An Engineered Yeast Efficiently Secreting Penicillin

This study aimed at developing an alternative host for the production of penicillin (PEN). As yet, the industrial production of this β-lactam antibiotic is confined to the filamentous fungus Penicillium chrysogenum. As such, the yeast Hansenula polymorpha, a recognized producer of pharmaceuticals, represents an attractive alternative. Introduction of the P. chrysogenum gene encoding the non-ribosomal peptide synthetase (NRPS) δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) in H. polymorpha, resulted in the production of active ACVS enzyme, when co-expressed with the Bacillus subtilis sfp gene encoding a phosphopantetheinyl transferase that activated ACVS. This represents the first example of the functional expression of a non-ribosomal peptide synthetase in yeast. Co-expression with the P. chrysogenum genes encoding the cytosolic enzyme isopenicillin N synthase as well as the two peroxisomal enzymes isopenicillin N acyl transferase (IAT) and phenylacetyl CoA ligase (PCL) resulted in production of biologically active PEN, which was efficiently secreted. The amount of secreted PEN was similar to that produced by the original P. chrysogenum NRRL1951 strain (approx. 1 mg/L). PEN production was decreased over two-fold in a yeast strain lacking peroxisomes, indicating that the peroxisomal localization of IAT and PCL is important for efficient PEN production. The breakthroughs of this work enable exploration of new yeast-based cell factories for the production of (novel) β-lactam antibiotics as well as other natural and semi-synthetic peptides (e.g. immunosuppressive and cytostatic agents), whose production involves NRPS's

Development and application of a differential method for reliable metabolome analysis in Escherichia coli

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Development of an accurate method for intracellular metabolome analysis in Escherichia coli for in vivo kinetic analysis

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Global profiling of the muscle metabolome: method optimization, validation and application to determine exercise-induced metabolic effects

Skeletal muscle represents a crucial metabolic organ in the body characterized by a tremendous metabolic plasticity and the ability to influence important metabolic events elsewhere in the body. In order to understand the metabolic implications of skeletal muscle, it is imperative to characterize the metabolites within the tissue itself. In this work we aimed at developing a suitable analytical pipeline to analyze the metabolome of muscle tissue. Methanol/chloroform/water at neutral pH was selected as the method of choice for metabolite extraction prior to analysis by chromatographic-mass spectrometry systems in five different platforms covering a relevant part of the muscle metabolome: organic acids, amines, nucleotides, coenzymes, acylcarnitines and oxylipins. This analytical pipeline was extensively validated and proved to be robust, precise, accurate and biologically sound. The capability of our analytical method to capture metabolic alterations upon challenges was finally tested using a small proof-of-concept study involving an exercise intervention. Mild but consistent metabolic patterns were observed, allowing the discrimination between non-exercised and exercised muscles. Despite the low numbers of subjects enrolled in this study (5), these results are indicative that our method is suitable to determine intervention effects in skeletal muscle tissue whenever applied to adequately powered and well characterized studies.</p

Quantification of produced β-lactam compounds in HpPen3 and HpPen4 cultures.

<p>Strains HpPen3 and HpPen4 were grown in glucose/methanol-limited chemostat cultures in the presence of 1 mM AAA and 1 mM PAA. β-lactam compounds were quantified by IP-LC–ESI-ID-MS/MS in spent medium and cell extracts. PenG could not be detected in HpPen3 cultures, but was clearly detectable in HpPen4 cultures, where it was efficiently excreted into the medium. The PenG precursor IPN was present in both HpPen3 and HpPen4 cells; however, the amount was significantly lower in the PenG producing HpPen4 cells. Concentrations are expressed as µmol/l culture. Samples were taken in triplicate. The bars represent the standard error (SE).</p

Subcellular localization of ACVS in <i>H. polymorpha</i>.

<p>Immunocytochemistry using anti-ACVS antibodies showing the presence of ACVS protein in the cytosol of strain HpPen4. Cells were fixed in 3% glutaraldehyde for 1 h on ice, dehydrated in an ethanol series and embedded in Lowicryl, polymerized by UV light. Post-staining was with 0.5% uranylacetate. M – mitochondrion; P – peroxisome; V – vacuole. The bar represents 0.5 µm.</p