OBJECTIVETo identify pathogenic mutations in a Chinese pedigree affected with methylmalonic academia for genetic counseling and prenatal diagnosis.

METHODSMolecular analysis of the MUT, MMACHC, MMAA and MMAB genes was performed for the proband with methylmalonic academia by Ion Torrent semiconductor sequencing. Candidate mutations were validated by Sanger sequencing. The couple was offered prenatal diagnosis via analyzing of the fetal DNA through amniocentesis.

RESULTSThe proband was found to be compound heterozygous for c.609G>A (p.Trp203X) and c.658-660del AAG (p.Lys220del) mutations, which were inherited respectively from each of his parents. Prenatal diagnosis showed that the fetus has inherited two wild-type parental alleles.

CONCLUSIONThe targeted Ion Torrent PGM sequencing has detected pathogenic mutations in the Chinese pedigree affected with methylmalonic academia, which has provided molecular evidence for clinical diagnosis, genetic counseling and prenatal diagnosis for the family.

Adult ; Alkyl and Aryl Transferases ; genetics ; Amino Acid Metabolism, Inborn Errors ; embryology ; genetics ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; Carrier Proteins ; genetics ; China ; Female ; High-Throughput Nucleotide Sequencing ; instrumentation ; methods ; Humans ; Infant ; Male ; Methylmalonyl-CoA Mutase ; genetics ; Mitochondrial Membrane Transport Proteins ; genetics ; Molecular Sequence Data ; Mutation ; Pedigree ; Pregnancy ; Prenatal Diagnosis ; instrumentation ; methods

Coenzyme Q10 (CoQ10) is a lipophilic antioxidant that improves human immunity, delays senility and enhances the vitality of the human body and has wide applications in pharmaceutical and cosmetic industries. Microbial fermentation is a sustainable way to produce CoQ10, and attracts increased interest. In this work, the native CoQ8 synthetic pathway of Escherichia coli was replaced by the CoQ10 synthetic pathway through integrating decaprenyl diphosphate synthase gene (dps) from Rhodobacter sphaeroides into chromosome of E. coli ATCC 8739, followed by deletion of the native octaprenyl diphosphate synthase gene (ispB). The resulting strain GD-14 produced 0.68 mg/L CoQ10 with a yield of 0.54 mg/g DCW. Modulation of dxs and idi genes of the MEP pathway and ubiCA genes in combination led to 2.46-fold increase of CoQ10 production (from 0.54 to 1.87 mg/g DCW). Recruiting glucose facilitator protein of Zymomonas mobilis to replace the native phosphoenolpyruvate: carbohydrate phosphotransferase systems (PTS) further led to a 16% increase of CoQ10 yield. Finally, fed-batch fermentation of the best strain GD-51 was performed, which produced 433 mg/L CoQ10 with a yield of 11.7 mg/g DCW. To the best of our knowledge, this was the highest CoQ10 titer and yield obtained for engineered E. coli.
Alkyl and Aryl Transferases ; genetics ; Bacterial Proteins ; genetics ; Batch Cell Culture Techniques ; Escherichia coli ; genetics ; metabolism ; Fermentation ; Gene Deletion ; Industrial Microbiology ; Metabolic Engineering ; Rhodobacter sphaeroides ; enzymology ; genetics ; Ubiquinone ; analogs & derivatives ; biosynthesis ; Zymomonas ; genetics

cblB defect is a rare type of methylmalonic aciduria. In this study, a Chinese boy was diagnosed with methylmalonic aciduria cblB type and a novel mutation in the MMAB gene. The clinical presentations, blood acylcarnitines profiles, urine organic acids and genetic features of the patient were reported. The boy presented with fever, feeding difficulty and lethargy at the age of 2 months. Seven days later, he had coma, cold limb, thrombocytopenia, metabolic acidosis and liver damage. His blood propionylcarnitine and urinary methylmalonic acid levels increased significantly, but the plasma total homocysteine level was in the normal range, which supported the diagnosis of isolated methylmalonic aciduria. Gene analysis was performed by direct sequencing. No mutation in the MUT gene was found. However, a reported mutation c.577G>A (p.E193K) and a novel mutation c.562G>A (p.V188M) in the MMAB gene were identified, which confirmed the diagnosis of methylmalonic aciduria cblB type. Progressive clinical and biochemical improvement has been observed after hydroxylcobalamin injection, protein-restricted diet with the supplements of special formula and L-carnitine. He is currently 3 years and 11 months old and has a normal development condition. The phenotypes of the patients with cblB defect are nonspecific. Metabolic analysis and MMAB gene analysis are keys for the diagnosis of the disorder.
Alkyl and Aryl Transferases ; genetics ; Amino Acid Metabolism, Inborn Errors ; genetics ; Humans ; Infant ; Male ; Mutation

A sesquiterpene synthase (AsSS4) full-length open reading frame (ORF) cDNA was cloned from wounded stems of Aquilaria sinensis by RT-PCR method. The result showed that the ORF of AsSS4 was 1,698 bp encoding 565 amino acids. Prokaryotic expression vector pET28a-AsSS4 was constructed and transformed into E. coli BL21 (DE3) pLysS. Recombinant AsSS4 protein was obtained after induction by IPTG and SDS-PAGE analysis with a MW of 64 kD. Enzymatic reactions using farnesyl pyrophosphate showed that recombinant AsSS4 protein purified by Ni-agarose gel yielded five sesquiterpene compounds, cyclohexane, 1-ethenyl-1-methyl-2, 4-bis(1-methylethenyl)-, β-elemene, α-guaiene, α-caryophyllene and δ-guaiene. This paper reported the first cloning and functional characterization of AsSS4 gene from A. sinensis, which will establish a foundation for future studies on the molecular mechanisms of wound-induce agarwood formation in A. sinensis
Alkyl and Aryl Transferases ; biosynthesis ; genetics ; Azulenes ; Cloning, Molecular ; DNA, Complementary ; Escherichia coli ; Open Reading Frames ; Polyisoprenyl Phosphates ; Recombinant Proteins ; biosynthesis ; Sesquiterpenes ; metabolism ; Sesquiterpenes, Guaiane ; Thymelaeaceae ; enzymology ; genetics

Alkyl and Aryl Transferases ; genetics ; Child ; DNA, Mitochondrial ; genetics ; Fibroblasts ; metabolism ; Glomerulosclerosis, Focal Segmental ; diagnosis ; drug therapy ; genetics ; Humans ; Kidney Diseases ; diagnosis ; drug therapy ; genetics ; Mitochondrial Diseases ; diagnosis ; drug therapy ; genetics ; Mutation ; Nephrotic Syndrome ; diagnosis ; drug therapy ; genetics ; Protein Kinases ; genetics ; Ubiquinone ; analogs & derivatives ; biosynthesis ; deficiency ; therapeutic use

Sclareol is a member of labdane type diterpenes mostly used as fragrance ingredient. To enable microbial production of sclareol, synthetic pathways were constructed by incorporating labdenediol diphosphate synthase (LPPS) and terpene synthase (TPS) of the plant Salvia sclarea into Saccharomyces cerevisiae. It was found that sclareol production could be benefited by overexpression of key enzyme for precursor biosynthesis, construction of fusion protein for substrate channeling, and removal of signal peptides from LPPS and TPS. Under optimal shake flask culture conditions, strain S6 produced 8.96 mg/L sclareol. These results provided useful information for development of heterologous hosts for production of terpenoids.
Alkyl and Aryl Transferases ; biosynthesis ; genetics ; Diterpenes ; metabolism ; Metabolic Engineering ; methods ; Metabolic Networks and Pathways ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Saccharomyces cerevisiae ; genetics ; metabolism ; Salvia ; chemistry ; enzymology ; genetics

OBJECTIVETo try to find the ways to enhance the expression of ADS gene encoding amorpha-4,11-diene synthase, a key enzyme in artemisinin biosynthesis pathway catalyzing the formation of amorpha-4,11-diene from farnesyl diphosphate, and accelerate the artemisinin synthesis, the promoter of ADS was isolated and characterized.

METHOD5' untranslated regions of ADS were isolated from Artemisia annua with PCR. For functional characterization, the isolated fragment was fused with GUS reporter gene and introduced into Nicotiana tabacum by Agrobacterium-mediated transformation. The GUS expression regulated by 5' untranslated regions of ADS in transgenic N. tabacum under the normal or stressed conditions were detected by histochemical staining and quantitative spectrophotometry assay.

RESULTThe 2 448 bp DNA fragment upstream of ADS coding sequence was isolated from A. annua and introduced into N. tabacum. Histochemical staining showed that the isolated fragment conferred stable GUS expression in transgenic plants. The quantitative results showed that the GUS activity in transgenic tobacco plants treated by low-temperature (4 degrees C) and ultraviolet irradiation were 1. 6 and 2.2 folds higher than that in the controls.

CONCLUSIONIt was suggested that the isolated fragment had promoter activity and maybe responsive to adverse environmental stresses.

5' Untranslated Regions ; genetics ; Alkyl and Aryl Transferases ; genetics ; metabolism ; Artemisia annua ; enzymology ; genetics ; Gene Expression Regulation, Plant ; Genetic Vectors ; genetics ; Molecular Sequence Data ; Promoter Regions, Genetic ; genetics

Diterpenes, an important class of natural compounds, are widely distributed in nature. As the valuable diterpenoids continue to be found, diterpene synthase in the course of diterpene synthesis get as much attention as possible. The multiformity of end-product-diterpenoids were also due to the diversity of diterpene synthase. This paper focuses on the advances in recent biosynthesis pathway of diterpene and types, cloning, catalytic mechanism, synthetic biology application.
Alkyl and Aryl Transferases ; metabolism ; Biosynthetic Pathways ; Diterpenes ; metabolism ; Isomerases ; metabolism ; Phosphorus-Oxygen Lyases ; metabolism ; Plant Proteins ; metabolism

Artemisinin-based combination therapies (ACTs) are recommended to be the most effective therapies for the first-line treatment of uncomplicated falciparum malaria. However, artemisinin is often in short supply and unaffordable to most malaria patients, which limits the wide use of ACTs. Production of amorpha-4,11-diene, an artemisinin precursor, was investigated by engineering a heterologous isoprenoid biosynthetic pathway in Escherichia coli. The production of amorpha-4,11-diene was achieved by expression of a synthetic amorpha-4,11-diene synthase gene in Escherichia coli DHGT7 and further improved by about 13.3 fold through introducing the mevalonate pathway from Enterococcus faecalis. After eliminating three pathway bottlenecks including amorpha-4,11-diene synthase, HMG-CoA reducase and mevalonate kinase by optimizing the metabolic flux, the yield of amorpha-4,11-diene was increased by nearly 7.2 fold and reached at 235 mg/L in shaking flask culture. In conclusion, an engineered Escherichia coli was constructed for high-level production of amorpha-4,11-diene.
Alkyl and Aryl Transferases ; genetics ; Antimalarials ; metabolism ; Artemisinins ; metabolism ; Enterococcus faecalis ; genetics ; Escherichia coli ; genetics ; metabolism ; Metabolic Engineering ; methods ; Phosphotransferases (Alcohol Group Acceptor) ; metabolism ; Sesquiterpenes ; metabolism ; Transformation, Bacterial

Blakeslea trispora CarRA has both lycopene cyclase and phytoene synthase activity. In order to analyze the double functional activity of CarRA proteins and to detect the active sites of lycopene cyclase, we constructed two detection systems in Escherichia coli by color complementary. Through PCR-driven overlap extension we got carRA gene cDNA, then constructed prokaryotes expression vector pET28a-carRA. pET28a-carRA with plasmid pAC-LYC carrying crtl/crtB/crtE gene clusters were co-transformed to BL21(DE3) to validate lycopene cyclase activity. We constructed the plasmid pAC-LYC delta (crtB) carrying crtl/crtE gene clusters, then co-transtormed them with pET28a-carRA to BL21(DE3) to validate phytoene synthase activity. Based on color complementary, and HPLC analysis of metabolites, we confirmed that the CarRA protein activity detection system was reliable. Our study provides a screening model for specific mutation of lycopene cyclase without affecting phytoene synthase activity.
Alkyl and Aryl Transferases ; genetics ; metabolism ; Carotenoids ; biosynthesis ; Cloning, Molecular ; DNA, Complementary ; genetics ; Escherichia coli ; genetics ; metabolism ; Fungal Proteins ; genetics ; metabolism ; Genetic Vectors ; genetics ; Geranylgeranyl-Diphosphate Geranylgeranyltransferase ; Intramolecular Lyases ; genetics ; metabolism ; Mucorales ; enzymology ; genetics ; Mutation ; Polymerase Chain Reaction