Paola Branduardi

Bio: Paola Branduardi is an academic researcher from University of Milano-Bicocca. The author has contributed to research in topics: Saccharomyces cerevisiae & Medicine. The author has an hindex of 20, co-authored 23 publications receiving 2680 citations. Previous affiliations of Paola Branduardi include University of Milan.

Recombinant Protein Production in Yeasts

Abstract: Recombinant protein production is a multibillion-dollar market. The development of a new product begins with the choice of a production host. While one single perfect host for every protein does not exist, several expression systems ranging from bacterial hosts to mammalian cells have been established. Among them, yeast cell factories combine the advantages of being single cells, such as fast growth and easy genetic manipulation, as well as eukaryotic features including a secretory pathway leading to correct protein processing and post-translational modifications. In this respect, especially the engineering of yeast glycosylation to produce glycoproteins of human-like glycan structures is of great interest. Additionally, different attempts of cellular engineering as well as the design of different production processes that are leading to improved productivities are presented. With the advent of cheaper next-generation sequencing techniques, systems biotechnology approaches focusing on genome scale analyses will advance and accelerate yeast cell factories and thus recombinant protein production processes in the near future. In this review we summarize advantages and limitations of the main and most promising yeast hosts, including Saccharomyces cerevisiae, Pichia pastoris, and Hansenula polymorpha as those presently used in large scale production of heterologous proteins.

Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview

Abstract: Different species of microorganisms including yeasts, filamentous fungi and bacteria have been used in the past 25 years for the controlled production of foreign proteins of scientific, pharmacological or industrial interest. A major obstacle for protein production processes and a limit to overall success has been the abundance of misfolded polypeptides, which fail to reach their native conformation. The presence of misfolded or folding-reluctant protein species causes considerable stress in host cells. The characterization of such adverse conditions and the elicited cell responses have permitted to better understand the physiology and molecular biology of conformational stress. Therefore, microbial cell factories for recombinant protein production are depicted here as a source of knowledge that has considerably helped to picture the extremely rich landscape of in vivo protein folding, and the main cellular players of this complex process are described for the most important cell factories used for biotechnological purposes.

Recombinant protein production in yeasts.

Abstract: Recombinant DNA (rDNA) technologies (genetic, protein, and metabolic engineering) allow the production of a wide range of peptides, proteins, and biochemicals from naturally nonproducing cells. These technologies, now approx 25 yr old, have become one of the most important technologies developed in the twentieth century. Pharmaceutical products and industrial enzymes were the first biotech products on the world market made by means of rDNA. Despite important advances in rDNA applications in mammalian cells, yeasts still represent attractive hosts for the production of heterologous proteins. In this review we summarize advantages and limitations of the main and most promising yeast hosts.

Improvement of Lactic Acid Production in Saccharomyces cerevisiae by Cell Sorting for High Intracellular pH

Abstract: Yeast strains expressing heterologous l-lactate dehydrogenases can produce lactic acid. Although these microorganisms are tolerant of acidic environments, it is known that at low pH, lactic acid exerts a high level of stress on the cells. In the present study we analyzed intracellular pH (pHi) and viability by staining with cSNARF-4F and ethidium bromide, respectively, of two lactic-acid-producing strains of Saccharomyces cerevisiae, CEN.PK m850 and CEN.PK RWB876. The results showed that the strain producing more lactic acid, CEN.PK m850, has a higher pHi. During batch culture, we observed in both strains a reduction of the mean pHi and the appearance of a subpopulation of cells with low pHi. Simultaneous analysis of pHi and viability proved that the cells with low pHi were dead. Based on the observation that the better lactic-acid-producing strain had a higher pHi and that the cells with low pHi were dead, we hypothesized that we might find better lactic acid producers by screening for cells within the highest pHi range. The screening was performed on UV-mutagenized populations through three consecutive rounds of cell sorting in which only the viable cells within the highest pHi range were selected. The results showed that lactic acid production was significantly improved in the majority of the mutants obtained compared to the parental strains. The best lactic-acid-producing strain was identified within the screening of CEN.PK m850 mutants.

Osmotic Stress Signaling and Osmoadaptation in Yeasts

Abstract: The ability to adapt to altered availability of free water is a fundamental property of living cells. The principles underlying osmoadaptation are well conserved. The yeast Saccharomyces cerevisiae is an excellent model system with which to study the molecular biology and physiology of osmoadaptation. Upon a shift to high osmolarity, yeast cells rapidly stimulate a mitogen-activated protein (MAP) kinase cascade, the high-osmolarity glycerol (HOG) pathway, which orchestrates part of the transcriptional response. The dynamic operation of the HOG pathway has been well studied, and similar osmosensing pathways exist in other eukaryotes. Protein kinase A, which seems to mediate a response to diverse stress conditions, is also involved in the transcriptional response program. Expression changes after a shift to high osmolarity aim at adjusting metabolism and the production of cellular protectants. Accumulation of the osmolyte glycerol, which is also controlled by altering transmembrane glycerol transport, is of central importance. Upon a shift from high to low osmolarity, yeast cells stimulate a different MAP kinase cascade, the cell integrity pathway. The transcriptional program upon hypo-osmotic shock seems to aim at adjusting cell surface properties. Rapid export of glycerol is an important event in adaptation to low osmolarity. Osmoadaptation, adjustment of cell surface properties, and the control of cell morphogenesis, growth, and proliferation are highly coordinated processes. The Skn7p response regulator may be involved in coordinating these events. An integrated understanding of osmoadaptation requires not only knowledge of the function of many uncharacterized genes but also further insight into the time line of events, their interdependence, their dynamics, and their spatial organization as well as the importance of subtle effects.