Synthetic Modification of the Escherichia coli Chromosome:  Enhancing the Biocatalytic Conversion of Glucose into Aromatic Chemicals

The yield of l-phenylalanine, racemic phenyllactic acid, and prephenic acid synthesized from glucose has been doubled and contamination of these aromatic end products by biosynthetic intermediates drastically reduced. These improvements resulted from increasing the in vivo catalytic activity of specific enzymes in the common pathway of aromatic amino acid biosynthesis by chromosomal modification of Escherichia coli. The centerpiece of these changes was the synthesis of a multigene cassette carrying aroA (encoding EPSP synthase), aroC (encoding chorismate synthase), and aroB (encoding DHQ synthase). Chromosomal insertion of the synthesized multigene cassette into E. coli KAD29B, a strain having a mutation in the tyrR locus which relieves transcriptional repression of aroL (encoding shikimate kinase), resulted in biocatalysts KAD1D and KAD11D. Improved catalytic activities of individual common pathway enzymes have previously been accomplished with extrachromosomal plasmids encoding the appropriate loci. By contrast, the chromosomal alterations possessed by KAD1D and KAD11D circumvent potential problems associated with plasmid instability and unnecessary overexpression of plasmid-encoded, common pathway enzymes. The described modifications of the E. coli chromosome and the methods utilized to achieve these changes will also simplify construction of future generations of aromatic-synthesizing biocatalysts.