Directed Evolution Of Linoleate Isomerase

Conjugated Linoleic Acid(CLA)is a general term for a kind of octadecadienoic acid with different positions and geometric isomers.CLA have different physiological functions because of its various structures.Currently,c9,t11-CLA and t10,c12-CLA are the active isomers that are investigated much.Biosynthesis method uses linoleic acid isomerase to catalyze LA to different CLA and it is favored by researchers because of its uni-structure and higher safety,but catalytic ability of linoleic acid isomerase also needs to be improved.In this study,the genomes of Lactobacillus plantarum isolated from fermented milk and commercially available Propionibacterium acnes were used as templates,to clone and express the mcra gene of Lactobacillus plantarum and the linoleic acid isomerase gene of Propionibacterium acnes.Error-prone PCR was employed to obtain mutant two kinds of genes so as to achieve strains with high absorbance values and high-yield CLA.The recombinant plasmid carrying green fluorescent protein label was screened by flow cytometry to achieve the positive mutants quickly.Certain mutant strains with increased enzyme activity were sequenced to analyze linoleic isomerase gene before and after mutation and to explore the effect of heterotopic site on enzyme activity.Ten strains are screened and identified from five traditional fermentation yogurts of Inner Mongolia and the fermentation performance of Lactobacillus plantarum HAC01 with high yield of CLA was determined.The optimal fermentation conditions by uniform design are as follows: 4% inoculum,0.7 mg/m L LA,fermentation for 32 h at p H 6.5 and 37 ?,ripening for 12 h at 4 ?.The mcra gene of Lactobacillus plantarum and linoleic acid isomerase gene of Propionibacterium acnes were cloned and named as lai-pl(ORF1695bp)and lai-p(ORF1275bp),whose gene similarity was 100 % as corresponding strains according to gene sequence,indicating that there was no gene mutation and the clone was successful.Two linoleic isomerase genes were connected with p Cold-SUMO soluble prokaryotic expression vector and transformed into E.coli.The protein molecular expressed in Lactobacillus plantarum is 70 k Da and that of Propionibacterium acne is 55 k Da.Use error-prone PCR technology to mutate lai-pl and lai-p,and connect the green fluorescent protein gfp gene toconstruct label expression vectors named p Cold-gfp-yclai-pl and p Cold-gfp-yclai-p.Then screen the positive mutants by flow cytometry.Among 282 p Cold-gfp-yclai-pl recombinant strains,there were 243 strains with improved absorbance values,with an increase rate of 86.2 %.Thereinto,the strain with highest absorbance values production is No.155,whose CLA production is 0.1746 ± 0.0002 before mutation and 1.0506 ± 0.0004 after mutation,increased by 6.02 times.Among 174 p Cold-gfp-yclai-p recombinant strains,243 strains exhibited improved CLA production,with an increase rate of 89.6%.The highest CLA production is No.39 with the yield of 11.62 ± 0.0036 ?g/m L after mutation,which is 3.99 times higher than that before mutation(2.91 ± 0.0000 ?g/m L).After sequencing of p Cold-gfp-yclai-pl,No.53 strain obtained the complete sequences.The amino acid mutation sites of the strain are Y-C(69),L-P(77),F-L(174)and N-S(486).Biological information analysis suggested that the increase in the overall activity of linoleic isomerase family MCRA protein was related to the configuration change of 38-69 amino acids,and the No.69 amino acid was mutated into the beneficial mutation sites of linoleic isomerase family mcra.In this paper,bioinformatics analysis om the primary,secondary and tertiary structures of MCRA protein in increased absorbance values production strains were carried out,providing theoretical basis for further elucidating the mechanism of linoleic isomerase action and site-specific mutation to improve CLA production.