Genetic Characterization and Manipulation of Solvent -Producing Clostridia
Jesse, Troy William
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https://hdl.handle.net/2142/83687
Description
Title
Genetic Characterization and Manipulation of Solvent -Producing Clostridia
Author(s)
Jesse, Troy William
Issue Date
2003
Doctoral Committee Chair(s)
Hans P. Blaschek
Department of Study
Food Science and Human Nutrition
Discipline
Food Science and Human Nutrition
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Agriculture, Food Science and Technology
Language
eng
Abstract
The sol operons from Clostridium beijerinckii NCIMB 8052 and C. beijerinckii BA101 were cloned and sequenced to determine if differences at the sequence level were responsible for the increased butanol production exhibited by C. beijerinckii BA101 relative to the wild type C. beijerinckii NCMIB 8052. No observable differences were detected in the sequences of the structural genes and regions upstream of the sol operons. The shuttle vector pYL102E was altered by the addition of a multiple cloning site to produce plasmids pTJ1 and pTJ2. The sol operon from C. beijerinckii BA101 was sub-cloned into pTJ1 and pTJ2 to form plasmids pTJ1sol and pTJ2sol. These constructs were used to transform the degenerate strain C. beijerinckii SA2, producing C. beijerinckii SA2TJ1, C. beijerinckii SA2TJ1sol, C. beijerinckii SA2TJ2, and C. beijerinckii SA2TJ2sol. The solvent production of these strains was examined. C. beijerinckii SA2TJ1sol, C. beijerinckii SA2TJ2, and C. beijerinckii SA2TJ2sol produced 16.50 +/- 0.55 g/L, 15.63 +/- 0.82 g/L, and 17.39 +/- 1.91 g/L total solvents, respectively. This is greater than the parent C. beijerinckii SA2 which produced 0.47 +/- 0.01 g/L total solvents. Southern hybridizations of total DNA from C. beijerinckii SA2TJ1sol, C. beijerinckii SA2TJ2, and C. beijerinckii SA2TJ2sol, revealed two distinct fragments homologous to the ermAM gene. These fragments were cloned and sequenced. A total of 20 putative Orfs were found in the cloned 5.5-kb and 7.6-kb fragments. Putative Orfs 1, 2, 3, 4, 16, 17, 18, 19, and 20 from the 5.5- and 7.6 kb fragments were found to have homology to the Tn916 Orfs 16, 17, 18, 19, 20, 21, 22, 23, and 24, respectively. The sequences of these Orfs are not found within the plasmids pTJ1 and pTJ2. PCR data suggested that these sequences were not present in the parent C. beijerinckii SA2. Results of rep-PCR experiments indicated that C. beijerinckii SA2TJ1, C. beijerinckii SA2TJ1 sol, C. beijerinckii SA2TJ2, and C. beijerinckii SA2TJ2sol are all derived from C. beijerinckii SA2. These results suggested that there may be an unknown source of the transposon-like elements found in the C. beijerinckii SA2TJ1sol, SA2TJ2, and SA2TJ2sol transformants.
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