Systems metabolic engineering of Escherichia coli for L-threonine production

Abstract

Amino-acid producers have traditionally been developed by repeated random mutagenesis owing to the difficulty in rationally engineering the complex and highly regulated metabolic network. Here, we report the development of the genetically defined L-threonine overproducing Escherichia coli strain by systems metabolic engineering. Feedback inhibitions of aspartokinase I and III (encoded by thrA and lysC, respectively) and transcriptional attenuation regulations (located in thrL) were removed. Pathways for Thr degradation were removed by deleting tdh and mutating ilvA. The metA and lysA genes were deleted to make more precursors available for Thr biosynthesis. Further target genes to be engineered were identified by transcriptome profiling combined with in silico flux response analysis, and their expression levels were manipulated accordingly. The final engineered E. coli strain was able to produce Thr with a high yield of 0.393 g per gram of glucose, and 82.4 g/l Thr by fed-batch culture. The systems metabolic engineering strategy reported here may be broadly employed for developing genetically defined organisms for the efficient production of various bioproducts.

Figure 1

Overall systems metabolic engineering strategies employed for the development of a genetically defined Thr-overproducing E. coli strain. Central metabolic pathways that lead to biosynthesis of Thr together with the regulatory circuits and competing pathways are shown. The shaded boxes represent the targeted mutations introduced into the genome. The gray Xs indicate that the genes are knocked out or the inhibition/repression is removed. Thick red arrows indicate the increased flux or activity by directly overexpressing the corresponding genes. Dashed line indicates repression regulation. Dotted lines indicate feedback inhibition.

Figure 2

Flux response analysis during production of Thr using the in silico genome-scale metabolic model. (A) The response of Thr production rate to varying PPC flux. (B) The response of Thr production rate to varying ICL flux. The predicted flux distributions belonging to the region i, ii, iii, and iv are described with colored arrows; red, blue, and black arrows indicate the fluxes that are predicted to be increased, decreased, and remain unchanged, respectively, in each region. (C) The response of acetic acid formation rate to the varying flux of the individual central metabolic reaction. Those reactions that upon increasing their fluxes result in decreasing the acetic acid formation rate are shown in blue. In contrast, those reactions that upon increasing their fluxes result in increasing the acetic acid formation rate are shown in red. In the two graphs inside, the x-axis denotes the normalized flux of one of the color-coded reactions, whereas the y-axis denotes the normalized acetic acid formation rate. Abbreviations are as follows: G6P, glucose-6-phosphate; RL5P, ribulose-5-phosphate; X5P, xylulose-5-phosphate; R5P, ribose-5-phosphate; E4P, erythrose-4-phosphate; F6P, fructose-6-phosphate; F1,6dP, fructose-1, 6-bisphosphate; G3P, glyceraldehyde-3-phosphate; DHAP, dihydroxyacetonephosphate; PEP, phosphoenolpyruvate; PYR, pyruvate; ICT, isocitrate; α-KG, α-ketoglutarate; SUC, succinate; MAL, malate; OAA, oxaloacetate; THR, L-threonine; ACE, acetate.

Figure 3

Time profiles of cell growth, Thr production, acetic acid accumulation during the fed-batch culture of (A) TH27C (pBRThrABCR3), and (B) TH28C (pBRThrABCR3). The arrows indicate the sampling points for real time RT–PCR analysis. Symbols are as follows: ○, cell growth (OD₆₀₀); ▪, L-threonine (g/l); ▴, glucose (g/l); □, acetic acid (g/l); ⋄, lactic acid (g/l).