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Targeted mutagenesis of the Clostridium acetobutylicum acetone–butanol–ethanol fermentation pathway

Paper ID Volume ID Publish Year Pages File Format Full-Text
31714 44832 2012 12 PDF Available
Title
Targeted mutagenesis of the Clostridium acetobutylicum acetone–butanol–ethanol fermentation pathway
Abstract

The production of the chemical solvents acetone and butanol by the bacterium Clostridium acetobutylicum was one of the first large-scale industrial processes to be developed, and in the first part of the last century ranked second in importance only to ethanol production. After a steep decline in its industrial use, there has been a recent resurgence of interest in the acetone–butanol–ethanol (ABE) fermentation process, with a particular emphasis on butanol production. In order to generate strains suitable for efficient use on an industrial scale, metabolic engineering is required to alter the AB ratio in favour of butanol, and eradicate the production of unwanted products of fermentation. Using ClosTron technology, a large-scale targeted mutagenesis in C. acetobutylicum ATCC 824 was carried out, generating a set of 10 mutants, defective in alcohol/aldehyde dehydrogenases 1 and 2 (adhE1, adhE2), butanol dehydrogenases A and B (bdhA, bdhB), phosphotransbutyrylase (ptb), acetate kinase (ack), acetoacetate decarboxylase (adc), CoA transferase (ctfA/ctfB), and a previously uncharacterised putative alcohol dehydrogenase (CAP0059). However, inactivation of the main hydrogenase (hydA) and thiolase (thl) could not be achieved. Constructing such a series of mutants is paramount for the acquisition of information on the mechanism of solvent production in this organism, and the subsequent development of industrial solvent producing strains. Unexpectedly, bdhA and bdhB mutants did not affect solvent production, whereas inactivation of the previously uncharacterised gene CAP0059 resulted in increased acetone, butanol, and ethanol formation. Other mutants showed predicted phenotypes, including a lack of acetone formation (adc, ctfA, and ctfB mutants), an inability to take up acids (ctfA and ctfB mutants), and a much reduced acetate formation (ack mutant). The adhE1 mutant in particular produced very little solvents, demonstrating that this gene was indeed the main contributor to ethanol and butanol formation under the standard batch culture conditions employed in this study. All phenotypic changes observed could be reversed by genetic complementation, with exception of those seen for the ptb mutant. This mutant produced around 100 mM ethanol, no acetone and very little (7 mM) butanol. The genome of the ptb mutant was therefore re-sequenced, together with its parent strain (ATCC 824 wild type), and shown to possess a frameshift mutation in the thl gene, which perfectly explained the observed phenotype. This finding reinforces the need for mutant complementation and Southern Blot analysis (to confirm single ClosTron insertions), which should be obligatory in all further ClosTron applications.

► A large-scale targeted mutagenesis in Clostridium acetobutylicum ATCC 824 was carried out. ► Generating mutants: adhE1, adhE2, bhdA, bdhB, ptb, ack, adc, ctfA, ctfB, CAP0059. ► Mutant complementation and Southern Blot should be obligatory in ClosTron applications. ► Gene adhE1 is the main contributor to ethanol and butanol formation. ► bdhA and bdhB mutants were not affected in solvent production, other mutants showed predicated phenotypes.

Keywords
Clostridium; ClosTron; Phosphotransbutyrylase; Alcohol/aldehyde dehydrogenases; Acetate kinase; Acetoacetate decarboxylase; CoA transferase; CAP0059; Butanol dehydrogenases; Thiolase
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Targeted mutagenesis of the Clostridium acetobutylicum acetone–butanol–ethanol fermentation pathway
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Publisher
Database: Elsevier - ScienceDirect
Journal: Metabolic Engineering - Volume 14, Issue 6, November 2012, Pages 630–641
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us