Modification Of Tyrosine Phenol Lyase To Enhance Whole-Cell Catalytic Synthesis Of L-DOPA

3,4-2dihydroxylphenylalanine?L-DOPA?is the main drug for the treatment of Parkinson's disease.With the aging of population and increase of modern society pressure,the demand for L-DOPA among the global market increased rapidly.At present,the L-DOPA synthesis in the industry mainly includes chemical synthesis and microbial enzymatic method.The chemical synthesis method has the advantages of high purity and high yield,however,it also has the problems of complicated steps,serious environmental pollution and high cost.The synthesis of L-DOPA by biocatalysis has broad industrial application prospects with the advantages of simple process,stable yield and mild conditions.The activity of tyrosine phenol-lyase?TPL?in the original strain is generally low.In order to increase the expression and activity of tyrosine phenol-lyase,a recombinant strain of E.coli expressing TPL derived from Erwinia herbicola was used as a start strain in this study.The error-prone PCR was used to introduce random mutagenesis to the TPL gene.Then the culture catalytic synthesis conditions were optimized at the shake flask level.Finally,the high density of the recombinant bacteria was achieved by optimizing the fermentation conditions on the bioreactor.The main findings of the paper are as follows:?1?Tyrosine phenol lyase was directed to evolve by busing error-prone PCR technology,and a mutant strain with higher TPL activity was obtained through high-throughput screening technology.The enzymatic properties of the mutant enzyme were further analyzed.First,the conditions of error-prone PCR were optimized:the concentrations of Mg²⁺and Mn²⁺were determined to be 4 mmol/L and 0.2 mmol·L^-1,respectively.After two rounds of error-prone PCR and high-throughput screening,a positive mutant strain 3-2F9 was screened from about 2×10⁴ mutants.The L-DOPA titer was increased by 36.5%compared with the original strain after whole cell catalysis.The enzymatic properties of the mutant TPL were further determined.Results showed that the optimum reaction temperature was 30^oC,the optimum pH was 9.0.In addition,the enzyme was relatively stable at temperature of 20-35^oC and pH of 8.5-10.Compared with wild-type TPL,the temperature range of the effective enzyme activity of the mutant TPL is increased,and the alkali resistance is strong.?2?The conditions of mutant strain cultivation,TPL expression and the L-DOPA synthesis were optimized at the shake flask level.The optimum medium conditions were as following:1.5 g·L^-1 of MgSO₄·7H₂O and 4 mL·L^-1 of trace element mother liquor were added based on TB medium.The optimum culture conditions were as following:optimum culture time of the seed was 8-10 h,induction time was 12 h at 25^oC;IPTG concentration was 0.2 mmol·L^-1.And the optimum transformation conditions were as following:The initial concentrations of pyruvic acid,catechol and ammonium acetate were 15 g·L^-1,10 g·L^-1 and 30 g·L^-1,respectively,and pyruvate and catechol were added every 1.5 h to a final concentration of 6 g·L^-1 and 4 g·L^-1 with three times feeding,respectively.After 8 h of reaction at 15^oC,the titer of L-DOPA was up to 30 g·L^-1,which is 72.4%higher than before optimization.?3?Conditions for high-density fermentation production of TPL and catalytic synthesis of L-DOPA were optimized on 5 L fermentors.The optimum inoculum size was 2%.And a three-stage fermentation culture mode for controlling dissolved oxygen was established by using the DO-stat feeding strategy.The specific fed-batch fermentation process was as following:The dissolved oxygen was controlled at 20%before induction,then controlled at 30%for the first 2 hours after induction,the dissolved oxygen was finally controlled at 40%or more.The added glycerin concentration was300 g·L^-1.Under this condition,the TPL activity reached 2.4 U·mL^-11 at 10 h,the bacterial concentration reached 51.2,and the L-DOPA titer was 69.1 g·L^-1.