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| Author(s): |
Jonatã Bortolucci
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | Ribeirão Preto. |
| Institution: | Universidade de São Paulo (USP). Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (PCARP/BC) |
| Defense date: | 2023-01-25 |
| Examining board members: |
Valeria Reginatto Spiller;
María Eugenia Guazzaroni;
Adilson José da Silva
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| Advisor: | Valeria Reginatto Spiller |
| Abstract | |
Bioeconomy as a substitute for the current economic model is the most discussed strategy in recent decades, as a way to diminish dependence on fossil resources, which are considered as pollutants and non-renewable. Biotechnological processes carried out at biorefineries are considered as one of the most attractive alternatives for valorization of biomasses, by converting them into bioproducts, biofuels and bioenergy. For example, biodiesel can be obtained from oils and grease, but generates glycerol as byproduct. Glycerol recycling as carbon source has been studied in several bioprocesses, one of them being its conversion to 1,3-propanediol (1,3-PDO) by bacteria from Clostridium genus. Glycerol metabolization occurs by two routes: the oxidative one, by glycerol dehydrogenase (GDH) and dihydroxyacetone kinase (DHAK) enzymes, or glycerol kinase (GK) and glycerol-3-phosphate dehydrogenase (GPDH) enzymes; and the reductive one, by glycerol dehydratase (GDHt) and 1,3-propanediol dehydrogenase (PDODH) enzymes. The final 1,3-PDO productivity is dependent on the gene expression of these enzymes. In this work, the isolate Clostridium beijerinckii Br21 was studied, for which the fermentation nutritional conditions and gene expression of enzymes involved in glycerol oxidation/reduction were investigated. These investigations were also carried out for a reference strain, Clostridium pasteurianum DSM 525. Finally, genetic engineering was studied for C. beijerinckii Br21, to improve 1,3-PDO synthesis. Comparing the Br21 strain fermentation at highly nutritious (RCM) and lowly nutritious medium (WIS, described by WISCHRAL et al., 2015), 1,3-PDO attainment was only observed at lower availability of nutrients. The final concentration of 1,3-PDO is similar to the one obtained by C. pasteurianum DSM 525. In addition, GPDH gene expression on the Br21 strain cultivated in WIS medium was low, possibly indicating rate limitations of glycerol oxidation via GK. Thus, this oxidation probably occurs via GDH/DHAK. As for glycerol reduction, gene expression of PDODH was similar for both strains, Br21 and DSM 525. To increase gene expression related to glycerol reduction, C. beijerinckii Br21 transformation was performed. pMTL83251 plasmid was used, containing the genes encoding for GDHt, cobalamin adenosyltransferase and PDODH, under control of the promoter coding for phosphotransacetylase/acetate kinase enzymes. After selection and improvement of a literature described protocol, transformation by electroporation to promote the gene overexpression of GDHt, cobalamin adenosyltransferase and PDODH was successful. The overexpression of these genes increased 1,3-PDO productivity, but there was no increase in the final concentration of this compound. This work contributed to better understanding of the metabolic pathways of glycerol conversion to 1,3-PDO by a C. beijerinckii isolate, as well as made possible to adapt a transformation method for this strain. (AU) | |
| FAPESP's process: | 20/03168-3 - Expression of enzymes related to 1,3-propanediol sustainable production by Clostridium strains from glycerol and other substrates |
| Grantee: | Jonatã Bortolucci |
| Support Opportunities: | Scholarships in Brazil - Master |