Untersuchungen zum peroxisomalen Import von Isocitrat-Lyasen in Saccharomyces cerevisiae

Peroxisomes play a central role in the metabolism of fatty acids and the detoxication of reactive oxygen species, e.g. $H_{2}O_{2}$, emerging from oxidation processes. Peroxisomal disorders cause diseases, often due to an import defect of certain enzymes. The two major import pathways of peroxisomal matrix proteins are based on conserved signal sequences. The most common signal is the so-called PTS1, a tripeptide at the end of the C-terminus. More rarely an N-terminal nonapeptide (PTS2) occurs. Moreover some proteins contain an additional signal and/or use a hitherto unknown import pathway. For instance only some peroxisomal isocitrate lyases have a PTS1 signal. Isocitrate lyase (ICL) is a key enzyme of the glyoxylate pathway and thus essential for growth on non-fermentable carbon sources. Eukaryotic ICLs are usually localized in the peroxisomes, with the exception of the cytosolic ICL of the baker’s yeast S.cerevisiae. In contrast to this cytosolic Sc ICL the peroxisomal Ashbya gossypii -ICL contains a PTS1 signal. Therefore the question arose, whether this signal was sufficient to mediate the peroxisomal ICL-import or whether an additional signal was necessary. By fusion of the PTS1-tripeptide AKL or exchange of the C-terminus with that of Ag ICL an import of 3 - 4% of the modified Sc ICL was achieved. In the course of this the peroxisomal localization was independently shown by sucrose density centrifugation as well as immuno electron microscopy. Interestingly, the import of eterologously expressed Ag ICL was far more efficient with approx. 20%. This difference pointed to an additional peroxisomal signal of the Ag ICL compared with the Sc ICL. The thesis was supported by muteins without a functional PTS1, which were imported to 20% (Ag ICL extended by 39 amino acids) or 9% (PTS1 deleted). The signal needed for this import was assumed within the region of the structural domain II of the Ag ICL and researched with a hybrid protein of the E.coli -ICL. The ICL structural domain II is an internal region of about 100 amino acids, which is missing in prokaryotes. Thus the hybrid protein Ec ICLAg DII was made by insertion of the Ag ICL structural domain II (Ag DII). Differential centrifugations displayed a significant peroxisomal localization of this hybrid protein. While - according to activity assays - only 0.7% of the wild type Ec ICL located in the yeast organelles, the proportion of the hybrid protein was 33%. The result was confirmed by Western blots presenting a clear band of Ec ICLAg DII within the organellar pellet. Thereby the functionality of an ICL structural domain II sequence as an organellar signal was shown for the first time. In castor bean ICL the PTS1 region is also dispensable for peroxisomal import, which nevertheless requires the PTS1 receptor PEX5 (Parkes et al, 2003). To check whether this relationship also exists for the PTS1-less Ag ICL and the hybrid Ec ICLAg DII, a PEX5 disruption mutant was cloned. The unaltered subcellular distribution of both ICL muteins in this strain background contradicted a dependency of the import on PEX5. Enzyme assays demonstrated 8% of the PTS1-less Ag ICL or 34% of the hybrid Ec ICLAg DII within the organellar pellet. The localization of the ICLs within the organelles as well as the mislocalization of the PEX5-dependent catalase A were confirmed by Western blots. Immuno-detection within the fractions of sucrose density gradients showed the co-localization of both ICL muteins with thiolase, a PTS2 peroxisomal marker. Hence the Ag ICL contained a novel, PEX5-independent peroxisomal signal within region of its structural domain II

Comparative expression of lipase CAL-A in the yeasts Saccharomyces cerevisiae, Kluyveromyces lactis and Hansenula polymorpha to investigate a possible host influence

Yeasts are an attractive expression platform, as they combine the ease of handling with the eukaryotic ability to process the produced protein. Important aspects of eukaryotic protein expression are posttranslational modifications, which can be required for functional expression of the protein of interest and can only be performed by eukaryotes. Each organism has its own modification pattern: for instance, the same protein produced in different hosts is subjected to various glycosylation pathways. It is amenable that these kinds of modifications can have an influence on the biochemical properties of the protein. To verify this thesis, the well-studied lipase CAL-A from Candida antarctica was chosen as a model enzyme. The codon bias of the gene sequence was uniformly optimized and expressed in three industrially relevant yeast hosts: Saccharomyces cerevisiae, Kluyveromyces lactis, and Hansenula polymorpha. The capacity of the expression systems to produce the enzyme was analyzed, as well as the biochemical properties of the produced and purified CAL-A. All hosts produced active enzyme; however, significant differences in the obtained yield were observed. H. polymorpha appeared to be the most productive host with a tenfold increase in productivity in comparison to S. cerevisiae. Studies on thermostability and activity of the purified enzymes towards various substrates showed a significant impact of the host on the biochemical properties of the produced protein. The most thermostable CAL-A from K. lactis retained 70% of its activity after incubation at 60 °C, in comparison to 45% remaining activity for the enzyme purified from S. cerevisiae. In the screenings with different substrates, a fourfold increase in activity between the enzymes from H. polymorpha and S. cerevisiae was found. Altogether, we herein exemplify how the selection of the host even within one taxonomic family (Saccharomycetaceae) significantly affects the produced enzyme's characteristics

Evaluation of coumarin-based fluorogenic P450 BM3 substrates and prospects for competitive inhibition screenings

Fluorescence-based assays for the cytochrome P450 BM3 monooxygenase from Bacillus megaterium address an attractive biotechnological challenge by facilitating enzyme engineering and the identification of potential substrates of this highly promising biocatalyst. In the current study, we used the scarcity of corresponding screening systems as an opportunity to evaluate a novel and continuous high-throughput assay for this unique enzyme. A set of nine catalytically diverse P450 BM3 variants was constructed and tested toward the native substrate-inspired fluorogenic substrate 12-(4-trifluoromethylcoumarin-7-yloxy)dodecanoic acid. Particularly high enzyme-mediated O-dealkylation yielding the fluorescent product 7-hydroxy-4-trifluoromethylcoumarin was observed with mutants containing the F87V substitution, with A74G/F87V showing the highest catalytic efficiency (0.458 min−1 μM−1). To simplify the assay procedure and show its versatility, different modes of application were successfully demonstrated, including (i) the direct use of NADPH or its oxidized form NADP+ along with diverse NADPH recycling systems for electron supply, (ii) the use of cell-free lysates and whole-cell preparations as the biocatalyst source, and (iii) its use for competitive inhibition screens to identify or characterize substrates and inhibitors. A detailed comparison with known, fluorescence-based P450 BM3 assays finally emphasizes the relevance of our contribution to the ongoing research

Lights on and action! Controlling microbial gene expression by light

Light-mediated control of gene expression and thus of any protein function and metabolic process in living microbes is a rapidly developing field of research in the areas of functional genomics, systems biology, and biotechnology. The unique physical properties of the environmental factor light allow for an independent photocontrol of various microbial processes in a noninvasive and spatiotemporal fashion. This mini review describes recently developed strategies to generate photo-sensitive expression systems in bacteria and yeast. Naturally occurring and artificial photoswitches consisting of light-sensitive input domains derived from different photoreceptors and regulatory output domains are presented and individual properties of light-controlled expression systems are discussed