TABLE 3.

Origin of the CFE, buffer, substrates, controls, and references used for each enzymatic assay

CFE	Tested enzyme	Buffer	Substrates	Controls	Reference
SBR-1	Acetoacetyl-CoA reductase	1X, supplemented	200 μM acetoacetyl-CoA, 200 μM NADH, or 200 μM NADPH	No acetoacetyl-CoA; E. coli MG1655; E. coli MG1655 + p-phaCABa	76
SBR-2	Acetoacetyl-CoA reductase	1X, supplemented	40 μM acetoacetyl-CoA, 100 μM NADH, or 200 μM NADPH	No acetoacetyl-CoA; isolated/purified enzyme	77
SBR-2	3-Hydroxybutyryl-CoA dehydrogenase	1X, supplemented	100 μM 3-hydroxybutyryl-CoA, 1,000 μM NAD(P)+	No hydroxybutyryl-CoA	77
SBR-1	Glucose-6-phosphate dehydrogenase	1X, supplemented	2 mM glucose-6-phosphate, 2,000 μM NAD+, or 200 μM NADP+	No glucose-6-phosphate; no CFE	78
SBR-2	Glucose-6-phosphate dehydrogenase	1X, supplemented	4 mM glucose-6-phosphate, 400 μM NAD(P)+	No glucose-6-phosphate; no CFE	78
SBR-2	Isocitrate dehydrogenase	1X, supplemented	400 μM NAD(P)+, 400 μM isocitrate	No substrate; no CFE	79
	Malate dehydrogenase	1X, supplemented	200 μM NAD(P)H, 200 μM oxaloacetate	No substrate; no CFE	16
	Malic enzyme	1X, supplemented	200 μM NADP, 200 μM l-malate	No substrate; no CFE	80
	Isocitrate lyase	40 mM HEPES (pH 7), 6 mM MgCl2	4 mM isocitrate, 280 μM NADH, 45 U of lactate dehydrogenase from rabbit muscle (Roche)	No isocitrate; no CFE; E. coli MG1655 grown on acetate, E. coli JW3975b grown on glucose	81
	Fumarate reductase	50 mM HEPES (pH 7)	15 mM fumarate, 250 μM NADH	No substrate; no CFE	82
	α-Ketoglutarate dehydrogenase	50 mM HEPES (pH 7), 1 mM MgCl2, 1 mM thiamine pyrophosphate, 2.5 mM DTT	2 mM α-ketoglutaric acid, 100 μM coenzyme A, 2.5 mM NAD(P)+	No substrate; no CFE	83

^aPlasmid bearing the genes phaCAB from C. necator under the control of its native promoter. These genes encode the enzymes of the pathway leading to polyhydroxybutyrate (PHB) synthesis, and the plasmid was kindly donated by J. G. Cabrera Gomez from Universidade de Sao Paulo, Brazil.

^bStrain without isocitrate lyase from the Keio collection (84): F⁻ Δ(araD-araB)567 ΔlacZ4787(::rrnB-3) λ⁻ rph-1 Δ(rhaD-rhaB)568 ΔaceA782::kan hsdR514.