Biotechnologische Erzeugung natürlicher Aromastoffe aus Submerskulturen von Asco- und Basidiomyceten

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Crosses between monokaryons of Pleurotus sapidus or Pleurotus florida show an improved biotransformation of (+)-valencene to (+)-nootkatone

Several hundred monokaryotic and new dikaryotic strains derived thereof were established from (+)-valencene tolerant Pleurotus species. When grouped according to their growth rate on agar plates and compared to the parental of Pleurotus sapidus 69, the slowly growing monokaryons converted (+)-valencene more efficiently to the grapefruit flavour compound (+)-nootkatone. The fast growing monokaryons and the slow × slow and the fast × fast dikaryotic crosses showed similar or inferior yields. Some slow × fast dikaryons, however, exceeded the biotransformation capability of the parental dikaryon significantly. The activity of the responsible enzyme, lipoxygenase, showed a weak correlation with the yields of (+)-nootkatone indicating that the determination of enzyme activity using the primary substrate linoleic acid may be misleading in predicting the biotransformation efficiency. This exploratory study indicated that a classical genetics approach resulted in altered and partly improved terpene transformation capability (plus 60%) and lipoxygenase activity of the strains.Fil: Omarini, Alejandra Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Leibniz Universität Hannover. Hannover; AlemaniaFil: Plagemann, Ina. Leibniz Universität Hannover. Hannover; AlemaniaFil: Schimanski, Silke. Leibniz Universität Hannover. Hannover; AlemaniaFil: Krings, Ulrich. Leibniz Universität Hannover. Hannover; AlemaniaFil: Berger, Ralf G.. Leibniz Universität Hannover. Hannover; Alemani

The highly diverged trypanosomal MICOS complex is organized in a non‐essential integral membrane and an essential peripheral module

The mitochondrial contact site and cristae organization system (MICOS) mediates the formation of cristae, invaginations in the mitochondrial inner membrane. The highly diverged MICOS complex of the parasitic protist Trypanosoma brucei consists of 9 subunits. Except for two Mic10-like and a Mic60-like protein, all subunits are specific for kinetoplastids. Here we determined on a proteome-wide scale how ablation of individual MICOS subunits affects the levels of the other subunits. The results reveal co-regulation of TbMic10-1, TbMic10-2, TbMic16 and TbMic60, suggesting that these non-essential, integral inner membrane proteins form an interdependent network. Moreover, the ablation of TbMic34 and TbMic32 reveals another network consisting of the essential, intermembrane space-localized TbMic20, TbMic32, TbMic34 and TbMic40, all of which are peripherally associated with the inner membrane. The downregulation of TbMic20, TbMic32 and TbMic34 also interferes with mitochondrial protein import and reduces the size of the TbMic10-containing complexes. Thus, the diverged MICOS of trypanosomes contains two subcomplexes: a non-essential membrane-integrated one, organized around the conserved Mic10 and Mic60, that mediates cristae formation, and an essential membrane-peripheral one consisting of four kinetoplastid-specific subunits, that is required for import of intermembrane space proteins

TAC42 and TAC40 contain C-terminal β-signals.

<p>(A) Amino acid sequence of the C-termini of the indicated β-barrel proteins in different trypanosomatids. Tb, <i>T</i>. <i>brucei</i>; Tv, <i>T</i>. <i>viva</i>x; Tc, <i>T</i>. <i>cruzi</i>; Lm, <i>L</i>. <i>major</i> and Li, <i>L</i>. <i>infantum</i>. The residues contributing to the predicted β-signal are indicated by the grey bars. The β-signal consenus sequence and the sequence of the two mutated variants (1mut, 4mut) of the β-signal consensus sequence are indicated. Changed residues are shown in red. P_O, polar amino acid; Hy, hydrophobic amino acid. (B) TAC42 top panel: immunoblot analysis of whole cells (Tot), soluble (Cyt) and digitonin-extracted mitochondria-enriched pellet (Mit) fractions of cell lines expressing C-terminally Myc-tagged full length (wt), 1mut and 4mut variants of TAC42, respectively. Lipoamide dehydrogenase (LipDH) and EF1a serve as mitochondrial and cytosolic markers, respectively. TAC42 bottom panel: carbonate extraction at pH 11.5 of the mitochondria-enriched pellet fraction (Mit) of the cell lines depicted above. The pellet (Pe) and the supernatant (Sup) fractions correspond integral membrane and soluble proteins, respectively. ATOM40 and Cyt C serve as markers for integral and peripheral membrane proteins, respectively. TAC40 top and bottom panels, same experiments as for TAC42 and its variants were performed for TAC40 and its two mutated variants. (C) Graphs showing the mean and the standard errors of a quantification of three biological replicates of the experiment shown in (B).</p

Trypanosomal β-signals are functional in yeast and knock-down of trypanosomal Sam50 causes a kDNA segregation defect.

<p>(A) In vitro translated ³⁵S-labeled TAC42 and TAC42-mut4 were incubated for the indicated time with mitochondria either isolated from wildtype (WT) yeast or from a yeast strain lacking the SAM complex subunit sam37 (Δ<i>sam37</i>). Subsequently all samples were analyzed by BN-PAGE and analyzed by autoradiography. A mock reaction lacking organelles was also analyzed. The putative TAC42/Sam50 complex is indicated. (B) Left graph: growth and loss of kDNA in the procyclic <i>T</i>. <i>brucei</i> Sam50-RNAi cell line. Red lines depict percentage of cells still having the kDNA. Right graph: fluorescent intensities of kDNA networks were measured after Sam50 knock down. Red lines mark the median.</p

Biogenesis of the mitochondrial DNA inheritance machinery in the mitochondrial outer membrane of <i>Trypanosoma brucei</i>

<div><p>Mitochondria cannot form de novo but require mechanisms that mediate their inheritance to daughter cells. The parasitic protozoan <i>Trypanosoma brucei</i> has a single mitochondrion with a single-unit genome that is physically connected across the two mitochondrial membranes with the basal body of the flagellum. This connection, termed the tripartite attachment complex (TAC), is essential for the segregation of the replicated mitochondrial genomes prior to cytokinesis. Here we identify a protein complex consisting of three integral mitochondrial outer membrane proteins—TAC60, TAC42 and TAC40—which are essential subunits of the TAC. TAC60 contains separable mitochondrial import and TAC-sorting signals and its biogenesis depends on the main outer membrane protein translocase. TAC40 is a member of the mitochondrial porin family, whereas TAC42 represents a novel class of mitochondrial outer membrane β-barrel proteins. Consequently TAC40 and TAC42 contain C-terminal β-signals. Thus in trypanosomes the highly conserved β-barrel protein assembly machinery plays a major role in the biogenesis of its unique mitochondrial genome segregation system.</p></div

TAC40, TAC60 and TAC42 form a complex.

<p>(A) SILAC-IP of C-terminally HA-tagged TAC40 from digitonin-solubilized whole cell lysates. Mean log₁₀ ratios (TAC40-HA/wt) of proteins detected by quantitative MS in at least two of three independent biological replicates are plotted against the corresponding log₁₀ <i>P</i> values (one-sided t-test). Horizontal dashed line indicates a t-test significance level of 0.05, while vertical dashed lines mark a fivefold enrichment. The bait protein TAC40 is marked in green. The co-precipitated proteins are marked in red. (B) and (C) Cell lines expressing Myc- and HA-tagged versions of the TAC40 interactors, TAC60-Myc and TAC42-HA, respectively, were used for reciprocal SILAC-IPs. For complete lists of proteins for all three IPs, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006808#ppat.1006808.s003" target="_blank">S1</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006808#ppat.1006808.s005" target="_blank">S3</a> Tables. (D) Table indicating the enrichment factors of TAC40, TAC60 and TAC42 in the reciprocal IPs.</p

TAC60 contains distinct mitochondrial and TAC targeting signals.

<p>(A) To scale schematic drawing of the variants used to analyze TAC60 targeting. The predicted transmembrane domains of TAC60 are indicated in black. ΔNx or ΔCx indicate the number of aa deleted from the N- or/and the C-termini. All variants were C-terminally Myc-tagged. The constructs are grouped according to the four distinct localization pattern observed for the TAC60-variants: TAC, TAC and general mitochondrial (TAC/MITO or T/M), mitochondrial (MITO) and cytosolic (CYTO) localization. (B, C, D and E) IF analysis of cells lines expressing the TAC60 variants listed in (A). The cell lines are grouped as in (A). DIC, differential interference contrast picture. DNA is stained with DAPI (blue). The Myc-tagged TAC60 variants are indicated in red. The merged picture (Merge) shows an overlay of the DAPI and the Myc staining. Bar, 5 μm. Inset shows an enlargement of the kDNA region. Bar inset, 1μm. Co-staining with a mitochondrial marker is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006808#ppat.1006808.s001" target="_blank">S1B Fig</a>.</p

TAC60 is an OM protein whose N- and C-termini face the cytosol.

<p>(A) Top panel: immunoblot analysis of whole cells (Tot), soluble (Cyt) and digitonin-extracted mitochondria-enriched pellet (Mit) fractions of cells expressing C-terminally Myc-tagged TAC60. ATOM40 and EF1a served as mitochondrial and cytosolic markers, respectively. Middle panel: carbonate extraction at pH 11.5 of the mitochondria-enriched pellet fraction (Mit). The pellet (Pe) and the supernatant (Sup) fractions correspond integral membrane and soluble proteins. ATOM40 and cytochrome C (Cyt C) serve as markers for integral and peripheral membrane proteins, respectively. Bottom panel: normalized abundance profile of TAC60 over six subcellular fractions, produced in a previous proteomic analysis [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006808#ppat.1006808.ref035" target="_blank">35</a>]. (B) Immunoblots of total cellular extracts from procyclic ATOM40-RNAi (top panel) and TbTim17-RNAi (bottom panel) cells that constitutively express Myc-tagged TAC60. Time of induction is indicated. The OM protein VDAC, the IM protein CoxIV and cytosolic EF1a serve as controls. The positions of the precursor (p) and mature forms (m) of CoxIV are indicated. (C) Top left: conceptual depiction of the split GFP approach [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006808#ppat.1006808.ref059" target="_blank">59</a>]. Top right: immunoblot analysis of whole cells (Tot), soluble (Cyt) and digitonin-extracted mitochondria-enriched pellet (Mit) fractions of cells expressing N-terminally HA-tagged GFP fusion lacking the last β-barrel strand (HA-GFP1-10). ATOM40 and EF1a served as mitochondrial and cytosolic markers, respectively. IF analysis of cells lines allowing tetracycline-inducible expression of HA-GFP1-10 and TAC60 variants fused to the last β-strand of GFP at the C-terminus (TAC60-GFP11) or N-terminus (GFP11-TAC60), respectively. DNA is stained with DAPI (blue). ATOM40 is shown in red. GFP in green. Bar, 5 μm.</p