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

Abstract

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 An TrpC 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.

Tryptophan Production Maximization in a Fed-Batch Bioreactor with Modified E. coli Cells, by Optimizing Its Operating Policy Based on an Extended Structured Cell Kinetic Model

“…These authors obtained a promising E. coli T5 strain which, tested in an FBR, showed an increased GLC import capacity together with an increased TRP yield by ca. 20% compared to an initial mutant S028 strain (i.e., 0.164 vs. 0.137 g TRP/g GLC), while the specific production rate was increased by 53% [ 73 ]. The cell flux analysis by Chen [ 74 , 75 ] indicated ( Figure 2 ) the doubling of fluxes responsive to TRP synthesis.…”

“…This paper uses a hybrid dynamic model built up by Maria [ 35 ] by linking a CCM-based structured kinetic model with an FBR simple dynamic model. The resulting hybrid FBR model was used to computationally determine the optimal (time stepwise) feeding policy of the FBR used by Chen et al [ 73 ] to study TRP synthesis using a modified E. coli T5 strain culture. The thus obtained optimal operating policy of the FBR has proven to be very effective, by ensuring maximization of TRP production involving a few key control variables (i.e., the feed flow-rate and the feeding GLC concentration), and it reported better performance compared to the non-optimally operated FBR of Maria et al [ 34 , 35 ] or of Chen [ 74 ].…”

“…The structured modular kinetic model of Maria [ 35 , 48 ] used in this numerical analysis includes modules characterizing the dynamics of the concerned cell pathways involved in TRP synthesis, i.e., glycolysis, ATP recovery system, TRP operon expression, and biomass growth. This bioprocess model was experimentally identified and checked over extensive experiments conducted by several authors, i.e., [ 33 , 48 , 52 , 68 ] for glycolysis, and by Chen et al [ 73 , 74 ] and Maria [ 35 ] for TRP synthesis. Experimental data of Chen [ 74 ] for TRP synthesis were also used to compare the derived predictions of the hybrid model.…”

“…The characteristics of this strain were reflected in the rate constants estimated by Maria [ 35 ]. This T5 strain was produced by Chen [ 75 ] and Chen et al [ 73 ] to increase TRP production in their bench-scale FBR. They performed genetic modifications of the TRP-producing “wild” strain S028.…”

“…Basically, “they removed the PTS import-system of GLC, by replacing it with a more effective one based on the galactose permease/glucokinase (GalP/Glk) uptake system, by modulating the gene expression of GalP/Glk. The resulting T5 strain showed an increase of the specific TRP production rate in a nonoptimal FBR by 52.93% (25.3 mg/gDW biomass /h) compared to the initial strain” [ 73 ] and by ca. 70% if the used FBR was optimally operated (this paper); (iv) the results reveal the close link between the cell key metabolites and the FBR operating conditions; (v) the used hybrid bilevel kinetic model is complex enough to adequately represent the dynamics of the FBR state variables (i.e., the biomass growth, the GLC depletion, and the excreted TRP and PYR in the bulk phase), as well as the dynamics of the cell key species involved in the concerned reaction pathway modules, i.e., (a) glycolysis, (b) ATP recovery system, and (c) TRP operon expression.…”