Metabolic engineering for butanol yield enhancement in Clostridium acetobutylicum

Hope, Ryan (2022) Metabolic engineering for butanol yield enhancement in Clostridium acetobutylicum. PhD thesis, University of Nottingham.

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Abstract

Clostridium acetobutylicum is the model solventogenic saccharolytic Clostridium spp. representing a group of bacteria which exclusively produce acetone and n-butanol along with the common solvent, ethanol; known as the ABE pathway. There is broad utility for n-butanol, particularly as a transport fuel but also as an industrial solvent and as a platform chemical. Hydrogen is also a major product of this organism by way of reduction of protons via ferredoxin coupled hydrogenase activity, where electron flux to this product is mediated by the oxidation of organic metabolic intermediates by the enzymes pyruvate ferredoxin oxidoreductase (PFOR) and the electron bifurcating activity of butyryl-CoA dehydrogenase (BCD). The role of BCD was explored utilising homologous recombination in-frame deletion methods, however, the apparent essentiality of the gene resulted in maintenance of the vector and the target gene in the genome, likely as a result of a random vector integration event. Replacing BCD with trans-2-enoyl-CoA reductase (TER) presents a metabolic engineering opportunity by subversion of electron flux to ferredoxin, and ultimately hydrogen gas production, furthermore, it allows us to investigate the importance of the bifurcating role of BCD. Hypothetically, successful replacement of BCD with TER should result in an alcohologenic fermentation, as the cells attempt to maintain redox cofactor homeostasis. The expression of TER resulted in a significant improvement in solvent productivity. Nevertheless, the electron bifurcating activity of BCD appears to be an essential metabolic function for C. acetobutylicum, and DNA-seq data from a mutant strain obtained from a third party suggests that this is due to the role of hydrogenase in maintaining the proton motive force - in which case a complementary mutation interrupting the function of the proton powered flagella will ultimately facilitate the replacement of BCD with TER.

A prototypic lactose inducible orthogonal expression system was applied in order to maximise the flux to butanol in the TER expressing parent strain. A control study using a strain expressing the lactose binding transcriptional activator and the TcdR sigma factor produced an altered phenotype where enhanced solvent production was observed and a computational approach was used to try to identify TcdR promotor binding sites in the C. acetobutylicum genome offering some insight as to the cause of the adjusted phenotype and a new regulator of solventogenesis is proposed.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Minton, Nigel
Winzer, Klaus
Keywords: Synthetic biology, Metabolic engineering, Biofuels, Butanol
Subjects: Q Science > QR Microbiology > QR 75 Bacteria. Cyanobacteria
T Technology > TP Chemical technology
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID: 67130
Depositing User: Hope, Ryan
Date Deposited: 31 Jul 2022 04:40
Last Modified: 31 Jul 2022 04:40
URI: http://eprints.nottingham.ac.uk/id/eprint/67130

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