## 4 Materials and Methods
### Total Proteins Extraction and Digestion  
A volume containing 1 ug of protein was injected for each of the 32 samples (2 strains \(\times\) 2 media \(\times\) 2 culture times \(\times\) 4 biological replicates) and subjected to an in-depth shotgun label-free analysis. The samples were alkylated before digestion by Lysyl-Endopeptidase (Wako Chemicals, Richmond, VA, USA) and sequencing-grade-modified trypsin (Promega, Charbonnieres-les-Bains, France). The resulting proteolytic peptides were pre-cleaned, concentrated under vacuum, and stored before mass spectrometry analysis as described previously [(8)]. Trypsin-generated peptides were analyzed by nanoLC-MSMS using a nanoElute liquid chromatography system (Bruker, Billerica, MA, USA) coupled to a timsTOF pro mass spectrometer (Bruker, Billerica, MA, USA). Then, 1 ug of protein digest in 2 \(\mu\)L of loading buffer (2% acetonitrile and 0.05% trifluoroacetic acid in water) were loaded on an Aurora analytical column (ION OPTIK, 25 cm \(\times\) 75 \(\mu\)m, C18, 1.6 \(\mu\)m, Bruker, France SAS) and eluted with a gradient of 0-35% of solvent B for 100 min. Solvent A was 0.1% formic  
\begin{table}
\begin{tabular}{l l} \hline \hline
**List of Transcriptional Regulators and Regulatory Protein Kinases** & **Impact on Act Biosynth** \\ \hline AbrC1/ SC04598 (TCS sensory histidine kinase) & Pos [(71)] \\ SCO6992 & Pos [(102)] \\ SCO3932 & Pos [(127)] \\ Spa/SC07629 (starvation sensing protein) & Pos [(117)] \\
**MacR/SC0212** (TCS regulator) & Pos [(109)] \\ BldC/SC04091 & Pos [(111,112)] \\ TCS **BraR/Dra** K-SC03063/SC03062 & Pos [(62)] \\ RoR/T/SC0608 (regulator of the xylose transport operon _xyl/FGH_) & Pos [(105,106)] \\ SCO5785 (TCS response regulator) & Pos [(128)] \\ ASK/SC04423 (ESTPK) & Pos [(195)] \\ Rev/SC03320 (Sensor of NAD+/NADH poise) &? \\ SCO5556 (HU DNA-binding protein) &? \\ LexA/SC05803 (SOS regulatory protein) &? \\ OrnA/SC07293 (Oligribonuclease) &? \\ SCO3209 (regulator of para-hydroxybenzoate catabolism) &? \\ SCO1614 (regulator of the ethanolamine utilization pathway) &? \\ PkA/SC04735 (ESTPK, regulation of sporulation) &? \\ PgW/SC06626 (phage resistance) & Pos [(161)] \\ SCO1678 & Pos [(158)] \\ Serl/SC0441 & Pos [(158)] \\ CcPh/SC0671 (regulator of \(\gamma\)-butylrolactone biosynthesis) & Neg [(162,163)] \\ NsdA/SC05582 (Nitrogen metabolism?) & Neg [(167,168)] \\ NsdB/SC07252 & Neg [(164)] \\ Cabb/SC05464 (calcium homeostasis) &? \\ SCO2823 (regulator of probable amino acid ABC transporter protein) &? \\ BldH/SC02792 (Activator of aerial mycelium formation) &? \\ SCO2775 &? \\ SCO5804/NrdR (Regulator of ribonucleotide reductase) &? \\ RamC/SC06681 (ESIPK, activator of aerial mycelium formation) &? \\ \hline \hline \end{tabular}
\end{table}
Table 1: Cont.  
acid and 2% acetonitrile in water, and solvent B was 99.9% acetonitrile with 0.1% formic acid. MS and MS/MS spectra were recorded from \(m/z\) 100 to 1700 with a mobility scan range from 0.6 to 1.5 V\(\cdot\)s/cm\({}^{2}\). MS/MS spectra were acquired with the PASEF (parallel accumulation--serial fragmentation (PASEF)) ion mobility-based acquisition mode using a number of PASEF MS/MS scans set as 10. MS and MSMS raw data were processed and converted into mgf files with DataAnalysis software (Bruker, Billerica, MA, USA).