Publicação
Engineering Saccharomyces cerevisiae for the production of value-added compounds from lignocellulosic-derived furans
| Resumo: | Furfural is a molecule derived from hemicellulose that can be further reduced into furfuryl alcohol. This last molecule is used to produce a plethora of compounds in industries such as foundry, chemical, wood, pharmaceutical, etc.. Industrial production of furfuryl alcohol uses Cu-Cr catalysts which not only achieve low yields but are also difficult to dispose of, since it causes severe environmental problems. Whole-cell biocatalysis appears as an excellent solution to these problems since it can achieve high titers, yields, and productivity while also being environmentally friendly. However, whole-cell biocatalysis is hindered by the fact that furfural is toxic to cells, even at low concentrations. Saccharomyces cerevisiae is one of the most promising biocatalysts for furfuryl alcohol production. Industrial strains have evolved in harsh environments and present high tolerance for inhibitory compounds. Strains grown in different conditions also have different genetic backgrounds and hence, different furfural resistance capacities. Genetic modification has shown to be an effective way to increase furfural resistance and/or furfuryl alcohol production. To determine the best strain to produce furfuryl alcohol, in this work several industrial and laboratorial strains were screened. To this end, the strains were grown in different concentrations of furfural and their growth profiles were analyzed. Furfural reducing activity using NADH and NADPH as a co-factor was also studied as well as furfuryl alcohol production. Finally, the gene FURX from Cupriavidus necator was inserted into the chosen strain’s genome using CRISPR-CAS9 and its effects on furfuryl alcohol production in oxygen-limited conditions were also explored. A variety of profiles were observed between the strains. The results show that industrial strains have higher furfural resistance than laboratory strains. Similarly, industrial strains showed the highest furfuryl alcohol production. All strains exhibited higher furfural reducing activity when using NADH when compared to NADPH and the laboratory strain CEN.PK 113-7D had the highest reducing activity. One industrial strain was chosen for further genetic modifications due to its increased growth performance in the presence of furfural and high furfuryl alcohol production when compared to the other strains. When using glucose as co-substrate, the modified strain had better furfuryl alcohol production while when using ethanol as co-substrate the wild type strain presented better furfuryl alcohol production. |
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| Autores principais: | Lima, Henrique Gonçalves de |
| Assunto: | Furfural Furfuryl alcohol Saccharomyces cerevisiae Whole-cell biocatalysis Álcool furfurílico Biocatálise com células inteiras Furfural |
| Ano: | 2021 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Furfural is a molecule derived from hemicellulose that can be further reduced into furfuryl alcohol. This last molecule is used to produce a plethora of compounds in industries such as foundry, chemical, wood, pharmaceutical, etc.. Industrial production of furfuryl alcohol uses Cu-Cr catalysts which not only achieve low yields but are also difficult to dispose of, since it causes severe environmental problems. Whole-cell biocatalysis appears as an excellent solution to these problems since it can achieve high titers, yields, and productivity while also being environmentally friendly. However, whole-cell biocatalysis is hindered by the fact that furfural is toxic to cells, even at low concentrations. Saccharomyces cerevisiae is one of the most promising biocatalysts for furfuryl alcohol production. Industrial strains have evolved in harsh environments and present high tolerance for inhibitory compounds. Strains grown in different conditions also have different genetic backgrounds and hence, different furfural resistance capacities. Genetic modification has shown to be an effective way to increase furfural resistance and/or furfuryl alcohol production. To determine the best strain to produce furfuryl alcohol, in this work several industrial and laboratorial strains were screened. To this end, the strains were grown in different concentrations of furfural and their growth profiles were analyzed. Furfural reducing activity using NADH and NADPH as a co-factor was also studied as well as furfuryl alcohol production. Finally, the gene FURX from Cupriavidus necator was inserted into the chosen strain’s genome using CRISPR-CAS9 and its effects on furfuryl alcohol production in oxygen-limited conditions were also explored. A variety of profiles were observed between the strains. The results show that industrial strains have higher furfural resistance than laboratory strains. Similarly, industrial strains showed the highest furfuryl alcohol production. All strains exhibited higher furfural reducing activity when using NADH when compared to NADPH and the laboratory strain CEN.PK 113-7D had the highest reducing activity. One industrial strain was chosen for further genetic modifications due to its increased growth performance in the presence of furfural and high furfuryl alcohol production when compared to the other strains. When using glucose as co-substrate, the modified strain had better furfuryl alcohol production while when using ethanol as co-substrate the wild type strain presented better furfuryl alcohol production. |
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