Table 2.

Optimization of precursor concentration (mg per 100 ml culture)

	Sample number
	1	2	3	4	5	6	7	8	9	10	11
α-ketoisovaleric (mg/100 ml):	0	5.6	11.2	18.6	24.8	31.3	38.2	45.0	51.7	58.5	67.5
α-ketobutyric acid (mg/100 ml):	0	1.8	5.6	9.3	12.4	15.7	19.1	22.5	25.9	29.3	33.3

α-ketoisovaleric acid, sodium salt (1,2,3,4-¹³C₄, 98%; 3,4′,4′,4′-d₄, 98%) and α-ketobutyric acid, sodium salt (¹³C₄, 98%; 3,3-d₂, 98%) were purchased from Cambridge isotope laboratories (catalog numbers, CDLM-8100-PK and CDLM-4611-PK, respectively). Precursor stock solutions containing 225 μg/μl α-ketoisovaleric acid and 112.5 μg/μl α-ketobutyric acid were prepared. A 150 ml culture in M9 ++/D₂O medium containing 0.75 g ¹⁵NH₄Cl and 2.7 g d₇-D-glucose was grown to an OD₆₀₀ ~ 8 in a 2.8 L flask, as described previously (Cai et al. 2016). Eleven 10 ml cultures were prepared by aliquoting 10 ml of the above culture to eleven 250 ml Corning disposable flasks to which were added 0, 2.5, 5, 8, 11, 14, 17, 20, 23, 26 and 30 μl of the above precursor stock solution. The 11 cell cultures were allowed to continue growing at 37 °C for 1 h (at which point the OD₆₀₀ ~ 10) and induced with 1 mM IPTG for 20 h at 25 °C. Cell pellets from these cultures were resuspended in 10 ml lysis buffer containing 50 mM Tris–HCl, pH 7.5 and 200 mM NaCl. The cells suspensions were sonicated for three minutes in a cold room with the samples immersed in ice water. The supernatant from each cell lysate was loaded onto a 1 ml Ni-affinity column, and washed with 20 mL of buffer A (50 mM Tris–HCl, pH 7.5, 200 mM NaCl and 40 mM imidazole). The ngMinE was eluted with an imidazole gradient from 40 mM (buffer A) to 1 M imidazole (buffer B = buffer A plus 1 M imidazole). The protein fractions were pooled, and further purified by gel filtration on a Superdex75 column in 25 mM potassium phosphate buffer, pH 6.5 and 200 mM NaCl. The fractions containing ngMinE were pooled and concentrated using a centriprep with a 3.5 kDa cutoff