Establishment of markerless gene deletion tools in thermophilic Bacillus smithii and construction of multiple mutant strains

Table 1 HPLC analysis of B. smithii ET 138 and DSM 4216^T wild-type and mutant strains

Strain	Products (mM) and measurement method
	Enzymatic		HPLC
	l-lac	d-lac	Lac	Ace	Pyr	Mal	Suc	OD₆₀₀
DSM 4216 wild-type	15.85 ± 2.22	0.73 ± 0.08	20.67 ± 1.25	9.12 ± 0.49	0.21 ± 0.11	0.06 ± 0.02	0.27 ± 0.10	0.785 ± 0.209
DSM 4216 ΔldhL	0.02 ± 0.03	4.11 ± 0.86	4.95 ± 3.35	12.40 ± 3.16	1.48 ± 1.14	0.35 ± 0.07	0.57 ± 0.22	0.466 ± 0.057
ET 138 wild-type	20.55 ± 4.09	0.87 ± 0.05	26.18 ± 7.66	8.25 ± 2.59	0.78 ± 0.31	0.05 ± 0.08	0.61 ± 0.16	1.036 ± 0.136
ET 138 ΔldhL	0.08 ± 0.09	1.08 ± 0.32	1.73 ± 0.48	14.33 ± 5.54	0.76 ± 0.82	0.33 ± 0.15	0.72 ± 0.22	0.581 ± 0.289
ET 138 ΔldhL ΔsigF	0.06 ± 0.05	0.95 ± 0.20	1.58 ± 0.62	10.97 ± 2.13	0.70 ± 0.41	0.27 ± 0.08	0.60 ± 0.16	0.672 ± 0.102
ET 138 ΔldhL ΔsigF ΔpdhA ^a	0.39 ± 0.02	11.17 ± 0.47	11.50 ± 2.07	−3.19^a ± 1.24	8.26 ± 0.82	1.12 ± 0.14	1.85 ± 0.37	0.696 ± 0.119
ET 138 ΔldhL ΔsigF + pWUR736	18.13 ± 3.82	1.11 ± 0.40	18.99 ± 5.19	7.32 ± 1.33	0.24 ± 0.16	0.08 ± 0.18	0.39 ± 0.17	0.831 ± 0.200
ET 138 ΔldhL ΔsigF + pNW33n	0.14 ± 0.04	2.57 ± 1.30	3.17 ± 1.13	10.14 ± 0.91	0.94 ± 0.53	0.07 ± 0.12	0.36 ± 0.08	0.535 ± 0.062
ET 138 wild-type + pNW33n	22.12 ± 3.69	0.69 ± 0.10	21.42 ± 1.51	5.63 ± 1.15	0.65 ± 0.07	0.05 ± 0.12	0.34 ± 0.14	0.849 ± 0.076

Strains were grown in 25 mL TVMY supplemented with 10 g/L glucose in 50 mL Greiner tubes at 55°C for 24 h after transfer from a 10 mL-overnight culture. d- and l-lactate were distinguished via enzymatic assays, for which the lowest detection limit was 0.04 mM. The values shown are the results of three to fourteen independent experiments; numbers in italics are standard deviations.
l -lac l-lactate, d -lac d-lactate, Ace acetate, Pyr pyruvate, Mal malate, Suc succinate.
^aThese cultures were supplemented with 3 g/L ammonium acetate.

ISSN: 1475-2859