Metabolic network capacity of Escherichia coli for Krebs cycle-dependent proline hydroxylation

Table 1 Physiological comparison of aerobically growing recombinant E. coli BL21(DE3)(pLysS) strains

	wt_pET		ΔputA_pET		wt_p4h1of		ΔputA_p4h1of
	−Pro	+Pro	−Pro	+Pro	−Pro	+Pro	−Pro	+Pro
μ (h⁻¹)	0.44 ± 0.01	0.48 ± 0.01	0.42 ± 0.01	0.43 ± 0.01	0.32 ± 0.02	0.33 ± 0.00	0.3 ± 0.01	0.32 ± 0.01
Final biomass (g_CDW L⁻¹)	1.9 ± 0.1	2.2 ± 0.1	2.0 ± 0.1	2.0 ± 0.1	1.4 ± 0.1	1.8 ± 0.1	1.5 ± 0.1	1.5 ± 0.1
r _Glucose (mmol g⁻¹ h⁻¹)	−5.3 ± 0.3	−5.1 ± 0.1	−5.2 ± 0.2	−5.1 ± 0.2	−4.8 ± 0.3	−4.4 ± 0.3	−4.7 ± 0.2	−3.9 ± 0.1
r _Acetate (mmol g h⁻¹)	1.33 ± 0.1	1.59 ± 0.2	0.98 ± 0.1	0.76 ± 0.1	0.92 ± 0.1	1.24 ± 0.1	1.09 ± 0.04	0.93 ± 0.1
r _Proline (mmol g h⁻¹)	NA	−1.08 ± 0.01	NA	ND	NA	−0.68 ± 0.01	NA	−0.75 ± 0.01
r _Hyp (mmol g h⁻¹)	ND	ND	ND	ND	0.09 ± 0.01	0.37 ± 0.01	0.14 ± 0.01	0.75 ± 0.01
Y _x/glc (g_CDW g ⁻¹_glc )	0.46 ± 0.02	0.52 ± 0.01	0.45 ± 0.02	0.47 ± 0.01	0.39 ± 0.02	0.42 ± 0.03	0.36 ± 0.01	0.45 ± 0.01
Y _ace/glc (mol_ace mol ⁻¹_glc )	0.25 ± 0.03	0.31 ± 0.04	0.19 ± 0.03	0.15 ± 0.01	0.19 ± 0.01	0.28 ± 0.01	0.23 ± 0.01	0.24 ± 0.03
Y _hyp/pro (mol_hyp mol ⁻¹_pro )	NA	ND	NA	ND	NA	0.55 ± 0.01	NA	1 ± 0.01
Y _hyp/glc (mol_hyp mol ⁻¹_glc )	ND	ND	ND	ND	0.019 ± 0.001	0.084 ± 0.003	0.030 ± 0.001	0.192 ± 0.002
r _Hyp (U g_CDW ⁻¹)	NA	NA	NA	NA	1.35 ± 0.02	6.1 ± 0.2	2.20 ± 0.02	12.6 ± 0.1

Cultivation was performed at 30°C in M9 medium with 5 g L⁻¹ glucose in the absence (−Pro) or presence (+Pro) of 5 mM proline. wt_pET, E. coli BL21(DE3)(pLysS)(pET-24a); ΔputA_pET, E. coli BL21ΔputA(DE3)(pLysS)(pET-24a); wt_p4h1of, E. coli BL21(DE3)(pLysS)(pET_p4h1of); ΔputA_p4h1of, E. coli BL21ΔputA(DE3)(pLysS)(pET_p4h1of); μ, specific growth rate; r, specific rates for substrate uptake (negative values) or product formation; CDW, cell dry weight; pro, proline; ace, acetate; hyp, trans-4-hydroxy-l-proline; Y_x/glc, Y_ace/glc, Y_hyp/pro, and Y_hyp/glc, yield coefficients for biomass and acetate on glucose and for hyp on proline and glucose, respectively; ND, not detected; NA, not applicable; 1 U = 1 μmol of product formed per min. Values represent the average from three different experiments (biological replicates) with standard deviations displayed as error.

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