Heterologous Expression and Polyphasic Analysis of CLA-Converting Linoleic Acid Isomerase from <i>Bifidobacterium breve</i> JKL2022

The probiotic <i>Bifidobacterium breve</i> is known for its efficient conjugated linoleic acid (CLA) conversion, yet their CLA conversion pathway remains underexplored. This study investigated <i>B. breve</i> JKL2022 for its CLA conversion in actively growing cells, washed cell states, and in crude protein extracts. Moreover, the study aimed to confirm the CLA-converting enzyme in strain JKL2022 and optimize its purification through heterologous expression of fusion proteins (LAI_sGFP and MBP_LAI). JKL2022 exhibited superior CLA conversion compared to genetically similar <i>B. breve</i> strains (JCM7017, JCM7019, JCM1192, and JCM1273), particularly the observed CLA conversion in washed cells (60.14 ± 7.60%) and crude protein fractions (96.11 ± 6.63%). The multipass transmembrane linoleic acid isomerase (LAI) was cloned into the <i>E. coli</i> BL21(DE3) as free LAI or modified with superfolder-GFP or MBP tags and expressed with 0.01 mM IPTG at 37 °C, resulting in highly active protein fractions. LAI was characterized by predictive modeling, molecular docking, and phylogenetic analyses. Moreover, reverse transcription-quantitative PCR analysis revealed upregulation (20-140× higher expression) of <i>lai</i> in JKL2022 compared with that in the JCM strains. Nevertheless, upscaling the production and purification of LAI for downstream applications remains a challenge, primarily because of their membrane-spanning configuration.