were analyzed for 16S rRNA sequencing and the butyryl-CoA:acetateCoA transferase (BCoAT) gene expression using real-time PCR. Plasma lipopolysaccharide binding protein (LBP) and intestinal fatty acid binding protein (I-FABP) were quantified by ELISA assays. SVR rates in mono- and coinfected patients were comparable (94% vs. 100%). The improvement of gut dysbiosis and microbial translocation was found in responders but was not in non-responders. Among responders, significant restoration of alpha-diversity, BCoAT and LBP were observed in HCV patients with low-grade fibrosis (F0-F1), while HCV/HIV patients exhibited partial improvement at FUw72. I-FABP did not decline significantly in responders. Treatment induced microbiota changes with increasing abundance of SCFAs-producing bacteria, including Blautia, Fusicatenibacter, Subdoligranulum and Bifidobacterium. In conclusion, long-term effect of DAAs impacted the restoration of gut dysbiosis and microbial translocation. However, early initiation of DAAs required for an alteration of gut microbiota, enhanced SCFAs-producing bacteria, and could reduce HCV-related complications.

Near-real time determination of BAHD acyl-coenzyme A transferase reaction rates and kinetic parameters using Ellman's reagent.

BAHD acyl-coenzyme A (CoA) acyltransferases play key roles in a large number of biosynthetic reactions involved in plant specialized metabolism. One approach to measure reaction rates for these enzymes is to quantify the amide or ester reaction products following chromatographic separation of reaction components, an approach that can be labor intensive and time consuming, and complicated by a lack of pure standards. We previously developed and validated an alternative approach using 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB, Ellman's reagent) to spectrophotometrically monitor reaction progress by the release of free CoA in the reaction. This approach allows near-real time measurement of reaction rates, permitting reaction conditions (buffer, reactant, and enzyme concentrations, etc.) to be changed "on the fly." The ease and rapidity of data collection allows a high density of data points to be collected for determination of kinetic parameters. Here we provide a detailed procedure for using DTNB to measure BAHD acyl-CoA acyltransferase reaction rates, and as an example, use it to determine kinetic parameters for red clover hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase, a BAHD acyl-CoA hydroxycinnamoyltransferase not previously characterized with respect to kinetic parameters. This approach may be more generally applicable to transferases using CoA donors.

Structural Basis for a Cork-Up Mechanism of the Intra-Molecular Mesaconyl-CoA Transferase.

Mesaconyl-CoA transferase (Mct) is one of the key enzymes of the 3-hydroxypropionate (3HP) bi-cycle for autotrophic CO2 fixation. Mct is a family III/Frc family CoA transferase that catalyzes an unprecedented intra-molecular CoA transfer from the C1-carboxyl group to the C4-carboxyl group of mesaconate at catalytic efficiencies >106 M-1 s-1. Here, we show that the reaction of Mct proceeds without any significant release of free CoA or the transfer to external acceptor acids. Mct catalyzes intra-molecular CoA transfers at catalytic efficiencies that are at least more than 6 orders of magnitude higher compared to inter-molecular CoA transfers, demonstrating that the enzyme exhibits exquisite control over its reaction. To understand the molecular basis of the intra-molecular CoA transfer in Mct, we solved crystal structures of the enzyme from Chloroflexus aurantiacus in its apo form, as well as in complex with mesaconyl-CoA and several covalently enzyme-bound intermediates of CoA and mesaconate at the catalytically active residue Asp165. Based on these structures, we propose a reaction mechanism for Mct that is similar to inter-molecular family III/Frc family CoA transferases. However, in contrast to the latter that undergo opening and closing cycles during the reaction to exchange substrates, the central cavity of Mct remains sealed ("corked-up") by the CoA moiety, strongly favoring the intra-molecular CoA transfer between the C1 and the C4 position of mesaconate.

The Antipsychotic Dopamine 2 Receptor Antagonist Diphenylbutylpiperidines Improve Glycemia in Experimental Obesity by Inhibiting Succinyl-CoA:3-Ketoacid CoA Transferase.

Despite significant progress in understanding the pathogenesis of type 2 diabetes (T2D), the condition remains difficult to manage. Hence, new therapeutic options targeting unique mechanisms of action are required. We have previously observed that elevated skeletal muscle succinyl CoA:3-ketoacid CoA transferase (SCOT) activity, the rate-limiting enzyme of ketone oxidation, contributes to the hyperglycemia characterizing obesity and T2D. Moreover, we identified that the typical antipsychotic agent pimozide is a SCOT inhibitor that can alleviate obesity-induced hyperglycemia. We now extend those observations here, using computer-assisted in silico modeling and in vivo pharmacology studies that highlight SCOT as a noncanonical target shared among the diphenylbutylpiperidine (DPBP) drug class, which includes penfluridol and fluspirilene. All three DPBPs tested (pimozide, penfluridol, and fluspirilene) improved glycemia in obese mice. While the canonical target of the DPBPs is the dopamine 2 receptor, studies in obese mice demonstrated that acute or chronic treatment with a structurally unrelated antipsychotic dopamine 2 receptor antagonist, lurasidone, was devoid of glucose-lowering actions. We further observed that the DPBPs improved glycemia in a SCOT-dependent manner in skeletal muscle, suggesting that this older class of antipsychotic agents may have utility in being repurposed for the treatment of T2D.

New insights into microbial interactions and putative competitive mechanisms during the hydrogen production from tequila vinasses.

This study aimed to characterize the prokaryotic community and putative microbial interactions involved in hydrogen (H2) production during the dark fermentation (DF) process, applying principal components analysis (PCA) to correlate changes in operational, physicochemical, and biological variables. For this purpose, a continuous stirred-tank reactor-type digester fed with tequila vinasses was operated at 24, 18, and 12 h of hydraulic retention times (HRTs) to apply organic loading rates of 20, 36, and 54 g-COD L-1 d-1, corresponding to stages I, II, and III, respectively. Results indicated high population dynamics for Archaea during the DF process toward a decrease in total sequences from 6299 to 99. Concerning the Bacteria community, lactic acid bacteria (LAB) were dominant reaching a relative abundance of 57.67%, while dominant H2-producing bacteria (HPB) decreased from 25.76% to 21.06% during stage III. Putative competitive exclusion mechanisms such as competition for substrates, bacteriocins production, and micronutrient depletion carried out by Archaea and non-H2-producing bacteria (non-HPB), especially LAB, could negatively impact the dominance of HPB such as Ethanoligenens harbinense and Clostridium tyrobutyricum. As a consequence, low maximal volumetric H2 production rate (672 mL-H2 L-1 d-1) and yield (3.88 mol-H2 assimilated sugars-1) were obtained. The global scenario obtained by PCA correlations suggested that C. tyrobutyricum positively impacted H2 molar yield through butyrate fermentation using the butyryl-CoA:acetate CoA transferase pathway, while the most abundant HPB E. harbinense decreased its relative abundance at the shortest HRT toward the dominance of non-HPB. This study provides new insights into the microbial interactions and helps to better understand the DF performance for H2 production using tequila vinasses as substrate. KEY POINTS: • E. harbinense and C. tyrobutyricum were responsible for H2 production. • Clostridiales used acetate and butyrate fermentations for H2 production. • LAB won the competition for sugars against Clostridiales during DF. • Putative bacteriocins production and micronutrients depletion could favor LAB.

An acetate-independent pathway for isopropanol production via HMG-CoA in Escherichia coli.

Isopropanol has a good potential as a new fuel substitution. In the model biosynthesis pathway of isopropanol synthesis, acetoacetyl-CoA is converted to acetoacetate by acetoacetyl-CoA transferases, which requires an acetate molecule as a substrate. Herein, a novel isopropanol synthesis pathway based on mammalian ketone metabolic pathway was developed. In this pathway, acetoacetyl-CoA is condensed with acetyl-CoA to generate 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) by HMG-CoA synthase, and then catalyzed by HMG-CoA lyase to generate acetoacetate. This process is acetate-independent. Under the same experimental system using glycerol as carbon source, the E. coli strain MG::ISOP1 containing the novel pathway produced 11.7 times more isopropanol than the strain MG::ISOP0 containing the model pathway. The pta-ackA knockout mutant strain MG∆pta-ackA::ISOP1, which reduced the conversion of acetyl-CoA to acetate, further increased the production from 76 mg/L to 360 mg/L. In another strategy, knocking out atoDA to block the acetoacetate degradation pathway in strain MG∆atoDA::ISOP1 increased the production to 680 mg/L. By knocking out both of pta-ackA and atoDA, strain MGΔpta-ackAΔatoDA::ISOP1 produced 964 mg/L of isopropanol, which was 12.7 times that of MG::ISOP1. This study indicated that the novel pathway is competent for isopropanol synthesis, and provides a new perspective for biosynthesis of isopropanol.

The sodium-glucose co-transporter-2 inhibitor ertugliflozin modifies the signature of cardiac substrate metabolism and reduces cardiac mTOR signalling, endoplasmic reticulum stress and apoptosis.

AIM: To investigate cardiac signalling pathways connecting substrate utilization with left ventricular remodelling in a murine pressure overload model. METHODS: Cardiac hypertrophy was induced by transverse aortic constriction surgery in 20-week-old C57BL/6J mice treated with or without the sodium-glucose co-transporter 2 (SGLT2) inhibitor ertugliflozin (225 mg kg-1 chow diet) for 10 weeks. RESULTS: Ertugliflozin improved left ventricular function and reduced myocardial fibrosis. This occurred simultaneously with a fasting-like response characterized by improved glucose tolerance and increased ketone body concentrations. While cardiac insulin signalling was reduced in response to SGLT2 inhibition, AMP-activated protein kinase (AMPK) signalling was increased with induction of the fatty acid transporter cluster of differentiation 36 and phosphorylation of acetyl-CoA carboxylase (ACC). Further, enzymes responsible for ketone body catabolism (ß-hydroxybutyrate dehydrogenase, succinyl-CoA:3-oxoacid-CoA transferase and acetyl-CoA acetyltransferase 1) were induced by SGLT2 inhibition. Ertugliflozin led to more cardiac abundance of fatty acids, tricarboxylic acid cycle metabolites and ATP. Downstream mechanistic target of rapamycin (mTOR) pathway, relevant for protein synthesis, cardiac hypertrophy and adverse cardiac remodelling, was reduced by SGLT2 inhibition, with alleviation of endoplasmic reticulum (ER) stress and unfolded protein response (UPR) providing a potential mechanism for abundant reduced left ventricular apoptosis and fibrosis. CONCLUSION: SGLT2 inhibition reduced left ventricular fibrosis in a murine model of cardiac hypertrophy. Mechanistically, this was associated with reduced cardiac insulin and increased AMPK signalling as a potential mechanism for less cardiac mTOR activation with alleviation of downstream ER stress, UPR and apoptosis.