Characterization of Halophilic Acyl-CoA Thioesterase from Chromohalobacter salexigens for Use in Biofuel Production

Abstract

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 biofuel systems.