The fossil energy resources decrease and climate changes, caused by carbon dioxide (CO2) emissions, have led most industrialized countries to undertake policies aimed at the development and use of renewable energy sources. Among the renewable energies, vegetal biomasses play a key role because widely available and potentially able to cover up to 200% of the global energy demand. Vegetal biomasses can be used mainly as raw materials for the production of chemicals, biofuels and energy, in the increasingly important green economy concept based on biorefineries creation. Although the vegetal biomasses result widely available, rising costs of food raw material such as wheat, corn and sugar beet have raised a serious ethical problem using these resources. To avoid the use of such raw materials, the exploitation of lignocellulosic biomasses plays a fundamental role in the industry. However, for an efficient utilization of lignocellulosic biomasses, new technologies are required in order to transform the starting biomass into simple molecules, such as pentoses and hexoses sugars, more easily to use by the microrganism, which will have the task of producing both fine chemicals and bulk chemicals in an economically and environmentally sustainable processes. In this regard, industrial biotechnologies should be able to develop new microrganisms capable to face the harsh environmental conditions that occur during an industrial production process. For many of these productions the yeast Saccharomyces cerevisiae is largely used, not only because of its naturally ability to produce large ethanol amount, but also is widely known both at genetic and metabolic level, outlining a good starting point for the development of producers strains with high tolerance against different stresses occur during an industrial process. This is the view adopted by NEMO project (Novel high performance Enzymes and Microrganisms for conversion of lignocellulosic biomass to ethanol), belonging to the European Union seventh framework program, where it become of primary importance the development of microrganisms, especially S.cerevisiae, for the second generation ethanol production. Microrganisms must be, on the one hand able to efficiently utilize all the sugars released from lignocellulosic biomass pre-treatment, on the other hand should be more tolerant against process conditons, such as inhibitory compounds and environmental stresses. A point of relevant importance is the ability to utilize pentose sugars, like D-xylose, released in large amount after lignocellulose pre-treatment. Currently, worldwide researches are focused on the development of yeast strains engineered with xylose degradation pathways involving the pentose phosphate pathway. In fact the fungal pathway exploits xylose reductase and the xylitol dehydrogenase while the bacterial pathway exploits xylose isomerase; both pathways degrade D-xylose into D-xylulose, which will enter into pentose phosphate pathway. In addition to these two pathways studied since the ‘80s of the last century, there also two other poorly known metabolisms, described for the first time in the ‘70s, which produce alpha-ketoglutarate or pyruvate and glycolaldehyde through an oxidative xylose degradation. These pathways are composed of 5 enzymatic reactions by the Weimberg’s pathway and of 4 enzymatic reactions by the Dahms’ pathway, however they share the first 3 enzymatic reactions. After bioinformatics we were identified the presence of Weimberg’s pathway into Burkholderia xenovorans, while the reaction that characterizes the Dahms’ pathway has been identified in Escherichia coli. The encoding genes for these enzymatic activities were expressed in S.cerevisiae, and the capacity to grow on D-xylose as carbon source are evaluated. The reconstruction of these two pathways showed a poorly growth capacity on xylose. Such growth limitation seems to be related to several factors: the presence of bottlenecks associated to enzymes functionality, like D-xylonate dehydratase activity; the yeast ability to internalize xylose efficiently; the involved genes optimization. Another important aspect is the yeast ability to face and overcome environmental stresses encountered during an industrial process. The cytoplasmic membrane plays a key role in cellular homeostasis, being at the interface between the cell and the external environment, and reacting at environmental changes. The plant membrane protein TIL gives particular strength to the yeast cells when these are subjected to environmental stresses of industrial relevance, such as the presence of oxidative agents or during temperature changes. However, when TIL is expressed in an industrial and/or in an engineered laboratory strains, for industrial use, the protective effect against prolonged stress exposure and process conditions disappear. Finally, a further important aspect during an industrial process is the S.cerevisiae ability to tolerate the growth inhibitory compounds presence into pre-treated lignocellulose. In fact has been largely described how chemical compounds like aldehydes, organic acids and phenolic compounds, released during lignocellulose pre-treatment process, are toxic at certain concentration, inhibiting S.cerevisiae growth or causing yeast death. The growth performance of different wild type or engineered yeast strains are evaluated on spruce and giant cane lignocellulose pre-treated: in addition the same strains were tested on minimal formulated medium according to the spruce pre-treated composition. The results showed that the combination of low pH and the presence of organic acids, especially acetic acid and formic acid, are dramatically harmful for growth of both industrial strain, naturally more tolerant, and engineered strain, for the production and recycle of L-ascorbic acid. However, the behavior of engineered strain for production and recycle of L-ascorbic acid is interesting at low pH, because showed higher tolerance than other strains in terms of growth rate and ethanol production and productivity. Despite the positive results obtained by engineering microrganisms, especially S.cerevisiae, in laboratory, their industrial uses still remain limited. Therefore, appears extremely important the construction of more robustness strains, able to withstand different environmental conditions along an entire industrial process, with consequent influence on yields, production and productivity. For these reasons, the research is aimed to combine these aspects to provide the best microrganism possible to industry productions.

(2012). Improving robustness and metabolic profile of saccharomyces cerevisiae for industrial bioprocesses. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2012).

Improving robustness and metabolic profile of saccharomyces cerevisiae for industrial bioprocesses

BERTAGNOLI, STEFANO
2012

Abstract

The fossil energy resources decrease and climate changes, caused by carbon dioxide (CO2) emissions, have led most industrialized countries to undertake policies aimed at the development and use of renewable energy sources. Among the renewable energies, vegetal biomasses play a key role because widely available and potentially able to cover up to 200% of the global energy demand. Vegetal biomasses can be used mainly as raw materials for the production of chemicals, biofuels and energy, in the increasingly important green economy concept based on biorefineries creation. Although the vegetal biomasses result widely available, rising costs of food raw material such as wheat, corn and sugar beet have raised a serious ethical problem using these resources. To avoid the use of such raw materials, the exploitation of lignocellulosic biomasses plays a fundamental role in the industry. However, for an efficient utilization of lignocellulosic biomasses, new technologies are required in order to transform the starting biomass into simple molecules, such as pentoses and hexoses sugars, more easily to use by the microrganism, which will have the task of producing both fine chemicals and bulk chemicals in an economically and environmentally sustainable processes. In this regard, industrial biotechnologies should be able to develop new microrganisms capable to face the harsh environmental conditions that occur during an industrial production process. For many of these productions the yeast Saccharomyces cerevisiae is largely used, not only because of its naturally ability to produce large ethanol amount, but also is widely known both at genetic and metabolic level, outlining a good starting point for the development of producers strains with high tolerance against different stresses occur during an industrial process. This is the view adopted by NEMO project (Novel high performance Enzymes and Microrganisms for conversion of lignocellulosic biomass to ethanol), belonging to the European Union seventh framework program, where it become of primary importance the development of microrganisms, especially S.cerevisiae, for the second generation ethanol production. Microrganisms must be, on the one hand able to efficiently utilize all the sugars released from lignocellulosic biomass pre-treatment, on the other hand should be more tolerant against process conditons, such as inhibitory compounds and environmental stresses. A point of relevant importance is the ability to utilize pentose sugars, like D-xylose, released in large amount after lignocellulose pre-treatment. Currently, worldwide researches are focused on the development of yeast strains engineered with xylose degradation pathways involving the pentose phosphate pathway. In fact the fungal pathway exploits xylose reductase and the xylitol dehydrogenase while the bacterial pathway exploits xylose isomerase; both pathways degrade D-xylose into D-xylulose, which will enter into pentose phosphate pathway. In addition to these two pathways studied since the ‘80s of the last century, there also two other poorly known metabolisms, described for the first time in the ‘70s, which produce alpha-ketoglutarate or pyruvate and glycolaldehyde through an oxidative xylose degradation. These pathways are composed of 5 enzymatic reactions by the Weimberg’s pathway and of 4 enzymatic reactions by the Dahms’ pathway, however they share the first 3 enzymatic reactions. After bioinformatics we were identified the presence of Weimberg’s pathway into Burkholderia xenovorans, while the reaction that characterizes the Dahms’ pathway has been identified in Escherichia coli. The encoding genes for these enzymatic activities were expressed in S.cerevisiae, and the capacity to grow on D-xylose as carbon source are evaluated. The reconstruction of these two pathways showed a poorly growth capacity on xylose. Such growth limitation seems to be related to several factors: the presence of bottlenecks associated to enzymes functionality, like D-xylonate dehydratase activity; the yeast ability to internalize xylose efficiently; the involved genes optimization. Another important aspect is the yeast ability to face and overcome environmental stresses encountered during an industrial process. The cytoplasmic membrane plays a key role in cellular homeostasis, being at the interface between the cell and the external environment, and reacting at environmental changes. The plant membrane protein TIL gives particular strength to the yeast cells when these are subjected to environmental stresses of industrial relevance, such as the presence of oxidative agents or during temperature changes. However, when TIL is expressed in an industrial and/or in an engineered laboratory strains, for industrial use, the protective effect against prolonged stress exposure and process conditions disappear. Finally, a further important aspect during an industrial process is the S.cerevisiae ability to tolerate the growth inhibitory compounds presence into pre-treated lignocellulose. In fact has been largely described how chemical compounds like aldehydes, organic acids and phenolic compounds, released during lignocellulose pre-treatment process, are toxic at certain concentration, inhibiting S.cerevisiae growth or causing yeast death. The growth performance of different wild type or engineered yeast strains are evaluated on spruce and giant cane lignocellulose pre-treated: in addition the same strains were tested on minimal formulated medium according to the spruce pre-treated composition. The results showed that the combination of low pH and the presence of organic acids, especially acetic acid and formic acid, are dramatically harmful for growth of both industrial strain, naturally more tolerant, and engineered strain, for the production and recycle of L-ascorbic acid. However, the behavior of engineered strain for production and recycle of L-ascorbic acid is interesting at low pH, because showed higher tolerance than other strains in terms of growth rate and ethanol production and productivity. Despite the positive results obtained by engineering microrganisms, especially S.cerevisiae, in laboratory, their industrial uses still remain limited. Therefore, appears extremely important the construction of more robustness strains, able to withstand different environmental conditions along an entire industrial process, with consequent influence on yields, production and productivity. For these reasons, the research is aimed to combine these aspects to provide the best microrganism possible to industry productions.
BRANDUARDI, PAOLA
Xylose; Robustness; Lignocellulose; Entner-Doudoroff; Xylose metabolism; TIL, Lipocalin; Process stresses; Biomass pre-treatment; Lignocellulosic inhibitors; Industrial processes; Saccharomyces cerevisiae
CHIM/11 - CHIMICA E BIOTECNOLOGIA DELLE FERMENTAZIONI
English
14-feb-2012
Scuola di dottorato di Scienze
BIOTECNOLOGIE INDUSTRIALI - 15R
24
2010/2011
open
(2012). Improving robustness and metabolic profile of saccharomyces cerevisiae for industrial bioprocesses. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2012).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/28926
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