Dissection of central carbon metabolism of hemoglobin-expressing Escherichia coli by 13C nuclear magnetic resonance flux distribution analysis in microaerobic bioprocesses

Abstract

Escherichia coli MG1655 cells expressing Vitreoscilla hemoglobin (VHb), Alcaligenes eutrophus flavohemoprotein (FHP), the N-terminal hemoglobin domain of FHP (FHPg), and a fusion protein which comprises VHb and the A. eutrophus C-terminal reductase domain (VHb-Red) were grown in a microaerobic bioreactor to study the effects of low oxygen concentrations on the central carbon metabolism, using fractional (13)C-labeling of the proteinogenic amino acids and two-dimensional [(13)C, (1)H]-correlation nuclear magnetic resonance (NMR) spectroscopy. The NMR data revealed differences in the intracellular carbon fluxes between E. coli cells expressing either VHb or VHb-Red and cells expressing A. eutrophus FHP or the truncated heme domain (FHPg). E. coli MG1655 cells expressing either VHb or VHb-Red were found to function with a branched tricarboxylic acid (TCA) cycle. Furthermore, cellular demands for ATP and reduction equivalents in VHb- and VHb-Red-expressing cells were met by an increased flux through glycolysis. In contrast, in E. coli cells expressing A. eutrophus hemeproteins, the TCA cycle is running cyclically, indicating a shift towards a more aerobic regulation. Consistently, E. coli cells displaying FHP and FHPg activity showed lower production of the typical anaerobic by-products formate, acetate, and D-lactate. The implications of these observations for biotechnological applications are discussed.

FIG. 1

Determination of the degree of overall ¹³C labeling of the biomass from a 1D ¹H-NMR spectrum. (a) Region of the 1D ¹H-NMR spectrum recorded for the hydrolyzed biomass comprising the aromatic resonances. Several well-resolved peaks allow observation of the ¹³C satellites. (b) Expansion showing a well-resolved resonance (unassigned). The satellite doublet arising from protons bound to ¹³C and the corresponding ¹²C-H peak are indicated by arrows. The ratio of the sum of the integrals of the two satellites to the total integral of all three peaks yields the overall labeling degree of the biomass (4.5% in this case).

FIG. 2

Growth trajectories of the four hemoglobin-expressing E. coli MG1655 strains used for this study, when grown in a minimal medium under hypoxic conditions. The expression of the proteins was induced at an A₆₀₀ of 1, and the fractional ¹³C-labeling was started at an A₆₀₀ of ≈4.5. The cells were harvested for NMR analysis at the end of the cultivations, with an A₆₀₀ of 7. ●, MG1655:pPPC1, which expresses the VHb protein; ■, MG1655:pAX5, which expresses the FHP protein; ○, MG1655:pAX1, which expresses the FHPg protein; ×, MG1655:pAX4, which expresses the VHb-Red protein.

FIG. 3

Flux ratios and interaction of glycolysis and the TCA cycle in the central carbon metabolism of hemoglobin-expressing microaerobic E. coli MG1655 cells. (A) Flux ratios of wild-type E. coli cells (11), VHb-expressing cells, and VHb-Red-expressing cells (from top to bottom of the boxes). (B) Flux ratios of FHPg- and FHP-expressing E. coli cells (from top to bottom of the boxes). Glycolysis (left) and the TCA cycle (right) are highlighted. Enzymatically measured by-products and glucose are given in bold italics. Carbon fragment patterns of the boxed metabolites were directly determined by [¹³C, ¹H]-COSY of proteinogenic amino acids. The fractions of molecules given in boxes are synthesized via the reactions pointing at them, and numbers in ellipses indicate the amount of reversible interconversion of the molecules. Enzyme names are given in italics. Active pathways are shown with solid arrows. Dashed arrows indicate that the represented enzymatic conversion is inactive. Abbreviations: CIT, citrate; MAL, malate; OAA, oxaloacetate; OGA, oxoglutarate; PEP, phosphoenolpyruvate; PYR, pyruvate; ME, malic enzyme; PFL, pyruvate-formate lyase; PEP-CARB, phosphoenolpyruvate carboxylase; CIT-SYN, citrate synthase; GOX shunt, glyoxylate shunt; OGA-DH, oxoglutarate dehydrogenase.