Synthetic Levodopa By Escherihia Coli And Medium Optimization

Parkinson’s disease is a kind of extrapyramidal dysfunction caused by chronicneurodegenerative diseases. The brain black and striatum in the nerve cells of patientare damaged, leading to the low level of dopamine tyrosine enzyme and consequentlycause the insufficient dopamine in the brain. Nowadays, the most effective treatmentfor Parkinson’s disease and the most common clinical drug is levodopa. This paper isaimed to construct the dopamine production engineering bacterium, optimizefermentation conditions to achieve the maximum levodopa production, and improvelmbB2gene expression activity by using error-PCR.The paper constructs different recombinant plasmids and selects pCDFDuet-1asoptimal lmbB2expression vector. Optimization expression of the gene lmbB2is done,finding that with1mmol/L IPTG and induction of8hours, the expression is the best.The method of detection tyrosine and levodopa using HPLC is established. Thespectrogram of HPLC-MS acknowledges the existing of levodopa in broth.Through the selection of medium, M9using glycerol as carbon source is chosen.The tyrosine level far cannot satisfy the hydroxyl speed of the lmbB2gene product inthe wild E.coli. And addition of substrate can increase the level of levodopa during thefermentation. Ascorbic acid can significantly improve the tyrosine conversion rate by92.5%while3g/l ascorbic acid joins. The addition of Fe²⁺and H₂O₂is favorable tothe levodopa production.Mutation library is carried out by error-PCR, after setting up the quick scanningmethod, lots of bacterium producing more levodopa can be found and be sequenced.Through sequencing the high yield E.coli and bioinformatics, the coenzyme oflmbB2gene product might be heme, and the catalytic activity sites might be96Gln，133Tyr，139Tyr，148Tyr，160His，170Asp，202His，238Tyr，239Asp，268Asp.This paper establishes a genetic stable engineering bacterium with high expressionefficiency. This work laid a foundation to further optimize levodopa fermentationprocesses and the prediction the active site of gene lmbB2.