Brian McNeil

Heterologous protein production using the Pichia pastoris expression system

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

Near Infrared Spectroscopy for Bioprocess Monitoring and Control: Current Status and Future Trends

ABSTRACT The development of Near Infrared Spectroscopy has paralleled that of the PC, and the application of NIR in many industries has undergone explosive growth in recent years. This has been particularly apparent in the area of microbial and cell culture system monitoring and control. Potentially, NIR offers the prospect of real-time control of the physiology of cultured cells in fermenters, leading to marked improvements in authenticity, purity and production efficiency. Despite this, NIR is not yet as widely applied within the bioprocessing industry as its potential might suggest. This review critically evaluates the development of this rapidly moving area as it pertains to microbial and cell culture system control and highlights the critical stages in the development of the technology. It indicates the work that must still be carried out if the full potential of NIR is to be exploited in making proteins, hormones and antibiotics by the fermentation route. The review comes at a particularly timely moment when NIR stands on the threshold of widespread acceptance in bioprocessing. This is the ideal moment to assess what the technology can offer the microbiologist, and where it may develop in the future.

The Acetone Butanol Fermentation

Publisher Summary The list of products produced by the fermentation route is wide, and includes, or has included, glycerol, ethanol, 2,3-butanediol, isopropanol, lactic acid, acetone, and butanol. The most important of these fermentations has been the acetone/butanol fermentation carried out by microbes of the genus Clostridium. This process is carried out on an industrial scale only where special conditions permit it to rival feedstock produced from a petrochemical source. The knowledge of the organisms vitamin requirements allowed formulation of the synthetic media in common use. Economic pressure on the fermentation led to the necessity to recover all possible products of the fermentation; thus, the dried biomass (being high in riboflavin), was used as a feed additive for ruminants, and the fermentation gases hydrogen and carbon dioxide were recovered. Such integrated plants achieved a high degree of efficiency in operation. Similarly, rising molasses prices led to investigations into alternative, cheaper sources of fermentable carbohydrates that proved successful at least to the pilot plant stage.

At-line monitoring of a submerged filamentous bacterial cultivation using near-infrared spectroscopy

The use of near infra-red spectroscopy (NIRS) to monitor a submerged filamentous bacterial bioprocess was investigated. An industrial strain of the filamentous bacterium Streptomyces fradiae was cultured in a 12 litre stirred tank reactor (STR) using a complex medium. This mycelial 4 phase (oil, water, gas and solid) system produced highly complex and variable matrices, therefore monitoring such a complex fluid with NIRS represented a considerable challenge. Nevertheless, successful models for four key analytes (methyl oleate, glucose, glutamate and ammonium) were built at-line (rapid off-line) using NIRS. In the present study, the methods used to formulate, select and validate the models for the key analytes are discussed, with particular emphasis on how the model performance can be critically evaluated. Since previous reports on NIRS in monitoring bioprocesses have either involved simpler matrices, or, in filamentous systems, have not discussed how NIRS models can be critically assessed, the emphasis in the present study on providing an insight into the modelling process in such a complex matrix, may be particularly important to the applicability of NIRS to such industrial bioprocesses.

Oxidative stress in industrial fungi

Fungi are amongst the most industrially important microorganisms in current use within the biotechnology industry. Most such fungal cultures are highly aerobic in nature, a character that has been frequently referred to in both reactor design and fungal physiology. The most fundamentally significant outcome of the highly aerobic growth environment in fermenter vessels is the need for the fungal culture to effectively combat in the intracellular environment the negative consequences of high oxygen transfer rates. The use of oxygen as the respiratory substrate is frequently reported to lead to the development of oxidative stress, mainly due to oxygen-derived free radicals, which are collectively termed as reactive oxygen species (ROS). Recently, there has been extensive research on the occurrence, extent, and consequences of oxidative stress in microorganisms, and the underlying mechanisms through which cells prevent and repair the damage caused by ROS. In the present study, we critically review the current understanding of oxidative stress events in industrially relevant fungi. The review first describes the current state of knowledge of ROS concisely, and then the various antioxidant strategies employed by fungal cells to counteract the deleterious effects, together with their implications in fungal bioprocessing are also discussed. Finally, some recommendations for further research are made.

Submerged Culture Fermentation of “Higher Fungi”: The Macrofungi

Publisher Summary This chapter evaluates recent advances in submerged liquid cultivation of the vegetative or mycelial forms of those members of the “higher fungi” that produce macroscopic spore-bearing structures, namely the macrofungi. The chapter focuses on those producing potentially useful natural products and novel compounds with interesting biological activities by means of submerged culture fermentation processes. The taxonomy of the fungi is in a state of rapid flux arising from the widespread use of molecular genetics to characterize and classify the organisms, and many of earlier ideas on their phylogenetic relationships must be revised. Fungi can be defined as being eukaryotic, characteristically mycelial organisms, with chitin-based cell walls, an absorptive nutrition, and which reproduce by production of spores. Some of the macrofungi are edible, and some also have biotechnological and environmental applications. Edible macrofungi have long been consumed both as valuable protein and energy sources, and also for improving human health and longevity.

The development and application of high throughput cultivation technology in bioprocess development

This review focuses on recent progress in the technology of high throughput (HTP) cultivation and its increasing application in quality by design (QbD) -driven bioprocess development. Several practical HTP strategies aimed at shortening process development (PD) timelines from DNA to large scale processes involving commercially available HTP technology platforms, including microtiter plate (MTP) culture, micro-scale bioreactors, and in parallel fermentation systems, etc., are critically reviewed in detail. This discussion focuses upon the relative strengths and weaknesses or limitations of each of these platforms in this context. Emerging prototypes of micro-bioreactors reported recently, such as milliliter (mL) scale stirred tank bioreactors, and microfludics integrated micro-scale bioreactors, and their potential for practical application in QbD-driven HTP process development are also critically appraised. The overall aim of such technology is to rapidly gain process insights, and since the analytical technology deployed in HTP systems is critically important to the achievement of this aim, this rapidly developing area is discussed. Finally, general future trends are critically reviewed.

Effect of process temperature, pH and suspended solids content upon pasteurization of a model agricultural waste during thermophilic aerobic digestion

Thermophilic aerobic digestion(TAD), or liquid composting, is a versatile new process for the treatment and stabilization of high strength wastes of liquid or, perhaps more importantly, slurry consistency.

Deconvolution of near-infrared spectral information for monitoring mycelial biomass and other key analytes in a submerged fungal bioprocess

Near-infrared spectroscopy is a promising technique for the rapid monitoring of submerged culture bioprocesses. However, despite the key role of mycelial (filamentous fungal and bacterial) micro-organisms in the manufacture of antibiotics and other valuable therapeutics, there is little information on the application of the technique to monitor mycelial bioprocesses. In part, this is due to the complex and spectroscopically challenging matrices, which result from the growth of these micro-organisms. Moreover, there is a particular lack of any detailed mechanistic information on how models for the prediction of the concentration of key analytes (e.g. biomass, substrates, product) can be constructed, evaluated and improved using the spectral data arising from such complex matrices. We investigated the near-infrared spectra of culture fluid from a submerged fungal bioprocess, for monitoring the concentrations of mycelial biomass and other key analytes. Several empirical models were developed for predicting the concentration of the analytes, using multivariate statistical techniques. Despite the filamentous nature of the biomass and the resulting complexity of the spectral variations, empirical models could be developed for the prediction of this analyte, using biomass ‘specific’ information. SEP values of <1 g/l could be achieved on external validation, for models developed in the concentration range of 0–20 g/l. The concentrations of the substrates, total sugars (as glucose equivalents) and ammonium, could also be predicted, simultaneously. However, the product (penicillin) and by product (extracellular proteins) levels had to be monitored on the cell free culture fluid, due to their relatively low concentration. Here we report upon how the spectral information can be deconvoluted for predicting the levels of the analytes and upon how the ‘analyte specific’ information in the spectral data can be used to inform and assist the modelling process, in order to increase confidence in exactly what is being modelled.

Operating bioreactors for microbial exopolysaccharide production

There is considerable interest in exploiting the novel physical and biological properties of microbial exopolysaccharides in industry and medicine. For economic and scientific reasons, large scale production under carefully monitored and controlled conditions is required. Producing exopolysaccharides in industrial fermenters poses several complex bioengineering and microbiological challenges relating primarily to the very high viscosities of such culture media, which are often exacerbated by the producing organism’s morphology. What these problems are, and the strategies for dealing with them are discussed critically in this review, using pullulan, curdlan, xanthan, and fungal β-glucans as examples of industrially produced microbial exopolysaccharides. The role of fermenter configuration in their production is also examined.