Metabolic engineering of Escherichia coli for the production of L-valine based on transcriptome analysis and in silico gene knockout simulation

Abstract

The L-valine production strain of Escherichia coli was constructed by rational metabolic engineering and stepwise improvement based on transcriptome analysis and gene knockout simulation of the in silico genome-scale metabolic network. Feedback inhibition of acetohydroxy acid synthase isoenzyme III by L-valine was removed by site-directed mutagenesis, and the native promoter containing the transcriptional attenuator leader regions of the ilvGMEDA and ilvBN operon was replaced with the tac promoter. The ilvA, leuA, and panB genes were deleted to make more precursors available for L-valine biosynthesis. This engineered Val strain harboring a plasmid overexpressing the ilvBN genes produced 1.31 g/liter L-valine. Comparative transcriptome profiling was performed during batch fermentation of the engineered and control strains. Among the down-regulated genes, the lrp and ygaZH genes, which encode a global regulator Lrp and L-valine exporter, respectively, were overexpressed. Amplification of the lrp, ygaZH, and lrp-ygaZH genes led to the enhanced production of L-valine by 21.6%, 47.1%, and 113%, respectively. Further improvement was achieved by using in silico gene knockout simulation, which identified the aceF, mdh, and pfkA genes as knockout targets. The VAMF strain (Val DeltaaceF Deltamdh DeltapfkA) overexpressing the ilvBN, ilvCED, ygaZH, and lrp genes was able to produce 7.55 g/liter L-valine from 20 g/liter glucose in batch culture, resulting in a high yield of 0.378 g of L-valine per gram of glucose. These results suggest that an industrially competitive strain can be efficiently developed by metabolic engineering based on combined rational modification, transcriptome profiling, and systems-level in silico analysis.

Fig. 1.

The biosynthetic pathways of branched-chain amino acids in E. coli, regulations involved, and the strategies for constructing the l-valine-producing base strain. The shaded boxes represent mutations introduced into the genome, and the gray bars indicate the genes that were knocked out. Thin red arrows indicate the increased flux by knocking out the genes suggested by in silico simulation. Thick red arrows indicate the increased flux or activity by directly overexpressing the corresponding genes. Thick blue arrows indicate repression of gene expression by Lrp. Thin blue arrows indicate the decreased flux by knocking out the corresponding genes. Dotted lines indicate feedback inhibition. The black X indicates that the inhibition or repression is removed. The plus (+) and minus (−) symbols indicate activation and repression of gene expression, respectively.

Fig. 2.

Results of comparative transcriptome profiling. The numbers are the ratios of the expression levels in Val (pKKilvBN) vs. WL3110 (pKK223–3). The shaded and boxed numbers indicate significantly up- and down-regulated genes, respectively, in l-valine-producing strain Val (pKKilvBN). GLC, glucose; G6P, glucose-6-phosphate; 6PGL, gluconolactone-6-phosphate; 6PGC, 6-phospho gluconate; RL5P, ribulose-5-phosphate; X5P, xylurose-5-phosphate; R5P, ribose-5-phosphate; S7P, sedoheptulose-7-phosphate; E4P, erythrose-4-phosphate; F6P, fructose-6-phosphate; FBP, fructose-1, 6-bisphosphate; GAP, glyceraldehyde-3-phosphate; DHAP, dihydroxyacetonephosphate; GBP, 1,3-bisphosphoglycerate; PEP, phosphoenolpyruvate; PYR, pyruvate; ACA, acetyl-CoA; ACP, acetyl-phosphate; Ace, acetate; ACL, 2-acetolactate; DHV, 2,3-dihydroxyisovalerate; KIV, 2-ketoisovalerate; Val, l-valine; Leu, l-leucine; Pan, pantothenate; Ala, l-alanine; CIT, citrate; ICIT, isocitrate; AKG, α-ketoglutarate; SUCOA, succinyl-CoA; SUC, succinate; FUM, fumarate; MAL, malate; OAA, oxaloacetate.

Fig. 3.

Results of in silico gene knockout simulations by using the genome-scale metabolic model of E. coli MBEL979. The results of single (A), double (B), and triple (C) gene knockout simulations with respect to l-valine production and growth rates are shown. Only the five best candidates with respect to the l-valine production rate are shown for each stage of knockout simulation. Slashes indicate isoenzymes or subunits of the enzyme complex. The l-valine production and growth rates of the control Val strain harboring pKKilvBN are also indicated for comparison.