Expression of lignocellulolytic enzymes in Pichia pastoris

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 we could develop strains capable of secreting gram quantities of enzyme per liter in fed-batch cultivations. Additionally, we compared our achieved yields of secreted enzymes and the corresponding activities to literature data. CONCLUSION: In our experiments we could clearly show the importance of gene optimization and strain characterization for successfully improving secretion levels. We also present a basic guideline how to correctly interpret the interplay of promoter strength and gene dosage for a successful improvement of the secretory production of lignocellulolytic enzymes in Pichia pastoris

Distinct ATOH1 and Neurog3 requirements define tuft cells as a new secretory cell type in the intestinal epithelium

Tuft cells represent a fourth type of intestinal secretory cell that constitutes the primary source of endogenous intestinal opioids and are the only epithelial cell that constitutively express cyclooxygenases

Transient expression of Ngn3 in Xenopus endoderm promotes early and ectopic development of pancreatic beta and delta cells

Author Posting. © The Author(s), 2011. This is the author's version of the work. It is posted here by permission of John Wiley & Sons for personal use, not for redistribution. The definitive version was published in genesis 50 (2012): 271-285, doi:10.1002/dvg.20828.Promoting ectopic development of pancreatic beta cells from other cell types is one of the strategies being pursued for the treatment of diabetes. To achieve this, a detailed outline of the molecular lineage that operates in pancreatic progenitor cells to generate beta cells over other endocrine cell types is necessary. Here, we demonstrate that early transient expression of the endocrine progenitor bHLH protein Neurogenin 3 (Ngn3) favors the promotion of pancreatic beta and delta cell fates over an alpha cell fate, while later transient expression promotes ectopic development of all three endocrine cell fates. We found that short-term activation of Ngn3 in Xenopus laevis endoderm just after gastrulation was sufficient to promote both early and ectopic development of beta and delta cells. By examining gene expression changes four hours after Ngn3 activation we identified several new downstream targets of Ngn3. We show that several of these are required for the promotion of ectopic beta cells by Ngn3 as well as for normal beta cell development. These results provide new detail regarding the Ngn3 transcriptional network operating in endocrine progenitor cells to specify a beta cell phenotype and should help define new approaches to promote ectopic development of beta cells for diabetes therapy.National Institutes of Health (DK077197

Inhibition of Gap Junction Communication at Ectopic Eph/ephrin Boundaries Underlies Craniofrontonasal Syndrome

Mutations in X-linked ephrin-B1 in humans cause craniofrontonasal syndrome (CFNS), a disease that affects female patients more severely than males. Sorting of ephrin-B1–positive and –negative cells following X-inactivation has been observed in ephrin-B1(+/−) mice; however, the mechanisms by which mosaic ephrin-B1 expression leads to cell sorting and phenotypic defects remain unknown. Here we show that ephrin-B1(+/−) mice exhibit calvarial defects, a phenotype autonomous to neural crest cells that correlates with cell sorting. We have traced the causes of calvarial defects to impaired differentiation of osteogenic precursors. We show that gap junction communication (GJC) is inhibited at ectopic ephrin boundaries and that ephrin-B1 interacts with connexin43 and regulates its distribution. Moreover, we provide genetic evidence that GJC is implicated in the calvarial defects observed in ephrin-B1(+/−) embryos. Our results uncover a novel role for Eph/ephrins in regulating GJC in vivo and suggest that the pleiotropic defects seen in CFNS patients are due to improper regulation of GJC in affected tissues

Wnt5b–Ryk pathway provides directional signals to regulate gastrulation movement

The soluble ligand Wnt5b repels cells expressing the Ryk (related to tyrosine kinase) receptor, establishing directional motility during gastrulation

PTP1B regulates Eph receptor function and trafficking

Changes in protein tyrosine phosphatase 1B expression affect duration and amplitude of EphA3 phosphorylation and cell surface concentration

Insulin-Producing Cells Generated from Dedifferentiated Human Pancreatic Beta Cells Expanded In Vitro

Expansion of beta cells from the limited number of adult human islet donors is an attractive prospect for increasing cell availability for cell therapy of diabetes. However, attempts at expanding human islet cells in tissue culture result in loss of beta-cell phenotype. Using a lineage-tracing approach we provided evidence for massive proliferation of beta-cell-derived (BCD) cells within these cultures. Expansion involves dedifferentiation resembling epithelial-mesenchymal transition (EMT). Epigenetic analyses indicate that key beta-cell genes maintain open chromatin structure in expanded BCD cells, although they are not transcribed. Here we investigated whether BCD cells can be redifferentiated into beta-like cells.Redifferentiation conditions were screened by following activation of an insulin-DsRed2 reporter gene. Redifferentiated cells were characterized for gene expression, insulin content and secretion assays, and presence of secretory vesicles by electron microscopy. BCD cells were induced to redifferentiate by a combination of soluble factors. The redifferentiated cells expressed beta-cell genes, stored insulin in typical secretory vesicles, and released it in response to glucose. The redifferentiation process involved mesenchymal-epithelial transition, as judged by changes in gene expression. Moreover, inhibition of the EMT effector SLUG (SNAI2) using shRNA resulted in stimulation of redifferentiation. Lineage-traced cells also gave rise at a low rate to cells expressing other islet hormones, suggesting transition of BCD cells through an islet progenitor-like stage during redifferentiation.These findings demonstrate for the first time that expanded dedifferentiated beta cells can be induced to redifferentiate in culture. The findings suggest that ex-vivo expansion of adult human islet cells is a promising approach for generation of insulin-producing cells for transplantation, as well as basic research, toxicology studies, and drug screening