Integrated laboratory evolution and rational engineering of GalP/Glk-dependent Escherichia coli for higher yield and productivity of L-tryptophan biosynthesis.

L-Tryptophan (Trp) is a high-value aromatic amino acid with diverse applications in food and pharmaceutical industries. Although production of Trp by engineered Escherichia coli has been extensively studied, the need of multiple precursors for its synthesis and the complex regulations of the biosynthetic pathways make the achievement of a high product yield still very challenging. Metabolic flux analysis suggests that the use of a phosphoenolpyruvate:sugar phosphotransferase system (PTS) independent glucose uptake system, i.e. the galactose permease/glucokinase (GalP/Glk) system, can theoretically double the Trp yield from glucose. To explore this possibility, a PTS(-) and GalP/Glk-dependent E. coli strain was constructed from a previously rationally developed Trp producer strain S028. However, the growth rate of the S028 mutant was severely impaired. To overcome this problem, promoter screening for modulated gene expression of GalP/Glk was carried out, following by a batch mode of adaptive laboratory evolution (ALE) which resulted in a strain K3 with a similar Trp yield and concentration as S028. In order to obtain a more efficient Trp producer, a novel continuous ALE system was developed by combining CRISPR/Cas9-facilitated in vivo mutagenesis with real-time measurement of cell growth and online monitoring of Trp-mediated fluorescence intensity. With the aid of this automatic system (auto-CGSS), a promising strain T5 was obtained and fed-batch fermentations showed an increase of Trp yield by 19.71% with this strain compared with that obtained by the strain K3 (0.164 vs. 0.137 ​g/g). At the same time, the specific production rate was increased by 52.93% (25.28 vs. 16.53 ​mg/g (DCW)/h). Two previously engineered enzyme variants AroG(D6G-D7A) and AnTrpC(R378F) were integrated into the strain T5, resulting in a highly productive strain T5AA with a Trp yield of 0.195 ​g/g and a specific production rate of 28.83 ​mg/g (DCW)/h.