Chromosome engineering to generate plasmid-free phenylalanine- and tyrosine-overproducing Escherichia coli which can be applied in generation of aromatic compound-producing strains.

Many phenylalanine- and tyrosine-producing strains have used plasmid-based overexpression of pathway genes. The resulting strains achieved high titer and yield of phenylalanine and tyrosine. Chromosomally-engineered, plasmid-free producers have shown lower titer and yield than plasmid-based strains, but the former are advantageous in terms of cultivation cost and public health/environmental risk. Therefore, here, we engineered the Escherichia coli chromosome to create superior phenylalanine- and tyrosine-overproducing strains that did not depend on plasmid-based expression. Integration into the E. coli chromosome of two central metabolic pathway genes (ppsA and tktA) and eight shikimate pathway genes (aroA, aroB, aroC, aroD, aroE, aroGfbr, aroL, and pheAfbr), controlled by the T7lac promoter, resulted in excellent titer and yield of phenylalanine; fbr indicates that the enzyme encoded by the gene was feedback-resistant. The generated strain could be changed to be a superior tyrosine-producing strain by substitution of pheAfbr to tyrAfbr. A rational approach revealed that integration of seven genes (ppsA, tktA, aroA, aroB, aroC, aroGfbr, and pheAfbr) was necessary as the minimum gene set for high-yield phenylalanine production in E. coli MG1655 (tyrR, adhE, ldhA, pykF, pflDC, and ascF deletant). The phenylalanine- and tyrosine-producing strains were further applied to generate phenyllactic acid-, 4-hydroxyphenyllactic acid-, tyramine-, and tyrosol-producing strains; yield of these aromatic compounds increased proportionally to the increase in phenylalanine and tyrosine yields. Importance Plasmid-free strains for aromatic compound production are desired in the aspect of industrial application. However, yield of phenylalanine and tyrosine have been considerably lower in plasmid-free strains than in plasmid-based strains. The significance of this research is that we succeeded to generate superior plasmid-free phenylalanine- and tyrosine-producing strains, by engineering E. coli chromosome, which was comparable to plasmid-based strains. The generated strains have a potential to generate superior strains for production of aromatic compounds. Actually, we demonstrated that 4 kinds of aromatic compounds could be produced from glucose with high yield (eg. 0.28 g-tyrosol/g-glucose).