Unique processes yielding pure azaphilones in Talaromyces atroroseus.

Azaphilones are a class of fungal pigments, reported mostly in association with Monascus species. In Asian countries, they are used as food colourants under the name of "red yeast rice" and their production process is well described. One major limitation of current production techniques of azaphilones is that they always occur in a mixture of yellow, orange and red pigments. These mixtures are difficult to control and to quantify. This study has established a controlled and reproducible cultivation protocol to selectively tailor production of individual pigments during a submerged fermentation using another fungal species capable of producing azaphilone pigments, Talaromyces atroroseus, using single amino acids as the sole nitrogen source. The produced azaphilone pigments are called atrorosins and are amino acid derivatives of the known azaphilone pigment Penicillium purpurogenum-orange (PP-O), with the amino acid used as nitrogen source incorporated into the core skeleton of the azaphilone. This strategy was successfully demonstrated using 18 proteinogenic amino acids and the non-proteinogenic amino acid ornithine. Two cultivation methods for production of the pure serine derivative (atrorosin S) have been further developed, with yields of 0.9 g/L being obtained. Yielding pure atrorosins through switching from KNO3 to single amino acids as nitrogen source allows for considerably easier downstream processing and thus further enhances the commercial relevance of azaphilone producing fungal cell factories.

A modular metabolic engineering approach for the production of 1,2-propanediol from glycerol by Saccharomyces cerevisiae.

Compared to sugars, a major advantage of using glycerol as a feedstock for industrial bioprocesses is the fact that this molecule is more reduced than sugars. A compound whose biotechnological production might greatly profit from the substrate's higher reducing power is 1,2-propanediol (1,2-PDO). Here we present a novel metabolic engineering approach to produce 1,2-PDO from glycerol in S. cerevisiae. Apart from implementing the heterologous methylglyoxal (MG) pathway for 1,2-PDO formation from dihydroxyacetone phosphate (DHAP) and expressing a heterologous glycerol facilitator, the employed genetic modifications included the replacement of the native FAD-dependent glycerol catabolic pathway by the 'DHA pathway' for delivery of cytosolic NADH and the reduction of triosephosphate isomerase (TPI) activity for increased precursor (DHAP) supply. The choice of the medium had a crucial impact on both the strength of the metabolic switch towards fermentation in general (as indicated by the production of ethanol and 1,2-PDO) and on the ratio at which these two fermentation products were formed. For example, virtually no 1,2-PDO but only ethanol was formed in synthetic glycerol medium with urea as the nitrogen source. When nutrient-limited complex YG medium was used, significant amounts of 1,2-PDO were formed and it became obvious that the concerted supply of NADH and DHAP are essential for boosting 1,2-PDO production. Additionally, optimizing the flux into the MG pathway improved 1,2-PDO formation at the expense of ethanol. Cultivation of the best-performing strain in YG medium and a controlled bioreactor set-up resulted in a maximum titer of > 4gL-1 1,2-PDO which, to the best of our knowledge, has been the highest titer of 1,2-PDO obtained in yeast so far. Surprisingly, significant 1,2-PDO production was also obtained in synthetic glycerol medium after changing the nitrogen source towards ammonium sulfate and adding a buffer.

Heterologous expression of peptidyl-Lys metallopeptidase of Armillaria mellea and mutagenic analysis of the recombinant peptidase.

A method to express, purify and modify the Peptidyl-Lys metallopeptidase (LysN) ofArmillaria melleainPichia pastoriswas developed to enable functional studies of the protease. Based on prior work, we propose a mechanism of action of LysN. Catalytic residues were investigated by site-directed mutagenesis. As anticipated, these mutations resulted in significantly reduced catalytic rates. Additionally, based on molecular modelling eleven mutants were designed to have altered substrate specificity. The S1' binding pocket of LysN is quite narrow and lined with negative charge to specifically accommodate lysine. To allow for arginine specificity in S1', it was proposed to extend the S1' binding pocket by mutagenesis, however the resulting mutant did not show any activity with arginine in P1'. Two mutants, A101D and T105D, showed increased specificity towards arginine in subsites S2'-S4' compared to the wild type protease. We speculate that the increased specificity to result from the additional negative charge which attract and interact with positively charged residues better than the wild type.

Novel Application of Peptidyl-Lys Metallopeptidase as a C-Terminal Processing Protease.

Adding fusion partners to proteins or peptides can aid or be a necessity to facilitate recombinant expression, folding, or purification. Independent of the reason it is desirable to remove the fusion partner to restore native functionality. Processing proteases catalyze the removal of fusion partners, however, most of these proteases have substrate specificity for the N-terminal of the scissile bond, leaving non-native termini if fusions are added to the C-terminal. The peptidyl-lys metallopeptidease of Armillaria mellea (Am-LysN) is unusual by having substrate specificity for the C-terminal side of the scissile peptide bond, allowing it to generate native C-termini. Am-LysN has strict specificity for lysine in P1', making all lysines of a protein or peptide a potential degradation site, however there are a number of amino acid side chains which lower hydrolysis significantly when located adjacent to the lysine. In this study we show that Am-LysN can be used as a processing protease to remove C-terminal extensions of peptides with no internal lysine to generate native Ctermini. Furthermore we show that removal of C-terminal extensions on peptides containing internal lysines can be achieved with little degradation of the product depending on the adjacent amino acids. These results demonstrate the utility of LysN allowing for novel ways to use fusion technology in the production of recombinant proteins.

Substrate Specificity Profiling of Peptidyl-Lys Metallopeptidase of Armillaria mellea by FRET Based Peptide Library.

Determining the substrate specificity of a protease is essential for developing assays, inhibitors and understanding the mechanisms of the enzyme. In this work, we have profiled the specificity of Peptidyl-Lys metallopeptidase, (LysN), of Armillaria mellea, by a synthetic fluorescence resonance energy transfer (FRET) positional-scanning library. The library was based on a reference sequence K(Abz)-S-A-Q-K-M-V-S-K(Dnp), where the fluorescent donor is 2-aminobenzamide and the quencher is N-2,4-dinitrophenyl. Each position was varied between 19 different amino acids one by one, to reveal the specificity of the protease. LysN exhibits strict specificity for lysine in S1', and has less specificity moving further away from the scissile bond. Additivity between the subsites was observed and the best substrate identified was K(Abz)-M-R-F-K-R-R-R-K(Dnp) with a kcat/KM of 42.6 µM/s. Based on a homology structure model the reference substrate was fitted into the active site using molecular dynamics to propose peptide-enzyme interactions.