13C-Methanol Labeling of E. coli Mutant

Freshly transformed
colonies of MG1655(DE3) ΔfrmA^{32 (link)} were precultured overnight in Medium A (LB + 5 g/L xylose,
20 mM MgSO₄, 100 μM ZnCl₂, 50 μg/mL
kanamycin from 1000× aqueous stock solution), then diluted 100-fold
into fresh Medium A and incubated with shaking (200 rpm) at 37 °C
until OD₆₀₀ 0.5–0.6. Pathway enzyme expression was
induced with 100 μM IPTG, and growth continued for another 2
h. The culture was then centrifuged (10 min at 3500 rcf), washed once
with an equal volume of M9 medium, and resuspended in an equal volume
of M9 containing 5 g/L d-xylose, and 250 mM ¹³C methanol (Cambridge Isotope Laboratories, 99%). Cells were incubated
with shaking for a further hour before intracellular metabolites were
extracted as described previously.^{50 (link)} Briefly,
1 mL of liquid culture was filtered through a 0.45 μm nylon
filter, washed with 10 mL room-temperature water, and then the filter
was transferred into a 50 mL falcon tube containing 5 mL of extraction
solution (40:40:20 acetonitrile:methanol:water) at −20 °C.
After 30 min, the filter was removed, the samples were centrifuged,
and the supernatants dried overnight under air. The next morning,
dried metabolites were resuspended in 150 μL water, centrifuged
at 13 000 rpm for 40 min, and injected into an LC–MS/MS
system as previously described.^{50 (link)}