Construction Of The Engineered Strain For Effective Bioconversion Of Phenylalanine To D-phenyllactic Acid

Phenyllactic acid(PLA)is widely found in kimchi,honey and other foods.It is a new type of natural antibacterial substance and preservative that can inhibit a series of gram-negative/positive bacteria,and fungi.In addition,PLA has obvious improvement and protection of vascular system,which has been widely used in food and pharmaceutical industries.There are two isomers of phenyllactic acid,and D-phenyllactic acid(D-PLA)has higher antibacterial activity.In this study,Escherichia coli was used as a whole-cell catalyst,through the efficient expression of D-lactate dehydrogenase and phenylalanine aminotransferase,the introduction of the cofactor self-sufficient system,the optimization of the transformation system,the elimination of product inhibition,and the improvement of membrane permeability,D-PLA was efficiently produced using phenylalanine as a substrate.Firstly,D-lactate dehydrogenase was over-expressed using a high-copy plasmid with an arabinose-inducible promoter,and the recombinant strain BW/pRB1s-Dldh was constructed based on E.coli BW25113.About 0.35 mM D-PLA was successfully produced by whole-cell bioconversion,and the conversion rate was 0.6%.Then,the phenylalanine aminotransferase was introduced into the recombinant strain to accelerate the biosynthesis of phenylalanine to phenylpyruvate,and the recombinant strain BW/pRB1s-Dldh-Tyrb was constructed.The yield of D-PLA reached 3.2 mM,and the conversion rate was about 6%.Furthermore,the site-directed mutagenesis of D-lactate dehydrogenase was performed to construct the recombinant strain BW/pRB1s-MDldh-Tyrb with high enzyme activity and D-PLA production was increased to 5.5 mM,and the conversion rate was 11%.Since the D-lactate dehydrogenase is a NADH-dependent oxidoreductase,glutamate dehydrogenase was then introduced to establish a cofactor NADH regeneration system,and the recombinant strain BW/pRB 1 s-MDldh-Tyrb-rocG was constructed,resulting in an increase in D-PLA production(about 13 mM),and the conversion rate reached 27%.Secondly,it was found that excessive expression of D-lactate dehydrogenase significantly inhibited the expression of phenylalanine aminotransferase and glutamate dehydrogenase in recombinant strain.Therefore,the expression level of D-Lactate dehydrogenase was reduced by adding different numbers of arginine(Arg)rare codons.The results showed 17.5 mM D-PLA was achieved by recombinant strain containing four Arg rare codons with a conversion rate of 35%.In addition,the transformation conditions were optimized to determine 50 mM Tris-HCl pH 8.0 buffer,30 OD broth concentration,50 mM phenylalanine as the substrate,with 5 mM alpha-ketoglutarate as the most favorable system for the synthesis of D-PLA.After optimization,D-PLA production was up to 20 mM with 40 mol%conversion during 1 h bioconversion,and the productivity was 3.4 g/L/h.Finally,in order to eliminate the product inhibition in the bioconversion system and to study the effect of membrane permeability of E.coli on the production of D-PLA,the ion exchange resins as well as different concentrations of organic solvents and surfactants were added to the conversion system.When D-PLA was adsorbed by the resin,the concentration of the phenylalanine remained basically unchanged,indicating that the product inhibition did not exist in the system.Furthermore,the results of the addition of organic solvents and surfactants showed that hexane had the most obvious effect(1%for 20 min).The final yield of D-PLA reached up to 30 mM with 60 mol%conversion by increasing the intake of phenylalanine.The productivity was about 5 g/L/h,which was the highest level of D-PLA productivity catalyzed by phenylalanine as the substrate by whole-cell conversion.