14 research outputs found

    Optimization of Îł-PGA biosynthesis supported by synthetic biology and metabolic engineering strategies

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    Poly-Îł-glutamate (Îł-PGA) is a natural polymer composed by glutamic acid residues, synthesized by the pgs operon of Bacillus subtilis. Îł-PGA has a wide range of applications as food, cosmetic and pharmaceutical additive. However, to increase its industrial attractiveness, it is necessary to cut production costs utilizing cost-competitive feedstocks for fermentation. A low-cost by-product that can be used as feedstock is raw glycerol, that accounts for 10% (w/w) of the total biodiesel production. To achieve cost-competitive Îł-PGA production from glycerol a multifaceted approach has been set up that includes: 1) improvement of pgs expression; 2) accumulation of Îł-PGA precursors by metabolic engineering; 3) enhancement of glycerol metabolism. 1) The strength of the pgs operon regulatory elements has been analysed both by a synthetic biology approach, exploiting the well-characterized expression operating unit (EOU) inserted in amyE, and by a classical in-locus transcriptional fusion. Results from the two settings will be compared. These data will be then used to finely tune pgs expression and optimize Îł-PGA yield. To this end, an inducible pgs operon has been constructed. 2) A genome-scale metabolic model was used to identify suitable targets for enhancing central carbon pathway flux toward Îł-PGA synthesis. The first two B. subtilis strains, engineered following this analysis, showed enhanced polymer production. Other target genes are currently under investigation. 3) B. subtilis tolerance to raw glycerol obtained from a biodiesel plant (from both vegetable and animal origin) was verified. Further investigations are underway to improve glycerol uptake and consumption

    Integration of enzymatic data in <i>Bacillus subtilis</i> genome-scale metabolic model improves phenotype predictions and enables in silico design of poly-Îł-glutamic acid production strains

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    Abstract Background Genome-scale metabolic models (GEMs) allow predicting metabolic phenotypes from limited data on uptake and secretion fluxes by defining the space of all the feasible solutions and excluding physio-chemically and biologically unfeasible behaviors. The integration of additional biological information in genome-scale models, e.g., transcriptomic or proteomic profiles, has the potential to improve phenotype prediction accuracy. This is particularly important for metabolic engineering applications where more accurate model predictions can translate to more reliable model-based strain design. Results Here we present a GEM with Enzymatic Constraints using Kinetic and Omics data (GECKO) model of Bacillus subtilis, which uses publicly available proteomic data and enzyme kinetic parameters for central carbon (CC) metabolic reactions to constrain the flux solution space. This model allows more accurate prediction of the flux distribution and growth rate of wild-type and single-gene/operon deletion strains compared to a standard genome-scale metabolic model. The flux prediction error decreased by 43% and 36% for wild-type and mutants respectively. The model additionally increased the number of correctly predicted essential genes in CC pathways by 2.5-fold and significantly decreased flux variability in more than 80% of the reactions with variable flux. Finally, the model was used to find new gene deletion targets to optimize the flux toward the biosynthesis of poly-γ-glutamic acid (γ-PGA) polymer in engineered B. subtilis. We implemented the single-reaction deletion targets identified by the model experimentally and showed that the new strains have a twofold higher γ-PGA concentration and production rate compared to the ancestral strain. Conclusions This work confirms that integration of enzyme constraints is a powerful tool to improve existing genome-scale models, and demonstrates the successful use of enzyme-constrained models in B. subtilis metabolic engineering. We expect that the new model can be used to guide future metabolic engineering efforts in the important industrial production host B. subtilis

    The still under-investigated role of cognitive deficits in PML diagnosis

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    Background: Despite cognitive deficits frequently represent the first clinical manifestations of Progressive Multifocal Leukoencephalopathy (PML) in Natalizumab-treated MS patients, the importance of cognitive deficits in PML diagnosis is still under-investigated. The aim of the current study is to investigate the cognitive deficits at PML diagnosis in a group of Italian patients with PML. Methods: Thirty-four PML patients were included in the study. The demographic and clinical data, the lesion load and localization, and the longitudinal clinical course was compared between patients with (n = 13) and without (n = 15) cognitive deficit upon PML suspicion (the remaining six patients were asymptomatic). Clinical presentation of cognitive symptoms was described in detail. Result: After symptoms detection, the time to diagnosis resulted to be shorter for patients presenting with cognitive than for patients with non cognitive onset (p = 0.03). Within patients with cognitive onset, six patients were presenting with language and/or reading difficulties (46.15%); five patients with memory difficulties (38.4%); three patients with apraxia (23.1%); two patients with disorientation (15.3%); two patients with neglect (15.3%); one patients with object agnosia (7.7%), one patient with perseveration (7.7%) and one patient with dementia (7.7%). Frontal lesions were less frequent (p = 0.03), whereas temporal lesions were slightly more frequent (p = 0.06) in patients with cognitive deficits. The longitudinal PML course seemed to be more severe in cognitive than in non cognitive patients (F = 2.73, p = 0.03), but differences disappeared (F = 1.24, p = 0.29) when balancing for the incidence of immune reconstitution syndrome and for other treatments for PML (steroids, plasma exchange (PLEX) and other therapies (Mefloquine, Mirtazapine, Maraviroc). Conclusion: Cognitive deficits at PML onset manifest with symptoms which are absolutely rare in MS. Their appearance in MS patients should strongly suggest PML. Clinicians should be sensitive to the importance of formal neuropsychological evaluation, with particular focus on executive function, which are not easily detected without a formal assessment

    OPTIMIZATION OF GAMMA-PGA BIOSYNTHESIS SUPPORTED BY SYNTHETIC BIOLOGY AND METABOLIC ENGINEERING STRATEGIES

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    Poly-Îł-glutamate (Îł-PGA) is a natural polymer made of glutamic acid residues, synthesized by the pgs operon of Bacillus subtilis. Îł-PGA has a wide range of applications as food, cosmetics and pharmaceutical additive. However, to increase its industrial attractiveness, it is necessary to cut production costs utilizing cost-competitive feedstocks for fermentation. Raw glycerol is a low-cost by-product of biodiesel plants (it accounts for 10% of the final product) that can be used as feedstock. To achieve cost-competitive Îł-PGA production from glycerol a multifaceted approach has been set up that includes: 1) Characterization and optimization of pgs operon regulation: the strength of the pgs operon regulatory elements has been analysed both by a synthetic biology approach, exploiting the well-characterized expression operating unit (EOU) inserted in amyE, and by a classical in-locus transcriptional fusion. Results from the two settings will be compared. These data will now be used to finely tune pgs expression through an inducible promoter to optimize Îł-PGA yield. 2) Accumulation of Îł-PGA precursors by metabolic engineering: a genome-scale metabolic model was used to identify suitable targets for enhancing central carbon pathway flux toward Îł-PGA synthesis. The first two B. subtilis strains, engineered according to this analysis, showed enhanced polymer production. Other target genes are under investigation. 3) Enhancement of glycerol metabolism: B. subtilis tolerance to raw glycerol obtained from a biodiesel plant (from both vegetable and animal origin) was verified. Further investigations are underway to improve glycerol uptake and consumption

    Lack of sterol regulatory element binding factor-1c imposes glial fatty acid utilization leading to peripheral neuropathy

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    Myelin is a membrane characterized by high lipid content to facilitate impulse propagation. Changes in myelin fatty acid (FA) composition have been associated with peripheral neuropathy, but the specific role of peripheral nerve FA synthesis in myelin formation and function is poorly understood. We have found that mice lacking sterol regulatory element-binding factor-1c (Srebf1c) have blunted peripheral nerve FA synthesis that results in development of peripheral neuropathy. Srebf1c-null mice develop Remak bundle alterations and hypermyelination of small-caliber fibers that impair nerve function. Peripheral nerves lacking Srebf1c show decreased FA synthesis and glycolytic flux, but increased FA catabolism and mitochondrial function. These metabolic alterations are the result of local accumulation of two endogenous peroxisome proliferator-activated receptor-α (Pparα) ligands, 1-palmitoyl-2-oleyl-sn-glycerol-3-phosphatidylcholine and 1-stearoyl-2-oleyl-sn-glycerol-3-phosphatidylcholine. Treatment with a Pparα antagonist rescues the neuropathy of Srebf1c-null mice. These findings reveal the importance of peripheral nerve FA synthesis to sustain myelin structure and function.These studies were supported by funding from Giovanni Armenise-Harvard Foundation Career Development Grant (N.M.), Fondazione CARIPLO 2014-0991 (N.M.), Fondazione CARIPLO 2012-0547 (R.C.M.), Italian Ministry of Health GR-2011-02346791 (M.D. and N.M.) and Research Center for the Characterization and Safe Use of Natural Compounds—“Giovanni Galli” directed by D.C. S.S. is an employee and founder of DASP s.r.l.; all other authors declare no competing financial interests

    Risk Factors and Outcomes of Thalidomide-induced Peripheral Neuropathy in a Pediatric Inflammatory Bowel Disease Cohort

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    Background: Thalidomide is an effective therapy in children with inflammatory bowel disease refractory to standard treatments, but thalidomide-induced peripheral neuropathy (TiPN) limits its long-term use. We aimed to investigate the risk factors and the outcome of TiPN in children with inflammatory bowel disease. Methods: Within a retrospective multicenter cohort study, we evaluated prevalence and evolution of TiPN. Clinical data and candidate genetic profiles of patients with and without TiPN were compared with detect predisposing factors. Results: One hundred forty-two patients were identified. TiPN was found in 72.5% of patients (38.7% clinical and instrumental alterations, 26.8% exclusive electrophysiological anomalies, and 7.0% exclusive neurological symptoms). Median TiPN-free period of treatment was 16.5 months; percentage of TiPN-free patients was 70.0% and 35.6% at 12 and 24 months of treatment, respectively. The risk of TiPN increased depending on the mean daily dose (50-99 mg/d adjusted hazard ratio 2.62; 95% confidence interval [CI], 1.31-5.21; 100-149 mg/d adjusted hazard ratio 6.16; 95% CI, 20.9-13.06; >150 mg/d adjusted hazard ratio 9.57; 95% CI, 2.6-35.2). Single nucleotide polymorphisms in ICAM1 (rs1799969) and SERPINB2 (rs6103) genes were found to be protective against TiPN (odds ratio 0.15; 95% CI, 0.03-0.82 and 0.36; 95% CI, 0.14-0.88, respectively). TiPN was the cause of drug suspension in 41.8% of patients. Clinical symptoms resolved in 89.2% of cases, whereas instrumental alteration persisted in more than half of the patients during a short follow-up. Conclusions: In children with inflammatory bowel disease, TiPN is common but mild and generally reversible. Cumulative dose seems to be the most relevant risk factor, whereas polymorphisms in genes involved in neuronal inflammation may be protective
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