Base medium used contained 10 g glucose, 5 g (NH4)2SO4, 3 g KH2PO4 and 0.5 g MgSO4 per litre, in addition to 1 mL of trace elements solution and 1 mL of vitamin solution. The trace element solution contained, per litre (pH=4): EDTA (sodium salt), 15.0 g; ZnSO4⋅7H2O, 4.5 g; MnCl2⋅2H2O, 0.84 g; CoCl2⋅6H2O, 0.3 g; CuSO4⋅5H2O, 0.3 g; Na2MoO4⋅2H2O, 0.4 g; CaCl2⋅2H2O, 4.5 g; FeSO4⋅7H2O, 3.0 g; H3BO3, 1.0 g; and KI, 0.10 g.). The vitamin solution contained, per liter (pH=6.5): biotin, 0.05 g; p-amino benzoic acid, 0.2 g; nicotinic acid, 1 g; Ca-pantothenate, 1 g; pyridoxine-HCl, 1 g; thiamine-HCl, 1 g and myo-inositol, 25 g.

Quantitative Proteome Measurements: The peak intensity-based absolute quantification method iBAQ was chosen for protein quantification [1]. First, a lysine auxotrophic strain was created by deleting the LYS1 gene. Latter strain was cultivated with a labelled heavy 15N, 13C-l ... ysine (Cambridge Isotope Laboratories), and fully labelled biomass was produced (in which 98% of proteogenic lysine was labelled, data not shown) and used as an internal standard in the measurements.
Nano-LC/MS/MS Analysis: Injected peptides (2 μg) were separated on an Ultimate 3000 RSLCnano system (Dionex, Sunnyvale, California, United States) using a C18 cartridge trap-column in a backflush configuration and an in-house packed (3 μm C18 particles, Dr Maisch) analytical 50 cm x 75 μm emitter-column (New Objective). The peptides were eluted at 200 nL min−1 with an 8%–40% B 240 min gradient (buffer B: 80% acetonitrile + 0.1% formic acid, buffer A: 0.1% formic acid) to a Q Exactive (Thermo Fisher Scientific) tandem mass spectrometer operating with a top-10 strategy and a cycle time of 0.9 seconds. Briefly, one 350-1 400 m/z MS scan at a resolution of R = 70,000 was followed by higher-energy collisional dissociation fragmentation (normalized collision energy of 25) of the 10 most-intense ions (charge states +2 to +6) at R = 17,500. The MS and MS/MS ion target values were 3x 106 and 5x 104, respectively. Dynamic exclusion was limited to 80 seconds.
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[1] Schwanhäusser B., Busse D., Li N., Dittmar G., Schuchhardt J., Wolf J., Chen W., Selbach M.Nature. 2011; 473: 337-342. http://doi.org/bgjkh9