Characterization of Halophilic Acyl-CoA Thioesterase from Chromohalobacter salexigens for Use in Biofuel Production
Steven Schreck, Rushyannah R. Killens-Cade and Amy M. Grunden
Affiliation: Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC 27695- 7615, USA.
Keywords: Algae, biofuel, Chromohalobacter salexigens, esterase, extremophile, halophile, lipase, lipids, thioesterase.
Acyl-CoA thioesterases are enzymes which catalyze the hydrolysis of acyl-CoAs at thioester linkages to form
free fatty acids and coenzyme A. These enzymes, along with lipases, have a wide spectrum of use in industrial
applications including the production of fatty acids from oil-producing microorganisms for use as biofuel feed-stocks.
This technology coupled with an increasing interest in sustainable biofuels has led researchers to investigate algae based
systems of fuel production. Since a number of marine algae that are being evaluated for biofuel production prefer
moderately halophilic environments, it follows that thioesterases/lipases originating from a halophilic organism may be
best suited for use with a marine algae-based biofuel system. Recent studies have shown increased production of free fatty
acids in Escherichia coli and cyanobacteria following overexpression of recombinant, leaderless thioesterase I (TesA)
from E. coli. A homolog of TesA from the moderate halophilic bacterium Chromohalobacter salexigens has been
identified, cloned, and recombinantly expressed in E. coli strains BL21 and M15. Because previous studies indicated that
histidine tag position alters TesA substrate specificity, three different recombinant versions of the C. salexigens TesA
were produced for this study; an N-terminal histidine-tagged enzyme, a C-terminal histidine-tagged enzyme, and one in
which the N-terminal histidine-tag was removed via DAPase digestion. Introduction of a C-terminal histidine tag shifted
substrate preference to shorter (<C10) carbon chain lengths, while introduction of an N-terminal tag resulted in overall
reduced activity. Finally, removal of the N-terminal tag restored overall activity levels while slightly increasing enzyme
preference for longer (C10/C12) carbon chain substrates. Given the requirement of lipolytic activity to generate free fatty
acids for biofuel synthesis, this study has important implications for the use of thioesterases/lipases in marine algae based
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