Building Of Engineering Bacterias To Produce L-phenylalanine And Optimizing The Fermentation Conditions Of The Strains

Phenylalanine is widely used in the production of medicine, food and feed addictives. But now, the practical yield of Phenylalanine from domestic enterprise can never ever meet the increasing demands. For this reasion, construction of engineered strain which can produce high yield of L-Phenylalanine by molecular biology technology has great practical significance, lowering the cost, cuting down the import volume and increasing the competitive power.GlpK gene and glpD gene which involved in L-Phenylalanine biosynthesis pathway were amplified by PCR, then attached them to pAF(C), pAF(L), pZE12-GFP, pZA31respectively, pAF(C)D, pAF(C)K I, pAF(L)K, pAF(C)KⅡ, p31K were obtained. Transfoumed one or two vector to E.coliMGΔ, to acquire enjineering bacterias of E.coliMGΔpAF(C)D, E.coliMGΔpAF(C)K I,E.coliMGΔpAF(L)K I, E.coliMGΔpAF(C)K Ⅱ, E.coliMG Ap31K-pAF(C), E.coliMGΔpAF(C)K Ⅱ-p31PT. So for the first time, we fulfill the coexpression of pheA、aroF and glpD, pheA、aroF and glpK, glpK、pheA、aroF、ppsA and tktA.The analysis performed by SDS-PAGE, both glpK gene and glpD gene show target band in related engineering bacterias.On condition that37℃、240rpm/min、volume of250ml, when use15g/L glucose or10g/L glycerol as carbon source separately, result show that L-Phenylalainne production of all strains was higher when use10g/L glycerol as the carbon source than15g/L glucose, the productive power of strain E.coliMGΔpAF(C)K Ⅱ is higher.When10g/L glycerol was used as carbon source, the production of L-Phenylalainne in engineering strain E.coliMGΔpAF(C)K Ⅱ was3.33times higher than pAF(C),62.36times higher than E.coliMGΔ, which illustrated that the extra express of glpK gene could increase the output of L-Phenylalanine.On condition we mentioned above, the production of L-Phenylalainne in engineering strain E.coliMGApAF(C)K Ⅱ is the best. Then we use this strain to make our further study, we found that37℃is the best growth temperature for E.coli; also, to increase the fermentation time and ventilation can increase the output of L-Phenylalanine. On condition of37℃,240rpm/min, and volume of500ml, the production of L-Phenylalainne in engineering strain E.coliMGΔpAF(C)KⅡ can reach3.94g/L.Our study of the construction of E.coliMGΔpAF(C)KⅡ, make the increase of the production of L-Phenylalainne come true. This will give us the guidance of the further study of high output strains of L-Phenylalainne.