Mechanism Of Missense Mutation Affecting Linoleic Acid Isomerase Activity

Conjugated linoleic acid(CLA)has the functions of preventing hyperglycemia,hyperlipidemia,hypertension,malignant tumors,atherosclerosis,and inhibiting fat growth,which has attracted the attention of scientific and technological workers.CLA product produced by microbial catalysis is single,high safety factor and stable,but the biocatalytic performance of linoleic acid isomerase(LAI)needs to be improved.In this study,the linoleic acid isomerase gene of Propionibacterium acnes CICC 10864(referred to as P.acnes)was used as a template,and a high-yielding CLA mutant was obtained by using error-prone PCR mutation.In order to explore the generation of CLA and LAI activity,the relationship between using proteomics technology to mutant strain and the mutant strains of differentially expressed proteins were analyzed.Metabolomics analysis was used to find differential metabolites,explore the correlation and change rules,and elucidate the effect of protein differences on the activity and function of P.acnes linoleic acid isomerase.The result is as follows:Using directed evolution technology and multiple rounds of error-prone PCR,a mutant library of P.acnes mutants was constructed,and the highest yield of CLA of P229 was17.4609±0.0007 μg/m L by flow cytometry.The enzyme activity of 52.3827 U/m L,4.1454 times is not mutated bacteria.The sequence of mutant P229 is complete,and homology modeling analysis found that there are five amino acid mutation sites,namely D101 Y,Q138R,F214 I,W227R,and F256 I.The hydrogen bonds before and after the mutation have changed significantly,which is a direct result of the improvement of catalytic activity.reason.Proteomics technology was carried out on the P229 strain,identified 2146 proteins,of which 2110 protein is quantifiable;Select 104 differentially expressed proteins,including lower protein 47,increase protein 57.Metabolomics analysis found that a variety of metabolites,distributed in different metabolic pathways,play an indispensable role.The differentially expressed proteins were mainly distributed in starch and sucrose metabolism,galactose metabolism and phosphotransferase system.According to the glycolysis and tricarboxylic acid cycle pathways,three differential metabolites were annotated,namely phosphoenolpyruvate,acetyl-Co A,and glutamate.Five differentially expressed proteins were up-regulated,namely epd,crr,pfk B,tre B,and gdh A.The expression of proteins gdh A and mtr related to glutamate metabolism were up regulated,which was beneficial to promote the conversion of linoleic acid to conjugated linoleic acid.Analysis of the CLA synthesis pathway of genetically engineered strains found that up-regulating key proteins in carbohydrate and amino acid metabolism can increase the production of CLA,which lays a theoretical foundation for the industrialized production of CLA.