Abstract: In order to obtain high-yield conjugated linoleic acid (CLA)-producing strains, the myosin-cross-reactive antigen gene (mcra) from Lactobacillus plantarum and the linoleic acid isomerase gene (lai-p) from Propionibacterium acnes were separately cloned and ligated to pCold-SUMO skeleton plasmid to construct recombinant E. coli strains. The expression of the positive recombinant strains was induced with isopropyl-β-D-thiogalactopyranoside (IPTG) at low temperature. The target proteins were validated by SDS-PAGE, indicating successful expression of the two genes. Error-prone polymerase chain reaction was employed to establish a library of recombinant mutant strains with mcra or lai-p. Recombinant mutants with higher absorbance and CLA production were identified by flow cytometry, including 243 recombinant pCold-ycmcra-gfp mutants, among which the one numbered 155 displayed the highest absorbance value (1.050 6 ± 0.000 4), 6.02 times higher than that of the original strain; and 156 recombinant pCold-yclai-p-gfp mutants, among which the one numbered 39 showed the highest yield of (11.62 ± 0.003 6) μg/mL, 3.99 times higher than the original strain. This study can be helpful for further understanding the mechanism for increased CLA production in the mutant strains.

Key words: linoleic acid isomerase gene; error-prone polymerase chain reaction; recombinant plasmid; flow cytometry