Publicação
Engineering of core Pentose Metabolism in Saccharomyces cerevisiae for Bio-ethanol Production
| Resumo: | Renewable fuels that do not contribute to atmospheric carbon dioxide have gained increased attention due to peak oil and the possibility of carbon dioxide induced climate change. Bioethanol is the currently largest biofuel in terms of annual production and is mainly produce by fermentation of hexose sugars in sucrose or starch from sugarcane or corn by the yeast Saccharomyces cerevisiae. Second generation biofuel is based on a low value carbon source and production costs are sensitive to ethanol yield. One obvious way to improve yield would be to also ferment the pentose sugars such as D-xylose and L-arabinose from the hemicellulose fraction of the biomass. S. cerevisiae does not naturally ferment D-xylose and L-arabinose, but can be made to do it by metabolic engineering with heterologous genes from yeasts, fungi or bacteria. Aerobic growth on D-xylose requires the expression of xylose reductase (XR) and xylitol dehydrogenase (XDH) or xylose isomerase, both resulting in the conversion of D xylose to D-xylulose. Further efficient D-xylose catabolism to ethanol requires the overproduction of D-xylulose kinase and genes encoding pentose phosphate pathway enzymes. Initial L-arabinose metabolism requires on the other hand, the expression of the three genes in the bacterial AraBAD operon or two fungal L-arabinitol-4 dehydrogenase, L-xylulose-reductase. The number of metabolic engineering target genes for efficient pentose fermentation has increased to about ten or more, making the classic expression of genes, one by one, using plasmid vectors or genetic integration an impractical strategy. In this thesis, a new pathway assembly tool that allows the simultaneous expression of at least eight genes was developed and named The Yeast Pathway Kit (YPK). This tool has several advantages over alternative pathway assembly protocols, notably that it allows reuse of genetic elements and both rational and random assembly of pathway components. We used the YPK to construct several yeast strains expressing the initial D xylose metabolism (XR and XDH) from the D-xylose utilising yeast Scheffersomyces stipitis together with overexpression of D-xylulose kinase (XKS1) and transaldolase (TAL1), four genes in total. A pathway additionally expressing D-ribulose-5-phosphate 3-epimerase (RPE1), Ribose-5-phosphate ketol-isomerase (RKI1) and Transketolase (TKL1) and the Candida intermedia GXF1 D-xylose transporter was also constructed, resulting in an eight gene pathway. A rationally designed four gene pathway enabled an aerobic growth rate of 0.21 h-1, which is the highest reported to date, possibly because all participating genes are present on a multicopy construct, in contrast with previously described strains where all or a part of the pathways were integrated into the genome in one or a few copies. Overexpressing the additional four genes in the eight gene pathway did not further increase growth rate, but led to fermentation with higher ethanol and lower xylitol yields. The manual planning of assembly of large metabolic pathways using DNA editor software is an error prone and time consuming task that is also difficult to properly document. We therefore developed a software package that allows the simulation of the molecular biology unit operations involved in the assembly of metabolic pathways using the YPK. An assembly protocol is expressed in a scripting language that allows the simulation of the assembly and also serves as a verifiable documentation of the experiment. A key metabolic step for effective L-arabinose and D-xylose metabolism is the initial transport into the cell. A number of fungal L-arabinose and D-xylose transporters have been identified and used in metabolic engineering for more efficient catabolism of these sugars. Transporters found by classical biochemistry or functional screening with recombinant pentose utilizing S. cerevisiae have identified transporters with poor kinetics when expressed in S. cerevisiae. Therefore, we have also developed a novel functional screening strain based on the glucose negative phenotype of the S. cerevisiae phosphoglucose isomerase (pgi1) mutant expressing XR. D-xylose or L-arabinose transport into the cells led to reversal of the glucose negative phenotype. The addition of a D-xylose or L-arabinose transporter was readily visible as a faster growth on a mixed glucose/pentose medium. This strain is a valuable future tool to screen for efficient D xylose or L-arabinose transporters able for the uptake of these nutrients in the presence of glucose. Finally, the MX4blaster cassette was developed for clean S. cerevisiae genome modifications. This tool allows the complete removal of kanMX4 and other MX4 based markers so that the genome wide gene deletion collection can be used as a tool for multiple gene deletions. |
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| Autores principais: | Pereira, Filipa Alexandra Barroso |
| Assunto: | Ciências Naturais::Ciências Biológicas |
| Ano: | 2013 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso restrito |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Renewable fuels that do not contribute to atmospheric carbon dioxide have gained increased attention due to peak oil and the possibility of carbon dioxide induced climate change. Bioethanol is the currently largest biofuel in terms of annual production and is mainly produce by fermentation of hexose sugars in sucrose or starch from sugarcane or corn by the yeast Saccharomyces cerevisiae. Second generation biofuel is based on a low value carbon source and production costs are sensitive to ethanol yield. One obvious way to improve yield would be to also ferment the pentose sugars such as D-xylose and L-arabinose from the hemicellulose fraction of the biomass. S. cerevisiae does not naturally ferment D-xylose and L-arabinose, but can be made to do it by metabolic engineering with heterologous genes from yeasts, fungi or bacteria. Aerobic growth on D-xylose requires the expression of xylose reductase (XR) and xylitol dehydrogenase (XDH) or xylose isomerase, both resulting in the conversion of D xylose to D-xylulose. Further efficient D-xylose catabolism to ethanol requires the overproduction of D-xylulose kinase and genes encoding pentose phosphate pathway enzymes. Initial L-arabinose metabolism requires on the other hand, the expression of the three genes in the bacterial AraBAD operon or two fungal L-arabinitol-4 dehydrogenase, L-xylulose-reductase. The number of metabolic engineering target genes for efficient pentose fermentation has increased to about ten or more, making the classic expression of genes, one by one, using plasmid vectors or genetic integration an impractical strategy. In this thesis, a new pathway assembly tool that allows the simultaneous expression of at least eight genes was developed and named The Yeast Pathway Kit (YPK). This tool has several advantages over alternative pathway assembly protocols, notably that it allows reuse of genetic elements and both rational and random assembly of pathway components. We used the YPK to construct several yeast strains expressing the initial D xylose metabolism (XR and XDH) from the D-xylose utilising yeast Scheffersomyces stipitis together with overexpression of D-xylulose kinase (XKS1) and transaldolase (TAL1), four genes in total. A pathway additionally expressing D-ribulose-5-phosphate 3-epimerase (RPE1), Ribose-5-phosphate ketol-isomerase (RKI1) and Transketolase (TKL1) and the Candida intermedia GXF1 D-xylose transporter was also constructed, resulting in an eight gene pathway. A rationally designed four gene pathway enabled an aerobic growth rate of 0.21 h-1, which is the highest reported to date, possibly because all participating genes are present on a multicopy construct, in contrast with previously described strains where all or a part of the pathways were integrated into the genome in one or a few copies. Overexpressing the additional four genes in the eight gene pathway did not further increase growth rate, but led to fermentation with higher ethanol and lower xylitol yields. The manual planning of assembly of large metabolic pathways using DNA editor software is an error prone and time consuming task that is also difficult to properly document. We therefore developed a software package that allows the simulation of the molecular biology unit operations involved in the assembly of metabolic pathways using the YPK. An assembly protocol is expressed in a scripting language that allows the simulation of the assembly and also serves as a verifiable documentation of the experiment. A key metabolic step for effective L-arabinose and D-xylose metabolism is the initial transport into the cell. A number of fungal L-arabinose and D-xylose transporters have been identified and used in metabolic engineering for more efficient catabolism of these sugars. Transporters found by classical biochemistry or functional screening with recombinant pentose utilizing S. cerevisiae have identified transporters with poor kinetics when expressed in S. cerevisiae. Therefore, we have also developed a novel functional screening strain based on the glucose negative phenotype of the S. cerevisiae phosphoglucose isomerase (pgi1) mutant expressing XR. D-xylose or L-arabinose transport into the cells led to reversal of the glucose negative phenotype. The addition of a D-xylose or L-arabinose transporter was readily visible as a faster growth on a mixed glucose/pentose medium. This strain is a valuable future tool to screen for efficient D xylose or L-arabinose transporters able for the uptake of these nutrients in the presence of glucose. Finally, the MX4blaster cassette was developed for clean S. cerevisiae genome modifications. This tool allows the complete removal of kanMX4 and other MX4 based markers so that the genome wide gene deletion collection can be used as a tool for multiple gene deletions. |
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