| Summary: | The limited abundance of fossil fuels and its harmful effects on the environment has led to the increased global interest in more environmentally friendly sources of chemicals and fuels. The solventogenic Clostridium acetobutylicum represents a model organism for solvent production in this genus and in the so-called ABE fermentation produces acetone, butanol and ethanol in a 3:6:1 ratio.
The identification of those factors that influence solvent production through mutation of the encoding genes is key to the rational metabolic engineering and development of an industrial solvent producing C. acetobutylicum strain. Consequently, an attempt to knock-out genes previously impossible to knock-out was carried out, using a conditional variant of the ClosTron intron re-targeting system. While the knock-out of hydrogenase (hydA), butyryl CoA dehydrogenase (bcd) and crotonase (crt) genes were not possible in this thesis, the isolation of a null 3-hydroxybutyryl CoA dehydrogenase (hbd) mutant was demonstrated.
To further enhance metabolic engineering of this organism, it was also desirable to put in place a system for the introduction of genes encoding pivotal metabolic enzymes at discrete loci around the chromosome, similar to pyrE, that may be used to implement new metabolic pathways and to exemplify its utility by extending product formation in C. acetobutylicum to isopropanol. This will include the introduction of an inducible orthogonal expression system at a locus, which would control the expression of additional gene sets introduced at the other loci. A triple auxotrophic mutant of C. acetobutylicum was created and the utility of the pheA and argH loci in addition to the pyrE locus described. Integration at the pyrE and pheA loci were relatively easily accomplished compared to the argH locus which proved difficult despite several attempts. With the introduction of a synthetic acetone operon and a gene encoding a secondary dehydrogenase from Clostridium beijeirinckii NRRL B593 both under the control of a lactose inducible tcdR system, up to 18.47 g/l Isopropanol-Butanol-Ethanol (8.47 g/l isopropanol, 10.02 g/l butanol, 0.21 g/l ethanol) was produced. This represents the highest recorded production of isopropanol from C. acetobutylicum to date.
|
|---|