Application of a wide-range yeast vector (CoMed™) system to recombinant protein production in dimorphic Arxula adeninivorans, methylotrophic Hansenula polymorpha and other yeasts

BACKGROUND: Yeasts provide attractive expression platforms in combining ease of genetic manipulation and fermentation of a microbial organism with the capability to secrete and to modify proteins according to a general eukaryotic scheme. However, early restriction to a single yeast platform can result in costly and time-consuming failures. It is therefore advisable to assess several selected systems in parallel for the capability to produce a particular protein in desired amounts and quality. A suitable vector must contain a targeting sequence, a promoter element and a selection marker that function in all selected organisms. These criteria are fulfilled by a wide-range integrative yeast expression vector (CoMed™) system based on A. adeninivorans- and H. polymorpha-derived elements that can be introduced in a modular way. RESULTS: The vector system and a selection of modular elements for vector design are presented. Individual single vector constructs were used to transform a range of yeast species. Various successful examples are described. A vector with a combination of an rDNA sequence for genomic targeting, the E. coli-derived hph gene for selection and the A. adeninivorans-derived TEF1 promoter for expression control of a GFP (green fluorescent protein) gene was employed in a first example to transform eight different species including Hansenula polymorpha, Arxula adeninivorans and others. In a second example, a vector for the secretion of IL-6 was constructed, now using an A. adeninivorans-derived LEU2 gene for selection of recombinants in a range of auxotrophic hosts. In this example, differences in precursor processing were observed: only in A. adeninivorans processing of a MFα1/IL-6 fusion was performed in a faithful way. CONCLUSION: rDNA targeting provides a tool to co-integrate up to 3 different expression plasmids by a single transformation step. Thus, a versatile system is at hand that allows a comparative assessment of newly introduced metabolic pathways in several organisms or a comparative co-expression of bottleneck genes in cases where production or secretion of a certain product is impaired

Expression pattern of a nuclear encoded mitochondrial arginine-ornithine translocator gene from Arabidopsis

BACKGROUND: Arginine and citrulline serve as nitrogen storage forms, but are also involved in biosynthetic and catabolic pathways. Metabolism of arginine, citrulline and ornithine is distributed between mitochondria and cytosol. For the shuttle of intermediates between cytosol and mitochondria transporters present on the inner mitochondrial membrane are required. Yeast contains a mitochondrial translocator for ornithine and arginine, Ort1p/Arg11p. Ort1p/Arg11p is a member of the mitochondrial carrier family (MCF) essential for ornithine export from mitochondria. The yeast arg11 mutant, which is deficient in Ort1p/Arg11p grows poorly on media lacking arginine. RESULTS: High-level expression of a nuclear encoded Arabidopsis thaliana homolog (AtmBAC2) of Ort1p/Arg11p was able to suppress the growth deficiency of arg11. RT-PCR analysis demonstrated expression of AtmBAC2 in all tissues with highest levels in flowers. Promoter-GUS fusions showed preferential expression in flowers, i.e. pollen, in the vasculature of siliques and in aborted seeds. Variable expression was observed in leaf vasculature. Induction of the promoter was not observed during the first two weeks in seedlings grown on media containing NH(4)NO(3), arginine or ornithine as sole nitrogen sources. CONCLUSION: AtmBAC2 was isolated as a mitochondrial transporter for arginine in Arabidopsis. The absence of expression in developing seeds and in cotyledons of seedlings indicates that other transporters are responsible for storage and mobilization of arginine in seeds

Aadh2p: an Arxula adeninivorans alcohol dehydrogenase involved in the first step of the 1-butanol degradation pathway

Additional file 3: Figures S3. Key compounds of the ß-oxidation - microarray studies. The SBGN style metabolic network depicts reversible (double headed arrow) and irreversible (single headed arrow) reactions catalyzed by the corresponding enzymes (rectangular square). Enzymes are enriched with color-coded fold change values of time resolved expression data of the respective genes. The colors represent upregulation (blue) and downregulation (red) of genes in cells shifted to medium containing 1-butanol as the carbon source compared to cells grown with glucose. Metabolites or enzymes occurring multiple times in the metabolic network are decorated with a clone marker (e.g. CoA) (produced using VANTED [2, 3])

Agdc1p - a gallic acid decarboxylase involved in the degradation of tannic acid in the yeast Blastobotrys (Arxula) adeninivorans

Tannins and hydroxylated aromatic acids, such as gallic acid (3,4,5-trihydroxybenzoic acid), are plant secondary metabolites which protect plants against herbivores and plant-associated microorganisms. Some microbes, such as the yeast Arxula adeninivorans are resistant to these antimicrobial substances and are able to use tannins and gallic acid as carbon sources. In this study, the Arxula gallic acid decarboxylase (Agdc1p) which degrades gallic acid to pyrogallol was characterized and its function in tannin catabolism analyzed. The enzyme has a higher affinity for gallic acid (Km -0.7 ± 0.2 mM, kcat -42.0 ± 8.2 s-1) than to protocatechuic acid (3,4-dihydroxybenzoic acid) (Km -3.2 ± 0.2 mM, kcat -44.0 ± 3.2 s-1). Other hydroxylated aromatic acids, such as 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid are not gallic acid decarboxylase substrates. A. adeninivorans G1212/YRC102-AYNI1-AGDC1, which expresses the AGDC1 gene under the control of the strong nitrate inducible AYNI1 promoter achieved a maximum gallic acid decarboxylase activity of 1064.4 U/l and 97.5 U/g of dry cell weight in yeast grown in minimal medium with nitrate as nitrogen source and glucose as carbon source. In the same medium, gallic acid decarboxylase activity was not detected for the control strain G1212/YRC102 with AGDC1 expression under the control of the endogenous promoter. Gene expression analysis showed that AGDC1 is induced by gallic acid and protocatechuic acid. In contrast to G1212/YRC102-AYNI1-AGDC1 and G1212/YRC102, A. adeninivorans G1234 [δagdc1] is not able to grow on medium with gallic acid as carbon source but can grow in presence of protocatechuic acid. This confirms that Agdc1p plays an essential role in the tannic acid catabolism and could be useful in the production of catechol and cis, cis-muconic acid. However, the protocatechuic acid catabolism via Agdc1p to catechol seems to be not the only degradation pathway

Influence of Solder Pads to PERC Solar Cells for Module Integration

AbstractThe majority of screen printed solar cells has silver pads at the rear side to enable soldering for the module manufacturing. The pads increase the recombination at the silicon/metal interface due to the absence of a back surface field (BSF) at the solder pads. This reduces the efficiency of full-area Al-BSF solar cells. For passivated emitter and rear cells (PERC), a large area fraction of the rear side is covered with the passivation layer. When using specially designed Ag pastes for the rear side of PERC cells, the passivation of this layer is maintained, and the rear recombination is reduced.A comparison of solar cells with and without solder pads confirms that there is no loss in solar cell performance, both cell types achieve an efficiency of 19.6%. We investigate the influence of solder pads to PERC solar cells by calculating the effective rear surface recombination. The calculations confirm that there is a loss in open circuit voltage of less than 2mV due to the solder pads.A 54-cell PERC PV module is manufactured. The cell-to-module loss reveals that the module process is still to be optimized. Comparable modules made from 9 solar cells lost less than 1% relative in all J-V parameters after a 1000h damp-heat test

Synergistic NGF/B27 Gradients Position Synapses Heterogeneously in 3D Micropatterned Neural Cultures

Native functional brain circuits show different numbers of synapses (synaptic densities) in the cerebral cortex. Until now, different synaptic densities could not be studied in vitro using current cell culture methods for primary neurons. Herein, we present a novel microfluidic based cell culture method that combines 3D micropatterning of hydrogel layers with linear chemical gradient formation. Micropatterned hydrogels were used to encapsulate dissociated cortical neurons in laminar cell layers and neurotrophic factors NGF and B27 were added to influence the formation of synapses. Neurotrophic gradients allowed for the positioning of distinguishable synaptic densities throughout a 3D micropatterned neural culture. NGF and B27 gradients were maintained in the microfluidic device for over two weeks without perfusion pumps by utilizing a refilling procedure. Spatial distribution of synapses was examined with a pre-synaptic marker to determine synaptic densities. From our experiments, we observed that (1) cortical neurons responded only to synergistic NGF/B27 gradients, (2) synaptic density increased proportionally to synergistic NGF/B27 gradients; (3) homogeneous distribution of B27 disturbed cortical neurons in sensing NGF gradients and (4) the cell layer position significantly impacted spatial distribution of synapses

DNA methylation-based measures of biological age:meta-analysis predicting time to death

Estimates of biological age based on DNA methylation patterns, often referred to as "epigenetic age", "DNAm age", have been shown to be robust biomarkers of age in humans. We previously demonstrated that independent of chronological age, epigenetic age assessed in blood predicted all-cause mortality in four human cohorts. Here, we expanded our original observation to 13 different cohorts for a total sample size of 13,089 individuals, including three racial/ethnic groups. In addition, we examined whether incorporating information on blood cell composition into the epigenetic age metrics improves their predictive power for mortality. All considered measures of epigenetic age acceleration were predictive of mortality (p ≤ 8.2 x 10-9), independent of chronological age, even after adjusting for additional risk factors (p < 5.4 x 10-4), and within the racial/ethnic groups that we examined (non-Hispanic whites, Hispanics, African Americans). Epigenetic age estimates that incorporated information on blood cell composition led to the smallest p-values for time to death (p≤ 7.5 x 10-43). Overall, this study a) strengthens the evidence that epigenetic age predicts all-cause mortality above and beyond chronological age and traditional risk factors, and b) demonstrates that epigenetic age estimates that incorporate information on blood cell counts lead to highly significant associations with all-cause mortality

Micro-engineering the Cerebral Cortical Cell Niche:A new Cell Culture Tool for Neuroscience Research

A major problem in traditional cell culture methods, such as Petri dishes and culture flasks, is the very simplified artificial environment around the cells. Traditional cell culture methods lack features of the native cell niche, such as gradients and cell organization. This lack probably explains why pharmaceutics against the neurodegenerative Alzheimer's disease successfully stop the propagation of the disease in the Petri dish, but fail so far in clinical trials. This thesis intends to improve cell culture methods for neuroscience research related to neural developmental questions and neurodegenerative diseases. As the cortex is the main part in our brain, related to memory, emotions and perception, this thesis does focus on cell culture tools and protocols for primary cortical neurons. Currently, dissociated neurons are cultured in pure or co-culture of neural and non-neural cells, but structuring elements and controlled gradient formation is missing. The first part of this thesis discusses different studies that implicate environmental components for neural cells in their native neural cell niche. We will establish a generic neural cell niche, which consists of different neural and non-neural cells, a structured 3D environment, molecular gradients and oriented neurite networks, in a nutshell. Additionally, a simplified model of the generic neural cell niche is introduced that is implemented in a microfluidic base cell culture tool. This novel artificial neural cell niche will provide cell layer structure in 3D and local control of molecular gradients at the microscale. We will use microfluidic technology to integrate missing features in cell culture techniques for primary cortical neurons. The microfluidic device will consist of three parts: (1) a main cell culture channel that is used to organize neural cells in 3D hydrogel layers, side-by-side; (2) parallel perfusion channels to mimic nutrient supply and to control stable gradient formation, (3) interconnecting microchannels, called junction channels, that separate perfusion driven molecular transport from diffusive molecular transport. The perfusion channels are connected to device-incorporated reservoirs that allow maintenance of stable molecular gradients based on perfusion and diffusion without the use of peristaltic or syringe pumps. By injecting cortical neurons entrapped in an agarose-alginate solution in the microfluidic device, we generated 3D micropatterned neural cell layers with stable gradients perpendicular to the layer orientation. We demonstrated neurite outgrowth behavior until three weeks in culture. The application of different cell organization patterns revealed an influence of the pattern on the cell culture response. Using neurotrophic gradients of nerve growth factor (NGF) and nutrient supplements (B27), we showed that neurite guidance and synapse formation followed synergistic NGF/B27 gradients. We found that the gradient induced effects are very sensitive to changes in the environmental structure, such as the cell layer organization. Using a gradient of a phosphatase inhibitor, okadaic acid, we generated locally diseased states of the protein Tau in both 2D and 3D micropatterned neural cell cultures. The diseased form of Tau, hyper-phosphorylated Tau, is a major hallmark of Alzheimer's disease. For the first time, local formation of hyper-phosphorylated Tau was demonstrated to affect a defined cell population in a compartmentalized 2D or 3D neural cell culture. Our results revealed that the propagation mechanism of this Alzheimer's diseased lesion is determined through the formation of the 2D or 3D environment. We think that this new neural cell culture tool has the potential to answer biological questions related to environmental and structural parameters involved in the formation of the cerebral cortex and in the propagation of neurodegenerative diseases. Future studies in modern neuroscience research can now better investigate the effect of gradient parameters such as the slope, average concentration or gradient profile on cell culture and disease propagation in a controlled manner. Furthermore, the influence of cell patterns in 2D and 3D is addressed with the ability to modify cell position, density and type at the microscale. The local formation of neurodegenerative disease lesions in a micropatterned neural cell culture is generic, which makes the integration of other neurodegenerative disease models, such as Parkinson's disease, Amyotrophic lateral sclerosis or Huntington's disease, possible