Natamycin is a safe and efficient antimycotics which is widely used in food and medicine industry. The polyene macrolide compound, produced by several bacterial species of the genus Streptomyces, is synthesized by type Ⅰ polyketide synthases using acetyl-CoA, malonyl-CoA, and methylmalonyl-CoA as substrates. In this study, four pathways potentially responsible for the supply of the three precursors were evaluated to identify the effective precursor supply pathway which can support the overproduction of natamycin in Streptomyces gilvosporeus, a natamycin-producing wild-type strain. The results showed that over-expressing acetyl-CoA synthetase and methylmalonyl-CoA mutase increased the yield of natamycin by 44.19% and 20.51%, respectively, compared with the wild type strain under shake flask fermentation. Moreover, the yield of natamycin was increased by 66.29% compared with the wild-type strain by co-overexpression of acetyl-CoA synthetase and methylmalonyl-CoA mutase. The above findings will facilitate natamycin strain improvement as well as development of strains for producing other polyketide compounds.
Natamycin/metabolism* ; Methylmalonyl-CoA Mutase/metabolism* ; Acetyl Coenzyme A/metabolism* ; Streptomyces/genetics* ; Polyketide Synthases/metabolism*

Child ; Humans ; Multiple Acyl Coenzyme A Dehydrogenase Deficiency/genetics* ; Mutation

Raspberry ketones have important therapeutic properties such as anti-influenza and prevention of diabetes. In order to obtain raspberry ketone from Chlamydomonas reinhardtii, two enzymes catalyzing the last two steps of raspberry ketone synthesis, i.e. 4-coumaryl-CoA ligase (4CL) and polyketide synthase (PKS1), were fused using a glycine-serine-glycine (GSG) tripeptide linker to construct an expression vector pChla-4CL-PKS1. The fusion gene 4CL-PKS1 driven by a PSAD promoter was transformed into a wild-type (CC125) and a cell wall-deficient C. reinhardtii (CC425) by electroporation. The results showed the recombinant C. reinhardtii strain CC125 and CC425 with 4CL-PKS1 produced raspberry ketone at a level of 6.7 μg/g (fresh weight) and 5.9 μg/g (fresh weight), respectively, both were higher than that of the native raspberry ketone producing plants (2-4 μg/g).
Acyl Coenzyme A ; Butanones ; Chlamydomonas reinhardtii/genetics* ; Ligases ; Polyketide Synthases

BACKGROUND: Late-onset multiple acyl-coA dehydrogenase deficiency (MADD) is an autosomal recessive inherited metabolic disorder. It is still unclear about the muscle magnetic resonance image (MRI) pattern of the distal lower limb pre- and post-treatment in patients with late-onset MADD. This study described the clinical and genetic findings in a cohort of patients with late-onset MADD, and aimed to characterize the MRI pattern of the lower limbs. METHODS: Clinical data were retrospectively collected from clinic centers of Peking University People's Hospital between February 2014 and February 2018. Muscle biopsy, blood acylcarnitines, and urine organic acids profiles, and genetic analysis were conducted to establish the diagnosis of MADD in 25 patients. Muscle MRI of the thigh and leg were performed in all patients before treatment. Eight patients received MRI re-examinations after treatment. RESULTS: All patients presented with muscle weakness or exercise intolerance associated with variants in the electron transfer flavoprotein dehydrogenase gene. Muscle MRI showed a sign of both edema-like change and fat infiltration selectively involving in the soleus (SO) but sparing of the gastrocnemius (GA) in the leg. Similar sign of selective involvement of the biceps femoris longus (BFL) but sparing of the semitendinosus (ST) was observed in the thigh. The sensitivity and specificity of the combination of either "SO+/GA-" sign or "BFL+/ST-" sign for the diagnosis of late-onset MADD were 80.0% and 83.5%, respectively. Logistic regression model supported the findings. The edema-like change in the SO and BFL muscles were quickly recovered at 1 month after treatment, and the clinical symptom was also relieved. CONCLUSIONS This study expands the clinical and genetic spectrums of late-onset MADD. Muscle MRI shows a distinct pattern in the lower limb of patients with late-onset MADD. The dynamic change of edema-like change in the affected muscles might be a potential biomarker of treatment response.
Adolescent ; Adult ; Biopsy ; methods ; Carnitine ; analogs & derivatives ; blood ; Electron-Transferring Flavoproteins ; genetics ; Female ; Hamstring Muscles ; diagnostic imaging ; metabolism ; pathology ; Humans ; Iron-Sulfur Proteins ; genetics ; Magnetic Resonance Imaging ; methods ; Male ; Middle Aged ; Multiple Acyl Coenzyme A Dehydrogenase Deficiency ; diagnostic imaging ; genetics ; pathology ; Muscle, Skeletal ; diagnostic imaging ; metabolism ; pathology ; Oxidoreductases Acting on CH-NH Group Donors ; genetics ; Retrospective Studies ; Young Adult

To study the effect of nicorandil pretreatment on ketone body metabolism and Acetyl-CoA acetyltransferase (ACAT1) activity in hypoxia/reoxygenation (H/R)-induced cardiomyocytes. In our study, we applied H9c2 cardiomyocytes cell line to evaluate the cardioprotective effects of nicorandil. We detected mitochondrial viability, cellular apoptosis, reactive oxygen species (ROS) production and calcium overloading in H9c2 cells that exposed to H/R-induced cytotoxicity. Then we evaluated whether nicorandil possibly regulated ketone body, mainly β-hydroxybutyrate (BHB) and acetoacetate (ACAC), metabolism by regulating ACAT1 and Succinyl-CoA:3-keto-acid coenzyme A transferase 1 (OXCT1) protein and gene expressions. Nicorandil protected H9c2 cardiomyocytes against H/R-induced cytotoxicity dose-dependently by mitochondria-mediated anti-apoptosis pathway. Nicorandil significantly decreased cellular apoptotic rate and enhanced the ratio of Bcl-2/Bax expressions. Further, nicorandil decreased the production of ROS and alleviated calcium overloading in H/R-induced H9c2 cells. In crucial, nicorandil upregulated ACAT1 and OXCT1 protein expressions and either of their gene expressions, contributing to increased production of cellular BHB and ACAC. Nicorandil alleviated cardiomyocytes H/R-induced cytotoxicity through upregulating ACAT1/OXCT1 activity and ketone body metabolism, which might be a potential mechanism for emerging study of nicorandil and other K(ATP) channel openers.
Acetyl-CoA C-Acetyltransferase ; Apoptosis ; Calcium ; Cell Line ; Coenzyme A ; Gene Expression ; Metabolism* ; Myocytes, Cardiac ; Nicorandil* ; Reactive Oxygen Species ; Transferases

This article reports the results of tandem mass spectrometry and the mutation features of the ETFDH gene for an infant with multiple acyl-CoA dehydrogenase deficiency. The results of tandem mass spectrometry showed that C14 : 1, C8, C6, C10, and C12 increased. Exon sequencing was performed on this infant and his parents and revealed double heterozygous mutations in the ETFDH gene of the infant: c.992A>T and c.1450T>C. The former was inherited from his mother, and the latter was inherited from his father. c.1450T>C was shown to be the pathogenic mutation in the HGMD database. PolyPhen2, SIFT, and PROVEAN all predicted that the novel mutation c.992A>T might be pathogenic, and the mutant amino acids were highly conserved across various species. The findings expand the mutation spectrum of the ETFDH gene, and provide molecular evidence for the etiological diagnosis of the patient with multiple acyl-CoA dehydrogenase deficiency as well as for the genetic counseling and prenatal diagnosis in the family.
Base Sequence ; Electron-Transferring Flavoproteins ; genetics ; Exons ; Humans ; Infant, Newborn ; Iron-Sulfur Proteins ; genetics ; Male ; Multiple Acyl Coenzyme A Dehydrogenase Deficiency ; enzymology ; genetics ; Mutation ; Oxidoreductases Acting on CH-NH Group Donors ; genetics

No abstract available.
Multiple Acyl Coenzyme A Dehydrogenase Deficiency*

Mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase deficiency (HMCSD) is caused by HMGCS2 gene mutation. This paper reports the clinical and genetic features of an infant with this disease. The 8-month-old female infant was admitted to the hospital with diarrhea for 1 week and fever and convulsion for 1 day. The child presented with seizures, acidosis, hypoglycemia, abnormal liver function, myocardial injury and coagulation dysfunction. The new homozygous mutation c.1502G>A(p.R501Q) in the HMGCS2 gene was found in the infant by genetic testing. The mutant gene was found to be harmful by bioinformatics software analysis. Urine organic acid analysis indicated that 4-hydroxy-6-methyl-2-pyranone was significantly increased, which was consistent with the results of genetic testing. The infant was definitely diagnosed with HMCSD.
Acyl Coenzyme A ; Female ; Humans ; Hypoglycemia ; Infant ; Mitochondria ; Mutation

A boy aged 11 years was admitted due to intermittent weakness and difficulty in walking for 6 years, and hepatomegaly, glycopenia and unconsciousness for 4 years. The laboratory examinations showed severe metabolic acidosis, hypoglycemia, and abnormal liver function. CT scan showed marked liver enlargement with fat density shadow. The boy was given fluid infusion, correction of acidosis, intravenous injection of glucose, L-carnitine, compound vitamin B, and coenzyme Q10, but he was in a persistent coma and it was difficult to correct refractory metabolic acidosis and hypoglycemia. The boy died. Blood and urinary organic acid screening and gene detection confirmed that the boy had late-onset glutaric aciduria type II (GAIIc) caused by electron-transferring-flavoprotein dehydrogenase (ETFDH) gene defect. GAIIc is an inherited metabolic disease with a low incidence, resulting in a high misdiagnosis rate. GAIIc should be considered for children with recurrent weakness or reduced activity endurance, hypoglycemia, and marked liver enlargement with abnormal liver function. Urinary organic acid analysis and blood tandem mass spectrometry can help with the early diagnosis of GAIIc, and ETFDH gene analysis helps to make a confirmed diagnosis.
Child ; Hepatomegaly ; etiology ; Humans ; Hypoglycemia ; etiology ; Male ; Multiple Acyl Coenzyme A Dehydrogenase Deficiency ; diagnosis ; Muscle Weakness ; etiology

OBJECTIVETo investigate the clinical and genetic features of two families with late-onset glutaric aciduria type II caused by ETFDH mutations.

METHODSTarget gene sequence capture and next generation sequencing were used for sequencing of suspected patients and their family members. The patients' clinical features were retrospectively analyzed and literature review was performed.

RESULTSThe probands of the two families had a clinical onset at the ages of 10 years and 5.5 years respectively, with the clinical manifestations of muscle weakness and muscle pain. Laboratory examinations revealed significant increases in the serum levels of creatine kinase, creatine kinase-MB, and lactate dehydrogenase. Tandem mass spectrometry showed increases in various types of acylcarnitines. The analysis of urine organic acids showed an increase in glutaric acid. Electromyography showed myogenic damage in both patients. Gene detection showed two novel mutations in the ETFDH gene (c.1331T>C from the mother and c.824C>T from the father) in patient 1, and the patient's younger brother carried the c.1331T>C mutation but had a normal phenotype. In patient 2, there was a novel mutation (c.177insT from the father) and a known mutation (c.1474T>C from the mother) in the ETFDH gene. Several family members carried such mutations. Both patients were diagnosed with glutaric aciduria type II. Their symptoms were improved after high-dose vitamin B2 treatment.

CONCLUSIONSFor patients with unexplained muscle weakness and pain, serum creatine kinase, acylcarnitines, and urinary organic acids should be measured, and the possibility of glutaric aciduria type II should be considered. Genetic detection is helpful to make a confirmed diagnosis.

Child ; Computational Biology ; Electron-Transferring Flavoproteins ; genetics ; Female ; Humans ; Iron-Sulfur Proteins ; genetics ; Male ; Multiple Acyl Coenzyme A Dehydrogenase Deficiency ; drug therapy ; genetics ; Mutation ; Oxidoreductases Acting on CH-NH Group Donors ; genetics