The CreC Regulator of Escherichia coli, a New Target for Metabolic Manipulations

Abstract

The CreBC (carbon source-responsive) two-component regulation system of Escherichia coli affects a number of functions, including intermediary carbon catabolism. The impacts of different creC mutations (a ΔcreC mutant and a mutant carrying the constitutive creC510 allele) on bacterial physiology were analyzed in glucose cultures under three oxygen availability conditions. Differences in the amounts of extracellular metabolites produced were observed in the null mutant compared to the wild-type strain and the mutant carrying creC510 and shown to be affected by oxygen availability. The ΔcreC strain secreted more formate, succinate, and acetate but less lactate under low aeration. These metabolic changes were associated with differences in AckA and LdhA activities, both of which were affected by CreC. Measurement of the NAD(P)H/NAD(P)(+) ratios showed that the creC510 strain had a more reduced intracellular redox state, while the opposite was observed for the ΔcreC mutant, particularly under intermediate oxygen availability conditions, indicating that CreC affects redox balance. The null mutant formed more succinate than the wild-type strain under both low aeration and no aeration. Overexpression of the genes encoding phosphoenolpyruvate carboxylase from E. coli and a NADH-forming formate dehydrogenase from Candida boidinii in the ΔcreC mutant further increased the yield of succinate on glucose. Interestingly, the elimination of ackA and adhE did not significantly improve the production of succinate. The diverse metabolic effects of this regulator on the central biochemical network of E. coli make it a good candidate for metabolic-engineering manipulations to enhance the formation of bioproducts, such as succinate.

FIG 1

Determination of the NADH/NAD⁺ and NADPH/NADP⁺ ratios in E. coli K1060 (wild-type strain), K1060C (carrying the constitutive creC510 allele), and DC1060 (ΔcreC). Cells were grown in M9 minimal medium containing 30 g liter⁻¹ glucose under high aeration (A), low aeration (B), and no aeration (C). Cells were harvested at mid-exponential phase. The results represent the averages ± standard deviations of duplicate measurements of at least two independent cultures.

FIG 2

Acetate kinase (A) and lactate dehydrogenase (B) in vitro specific activities (sp act) of cells grown in M9 minimal medium containing 30 g liter⁻¹ glucose under high aeration, low aeration, and no aeration. The samples were harvested at mid-exponential phase, except under anaerobic conditions, where the activity was measured both in the exponential (E) and early stationary (S) phases (see Fig. S1 in the supplemental material for detailed information on sampling times). The results represent the averages ± standard deviations of duplicate measurements of at least two independent cultures.

FIG 3

Diagram of the main metabolic pathways leading to succinate formation from glucose in E. coli. The genes encoding Ppc^Ec (phosphoenolpyruvate carboxylase) from E. coli and FDH1^Cb (NADH-forming formate dehydrogenase) from C. boidinii were overexpresed in plasmids pEcPpc and pSBF2, respectively (the corresponding reactions are highlighted in green). The genes encoding AdhE (alcohol dehydrogenase) and AckA (acetate kinase) were knocked out in an attempt to enhance succinate accumulation (indicated by the red arrowheads). Note that several reactions within the biochemical network were grouped for the sake of simplicity. EMP pathway, Embden-Meyerhof-Parnas pathway; PEP, phosphoenolpyruvate; Acetyl-CoA, acetyl coenzyme A.

FIG 4

Profile of succinate formation in the E. coli strains under study. Cells were grown in M9 minimal medium containing 30 g liter⁻¹ glucose and 100 mM NaHCO₃ under low aeration for 48 h. The E. coli strains tested were K1060 (wild-type strain), DC1060 (ΔcreC), CE1060 (ΔcreC ΔadhE), and CEA1060 (ΔcreC ΔadhE ΔackA). All the bacteria were transformed with plasmids pSBF2 (carrying FDH1^Cb, encoding a NADH-forming formate dehydrogenase from C. boidinii) and pEcPpc (carrying ppc^Ec, encoding the endogenous phosphoenolpyruvate carboxylase from E. coli). The expression of the genes in these plasmids was induced by addition of IPTG at two concentrations (0.1 mM and 1 mM). Succinate was assayed in culture supernatants, and the results are reported as the final concentration (A) and yield of succinate on glucose (Y_Succ/Glc) (B). The results represent the averages ± standard deviations of duplicate measurements of at least two independent cultures.