Rational design of a synthetic Entner–Doudoroff pathway for enhancing glucose transformation to isobutanol in Escherichia coli

Abstract

Isobutanol as a more desirable biofuel has attracted much attention. In our previous work, an isobutanol-producing strain Escherichia coli LA09 had been obtained by rational redox status improvement under guidance of the genome-scale metabolic model. However, the low transformation from sugar to isobutanol is a limiting factor for isobutanol production by E. coli LA09. In this study, the intracellular metabolic profiles of the isobutanol-producing E. coli LA09 with different initial glucose concentrations were investigated and the metabolic reaction of fructose 6-phosphate to 1, 6-diphosphate fructose in glycolytic pathway was identified as the rate-limiting step of glucose transformation. Thus, redesigned carbon catabolism was implemented by altering flux of sugar metabolism. Here, the heterologous Entner-Doudoroff (ED) pathway from Zymomonas mobilis was constructed, and the adaptation of upper and lower parts of ED pathway was further improved with artificial promoters to alleviate the accumulation of toxic intermediate metabolite 2-keto-3-deoxy-6-phospho-gluconate (KDPG). Finally, the best isobutanol-producing E. coli ED02 with higher glucose transformation and isobutanol production was obtained. In the fermentation of strain E. coli ED02 with 45 g/L initial glucose, the isobutanol titer, yield and average producing rate were, respectively, increased by 56.8, 47.4 and 88.1% to 13.67 g/L, 0.50 C-mol/C-mol and 0.456 g/(L × h) in a shorter time of 30 h, compared with that of the starting strain E. coli LA09.

Engineering Improves Enzymatic Synthesis of L-Tryptophan by Tryptophan Synthase from Escherichia coli

“…According to the structural information of proteins and sequence comparisons of homologous proteins, using a rational selection of amino acid residues as targets combined with site-specific mutation of key amino acids, we can screen and obtain target mutants [26][27][28]. Rational design can improve the substrate specificity of an enzyme by remodeling its active pocket, which is the structural domain of the enzyme responsible for its catalytic function and usually consists of a hydrophobic core.…”

Reconstruction of metabolic pathway for isobutanol production in Escherichia coli

“…In these reports, the inactivation of genes concerning byproducts formation, direct evolution of the enzymes in the biosynthetic pathway of isobutanol, and optimization of culture conditions were performed as the general strategies to improve isobutanol production. A study on isobutanol production has demonstrated the use of partially ED pathway-dependent E. coli, which adopted the ED pathway derived from Z. mobilis [16]. However, the strain used in that study was simply introduced to the genes concerning the ED pathway, and the redox balance in the biosynthesis pathway involved in converting glucose to isobutanol was not completely considered.…”

“…1). Although isobutanol has reportedly been produced using E. coli carrying a synthetic ED pathway of Z. mobilis [16], the EM pathway and pentose phosphate pathway (PPP) has remained in the metabolic pathway of E. coli used in the report; this indicates that the metabolic pathway for isobutanol production involving the ED pathway should be improved from the perspective of redox balance.

Fig. 1The synthetic metabolic pathway for isobutanol production in E. coli.

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Synthetic Biology and Metabolic Engineering Employing Escherichia coli for C2–C6 Bioalcohol Production

“…Other efforts have focused on increasing isobutanol production from glucose. Liang et al (2018) introduced the heterologous Entner-Doudoroff (ED) pathway from Z. mobilis to increase glucose transformation to pyruvate for enhanced precursor accumulation. The resulting E. coli strain, ED02, produced 13.67 g/L isobutanol with a productivity of 0.456 g/L/h, a 56.8 and 88.1% improvement over the parent strain, respectively.…”