Genomic mutational analysis of the impact of the classical strain improvement program on β-lactam producing Penicillium chrysogenum

BACKGROUND: Penicillium chrysogenum is a filamentous fungus that is employed as an industrial producer of β-lactams. The high β-lactam titers of current strains is the result of a classical strain improvement program (CSI) starting with a wild-type like strain more than six decades ago. This involved extensive mutagenesis and strain selection for improved β-lactam titers and growth characteristics. However, the impact of the CSI on the secondary metabolism in general remains unknown. RESULTS: To examine the impact of CSI on secondary metabolism, a comparative genomic analysis of β-lactam producing strains was carried out by genome sequencing of three P. chrysogenum strains that are part of a lineage of the CSI, i.e., strains NRRL1951, Wisconsin 54-1255, DS17690, and the derived penicillin biosynthesis cluster free strain DS68530. CSI has resulted in a wide spread of mutations, that statistically did not result in an over- or underrepresentation of specific gene classes. However, in this set of mutations, 8 out of 31 secondary metabolite genes (20 polyketide synthases and 11 non-ribosomal peptide synthetases) were targeted with a corresponding and progressive loss in the production of a range of secondary metabolites unrelated to β-lactam production. Additionally, key Velvet complex proteins (LeaA and VelA) involved in global regulation of secondary metabolism have been repeatedly targeted for mutagenesis during CSI. Using comparative metabolic profiling, the polyketide synthetase gene cluster was identified that is responsible for sorbicillinoid biosynthesis, a group of yellow-colored metabolites that are abundantly produced by early production strains of P. chrysogenum. CONCLUSIONS: The classical industrial strain improvement of P. chrysogenum has had a broad mutagenic impact on metabolism and has resulted in silencing of specific secondary metabolite genes with the concomitant diversion of metabolism towards the production of β-lactams

Metabolic characterization of the natural progression of chronic hepatitis B

Background: Worldwide, over 350 million people are chronically infected with the hepatitis B virus (HBV) and are at increased risk of developing progressive liver diseases. The confinement of HBV replication to the liver, which also acts as the central hub for metabolic and nutritional regulation, emphasizes the interlinked nature of host metabolism and the disease. Still, the metabolic processes operational during the distinct clinical phases of a chronic HBV infection-immune tolerant, immune active, inactive carrier, and HBeAg-negative hepatitis phases-remains unexplored. Methods: To investigate this, we conducted a targeted metabolomics approach on serum to determine the metabolic progression over the clinical phases of chronic HBV infection, using patient samples grouped based on their HBV DNA, alanine aminotransferase, and HBeAg serum levels. Results: Our data illustrate the strength of metabolomics to provide insight into the metabolic dysregulation experienced during chronic HBV. The immune tolerant phase is characterized by the speculated viral hijacking of the glycerol-3-phosphate-NADH shuttle, explaining the reduced glycerophospholipid and increased plasmalogen species, indicating a strong link to HBV replication. The persisting impairment of the choline glycerophospholipids, even during the inactive carrier phase with minimal HBV activity, alludes to possible metabolic imprinting effects. The progression of chronic HBV is associated with increased concentrations of very long chain triglycerides together with citrulline and ornithine, reflective of a dysregulated urea cycle peaking in the HBV envelope antigen-negative phase. Conclusions: The work presented here will aid in future studies to (i) validate and understand the implication of these metabolic changes using a thorough systems biology approach, (ii) monitor and predict disease severity, as well as (iii) determine the therapeutic value of the glycerol-3-phosphate-NADH shuttle

Metabolomics of cerebrospinal fluid reveals changes in the central nervous system metabolism in a rat model of multiple sclerosis

Experimental Autoimmune Encephalomyelitis (EAE) is the most commonly used animal model for Multiple Sclerosis (MScl). CSF metabolomics in an acute EAE rat model was investigated using targetted LC–MS and GC–MS. Acute EAE in Lewis rats was induced by co-injection of Myelin Basic Protein with Complete Freund’s Adjuvant. CSF samples were collected at two time points: 10 days after inoculation, which was during the onset of the disease, and 14 days after inoculation, which was during the peak of the disease. The obtained metabolite profiles from the two time points of EAE development show profound differences between onset and the peak of the disease, suggesting significant changes in CNS metabolism over the course of MBP-induced neuroinflammation. Around the onset of EAE the metabolome profile shows significant decreases in arginine, alanine and branched amino acid levels, relative to controls. At the peak of the disease, significant increases in concentrations of multiple metabolites are observed, including glutamine, O-phosphoethanolamine, branched-chain amino acids and putrescine. Observed changes in metabolite levels suggest profound changes in CNS metabolism over the course of EAE. Affected pathways include nitric oxide synthesis, altered energy metabolism, polyamine synthesis and levels of endogenous antioxidants

2-Methyl-pentanoyl-carnitine (2-MPC) : a urine biomarker for patent Ascaris lumbricoides infection

Infections with intestinal worms, such as Ascaris lumbricoides, affect hundreds of millions of people in all tropical and subtropical regions of the world. Through large-scale deworming programs, World Health Organization aims to reduce moderate-to-heavy intensity infections below 1%. Current diagnosis and monitoring of these control programs are solely based on the detection of worm eggs in stool. Here we describe how metabolome analysis was used to identify the A. lumbricoides-specific urine biomarker 2-methyl pentanoyl carnitine (2-MPC). This biomarker was found to be 85.7% accurate in determining infection and 90.5% accurate in determining a moderate-to-heavy infection. Our results also demonstrate that there is a correlation between 2-MPC levels in urine and A. lumbricoides DNA detected in stool. Furthermore, the levels of 2-MPC in urine were shown to rapidly and strongly decrease upon administration of a standard treatment (single oral dose of 400 mg albendazole). In an Ascaris suum infection model in pigs, it was found that, although 2-MPC levels were much lower compared to humans, there was a significant association between urinary 2-MPC levels and both worm counts (p=0.023) and the number of eggs per gram (epg) counts (p<0.001). This report demonstrates that urinary 2-MPC can be considered an A. lumbricoides-specific biomarker that can be used to monitor infection intensity

Enhanced performance for the analysis of prostaglandins and thromboxanes by liquid chromatography-tandem mass spectrometry using a new atmospheric pressure ionization source

Eicosanoids, including prostaglandins and thromboxanes are lipid mediators synthetized from polyunsaturated fatty acids. They play an important role in cell signaling and are often reported as inflammatory markers. LC-MS/MS is the technique of choice for the analysis of these compounds, often in combination with advanced sample preparation techniques. Here we report a head to head comparison between an electrospray ionization source (ESI) and a new atmospheric pressure ionization source (UniSpray). The performance of both interfaces was evaluated in various matrices such as human plasma, pig colon and mouse colon. The UniSpray source shows an increase in method sensitivity up to a factor 5. Equivalent to better linearity and repeatability on various matrices as well as an increase in signal intensity were observed in comparison to ESI.publisher: Elsevier articletitle: Enhanced performance for the analysis of prostaglandins and thromboxanes by liquid chromatography-tandem mass spectrometry using a new atmospheric pressure ionization source journaltitle: Journal of Chromatography A articlelink: http://dx.doi.org/10.1016/j.chroma.2016.02.055 content_type: article copyright: Copyright © 2016 Elsevier B.V. All rights reserved.status: publishe

Development and application of a UHPLC-MS/MS metabolomics based comprehensive systemic and tissue-specific screening method for inflammatory, oxidative and nitrosative stress

Oxidative stress and inflammation are underlying pathogenic mechanisms associated with the progression of several pathological conditions and immunological responses. Elucidating the role of signalling lipid classes, which include, among others, the isoprostanes, nitro fatty acids, prostanoids, sphingoid bases and lysophosphatidic acids, will create a snapshot of the cause and effect of inflammation and oxidative stress at the metabolic level. Here we describe a fast, sensitive, and targeted ultra-high-performance liquid chromatography-tandem mass spectrometry metabolomics method that allows the quantitative measurement and biological elucidation of 17 isoprostanes as well as their respective isomeric prostanoid mediators, three nitro fatty acids, four sphingoid mediators, and 24 lysophosphatidic acid species from serum as well as organ tissues, including liver, lung, heart, spleen, kidney and brain. Application of this method to paired mouse serum and tissue samples revealed tissue- and serum-specific stress and inflammatory readouts. Little correlation was found between localized (tissue) metabolite levels compared with the systemic (serum) circulation in a homeostatic model. The application of this method in future studies will enable us to explore the role of signalling lipids in the metabolic pathogenicity of stress and inflammation during health and diseas