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From scratch to value: engineering Escherichia coli wild type cells to the production of L-phenylalanine and other fine chemicals derived from chorismate.

Applied microbiology and biotechnology (2007-04-17)
Georg A Sprenger
ABSTRACT

Recombinant strains of Escherichia coli K-12 for the production of the three aromatic amino acids (L-phenylalanine, L-tryptophan, L-tyrosine) have been constructed. The largest demand is for L-phenylalanine (L-Phe), as it can be used as a building block for the low-calorie sweetener, aspartame. Besides L-Phe, an increasing number of shikimic acid pathway intermediates can be produced from appropriate E. coli mutants with blocks in this pathway. The last common intermediate, chorismate, in E. coli not only serves for production of aromatic amino acids but can also be used for high-titer production of non-aromatic compounds, e.g., cyclohexadiene-transdiols. In an approach to diversity-oriented metabolic engineering (metabolic grafting), platform strains with increased flux through the general aromatic pathway were created by suitable gene deletions, additions, or rearrangements. Examples for rational strain constructions for L-phenylalanine and chorismate derivatives are given with emphasis on genetic engineering. As a result, L-phenylalanine producers are available, which were derived through several defined steps from E. coli K-12 wild type. These mutant strains showed L-phenylalanine titers of up to 38 g/l of L-phenylalanine (and up to 45.5 g/l using in situ product recovery). Likewise, two cyclohexadiene-transdiols could be recovered.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
L-Phenylalanine, from non-animal source, meets EP, JP, USP testing specifications, suitable for cell culture, 98.5-101.0%
Sigma-Aldrich
L-Phenylalanine, reagent grade, ≥98%
Sigma-Aldrich
L-Phenylalanine, BioUltra, ≥99.0% (NT)
Supelco
L-Phenylalanine, analytical standard, for Nitrogen Determination According to Kjeldahl Method
Supelco
L-Phenylalanine, certified reference material, TraceCERT®
Phenylalanine, European Pharmacopoeia (EP) Reference Standard
Supelco
L-Phenylalanine, Pharmaceutical Secondary Standard; Certified Reference Material
USP
L-Phenylalanine, United States Pharmacopeia (USP) Reference Standard
Sigma-Aldrich
L-Phenylalanine, 99%, FCC