43 research outputs found
Die EU-Zinsenrichtlinie: Ein Schuss in den Ofen?
Die EU hat durch die Zinsenrichtlinie einerseits eindeutig von einer Politik, die auf einem Vertrauen gegenüber dem Bürger und einer gewissen Selbstbestimmung beruhte, zu einer Vorgehensweise gewechselt die durch Misstrauen und Kontrolle geprägt ist. Andererseits erscheinen die Regelungen der Zinsenrichtlinie, die auf die klassischen Verhaltensdeterminanten der Steuerhinterziehung abzielen, aber unzureichend. In Summe ist es daher möglich, dass die Zinsenrichtlinie ihr eigentliches Ziel der effektiven Besteuerung von Zinserträgen nicht erreicht. Da steuerbezogener Informationsaustausch eine strategische Variable der Mitgliedstaaten ist um die Attraktivität ihrer Finanzmärkte zu regulieren, ist es zumindest diskutabel, ob innerhalb der nationalen Umsetzungen der Richtlinie vorhandene Schlupflöcher nicht auf ein verschleiertes nichtkooperatives Verhalten der Mitgliedsstaaten zurückzuführen sind.Steuerhinterziehung; Steuermoral; steuerbezogener Informationsaustausch
Multi-omics view of recombinant Yarrowia lipolytica: Enhanced ketogenic amino acid catabolism increases polyketide-synthase-driven docosahexaenoic production to high selectivity at the gram scale
DHA is a marine PUFA of commercial value, given its multiple health benefits. The worldwide emerging shortage
in DHA supply has increased interest in microbial cell factories that can provide the compound de novo. In this
regard, the present work aimed to improve DHA production in the oleaginous yeast strain Y. lipolytica Af4, which
synthetized the PUFA via a heterologous myxobacterial polyketide synthase (PKS)-like gene cluster. As starting
point, we used transcriptomics, metabolomics, and 13C-based metabolic pathway profiling to study the cellular
dynamics of Y. lipolytica Af4. The shift from the growth to the stationary DHA-production phase was associated
with fundamental changes in carbon core metabolism, including a strong upregulation of the PUFA gene cluster,
as well as an increase in citrate and fatty acid degradation. At the same time, the intracellular levels of the two
DHA precursors acetyl-CoA and malonyl-CoA dropped by up to 98% into the picomolar range. Interestingly, the
degradation pathways for the ketogenic amino acids L-lysine, L-leucine, and L-isoleucine were transcriptionally
activated, presumably to provide extra acetyl-CoA. Supplementation with small amounts of these amino acids at
the beginning of the DHA production phase beneficially increased the intracellular CoA-ester pools and boosted
the DHA titer by almost 40%. Isotopic 13C-tracer studies revealed that the supplements were efficiently directed
toward intracellular CoA-esters and DHA. Hereby, L-lysine was found to be most efficient, as it enabled long-term
activation, due to storage within the vacuole and continuous breakdown. The novel strategy enabled DHA
production in Y. lipolytica at the gram scale for the first time. DHA was produced at a high selectivity (27% of
total fatty acids) and free of the structurally similar PUFA DPA, which facilitates purification for high-value
medical applications that require API-grade DHA. The assembled multi-omics picture of the central metabolism of Y. lipolytica provides valuable insights into this important yeast. Beyond our work, the enhanced
catabolism of ketogenic amino acids seems promising for the overproduction of other compounds in Y. lipolytica,
whose synthesis is limited by the availability of CoA ester precursors
A common approach for absolute quantification of short chain CoA thioesters in prokaryotic and eukaryotic microbes
Background
Thioesters of coenzyme A participate in 5% of all enzymatic reactions. In microbial cell factories, they function as building blocks for products of recognized commercial value, including natural products such as polyketides, polyunsaturated fatty acids, biofuels, and biopolymers. A core spectrum of approximately 5–10 short chain thioesters is present in many microbes, as inferred from their genomic repertoire. The relevance of these metabolites explains the high interest to trace and quantify them in microbial cells.
Results
Here, we describe a common workflow for extraction and absolute quantification of short chain CoA thioesters in different gram-positive and gram-negative bacteria and eukaryotic yeast, i.e. Corynebacterium glutamicum, Streptomyces albus, Pseudomonas putida, and Yarrowia lipolytica. The approach assessed intracellular CoA thioesters down to the picomolar level and exhibited high precision and reproducibility for all microbes, as shown by principal component analysis. Furthermore, it provided interesting insights into microbial CoA metabolism. A succinyl-CoA synthase defective mutant of C. glutamicum exhibited an unaffected level of succinyl-CoA that indicated a complete compensation by the L-lysine pathway to bypass the disrupted TCA cycle. Methylmalonyl-CoA, an important building block of high-value polyketides, was identified as dominant CoA thioester in the actinomycete S. albus. The microbe revealed a more than 10,000-fold difference in the abundance of intracellular CoA thioesters. A recombinant strain of S. albus, which produced different derivatives of the antituberculosis polyketide pamamycin, revealed a significant depletion of CoA thioesters of the ethylmalonyl CoA pathway, influencing product level and spectrum.
Conclusions
The high relevance of short chain CoA thioesters to synthetize industrial products and the interesting insights gained from the examples shown in this work, suggest analyzing these metabolites in microbial cell factories more routinely than done so far. Due to its broad application range, the developed approach appears useful to be applied this purpose. Hereby, the possibility to use one single protocol promises to facilitate automatized efforts, which rely on standardized workflows
Microparticles globally reprogram Streptomyces albus toward accelerated morphogenesis, streamlined carbon core metabolism, and enhanced production of the antituberculosis polyketide pamamycin
Streptomyces spp. are a rich source for natural products with recognized industrial value, explaining the high interest to improve and streamline the performance of in these microbes. Here, we studied the production of pamamycins, macrodiolide homologs with a high activity against multiresistant pathogenic microbes, using recombinant Streptomyces albus J1074/R2. Talc particles (hydrous magnesium silicate, 3MgO·4SiO2·H2O) of micrometer size, added to submerged cultures of the recombinant strain, tripled pamamycin production up to 50 mg/L. Furthermore, they strongly affected morphology, reduced the size of cell pellets formed by the filamentous microbe during the process up to sixfold, and shifted the pamamycin spectrum to larger derivatives. Integrated analysis of transcriptome and precursor (CoA thioester) supply of particle‐enhanced and control cultures provided detailed insights into the underlying molecular changes. The microparticles affected the expression of 3,341 genes (56% of all genes), revealing a global and fundamental impact on metabolism. Morphology‐associated genes, encoding major regulators such as SsgA, RelA, EshA, Factor C, as well as chaplins and rodlins, were found massively upregulated, indicating that the particles caused a substantially accelerated morphogenesis. In line, the pamamycin cluster was strongly upregulated (up to 1,024‐fold). Furthermore, the microparticles perturbed genes encoding for CoA‐ester metabolism, which were mainly activated. The altered expression resulted in changes in the availability of intracellular CoA‐esters, the building blocks of pamamycin. Notably, the ratio between methylmalonyl CoA and malonyl‐CoA was increased fourfold. Both metabolites compete for incorporation into pamamycin so that the altered availability explained the pronounced preference for larger derivatives in the microparticle‐enhanced process. The novel insights into the behavior of S. albus in response to talc appears of general relevance to further explore and upgrade the concept of microparticle enhanced cultivation, widely used for filamentous microbes
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A bio-based route to the carbon-5 chemical glutaric acid and to bionylon-6,5 using metabolically engineered Corynebacterium glutamicum
In the present work, we established the bio-based production of glutarate, a carbon-5 dicarboxylic acid with recognized value for commercial plastics and other applications, using metabolically engineered Corynebacterium glutamicum. The mutant C. glutamicum AVA-2 served as a starting point for strain development, because it secreted small amounts of glutarate as a consequence of its engineered 5-aminovalerate pathway. Starting from AVA-2, we overexpressed 5-aminovalerate transaminase (gabT) and glutarate semialdehyde dehydrogenase (gabD) under the control of the constitutive tuf promoter to convert 5-aminovalerate further to glutarate. The created strain GTA-1 formed glutarate as a major product, but still secreted 5-aminovalerate as well. This bottleneck was tackled at the level of 5-aminovalerate re-import. The advanced strain GTA-4 overexpressed the newly discovered 5-aminovalerate importer NCgl0464 and formed glutarate from glucose in a yield of 0.27 mol mol−1. In a fed-batch process, GTA-4 produced more than 90 g L−1 glutarate from glucose and molasses based sugars in a yield of up to 0.70 mol mol−1 and a maximum productivity of 1.8 g L−1 h−1, while 5-aminovalerate was no longer secreted. The bio-based glutaric acid was purified to >99.9% purity. Interfacial polymerization and melt polymerization with hexamethylenediamine yielded bionylon-6,5, a polyamide with a unique structure
Systems biology of industrial oxytetracycline production in Streptomyces rimosus: the secrets of a mutagenized hyperproducer
Background Oxytetracycline which is derived from Streptomyces rimosus, inhibits a wide range of bacteria
and is industrially important. The underlying biosynthetic processes are complex and hinder rational engineering,
so industrial manufacturing currently relies on classical mutants for production. While the biochemistry underlying
oxytetracycline synthesis is known to involve polyketide synthase, hyperproducing strains of S. rimosus have not been
extensively studied, limiting our knowledge on fundamental mechanisms that drive production.
Results In this study, a multiomics analysis of S. rimosus is performed and wild-type and hyperproducing strains are
compared. Insights into the metabolic and regulatory networks driving oxytetracycline formation were obtained.
The overproducer exhibited increased acetyl-CoA and malonyl CoA supply, upregulated oxytetracycline biosynthe‑
sis, reduced competing byproduct formation, and streamlined morphology. These features were used to synthesize
bhimamycin, an antibiotic, and a novel microbial chassis strain was created. A cluster deletion derivative showed
enhanced bhimamycin production.
Conclusions This study suggests that the precursor supply should be globally increased to further increase
the expression of the oxytetracycline cluster while maintaining the natural cluster sequence. The mutagenized hyper‑
producer S. rimosus HP126 exhibited numerous mutations, including large genomic rearrangements, due to natural
genetic instability, and single nucleotide changes. More complex mutations were found than those typically observed
in mutagenized bacteria, impacting gene expression, and complicating rational engineering. Overall, the approach
revealed key traits infuencing oxytetracycline production in S. rimosus, suggesting that similar studies for other antibi‑
otics could uncover general mechanisms to improve production
Large-scale genome-wide association studies and meta-analyses of longitudinal change in adult lung function.
BACKGROUND: Genome-wide association studies (GWAS) have identified numerous loci influencing cross-sectional lung function, but less is known about genes influencing longitudinal change in lung function.
METHODS: We performed GWAS of the rate of change in forced expiratory volume in the first second (FEV1) in 14 longitudinal, population-based cohort studies comprising 27,249 adults of European ancestry using linear mixed effects model and combined cohort-specific results using fixed effect meta-analysis to identify novel genetic loci associated with longitudinal change in lung function. Gene expression analyses were subsequently performed for identified genetic loci. As a secondary aim, we estimated the mean rate of decline in FEV1 by smoking pattern, irrespective of genotypes, across these 14 studies using meta-analysis.
RESULTS: The overall meta-analysis produced suggestive evidence for association at the novel IL16/STARD5/TMC3 locus on chromosome 15 (P = 5.71 × 10(-7)). In addition, meta-analysis using the five cohorts with ≥3 FEV1 measurements per participant identified the novel ME3 locus on chromosome 11 (P = 2.18 × 10(-8)) at genome-wide significance. Neither locus was associated with FEV1 decline in two additional cohort studies. We confirmed gene expression of IL16, STARD5, and ME3 in multiple lung tissues. Publicly available microarray data confirmed differential expression of all three genes in lung samples from COPD patients compared with controls. Irrespective of genotypes, the combined estimate for FEV1 decline was 26.9, 29.2 and 35.7 mL/year in never, former, and persistent smokers, respectively.
CONCLUSIONS: In this large-scale GWAS, we identified two novel genetic loci in association with the rate of change in FEV1 that harbor candidate genes with biologically plausible functional links to lung function
The clinical course of idiopathic pulmonary fibrosis and its association to quality of life over time: longitudinal data from the INSIGHTS-IPF registry
Background: Quality of life (QoL) is profoundly impaired in patients with idiopathic pulmonary fibrosis (IPF). However, data is limited regarding the course of QoL. We therefore analysed longitudinal data from the German INSIGHTS-IPF registry.
Methods: Clinical status and QoL were assessed at enrollment and subsequently at 6- to 12-months intervals. A range of different QoL questionnaires including the St. George’s Respiratory Questionnaire (SGRQ) were used.
Results: Data from 424 patients were included; 76.9% male; mean age 68.7 ± 9.1 years, mean FVC% predicted 75.9 ± 19.4, mean DLCO% predicted 36.1 ± 15.9. QoL worsened significantly during follow-up with higher total SGRQ scores (increased by 1.47 per year; 95% CI: 1.17 to 1.76; p < 0.001) and higher UCSD-SOBQ scores and lower EQ-5D VAS and WHO-5 scores. An absolute decline in FVC% predicted of > 10% was associated with a significant deterioration in SGRQ (increasing by 9.08 units; 95% CI: 2.48 to 15.67; p = 0.007), while patients with stable or improved FVC had no significantly change in SGRQ. Patients with a > 10% decrease of DLCO % predicted also had a significant increase in SGRQ (+ 7.79 units; 95% CI: 0.85 to 14.73; p = 0.028), while SQRQ was almost stable in patients with stable or improved DLCO. Patients who died had a significant greater increase in SGRQ total scores (mean 11.8 ± 18.6) at their last follow-up visit prior to death compared to survivors (mean 4.2 ± 18.9; HR = 1.03; 95% CI: 1.01 to 1.04; p < 0.001). All QoL scores across the follow-up period were significantly worse in hospitalised patients compared to non-hospitalised patients, with the worst scores reported in those hospitalised for acute exacerbations.
Conclusions: QoL assessments in the INSIGHTS-IPF registry demonstrate a close relationship between QoL and clinically meaningful changes in lung function, comorbidities, disease duration and clinical course of IPF, including hospitalisation and mortality
Health related quality of life in patients with idiopathic pulmonary fibrosis in clinical practice: insights-IPF registry
Background: The INSIGHTS-IPF registry provides one of the largest data sets of clinical data and self-reported patient related outcomes including health related quality of life (QoL) on patients with idiopathic pulmonary fibrosis (IPF). We aimed to describe associations of various QoL instruments between each other and with patient characteristics at baseline. Methods: Six hundred twenty-three IPF patients with available QoL data (St George's Respiratory Questionnaire SGRQ, UCSD Shortness-of-Breath Questionnaire SoB, EuroQol visual analogue scale and index EQ-5D, Well-being Index WHO-5) were analysed. Mean age was 69.6 +/- 8.7 years, 77% were males, mean disease duration 2.0 +/- 3.3 years, FVC pred was 67.5 +/- 17.8%, DLCO pred 35.6 +/- 17%. Results: Mean points were SGRQ total 48.3, UCSD SoB 47.8, EQ-5D VAS 66.8, and WHO-5 13.9. These instruments had a high or very high correlation (exception WHO-5 to EQ-5D VAS with moderate correlation). On bivariate analysis, QoL by SGRQ total was statistically significantly associated with clinical symptoms (NYHA;p < 0.001), number of comorbidities (p < 0.05), hospitalisation rate (p < 0.01) and disease severity (as measured by GAP score, CPI, FVC and 6-min walk test;p < 0.05 each). Multivariate analyses showed a significant association between QoL (by SGRQ total) and IPF duration, FVC, age, NYHA class and indication for long-term oxygen treatment. Conclusions: Overall, IPF patients under real-life conditions have lower QoL compared to those in clinical studies. There is a meaningful relationship between QoL and various patient characteristics
Game laboratory studies
Prof. Dr. Jens Schröter ist Herausgeber der Reihe und die Herausgeber der einzelnen Hefte sind renommierte Wissenschaftler und -innen aus dem In- und Ausland.Um die Analyse von Computerspielen aus produktionsästhetischer Perspektive
zu erproben, lehnt sich der vorliegende Band an die Akteur-Netzwerk-Theorie
(ANT) an. Mit ihr geht es ihm um die Frage nach den Aktanten des Game Design –
etwa: Welche Hard- und Softwarekomponenten kommen wann und wofür zum
Einsatz; wie und mittels welcher Medien notieren Level-Designer ihre Ideen, und
wie werden die Aufzeichnungen später von Programmierern implementiert; und
welche Rolle spielt eigentlich eine Action-Figur auf dem Schreibtisch eines Textur-Artists