Fatty alcohols production by oleaginous yeast.

We have engineered Rhodosporidium toruloides to produce fatty alcohols by expressing a fatty acyl-CoA reductase from Marinobacter aquaeolei VT8. Production of fatty alcohols in flasks was achieved in different fermentation media at titers ranging from 0.2 to 2 g/L. In many of the conditions tested, more than 80 % of fatty alcohols were secreted into the cultivation broth. Through fed-batch fermentation in 7 L bioreactors, over 8 g/L of C(16)-C(18) fatty alcohols were produced using sucrose as the substrate. This is the highest titer ever reported on microbial production of fatty alcohols to date.

Activity of maize transglutaminase overexpressed in Escherichia coli inclusion bodies: an alternative to protein refolding.

Transglutaminases (TGases) catalyze protein post-translational modification by ε-(γ-glutamyl) links and covalent polyamine conjugation. In plants, this enzyme is poorly characterized and only the maize plastidial TGase gene (tgz) has been cloned. The tgz gene (Patent WWO03102128) had been subcloned and overexpressed in Escherichia coli cells, and the recombinant protein (TGZp) was present mainly in inclusion bodies (IB) fraction. In this work, after overexpression of TGZ15p and SDS-PAGE IB fraction analysis, bands about 65 and 56 kDa were obtained. Western blot, alkylation and MALDI-TOF/TOF analyses indicated that the 56 kDa band corresponded to a truncated sequence from the native TGZ15p (expected MW 65 kDa), by elimination of a chloroplast signal peptide fragment during expression processing. So that large-scale protein production and protein crystallization can be applied, we characterized the TGZ15p enzyme activity in the IB protein fraction, with and without refolding. Results indicate that it presented the biochemical characteristics of other described TGases, showing a certain plant-substrate preference. Solubilization of the IB fraction with Triton X-100 as nondenaturing detergent yielded active TGZ without the need for refolding, giving activity values comparable to those of the refolded protein, indicating that this is a valuable, faster way to obtain TGZ active protein.

Amyloid-like protein inclusions in tobacco transgenic plants.

The formation of insoluble protein deposits in human tissues is linked to the onset of more than 40 different disorders, ranging from dementia to diabetes. In these diseases, the proteins usually self-assemble into ordered ß-sheet enriched aggregates known as amyloid fibrils. Here we study the structure of the inclusions formed by maize transglutaminase (TGZ) in the chloroplasts of tobacco transplastomic plants and demonstrate that they have an amyloid-like nature. Together with the evidence of amyloid structures in bacteria and fungi our data argue that amyloid formation is likely a ubiquitous process occurring across the different kingdoms of life. The discovery of amyloid conformations inside inclusions of genetically modified plants might have implications regarding their use for human applications.

Dark-lumen MR colonography with fecal tagging: a comparison of water enema and air methods of colonic distension for detecting colonic neoplasms.

The purpose was to evaluate MR colonography (MRC) with barium fecal tagging in detecting colorectal pathology and to determine how air-based and water-based colonic distension influences MRC. We studied 83 patients with high risk of colonic neoplasms. All received oral barium sulfate for colonic preparation before unenhanced and enhanced T1-weighted gradient-echo MRC using either water (n=54) or air (n=29) for colonic distension. Fecal tagging, distension, and artifacts were recorded. All patients underwent conventional colonoscopy within 2 weeks of MRC; the techniques were compared for detection of malignant neoplasms and polyps >or=1 cm, 6-9 mm, and or=1 cm, but more air-distended MRC were excluded for poor quality. MRC with fecal tagging is useful for detecting lesions >or=1 cm. Air distension was inferior to water distension in most aspects. Water-based colonic distension should be used for barium-tagging MRC.