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Optimisation du métabolisme d'une souche d'Escherichia coli génétiquement modifiée pour la production de n-butanol à haut titre et rendement

Abstract : When it comes to sustainable development, n-butanol production by biological means represents an environmentally friendly alternative compared to petrochemical means. Some organisms are able to produce butanol naturally, such as the Clostridiae family, which are Gram-positive bacteria, growing in strict anaerobic conditions. They produce butanol in mixture with acetone and ethanol, which reduce the production yield of butanol and increase the purification costs. In 2015, the PEEP team of TBI built a genetically modified strain of E. coli, expressing the complete conversion pathway from pyruvate to butanol from C. acetobutylicum. This strain’s metabolism was built so that the growth and glucose consumption speeds are coupled with the butanol production speed. In strict anaerobic growth in batch culture, in a chemically defined medium based on glucose and supplemented with yeast extracts (YE) and nitrate (NO3-), this strain produces 3,3 g/L of n-butanol, with a yield of 0,23 g butanol/g glucose, among other minority coproducts (succinate, lactate, acetate, butyrate). These coproducts lower the butanol production yield, and remain unexpected, because the corresponding metabolic pathways were deleted. The goal of this PhD project consists in identifying the metabolic pathways involved in the coproducts production and improving the understanding of the strain, in order to optimize it, improve the butanol/glucose yield, and simplify the culture medium. Several aspects are considered: expressing ferredoxin NAD(P)+ oxidoreductases (FNOR) from organisms other than C. acetobutylicum, in order to combine the reoxidation of the reduced ferredoxin to the generation of ATP; ii) inactivating the metabolic pathways from E. coli which may be involved in the coproducts synthesis; iii) performing an evolutionary in-vivo adaptation of the strain, in order to improve its performances and increase its tolerance to butanol in a chemically defined medium supplemented with YE and NO3-, and then iv) performing an evolutionary adaptation of the Butanol strain to allow its growth in the chemically defined medium without any supplementation. These strategies lead to i) the construction of a new E. coli strain, with its anaerobic growth depending on the functionality of an FNOR: the FNOR from C. acetobutylicum, the Rnf complex from C. ljungdahlii and the FNOR from C. tepidum were assessed. This work lead to the selection of a mutant of the FNOR from C. tepidum, which has a ferredoxin NAD+ reductase activity 2,2 times higher than the native enzyme. This mutant FNOR was then expressed in the E. coli strain producing butanol, as a replacement of C. acetobutylicum’s. The characterization of its phenotype demonstrated its capacity to produce 3 g/L of butanol, with a yield of 0,26 g/g; ii) the deletion of the genes zwf and mdh in the Butanol strain also lead to an improvement of the production in butanol up to 6 g/L , and of the yield up to 0,33 g/g; iii) the in-vivo evolution of the Butanol strain, in continuous culture (chemostat) with regulated pH, in the medium supplemented with YE and Ni, lead to the selection of an evolved strain producing up to 11 g/L of butanol, and a yield of 0,34 g/g. The complete sequencing of the genomes of the clones isolated from this population lead to the identification of mutations in two genes, yqhC and lptG, potentially involved in the strain’s performances; iv) the in-vivo adaptation of the Butanol strain in the medium without any supplementation lead to the selection of a evolved clones producing 2,5 g/L of butanol, and a yield of 0,28 g/g in batch culture. To our knowledge, the highest production of butanol observed coupled with anaerobic growth in E. coli does not exceed 0,6g/L, in a chemically defined medium base on glucose without any supplementation.
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Submitted on : Wednesday, March 30, 2022 - 3:38:08 PM
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Antoine Boullet. Optimisation du métabolisme d'une souche d'Escherichia coli génétiquement modifiée pour la production de n-butanol à haut titre et rendement. Biotechnologies. INSA de Toulouse, 2019. Français. ⟨NNT : 2019ISAT0044⟩. ⟨tel-03624950⟩

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