Studies On Coordinate Expression Of Multienzyme-genes In Phenylalanine Biosynthesis Pathway

Phenylalanine (Phe), being one of the essential amino acid, has been widely utilized in medicine, nutrition, food and feed industry, especially, L-Phe is one of key material for synthesis of aspartame, a sweetener utilized in mass, the market demand is increasing. However, for lack of advanced and efficient technology for production, L-Phe demand of our country mainly rely on imports, thus the studies on development of engineering bacteria strains for L-Phe production is of great practical significance.To improve the yield of L-Phe producing Engineering Bacteria, coordinate expression of the key enzymes genes, pheA, aroF and ppsA, that involved in L-Phe biosynthesis pathway were studied as follows: [1] Regulatory sequence of enzyme gene pheA, aroF and ppsA was designed and optimized respectively and single recombinant plasmids was constructed separately to achieve high expression of correlating enzyme protein. [2] Recombinant plasmids pZE12-AF and pZE12-RBS-AF were designed and developed, the former with two enzyme genes pheA and aroF tandem, and the latter was adjusted the DNA sequences of intergenic regulatory region between pheA and aroF, and the plasmids were transformed into an auxotrophic E.coli strain MGâ–³pheA...aroF respectively and obtained two gene engineering stains MGâ–³pZE12-AF andâ–³pZE12-RBS-AF. [3] Three enzyme genes pheA, aroF and ppsA were connected in series to develop multi-enzyme genes recombinant plasmids pZE12-AFP and pZE12-RBS-AFP, the latter was modulated the regulatory sequences of ppsA to optimized the expression of correlating enzyme proteins, and two correlating engineering stains MGâ–³pZE12-AFP and MGâ–³pZE12-RBS-AFP were constructed.Enzyme proteins in fermentation broths were observed by SDS-PAGE and L-Phe contents were analyzed. Adjusting of intergenic regulatory sequences between the tandem genes made the enzyme expression of the recombinant plasmids pZE12-RBS-AF and pZE12-RBS-AFP significantly increased, and that made the L-Phe yield of strain MGâ–³pZE12-RBS-AF was 1.5 times that of MGâ–³pZE12-AF; And correspondingly, the yield of MGâ–³pZE12-RBS-AFP was also increased 2 times than that of MGâ–³pZE12-AFP. The successful co-expression of tandem genes cluster of pheA, aroF and ppsA, have maximized the metabolic flow leaning to the biosynthesis of L-Phe. This somehow is a creative attempt to improve the L-Phe yields of engineering strain. In addition, low copy plasmids pZE12 vectors, compared to high copy plasmids (such as pTrc-99a), showed better in enzyme expression, stable, not easy to form inclusion bodies and higher activity.