Construction and application of novel feedback-resistant 3-deoxy-D-arabino-heptulosonate-7-phosphate synthases by engineering the N-terminal domain for L-phenylalanine synthesis

Abstract

3-Deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAHP synthase) encoded by aroF is the first enzyme of the shikimate pathway. In the present study, an AroF variant with a deficiency in residue Ile11 (named AroF́) was shown to be insensitive to l-tyrosine. According to three-dimensional structure analysis, nine AroF variants were constructed with truncation of different N-terminal fragments, and overexpression of the variants AroFΔ(1-9), AroFΔ(1-10), AroFΔ(1-12) and, in particular, AroFΔ(1-11) significantly increased the accumulation of l-phenylalanine (l-Phe). However, the AroG and AroH variants with similar truncations of the N-terminal fragments decreased the production of l-Phe. By co-overexpressing AroFΔ(1-11) and PheAfbr, the production of l-Phe was increased from 2.36 ± 0.07 g L-1 (co-overexpression of the wild-type AroF and PheAfbr) to 4.29 ± 0.06 g L-1. The novel variant AroFΔ(1-11) showed great potential for the production of aromatic amino acids and their derivatives. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.