The research in the field of industrial biotechnology, especially regards bioproducts and bioprocesses, are aimed at developing innovative technologies that lead to obtaining compounds with the use of microorganisms, or seeking to enhance existing processes to increase yield, production and productivity, trying to ensure a higher degree of sustainability and reducing environmental impact. To pursue these goals is possible to intervene by adopting a “technologic” approach that includes the development of systems capable of ensuring a more effective control of the parameters that govern the production processes or, with a “molecular / metabolic” approach, acting directly on the host system, namely by working on production capacity of the microorganism itself. To achieve this goal the pathways responsible for the processes of synthesis or secretion of products of interest must be identified and, if necessary, modified, or the environmental conditions in which the organism is operating during the process must be considered, to study how to improve its production capacity even in non-optimal physiological condition. The process conditions that characterize industrial production processes often put the cells through a series of stress that inevitably act negatively on yields. It therefore becomes necessary to identify the limiting factors in relation to the host organism and, on this basis, to act in an appropriate manner. To do this it is possible to adopt different strategies, complementary and not mutually exclusive. The most immediate is the exploration of biodiversity in order to choose a host that is intrinsically and naturally more resistant to the type of stress imposed by the process. This way is not always easy to follow, given the extent of the possible solutions and the lack of resources that allow the exploration and characterization in a reasonable time for the development of a biotechnological process. An alternative strategy is focused on the characterization of the cellular response to these stress conditions to identify the key factors involved in the mechanism. In this way, for example, through genetic manipulation on these factors it could be possible to improve the resistance of the cell itself, or by the transfer of these specific genetic traits to improve the resistance of other micro-organisms selected as host system. The yeast Saccharomyces cerevisiae is one of the most widely used microorganisms for the production of compounds of biotechnological interest because of the available knowledge on the physiology, genetics, biochemistry, and on the existence of technological and molecular tools suitable for its manipulation in order to optimize the production by fermentation. It is important to underline that S. cerevisiae is recognized as a GRAS organism (generally regarded as safe) by the Food & Drug Administration that has allowed the use for the production of pharmaceutical compounds for human use. While it is now increasingly clear the potential of S. cerevisiae as a platform for metabolic engineering, for some heterologous proteins production on a large scale this yeast is not the ideal host system. Very often the expressed proteins are hyper-glycosilated or, if retained in the periplasmic space, they suffer significant degradation. For other industrial productions, especially where the product of interest has to be cheap, the fermentation technologies needed are too complex and sophisticated (and expensive) to be implemented on a large scale. With these assumptions in recent years it has been explored the opportunity of adopting other yeasts, called "unconventional yeast" by developing new systems of expression. In this thesis project an "alternative" yeast, Zygosaccharomyces bailii, is considered. Although it is not well characterized from molecular and genetic point of view, it presents interesting features in view of potential biotechnological production: it allows high yields of biomass, it has a high specific growth rate and a higher resistance compared to S. cerevisiae, to certain types of stress, and in particular to stresses generated by an acid medium. The cell surface, as an area of communication and exchange between the extracellular environment and the cell itself is one of the targets of this study. In S. cerevisiae, thanks to the availability of molecular tools and the knowledge of the entire genome sequence, it is possible the design of in-depth studies and the engineering of the cellular metabolic network. Given the difficulties in its genetic manipulation, in the case of Z. bailii was first necessary to address another preliminary issue. In this diploid yeast the deletion of an essential gene was never been made. It has been developed a reproducible protocol for gene deletion by a gene-targeting approach and it was obtained the deletion of the gene ZbLEU2, who gave birth to the first auxotroph strain of Z. bailii (leuˉ). This represents an important step for a possible use of Z. bailii as host system. Thanks to this protocol a mutant strain of Z. bailii of potential interest for heterologous protein production was also obtained, in analogy with what reported in the literature for S. cerevisiae. The deletion concerns the homologue of ScGAS1, coding for the enzyme β-1 ,3-glucanosyltransferase which catalyzes the crosslinking of cell wall glucans. ZbGAS1. The gene was cloned by PCR and sequenced. The deleted mutant of Z. bailii has morphological and phenotypic characteristics very similar to the correspondent in S. cerevisiae, showing an alteration of the cell wall structure, and enhanced secretive capacity than the wild type strain for some heterologous proteins that have been considered. In parallel to these studies, populations of Z. bailii growing on different carbon sources were analyzed by flow cytometry. The analysis of DNA and protein content was performed to better characterize this yeast not only from molecular point of view, but also to explore its cellular features. The characterization of this unconventional yeast confirmed once again one of the most appreciated features for yeasts used as cell factories: versatility. This property is so strong that yeasts has been exploited for natural abilities, such as production of ethanol, and also for processes where a targeted manipulation was introduced, for example in lactic acid production, just to cite a pair of biotechnological production of industrial relevance. To make this process competitive on the market, in terms of yield, production and productivity, yeasts were pushed to their physiological limits. These limits are given by the accumulation of vast amounts of product that, in the case of ethanol, can cause damage to the lipid component of the plasma membrane, in the case of lactic acid can result in a loss of proper cellular homeostasis with the fall of the intracellular pH. It is therefore necessary to assess whether these limitations can be overcome by acting in particular on the plasma membrane, whose role in controlling the transport and the cellular homeostasis makes it a target of interest to improve the robustness of cells in response to stress generated by the process, such as oxidative stress or generated by low pH. The optimization of transport through the membrane plays a key role in the mechanisms of adaptation to these stresses. In particular, improving the flux of nutrients entering the cell could allow an optimal uptake of nutrient in the cytoplasm (as in the case of bioethanol production), an improvement of outflows from the cells might instead allow effective removal of compounds that may be deleterious for cell viability when present beyond a threshold value (eg organic acids). By focusing on the protein fraction that characterizes the cytoplasmic membrane, we have studied the effects of modulation of the expression of H +-ATPase pump of the plasma membrane (Pma1p), involved in the intracellular pH homeostasis. In particular, the gene ScPMA1 was overexpressed in S. cerevisiae and this overexpression was able to confer a greater resistance to acid stress, evidenced by growth kinetics in the presence of lactic acid. The increased cell viability under restrictive conditions in respect to the wild type strain was also checked by flow cytometry. The use of this tool has enabled the development of a system able to assess quantitatively the degree of robustness of the cells in stressful conditions. With this instrument able to assess the robustness of the cells as a function of various types of stress (oxidative, pH, ...) it is possible to design new interventions of metabolic engineering in order to provide greater resistance to yeast in restrictive process conditions, similar to conditions prevailing in the processes of production of compounds of biotech interest. It will be possible to evaluate the effectiveness of these interventions with the flow cytometer by assessing the response of the engineered cells under restrictive conditions by measuring the ability of cells to increase their robustness. The robustness remains one of the key features of yeasts as microbial cell factories, in particular to reach one of the main goal of White Biotechnology: to provide value products from renewable resources through sustainable processes with low environmental impact.

(2009). Matching biotech needs and yeast physiology. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2009).

Matching biotech needs and yeast physiology

PASSOLUNGHI, SIMONE
2009-12-15

Abstract

The research in the field of industrial biotechnology, especially regards bioproducts and bioprocesses, are aimed at developing innovative technologies that lead to obtaining compounds with the use of microorganisms, or seeking to enhance existing processes to increase yield, production and productivity, trying to ensure a higher degree of sustainability and reducing environmental impact. To pursue these goals is possible to intervene by adopting a “technologic” approach that includes the development of systems capable of ensuring a more effective control of the parameters that govern the production processes or, with a “molecular / metabolic” approach, acting directly on the host system, namely by working on production capacity of the microorganism itself. To achieve this goal the pathways responsible for the processes of synthesis or secretion of products of interest must be identified and, if necessary, modified, or the environmental conditions in which the organism is operating during the process must be considered, to study how to improve its production capacity even in non-optimal physiological condition. The process conditions that characterize industrial production processes often put the cells through a series of stress that inevitably act negatively on yields. It therefore becomes necessary to identify the limiting factors in relation to the host organism and, on this basis, to act in an appropriate manner. To do this it is possible to adopt different strategies, complementary and not mutually exclusive. The most immediate is the exploration of biodiversity in order to choose a host that is intrinsically and naturally more resistant to the type of stress imposed by the process. This way is not always easy to follow, given the extent of the possible solutions and the lack of resources that allow the exploration and characterization in a reasonable time for the development of a biotechnological process. An alternative strategy is focused on the characterization of the cellular response to these stress conditions to identify the key factors involved in the mechanism. In this way, for example, through genetic manipulation on these factors it could be possible to improve the resistance of the cell itself, or by the transfer of these specific genetic traits to improve the resistance of other micro-organisms selected as host system. The yeast Saccharomyces cerevisiae is one of the most widely used microorganisms for the production of compounds of biotechnological interest because of the available knowledge on the physiology, genetics, biochemistry, and on the existence of technological and molecular tools suitable for its manipulation in order to optimize the production by fermentation. It is important to underline that S. cerevisiae is recognized as a GRAS organism (generally regarded as safe) by the Food & Drug Administration that has allowed the use for the production of pharmaceutical compounds for human use. While it is now increasingly clear the potential of S. cerevisiae as a platform for metabolic engineering, for some heterologous proteins production on a large scale this yeast is not the ideal host system. Very often the expressed proteins are hyper-glycosilated or, if retained in the periplasmic space, they suffer significant degradation. For other industrial productions, especially where the product of interest has to be cheap, the fermentation technologies needed are too complex and sophisticated (and expensive) to be implemented on a large scale. With these assumptions in recent years it has been explored the opportunity of adopting other yeasts, called "unconventional yeast" by developing new systems of expression. In this thesis project an "alternative" yeast, Zygosaccharomyces bailii, is considered. Although it is not well characterized from molecular and genetic point of view, it presents interesting features in view of potential biotechnological production: it allows high yields of biomass, it has a high specific growth rate and a higher resistance compared to S. cerevisiae, to certain types of stress, and in particular to stresses generated by an acid medium. The cell surface, as an area of communication and exchange between the extracellular environment and the cell itself is one of the targets of this study. In S. cerevisiae, thanks to the availability of molecular tools and the knowledge of the entire genome sequence, it is possible the design of in-depth studies and the engineering of the cellular metabolic network. Given the difficulties in its genetic manipulation, in the case of Z. bailii was first necessary to address another preliminary issue. In this diploid yeast the deletion of an essential gene was never been made. It has been developed a reproducible protocol for gene deletion by a gene-targeting approach and it was obtained the deletion of the gene ZbLEU2, who gave birth to the first auxotroph strain of Z. bailii (leuˉ). This represents an important step for a possible use of Z. bailii as host system. Thanks to this protocol a mutant strain of Z. bailii of potential interest for heterologous protein production was also obtained, in analogy with what reported in the literature for S. cerevisiae. The deletion concerns the homologue of ScGAS1, coding for the enzyme β-1 ,3-glucanosyltransferase which catalyzes the crosslinking of cell wall glucans. ZbGAS1. The gene was cloned by PCR and sequenced. The deleted mutant of Z. bailii has morphological and phenotypic characteristics very similar to the correspondent in S. cerevisiae, showing an alteration of the cell wall structure, and enhanced secretive capacity than the wild type strain for some heterologous proteins that have been considered. In parallel to these studies, populations of Z. bailii growing on different carbon sources were analyzed by flow cytometry. The analysis of DNA and protein content was performed to better characterize this yeast not only from molecular point of view, but also to explore its cellular features. The characterization of this unconventional yeast confirmed once again one of the most appreciated features for yeasts used as cell factories: versatility. This property is so strong that yeasts has been exploited for natural abilities, such as production of ethanol, and also for processes where a targeted manipulation was introduced, for example in lactic acid production, just to cite a pair of biotechnological production of industrial relevance. To make this process competitive on the market, in terms of yield, production and productivity, yeasts were pushed to their physiological limits. These limits are given by the accumulation of vast amounts of product that, in the case of ethanol, can cause damage to the lipid component of the plasma membrane, in the case of lactic acid can result in a loss of proper cellular homeostasis with the fall of the intracellular pH. It is therefore necessary to assess whether these limitations can be overcome by acting in particular on the plasma membrane, whose role in controlling the transport and the cellular homeostasis makes it a target of interest to improve the robustness of cells in response to stress generated by the process, such as oxidative stress or generated by low pH. The optimization of transport through the membrane plays a key role in the mechanisms of adaptation to these stresses. In particular, improving the flux of nutrients entering the cell could allow an optimal uptake of nutrient in the cytoplasm (as in the case of bioethanol production), an improvement of outflows from the cells might instead allow effective removal of compounds that may be deleterious for cell viability when present beyond a threshold value (eg organic acids). By focusing on the protein fraction that characterizes the cytoplasmic membrane, we have studied the effects of modulation of the expression of H +-ATPase pump of the plasma membrane (Pma1p), involved in the intracellular pH homeostasis. In particular, the gene ScPMA1 was overexpressed in S. cerevisiae and this overexpression was able to confer a greater resistance to acid stress, evidenced by growth kinetics in the presence of lactic acid. The increased cell viability under restrictive conditions in respect to the wild type strain was also checked by flow cytometry. The use of this tool has enabled the development of a system able to assess quantitatively the degree of robustness of the cells in stressful conditions. With this instrument able to assess the robustness of the cells as a function of various types of stress (oxidative, pH, ...) it is possible to design new interventions of metabolic engineering in order to provide greater resistance to yeast in restrictive process conditions, similar to conditions prevailing in the processes of production of compounds of biotech interest. It will be possible to evaluate the effectiveness of these interventions with the flow cytometer by assessing the response of the engineered cells under restrictive conditions by measuring the ability of cells to increase their robustness. The robustness remains one of the key features of yeasts as microbial cell factories, in particular to reach one of the main goal of White Biotechnology: to provide value products from renewable resources through sustainable processes with low environmental impact.
PORRO, DANILO
Zygosaccharomyces bailii, recombinant protein production, secretion, robustness
CHIM/11 - CHIMICA E BIOTECNOLOGIA DELLE FERMENTAZIONI
English
Scuola di dottorato di Scienze
BIOTECNOLOGIE INDUSTRIALI - 15R
22
2008/2009
(2009). Matching biotech needs and yeast physiology. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2009).
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/10281/8206
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