PMID:18726959

Citation	Sillers, R, Al-Hinai, MA and Papoutsakis, ET (2009) Aldehyde-alcohol dehydrogenase and/or thiolase overexpression coupled with CoA transferase downregulation lead to higher alcohol titers and selectivity in Clostridium acetobutylicum fermentations. Biotechnol. Bioeng. 102:38-49
Abstract	Metabolic engineering (ME) of Clostridium acetobutylicum has led to increased solvent (butanol, acetone, and ethanol) production and solvent tolerance, thus demonstrating that further efforts have the potential to create strains of industrial importance. With recently developed ME tools, it is now possible to combine genetic modifications and thus implement more advanced ME strategies. We have previously shown that antisense RNA (asRNA)-based downregulation of CoA transferase (CoAT, the first enzyme in the acetone-formation pathway) results in increased butanol to acetone selectivity, but overall reduced butanol yields and titers. In this study the alcohol/aldehyde dehydrogenase (aad) gene (encoding the bifunctional protein AAD responsible for butanol and ethanol production from butyryl-CoA and acetyl-CoA, respectively) was expressed from the phosphotransbutyrylase (ptb) promoter to enhance butanol formation and selectivity, while CoAT downregulation was used to minimize acetone production. This led to early production of high alcohol (butanol plus ethanol) titers, overall solvent titers of 30 g/L, and a higher alcohol/acetone ratio. Metabolic flux analysis revealed the likely depletion of butyryl-CoA. In order to increase then the flux towards butyryl-CoA, we examined the impact of thiolase (THL, thl) overexpression. THL converts acetyl-CoA to acetoacetyl-CoA, the first step of the pathway from acetyl-CoA to butyryl-CoA, and thus, combining thl overexpression with aad overexpression decreased, as expected, acetate and ethanol production while increasing acetone and butyrate formation. thl overexpression in strains with asRNA CoAT downregulation did not significantly alter product formation thus suggesting that a more complex metabolic engineering strategy is necessary to enhance the intracellular butyryl-CoA pool and reduce the acetyl-CoA pool in order to achieve improved butanol titers and selectivity.
Links	PubMed Online version:10.1002/bit.22058
Keywords	Acetone/metabolism; Acetyl-CoA C-Acetyltransferase/biosynthesis; Acyl Coenzyme A/metabolism; Aldehyde Dehydrogenase/biosynthesis; Bacterial Proteins/biosynthesis; Butanols/metabolism; Clostridium acetobutylicum/genetics; Clostridium acetobutylicum/metabolism; Ethanol/metabolism; Fermentation; Gene Expression; Metabolic Networks and Pathways/genetics
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
CLOAB:ADHE		GO:0004022: alcohol dehydrogenase (NAD) activity	ECO:0000314:		F		Figure 2 shows that overexpression of the alcohol/aldehyde dehydrogenase gene increased the production of butanol and ethanol.	complete CACAO 4827
CLOAB:ADHE	enables	GO:0004022: alcohol dehydrogenase (NAD) activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete

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