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Plastics have become an indispensable material in many fields of human activities, with production increasing every year; however, most of the plastic waste is still incinerated or landfilled, and only 10% of the new plastic is recycled even once. Among all plastics, polyethylene terephthalate (PET) is the most produced polyester worldwide; ethylene glycol (EG) is one of the two monomers released by the biorecycling of PET. While most research focuses on bacterial EG metabolism, this work reports the ability of Saccharomyces cerevisiae and nine other common laboratory yeast species not only to consume EG, but also to produce glycolic acid (GA) as the main by-product. A two-step bioconversion of EG to GA by S. cerevisiae was optimized by a design of experiment approach, obtaining 4.51 +/- 0.12 g l-1 of GA with a conversion of 94.25 +/- 1.74% from 6.21 +/- 0.04 g l-1 EG. To improve the titer, screening of yeast biodiversity identified Scheffersomyces stipitis as the best GA producer, obtaining 23.79 +/- 1.19 g l-1 of GA (yield 76.68%) in bioreactor fermentation, with a single-step bioprocess. Our findings contribute in laying the ground for EG upcycling strategies with yeasts.This work aims at exploring the potential of yeasts to upcycle ethylene glycol - derived from PET hydrolysis - to glycolic acid, a chemical of industrial interest.

Pseudomonas putida, a microbial host widely adopted for metabolic engineering, processes glucose through convergent peripheral pathways that ultimately yield 6-phosphogluconate. The periplasmic gluconate shunt (PGS), composed by glucose and gluconate dehydrogenases, sequentially transforms glucose into gluconate and 2-ketogluconate. Although the secretion of these organic acids by P. putida has been extensively recognized, the mechanism and spatiotemporal regulation of the PGS remained elusive thus far. To address this challenge, we adopted a dynamic 13C- and 2H-metabolic flux analysis strategy, termed D-fluxomics. D-fluxomics demonstrated that the PGS underscores a highly dynamic metabolic architecture in glucose-dependent batch cultures of P. putida, characterized by hierarchical carbon uptake by the PGS throughout the cultivation. Additionally, we show that gluconate and 2-ketogluconate accumulation and consumption can be solely explained as a result of the interplay between growth rate-coupled and decoupled metabolic fluxes. As a consequence, the formation of these acids in the PGS is inversely correlated to the bacterial growth rate—unlike the widely studied overflow metabolism of Escherichia coli and yeast. Our findings, which underline survival strategies of soil bacteria thriving in their natural environments, open new avenues for engineering P. putida towards efficient, sugar-based bioprocesses.

Missense variants of hyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channels cause variable phenotypes, ranging from mild generalized epilepsy to developmental and epileptic encephalopathy (DEE). Although variants of HCN1 are an established cause of DEE, those of HCN2 have been reported in generalized epilepsies. Here we describe the first case of DEE caused by the novel de novo heterozygous missense variant c.1379G>A (p.G460D) of HCN2. Functional characterization in transfected HEK293 cells and neonatal rat cortical neurons revealed that HCN2 p.G460D currents were strongly reduced compared to wild-type, consistent with a dominant negative loss-of-function effect. Immunofluorescence staining showed that mutant channels are retained within the cell and do not reach the membrane. Moreover, mutant HCN2 also affect HCN1 channels, by reducing the Ih current expressed by the HCN1-HCN2 heteromers. Due to the persistence of frequent seizures despite pharmacological polytherapy, the patient was treated with a ketogenic diet, with a significant and long-lasting reduction of episodes. In vitro experiments conducted in a ketogenic environment demonstrated that the clinical improvement observed with this dietary regimen was not mediated by a direct action on HCN2 activity. These results expand the clinical spectrum related to HCN2 channelopathies, further broadening our understanding of the pathogenesis of DEE.

Nowadays, the yeast Saccharomyces cerevisiae is the platform of choice for demonstrating the proof of concept of the production of metabolites with a complex structure. However, introducing heterologous genes and rewiring the endogenous metabolism is still not standardized enough, affecting negatively the readiness-to-market of such metabolites. We developed the Easy Modular Integrative fuSion-ready Expression (Easy-MISE) toolkit, which is a novel combination of synthetic biology tools based on a single Golden Gate multiplasmid assembly meant to further ameliorate the rational predictability and flexibility of the process of yeast engineering. Thanks to an improved cloning screening strategy, double and independent transcription units are easily assembled and subsequently integrated into previously characterized loci. Moreover, the devices can be tagged for localization. This design allows for a higher degree of modularity and increases the flexibility of the engineering strategy. We show with a case study how the developed toolkit accelerates the construction and the analysis of the intermediate and the final engineered yeast strains, leaving space to better characterize the heterologous biosynthetic pathway in the final host and, overall, to improve the fermentation performances. Different S. cerevisiae strains were built harboring different versions of the biochemical pathway toward glucobrassicin (GLB) production, an indolyl-methyl glucosinolate. In the end, we could demonstrate that in the tested conditions the best-producing strain leads to a final concentration of GLB of 9.80 ± 0.267 mg/L, a titer 10-fold higher than the best result previously reported in the literature.

Plastic has become an indispensable material in many fields, with production increasing every year; however, most of the plastic waste is still incinerated or landfilled, and only 10% of the new plastic is recycled even once. Considering that plastic is derived from petroleum-based resources, this creates a worrying loss of resources and a cascade of environmental issues. Among all plastic, polyethylene terephthalate (PET) is the most produced polyester worldwide (56 Mt/year). This work focuses on the upcycling of PET monomers – terephthalic acid (TPA) and ethylene glycol (EG) – by yeast fermentation for the production of industrially relevant organic acids (protocatechuic acid, cis,cis-muconic acid, 3-carboxy-cis,cis-muconic acid, glycolic acid). Thanks to synthetic biology (EASY-MISE toolkit) and metabolic engineering, different Saccharomyces cerevisiae strains were created for the bioconversion of TPA and the use of EG as a carbon source. For the bioconversion of TPA, several genes from Ideonella sakaiensis were introduced; TPA toxicity on the new strains was assessed in microwell plates. Since EG assimilation has never been described in yeast, different medium combinations were tested for the assimilation of EG; in parallel, two synthetic metabolic pathways were introduced in S. cerevisiae for a more efficient assimilation. In silico constraint-based models are currently being developed to optimize growth and production on TPA and EG, and to shed light on the yeast native metabolism of the latter. Our research shows promising results in the biodegradation and upcycling of PET monomers by yeast fermentation, and it will become even more interesting thanks to the recent advances in enzymatic PET hydrolysis.

So far mutations in HCN2 gene, encoding for the hyperpolarization-activated cyclic nucleotide-gated channel 2, have been related to mild epileptic clinical phenotypes. In this study, a novel HCN2 mutation was found in a proband, now aged 7 years old, with a congenital encephalopathy characterized by drug resistant epilepsy, severe developmental delay, ataxia, dystonia and cerebral visual impairment. A convulsive status epilepticus at 5 months of age marked the onset of epilepsy. The HCN2 mutation affects an aminoacid located in the S6 transmembrane helix (p.Gly460Asp) and is carried in heterozygosis. To describe the functional consequences of the mutant channel, whole-cell patch-clamp experiments were performed in HEK293 cells expressing HCN2 wild type (WT) or p.Gly460Asp channel. A coexpression of the same amount of plasmid encoding for the wt or the mutant form of the channel mimed the heterozygous condition. A complete abolishment of the current was observed considering the mutant compared to the WT channel (-9.9±1.2 pA/pF, n=20 vs -31.2±8.4 pA/pF, n=44 respectively; p<0.05) and the heterozygous condition led to a significant reduction of the current density (-20.1±5.1 pA/pF n=35; p<0.05). WT and heterozygotic channels shared overlapping activation curves (V1/2 and k -92.9±0.3 mV and 5.6±0.3, n=29 vs -91.6±0.2 mV and 5.6±0.2, n=16 respectively) and no significant differences were present in their kinetics of both activation and deactivation. In conclusion, this is the first study linking HCN2 to progressive epileptic encephalopathy. The Gly460Asp mutation seems to act as a loss-of-function that could potentially affect the control of neuronal excitability and therefore explain the proband pathological condition.

To achieve growth, microbial organisms must cope with stresses and adapt to the envi- ronment, exploiting the available nutrients with the highest efficiency. In Saccharomyces cerevisiae, Ras/PKA and Snf1/AMPK pathways regulate cellular metabolism according to the supply of glucose, alternatively supporting fermentation or mitochondrial respiration. Many reports have highlighted crosstalk between these two pathways, even without providing a comprehensive mechanism of regulation. Here, we show that glucose-dependent inactivation of Snf1/AMPK is independent from the Ras/PKA pathway. Decoupling glucose uptake rate from glucose concentration, we highlight a strong coordination between glycolytic metabolism and Snf1/AMPK, with an inverse correlation between Snf1/AMPK phosphorylation state and glucose uptake rate, regardless of glucose concentra- tion in the medium. Despite fructose-1,6-bisphosphate (F1,6BP) being proposed as a glycolytic flux sensor, we demonstrate that glucose-6-phosphate (G6P), and not F1,6BP, is involved in the control of Snf1/AMPK phosphorylation state. Altogether, this study supports a model by which Snf1/AMPK senses glucose flux independently from PKA activity, and thanks to conversion of glucose into G6P.

Liver cancer is one of the most common cancer worldwide with a high mortality. Methionine is an essential amino acid required for normal development and cell growth, is mainly metabolized in the liver, and its role as an anti‐cancer supplement is still controversial. Here, we evaluate the effects of methionine supplementation in liver cancer cells. An integrative proteomic and metabolomic analysis indicates a rewiring of the central carbon metabolism, with an upregulation of the tricarboxylic acid (TCA) cycle and mitochondrial adenosine triphosphate (ATP) production in the presence of high methionine and AMP‐activated protein kinase (AMPK) inhibition. Methionine supplementation also reduces growth rate in liver cancer cells and induces the activation of both the AMPK and mTOR pathways. Interestingly, in high methionine concentration, inhibition of AMPK strongly impairs cell growth, cell migration, and colony formation, indicating the main role of AMPK in the control of liver cancer phenotypes. Therefore, regulation of methionine in the diet combined with AMPK inhibition could reduce liver cancer progression.

Robust biological systems are able to adapt to internal and environmental perturbations. This is ensured by a thick crosstalk between metabolism and signal transduction pathways, through which cell cycle progression, cell metabolism and growth are coordinated. Although several reports describe the control of cell signaling on metabolism (mainly through transcriptional regulation and post-translational modifications), much fewer information is available on the role of metabolism in the regulation of signal transduction. Protein-metabolite interactions (PMIs) result in the modification of the protein activity due to a conformational change associated with the binding of a small molecule. An increasing amount of evidences highlight the role of metabolites of the central metabolism in the control of the activity of key signaling proteins in different eukaryotic systems. Here we review the known PMIs between primary metabolites and proteins, through which metabolism affects signal transduction pathways controlled by the conserved kinases Snf1/AMPK, Ras/PKA and TORC1. Interestingly, PMIs influence also the mitochondrial retrograde response (RTG) and calcium signaling, clearly demonstrating that the range of this phenomenon is not limited to signaling pathways related to metabolism.

AIMS: Atrial fibrillation (AF) is the most common type of cardiac arrhythmias, whose incidence is likely to increase with the aging of the population. It is considered a progressive condition, frequently observed as a complication of other cardiovascular disorders. However, recent genetic studies revealed the presence of several mutations and variants linked to AF, findings that define AF as a multifactorial disease. Due to the complex genetics and paucity of models, molecular mechanisms underlying the initiation of AF are still poorly understood. Here we investigate the pathophysiological mechanisms of a familial form of AF, with particular attention to the identification of putative triggering cellular mechanisms, using patient's derived cardiomyocytes (CMs) differentiated from induced pluripotent stem cells (iPSCs). METHODS AND RESULTS: Here we report the clinical case of three siblings with untreatable persistent AF whose whole-exome sequence analysis revealed several mutated genes. To understand the pathophysiology of this multifactorial form of AF we generated three iPSC clones from two of these patients and differentiated these cells towards the cardiac lineage. Electrophysiological characterization of patient-derived CMs (AF-CMs) revealed that they have higher beating rates compared to control (CTRL)-CMs. The analysis showed an increased contribution of the If and ICaL currents. No differences were observed in the repolarizing current IKr and in the sarcoplasmic reticulum calcium handling. Paced AF-CMs presented significantly prolonged action potentials and, under stressful conditions, generated both delayed after-depolarizations of bigger amplitude and more ectopic beats than CTRL cells. CONCLUSIONS: Our results demonstrate that the common genetic background of the patients induces functional alterations of If and ICaL currents leading to a cardiac substrate more prone to develop arrhythmias under demanding conditions. To our knowledge this is the first rep

The Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels are highly expressed in the Central Nervous Systems, where they are responsible for the I h current. Together with specific accessory proteins, these channels finely regulate neuronal excitability and discharge activity. In the last few years, a substantial body of evidence has been gathered showing that modifications of I h can play an important role in the pathogenesis of epilepsy. However, the extent to which HCN dysfunction is spread among the epileptic population is still unknown. The aim of this work is to evaluate the impact of genetic mutations potentially affecting the HCN channels’ activity, using a NGS approach. We screened a large cohort of patients with epilepsy of unknown etiology for mutations in HCN1, HCN2 and HCN4 and in genes coding for accessory proteins (MiRP1, Filamin A, Caveolin-3, TRIP8b, Tamalin, S-SCAM and Mint2). We confirmed the presence of specific mutations of HCN genes affecting channel function and predisposing to the development of the disease. We also found several previously unreported additional genetic variants, whose contribution to the phenotype remains to be clarified. According to these results and data from literature, alteration of HCN1 channel function seems to play a major role in epilepsy, but also dysfunctional HCN2 and HCN4 channels can predispose to the development of the disease. Our findings suggest that inclusion of the genetic screening of HCN channels in diagnostic procedures of epileptic patients should be recommended. This would help pave the way for a better understanding of the role played by I h dysfunction in the pathogenesis of epilepsy.

HCN channels are highly expressed and functionally relevant in neurons and increasing evidence demonstrates their involvement in the etiology of human epilepsies. Among HCN isoforms, HCN4 is important in cardiac tissue, where it underlies pacemaker activity. Despite being expressed also in deep structures of the brain, mutations of this channel functionally shown to be associated with epilepsy have not been reported yet. Using Next Generation Sequencing for the screening of patients with idiopathic epilepsy, we identified the p.Arg550Cys (c.1648C>T) heterozygous mutation on HCN4 in two brothers affected by benign myoclonic epilepsy of infancy. Functional characterization in heterologous expression system and in neurons showed that the mutation determines a loss of function of HCN4 contribution to activity and an increase of neuronal discharge, potentially predisposing to epilepsy. Expressed in cardiomyocytes, mutant channels activate at slightly more negative voltages than wild-type (WT), in accordance with borderline bradycardia. While HCN4 variants have been frequently associated with cardiac arrhythmias, these data represent the first experimental evidence that functional alteration of HCN4 can also be involved in human epilepsy through a loss-of-function effect and associated increased neuronal excitability. Since HCN4 appears to be highly expressed in deep brain structures only early during development, our data provide a potential explanation for a link between dysfunctional HCN4 and infantile epilepsy. These findings suggest that it may be useful to include HCN4 screening to extend the knowledge of the genetic causes of infantile epilepsies, potentially paving the way for the identification of innovative therapeutic strategies.

HCN channels are highly expressed and functionally relevant in neurons and increasing evidence demonstrates their involvement in the etiology of human epilepsies. Among HCN isoforms, HCN4 is important in cardiac tissue, where it underlies pacemaker activity. Despite being expressed also in deep structures of the brain, mutations of this channel functionally shown to be associated with epilepsy have not been reported yet. Using Next Generation Sequencing for the screening of patients with idiopathic epilepsy, we identified the p.Arg550Cys (c.1648C>T) heterozygous mutation on HCN4 in two brothers affected by benign myoclonic epilepsy of infancy. Functional characterization in heterologous expression system and in neurons showed that the mutation determines a loss of function of HCN4 contribution to activity and an increase of neuronal discharge, potentially predisposing to epilepsy. Expressed in cardiomyocytes, mutant channels activate at slightly more negative voltages than wild-type (WT), in accordance with borderline bradycardia. While HCN4 variants have been frequently associated with cardiac arrhythmias, these data represent the first experimental evidence that functional alteration of HCN4 can also be involved in human epilepsy through a loss-of-function effect and associated increased neuronal excitability. Since HCN4 appears to be highly expressed in deep brain structures only early during development, our data provide a potential explanation for a link between dysfunctional HCN4 and infantile epilepsy. These findings suggest that it may be useful to include HCN4 screening to extend the knowledge of the genetic causes of infantile epilepsies, potentially paving the way for the identification of innovative therapeutic strategies.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels control neuronal excitability and their dysfunction has been linked to epileptogenesis but few individuals with neurological disorders related to variants altering HCN channels have been reported so far. In 2014, we described five individuals with epileptic encephalopathy due to de novo HCN1 variants. To delineate HCN1-related disorders and investigate genotype-phenotype correlations further, we assembled a cohort of 33 unpublished patients with novel pathogenic or likely pathogenic variants: 19 probands carrying 14 different de novo mutations and four families with dominantly inherited variants segregating with epilepsy in 14 individuals, but not penetrant in six additional individuals. Sporadic patients had epilepsy with median onset at age 7 months and in 36% the first seizure occurred during a febrile illness. Overall, considering familial and sporadic patients, the predominant phenotypes were mild, including genetic generalized epilepsies and genetic epilepsy with febrile seizures plus (GEFS+) spectrum. About 20% manifested neonatal/infantile onset otherwise unclassified epileptic encephalopathy. The study also included eight patients with variants of unknown significance: One adopted patient had two HCN1 variants, four probands had intellectual disability without seizures, and three individuals had missense variants inherited from an asymptomatic parent. Of the 18 novel pathogenic missense variants identified, 12 were associated with severe phenotypes and clustered within or close to transmembrane domains, while variants segregating with milder phenotypes were located outside transmembrane domains, in the intracellular N- and C-terminal parts of the channel. Five recurrent variants were associated with similar phenotypes. Using whole-cell patch-clamp, we showed that the impact of 12 selected variants ranged from complete loss-of-function to significant shifts in activation kinetics and/or volta

The causes of genetic epilepsies are unknown in the majority of patients. HCN ion channels have a widespread expression in neurons and increasing evidence demonstrates their functional involvement in human epilepsies. Among the four known isoforms, HCN1 is the most expressed in the neocortex and hippocampus and de novo HCN1 point mutations have been recently associated with early infantile epileptic encephalopathy. So far, HCN1 mutations have not been reported in patients with idiopathic epilepsy. Using a Next Generation Sequencing approach, we identified the de novo heterozygous p.Leu157Val (c.469C > G) novel mutation in HCN1 in an adult male patient affected by genetic generalized epilepsy (GGE), with normal cognitive development. Electrophysiological analysis in heterologous expression model (CHO cells) and in neurons revealed that L157V is a loss-of-function, dominant negative mutation causing reduced HCN1 contribution to net inward current and responsible for an increased neuronal firing rate and excitability, potentially predisposing to epilepsy. These data represent the first evidence that autosomal dominant missense mutations of HCN1 can also be involved in GGE, without the characteristics of epileptic encephalopathy reported previously. It will be important to include HCN1 screening in patients with GGE, in order to extend the knowledge of the genetic causes of idiopathic epilepsies, thus paving the way for the identification of innovative therapeutic strategies.

Aims Caveolinopathies are a family of genetic disorders arising from alterations of the caveolin-3 (cav-3) gene. The T78M cav-3 variant has been associated with both skeletal and cardiac muscle pathologies but its functional contribution, especially to cardiac diseases, is still controversial. Here, we evaluated the effect of the T78M cav-3 variant on cardiac ion channel function and membrane excitability. Methods and results We transfected either the wild type (WT) or T78M cav-3 in caveolin-1 knock-out mouse embryonic fibroblasts and found by immunofluorescence and electron microscopy that both are expressed at the plasma membrane and form caveolae. Two ion channels known to interact and co-immunoprecipitate with the cav-3, hKv1.5 and hHCN4, interact also with T78M cav-3 and reside in lipid rafts. Electrophysiological analysis showed that the T78M cav-3 causes hKv1.5 channels to activate and inactivate at more hyperpolarized potentials and the hHCN4 channels to activate at more depolarized potentials, in a dominant way. In spontaneously beating neonatal cardiomyocytes, the expression of the T78M cav-3 significantly increased action potential peak-to-peak variability without altering neither the mean rate nor the maximum diastolic potential. We also found that in a small cohort of patients with supraventricular arrhythmias, the T78M cav-3 variant is more frequent than in the general population. Finally, in silico analysis of both sinoatrial and atrial cell models confirmed that the T78M-dependent changes are compatible with a pro-arrhythmic effect. Conclusion This study demonstrates that the T78M cav-3 induces complex modifications in ion channel function that ultimately alter membrane excitability. The presence of the T78M cav-3 can thus generate a susceptible substrate that, in concert with other structural alterations and/or genetic mutations, may become arrhythmogenic

The hyperpolarization-activated I(h) current, coded for by hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, controls synaptic integration and intrinsic excitability in many brain areas. Because of their role in pacemaker function, defective HCN channels are natural candidates for contributing to epileptogenesis. Indeed, I(h) is pathologically altered after experimentally induced seizures, and several independent data indicate a link between dysfunctional HCN channels and different forms of epilepsy. However, direct evidence for functional changes of defective HCN channels correlating with the disease in human patients is still elusive. By screening families with epilepsy for mutations in Hcn1 and Hcn2 genes, we found a recessive loss-of-function point mutation in the gene coding for the HCN2 channel in a patient with sporadic idiopathic generalized epilepsy. Of 17 screened members of the same family, the proband was the only one affected and homozygous for the mutation. The mutation (E515K) is located in the C-linker, a region known to affect channel gating. Functional analysis revealed that homomeric mutant, but not heteromeric wild-type/mutant channels, have a strongly inhibited function caused by a large negative shift of activation range and slowed activation kinetics, effectively abolishing the HCN2 contribution to activity. After transfection into acutely isolated newborn rat cortical neurons, homomeric mutant, but not heteromeric wild type/mutant channels, lowered the threshold of action potential firing and strongly increased cell excitability and firing frequency when compared with wild-type channels. This is the first evidence in humans for a single-point, homozygous loss-of-function mutation in HCN2 potentially associated with generalized epilepsy with recessive inheritance.