APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 2007, p. 3061–3068
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 73, No. 9
Dynamics of Genomic-Library Enrichment and Identification of
Solvent Tolerance Genes for Clostridium acetobutylicum?†
Jacob R. Borden and Eleftherios Terry Papoutsakis*
Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208
Received 28 September 2006/Accepted 22 February 2007
A Clostridium acetobutylicum ATCC 824 genomic library was constructed using randomly sheared DNA.
Library inserts conferring increased tolerance to 1-butanol were isolated using two protocols. Protocol I
utilized a single round of butanol challenges in batch culture, while protocol II, which gave clearly superior
outcomes, was based on the serial transfer of stationary-phase cultures into progressively higher butanol
concentrations. DNA microarray analysis made a high-resolution assessment of the dynamic process of library
enrichment possible for the first time. Protocol I yielded a library insert containing the entire coding region of
the gene CAC0003 (which codes for a protein of unknown function) but also several DNA fragments containing
promoter regions. Protocol II enabled the successful identification of DNA fragments containing several intact
genes conferring preferential growth under conditions of butanol stress. Since expression using the employed
library is possible only from natural promoters, among the enriched genes, we identified 16 genes that
constitute the first cistron of a transcriptional unit. These genes include four transcriptional regulators
(CAC0977, CAC1463, CAC1869, and CAC2495). After subcloning plasmids carrying the CAC0003 and
CAC1869 genes, strains 824(pCAC0003) and 824(pCAC1869) exhibited 13% and an 81% increases, respec-
tively, in butanol tolerance relative to the plasmid control strain. 824(pCAC1869) consistently grew to higher
cell densities in challenged and unchallenged cultures and exhibited prolonged metabolism. Our serial
enrichment approach provided a more detailed understanding of the dynamic process of library enrichment
under conditions of selective growth. Further characterization of the genes identified in this study will likely
enhance our understanding of the complex phenotype of solvent tolerance.
Clostridium acetobutylicum ATCC 824 is important for the
biological production of butanol and acetone, two solvents
currently produced mainly from petrochemical feedstocks. Un-
til the mid-1950s, C. acetobutylicum was used in the profitable
ABE (acetone, butanol, and ethanol) fermentation (56). In
response to increasing demands for limited petroleum re-
sources, interest in ABE fermentation has been renewed to the
point that new fermentation facilities are again becoming eco-
nomically viable (15, 25).
An important part of biological research in this area is un-
derstanding and in turn ameliorating the impact of intermedi-
ate and final metabolite products on cell growth and product
formation. For instance, the switch in C. acetobutylicum fer-
mentation from the accumulation of acetic and butyric acids
during the acidogenic exponential phase to acid reassimilation
and butanol, acetone, and ethanol formation during the sol-
ventogenic stationary phase may be due to butyrate accumu-
lation (37, 40, 55, 58). Before long, however, the accumulation
of butanol also has a negative impact on cellular processes due
primarily to the partitioning of polar solvents into the hydro-
phobic region of the phospholipid bilayer (43).
Fundamental work on the impact of alcohols and ketones on
Escherichia coli showed that the intercalation of solvents within
the lipid bilayer increases membrane fluidity (21) and also
affects lipid-protein interactions integral to membrane func-
tion (22, 23). The microbial response to perturbations in mem-
brane fluidity, or “homeoviscous adaptation,” has been studied
extensively with regard to the response of E. coli to tempera-
ture upshifts, a stress phenomenon with an impact on mem-
brane fluidity similar to that of solvent toxicity (44). Responses
of various microorganisms to solvents include active solvent
expulsion by molecular pumps, alterations in the composition
of membrane-lipid headgroups, and the adjustment of the pro-
tein content within the cell membrane (53).
Butanol has a lower partition coefficient (0.8) than either
toluene (2.5) or benzene (2.0) and is therefore predictably
more toxic (43). When clostridia are exposed to butanol, the
ratio of saturated to unsaturated fatty acids incorporated in the
membrane lipid bilayer increases, presumably to compensate
for the fluidity increase imposed by the solvent (7, 8, 28).
Solvents inhibit membrane-bound ATPases, resulting in a drop
in internal pH and the abolishment of the ?pH gradient across
the membrane (10, 46). Butanol also inhibits glucose uptake,
thus inhibiting energy generation, which is compounded by an
independent drop in intracellular ATP levels (10).
Improvements to solvent tolerance and titers in C. acetobu-
tylicum are not limited to the metabolic engineering of mem-
brane functions. The overexpression of groESL, a class I stress
response operon, resulted in increased solvent tolerance and
solvent titers 33% higher than those of a plasmid control strain
(50), indicating a denaturing effect of solvents on functional
enzymes and a role for stress proteins in solvent tolerance.
Secondly, knockout of the metabolic intermediate enzyme bu-
tyrate kinase by chromosomal insertion resulted in a strain that
* Corresponding author. Mailing address: Department of Chemical
and Biological Engineering, Northwestern University, Evanston, IL
60208. Phone: (847) 491-7455. Fax: (847) 491-3728. E-mail: e-paps
† Supplemental material for this article may be found at http://aem
?Published ahead of print on 2 March 2007.
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3068BORDEN AND PAPOUTSAKISAPPL. ENVIRON. MICROBIOL.