Metabolic Engineering of Bacillus amyloliquefaciens to Efficiently Synthesize L-Ornithine From Inulin.

Bacillus amyloliquefaciens is the dominant strain used to produce γ-polyglutamic acid from inulin, a non-grain raw material. B. amyloliquefaciens has a highly efficient tricarboxylic acid cycle metabolic flux and glutamate synthesis ability. These features confer great potential for the synthesis of glutamate derivatives. However, it is challenging to efficiently convert high levels of glutamate to a particular glutamate derivative. Here, we conducted a systematic study on the biosynthesis of L-ornithine by B. amyloliquefaciens using inulin. First, the polyglutamate synthase gene pgsBCA of B. amyloliquefaciens NB was knocked out to hinder polyglutamate synthesis, resulting in the accumulation of intracellular glutamate and ATP. Second, a modular engineering strategy was applied to coordinate the degradation pathway, precursor competition pathway, and L-ornithine synthesis pathway to prompt high levels of intracellular precursor glutamate for l-ornithine synthesis. In addition, the high-efficiency L-ornithine transporter was further screened and overexpressed to reduce the feedback inhibition of L-ornithine on the synthesis pathway. Combining these strategies with further fermentation optimizations, we achieved a final L-ornithine titer of 31.3 g/L from inulin. Overall, these strategies hold great potential for strengthening microbial synthesis of high value-added products derived from glutamate.

Creatine metabolism in patients with urea cycle disorders.

The urea cycle generates arginine that is one of the major precursors for creatine biosynthesis. Here we evaluate levels of creatine and guanidinoacetate (the precursor in the synthesis of creatine) in plasma samples (ns = 207) of patients (np = 73) with different types of urea cycle disorders (ornithine transcarbamylase deficiency (ns = 22; np = 7), citrullinemia type 1 (ns = 60; np = 22), argininosuccinic aciduria (ns = 81; np = 31), arginase deficiency (ns = 44; np = 13)). The concentration of plasma guanidinoacetate positively correlated (p < 0.001, R2 = 0.64) with levels of arginine, but not with glycine in all patients with urea cycle defects, rising to levels above normal in most samples (34 out of 44) of patients with arginase deficiency. In contrast to patients with guanidinoacetate methyltransferase deficiency (a disorder of creatine synthesis characterized by elevated guanidinoacetate concentrations), creatine levels were normal (32 out of 44) or above normal (12 out of 44) in samples from patients with arginase deficiency. Creatine levels correlated significantly, but poorly (p < 0.01, R2 = 0.1) with guanidinoacetate levels and, despite being overall in the normal range in patients with all other urea cycle disorders, were occasionally below normal in some patients with argininosuccinic acid synthase and lyase deficiency. Creatine levels positively correlated with levels of methionine (p < 0.001, R2 = 0.16), the donor of the methyl group for creatine synthesis. The direct correlation of arginine levels with guanidinoacetate in patients with urea cycle disorders explains the increased concentration of guanidino compounds in arginase deficiency. Low creatine levels in some patients with other urea cycle defects might be explained by low protein intake (creatine is naturally present in meat) and relative or absolute intracellular arginine deficiency.

Hyperornithinemia-hyperammonemia-homocitrullinuria syndrome in pregnancy: Considerations for management and review of the literature.

Hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome is a rare metabolic autosomal recessive urea cycle disorder. Only about 100 patients have been reported in the literature. As the population survives into reproductive years, pregnancy management becomes a new challenge for this clinicians. To our knowledge, there are less than three patients with successful pregnancies and deliveries found in the literature with no specific consensus on management or recommendations for HHH syndrome. We reviewed the current literature regarding pregnancy outcomes, combine it with our experience managing a patient through two successful pregnancies and identify a new concern of fetal intrauterine growth restriction. From this, recommendations for pregnancy management are made, including a detailed protocol for clinicians to use for disease management at delivery and in the post-partum period.

[Clinical diagnosis and treatment of three cases with hyperornithinemia-hyperammonemia-homocitrullinuria syndrome].

Objective: To study the clinical characteristics, methods of diagnosis and treatment of hyperornithinemia-hyperammonemia- homocitrullinuria (HHH) syndrome. Method: From July 2011 to August 2016, 3 Chinese patients with HHH syndrome were enrolled in this study. The clinical course, biochemical features, brain MRI findings, and gene mutations were analyzed. Result: The three patients' age at onset of symptoms was 3 months to 7 years, and the age of diagonosis was 3 years and 10 months to 9 years and 10 months. All of them presented with intolerance to protein-rich foods from the infant period, development retardation and abnormal posture. Case 1 and 2 had moderate mental retardation. Serum ammonia 25-276 µmol/L (reference range<60 µmol/L), alanine aminotransferase (ALT) 20-139 IU/L (reference range 9-50 IU/L), ornithine 29.12-99.44 µmol/L(reference range 15-100 µmol/L), urinary orotic acid 1.49-29.75 mmol/mol Cr (reference range 0-7 mmol/mol Cr), uracil 6.09-103.97 mmol/mol Cr (reference range 0-1.5 mmol/mol Cr). The cranial MRI revealed lesions in the basal ganglia, abnormal white matter signal, progressive demyelination and cerebral atrophy. On their SLC25A15 gene, a novel homozygous missense mutation c. 416A>G (p.E139G) was identified in case 1, a known pathogenic homozygous nonsense mutation c. 535C>T was found in case 2 and 3. Liver transplantation had been performed when case 1 was 6 years old. Significant improvements were observed in dietary habit, mental and motor functions, and biochemical parameters. After the dietary intervention with the supplements of arginine, L-carnitine, case 2 was improved, spastic paraplegia of case 3 had no mitigation. Liver transplant was recommended. Conclusion: HHH syndrome has an aversion to protein-rich food, and the patients have recurrent vomiting and progressive neurological dysfunction. Clinical diagnosis of HHH syndrome is difficult and patients may present with incomplete biochemical phenotype. The genetic analysis is key for the diagnosis. Depending on their condition, individuals with HHH syndrome can be treated with a low-protein diet, drugs and liver transplantation.