Identification of novel metabolic interactions controlling carbon flux from xylose to ethanol in natural and recombinant yeasts

Trausinger, Gert; Gruber, Christoph; Krahulec, Stefan; Magnes, Christoph; Nidetzky, Bernd; Klimacek, Mario

doi:10.1186/s13068-015-0340-x

Biotechnology for Biofuels and Bioproducts

Table 2 Reaction-specific kinetic parameters used in kinetic modeling

From: Identification of novel metabolic interactions controlling carbon flux from xylose to ethanol in natural and recombinant yeasts

CtXR^a	NAD(H) (R ₁)	NADP(H) (R ₂)
E	PO^b	PO^b
V _f	1.0	1.3
V _f/V _r	13	25
K _iA (mM)	0.18 ± 0.04	0.0019 ± 0.0001^c
K _mA (mM)	0.04 ± 0.01	0.005 ± 0.002
K _mB (mM)	98 ± 22	108 ± 17
K _iB (mM)^d	392	3920
K _iQ (mM)	0.344 ± 0.03	0.0059 ± 0.0004^c
K _mQ (mM)	0.019 ± 0.005	0.008 ± 0.001
K _mP (mM)	529 ± 34	220 ± 18
K _iP (mM)	758 ± 48	556 ± 48
K ^{Haldane, e}_eq	134	142
K ^{exp, e}_eq	142	156

GmXDH^f(R ₃)		PP (R ₅), UG (R ₉), LG (R ₁₀)
E	PO^b	V _f	PO^b
V _f	1.0	K _m (mM)	0.1
V _r	12.6
\({K_{\rm{m,NAD}^{+}}}\) (mM)	0.14	PGI^h (R ₆)
K _m,xylitol (mM)	14	V _f	PO^b
\({K_{\rm{i,NAD}^{+}}}\) (mM)	0.78	K _m,F6P (mM)	0.15
K _i,xylitol (mM)^d	4.5	K _m,G6P (mM)	0.30
K _i,NADH (mM)	0.025	K _eq	3.23
K _m,NADH (mM)	0.073
K _m,xylulose (mM)	4.0	G6PDH (R ₇)
K _i,xylulose (mM)^g	1.0	V _f	PO^b
K ^{Haldane, e}_eq	7 × 10⁻⁴	\({K_{\rm{m,NADP}^{+}}}\) (mM)	0.1
K ^{exp, e}_eq	(4-7) × 10⁻⁴	K ^h_m,G6P (mM)	0.05
		K _i,NADPH (mM)	0.20 (0.05)ⁱ
XK (R ₄)
V _f	PO^b	GND (R ₈)
K _m,xylulose (mM)^h	0.31	V _f	PO^b
		K ⁺_m,NADP (mM)	0.02
		K ^h_m,6PG (mM)	0.05
		K _i,NADPH (mM)	0.02

^aA, B, P, Q relate to NAD(P)H, xylose, xylitol, and NAD(P)⁺, respectively
^bPO, values were found within a predefined range by parameter optimization (see main text and Additional file 1: Table S3)
^cValues obtained from ligand-binding analysis using fluorescence spectroscopy (this study)
^dValues calculated from Haldane relationship K _eq = V ²_f K _iP K _mQ/(V ²_r K _iB K _mA) [38]
^eEquilibrium constants were calculated in accordance with the Haldane relationship [K ^Haldane_eq = V _f K _mP K _iQ/(V _r K _mB K _iA)] [38]. Values can be compared to those experimentally obtained in this study (CtXR) or to reported K ^exp_eq [39, 40]. Calculated Gibbs free energies of reaction of 6 ± 1 kJ mol⁻¹ for the isomerization of xylose into xylulose [ΔG _r = −RTln(K ^Haldane_eqXR K ^Haldane_eqXDH ] or ΔG _r = −RTln(K ^exp_eqXR K ^exp_eqXDH ) are in excellence accordance with a value of 4.3 kJ mol⁻¹ calculated from standard transformed Gibbs free energies of formation [41]
^fReported kinetic parameters obtained from comprehensive full-kinetic study acquired at 25 °C in 50 mM potassium phosphate buffer pH 7.5 were applied [42]. A, B, P, and Q correspond to NAD⁺, xylitol, xylulose, and NADH, respectively. Note, to fulfill thermodynamic with respect to K ^exp_eq reported upper limits were used for V _r (1800 ± 350 s⁻¹), K _B (12 ± 2 mM) and lower limits for K _P (8 ± 4 mM)
^gValue was taken from [43]
^hValues of kinetic parameters for XK, G6PDH, 6PGDH, and PGI were from Refs. [33, 44–46], respectively
ⁱValue referred to CBS4435 (BP000). Note based on a sensitivity analysis implemented in Copasi K _i of both ScG6PDH and CtG6PDH did not significantly influence FCC and YCC

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