Increased malonyl coenzyme A biosynthesis by tuning the Escherichia coli metabolic network and its application to flavanone production

Abstract

Identification of genetic targets able to bring about changes to the metabolite profiles of microorganisms continues to be a challenging task. We have independently developed a cipher of evolutionary design (CiED) to identify genetic perturbations, such as gene deletions and other network modifications, that result in optimal phenotypes for the production of end products, such as recombinant natural products. Coupled to an evolutionary search, our method demonstrates the utility of a purely stoichiometric network to predict improved Escherichia coli genotypes that more effectively channel carbon flux toward malonyl coenzyme A (CoA) and other cofactors in an effort to generate recombinant strains with enhanced flavonoid production capacity. The engineered E. coli strains were constructed first by the targeted deletion of native genes predicted by CiED and then second by incorporating selected overexpressions, including those of genes required for the coexpression of the plant-derived flavanones, acetate assimilation, acetyl-CoA carboxylase, and the biosynthesis of coenzyme A. As a result, the specific flavanone production from our optimally engineered strains was increased by over 660% for naringenin (15 to 100 mg/liter/optical density unit [OD]) and by over 420% for eriodictyol (13 to 55 mg/liter/OD).

FIG. 1.

CiED simulations. Schematic representation of the CiED with the traditional genetic algorithm elements (gray box).

FIG. 2.

Metabolic reaction network. A reduced central carbon network and relevant pathways are shown, including genes identified by CiED for deletion (bold) and those overexpressed (underlined).

FIG. 3.

Genotype analysis. The genotype distribution for single gene deletions as calculated using CiED ordered into viable and productive (x), nonviable productive (□), and (non)viable nonproductive (○) is shown. The lower limits of 15% of wild-type growth for viability and 10% of maximum for productivity (gray lines) were used for classification. Deletions for sdhCDAB (▴), tpiA (▾), glyA (▪), and gdhA (♦) are also marked.

FIG. 4.

Intracellular concentrations of CoA thioesters. Shown are concentrations of malonyl-CoA (A), acetyl-CoA (B), and free CoA (C) from the following experimental strains: BL21Star, the commercial strain; E2M; Z24M (ΔsdhA ΔcitE); Z40M (ΔsdhA ΔadhE ΔcitE ΔbrnQ). Values are averages of three separate runs with standard deviations. Symbols indicate Z24M and Z40M are statistically significant (P > 0.05) from E2M at 3 h for all intracellular metabolites investigated.

FIG. 5.

Overexpression of cofactor and assimilation pathways. (A) Production levels with strains overexpressing CoA biosynthetic genes coaA (E2NC), dfp (E2ND), and coaD (E2NP) and the control (E2N). Production of naringenin (dark gray) or eriodictyol (white) is shown. Symbols indicate E2NC is statistically significantly different (P > 0.05) from the E2N strain. (B) Batch fermentation for flavanone production strains (E2, wild-type; Z24, ΔsdhA ΔcitE; Z40, ΔsdhA ΔadhE ΔcitE ΔbrnQ) either without (black; strains E2N, Z24N, and Z40N) or with (light gray; strains E2NAC, Z24NAC, and Z40NAC) overexpression of ACC, BPL, and both acs and coaA. Values are averages of three separate runs with standard deviations. Symbols indicate Z24 and Z40 strains were statistically significantly different (P > 0.05) from the E2 strain.