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The Pichia pastoris expression system is being used successfully for the production of various recombinant heterologous proteins. Recent developments with respect to the Pichia expression system have had an impact on not only the expression levels that can be achieved, but also the bioactivity of various heterologous proteins. We review here some of these recent developments, as well as strategies for reducing proteolytic degradation of the expressed recombinant protein at cultivation, cellular and protein levels. The problems associated with post-translational modifications performed on recombinant proteins by P. pastoris are discussed, including the effects on bioactivity and function of these proteins, and some engineering strategies for minimizing unwanted glycosylations. We pay particular attention to the importance of optimizing the physicochemical environment for efficient and maximal recombinant protein production in bioreactors and the role of process control in optimizing protein production is reviewed. Finally, future aspects of the use of the P. pastoris expression system are discussed with regard to the production of complex membrane proteins, such as G protein-coupled receptors, and the industrial and clinical importance of these proteins., (Copyright (c) 2005 John Wiley & Sons, Ltd.)

In the present study the relationship between oxidative stress and elevated culture temperature was examined in an industrially relevant fungal culture, Aspergillus niger B1-D. For the first time, both the intracellular levels of the main stressor species (superoxide radical [O(2) (.-)]) and activities of cellular defensive enzymes (superoxide dismutase [SOD], catalase [CAT], and glutathione peroxide [GPx]) were quantified at varying temperature (25, 30, 35, 40 degrees C) to more fully characterize culture response in different growth phases. Elevated culture temperature led to increased O(2) (.-) levels in various culture phases. In the exponential phase this was due to an enhanced generation of O(2) (.-), whereas in stationary phase a decreased dismutation rate may also have contributed. CAT activities generally increased with culture temperature, whereas GPx activity changed little as temperature rose, indicating that GPx played only a minor role in destroying H(2)O(2) in this A. niger. The combination of elevated temperature (35 degrees C) and increased O(2) supply (50% enrichment) led to decreased levels of O(2) (.-) compared to the cultivation at 35 degrees C gassed with air, probably due to enhanced activity of the alternative fungal respiratory pathway. Our findings indicate that while elevated cultivation temperature does clearly induce oxidative stress events, mechanistically, it does so by a rather more complex route than previous studies indicate. Elevated temperature caused a marked disparity in the activities of SOD and CAT, very distinct from the integrated increase in activity of these enzymes in response to oxidative stress., (Copyright 2003 Wiley Periodicals.)

One of the key goals in bioprocess monitoring is to achieve real-time knowledge of conditions within the bioreactor, i.e., in-situ. Near-infrared spectroscopy (NIRS), with its ability to carry out multi-analyte quantification rapidly with little sample presentation, is potentially applicable in this role. In the present study, the application of NIRS to a complex, fed-batch industrial E. coli (RV308/PHKY531) process was investigated. This process undergoes a series of temperature changes and is vigorously agitated and aerated. These conditions can pose added challenges to in-situ NIRS. Using the measurement of a key analyte (biomass) as an illustration, the details of the relationship between the at-line and in-situ use of NIRS are considered from the viewpoint of both theory and practical application. This study shows that NIRS can be used both at-line and in-situ in order to achieve good predictive models for biomass. There are particular challenges imposed by in-situ operation (loss of wavelength regions and noise) which meant the need for signal optimisation studies. This showed that whilst the at-line modelling process may provide some useful information for the in-situ process, there were distinct differences. This study shows that the in-situ use of NIRS in a highly challenging matrix (similar to those encountered in current industrial practice) is possible, and thus extends previous works in the area., (Copyright 2002 Wiley Periodicals, Inc.)