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Author

Mariana L. Fazenda

Other affiliations: University of Strathclyde
Bio: Mariana L. Fazenda is an academic researcher from Strathclyde Institute of Pharmacy and Biomedical Sciences. The author has contributed to research in topics: Heterologous expression & Calibration (statistics). The author has an hindex of 6, co-authored 8 publications receiving 1367 citations. Previous affiliations of Mariana L. Fazenda include University of Strathclyde.

Papers
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Journal ArticleDOI
01 Mar 2005-Yeast
TL;DR: The Pichia pastoris expression system is being used successfully for the production of various recombinant heterologous proteins and 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.
Abstract: 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.

1,237 citations

Book ChapterDOI
TL;DR: 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, those producing potentially useful natural products and novel compounds with interesting biological activities by means of submerged culture fermentation processes.
Abstract: 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.

89 citations

Journal ArticleDOI
TL;DR: This review uses pullulan, curdlan, xanthan, and fungal β-glucans as examples of industrially produced microbial exopolysaccharides, and the role of fermenter configuration in their production is examined.
Abstract: 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.

60 citations

Journal ArticleDOI
TL;DR: Morphologically, dispersed mycelium was predominant under controlled DO conditions, with highly branched hyphae, consistent with the enhanced culture growth noted under these conditions, and rheological terms, broths demonstrated shear-thinning behaviour with a yield stress under both DO conditions.
Abstract: Controlling the dissolved oxygen (DO) in the fed-batch culture of the medicinal mushroom Ganoderma lucidum led to a two-fold increase of the maximum biomass productivity compared to uncontrolled DO conditions. By contrast, extracellular polysaccharide (EPS) production was two times higher under oxygen limitation (uncontrolled DO) than under increased oxygen availability (controlled DO). Morphologically, dispersed mycelium was predominant under controlled DO conditions, with highly branched hyphae, consistent with the enhanced culture growth noted under these conditions. While in the uncontrolled DO processes mycelial clumps were the most common morphology throughout the culture. However, in both cultures clamp connections were found. This is an exciting new finding, which widens the applicability of this basidiomycete in submerged fermentation. In rheological terms, broths demonstrated shear-thinning behaviour with a yield stress under both DO conditions. The flow curves were best described by the Herschel-Bulkley model: flow index down to 0.6 and consistency coefficient up to 0.2 and 0.6 Pa sn in uncontrolled and controlled cultures DO, respectively. The pseudoplastic behaviour was entirely due to the fungal biomass, and not to the presence of EPS (rheological analysis of the filtered broth showed Newtonian behaviour). It is clear that dissolved oxygen tension is a critical process parameter that distinctly influences G. lucidum morphology and rheology, affecting the overall performance of the process. This study contributes to an improved understanding of the process physiology of submerged fermentation of G. lucidum.

35 citations

Journal ArticleDOI
TL;DR: The findings of this study show for the first time the potential of NIRS as an input generating for MFA models, contributing to the optimization of cell factory metabolism in real-time.
Abstract: Background: Novel analytical tools, which shorten the long and costly development cycles of biopharmaceuticals are essential. Metabolic flux analysis (MFA) shows great promise in improving our understanding of the metabolism of cell factories in bioreactors, but currently only provides information post-process using conventional off-line methods. MFA combined with real time multianalyte process monitoring techniques provides a valuable platform technology allowing real time insights into metabolic responses of cell factories in bioreactors. This could have a major impact in the bioprocessing industry, ultimately improving product consistency, productivity and shortening development cycles. Results: This is the first investigation using Near Infrared Spectroscopy (NIRS) in situ combined with metabolic flux modelling which is both a significant challenge and considerable extension of these techniques. We investigated the feasibility of our approach using the industrial workhorse Pichia pastoris in a simplified model system. A parental P. pastoris strain (i.e. which does not synthesize recombinant protein) was used to allow definition of distinct metabolic states focusing solely upon the prediction of intracellular fluxes in central carbon metabolism. Extracellular fluxes were determined using off-line conventional reference methods and on-line NIR predictions (calculated by multivariate analysis using the partial least squares algorithm, PLS). The results showed that the PLS-NIRS models for biomass and glycerol were accurate: correlation coefficients, R 2 , above 0.90 and the root mean square error of prediction, RMSEP, of 1.17 and 2.90 g/L, respectively. The analytical quality of the NIR models was demonstrated by direct comparison with the standard error of the laboratory (SEL), which showed that performance of the NIR models was suitable for quantifying biomass and glycerol for calculating extracellular metabolite rates and used as independent inputs for the MFA (RMSEP lower than 1.5 × SEL). Furthermore, the results for the MFA from both datasets passed consistency tests performed for each steady state, showing that the precision of on-line NIRS is equivalent to that obtained by the off-line measurements. Conclusions: The findings of this study show for the first time the potential of NIRS as an input generating for MFA models, contributing to the optimization of cell factory metabolism in real-time.

27 citations


Cited by
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Journal ArticleDOI
TL;DR: The most popular system for producing recombinant mammalian glycosylated proteins is that of mammalian cells while transgenic plants such as Arabidopsis thaliana and others can generate many recombinant proteins.

894 citations

Journal ArticleDOI
TL;DR: This review refers to established tools in protein expression in P. pastoris and highlights novel developments in the areas of expression vector design, host strain engineering and screening for high-level expression strains.
Abstract: Pichia pastoris is an established protein expression host mainly applied for the production of biopharmaceuticals and industrial enzymes. This methylotrophic yeast is a distinguished production system for its growth to very high cell densities, for the available strong and tightly regulated promoters, and for the options to produce gram amounts of recombinant protein per litre of culture both intracellularly and in secretory fashion. However, not every protein of interest is produced in or secreted by P. pastoris to such high titres. Frequently, protein yields are clearly lower, particularly if complex proteins are expressed that are hetero-oligomers, membrane-attached or prone to proteolytic degradation. The last few years have been particularly fruitful because of numerous activities in improving the expression of such complex proteins with a focus on either protein engineering or on engineering the protein expression host P. pastoris. This review refers to established tools in protein expression in P. pastoris and highlights novel developments in the areas of expression vector design, host strain engineering and screening for high-level expression strains. Breakthroughs in membrane protein expression are discussed alongside numerous commercial applications of P. pastoris derived proteins.

749 citations

Journal ArticleDOI
TL;DR: Phylogenetic analysis of the ABC pleiotropic drug resistance family has provided a new view of the evolution of this important class of efflux pumps, and potential therapeutic approaches that could overcome azole resistance are proposed.
Abstract: Fungi cause serious infections in the immunocompromised and debilitated, and the incidence of invasive mycoses has increased significantly over the last 3 decades. Slow diagnosis and the relatively few classes of antifungal drugs result in high attributable mortality for systemic fungal infections. Azole antifungals are commonly used for fungal infections, but azole resistance can be a problem for some patient groups. High-level, clinically significant azole resistance usually involves overexpression of plasma membrane efflux pumps belonging to the ATP-binding cassette (ABC) or the major facilitator superfamily class of transporters. The heterologous expression of efflux pumps in model systems, such Saccharomyces cerevisiae, has enabled the functional analysis of efflux pumps from a variety of fungi. Phylogenetic analysis of the ABC pleiotropic drug resistance family has provided a new view of the evolution of this important class of efflux pumps. There are several ways in which the clinical significance of efflux-mediated antifungal drug resistance can be mitigated. Alternative antifungal drugs, such as the echinocandins, that are not efflux pump substrates provide one option. Potential therapeutic approaches that could overcome azole resistance include targeting efflux pump transcriptional regulators and fungal stress response pathways, blockade of energy supply, and direct inhibition of efflux pumps.

518 citations

Journal ArticleDOI
TL;DR: The key aspects of microbial exopolysaccharide biosynthesis are summarized and the latest engineering approaches toward the production of tailor-made variants with the potential to be used as valuable renewable and high-performance products for medical and industrial applications are highlighted.
Abstract: Bacteria produce a wide range of exopolysaccharides which are synthesized via different biosynthesis pathways. The genes responsible for synthesis are often clustered within the genome of the respective production organism. A better understanding of the fundamental processes involved in exopolysaccharide biosynthesis and the regulation of these processes is critical toward genetic, metabolic and protein-engineering approaches to produce tailor-made polymers. These designer polymers will exhibit superior material properties targeting medical and industrial applications. Exploiting the natural design space for production of a variety of biopolymer will open up a range of new applications. Here, we summarize the key aspects of microbial exopolysaccharide biosynthesis and highlight the latest engineering approaches toward the production of tailor-made variants with the potential to be used as valuable renewable and high-performance products for medical and industrial applications.

387 citations

Journal ArticleDOI
TL;DR: Xylanases have been used in traditional fields such as food, feed and paper industries for a longer time but more and more attention has been paid to using them in producing sugars and other chemicals from lignocelluloses in recent years.

361 citations