Modification,Constitutive Expression And Synthesis Of L-DOPA Of Tyrosine Phenol-Lyase

Levodopa（3,4-dihydroxylphenylalandine,L-DOPA）is the most effective drug against Parkinson’s disease.Microbial enzyme synthesis of L-DOPA is an environmentally friendly,simple and efficient method with broad application prospects.Tyrosine phenol-lyase（TPL）can reversibly catalyze the synthesis of catechol,pyruvate and ammonium salts into L-DOPA.In this study,TPL from Erwinia herbicola was used as the starting enzyme to realize the molecular transformation and constitutive expression of TPL in Escherichia coli,and the optimization of culture medium and whole cell catalysis conditions were studied.The main research results were showed as follows:（1）The recombinant expression plasmid of pET-28a-tpl was constructed and expressed in E.coli BL21（DE3）.After sequence analysis and homologous modeling of TPL,molecular docking simulation showed that the amino acids around the active center of the enzyme were highly conserved,and four mutation sites of E44,M69,T127 and E198 were identified for site-directed mutation.A positive mutant strain T127P was screened.Under the condition of 20℃and pH 8.0,the yield of L-DOPA catalyzed by whole cell was 3.71 g L^-1 h^-1,which was 1.21times of wild type.（2）The constitutive expression of the TPL under the combination of six intensity and different composition promoters and ribosome binding sites（RBS）were studied in E.coli.The recombinant bacterium strain 1-6 is constructed from low to high according to the composition of the constituent promoter and the RBS combination.The strain 5 was screened by comparing the yield of the recombinant bacteria to catalyze the production of L-DOPA,and the expression of TPL can be greatly coupled with cell growth.Strain 5 was added into the system at a wet cell concentration of 6 g L^-1 for 1 h,and 3.68 g L^-1 L-DOPA was obtained.（3）Based on the strain 5,the whole cell catalytic reaction conditions and fermentation medium were optimized.The whole cell catalytic reaction temperature,reaction pH,and the concentration of catechol in the system were optimized.The results showed that the optimum reaction temperature was 20°C,the most suitable pH was 8.5,and the most adaptive concentration of catechol was 10 g L^-1.The fermentation medium was optimized,and the optimum ratio of culture medium was as follows:peptone 18 g·L^-1,yeast extract 28 g·L^-1,glycerol 10 g·L^-1.（4）After fed-batch whole cell transformation reaction in a 5 L bioreactor for 6 h,the concentration of L-DOPA could reach 67.90 g·L^-1.