Claus Maxel Henriksen

Bio: Claus Maxel Henriksen is an academic researcher from Technical University of Denmark. The author has contributed to research in topics: Penicillium chrysogenum & Penicillin. The author has an hindex of 8, co-authored 10 publications receiving 325 citations.

Purification and characterization of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Penicillium chrysogenum.

Abstract: delta-(L-alpha-Aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) from Penicillium chrysogenum was purified to homogeneity by a combination of (NH4)2SO4 precipitation, protamine sulphate treatment, ion-exchange chromatography, gel filtration and hydrophobic interaction chromatography The molecular mass of ACVS was estimated with native gradient gel electrophoresis and SDS/PAGE The native enzyme consisted of a single polymer chain with an estimated molecular mass of 470 kDa The denatured enzyme had an estimated molecular mass of 440 kDa The influence of different reaction parameters such as substrates, cofactors and pH on the activity of the purified ACVS was investigated The Km values for the three precursor substrates L-alpha-aminoadipic acid, L-cysteine and L-valine were determined as 45, 80 and 80 microM respectively, and the optimal assay concentration of ATP was found to be 5 mM (with 20 mM MgCl2) The dimer of the reaction product bis-delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (bisACV) gave feedback inhibition of the purified ACVS; the inhibition parameter KbisACV was determined as 14 mM Furthermore dithiothreitol was shown to inhibit the purified ACVS From the addition of a glucose pulse to a steady-state glucose-limited continuous culture of P chrysogenum it was found that there is glucose repression of the synthesis of ACVS and that there must be a constant turnover of ACVS owing to synthesis and degradation

Influence of the Dissolved Oxygen Concentration on the Penicillin Biosynthetic Pathway in Steady-State Cultures of Penicillium chrysogenum

Abstract: The influence the of dissolved oxygen concentration on penicillin biosynthesis was studied in steady-state continuous cultures of a high-yielding strain of Penicillium chrysogenum operated at a dilution rate of 0.05 h−1. The dissolved oxygen concentration was varied between 0.019 and 0.344 mM (corresponding to 7% and 131% air saturation at 1 bar) solely through manipulations of the inlet gas composition. At dissolved oxygen concentrations above 0.06–0.08 mM, a constant specific penicillin productivity of around 22 (μmol/g of DW) /h is maintained. At lower oxygen concentrations, the specific penicillin productivity decreases, and a value of 17 (μmol/g of DW) /h was obtained when the dissolved oxygen concentration was 0.042 mM. A further lowering of the dissolved oxygen concentration to 0.019 mM resulted in the loss of penicillin production. However, penicillin productivity was instantly recovered to its maximum value when the dissolved oxygen concentration was reset to a value above 0.08 mM. The specific formation rates of a number of typical byproducts of the penicillin production, i.e., δ-(l-α-aminoadipyl) -l-cysteinyl-d-valine, isopenicillin N, 6-aminopenicillanic acid, 8-hydroxypenillic acid and 6-oxopiperidine-2-carboxylic acid, the δ-lactam form of α-aminoadipic acid, all increased with decreasing dissolved oxygen concentration. A simultaneous increase in the secretion of glutathione was observed, and a link between the secretion of δ-(l-α-aminoadipyl) -l-cysteinyl-d-valine and glutathione is suggested. The intracellular pools of the pathway intermediates δ-(l-α-aminoadipyl) -l-cysteinyl-d-valine and isopenicillin N increased respectively 2- and 3-fold when the dissolved oxygen concentration was lowered from 0.344 to 0.042 mM, whereas the intracellular pools of glutathione and cysteine decreased at low dissolved oxygen concentrations. On the basis of the intracellular pool measurements, metabolic control analysis is performed, and the flux control coefficients for the first two enzymes in the penicillin biosynthetic pathway, i.e., δ-(l-α-aminoadipyl) -l-cysteinyl-d-valine synthetase and isopenicillin N synthetase, are calculated at different dissolved oxygen concentrations. It is found that for low dissolved oxygen concentrations, the flux control is mainly exerted by the isopenicillin N synthetase, whereas a more even distribution of the flux control by the two enzymes is encountered at high dissolved oxygen concentrations.

Metabolic Engineering of Saccharomyces cerevisiae

Abstract: Comprehensive knowledge regarding Saccharomyces cerevisiae has accumulated over time, and today S. cerevisiae serves as a widley used biotechnological production organism as well as a eukaryotic model system. The high transformation efficiency, in addition to the availability of the complete yeast genome sequence, has facilitated genetic manipulation of this microorganism, and new approaches are constantly being taken to metabolicially engineer this organism in order to suit specific needs. In this paper, strategies and concepts for metabolic engineering are discussed and several examples based upon selected studies involving S. cerevisiae are reviewed. The many different studies of metabolic engineering using this organism illustrate all the categories of this multidisciplinary field: extension of substrate range, improvements of producitivity and yield, elimination of byproduct formation, improvement of process performance, improvements of cellular properties, and extension of product range including heterologous protein production.

The RAVEN toolbox and its use for generating a genome-scale metabolic model for Penicillium chrysogenum

Abstract: We present the RAVEN (Reconstruction, Analysis and Visualization of Metabolic Networks) Toolbox: a software suite that allows for semi-automated reconstruction of genome-scale models. It makes use of published models and/or the KEGG database, coupled with extensive gap-filling and quality control features. The software suite also contains methods for visualizing simulation results and omics data, as well as a range of methods for performing simulations and analyzing the results. The software is a useful tool for system-wide data analysis in a metabolic context and for streamlined reconstruction of metabolic networks based on protein homology. The RAVEN Toolbox workflow was applied in order to reconstruct a genome-scale metabolic model for the important microbial cell factory Penicillium chrysogenum Wisconsin54-1255. The model was validated in a bibliomic study of in total 440 references, and it comprises 1471 unique biochemical reactions and 1006 ORFs. It was then used to study the roles of ATP and NADPH in the biosynthesis of penicillin, and to identify potential metabolic engineering targets for maximization of penicillin production.

Glutathione, altruistic metabolite in fungi.

Abstract: Glutathione (GSH; gamma-L-glutamyl-L-cysteinyl-glycine), a non-protein thiol with a very low redox potential (E'0 = 240 mV for thiol-disulfide exchange), is present in high concentration up to 10 mM in yeasts and filamentous fungi. GSH is concerned with basic cellular functions as well as the maintenance of mitochondrial structure, membrane integrity, and in cell differentiation and development. GSH plays key roles in the response to several stress situations in fungi. For example, GSH is an important antioxidant molecule, which reacts non-enzymatically with a series of reactive oxygen species. In addition, the response to oxidative stress also involves GSH biosynthesis enzymes, NADPH-dependent GSH-regenerating reductase, glutathione S-transferase along with peroxide-eliminating glutathione peroxidase and glutaredoxins. Some components of the GSH-dependent antioxidative defence system confer resistance against heat shock and osmotic stress. Formation of protein-SSG mixed disulfides results in protection against desiccation-induced oxidative injuries in lichens. Intracellular GSH and GSH-derived phytochelatins hinder the progression of heavy metal-initiated cell injuries by chelating and sequestering the metal ions themselves and/or by eliminating reactive oxygen species. In fungi, GSH is mobilized to ensure cellular maintenance under sulfur or nitrogen starvation. Moreover, adaptation to carbon deprivation stress results in an increased tolerance to oxidative stress, which involves the induction of GSH-dependent elements of the antioxidant defence system. GSH-dependent detoxification processes concern the elimination of toxic endogenous metabolites, such as excess formaldehyde produced during the growth of the methylotrophic yeasts, by formaldehyde dehydrogenase and methylglyoxal, a by-product of glycolysis, by the glyoxalase pathway. Detoxification of xenobiotics, such as halogenated aromatic and alkylating agents, relies on glutathione S-transferases. In yeast, these enzymes may participate in the elimination of toxic intermediates that accumulate in stationary phase and/or act in a similar fashion as heat shock proteins. GSH S-conjugates may also form in a glutathione S-transferases-independent way, e.g. through chemical reaction between GSH and the antifugal agent Thiram. GSH-dependent detoxification of penicillin side-chain precursors was shown in Penicillium sp. GSH controls aging and autolysis in several fungal species, and possesses an anti-apoptotic feature.