A Metabolic Basis for Endothelial-to-Mesenchymal Transition.

Endothelial-to-mesenchymal transition (EndoMT) is a cellular process often initiated by the transforming growth factor ß (TGF-ß) family of ligands. Although required for normal heart valve development, deregulated EndoMT is linked to a wide range of pathological conditions. Here, we demonstrate that endothelial fatty acid oxidation (FAO) is a critical in vitro and in vivo regulator of EndoMT. We further show that this FAO-dependent metabolic regulation of EndoMT occurs through alterations in intracellular acetyl-CoA levels. Disruption of FAO via conditional deletion of endothelial carnitine palmitoyltransferase II (Cpt2E-KO) augments the magnitude of embryonic EndoMT, resulting in thickening of cardiac valves. Consistent with the known pathological effects of EndoMT, adult Cpt2E-KO mice demonstrate increased permeability in multiple vascular beds. Taken together, these results demonstrate that endothelial FAO is required to maintain endothelial cell fate and that therapeutic manipulation of endothelial metabolism could provide the basis for treating a growing number of EndoMT-linked pathological conditions.

Type 1 Interferons Induce Changes in Core Metabolism that Are Critical for Immune Function.

Greater understanding of the complex host responses induced by type 1 interferon (IFN) cytokines could allow new therapeutic approaches for diseases in which these cytokines are implicated. We found that in response to the Toll-like receptor-9 agonist CpGA, plasmacytoid dendritic cells (pDC) produced type 1 IFNs, which, through an autocrine type 1 IFN receptor-dependent pathway, induced changes in cellular metabolism characterized by increased fatty acid oxidation (FAO) and oxidative phosphorylation (OXPHOS). Direct inhibition of FAO and of pathways that support this process, such as fatty acid synthesis, prevented full pDC activation. Type 1 IFNs also induced increased FAO and OXPHOS in non-hematopoietic cells and were found to be responsible for increased FAO and OXPHOS in virus-infected cells. Increased FAO and OXPHOS in response to type 1 IFNs was regulated by PPARα. Our findings reveal FAO, OXPHOS and PPARα as potential targets to therapeutically modulate downstream effects of type 1 IFNs.

Chromodomain Protein CDYL Acts as a Crotonyl-CoA Hydratase to Regulate Histone Crotonylation and Spermatogenesis.

Lysine crotonylation (Kcr) is a newly identified histone modification that is associated with active transcription in mammalian cells. Here we report that the chromodomain Y-like transcription corepressor CDYL negatively regulates histone Kcr by acting as a crotonyl-CoA hydratase to convert crotonyl-CoA to ß-hydroxybutyryl-CoA. We showed that the negative regulation of histone Kcr by CDYL is intrinsically linked to its transcription repression activity and functionally implemented in the reactivation of sex chromosome-linked genes in round spermatids and genome-wide histone replacement in elongating spermatids. Significantly, Cdyl transgenic mice manifest dysregulation of histone Kcr and reduction of male fertility with a decreased epididymal sperm count and sperm cell motility. Our study uncovers a biochemical pathway in the regulation of histone Kcr and implicates CDYL-regulated histone Kcr in spermatogenesis, adding to the understanding of the physiology of male reproduction and the mechanism of the spermatogenic failure in AZFc (Azoospermia Factor c)-deleted infertile men.

Structures of an unusual 3-hydroxyacyl dehydratase (FabZ) from a ladderane-producing organism with an unexpected substrate preference.

The genomes of anaerobic ammonium-oxidizing (anammox) bacteria contain a gene cluster comprising genes of unusual fatty acid biosynthesis enzymes that were suggested to be involved in the synthesis of the unique "ladderane" lipids produced by these organisms. This cluster encodes an acyl carrier protein (denoted as "amxACP") and a variant of FabZ, an ACP-3-hydroxyacyl dehydratase. In this study, we characterize this enzyme, which we call anammox-specific FabZ ("amxFabZ"), to investigate the unresolved biosynthetic pathway of ladderane lipids. We find that amxFabZ displays distinct sequence differences to "canonical" FabZ, such as a bulky, apolar residue on the inside of the substrate-binding tunnel, where the canonical enzyme has a glycine. Additionally, substrate screens suggest that amxFabZ efficiently converts substrates with acyl chain lengths of up to eight carbons, whereas longer substrates are converted much more slowly under the conditions used. We also present crystal structures of amxFabZs, mutational studies and the structure of a complex between amxFabZ and amxACP, which show that the structures alone cannot explain the apparent differences from canonical FabZ. Moreover, we find that while amxFabZ does dehydrate substrates bound to amxACP, it does not convert substrates bound to canonical ACP of the same anammox organism. We discuss the possible functional relevance of these observations in the light of proposals for the mechanism for ladderane biosynthesis.

ECHDC2 inhibits the proliferation of gastric cancer cells by binding with NEDD4 to degrade MCCC2 and reduce aerobic glycolysis.

BACKGROUND: The Enoyl-CoA hydratase/isomerase family plays a crucial role in the metabolism of tumors, being crucial for maintaining the energy balance and biosynthetic needs of cancer cells. However, the enzymes within this family that are pivotal in gastric cancer (GC) remain unclear. METHODS: We employed bioinformatics techniques to identify key Enoyl-CoA hydratase/isomerase in GC. The expression of ECHDC2 and its clinical significance were validated through tissue microarray analysis. The role of ECHDC2 in GC was further assessed using colony formation assays, CCK8 assay, EDU assay, Glucose and lactic acid assay, and subcutaneous tumor experiments in nude mice. The mechanism of action of ECHDC2 was validated through Western blotting, Co-immunoprecipitation, and immunofluorescence experiments. RESULTS: Our analysis of multiple datasets indicates that low expression of ECHDC2 in GC is significantly associated with poor prognosis. Overexpression of ECHDC2 notably inhibits aerobic glycolysis and proliferation of GC cells both in vivo and in vitro. Further experiments revealed that overexpression of ECHDC2 suppresses the P38 MAPK pathway by inhibiting the protein level of MCCC2, thereby restraining glycolysis and proliferation in GC cells. Ultimately, it was discovered that ECHDC2 promotes the ubiquitination and subsequent degradation of MCCC2 protein by binding with NEDD4. CONCLUSIONS: These findings underscore the pivotal role of the ECHDC2 in regulating aerobic glycolysis and proliferation in GC cells, suggesting ECHDC2 as a potential therapeutic target in GC.

Further delineation of short-chain enoyl-CoA hydratase deficiency in the Pacific population.

Short-chain enoyl-coA hydratase (SCEH) deficiency due to biallelic pathogenic ECHS1 variants was first reported in 2014 in association with Leigh syndrome (LS) and increased S-(2-carboxypropyl)cysteine excretion. It is potentially treatable with a valine-restricted, high-energy diet and emergency regimen. Recently, Simon et al. described four Samoan children harbouring a hypomorphic allele (c.489G > A, p.Pro163=) associated with reduced levels of normally-spliced mRNA. This synonymous variant, missed on standard genomic testing, is prevalent in the Samoan population (allele frequency 0.17). Patients with LS and one ECHS1 variant were identified in NZ and Australian genomic and clinical databases. ECHS1 sequence data were interrogated for the c.489G > A variant and clinical data were reviewed. Thirteen patients from 10 families were identified; all had Pacific ancestry including Samoan, Maori, Cook Island Maori, and Tokelauan. All developed bilateral globus pallidi lesions, excluding one pre-symptomatic infant. Symptom onset was in early childhood, and was triggered by illness or starvation in 9/13. Four of 13 had exercise-induced dyskinesia, 9/13 optic atrophy and 6/13 nystagmus. Urine S-(2-carboxypropyl)cysteine-carnitine and other SCEH-related metabolites were normal or mildly increased. Functional studies demonstrated skipping of exon four and markedly reduced ECHS1 protein. These data provide further support for the pathogenicity of this ECHS1 variant which is also prevalent in Maori, Cook Island Maori, and Tongan populations (allele frequency 0.14-0.24). It highlights the need to search for a second variant in apparent heterozygotes with an appropriate phenotype, and has implications for genetic counselling in family members who are heterozygous for the more severe ECHS1 alleles. SYNOPSIS: Short-chain enoyl-CoA hydratase deficiency is a frequent cause of Leigh-like disease in Maori and wider-Pacific populations, due to the high carrier frequency of a hypomorphic ECHS1 variant c.489G > A, p.[Pro163=, Phe139Valfs*65] that may be overlooked by standard genomic testing.

Microaerobic insights into production of polyhydroxyalkanoates containing 3-hydroxyhexanoate via native reverse ß-oxidation from glucose in Ralstonia eutropha H16.

BACKGROUND: Ralstonia eutropha H16, a facultative chemolitoautotroph, is an important workhorse for bioindustrial production of useful compounds such as polyhydroxyalkanoates (PHAs). Despite the extensive studies to date, some of its physiological properties remain not fully understood. RESULTS: This study demonstrated that the knallgas bacterium exhibited altered PHA production behaviors under slow-shaking condition, as compared to its usual aerobic condition. One of them was a notable increase in PHA accumulation, ranging from 3.0 to 4.5-fold in the mutants lacking of at least two NADPH-acetoacetyl-CoA reductases (PhaB1, PhaB3 and/or phaB2) when compared to their respective aerobic counterpart, suggesting the probable existence of (R)-3HB-CoA-providing route(s) independent on PhaBs. Interestingly, PHA production was still considerably high even with an excess nitrogen source under this regime. The present study further uncovered the conditional activation of native reverse ß-oxidation (rBOX) allowing formation of (R)-3HHx-CoA, a crucial precursor for poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)], solely from glucose. This native rBOX led to the natural incorporation of 3.9 mol% 3HHx in a triple phaB-deleted mutant (∆phaB1∆phaB1∆phaB2-C2). Gene deletion experiments elucidated that the native rBOX was mediated by previously characterized (S)-3HB-CoA dehydrogenases (PaaH1/Had), ß-ketothiolase (BktB), (R)-2-enoyl-CoA hydratase (PhaJ4a), and unknown crotonase(s) and reductase(s) for crotonyl-CoA to butyryl-CoA conversion prior to elongation. The introduction of heterologous enzymes, crotonyl-CoA carboxylase/reductase (Ccr) and ethylmalonyl-CoA decarboxylase (Emd) along with (R)-2-enoyl-CoA hydratase (PhaJ) aided the native rBOX, resulting in remarkably high 3HHx composition (up to 37.9 mol%) in the polyester chains under the low-aerated condition. CONCLUSION: These findings shed new light on the robust characteristics of Ralstonia eutropha H16 and have the potential for the development of new strategies for practical P(3HB-co-3HHx) copolyesters production from sugars under low-aerated conditions.

Strategic validation of variants of uncertain significance in ECHS1 genetic testing.

BACKGROUND: Enoyl-CoA hydratase short-chain 1 (ECHS1) is an enzyme involved in the metabolism of branched chain amino acids and fatty acids. Mutations in the ECHS1 gene lead to mitochondrial short-chain enoyl-CoA hydratase 1 deficiency, resulting in the accumulation of intermediates of valine. This is one of the most common causative genes in mitochondrial diseases. While genetic analysis studies have diagnosed numerous cases with ECHS1 variants, the increasing number of variants of uncertain significance (VUS) in genetic diagnosis is a major problem. METHODS: Here, we constructed an assay system to verify VUS function for ECHS1 gene. A high-throughput assay using ECHS1 knockout cells was performed to index these phenotypes by expressing cDNAs containing VUS. In parallel with the VUS validation system, a genetic analysis of samples from patients with mitochondrial disease was performed. The effect on gene expression in cases was verified by RNA-seq and proteome analysis. RESULTS: The functional validation of VUS identified novel variants causing loss of ECHS1 function. The VUS validation system also revealed the effect of the VUS in the compound heterozygous state and provided a new methodology for variant interpretation. Moreover, we performed multiomics analysis and identified a synonymous substitution p.P163= that results in splicing abnormality. The multiomics analysis complemented the diagnosis of some cases that could not be diagnosed by the VUS validation system. CONCLUSIONS: In summary, this study uncovered new ECHS1 cases based on VUS validation and omics analysis; these analyses are applicable to the functional evaluation of other genes associated with mitochondrial disease.

Molecular and in silico investigation of a novel ECHS1 gene mutation in a consanguine family with short-chain enoyl-CoA hydratase deficiency and Mt-DNA depletion: effect on trimer assembly and catalytic activity.

Short-chain enoyl-CoA hydratase deficiency (ECHS1D) is a rare congenital metabolic disorder that follows an autosomal recessive inheritance pattern. It is caused by mutations in the ECHS1 gene, which encodes a mitochondrial enzyme involved in the second step of mitochondrial ß-oxidation of fatty acids. The main characteristics of the disease are severe developmental delay, regression, seizures, neurodegeneration, high blood lactate, and a brain MRI pattern consistent with Leigh syndrome. Here, we report three patients belonging to a consanguineous family who presented with mitochondrial encephalomyopathy. Whole-exome sequencing revealed a new homozygous mutation c.619G > A (p.Gly207Ser) at the last nucleotide position in exon 5 of the ECHS1 gene. Experimental analysis showed that normal ECHS1 pre-mRNA splicing occurred in all patients compared to controls. Furthermore, three-dimensional models of wild-type and mutant echs1 proteins revealed changes in catalytic site interactions, conformational changes, and intramolecular interactions, potentially disrupting echs1 protein trimerization and affecting its function. Additionally, the quantification of mtDNA copy number variation in blood leukocytes showed severe mtDNA depletion in all probands.

An auxin signaling network translates low-sugar-state input into compensated cell enlargement in the fugu5 cotyledon.

In plants, the effective mobilization of seed nutrient reserves is crucial during germination and for seedling establishment. The Arabidopsis H+-PPase-loss-of-function fugu5 mutants exhibit a reduced number of cells in the cotyledons. This leads to enhanced post-mitotic cell expansion, also known as compensated cell enlargement (CCE). While decreased cell numbers have been ascribed to reduced gluconeogenesis from triacylglycerol, the molecular mechanisms underlying CCE remain ill-known. Given the role of indole 3-butyric acid (IBA) in cotyledon development, and because CCE in fugu5 is specifically and completely cancelled by ech2, which shows defective IBA-to-indoleacetic acid (IAA) conversion, IBA has emerged as a potential regulator of CCE. Here, to further illuminate the regulatory role of IBA in CCE, we used a series of high-order mutants that harbored a specific defect in IBA-to-IAA conversion, IBA efflux, IAA signaling, or vacuolar type H+-ATPase (V-ATPase) activity and analyzed the genetic interaction with fugu5-1. We found that while CCE in fugu5 was promoted by IBA, defects in IBA-to-IAA conversion, IAA response, or the V-ATPase activity alone cancelled CCE. Consistently, endogenous IAA in fugu5 reached a level 2.2-fold higher than the WT in 1-week-old seedlings. Finally, the above findings were validated in icl-2, mls-2, pck1-2 and ibr10 mutants, in which CCE was triggered by low sugar contents. This provides a scenario in which following seed germination, the low-sugar-state triggers IAA synthesis, leading to CCE through the activation of the V-ATPase. These findings illustrate how fine-tuning cell and organ size regulation depend on interplays between metabolism and IAA levels in plants.

Enoyl-CoA hydratase/3-hydroxyacyl CoA dehydrogenase is essential for the production of DHA in zebrafish.

Compared with other species, freshwater fish are more capable of synthesizing DHA via same biosynthetic pathways. Freshwater fish have a "Sprecher" pathway to biosynthesize DHA in a peroxisome-dependent manner. Enoyl-CoA hydratase/3-hydroxyacyl CoA dehydrogenase (Ehhadh) is involved in the hydration and dehydrogenation reactions of fatty acid ß-oxidation in peroxisomes. However, the role of Ehhadh in the synthesis of DHA in freshwater fish remains largely unclear. In this study, the knockout of Ehhadh significantly inhibited DHA synthesis in zebrafish. Liver transcriptome analysis showed that Ehhadh deletion significantly inhibited SREBF and PPAR signaling pathways and decreased the expression of PUFA synthesis-related genes. Our results from the analysis of transgenic zebrafish (Tg:Ehhadh) showed that Ehhadh overexpression significantly increased the DHA content in the liver and significantly upregulated the expression of genes related to PUFA synthesis. In addition, the DHA content in the liver of Tg:Ehhadh fed with linseed oil was significantly higher than that of wildtype, but the expression of PUFA synthesis-related genes fads2 and elovl2 were significantly lower, indicating that Ehhadh had a direct effect on DHA synthesis. In conclusion, our results showed that Ehhadh was essential for DHA synthesis in the "Sprecher" pathway, and Ehhadh overexpression could promote DHA synthesis. This study provides insight into the role of Ehhadh in freshwater fish.

Neuropathological Findings in Short-Chain enoyl-CoA Hydratase 1 Deficiency (ECHS1D): Case Report and Differential Diagnosis.

Short-chain enoyl-CoA hydratase 1 (ECHS1) is an enzyme that participates in the metabolism of valine, transforming methacrylyl-CoA in ß-hydroxy-isobutyryl-CoA. There is an accumulation of intermediate acids and ammonium as a consequence of its deficit. This background generates a harmful environment for the brain causing neuronal death and severe brain lesions. We present a case of a 39 weeks newborn that died at 31 hours old. We found vacuolization in basal areas, brain stem, cerebellum and spinal cord white matter (spongiform myelinopathy). These vacuoles were periodic acid-Schiff stain negative, there were neither acompanion gliosis nor macrophagic reaction. These findings were suggestive of metabolism acid disorders. The final diagnosis was confirmed by genetic study by massive parallel sequencing, showing 2 previously described pathogenic variants (c.160C > T and c.394G > A) of short-chain enoyl-CoA hydratase 1 gene. To our knowledge, this is the first case reporting the histological changes in short-chain enoyl-CoA hydratase 1 deficiency. Histological study provides useful information to orientate the diagnostic and clarify the clinical manifestations, especially in hospitals where urine or blood samples are not taking routinely or where genetic studies may not be performed.Synopsis: The main neuropathological findings in Short-chain enoyl-CoA hydratase 1 deficiency are the presence of whitte matter vacuoles in basal areas, brain stem and spinal cord.

Biosynthesis of Vanillin by Rational Design of Enoyl-CoA Hydratase/Lyase.

Vanillin holds significant importance as a flavoring agent in various industries, including food, pharmaceuticals, and cosmetics. The CoA-dependent pathway for the biosynthesis of vanillin from ferulic acid involved feruloyl-CoA synthase (Fcs) and enoyl-CoA hydratase/lyase (Ech). In this research, the Fcs and Ech were derived from Streptomyces sp. strain V-1. The sequence conservation and structural features of Ech were analyzed by computational techniques including sequence alignment and molecular dynamics simulation. After detailed study for the major binding modes and key amino acid residues between Ech and substrates, a series of mutations (F74W, A130G, A130G/T132S, R147Q, Q255R, ΔT90, ΔTGPEIL, ΔN1-11, ΔC260-287) were obtained by rational design. Finally, the yield of vanillin produced by these mutants was verified by whole-cell catalysis. The results indicated that three mutants, F74W, Q147R, and ΔN1-11, showed higher yields than wild-type Ech. Molecular dynamics simulations and residue energy decomposition identified the basic residues K37, R38, K561, and R564 as the key residues affecting the free energy of binding between Ech and feruloyl-coenzyme A (FCA). The large changes in electrostatic interacting and polar solvating energies caused by the mutations may lead to decreased enzyme activity. This study provides important theoretical guidance as well as experimental data for the biosynthetic pathway of vanillin.

Bioconversion of Phytosterols to 9-Hydroxy-3-Oxo-4,17-Pregadiene-20-Carboxylic Acid Methyl Ester by Enoyl-CoA Deficiency and Modifying Multiple Genes in Mycolicibacterium neoaurum.

Steroid drug precursors, including C19 and C22 steroids, are crucial to steroid drug synthesis and development. However, C22 steroids are less developed due to the intricacy of the steroid metabolic pathway. In this study, a C22 steroid drug precursor, 9-hydroxy-3-oxo-4,17-pregadiene-20-carboxylic acid methyl ester (9-OH-PDCE), was successfully obtained from Mycolicibacterium neoaurum by 3-ketosteroid-Δ1-dehydrogenase and enoyl-CoA hydratase ChsH deficiency. The production of 9-OH-PDCE was improved by the overexpression of 17ß-hydroxysteroid dehydrogenase Hsd4A and acyl-CoA dehydrogenase ChsE1-ChsE2 to reduce the accumulation of by-products. The purity of 9-OH-PDCE in fermentation broth was improved from 71.7% to 89.7%. Hence, the molar yield of 9-OH-PDCE was improved from 66.7% to 86.7%, with a yield of 0.78 g/L. Furthermore, enoyl-CoA hydratase ChsH1-ChsH2 was identified to form an indispensable complex in Mycolicibacterium neoaurum DSM 44704. IMPORTANCE C22 steroids are valuable precursors for steroid drug synthesis, but the development of C22 steroids remains unsatisfactory. This study presented a strategy for the one-step bioconversion of phytosterols to a C22 steroid drug precursor, 9-hydroxy-3-oxo-4,17-pregadiene-20-carboxylic acid methyl ester (9-OH-PDCE), by 3-ketosteroid-Δ1-dehydrogenase and enoyl-CoA hydratase deficiency with overexpression of 17ß-hydroxysteroid dehydrogenase acyl-CoA dehydrogenase in Mycolicibacterium. The function of the enoyl-CoA hydratase ChsH in vivo was revealed. Construction of the novel C22 steroid drug precursor producer provided more potential for steroid drug synthesis, and the characterization of the function of ChsH and the transformation of steroids further revealed the steroid metabolic pathway.

Crystal structure of multi-functional enzyme FadB from Cupriavidus necator: Non-formation of FadAB complex.

Cupriavidus necator H16 is a gram-negative chemolithoautotrophic bacterium that has been extensively studied for biosynthesis and biodegradation of polyhydroxyalkanoate (PHA) plastics. To improve our understanding of fatty acid metabolism for PHA production, we determined the crystal structure of multi-functional enoyl-CoA hydratase from Cupriavidus necator H16 (CnFadB). The predicted model of CnFadB created by AlphaFold was used to solve the phase problem during determination of the crystal structure of the protein. The CnFadB structure consists of two distinctive domains, an N-terminal enol-CoA hydratase (ECH) domain and a C-terminal 3-hydroxyacyl-CoA dehydrogenase (HAD) domain, and the substrate- and cofactor-binding modes of these two functional domains were identified. Unlike other known FadB enzymes that exist as dimers complexed with FadA, CnFadB functions as a monomer without forming a complex with CnFadA. Small angle X-ray scattering (SAXS) measurement further proved that CnFadB exists as a monomer in solution. The non-sequential action of FadA and FadB in C. necator appears to affect ß-oxidation and PHA synthesis/degradation.

ECHS1 deficiency and its biochemical and clinical phenotype.

ECHS1 gene encodes a mitochondrial enzyme, short-chain enoyl-CoA hydratase (SCEH). SCEH is involved in fatty acid oxidation ([Sharpe and McKenzie (2018); Mitochondrial fatty acid oxidation disorders associated with short-chain enoyl-CoA hydratase (ECHS1) deficiency, 7: 46]) and valine catabolism ([Fong and Schulz (1977); Purification and properties of pig heart crotonase and the presence of short chain and long chain enoyl coenzyme A hydratases in pig and guinea pig tissues, 252: 542-547]; [Wanders et al. (2012); Enzymology of the branched-chain amino acid oxidation disorders: The valine pathway, 35: 5-12]), and the dysfunction of SCEH leads to a severe Leigh or Leigh-like Syndrome phenotype in patients ([Haack et al. (2015); Deficiency of ECHS1 causes mitochondrial encephalopathy with cardiac involvement, 2: 492-509]; [Peters et al. (2014); ECHS1 mutations in Leigh disease: A new inborn error of metabolism affecting valine metabolism, 137: 2903-2908]; [Sakai et al. (2015); ECHS1 mutations cause combined respiratory chain deficiency resulting in Leigh syndrome, 36: 232-239]; [Tetreault et al. (2015); Whole-exome sequencing identifies novel ECHS1 mutations in Leigh, 134: 981-991]). This study aims to further describe the ECHS1 deficiency phenotype using medical history questionnaires and standardized tools assessing quality of life and adaptive skills. Our findings in this largest sample of ECHS1 patients in literature to date (n = 13) illustrate a severely disabling condition causing severe developmental delays (n = 11), regression (n = 10), dystonia/hypotonia and movement disorders (n = 13), commonly with symptom onset in infancy (n = 10), classical MRI findings involving the basal ganglia (n = 11), and variability in biochemical profile. Congruent with the medical history, our patients had significantly low composite and domain scores on Vineland Adaptive Behavior Scales, Third Edition. We believe there is an increasing need for better understanding of ECHS1 deficiency with an aim to support the development of transformative genetic-based therapies, driven by the unmet need for therapies for patients with this genetic disease.

Movement disorders in valine métabolism diseases caused by HIBCH and ECHS1 deficiencies.

BACKGROUND AND PURPOSE: HIBCH and ECHS1 genes encode two enzymes implicated in the critical steps of valine catabolism, 3-hydroxyisobutyryl-coenzyme A (CoA) hydrolase (HIBCH) and short-chainenoyl-CoA hydratase (ECHS1), respectively. HIBCH deficiency (HIBCHD) and ECHS1 deficiency (ECHS1D) generate rare metabolic dysfunctions, often revealed by neurological symptoms. The aim of this study was to describe movement disorders spectrum in patients with pathogenic variants in ECHS1 and HIBC. METHODS: We reviewed a series of 18 patients (HIBCHD: 5; ECHS1D: 13) as well as 105 patients from the literature. We analysed the detailed phenotype of HIBCHD (38 patients) and ECHS1D (85 patients), focusing on MDs. RESULTS: The two diseases have a very similar neurological phenotype, with an early onset before 10 years of age for three clinical presentations: neonatal onset, Leigh-like syndrome (progressive onset or acute neurological decompensation), and isolated paroxysmal dyskinesia. Permanent or paroxysmal MDs were recorded in 61% of HIBCHD patients and 72% of ECHS1D patients. Patients had a variable combination of either isolated or combined MD, and dystonia was the main MD. These continuous MDs included dystonia, chorea, parkinsonism, athetosis, myoclonus, tremors, and abnormal eye movements. Patients with paroxysmal dyskinesia (HIBCHD: 4; ECHS1D: 9) usually had pure paroxysmal dystonia with normal clinical examination and no major impairment in psychomotor development. No correlation could be identified between clinical pattern (especially MD) and genetic pathogenic variants. CONCLUSIONS: Movement disorders, including abnormal ocular movements, are a hallmark of HIBCHD and ECHS1D. MDs are not uniform; dystonia is the most frequent, and various types of MD are combined in single patient.

A Case of ECHS1 Deficiency with Severe Encephalopathy and Status Epilepticus after a Propofol Sedation: Case Report.

BACKGROUND: Short-chain enoyl-CoA hydratase (ECHS1) deficiency is a rare metabolic disorder. Concerned patients present with Leigh syndrome symptoms or a Leigh-like syndrome. Only 58 patients are known worldwide. The ECHS1 is a key component in ß-oxidation and valine catabolic pathways. CASE: Here we report a 6-month-old Lebanese boy born to consanguineous parents. He presented an increased muscle tone, hyperexcitability, feeding problems, horizontal nystagmus, and developmental delay. Magnetic resonance imaging of the brain revealed frontal brain atrophy, corpus callosum atrophy, and T2 hyperintensity in pallidum, internal capsule, pons, and thalamus. In the postsedation phase, the patient displayed a sudden generalized seizure with transition to status epilepticus. Therefore, we conducted metabolic examinations, which showed elevated levels of 2-methyl-2,3-DiOH-butyrate and 3-methylglutaconate in urine. Single exome sequencing revealed the homozygous mutation c.476A > G in the ECHS1 gene. CONCLUSION: This case report describes the clinical symptoms and the diagnostics of ECHS1 deficiency. It shows the importance of further metabolic and genetic testing of patients with motoric conspicuities and developmental delay. It is important to be cautious with propofol sedation of patients who present an unknown neurological disorder, when metabolic disturbance or especially mitochondriopathy is suspected.

Developing efficient vanillin biosynthesis system by regulating feruloyl-CoA synthetase and enoyl-CoA hydratase enzymes.

Vanillin is one of the most commonly used natural-occurring flavors in the world. This study successfully constructed an efficient whole-cell catalytic system for vanillin biosynthesis from ferulic acid by regulating feruloyl-CoA synthetase (FCS) and enoyl-CoA hydratase (ECH). First, we constructed an efficient cell-free catalytic system with FCS-Str (fcs from Streptomyces sp. V-1) and ECH-Str (ech from Streptomyces sp. V-1) combination at 1:1. The efficient cell-free catalytic system provided necessary strategies for optimizing the whole-cell catalytic system. Then, we constructed the recombinant Escherichia coli by heterologously expressing the fcs-Str and ech-Str combination. Moreover, E. coli JM109 was a better recombinant Escherichia coli than E. coli BL21 with higher vanillin production. Finally, we first adjusted the ratio of FCS and ECH in E. coli JM109 to 1:1 using two copies of fcs-Str. For higher vanillin production, we further optimized the induction conditions of E. coli JM109 to increase the amount of FCS and ECH. The optimized E. coli JM109-FE-F constructed in this study has the highest vanillin synthesis rate of converting 20 mM ferulic acid to 15 mM vanillin in 6 h among all of the E. coli catalytic systems. Our study made a significant contribution to the construction of the vanillin biosynthesis system and provided a valuable strategy for increasing vanillin production. KEY POINTS: • The efficient cell-free vanillin biosynthesis system was constructed by FCS-Str and ECH-Str combination at 1:1. • Escherichia coli JM109 was determined as a better recombinant Escherichia coli than E. coli BL21 with higher vanillin production. • Escherichia coli JM109-FE-F with two copies of fcs-Str and one copy of ech-Str has the highest catalytic efficiency for vanillin production.

Nitrogen Starvation Enhances the Production of Saturated and Unsaturated Fatty Acids in Aurantiochytrium sp. PKU#SW8 by Regulating Key Biosynthetic Genes.

Nitrogen deprivation is known to improve lipid accumulation in microalgae and thraustochytrids. However, the patterns of fatty acid production and the molecular mechanisms underlying the accumulation of unsaturated and saturated fatty acids (SFAs) under nitrogen starvation remain largely unknown for thraustochytrids. In this study, batch culture experiments under nitrogen replete and nitrogen starvation conditions were performed, and the changes in the transcriptome of Aurantiochytrium sp. PKU#SW8 strain between these conditions were investigated. Our results showed improved yields of total fatty acids (TFAs), total unsaturated fatty acids, and total SFAs under nitrogen starvation, which suggested that nitrogen starvation favors the accumulation of both unsaturated and saturated fatty acids in PKU#SW8. However, nitrogen starvation resulted in a more than 2.36-fold increase of SFAs whereas a 1.7-fold increase of unsaturated fatty acids was observed, indicating a disproportionate increase in these groups of fatty acids. The fabD and enoyl-CoA hydratase genes were significantly upregulated under nitrogen starvation, supporting the observed increase in the yield of TFAs from 2.63 ± 0.22 g/L to 3.64 ± 0.16 g/L. Furthermore, the pfaB gene involved in the polyketide synthase (PKS) pathway was significantly upregulated under nitrogen starvation. This suggested that the increased expression of the pfaB gene under nitrogen starvation may be one of the explanations for the increased yield of docosahexaenoic acid by 1.58-fold. Overall, our study advances the current understanding of the molecular mechanisms that underlie the response of thraustochytrids to nitrogen deprivation and their fatty acid biosynthesis.