Study On Biosynthesis Of Conjugated Linoleic Acid Monomers By Recombinant Yeast Strains

Conjugated linoleic acid(CLA)is a collective term for a group of octadecadienoic acids with a conjugated double bond,of which the two most physiologically active isomers are c9,t11-CLA and t10,c12-CLA,and each of the two isomers has a different physiological function.Currently chemically synthesized CLA is mostly a mixture of multiple isomers,while the biosynthesis of single isomers of CLA has received much attention in recent years as the study of conjugated linoleic acid monomers has become more advanced.The use of genetic engineering to heterologously express genes related to CLA synthesis can solve some of the problems associated with primary bacterial fermentation,which has low yields and stringent culture conditions.BBI,the linoleic acid isomerase from Bifidobacterium breve CCFM 683,is the only bacterial isomerase with a known sequence capable of synthesizing c9,t11-CLA by monoenzymatic catalysis,but BBI has a problem of low expression during heterologous expression.In order to solve the above problems,different BBI recombinants were constructed,and the most suitable host strain and expression forms of BBI were explored to provide a basis for the biological synthesis of c9,t11-CLA monomer.PAI,the linoleic acid isomerase from Propionibacterium acnes,can synthesize t10,c12-CLA by single enzyme catalytic synthesis,but the main problems of PAI in heterologous expression are easy to form inclusion bodies and low expression amount.In this study,we constructed PAI secretory expression vectors and soluble expression vectors,which provided a new direction for the efficient expression of PAI.The main findings of this paper are as follows:1.To achieve efficient expression of BBI,recombinant S.cerevisiae and recombinant Pichia pastoris were constructed.The protein expression and enzyme activity levels of each recombinant strains were analysed to determine the optimal host strain and the optimal His-tag position for BBI.The ability of resting cells to synthesise c9,t11-CLA and the enzymatic properties of the crude enzyme of BBI were investigated.Firstly,the recombinant S.cerevisiae BBI was constructed using S.cerevisiae as the host strain.The recombinant strain were analysed for protein expression level and no specific bands were detected;the recombinant bacteria were assayed for activity and the conversion rate of the substrate was only 5.12%.This indicates that BBI is heterologously expressed in S.cerevisiae and has catalytic activity,but the protein expression is extremely low.Secondly,three different recombinant strains of BBI were successfully constructed using Pichia pastorisas the host strain.The protein expression and enzyme activity levels of different recombinant strains were analysed,and it was found that Pichia pastoris is suitable for BBI heterologous expression,with the recombinant strain with His-tag at the C-terminus showing the highest expression of the target protein and the highest enzyme activity.The yield of c9,t11-CLA was 15.88 g/L with the addition of 25 g/L linoleic acid(LA)for 3 h.This is the highest yield of c9,t11-CLA monomer using resting cells of BBI recombinant strain as a catalyst,which was reported in the present study.The enzymatic properties of the crude enzyme of BBI recombinant strain were studied.The optimum reaction temperature of BBI was 37°C;the optimum p H was 8.0;there were significant differences in enzyme stability under different temperature conditions;most metal ions inhibited the enzyme activity to varying degrees.2.In order to achieve efficient expression of PAI,PAI-secreting recombinant strain and soluble expressing recombinant strain were constructed using Pichia pastoris as the host strain respectively.The protein expression and enzymatic activity levels of the recombinant strains were analysed to obtain a soluble PAI-expressing recombinant strain,to further optimise its protein expression levels and to investigate the ability of the recombinant resting cells to catalyse the production of t10,c12-CLA.Firstly,a recombinant PAI-secreting strain with ?-factor signal peptide was constructed.Analysis of the protein expression levels of the recombinant strain showed that the PAI recombinant protein was not expressed.The protein activity of the recombinant strain did not detect the production of t10,c12-CLA,which indicated that PAI was not secreted in Pichia pastoris.Secondly,the PAI soluble recombinant Pichia pastoris expression was constructed,and the protein expression level of the recombinant strain was optimized.The best induction time was 24 h,the best inducer methanol volume fraction was 2%,and the resting cells of the recombinant strain were used as the whole cell catalyst,adding 25 g/L of LA and reacting for 40 h,the yield of t10,c12-CLA is 8.39 g/L,and the conversion rate can reach 33.56%.