Heterologous Expression of Production of T10, C12-CLA Linoleic Acid Isomerase Gene from Propionibacterium acnes

The linoleic acid isomerase enzyme from Propionibacterium acnes, which catalyzed the isomerization of a methylene-interrupted double bond system to a conjugated double bond system, creating trans-10, cis-12 conjugated linoleic acid (t10, c12 CLA) was cloned and overexpressed in Escherichia coli using expression system of pCold-SUMO. The gene was expressed under T7 promotor with a fusion partner of 6×His. Tag at its 5'terminal. After induction by 0.2mM IPTG for 18 h, the recombinant enzyme are expressing in the cytoplasm. The 1275 bp gene from P. acnes encode a linoleic acid isomerase protein with 424 amino acids (55KD).

Molecular Cloning and Heterologous Expression of Linoleic Acid Isomerase Gene from Lactobacillus reuteri and Lactobacillus acidophilus

The linoleic acid isomerase enzyme from Lactobacillus reuteri and Lactobacillus acidophilus, which were responsible for bioconversion of linoleic acid to cis-9, trans-11 conjugated linoleic acid (c9, t11 CLA) was cloned and overexpressed in Escherichia coli using expression system of pCold-SUMO. The gene was expressed under T7 promotor with a fusion partner of 6×His. Tag at its 5'terminal. After induction by 0.2mM IPTG for 18 h, the recombinant enzyme are expressing in the cytoplasm. The 1776 bp gene from L. reuteri and L. acidophilus encode a linoleic acid isomerase protein with 591 amino acids (64KD).

Recombinant lactobacilli expressing linoleic acid isomerase can modulate the fatty acid composition of host adipose tissue in mice

We have previously demonstrated that oral administration of a metabolically active Bifidobacterium breve strain, with ability to form cis-9, trans-11 conjugated linoleic acid (CLA), resulted in modulation of the fatty acid composition of the host, including significantly elevated concentrations of c9, t11 CLA and omega-3 (n-3) fatty acids in liver and adipose tissue. In this study, we investigated whether a recombinant lactobacillus expressing linoleic acid isomerase (responsible for production of t10, c12 CLA) from Propionibacterium acnes (PAI) could influence the fatty acid composition of different tissues in a mouse model. Linoleic-acid-supplemented diets (2 %, w/w) were fed in combination with either a recombinant t10, c12 CLA-producing Lactobacillus paracasei NFBC 338 (Lb338), or an isogenic (vector-containing) control strain, to BALB/c mice for 8 weeks. A third group of mice received linoleic acid alone (2 %, w/w). Tissue fatty acid composition was assessed by GLC at the end of the trial. Ingestion of the strain expressing linoleic acid isomerase was associated with a 4-fold increase (P<0.001) in t10, c12 CLA in adipose tissues of the mice when compared with mice that received the isogenic non-CLA-producing strain. The livers of the mice that received the recombinant CLA-producing Lb338 also contained a 2.5-fold (albeit not significantly) higher concentration of t10, c12 CLA, compared to the control group. These data demonstrate that a single gene (encoding linoleic acid isomerase) expressed in an intestinal microbe can influence the fatty acid composition of host fat.