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Expression of the Saccharomyces cerevisiae FDH (formate dehydrogenase) in Escherichia coli: implications for recombinant protein production
S406
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
[P-I.172] Expression of the Saccharomyces cerevisiae FDH (formate dehydrogenase) in Escherichia coli: implications for recombinant protein production
ment yeast robustness; this characteristic makes then the obtained strains attractive for biotechnological applications. doi:10.1016/j.jbiotec.2010.09.539
V. Longo, D. Porro, P. Branduardi ∗
[P-I.174]
Department of Biotechnology and Bioscience, University of MilanoBicocca, Italy
Monitoring oxidative and acidic stress at single cell level
In recent years, the number of recombinant proteins used for therapeutic and industrial applications has enormously increased. Due to the well known biochemical and genetic tools, coupled with relative simplicity and inexpensive and fast high-density cultivation techniques, Escherichia coli is among the most common hosts for recombinant protein production. The main strategies to obtain high expression levels of recombinant proteins of interest imply several aspects of transcriptional and translational regulations, such as expression vectors design, gene dosage, promoter strength, mRNA stability, translation initiation and termination. Another strategy that can be considered to increase the product of interest is engineering the host system to obtain high level of biomass. In this approach we overexpressed a biologically active NAD(+) -dependent formate dehydrogenase from Saccharomyces cerevisiae (ScFDH) in two different E. coli strains (BL21 and Rosetta Tuner) under aerobic conditions. We observed that in the recombinant hosts the biomass accumulation is faster than in the control strains. The possible returns in terms of recombinant protein production will be also shown and discussed. doi:10.1016/j.jbiotec.2010.09.538 [P-I.173] Ascorbic acid producing yeasts learn from plants how to recycle it T. Fossati, N. Solinas, D. Porro, P. Branduardi ∗ Department of Biotechnology and Bioscience, University of MilanoBicocca, Italy Micro-organisms employed in industrial fermentation processes are often subjected to different stressful conditions that negatively affect growth and productivity. Therefore stress resistance is an important property for their application. Our group has previously developed Saccharomyces cerevisiae recombinant strains able to convert D-glucose in L-ascorbic acid (L-AA) through the introduction of the plant biosynthetic pathway; L-AA is an important antioxidant in higher eukaryotes but does not naturally occur in yeast cells, which accumulate an analogue molecule, D-erythroascorbic acid (D-EA). The L-AA producing strains were proved to be more resistant (also defined ‘robust’) to several stress conditions some of which can be often encountered in industrial fermentations. The aim of the present work is to increase, in these strains, the availability of intracellular antioxidants through their recycle, in order to obtain a stronger robustness. L-AA is regenerated in plants by the action of monodehydroascorbate reductase (MDAR) and dehydroascorbate reductase (DHAR). We have therefore introduced these activities in the L-AA producing strains: while MDAR has no effect in the tested conditions, DHAR expression led to an increase of both L-AA accumulated levels and strain robustness in the presence of H2O2 and acetic acid. In conclusion, this work shows that the implementation of antioxidant recycling systems is a successful strategy to aug-
S. Passolunghi, T. Fossati, P. Branduardi, D. Porro ∗ Department of Biotechnology and Bioscience, University of MilanoBicocca, Italy The ideal cell factory performs a bioprocess with high production, productivity and product yield. To fully get this goal, the cell factory and/or the cellular environment have to be often engineered or modified. Furthermore, the cell factory has also to be robust enough to overcome the physicochemical constraints often encountered during industrial processes. Looking for a robust host, we have developed a Saccharomyces cerevisiae strain able to convert D-glucose in L-Ascorbic Acid (L-AA). This goal has been obtained by implementing endogenous enzymatic activities with heterologous ones, fished from plant kingdom. Thanks to the endogenous production of L-AA, which is widely recognised to be a powerful oxygen scavenger, the recombinant strain becomes more tolerant to various stresses, including those driven by oxidative conditions, low inorganic or organic pH values. Aimed to shed more light on this better fitness, we challenged control and L-AA producing cells with different stresses caused by various organic acids and by hydrogen peroxide. We determined by flow cytometry the amount of reactive oxygen species (ROS) and the viability degree of any single cell in stationary and dynamic growth conditions in order to track the stressful pathway going from the appearance of the stress to the death or to the recovery of the cell. Moreover, we are optimizing methods for the ROS staining even in the presence of organic acids to extend the panel of observations and to evaluate if the different stresses could cause different responses that can be visualized over time also at the level of cellular subpopulations. Detailing the mechanisms of acid stress may be helpful to understand the network of tolerance and could be crucial for increasing yeast robustness to obtain cell factories able to guarantee competitive performance during acid production. doi:10.1016/j.jbiotec.2010.09.540 [P-I.175] Acid stress response and tolerance in vitamin C producing yeasts T. Fossati, S. Passolunghi, P. Branduardi, D. Porro ∗ Department of Biotechnology and Bioscience, University of MilanoBicocca, Italy Industrial Biotechnology represents a continuous developing field with multiple applications. Many heterologous proteins, enzymes and flavors are already produced through industrial fermentation processes and increasing attention is given to the production of biofuels and monomers for the construction of biomaterials starting from renewable resources. Unfortunately in industrial bioprocesses, microorganisms are subjected to limiting conditions (i.e. extreme temperatures, nonoptimal pH, high oxygenation, hypoxia and osmotic stress, among others), which stress the cell factory, challenging cellular responses and affecting production, productivity and yield of the bioprocess. Organic acids represent one example of such stressful agents, which can be regarded to both as final products (such as lactic
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
[P-I.172] Expression of the Saccharomyces cerevisiae FDH (formate dehydrogenase) in Escherichia coli: implications for recombinant protein production
ment yeast robustness; this characteristic makes then the obtained strains attractive for biotechnological applications. doi:10.1016/j.jbiotec.2010.09.539
V. Longo, D. Porro, P. Branduardi ∗
[P-I.174]
Department of Biotechnology and Bioscience, University of MilanoBicocca, Italy
Monitoring oxidative and acidic stress at single cell level
In recent years, the number of recombinant proteins used for therapeutic and industrial applications has enormously increased. Due to the well known biochemical and genetic tools, coupled with relative simplicity and inexpensive and fast high-density cultivation techniques, Escherichia coli is among the most common hosts for recombinant protein production. The main strategies to obtain high expression levels of recombinant proteins of interest imply several aspects of transcriptional and translational regulations, such as expression vectors design, gene dosage, promoter strength, mRNA stability, translation initiation and termination. Another strategy that can be considered to increase the product of interest is engineering the host system to obtain high level of biomass. In this approach we overexpressed a biologically active NAD(+) -dependent formate dehydrogenase from Saccharomyces cerevisiae (ScFDH) in two different E. coli strains (BL21 and Rosetta Tuner) under aerobic conditions. We observed that in the recombinant hosts the biomass accumulation is faster than in the control strains. The possible returns in terms of recombinant protein production will be also shown and discussed. doi:10.1016/j.jbiotec.2010.09.538 [P-I.173] Ascorbic acid producing yeasts learn from plants how to recycle it T. Fossati, N. Solinas, D. Porro, P. Branduardi ∗ Department of Biotechnology and Bioscience, University of MilanoBicocca, Italy Micro-organisms employed in industrial fermentation processes are often subjected to different stressful conditions that negatively affect growth and productivity. Therefore stress resistance is an important property for their application. Our group has previously developed Saccharomyces cerevisiae recombinant strains able to convert D-glucose in L-ascorbic acid (L-AA) through the introduction of the plant biosynthetic pathway; L-AA is an important antioxidant in higher eukaryotes but does not naturally occur in yeast cells, which accumulate an analogue molecule, D-erythroascorbic acid (D-EA). The L-AA producing strains were proved to be more resistant (also defined ‘robust’) to several stress conditions some of which can be often encountered in industrial fermentations. The aim of the present work is to increase, in these strains, the availability of intracellular antioxidants through their recycle, in order to obtain a stronger robustness. L-AA is regenerated in plants by the action of monodehydroascorbate reductase (MDAR) and dehydroascorbate reductase (DHAR). We have therefore introduced these activities in the L-AA producing strains: while MDAR has no effect in the tested conditions, DHAR expression led to an increase of both L-AA accumulated levels and strain robustness in the presence of H2O2 and acetic acid. In conclusion, this work shows that the implementation of antioxidant recycling systems is a successful strategy to aug-
S. Passolunghi, T. Fossati, P. Branduardi, D. Porro ∗ Department of Biotechnology and Bioscience, University of MilanoBicocca, Italy The ideal cell factory performs a bioprocess with high production, productivity and product yield. To fully get this goal, the cell factory and/or the cellular environment have to be often engineered or modified. Furthermore, the cell factory has also to be robust enough to overcome the physicochemical constraints often encountered during industrial processes. Looking for a robust host, we have developed a Saccharomyces cerevisiae strain able to convert D-glucose in L-Ascorbic Acid (L-AA). This goal has been obtained by implementing endogenous enzymatic activities with heterologous ones, fished from plant kingdom. Thanks to the endogenous production of L-AA, which is widely recognised to be a powerful oxygen scavenger, the recombinant strain becomes more tolerant to various stresses, including those driven by oxidative conditions, low inorganic or organic pH values. Aimed to shed more light on this better fitness, we challenged control and L-AA producing cells with different stresses caused by various organic acids and by hydrogen peroxide. We determined by flow cytometry the amount of reactive oxygen species (ROS) and the viability degree of any single cell in stationary and dynamic growth conditions in order to track the stressful pathway going from the appearance of the stress to the death or to the recovery of the cell. Moreover, we are optimizing methods for the ROS staining even in the presence of organic acids to extend the panel of observations and to evaluate if the different stresses could cause different responses that can be visualized over time also at the level of cellular subpopulations. Detailing the mechanisms of acid stress may be helpful to understand the network of tolerance and could be crucial for increasing yeast robustness to obtain cell factories able to guarantee competitive performance during acid production. doi:10.1016/j.jbiotec.2010.09.540 [P-I.175] Acid stress response and tolerance in vitamin C producing yeasts T. Fossati, S. Passolunghi, P. Branduardi, D. Porro ∗ Department of Biotechnology and Bioscience, University of MilanoBicocca, Italy Industrial Biotechnology represents a continuous developing field with multiple applications. Many heterologous proteins, enzymes and flavors are already produced through industrial fermentation processes and increasing attention is given to the production of biofuels and monomers for the construction of biomaterials starting from renewable resources. Unfortunately in industrial bioprocesses, microorganisms are subjected to limiting conditions (i.e. extreme temperatures, nonoptimal pH, high oxygenation, hypoxia and osmotic stress, among others), which stress the cell factory, challenging cellular responses and affecting production, productivity and yield of the bioprocess. Organic acids represent one example of such stressful agents, which can be regarded to both as final products (such as lactic