Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.719710
Title: Forward and reverse genetics in industrially important Clostridia
Author: Grosse-Honebrink, Alexander
Awarding Body: University of Nottingham
Current Institution: University of Nottingham
Date of Award: 2017
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Abstract:
The bacterial genus Clostridium is composed of Gram-positive, spore-forming rods with widespread biotechnological applications. This study focused mainly on Clostridium pasteurianum DSM 525, a saccharolytic species which is able to convert glycerol, the by-product of the biodiesel industry, into the valuable chemical commodities n-butanol, ethanol and 1,3-propanediol. The aim was to formulate reproducible methods for the creation of mutants, both directed and random, and use the tools developed to investigate genes, and their products, important in solvent production. A prerequisite for the deployment of the envisaged genetic tools was a reproducible means for their introduction into the cell. Following the observation of low frequencies of plasmid transfer by electroporation it was hypothesised that the low level of transformants observed were a consequence of the presence of rare hypertransformable variants within the population. Accordingly, successfully transformed clonal populations were cured of their acquired plasmid and retransformed. In a number of instances the cured cell lines proved hypertransformable, with plasmid transformation frequencies obtained that were 5 orders of magnitude higher than those obtained with the progenitor strain. All of the hypertransformable strains isolated were shown by whole genome sequence to contain single nucleotide polymorphisms (SNPs) in one or more genes. In one instance, the single SNP present was shown to be directly responsible for the increased transformation frequency by its deliberate restoration to wild type using the allelic exchange procedures subsequently developed. Having established reproducible, high frequencies of plasmid transformation reverse genetics was employed to establish gene function. Accordingly, allelic exchange gene knock-out procedures were used to target genes coding for enzymes of the central energy metabolism in C. pasteurianum and the phenotypes of the mutants obtained were analysed in laboratory scale fermentations. Strains in which the genes encoding the redox response regulator (rex) and a hydrogenase (hyd) were deleted showed increased n-butanol titres, representing first steps towards utilisation of C. pasteurianum as a chassis for this important chemical. With the inactivation of the dhaBCE gene, encoding glycerol dehydratase, production of 1,3-propanediol was entirely eliminated, demonstrating the importance of the reductive pathway for growth and redox homeostasis of this organism when grown on glycerol. In order to allow forward genetic approaches, a mariner-transposon system previously exemplified in Clostridium difficile was adapted for use in alternative clostridial hosts. In the absence of an efficient transformation system for C. pasteurianum, the initial exemplification of the system was undertaken in Clostridium acetobutylicum and Clostridium sporogenes. Successful transposon delivery was demonstrated through the use of a plasmid conditional for replication and through the insertion of a gene encoding an alternate sigma-factor, TcdR, into their genomes. Transposition was shown to be entirely random and the libraries obtained of sufficient size to allow the isolation of both auxotrophic and sporulation/germination deficient mutants. Steps were taken to develop the same system in C. pasteurianum which was successful by using a suicide delivery plasmid, which was only possible with the high transformation efficiency achieved as part of this study. This study presents an essential forward genetics procedure for industrially important Clostridium species and a comprehensive genetic engineering approach for the important biofuel producer C. pasteurianum.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.719710  DOI: Not available
Keywords: QR 75 Bacteria. Cyanobacteria
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