Chromosome Engineering To Generate Plasmid-Free Phenylalanine- and Tyrosine-Overproducing <i>Escherichia coli</i> Strains That Can Be Applied in the Generation of Aromatic-Compound-Producing Bacteria

Abstract

Many phenylalanine- and tyrosine-producing strains have used plasmid-based overexpression of pathway genes. The resulting strains achieved high titers and yields of phenylalanine and tyrosine. Chromosomally engineered, plasmid-free producers have shown lower titers and yields than plasmid-based strains, but the former are advantageous in terms of cultivation cost and public health/environmental risk. Therefore, we engineered here the <i>Escherichia coli</i> chromosome to create superior phenylalanine- and tyrosine-overproducing strains that did not depend on plasmid-based expression. Integration into the <i>E. coli</i> chromosome of two central metabolic pathway genes (<i>ppsA</i> and <i>tktA</i>) and eight shikimate pathway genes (<i>aroA</i>, <i>aroB</i>, <i>aroC</i>, <i>aroD</i>, <i>aroE</i>, <i>aroG^fbr</i> , <i>aroL</i>, and <i>pheA^fbr</i> ), controlled by the T7lac promoter, resulted in excellent titers and yields of phenylalanine; the superscript "<i>fbr</i>" 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 replacing <i>pheA^fbr</i> with <i>tyrA^fbr</i> A rational approach revealed that integration of seven genes (<i>ppsA</i>, <i>tktA</i>, <i>aroA</i>, <i>aroB</i>, <i>aroC</i>, <i>aroG^fbr</i> , and <i>pheA^fbr</i> ) was necessary as the minimum gene set for high-yield phenylalanine production in <i>E. coli</i> MG1655 (<i>tyrR</i>, <i>adhE</i>, <i>ldhA</i>, <i>pykF</i>, <i>pflDC</i>, and <i>ascF</i> 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.<b>IMPORTANCE</b> Plasmid-free strains for aromatic compound production are desired in the aspect of industrial application. However, the yields 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 in generating superior plasmid-free phenylalanine- and tyrosine-producing strains by engineering the <i>E. coli</i> chromosome, which was comparable to that in plasmid-based strains. The generated strains have a potential to generate superior strains for the production of aromatic compounds. Actually, we demonstrated that four kinds of aromatic compounds could be produced from glucose with high yields (e.g., 0.28 g tyrosol/g glucose).