Succinic Semialdehyde Dehydrogenase Research Articles

Delays in latencies of median-nerve evoked magnetic fields in patients with succinic semialdehyde dehydrogenase deficiency

ObjectiveSuccinic semialdehyde dehydrogenase deficiency (SSADHD) is a genetic disorder resulting in abnormal regulation of γ-aminobutyric acid, lipid metabolism, and myelin biogenesis, leading to ataxia, seizures, and cognitive impairment. Since the myelin sheath is thinner in a murine model of SSADHD compared to a wild type, we hypothesized that this also holds for human brain. We tested whether the conduction velocity in the somatosensory pathway is accordingly delayed. MethodsSomatosensory evoked magnetic fields (SEF) produced by transcutaneous electrical stimulation of the median nerve were measured in 13 SSADHD patients, 11 healthy and 14 disease controls with focal epilepsy. The peak latencies of the initial four components (M1, M2, M3 and M4) were measured. ResultsThe SEF waveforms and scalp topographies were comparable across the groups. The latencies were statistically significantly longer in the SSADHD group compared to the two controls. We found these latencies for the SSADHD, healthy and disease controls respectively to be: M1: (21.9 ± 0.8 ms [mean ± standard error of the mean], 20.4 ± 0.6 ms, and 21.0 ± 0.4 ms) (p < 0.05); M2: (36.1 ± 1.0 ms, 33.1 ± 0.6 ms, and 32.1 ± 1.1 ms) (p < 0.005); M3: (62.5 ± 2.4 ms, 54.7 ± 2.0 ms, and 49.9 ± 1.8 ms) (p < 0.005); M4: (86.2 ± 2.3 ms, 78.8 ± 2.8 ms, and 73.5 ± 2.9 ms) (p < 0.005). ConclusionsThe SEF latencies are delayed in patients with SSADHD compared with healthy controls and disease controls. SignificanceThis is the first study that compares conduction velocities in the somatosensory pathway in SSADHD, an inherited disorder of GABA metabolism. The longer peak latency implying slower conduction velocity supports the hypothesis that myelin sheath thickness is decreased in SSADHD.

Putative Molecular Aspects of Succinic Semialdehyde Dehydrogenase’s Operation in the Leaves of Wheat (Triticum aestivum L.) under Salt Stress

Putative Molecular Aspects of Succinic Semialdehyde Dehydrogenase’s Operation in the Leaves of Wheat (Triticum aestivum L.) under Salt Stress

Inherited Pediatric Neurotransmitter Disorders: Case Studies and Long-Term Outcomes

AbstractPrimary pediatric neurotransmitter disorders include genetic defects of neurotransmitter metabolism that may mimic common neurological conditions in children. Our objective was to evaluate the clinical experience and outcomes of affected patients. Five patients with primary neurotransmitter defects were identified in the neurometabolic database between 2004 and 2022. Two patients with 6-pyruvoyltetrahydropterin synthase deficiency and one with pyridoxine-dependent epilepsy (PDE) presented in the neonatal period. One patient with succinic semialdehyde dehydrogenase (SSADH) deficiency and one with aromatic l-amino acid decarboxylase (AADC) deficiency presented in later life. A diagnosis of cerebral palsy was revised following biochemical confirmation of SSADH deficiency. AADC deficiency was confirmed via exome sequencing and reduced activity on enzyme assay. Late diagnosis in the latter two cases was likely due to a low index of suspicion and lack of access to diagnostic tests in the country of origin. In two children with tetrahydrobiopterin deficiency, newborn screening results and atypical clinical features prompted investigations. An early diagnosis of PDE was established based on presenting features, a high index of suspicion, the presence of an identifiable biochemical marker and molecular genetic testing. Pediatric neurotransmitter disorders can be diagnosed based on a high clinical index of suspicion, availability of biochemical markers, and molecular genetic testing. These disorders, though rare, need to be included in the differential diagnosis of common neurological presentations in children as they may be potentially treatable. Outcomes and influencing factors in the present series are discussed in comparison to published data.

Rhizobacterial syntrophy between a helper and a beneficiary promotes tomato plant health.

Microbial interactions impact the functioning of microbial communities. However, microbial interactions within host-associated communities remain poorly understood. Here, we report that the beneficiary rhizobacterium Niallia sp. RD1 requires the helper Pseudomonas putida H3 for bacterial growth and beneficial interactions with the plant host. In the absence of the helper H3 strain, the Niallia sp. RD1 strain exhibited weak respiration and elongated cell morphology without forming bacterial colonies. A transposon mutant of H3 in a gene encoding succinate-semialdehyde dehydrogenase displayed much attenuated support of RD1 colony formation. Through the subsequent addition of succinate to the media, we found that succinate serves as a public good that supports RD1 growth. Comparative genome analysis highlighted that RD1 lacked the gene for sufficient succinate, suggesting its evolution as a beneficiary of succinate biosynthesis. The syntrophic interaction between RD1 and H3 efficiently protected tomato plants from bacterial wilt and promoted tomato growth. The addition of succinate to the medium restored complex II-dependent respiration in RD1 and facilitated the cultivation of various bacterial isolates from the rhizosphere. Taken together, we delineate energy auxotrophic beneficiaries ubiquitous in the microbial community, and these beneficiaries could benefit host plants with the aid of helpers in the rhizosphere.

The metabolism of amino acids, AsA and abscisic acid induced by strigolactone participates in chilling tolerance in postharvest zucchini fruit.

Zucchini fruit are notably susceptible to chilling injury when stored at low temperatures. The purpose of this experimental investigation was to assess the influence of strigolactone (ST) (5 μmol L-1) on mitigating chilling injury and the metabolic changes in amino acids, ascorbic acid, and abscisic acid in zucchini fruit stored at 4°C. Research findings demonstrated that ST-treated zucchini fruit displayed a significantly higher tolerance to chilling stress compared to the control group. Postharvest ST treatment led to a decrease in weight loss, accompanied by reduced levels of malondialdehyde and relative ion leakage compared to the untreated group. ST immersion significantly boosted the metabolic pathways associated with proline and arginine, affecting both the enzymatic reactions and gene expressions, thus cumulatively increasing the internal concentrations of these amino acids in zucchini fruit. Zucchini treated with ST exhibited an increased concentration of γ-aminobutyric acid (GABA) as a result of augmented activities and elevated transcriptional levels of glutamate decarboxylase (GAD), GABA transaminase (GAT), and succinate semialdehyde dehydrogenase (SSD). In the ST-treated sample, the elevated enzymatic activities and enhanced gene expressions within the ascorbic acid (AsA) biosynthesis pathway worked together to sustain AsA accumulation. The application of ST resulted in a rise in abscisic acid (ABA) concentration, which correspondingly correlated with the induction of both activities and gene expression levels of crucial enzymes involved in ABA metabolism. Our findings revealed that submerging zucchini fruit in ST could be a highly effective strategy for boosting their chilling tolerance. The alleviation in chilling injury induced by ST may be attributed to the modulation of proline, arginine, GABA, AsA and ABA metabolism.

Glymphatic dysfunction coincides with lower GABA levels and sleep disturbances in succinic semialdehyde dehydrogenase deficiency.

Succinic semialdehyde dehydrogenase deficiency (SSADHD) is an inherited metabolic disorder of γ-aminobutyrate (GABA) catabolism. Cerebral waste clearance along glymphatic perivascular spaces depends on aquaporin 4 (AQP4) water channels, the function of which was shown to be influenced by GABA. Sleep disturbances are associated independently with SSADHD and glymphatic dysfunction. This study aimed to determine whether indices of the hyperGABAergic state characteristic of SSADHD coincide with glymphatic dysfunction and sleep disturbances and to explicate the modulatory effect that GABA may have on the glymphatic system. The study included 42 individuals (21 with SSADHD; 21 healthy controls) who underwent brain MRIs and magnetic resonance spectroscopy (MRS) for assessment of glymphatic dysfunction and cortical GABA, plasma GABA measurements, and circadian clock gene expression. The SSADHD subjects responded to an additional Children's Sleep Habits Questionnaire (CSHQ). Compared with the control group, SSADHD subjects did not differ in sex and age but had a higher severity of enlarged perivascular spaces in the centrum semiovale (p < 0.001), basal ganglia (p = 0.01), and midbrain (p = 0.001), as well as a higher MRS-derived GABA/NAA peak (p < 0.001). Within the SSADHD group, the severity of glymphatic dysfunction was specific for a lower MRS-derived GABA/NAA (p = 0.04) and lower plasma GABA (p = 0.004). Additionally, the degree of their glymphatic dysfunction correlated with the CSHQ-estimated sleep disturbances scores (R = 5.18, p = 0.03). In the control group, EPVS burden did not correlate with age or cerebral and plasma GABA values. The modulatory effect that GABA may exert on the glymphatic system has therapeutic implications for sleep-related disorders and neurodegenerative conditions associated with glymphatic dysfunction.

The Optimization of Assay Conditions and Characterization of the Succinic Semialdehyde Dehydrogenase Enzyme of Germinated Tartary Buckwheat.

In this study, the conditions for optimizing the determination of succinic semialdehyde dehydrogenase (SSADH, EC 1.2.1.79) activity in germinated Tartary buckwheat were investigated. Based on a single-factor test, the effects of temperature, pH, and succinic semialdehyde (SSA) concentration on the enzyme activity of germinated buckwheat SSADH were investigated by using the response surface method, and optimal conditions were used to study the enzymatic properties of germinated buckwheat SSADH. The results revealed that the optimum conditions for determining SSADH enzyme activity are as follows: temperature-30.8 °C, pH-8.7, and SSA concentration-0.3 mmol/L. Under these conditions, SSADH enzyme activity was measured as 346 ± 9.61 nmol/min. Furthermore, the thermal stability of SSADH was found to be superior at 25 °C, and its pH stability remained comparable at pH levels of 7.6, 8.1, and 8.6 in germinated Tartary buckwheat samples; however, a decline in stability was observed at pH 9.1. Cu2+, Co2+, and Ni2+ exhibited an activating effect on SSADH activity in germinating Tartary buckwheat, with Cu2+ having the greatest influence (p < 0.05), which was 1.21 times higher than that of the control group. Zn2+, Mn2+, and Na+ inhibited SSADH activity in germinating Tartary buckwheat, with Zn2+ showing the strongest inhibitory effect (p < 0.05). On the other hand, the Km and Vmax of SSADH for SSA in germinated Tartary buckwheat were 0.24 mmol/L and 583.24 nmol/min. The Km and Vmax of SSADH for NAD+ in germinated Tartary buckwheat were 0.64 mmol/L and 454.55 nmol/min.

ALDH5A1-deficient iPSC-derived excitatory and inhibitory neurons display cell type specific alterations

Succinic semialdehyde dehydrogenase deficiency (SSADHD) is a neurometabolic disorder caused by ALDH5A1 mutations presenting with autism and epilepsy. SSADHD leads to impaired GABA metabolism and results in accumulation of GABA and γ-hydroxybutyrate (GHB), which alter neurotransmission and are thought to lead to neurobehavioral symptoms. However, why increased inhibitory neurotransmitters lead to seizures remains unclear. We used induced pluripotent stem cells from SSADHD patients (one female and two male) and differentiated them into GABAergic and glutamatergic neurons. SSADHD iGABA neurons show altered GABA metabolism and concomitant changes in expression of genes associated with inhibitory neurotransmission. In contrast, glutamatergic neurons display increased spontaneous activity and upregulation of mitochondrial genes. CRISPR correction of the pathogenic variants or SSADHD mRNA expression rescue various metabolic and functional abnormalities in human neurons. Our findings uncover a previously unknown role for SSADHD in excitatory human neurons and provide unique insights into the cellular and molecular basis of SSADHD and potential therapeutic interventions.

CmCML11 interacts with CmCAMTA5 to enhance γ-aminobutyric acid (GABA) accumulation by regulating GABA shunt in fresh-cut cantaloupe

The effect of calcium chloride (CaCl2) treatment on γ-aminobutyric acid (GABA) accumulation in fresh-cut cantaloupe and the involved mechanisms were investigated. The result showed that 1% (w/v) CaCl2 treatment increased GABA content and activities of glutamate decarboxylase (GAD) and succinate semialdehyde dehydrogenase (SSADH), while decreased glutamate (Glu) content and GABA transaminase (GABA-T) activities in fresh-cut cantaloupe. CmCML11 and CmCAMTA5 expressions of CaCl2-treated fruit increased by 187.4% and 165.6% than control fruit in the initial 6 h. Besides, expressions of GABA shunt genes, including CmGAD1, CmGAD2, CmGABA-T and CmSSADH were also up-regulated by CaCl2 treatment during early storage. Moreover, acting as a transcriptional activator, CmCAMTA5 could bind to the CG-box in promoters of CmGAD1, CmGABA-T and CmSSADH and activate their transcription. Furthermore, the interaction between CmCML11 and CmCAMTA5 could enhance the transcriptional activation on GABA shunt genes which were regulated by CmCAMTA5. Collectively, our findings revealed that CaCl2 treatment promoted GABA accumulation in fresh-cut cantaloupe via the combined effect of CmCML11 and CmCAMTA5 in the regulation of expressions of CmGAD1, CmGABA-T, and CmSSADH in GABA shunt.

Reduced evoked cortical beta and gamma activity and neuronal synchronization in succinic semialdehyde dehydrogenase deficiency, a disorder of γ-aminobutyric acid metabolism.

Succinic semialdehyde dehydrogenase deficiency is a rare autosomal recessively inherited metabolic disorder of γ-aminobutyric acid catabolism manifested by intellectual disability, expressive aphasia, movement disorders, psychiatric ailments and epilepsy. Subjects with succinic semialdehyde dehydrogenase deficiency are characterized by elevated γ-aminobutyric acid and related metabolites, such as γ-guanidinobutyric acid, and an age-dependent downregulation of cerebral γ-aminobutyric acid receptors. These findings indicate impaired γ-aminobutyric acid and γ-aminobutyric acid sub-type A (GABAA) receptor signalling as major factors underlying the pathophysiology of this neurometabolic disorder. We studied the cortical oscillation patterns and their relationship with γ-aminobutyric acid metabolism in 18 children affected by this condition and 10 healthy controls. Using high-density EEG, we recorded somatosensory cortical responses and resting-state activity. Using electrical source imaging, we estimated the relative power changes (compared with baseline) in both stimulus-evoked and stimulus-induced responses for physiologically relevant frequency bands and resting-state power. Stimulus-evoked oscillations are phase locked to the stimulus, whereas induced oscillations are not. Power changes for both evoked and induced responses as well as resting-state power were correlated with plasma γ-aminobutyric acid and γ-guanidinobutyric acid concentrations and with cortical γ-aminobutyric acid measured by proton magnetic resonance spectroscopy. Plasma γ-aminobutyric acid, γ-guanidinobutyric acid and cortical γ-aminobutyric acid were higher in patients than in controls (P < 0.001 for both). Beta and gamma relative power were suppressed for evoked responses in patients versus controls (P < 0.01). No group differences were observed for induced activity (P > 0.05). The mean gamma frequency of evoked responses was lower in patients versus controls (P = 0.002). Resting-state activity was suppressed in patients for theta (P = 0.011) and gamma (P < 0.001) bands. Evoked power changes were inversely correlated with plasma γ-aminobutyric acid and with γ-guanidinobutyric acid for beta (P < 0.001) and gamma (P < 0.001) bands. Similar relationships were observed between the evoked power changes and cortical γ-aminobutyric acid for all tested areas in the beta band (P < 0.001) and for the posterior cingulate gyrus in the gamma band (P < 0.001). We also observed a negative correlation between resting-state activity and plasma γ-aminobutyric acid and γ-guanidinobutyric acid for theta (P < 0.001; P = 0.003), alpha (P = 0.003; P = 0.02) and gamma (P = 0.02; P = 0.01) bands. Our findings indicate that increased γ-aminobutyric acid concentration is associated with reduced sensory-evoked beta and gamma activity and impaired neuronal synchronization in patients with succinic semialdehyde dehydrogenase deficiency. This further elucidates the pathophysiology of this neurometabolic disorder and serves as a potential biomarker for therapeutic trials.

GABase and glutaminase inhibitory activities of herbal extracts and acylated flavonol monoglycosides isolated from the leaves of Laurus nobilis L.

This study was to compare GABase [a mixture of γ-aminobutyric acid (GABA) aminotransferase and succinic semialdehyde dehydrogenase] and glutaminase inhibitory activities of 20 herbal extracts and investigate the isolation, structural elucidation and those inhibitory activities of three acylated flavonol monoglycosides from the selected extract of Laurus nobilis L. (laurel). On the basis of the NMR spectroscopic data and the ESI MS spectra together with the comparison with the literature values, three compounds were identified as kaempferol-3-O-(4″-E-p-coumaroyl)-α-l-rhamnopyranoside (1), kaempferol-3-O-(3″,4″-di-E-p-coumaroyl)-α-l-rhamnopyranoside (2) and kaempferol-3-O-(2″,4″-di-E-p-coumaroyl)-α-l-rhamnopyranoside (3), respectively. The IC50 values of GABase inhibitory activity of 1-3 and p-hydroxybenzaldehyde (HBA) as control were 0.24 mM, 0.14 mM, 0.12 mM and 0.43 mM, respectively. Additionally, the IC50 values of glutaminase inhibitory activity of 1-3 and 6-diazo-5-oxo-l-norleucine (DON) as control were 0.34 mM, 0.13 mM, 0.14 mM and 0.33 mM, respectively. The results suggest that the extract from laurel shows the strongest biological activities among 20 herbal extracts and three acylated flavonol monoglycosides may serve as potential lead compounds for the prevention and treatment of neurodegenerative and lifestyle-related diseases by targeting GABase and glutaminase. This is the first report on GABase and glutaminase inhibitory activities of 1-3.

Early Events after Herpes Simplex Virus-Type 1 Entry Are Necessary for the Release of Gamma-Hydroxybutyrate upon Acute Infection.

We reported that gamma-hydroxybutyrate (GHB) is released upon Herpes Simplex Virus Type-1 (HSV-1) acute infection. However, the cellular biochemical processes involved in the production of GHB in infected cells are unclear. This study aims to shed light on the biochemical pathway and the stage within the viral life cycle responsible for the release of GHB in infected cells. UV-inactivation, acyclovir (ACV), and cycloheximide (CHX) treatments were used to inhibit HSV-1 replication at various stages. Vero cells treated with UV-inactivated HSV-1 significantly decreased GHB production. However, ACV or CHX treatments did not affect GHB production. We also showed that inhibition of glycolytic enzyme enolase by sodium fluoride (NaF) significantly reduces GHB production upon infection. This finding suggests that suppression of glycolytic activity negatively affects cellular GHB production. Our data also indicated that succinic semialdehyde dehydrogenase, an enzyme involved in the shunt of the tricarboxylic acid (TCA) cycle to generate succinic acid, was decreased upon infection, suggesting that infection may trigger the accumulation of succinic semialdehyde, causing the production of GHB. Although the precise mechanism has yet to be defined, our results suggest that early events following infection modulates the release of GHB, which is generated through the metabolic pathways of glycolysis and TCA cycle.

Succinic semialdehyde dehydrogenase deficiency in mice and in humans: An untargeted metabolomics perspective.

Succinic semialdehyde dehydrogenase deficiency (SSADHD) is a rare neurometabolic disorder caused by disruption of the gamma-aminobutyric acid (GABA) pathway. A more detailed understanding of its pathophysiology, beyond the accumulation of GABA and gamma-hydroxybutyric acid (GHB), will increase our understanding of the disease and may support novel therapy development. To this end, we compared biochemical body fluid profiles from SSADHD patients with controls using next-generation metabolic screening (NGMS). Targeted analysis of NGMS data from cerebrospinal fluid (CSF) showed a moderate increase of aspartic acid, glutaric acid, glycolic acid, 4-guanidinobutanoic acid, and 2-hydroxyglutaric acid, and prominent elevations of GHB and 4,5-dihydroxyhexanoic acid (4,5-DHHA) in SSADHD samples. Remarkably, the intensities of 4,5-DHHA and GHB showed a significant positive correlation in control CSF, but not in patient CSF. In an established zebrafish epilepsy model, 4,5-DHHA showed increased mobility that may reflect limited epileptogenesis. Using untargeted metabolomics, we identified 12 features in CSF with high biomarker potential. These had comparable increased fold changes as GHB and 4,5-DHHA. For 10 of these features, a similar increase was found in plasma, urine and/or mouse brain tissue for SSADHD compared to controls. One of these was identified as the novel biomarker 4,5-dihydroxyheptanoic acid. The intensities of selected features in plasma and urine of SSADHD patients positively correlated with the clinical severity score of epilepsy and psychiatric symptoms of those patients, and also showed a high mutual correlation. Our findings provide new insights into the (neuro)metabolic disturbances in SSADHD and give leads for further research concerning SSADHD pathophysiology.

Succinic semialdehyde dehydrogenase deficiency: A model of neurocircuit imbalances in autism and potential insight into new biomarkers.

This commentary is on the original article by Latzer et al on pages 1596–1606 of this issue.

Establishment and validation of a clinical severity scoring system for succinic semialdehyde dehydrogenase deficiency.

Succinic semialdehyde dehydrogenase deficiency (SSADHD) is an inherited metabolic disorder with a variable phenotype and rate of progression. We aimed to develop and validate a clinical severity scoring (CSS) system applicable to the clinical setting and composed of five domains reflecting the principal manifestations of this disorder: cognitive, communication, motor, epilepsy, and psychiatry. A prospectively characterized cohort of 27 SSADHD subjects (55% females, median [IQR] age 9.2 [4.6-16.2] years) who enrolled in the SSADHD Natural History Study were included. The CSS was validated by comparison to an objective severity scoring (OSS) system based on comprehensive neuropsychologic and neurophysiologic assessments, which mirror and complement the domains of the CSS. The total CSS was sex and age-independent, and 80% of its domains lacked interdependence. With increasing age, there was a significant improvement in communication abilities (p = 0.05) and a worsening of epilepsy and psychiatric manifestations (p = 0.004 and p = 0.02, respectively). There was a significant correlation between all the CSS and OSS domain scores, as well as between the total CSS and OSS (R = 0.855, p < 0.001). Additionally, there were no significant demographic or clinical differences in the ratio of individuals in the upper quartile to the lower three quartiles of the CSS and OSS. The SSADHD CSS is validated using objective measures and offers a reliable condition-specific instrument universally applicable in clinical settings. This severity score may be utilized for family and patient counseling, genotype-phenotype correlations, biomarker development, clinical trials, and objective descriptions of the natural history of SSADHD.

Autism spectrum disorder and GABA levels in children with succinic semialdehyde dehydrogenase deficiency.

To elucidate the etiological aspects of autism spectrum disorder (ASD) in succinic semialdehyde dehydrogenase deficiency (SSADHD), related to dysregulation of γ-aminobutyric acid (GABA) and the imbalance of excitatory and inhibitory neurotransmission. In this prospective, international study, individuals with SSADHD underwent neuropsychological assessments, as well as biochemical, neurophysiological, and neuroimaging evaluations. Of the 29 individuals (17 females) enrolled (median age [IQR] 10 years 5 months [5 years 11 months-18 years 1 month]), 16 were diagnosed with ASD. ASD severity significantly increased with age (r = 0.67, p < 0.001) but was inversely correlated with plasma GABA (r = -0.67, p < 0.001) and γ-hydroxybutyrate levels (r = -0.538, p = 0.004), and resting motor threshold as measured by transcranial magnetic stimulation (r = -0.44, p = 0.03). A discriminative analysis indicated that an age older than 7 years 2 months (p = 0.004) and plasma GABA levels less than 2.47 μM (p = 0.01) are the threshold values beyond which the likelihood of ASD presenting in individuals with SSADHD is increased. ASD is prevalent but not universal in SSADHD, and it can be predicted by lower levels of plasma GABA and GABA-related metabolites. ASD severity in SSADHD increases with age and the loss of cortical inhibition. These findings add insight into the pathophysiology of ASD and may facilitate its early diagnosis and intervention in individuals with SSADHD.

Comparative genomic analysis of Halomonas campaniensis wild-type and ultraviolet radiation-mutated strains reveal genomic differences associated with increased ectoine production

Ectoine is a natural amino acid derivative and one of the most widely used compatible solutes produced by Halomonas species that affects both cellular growth and osmotic equilibrium. The positive effects of UV mutagenesis on both biomass and ectoine content production in ectoine-producing strains have yet to be reported. In this study, the wild-type H. campaniensis strain XH26 (CCTCCM2019776) was subjected to UV mutagenesis to increase ectoine production. Eight rounds of mutagenesis were used to generate mutated XH26 strains with different UV-irradiation exposure times. Ectoine extract concentrations were then evaluated among all strains using high-performance liquid chromatography analysis, alongside whole genome sequencing with the PacBio RS II platform and comparison of the wild-type strain XH26 and the mutant strain G8-52 genomes. The mutant strain G8-52 (CCTCCM2019777) exhibited the highest cell growth rate and ectoine yields among mutated strains in comparison with strain XH26. Further, ectoine levels in the aforementioned strain significantly increased to 1.51 ± 0.01 g L−1 (0.65 g g−1 of cell dry weight), representing a twofold increase compared to wild-type cells (0.51 ± 0.01 g L−1) when grown in culture medium for ectoine accumulation. Concomitantly, electron microscopy revealed that mutated strain G8-52 cells were obviously shorter than wild-type strain XH26 cells. Moreover, strain G8-52 produced a relatively stable ectoine yield (1.50 g L−1) after 40 days of continuous subculture. Comparative genomics analysis suggested that strain XH26 harbored 24 mutations, including 10 nucleotide insertions, 10 nucleotide deletions, and unique single nucleotide polymorphisms. Notably, the genes orf00723 and orf02403 (lipA) of the wild-type strain mutated to davT and gabD in strain G8-52 that encoded for 4-aminobutyrate-2-oxoglutarate transaminase and NAD-dependent succinate-semialdehyde dehydrogenase, respectively. Consequently, these genes may be involved in increased ectoine yields. These results suggest that continuous multiple rounds of UV mutation represent a successful strategy for increasing ectoine production, and that the mutant strain G8-52 is suitable for large-scale fermentation applications.

The presence and severity of epilepsy coincide with reduced γ-aminobutyrate and cortical excitatory markers in succinic semialdehyde dehydrogenase deficiency.

Succinic semialdehyde dehydrogenase deficiency (SSADHD) is a rare inherited metabolic disorder caused by a defect of γ-aminobutyrate (GABA) catabolism. Despite the resultant hyper-GABAergic environment facilitated by the metabolic defect, individuals with this disorder have a paradoxically high prevalence of epilepsy. We aimed to study the characteristics of epilepsy in SSADHD and its concordance with GABA-related metabolites and neurophysiologic markers of cortical excitation. Subjects in an international natural history study of SSADHD underwent clinical assessments, electroencephalography, transcranial magnetic stimulation (TMS), magnetic resonance spectroscopy for GABA/N-acetyl aspartate quantification, and plasma GABA-related metabolite measurements. A total of 61 subjects with SSADHD and 42 healthy controls were included in the study. Epilepsy was present in 49% of the SSADHD cohort. Over time, there was an increase in severity in 33% of the subjects with seizures. The presence of seizures was associated with increasing age (p = .001) and lower levels of GABA (p = .002), γ-hydroxybutyrate (GHB; p = .004), and γ-guanidinobutyrate (GBA; p = .003). Seizure severity was associated with increasing age and lower levels of GABA-related metabolites as well as lower TMS-derived resting motor thresholds (p = .04). The cutoff values with the highest discriminative ability to predict seizures were age > 9.2 years (p = .001), GABA < 2.57 μmol·L-1 (p = .002), GHB < 143.6 μmol·L-1 (p = .004), and GBA < .075 μmol·L-1 (p = .007). A prediction model for seizures in SSADHD was comprised of the additive effect of older age and lower plasma GABA, GHB, and GBA (area under the receiver operating characteristic curve of .798, p = .008). Epilepsy is highly prevalent in SSADHD, and its onset and severity correlate with an age-related decline in GABA and GABA-related metabolite levels as well as TMS markers of reduced cortical inhibition. The reduction of GABAergic activity in this otherwise hyper-GABAergic disorder demonstrates a concordance between epileptogenesis and compensatory responses. These findings may furthermore inform the timing of molecular interventions for SSADHD.

GABA shunt pathway is stimulated in response to early defoliation-induced carbohydrate limitation in Mandarin fruits

The regulation of sugar and organic acid metabolism during fruit development has a major effect on high-quality fruit production. The reduction of leaf area is a common feature in plant growth, induced by abiotic and biotic stresses and disturbing source/sink ratio, thus impacting fruit quality. Here, we induced carbohydrate limitation by partial leaf defoliation at the beginning of the second stage of mandarin development (before the citrate peak). Resulting changes were monitored in the short-term (48 h and 1 week) and long-term (7 weeks) after the defoliation. Short-term response to early defoliation implied metabolic settings to re-feed TCA for sustaining respiration rate. These features involved (i) vacuolar sucrose degradation (high acid invertase activity and mRNA expression level) and enhanced glycolytic flux (high ATP-phosphofructokinase activity), (ii) malic and citric acid utilization (increased phosphoenolpyruvate kinase and NADP-Isocitrate dehydrogenase) associated with vacuolar citric acid release (high mRNA expression of the transporter CsCit1) and (iii) stimulation of GABA shunt pathway (low GABA content and increased mRNA expression of succinate semialdehyde dehydrogenase). A steady-state proline level was found in ED fruits although an increase in P5CS mRNA expression level. These results contribute to a better knowledge of the molecular basis of the relationship between defoliation and sugar and organic acid metabolism in mandarin fruit.

Metabolic engineering to improve production of 3-hydroxypropionic acid from corn-stover hydrolysate in Aspergillus species

BackgroundFuels and chemicals derived from non-fossil sources are needed to lessen human impacts on the environment while providing a healthy and growing economy. 3-hydroxypropionic acid (3-HP) is an important chemical building block that can be used for many products. Biosynthesis of 3-HP is possible; however, low production is typically observed in those natural systems. Biosynthetic pathways have been designed to produce 3-HP from a variety of feedstocks in different microorganisms.ResultsIn this study, the 3-HP β-alanine pathway consisting of aspartate decarboxylase, β-alanine-pyruvate aminotransferase, and 3-hydroxypropionate dehydrogenase from selected microorganisms were codon optimized for Aspergillus species and placed under the control of constitutive promoters. The pathway was introduced into Aspergillus pseudoterreus and subsequently into Aspergillus niger, and 3-HP production was assessed in both hosts. A. niger produced higher initial 3-HP yields and fewer co-product contaminants and was selected as a suitable host for further engineering. Proteomic and metabolomic analysis of both Aspergillus species during 3-HP production identified genetic targets for improvement of flux toward 3-HP including pyruvate carboxylase, aspartate aminotransferase, malonate semialdehyde dehydrogenase, succinate semialdehyde dehydrogenase, oxaloacetate hydrolase, and a 3-HP transporter. Overexpression of pyruvate carboxylase improved yield in shake-flasks from 0.09 to 0.12 C-mol 3-HP C-mol−1 glucose in the base strain expressing 12 copies of the β-alanine pathway. Deletion or overexpression of individual target genes in the pyruvate carboxylase overexpression strain improved yield to 0.22 C-mol 3-HP C-mol−1 glucose after deletion of the major malonate semialdehyde dehydrogenase. Further incorporation of additional β-alanine pathway genes and optimization of culture conditions (sugars, temperature, nitrogen, phosphate, trace elements) for 3-HP production from deacetylated and mechanically refined corn stover hydrolysate improved yield to 0.48 C-mol 3-HP C-mol−1 sugars and resulted in a final titer of 36.0 g/L 3-HP.ConclusionsThe results of this study establish A. niger as a host for 3-HP production from a lignocellulosic feedstock in acidic conditions and demonstrates that 3-HP titer and yield can be improved by a broad metabolic engineering strategy involving identification and modification of genes participated in the synthesis of 3-HP and its precursors, degradation of intermediates, and transport of 3-HP across the plasma membrane.