In silico identification of gene amplification targets for improvement of lycopene production

Abstract

The identification of genes to be deleted or amplified is an essential step in metabolic engineering for strain improvement toward the enhanced production of desired bioproducts. In the past, several methods based on flux analysis of genome-scale metabolic models have been developed for identifying gene targets for deletion. Genome-wide identification of gene targets for amplification, on the other hand, has been rather difficult. Here, we report a strategy called flux scanning based on enforced objective flux (FSEOF) to identify gene amplification targets. FSEOF scans all the metabolic fluxes in the metabolic model and selects fluxes that increase when the flux toward product formation is enforced as an additional constraint during flux analysis. This strategy was successfully employed for the identification of gene amplification targets for the enhanced production of the red-colored antioxidant lycopene. Additional metabolic engineering based on gene knockout simulation resulted in further synergistic enhancement of lycopene production. Thus, FSEOF can be used as a general strategy for selecting genome-wide gene amplification targets in silico.

FIG. 1.

Concept of FSEOF framework to select the gene amplification targets for enhanced product formation. FSEOF searches for the candidate fluxes to be amplified through scanning for those fluxes that increase with enforced objective (product formation) flux under the objective function of maximizing biomass formation flux. During the FSEOF implementation, three types of intracellular flux profiles are typically identified, increased, decreased, and unchanged; oscillating flux profiles can be found in rare cases. Among them, FSEOF identifies the fluxes showing the increased profile as the primary amplification targets.

FIG. 2.

Results of FSEOF (gene amplification) simulation, flux variability analysis, and MOMA (gene knockout) simulation. (A) Overview of central metabolic pathways for lycopene production and the amplification target genes identified by FSEOF. Red, blue, and gray arrows indicate the fluxes that are increased, decreased and unchanged, respectively, with the enforced objective of increased lycopene production. Red-colored and blue-colored gene names indicate that these genes were amplified and deleted, respectively, in this study. Abbreviations of metabolites are as follows: 3C4MOP, 3-carboxy-4-methyl-2-oxopentanoate; AC_ex, acetate (extracellular); ACA, acetyl-CoA; ACGAM1P, N-acetyl-d-glucosamine 1-phosphate; ACGLU, N-acetyl-l-glutamate; ACGSSA, N-acetyl-l-glutamate-5-semialdehyde; ACSER, O-acetyl-l-serine; ACTP, acetyl phosphate; CYS-L, l-cysteine; DHAP, dihydroxyacetone phosphate; E4P, d-erythrose-4-phosphate; F6P, d-fructose 6-phosphate; FPP, trans,trans farnesyl pyrophosphate; FUM, fumarate; GLX, glyoxylate; GPP, geranyl diphosphate; l-Glu, l-glutamate; malACP, malonyl acyl carrier protein; OAA, oxaloacetate; P5P, alpha-d-ribose 5-phosphate; PEP, phosphoenolpyruvate; PGA, d-glycerate 2-phosphate; PYR, pyruvate; RL5P, d-ribulose 5-phosphate; S7P, sedoheptulose 7-phosphate; SER-L, l-serine; X5P, d-xylulose 5-phosphate. Other abbreviations which are not mentioned here are defined in the text and in Table 3. The detailed results of FSEOF are provided in Table S3B in the supplemental material. (B to E) Flux variability patterns of the targets selected by FSEOF calculation. The number above each plot indicates the number of fluxes out of the 35 fluxes identified by FSEOF that follow the corresponding pattern group. The remaining 5 fluxes resulted in an unbounded pattern and were not shown. The upper and bottom lines represent the maximum and minimum flux values, respectively. The various patterns are classified as fluxes increasing without variability (B), increasing in a narrow range in which the minimum of the upper line is lower than the maximum of the bottom line (C), increasing within a broad range (D), and showing no change or decreasing-increasing (E). (F) Results of MOMA simulation. The red circle represents the results for the E. coli wild-type strain. Red diamonds represent the results for single gene deletions. Green rectangles represent the results for the deletion of two genes.

FIG. 3.

Identification of gene targets for enhanced lycopene production by FSEOF, MOMA, and their combination. (A) Production of lycopene by recombinant E. coli strains engineered based on the results of FSEOF, MOMA, and their combination. All strains harbor the plasmid pLyc184 in addition to the plasmid shown. The lycopene concentrations (mg liter⁻¹) and contents (mg g DCW⁻¹) are shown by black and white bars, respectively. Error bars show standard deviations. Strains and plasmids used are described in Tables 1 and 2. All experiments were carried out in triplicate. The products of genes shown in the plasmid names are phosphofructokinase (pfkA), phosphoglucose isomerase (pgi), fructose-bisphosphate aldolase (fbaA), triosephosphate isomerase (tpiA), isocitrate dehydrogenase (icdA), malate dehydrogenase (mdh), and isopentenyl diphosphate isomerase (idi). (B) Lycopene production by batch-fed cultivation of WLGB-RPP(pTrcDx-idi-mdh, pLyc184). Closed circles, optical density at 600 nm (OD₆₀₀); open circles, glucose concentration (conc.); open triangles, acetic acid concentration; closed squares, lycopene concentration. Error bars represent standard deviations.