Expression of lignocellulolytic enzymes in Pichia pastoris

ABSTRACT: BACKGROUND: Sustainable utilization of plant biomass as renewable source for fuels and chemical building blocks requires a complex mixture of diverse enzymes, including hydrolases which comprise the largest class of lignocellulolytic enzymes. These enzymes need to be available in large amounts at a low price to allow sustainable and economic biotechnological processes. Over the past years Pichia pastoris has become an attractive host for the cost-efficient production and engineering of heterologous (eukaryotic) proteins due to several advantages. RESULTS: In this paper codon optimized genes and synthetic alcohol oxidase 1 promoter variants were used to generate Pichia pastoris strains which individually expressed cellobiohydrolase 1, cellobiohydrolase 2 and beta-mannanase from Trichoderma reesei and xylanase A from Thermomyces lanuginosus. For three of these enzymes even gram quantities of enzyme per liter were obtained by fed-batch cultivation. Additionally, we compared our achieved yields of secreted enzymes and the corresponding activities to literature data. CONCLUSION: In our experiments we could clearly see the importance of gene optimization and strain characterization for successfully improving secretion levels. We also give a basic guideline for understanding the interplay of promoter strength and gene dosage for a successful improvement of the secretory production of lignocellulolytic enzymes in Pichia pastoris.     

Direct Inhibition of GSK3β by the Phosphorylated Cytoplasmic Domain of LRP6 in Wnt/β-Catenin Signaling

Wnt/β-catenin signaling plays a central role in development and is also involved in a diverse array of diseases. Binding of Wnts to the coreceptors Frizzled and LRP6/5 leads to phosphorylation of PPPSPxS motifs in the LRP6/5 intracellular region and the inhibition of GSK3β bound to the scaffold protein Axin. However, it remains unknown how GSK3β is specifically inhibited upon Wnt stimulation. Here, we show that overexpression of the intracellular region of LRP6 containing a Ser/Thr rich cluster and a PPPSPxS motif impairs the activity of GSK3β in cells. Synthetic peptides containing the PPPSPxS motif strongly inhibit GSK3β in vitro only when they are phosphorylated. Microinjection of these peptides into Xenopus embryos confirms that the phosphorylated PPPSPxS motif potentiates Wnt-induced second body axis formation. In addition, we show that the Ser/Thr rich cluster of LRP6 plays an important role in LRP6 binding to GSK3β. These observations demonstrate that phosphorylated LRP6/5 both recruits and directly inhibits GSK3β using two distinct portions of its cytoplasmic sequence, and suggest a novel mechanism of activation in this signaling pathway.     

Zellphysiologische Untersuchungen über den Abblühvorgang beiIris undGladiolus

Ohne Zusammenfassung     

FLOW‐CYTOMETRIC CHARACTERIZATION OF THE CELL‐SURFACE GLYCANS OF SYMBIOTIC DINOFLAGELLATES (SYMBIODINIUM SPP.)1

Symbiodinium spp. dinoflagellates are common symbionts of marine invertebrates. The cell‐surface glycan profile may determine whether a particular Symbiodinium is able to establish and maintain a stable symbiotic relationship. To characterize this profile, eight Symbiodinium cultures were examined using eight glycan‐specific fluorescent lectin probes. Confocal imaging and flow‐cytometric analysis were used to determine significant levels of binding of each probe to the cell surface. No significant variation in glycan profile was seen within each Symbiodinium culture, either over time or over growth phase. No cladal trends in glycan profile were found, but of note, two different Symbiodinium cultures (from clades A and B) isolated from one host species had very similar profiles, and two other cultures (from clades B and F) from different host species had identical profiles. Two lectin probes were particularly interesting: concanavalin A (ConA) and Griffonia simplicifolia‐II (GS‐II). The ConA probe showed significant binding to all Symbiodinium cultures, suggesting the widespread presence of cell‐surface mannose residues, while the GS‐II probe, which is specific for glycans possessing N‐acetyl groups, showed significant binding to six of eight Symbiodinium cultures. Other probes showed significant binding to the following percentage of Symbiodinium cultures examined: wheat germ agglutinin (WGA), 37.5%; peanut agglutinin (PNA), 50%; Helix pomatia agglutinin (HPA), 50%; phytohemagglutinin‐L (PHA‐L), 62.5%; soybean agglutinin (SBA), 50%; and Griffonia simplicifolia‐IB₄ (GS‐IB₄), 12.5%. This study highlights the complexity of cell‐surface glycan assemblages and their potential role in the discrimination of different dinoflagellate symbionts by cnidarian hosts.     

Prostaglandin endoperoxide synthetase-mediated metabolism of carcinogenic aromatic amines and their binding to DNA and protein

The arachidonic acid-dependent metabolism of the carcinogens, 2-naphthylamine, 4-aminobiphenyl, 2-aminofluorene, benzidine, and N-methyl-4-aminoazobenzene was mediated by a prostaglandin endoperoxide synthetase preparation. Phenacetin, a suspected carcinogen, was not a substrate but its deacetylated metabolite, $\text{p}$-phenetidine, was rapidly oxidized. For each arylamine, extensive metabolism (14–81%) was observed, resulting in high levels of products bound covalently to protein. A low level of binding to added DNA was also detected for each substrate, except $\text{p}$-phenetidine and N-methyl-4-aminoazobenzene. Chromatography of the ethyl acetate-extractable metabolites indicated that the major products were N-oxidized and/or C-oxidized derivatives.     

Tolerance to Environmental Contaminants in the Mummichog,Fundulus heteroclitus

Mummichogs or killifish (Fundulus heteroclitus) are abundant estuarine fish that can tolerate widely varying environmental conditions. They are found in some highly contaminated sites, and their development of tolerance to toxicants has been studied. Populations have developed resistance to methylmercury, kepone, dioxins, polychlorinated biphenyls, and polyaromatic hydrocarbons. This article describes what is known about their tolerance, and discusses similarities and differences among these resistant populations. In some cases tolerance is seen only in early life stages, while in other cases tolerance is seen in adults also. In many of the populations, adults show signs of stress. The mechanism of embryo tolerance to meHg appears to be reduced chorionic permeability, and more rapid development through sensitive stages. One mechanism of resistance to dioxins, PCBs and PAHs is non-responsiveness (lack of inducibility) of CYP1A. In some populations it is already elevated, while in others it is not. Another mechanism in the PAH-resistant fish is elevation of the phase II metabolizing enzyme, GST. The tolerance appears to be genetic. In populations in which it has been studied, the age structure is skewed towards younger fish, and adults appear to put more energy into reproduction as a way of maintaining the population in the stressful environment.