Systems Metabolic Engineering of Escherichia coli

Abstract

Systems metabolic engineering, which recently emerged as metabolic engineering integrated with systems biology, synthetic biology, and evolutionary engineering, allows engineering of microorganisms on a systemic level for the production of valuable chemicals far beyond its native capabilities. Here, we review the strategies for systems metabolic engineering and particularly its applications in Escherichia coli. First, we cover the various tools developed for genetic manipulation in E. coli to increase the production titers of desired chemicals. Next, we detail the strategies for systems metabolic engineering in E. coli, covering the engineering of the native metabolism, the expansion of metabolism with synthetic pathways, and the process engineering aspects undertaken to achieve higher production titers of desired chemicals. Finally, we examine a couple of notable products as case studies produced in E. coli strains developed by systems metabolic engineering. The large portfolio of chemical products successfully produced by engineered E. coli listed here demonstrates the sheer capacity of what can be envisioned and achieved with respect to microbial production of chemicals. Systems metabolic engineering is no longer in its infancy; it is now widely employed and is also positioned to further embrace next-generation interdisciplinary principles and innovation for its upgrade. Systems metabolic engineering will play increasingly important roles in developing industrial strains including E. coli that are capable of efficiently producing natural and nonnatural chemicals and materials from renewable nonfood biomass.

Figure 1

Overview of systems metabolic engineering. Systems metabolic engineering is the recursive process of improving a candidate strain via pathway engineering, transporter engineering, omics tools, and in silico analysis in an effort to increase the production of desired chemicals to industrial scales.

Figure 2

Tools for systems metabolic engineering. List of tools for genetic modification of candidate strains: recombineering, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, global transcription machinery engineering (gTME), omics-based tools, multiplex automated genome engineering (MAGE), synthetic small regulatory RNA (sRNA), and scaffold proteins.

Figure 3

The endogenous metabolism of E. coli. The endogenous metabolic pathway of E. coli mapping products (metabolites in black boxes and amino acids in turquoise boxes) and the genes (italicized) of the enzymes responsible for the reactions based on EcoCyc E. coli database. Every overexpression (blue circle), downregulation (red circle), and all other miscellaneous modifications including feedback-release (asterisk) attempted in E. coli for systems metabolic engineering purposes are noted. Convergence and divergence of metabolites are denoted by circular nodes, where some reactions are reversible. As an example for reversible reaction, F6P and GAP converge to form E4P and Xu5P; conversely, E4P and Xu5P converge to form F6P and GAP. Glc, glucose; G6P, glucose 6-phosphate; F6P, fructose 6-phosphate; F1,6BP, fructose 1,6-bisphosphate; GAP, glyceraldehyde 3-phosphate; DHAP, dihydroxyacetone phosphate; 1,3-BPG, 1,3-bisphosphoglycerate; 3-PG, 3-phosphoglycerate; 2-PG, 2-phosphoglycerate; PEP, phosphoenolpyruvate; PYR, pyruvate; Ac-CoA, acetyl-CoA; CIT, citrate; I-CIT, isocitrate; α-KG, α-ketoglutarate; SUCC-CoA, succinyl-CoA; SUCC, succinate; FUM, fumarate; MAL, malate; OAA, oxaloacetate; GOX, glyoxylate; E4P, erythrose 4-phosphate; Xu5P, xylulose 5-phosphate; S7P, sedoheptulose 7-phosphate; R5P, ribose 5-phosphate; Ru5P, ribulose 5-phosphate; RuBP, ribulose 1,5-bisphosphate; PRPP, 5-phosphoribose 1-pyrophosphate; AICAR, 5-amino-1-(5-phosphoribosyl)imidazole-4-carboxamide; His, L-histidine; DAHP, 3-deoxy-arabino-heptulosonate 7-phosphate; DHQ, 3-dehydroquinate; DHS, dehydroshikimate; SHIK, shikimate; CHOR, chorismate; PPHN, prephenate; HPP, 4-hydroxyphenylpyruvate; Tyr, L-tyrosine; PHPYR, phenylpyruvate; Phe, L-phenylalanine; ANTH, anthranilate; Trp, L-tryptophan; 3-PH, 3-phosphohydroxypyruvate; P-Ser, 3-phosphoserine; Ser, L-serine; Ac-Ser, acetyl-serine; AcLAC, acetolactate; Val, L-valine; Leu, L-leucine; Ala, L-alanine; D-Ala, D-alanine; Ac-P, acetyl phosphate; AcAc-CoA, acetoacetyl-CoA; Glu, L-glutamate; Gln, L-glutamine; Arg, L-arginine; Pro, L-proline; Asp, L-aspartate; Asn, L-asparagine; AspSA, aspartate-semialdehyde; Lys, L-lysine; HMS, homoserine; Thr, L-threonine; Ile, L-isoleucine; SUCC-HMS, succinylhomoserine; CYST, cystathionine; HMC, homocysteine; Met, L-methionine; Ac-ACP, acetyl-acyl carrier protein (ACP); Mal-CoA, malonyl-CoA; Mal-ACP, malonyl-ACP; AcAc-ACP, acetoacetyl-ACP.

Figure 4

Examples of metabolic engineering strategies to produce chemicals of interest from central carbon metabolism. Target products included are (1) D- and L-lactic acid, (2) ethanol, (3) 1,2-propanediol, (4) 1,3-propanediol, (5) butyric acid and 1-butanol, (6) malic acid, (7) fumaric acid, (8) succinic acid, (9) adipic acid, (10) 1,4-butanediol, and (11) itaconic acid. Numbers labeled beside gene names indicate the products that targeted each gene for engineering during engineering. The colors of the numbers indicate modes of engineering: red, downregulation including knockout; blue, upregulation; black, all other miscellaneous modifications including feedback-release, heterologous gene introduction, and mutagenesis for modified enzyme activities. Convergence and divergence of metabolites are denoted by circular nodes, where some reactions are reversible. As an example for reversible reaction, F6P and GAP converge to form E4P and Xu5P; conversely, E4P and Xu5P converge to form F6P and GAP. For abbreviations, see Fig. 3.

Figure 5

Examples of metabolic engineering strategies to produce chemicals of interest derived from glycolytic intermediates. Target products included are (1) L-serine, (2) L-alanine, (3) L-valine, (4) isobutanol, (5) 3-methyl-1-butanol, and (6) glycine-rich spider silk protein. The colors of the numbers indicate modes of engineering: red, expression downregulation including knockout; blue, expression upregulation; black, all other miscellaneous modifications including feedback-release, heterologous gene introduction, and mutagenesis for modified enzyme activities. Convergence and divergence of metabolites are denoted by circular nodes, where some reactions are reversible. As an example for reversible reaction, F6P and GAP converge to form E4P and Xu5P; conversely, E4P and Xu5P converge to form F6P and GAP. For abbreviations, see Fig. 3.

Figure 6

Examples of metabolic engineering strategies to produce chemicals of interest derived from TCA cycle intermediates. Target products included are (1) L-threonine, (2) L-isoleucine, (3) L-homoalanine, (4) L-lysine, (5) cadaverine, (6) putrescine, (7) 1-propanol, (8) 1-heptanol, and (9) 3-methyl-1-propanol. The colors of the numbers indicate modes of engineering: red, expression downregulation including knockout; blue, expression upregulation; black, all other miscellaneous modifications including feedback-release, heterologous gene introduction, and mutagenesis for modified enzyme activities. Convergence and divergence of metabolites are denoted by circular nodes, where some reactions are reversible. As an example for reversible reaction, F6P and GAP converge to form E4P and Xu5P; conversely, E4P and Xu5P converge to form F6P and GAP. For abbreviations, see Fig. 3.

Figure 7

Examples of metabolic engineering strategies to produce chemicals of interest derived from PPP intermediates. Target products included are (1) L-phenylalanine, (2) L-tyrosine, (3) L-tryptophan, (4) catechol, (5) cis,cis-muconic acid, (6) p-hydroxybenzoic acid, (7) p-aminobenzoic acid, and (8) phenol. The colors of the numbers indicate modes of engineering: red, expression downregulation including knockout; blue, expression upregulation; black, all other miscellaneous modifications including feedback-release, heterologous gene introduction, and mutagenesis for modified enzyme activities. Convergence and divergence of metabolites are denoted by circular nodes, where some reactions are reversible. As an example for reversible reaction, F6P and GAP converge to form E4P and Xu5P; conversely, E4P and Xu5P converge to form F6P and GAP. For abbreviations, see Fig. 3.