Background
Trichoderma reesei is one of the best-known cellulolytic organisms, producing large quantities of a complete set of extracellular cellulases and hemicellulases for the degradation of lignocellulosic substances. Hence, T. reesei is a biotechnically important host and it is used commercially in enzyme production, of both native and foreign origin. Many strategies for producing enzymes in T. reesei rely on the cbh1 and other cellulase gene promoters for high level expression and these promoters require induction by sophorose, lactose or other inducers for high productivity during manufacturing.
Results
We described an approach for producing high levels of secreted proteins by overexpression of a transcription factor ACE3 in T. reesei. We refined the ace3 gene structure and identified specific ACE3 variants that enable production of secreted cellulases and hemicellulases on glucose as a sole carbon source (i.e. in the absence of an inducer). These specific ACE3 variants contain a full length Zn2Cys6 binuclear cluster domain at the N-terminus and a defined length of truncations the C-terminus. When expressed at a moderate level in the fungal cells, the ACE3 variants can induce high level expression of cellulases and hemicellulases on glucose (i.e. in the absence of an inducer), and further improve expression on lactose or glucose/sophorose (i.e. in the presence of an inducer). Finally, we demonstrated that this method is applicable to industrial strains and fermentation conditions, improving protein production both in the absence and in the presence of an inducer.
Conclusions
This study demonstrates that overexpression of ACE3 variants enables a high level of protein production in the absence of an inducer, and boosts protein production in the presence of an inducer. It is an efficient approach to increase protein productivity and to reduce manufacturing costs.
As part of the international Matroshka-R and Radi-N experiments, bubble detectors have been used on board the ISS in order to characterise the neutron dose and the energy spectrum of neutrons. Experiments using bubble dosemeters inside a tissue-equivalent phantom were performed during the ISS-16, ISS-18 and ISS-19 expeditions. During the ISS-20 and ISS-21 missions, the bubble dosemeters were supplemented by a bubble-detector spectrometer, a set of six detectors that was used to determine the neutron energy spectrum at various locations inside the ISS. The temperature-compensated spectrometer set used is the first to be developed specifically for space applications and its development is described in this paper. Results of the dose measurements indicate that the dose received at two different depths inside the phantom is not significantly different, suggesting that bubble detectors worn by a person provide an accurate reading of the dose received inside the body. The energy spectra measured using the spectrometer are in good agreement with previous measurements and do not show a strong dependence on the precise location inside the station. To aid the understanding of the bubble-detector response to charged particles in the space environment, calculations have been performed using a Monte-Carlo code, together with data collected on the ISS. These calculations indicate that charged particles contribute <2% to the bubble count on the ISS, and can therefore be considered as negligible for bubble-detector measurements in space.
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