Display of Linoleic Acid Isomerase on Bacillus subtilis Spores for Bioconversion of Linoleic Acid to t10,c12-Conjugated Linoleic Acid

Abstract

Conjugated linoleic acid (CLA) comprises positional and geometric isomers of linoleic acid (LA) that have attracted interest due to their reported biological activities. In particular, trans-10,cis-12-CLA (t10,c12-CLA) has been associated with distinct metabolic effects, but its selective production remains challenging because conventional chemical synthesis generates mixtures of CLA isomers. In this study, the linoleic acid isomerase from Propionibacterium acnes (PAI) was displayed on Bacillus subtilis spores by fusion to spore crust proteins to develop a biocatalyst for t10,c12-CLA production. Twenty-four recombinant B. subtilis strains were constructed by fusing PAI to six spore crust proteins with different linker architectures. All constructs produced detectable t10,c12-CLA from 5 mg/mL LA, demonstrating that spore-displayed PAI remained catalytically active and accessible to extracellular substrate. The CgeA-L2 construct showed the highest mean CLA production, approximately 1.33 mg/mL CLA under the tested conditions. Time-course experiments revealed that product formation increased during the initial phase of the reaction before reaching a plateau, while recovered spores retained catalytic activity after one reuse cycle. Finally, spore-displayed PAI converted LA released from safflower oil by soluble lipase. Together, these results establish B. subtilis spores as a functional display format for PAI and provide a basis for further development of a recoverable catalyst for CLA production.