Construction Of Multienzyme-genes Coordinate Expression Vectors In Phenylalanine Biosynthesis Pathway

Being one key material to synthesize sweetener aspartame, the market demanding for L-Phenylalanine (L-Phe) is having been significantly increased with the rapid increase of production of aspartame in recent years. But the domestic production of L-Phe could not meet the demand, and mainly relays on imports. Therefore, the studies on constructing high-yield strains are of great significance.To improve the yield of L-Phe, the following multi-gene vectors were developed to reform metabolic pathways of phenylalanine biosynthesis in E.coli strain:(1)The gene ppsA of phophoenol pyruvate synthase and transketolase tktA were cloned, and the recombinant plasmids pZE12-AFPT and pZE12-AGPT were constrcuted;(2)The plasmids pTrc-99a-ppsA and pTrc-99a-tktA were transformed into the competence of pZE21-AF respectively to obtain the recombinant vectors with two resistance;(3) The plasmid pZA31-ppsA-tktA was transformed into the competent cells of pZE21-AF, pZE12-AF and pZE12-AFK to obtain recombinant plasmids of multi-enzyme genes with double resistance.Fermentation parameters were optimized by single factor and orthogonal design experients. SDS-PAGE was applied to observe enzyme proteins yield. Results showed that the co-expression of tandem genes cluster of pheA, aroF, ppsA and tktA in recombinant plasmid pZE12-AFPT was realized, the L-Phe yield of engineering strain MGâ–³pZE12-AFPT increased about1.6times and25times when it compared to MGâ–³pZE12-AF and the auxotrophic E.coli strain MGâ–³pheA...aroF respectively; The L-Phe yield of some vectors with double resistance has increased than the former single vector.