Construction Of Recombinant Escherichia.coli And Biosynthesis Of L-DOPA

3,4-dihydroxyphenylalanine (L-DOPA) is the key therapy medicine for Parkinson'sdisease, and also has a wide prospect in the field of health protection, hairdressing, et. at.With the increase of proportion of the aged people, more and more L-DOPA would be neededin market. Biosynthesis of L-DOPA has the advantage of easier process and steadier yieldthan chemosynthesis or extraction from plant. Tyrosine phenol lyase(TPL) is a key enzymewhich could catalyze the biosynthesis of L-DOPA from pyrocatechol, sodium pyruvate andammonia.The tpl structural gene encoding Tyrosine phenol lyase was amplified by PCR usingCitrobacter freundii ATCC8090 chromosomal DNA as a template, then the obtained DNAfragment was ligated into an expression vector pQE30 harbouring strong promoter T5 toconstruct a new recombinant plasmid pQTPL, finally pQTPL was transformed into E.coliM15. The recombinant E.coli M15(pQTPL) could over express TPL under the inductioncondition at 18℃ with 0.2 mmol/L IPTG as inducer, the specific activity of TPL was 208U/mg protein of crude enzyme. L-DOPA synthesis by the cell of E.coli M15(pQTPL) couldreach 1.61 g/g cell/h.The conditions for E.coli M15(pQTPL)cell growth in shake flask were optimized. Theoptimized medium is composed of: glucose 10 g/L, (NH₄)₂SO₄ 3 g/L, peptone 1 g/L, citricacid 1 g/L, KH₂PO₄ 10 g/L, MgSO₄·7H₂O 1.5 g/L, trace elements solution (TES) 5 mL/L.4.18 g/L of dry cell weight was obtained. The effect of dissolved oxygen (DO) concentrationand the feeding of carbon and nitrogen source for the growth of E.coli DH5?(pQTPL) werestudied at a 3.7L fermenter. When the DO was controlled above 30%, the initial glucoseconcentration was 20g/L and 10g/L of glucose was feeded at log phase, dry cell weightreached 14.34 g/L after 7h incubation, and activity of TPL was 63.4 u/g cell at 8h.The conditions including temperature, pH, and the composition of reaction mixture forL-DOPA biosynthesis by E.coli DH5α(pQTPL) were optimized. The initial reactionmixture(20mL) contained 21g/L sodium pyruvate, 14g/L pyrocatechol, 30g/L ammoniumacetate, 1g/L EDTA, 2g/L sodium sulfite, the pH was adjusted to 8.2 by ammonia, and then15g/L E.coli DH5α(pQTPL)(DCW) was added into the mixture. The incubation temperaturewas 15℃. Sodium pyruvate and catechol were feeded into the mixture to maintain theconcentration at 9g/L and 6g/L, respectively once an hour in the first 4 hours and then onceper 2 hours. L-DOPA concentration reached 55.2g/L after 14 hours of incubation. The molconvert ratio of L-DOPA was 82.8% from catechol.