Metabolic Engineering of Ammonium Assimilation in Xylose-Fermenting Saccharomyces cerevisiae Improves Ethanol Production

Cofactor imbalance impedes xylose assimilation in Saccharomyces cerevisiae that has been metabolically engineered for xylose utilization. To improve cofactor use, we modified ammonia assimilation in recombinant S. cerevisiae by deleting GDH1, which encodes an NADPH-dependent glutamate dehydrogenase, and by overexpressing either GDH2, which encodes an NADH-dependent glutamate dehydrogenase, or GLT1 and GLN1, which encode the GS-GOGAT complex. Overexpression of GDH2 increased ethanol yield from 0.43 to 0.51 mol of carbon (Cmol) Cmol⁻¹, mainly by reducing xylitol excretion by 44%. Overexpression of the GS-GOGAT complex did not improve conversion of xylose to ethanol during batch cultivation, but it increased ethanol yield by 16% in carbon-limited continuous cultivation at a low dilution rate.     

Growth and enzyme production by three Penicillium species on monosaccharides

The growth and preference for utilisation of various sugar by the Penicillium species Penicillium pinophilum IBT 4186, Penicillium persicinum IBT 13226 and Penicillium brasilianum IBT 20888 was studied in batch cultivations using various monosaccharides as carbon source, either alone or in mixtures. P. pinophilum IBT 4186 and P. persicinum IBT 13226 had a micro(max) around 0.08-0.09 h(-1) using either glucose or xylose as carbon source. The micro(max) of P. brasilianum IBT 20888 was 0.16 and 0.14 h(-1) on glucose and xylose, respectively. Glucose was found to exert repression on the catabolism of mannose, galactose, xylose and arabinose. The three species were able to utilise all the tested monosaccharides, but arabinose was only slowly metabolised. Glucose was also found to repress the production of endoglucanases, endoxylanases and beta-xylosidases. After glucose depletion, the fungi started producing beta-glucosidase and endoglucanases. Xylose did not repress the enzyme production and it induced the production of endoxylanases and beta-xylosidases.     

Experimental investigations of multiple steady states in aerobic continuous cultivations of Saccharomyces cerevisiae

The steady-state behavior of a glucose-limited, aerobic, continuous cultivation of Saccharomyces cerevisiae CEN.PK113-7D was investigated around the critical dilution rate. Oxido-reductive steady states were obtained at dilution rates up to 0.09 h(-1) lower than the critical dilution rate by operating the bioreactor as a productostat, where the dilution rate was controlled on the basis of an ethanol measurement. Thus, the experimental investigations revealed that multiple steady states exist in a region of dilution rates below the critical dilution rate. The existence of multiple steady states was attributed to two distinct physiological effects occurring when growth changed from oxidative to oxido-reductive: (i) a decrease in the efficiency of ATP production and utilization (at ethanol concentrations below 3 g/L) and (ii) repression of the oxidative metabolism (at higher ethanol concentrations). The first effect was best observed at low ethanol concentrations, where multiple steady states were observed even when no repression of the oxidative metabolism was evident, i.e., the oxidative capacity was constant. However, at higher ethanol concentrations repression of the oxidative metabolism was observed (the oxidative capacity decreased), and this resulted in a broader range of dilution rates where multiple steady states could be found.     

Global and 3D Spatial Assessment of Neuroinflammation in Rodent Models of Multiple Sclerosis

Multiple Sclerosis (MS) is a progressive autoimmune inflammatory and demyelinating disease of the central nervous system (CNS). T cells play a key role in the progression of neuroinflammation in MS and also in the experimental autoimmune encephalomyelitis (EAE) animal models for the disease. A technology for quantitative and 3 dimensional (3D) spatial assessment of inflammation in this and other CNS inflammatory conditions is much needed. Here we present a procedure for 3D spatial assessment and global quantification of the development of neuroinflammation based on Optical Projection Tomography (OPT). Applying this approach to the analysis of rodent models of MS, we provide global quantitative data of the major inflammatory component as a function of the clinical course. Our data demonstrates a strong correlation between the development and progression of neuroinflammation and clinical disease in several mouse and a rat model of MS refining the information regarding the spatial dynamics of the inflammatory component in EAE. This method provides a powerful tool to investigate the effect of environmental and genetic forces and for assessing the therapeutic effects of drug therapy in animal models of MS and other neuroinflammatory/neurodegenerative disorders.     

394delTT: a Nordic cystic fibrosis mutation

In a systematic screening for mutations in the gene encoding the cystic fibrosis transmembrane regulator among Danish cystic fibrosis (CF) patients, we identified a mutation in exon 3 (394delTT); this mutation was found to be relatively common in Denmark. We therefore screened for 394delTT in Sweden and Norway, where it turned out to be the second most frequent mutation, accounting for 4% of all CF mutations. It also occurs with a high frequency in Finland, but has not been found in larger surveys of mutations in the CFTR gene. Thus, 394delTT seems to be a specific Nordic CF mutation.