SYNTHETIC MODIFICATION OF THE ESCHERICHIA-COLI CHROMOSOME - ENHANCINGTHE 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 t hese aromatic end products by biosynthetic intermediates drastically r educed. These improvements resulted from increasing the in vivo cataly tic activity of specific enzymes in the common pathway of aromatic ami no acid biosynthesis by chromosomal modification of Escherichia coli. The centerpiece of these changes was the synthesis of a multigene cass ette 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 havi ng a mutation in the tyrR locus which relieves transcriptional repress ion of aroL (encoding shikimate kinase), resulted in biocatalysts KAD1 D and KAD11D. Improved catalytic activities of individual common pathw ay enzymes have previously been accomplished with extrachromosomal pla smids encoding the appropriate loci. By contrast, the chromosomal alte rations possessed by KAD1D and KAD11D circumvent potential problems as sociated with plasmid instability and unnecessary overexpression of pl asmid-encoded, common pathway enzymes. The described modifications of the E. coil chromosome and the methods utilized to achieve these chang es will also simplify construction of future generations of aromatic-s ynthesizing biocatalysts.