Engineering the precursor supply pathway in Streptomyces gilvosporeus for overproduction of natamycin / 生物工程学报

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.

Screening and gene mutations analysis for neonates with methylmalonic aciduria / 中华围产医学杂志

Objective To investigate the characteristics of neonatal methylmalonic aciduria (MMA)regarding clinical manifestations,laboratory findings,gene mutations,treatments and prognosis.Methods Acylcamitine levels in blood samples of 207 308 neonates born from January 2016 to December 2017 in Xuzhou were detected by liquid chromatography tandem mass spectrometry and the abnormal results were further confirmed by detecting organic acids in urine samples with gas chromatography-mass spectrometry and gene sequencing analysis.Patients with isolated MMA were treated with dietary control and levocarnitine,while those complicated by homocysteinemia were treated with vitamin B12,levocarnitine,glycine betaine and calcium folinate.Clinical manifestations,laboratory findings,imaging features,genotypes,treatments and prognosis of patients with MMA were retrospectively analyzed.Paired sample t-test was applied for statistical analysis.Results MMA was eventually diagnosed in 12 patients,among which three were isolated MMA and nine were complicated by homocysteinemia.The three isolated MMA cases failed to response to vitamin B12 treatment without any symptoms on diagnosis.However,vitamin B12 was effective for the other nine patients,among which four had no clinical symptoms on diagnosis and five had manifestations such as slow response,recurrent vomiting,poor feeding,dyspnea,anemia and jaundice.Abnormal results of cranial MRI included bilateral basal ganglia damage,enlarged extracranial space,ventriculomegaly and changes in white matter.All patients underwent genetic analysis and three were found with MUT gene mutations and nine with MMACHC gene mutations.MUT gene mutations were classified into five types,including c.I106G＞A,c.1880A＞G,c.441T＞A,c.581C＞T and c.1741C＞T.Eight types of MMACHC gene mutations were identified,including c.609G＞A,c.658_660delAAG,c.482G＞A,c.1A＞G,c.567dupT,c.80A＞G,c.276+1G＞A and c.228_23 l delTGAC.Two mutations,c.276+lG＞A and c.228 23 ldelTGAC,were novel mutations.The most common mutation in MMACHC gene was c.609G＞A,followed by c.658_660delAAG and c.482G＞A.One of the isolated MMA patients died after refusing treatments and the other two showed significant decrease in serum propionylcarnitine,propionylcarnitine to acetylcarnitine ratio,serum homocysteine and methylmalonic acid and methylcitric acid in urine after treatment.Moreover,of the two patients who were alive at follow-up,one experienced normal growth and development and the other suffered from growth retardation.The ratio of propionylcamitine to acetylcarnitine and the levels of serum propionylcarnitine,serum homocysteine and methylmalonic acid and methylcitric acid in urine were significantly decreased in the nine patients with MMA complicated by homocystinuria after one month of treatment [0.88±0.35 vs 0.13±0.05,(7.12±1.90) μ mol/L vs (3.18±1.08) μ mol/L,(136.48±38.14) μ mol/L vs (34.41±17.33) μmol/L,103.51±69.62vs 5.35±2.15 and 7.95±6.88 vs 1.02±0.48,t=-6.166,-6.687,-12.941,-4.208 and-3.015,respectively,all P＜0.05].Two deaths,three asymptomatic and four psychomotor retardation patients were reported during follow-up.Conclusions Newborn screening with liquid chromatography tandem mass spectrometry is important for early diagnosis of MMA.MMACHC gene defects are the main causes of MMA in Xuzhou area and the predominant one is c.609G＞A mutation.Prognosis of MMA might be related to disease type,age of onset and patient's reactivity to vitamin B12.

A case of cerebral hemorrhage in patients with methylmalonic aciduria with hyperhomocystinuria / 中华神经科杂志

A case of late-onset methylmalonic aciduria combined with hyperhomocysteinemia (cblC type) is reported. The main manifestations were the reduction of intelligence,the instability of walking,and the inability to take care of oneself,with secondary cerebral hemorrhage. The effect of treatment was good. MMACHC gene mutation detection showed exon1 deletion, indicating that delExon1 is one of the causes of late onset methylmalonic aciduria, cblC type.

MUT genetic analysis in isolated methylmalonic acidemia pedigrees and its application in prenatal diagnosis / 中华围产医学杂志

Objective To analyze the mutation of MUT with Sanger sequencing technology to explore the feasibility of its application in prenatal diagnosis.Methods MUT sequencing was performed in 24 pedigrees who had history of isolated methylmalonic acidemia (MMA) babies and came to the First Affiliated Hospital of Zhengzhou University and Newborn Screening Center of Maternal and Child Health Hospital of He'nan Province between October 2012 and June 2015 for genetic counseling.Meanwhile,another 100 cases of normal controls also had their MUT gene sequence analyzed.After confirming the genotype of each pedigree,we collected the villi of nine high-risk fetuses in nine pedigrees whose parents were prepared for prenatal diagnosis.Results Totally,25 kinds of MUT gene mutations were identified among the 24 isolated MMA pedigrees,in which 11 were novel mutations including one nonsense mutation [c.616C＞T(p.Q206X)],six missense mutations [c.613G＞A(p.E205K),c.894T＞G(p.1298N),c.1009T＞C(p.F337L),c.1154G＞T(p.L385W),c.1663G＞A(p.A555T) and c.1675G＞A(p.R559G) and four frame shift mutations [c.626-627insC(p.P209Pfs*2),c.755-756insA(p.H252Qfs*6),c.756-757insA(p.M253Nfs*5) and c.1581-1582insA(p.A528Ifs*4)].None of the above mutations was detected in the controls.Finally,among the nine pedigrees for prenatal diagnosis,two were determined to have normal MUT gene,four were found to be heterozygous mutation carriers of MUT gene and three were confirmed as complex heterozygous or homozygous mutation carriers.Families of fetus who had normal MUT gene or fetuses who were carriers chose to continue the pregnancy,while those who had heterozygous mutation of MUT gene chose termination.The results of follow-up of newborns were consistent with that of prenatal diagnosis.Conclusions We found two novel mutations in MUT gene that might lead to isolated MMA.And Sanger sequencing technology for MUT gene sequencing analysis might effectively avoid the birth of isolated MMA children.

A Novel MUT Gene Mutation Detected in a Female Infant with Methylmalonic Acidemia

Methylmalonic acidemia (MMA) is an autosomal recessive metabolic disease. MMA results from a deficiency of L-methylmalonyl-CoA mutase (encoded by MUT), its cofactor 5-deoxyadenosylcobalamin (MMAA, MMAB, and MMADHC), or a deficiency of methylmalonyl CoA-epimerase (MCEE). We report the case of a 5-day-old infant with MMA in which a missense and a novel nonsense mutation in MUT were present. Direct sequencing analysis of MUT revealed a heterozygous c.1106G>A (p.Arg369His) mutation in exon 6 and a heterozygous c.362_368dupAGTTCTA (p.Tyr123*) mutation in exon 2; the latter results in a premature stop codon.

Novel mutation of methylmalonyl-CoA mutase gene in a Thai infant with methylmalonic acidemia (mut0).

Isolated methylmalonic acidemia is found in patients with mutations in the MUT gene causing partial methylmalonyl CoA mutase deficiency, mut-, or complete methylmalonyl CoA mutase deficiency, mut0. Most mut0 patients have an earlier and more severe presentation than the other groups such as mut- and cbl defect. We report a 6-month-old Thai male presenting with wide-anion gap metabolic acidosis after acute lower respiratory infection. Urine organic acids analysis demonstrated excretions of methylmalonic acid and methylcitrate, consistent with methylmalonic acidemia. He was then started on low protein diet with an appropriate metabolic formula, L-carnitine (100 mg/kg/day), and oral vitamin B12 (1 mg/day). He had only one single metabolic episode at 2 years of age. At present, he is doing well with normal growth and development. His methylmalonyl-CoA mutase activity was undetectable compatible with mut0. He was found to be homozygous for a novel IVS11-2A>G mutation causing two aberrantly spliced transcripts. The identified mutation and enzyme activity of this patient should cause severe phenotype, although, our patient has milder clinical manifestations. Therefore we hypothesize that there are other factors that may determine the clinical phenotype of mutase deficiency in the present case.