Publicação

Engineering of fatty acid production and secretion in Saccharomyces cerevisiae

Detalhes bibliográficos
Resumo:	Production of renewable liquid biofuels, that can substitute fossil fuels, has emerged as a major challenge for Biotechnology. Biodiesel, a more ecological alternative to petroleum based fuels, is currently obtained by the transesterification of vegetable oils with methanol or ethanol to produce fatty acid methyl esters (FAMEs) or ethyl esters (FAEEs), respectively. However, biodiesel production from plant oils requires intensive use of farmland, water, pesticides and fertilizers, and is restricted by geographical and seasonal issues that render the process economically, environmentally and ethically problematic. Biodiesel, produced by oleaginous microorganisms, could be an attractive alternative, since the utilization of diesel fuel is more efficient than, for example, ethanol. Oleaginous algae and yeast may accumulate very high (up to 80%) levels of intracellular lipids but two drawbacks are the relatively complicated extraction process and the subsequent transesterification with the accompanying glycerol by-product formation. The purpose of this work was to apply metabolic engineering of fatty acid synthesis and secretion, in the model yeast S. cerevisiae, in order to create a microorganism able to produce and secrete free fatty acids. S. cerevisiae is a suitable model, since lipid metabolism has been studied extensively and all genes encoding enzymes directly involved in lipid synthesis are known. In S. cerevisiae, activation of exogenous long-chain fatty acids to coenzyme A derivatives, prior to metabolic utilization, is mediated by the fatty acyl-CoA synthetases Faa1p and Faa4p. It has been shown previously that free fatty acids are secreted from a faa1faa4 double mutant. In this work, the “delitto perfetto” method was applied to delete these two fatty acyl-CoA synthetases generating genetically clean strains without markers or bacterial DNA. Results show that this technique can be used to generate multiple knockouts by recycling the marker gene. This modification was combined with engineering the pyruvate dehydrogenase bypass in order to enhance the supply of acetyl-CoA and NADPH to the fatty acid biosynthesis pathway. A strain where FAA1 and FAA4 were deleted and acetyl-CoA synthetase, glucose-6-phosphate dehydrogenase and acetaldehyde dehydrogenase were overexpressed secreted 2145mol.L-1 fatty acids to the extracellular media. In S. cerevisiae the acyl-CoA oxidase Pox1p, encoded by POX1, catalyses the first metabolic step of fatty acid -oxidation, and null mutants are unable to grow on fatty acids as sole carbon source. ScPox1p is the ortholog of the human acyl-CoA oxidase 1, and its expression is strongly induced by fatty acids. A triple knock-out mutant faa1/4  pox1  studied in this work secreted 2141mol.L-1 fatty acids to the extracellular media. Fatty acids form insoluble particles in cell culture media of the cells and these can be found as a white precipitate after centrifugation. In this work it was found that these particles scatter light in a way that can be measured with a spectrophotometer. The application of this property, as a method for rapid semiquantitative measurement of extracellular fatty acid concentration, shows a good correlation between the spectrophotometric and chromatographic analysis of the supernatants. The fatty acid production of the modified strains was analysed by optical density of the extracellular medium and gas chromatography. Strains constructed in this work have a 1.6 fold increased fatty acid secretion phenotype when compared with the faa1/4  mutant. However, secreted fatty acids disappear late in the growth phase, presumably metabolized by the cells. In summary, in this work it was possible to quantify and characterize fatty acids secreted to the extracellular medium by mutant Saccharomyces cerevisiae strains and the concentrations achieved are so far the highest reported in the literature.
Autores principais:	Ribeiro, Gabriela Filipa Lopes
Assunto:	Ciências Naturais::Ciências Biológicas
Ano:	2015
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

Descrição
Resumo:	Production of renewable liquid biofuels, that can substitute fossil fuels, has emerged as a major challenge for Biotechnology. Biodiesel, a more ecological alternative to petroleum based fuels, is currently obtained by the transesterification of vegetable oils with methanol or ethanol to produce fatty acid methyl esters (FAMEs) or ethyl esters (FAEEs), respectively. However, biodiesel production from plant oils requires intensive use of farmland, water, pesticides and fertilizers, and is restricted by geographical and seasonal issues that render the process economically, environmentally and ethically problematic. Biodiesel, produced by oleaginous microorganisms, could be an attractive alternative, since the utilization of diesel fuel is more efficient than, for example, ethanol. Oleaginous algae and yeast may accumulate very high (up to 80%) levels of intracellular lipids but two drawbacks are the relatively complicated extraction process and the subsequent transesterification with the accompanying glycerol by-product formation. The purpose of this work was to apply metabolic engineering of fatty acid synthesis and secretion, in the model yeast S. cerevisiae, in order to create a microorganism able to produce and secrete free fatty acids. S. cerevisiae is a suitable model, since lipid metabolism has been studied extensively and all genes encoding enzymes directly involved in lipid synthesis are known. In S. cerevisiae, activation of exogenous long-chain fatty acids to coenzyme A derivatives, prior to metabolic utilization, is mediated by the fatty acyl-CoA synthetases Faa1p and Faa4p. It has been shown previously that free fatty acids are secreted from a faa1faa4 double mutant. In this work, the “delitto perfetto” method was applied to delete these two fatty acyl-CoA synthetases generating genetically clean strains without markers or bacterial DNA. Results show that this technique can be used to generate multiple knockouts by recycling the marker gene. This modification was combined with engineering the pyruvate dehydrogenase bypass in order to enhance the supply of acetyl-CoA and NADPH to the fatty acid biosynthesis pathway. A strain where FAA1 and FAA4 were deleted and acetyl-CoA synthetase, glucose-6-phosphate dehydrogenase and acetaldehyde dehydrogenase were overexpressed secreted 2145mol.L-1 fatty acids to the extracellular media. In S. cerevisiae the acyl-CoA oxidase Pox1p, encoded by POX1, catalyses the first metabolic step of fatty acid -oxidation, and null mutants are unable to grow on fatty acids as sole carbon source. ScPox1p is the ortholog of the human acyl-CoA oxidase 1, and its expression is strongly induced by fatty acids. A triple knock-out mutant faa1/4  pox1  studied in this work secreted 2141mol.L-1 fatty acids to the extracellular media. Fatty acids form insoluble particles in cell culture media of the cells and these can be found as a white precipitate after centrifugation. In this work it was found that these particles scatter light in a way that can be measured with a spectrophotometer. The application of this property, as a method for rapid semiquantitative measurement of extracellular fatty acid concentration, shows a good correlation between the spectrophotometric and chromatographic analysis of the supernatants. The fatty acid production of the modified strains was analysed by optical density of the extracellular medium and gas chromatography. Strains constructed in this work have a 1.6 fold increased fatty acid secretion phenotype when compared with the faa1/4  mutant. However, secreted fatty acids disappear late in the growth phase, presumably metabolized by the cells. In summary, in this work it was possible to quantify and characterize fatty acids secreted to the extracellular medium by mutant Saccharomyces cerevisiae strains and the concentrations achieved are so far the highest reported in the literature.