The effect of modulating the quantity of enzymes in a model ethanol pathway on metabolic flux in Synechocystis sp. PCC 6803
Autor: | John G. Rowland, Geoff S. Baldwin, Nicole Prandi, Paulina Bartasun, Marko Storch, Yarin Aknin, Mark H. Bennett, Arianna Palma, Patrik R. Jones, Yumiko Sakuragi |
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Přispěvatelé: | Commission of the European Communities, Engineering & Physical Science Research Council (E |
Jazyk: | angličtina |
Rok vydání: | 2019 |
Předmět: |
0106 biological sciences
Optimization EXPRESSION TOOLBOX lcsh:Medicine Reductase Cyanobacteria 01 natural sciences General Biochemistry Genetics and Molecular Biology Metabolic engineering 03 medical and health sciences 010608 biotechnology Operon RBS 030304 developmental biology 2. Zero hunger chemistry.chemical_classification 0303 health sciences Science & Technology Ethanol Chemistry General Neuroscience lcsh:R General Medicine Metabolism CYANOBACTERIA Multidisciplinary Sciences Metabolic pathway SYNTHETIC BIOLOGY Enzyme Biochemistry Science & Technology - Other Topics General Agricultural and Biological Sciences Flux (metabolism) Pyruvate decarboxylase Pyruvate kinase Biotechnology |
Zdroj: | PeerJ Bartasun, P, Prandi, N, Storch, M, Aknin, Y, Bennett, M, Palma, A, Baldwin, G, Sakuragi, Y, Jones, P R & Rowland, J 2019, ' The effect of modulating the quantity of enzymes in a model ethanol pathway on metabolic flux in Synechocystis sp. PCC 6803 ', PeerJ, vol. 2019, no. 7, 7529, pp. 1-18 . https://doi.org/10.7717/peerj.7529 PeerJ, Vol 7, p e7529 (2019) |
DOI: | 10.7717/peerj.7529 |
Popis: | Synthetic metabolism allows new metabolic capabilities to be introduced into strains for biotechnology applications. Such engineered metabolic pathways are unlikely to function optimally as initially designed and native metabolism may not efficiently support the introduced pathway without further intervention. To develop our understanding of optimal metabolic engineering strategies, a two-enzyme ethanol pathway consisting of pyruvate decarboxylase and acetaldehyde reductase was introduced intoSynechocystissp. PCC 6803. We characteriseda new set of ribosome binding site sequences inSynechocystissp. PCC 6803 providing a range of translation strengths for different genes under test. The effect of ribosome-bindingsite sequence, operon design and modifications to native metabolism on pathway flux was analysed by HPLC. The accumulation of all introduced proteins was also quantified using selected reaction monitoring mass spectrometry. Pathway productivity was more strongly dependent on the accumulation of pyruvate decarboxylase than acetaldehyde reductase. In fact, abolishment of reductase over-expression resulted in the greatest ethanol productivity, most likely because strains harbouringsingle-gene constructs accumulated more pyruvate decarboxylase than strains carrying any of the multi-gene constructs. Overall, several lessons were learned. Firstly, the expression level of the first gene in anyoperon influenced the expression level of subsequent genes, demonstrating that translational coupling can also occur in cyanobacteria. Longer operons resulted in lower protein abundance for proximally-encoded cistrons. And, implementation of metabolic engineering strategies that have previously been shown to enhance the growth or yield of pyruvate dependent products, through co-expression with pyruvate kinase and/or fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase, indicated that other factors had greater control over growth and metabolic flux under the tested conditions. |
Databáze: | OpenAIRE |
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