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Additional File 1 Fermentation stage-dependent adaptations of Bacillus licheniformis during enzyme production Sandra Wiegand, Birgit Voigt, Dirk Albrecht, Johannes Bongaerts, Stefan Evers, Michael Hecker, Rolf Daniel and Heiko Liesegang Content Figure S1 Protease production and process parameters. ........................................................................... 2 Figure S2 Proteome of the amino acid metabolism – Part I. .................................................................... 3 Figure S3 Proteome of the amino acid metabolism – Part II. ................................................................... 4 Figure S4 Amino acid transport. ............................................................................................................... 5 Figure S5 Proteome of the central carbon metabolism. ............................................................................ 7 Figure S6 Carbohydrate transport. ............................................................................................................ 8 Figure S7 Acetoin utilization operon acuABC. ....................................................................................... 10 Figure S8 Most abundant proteins. ......................................................................................................... 10 Figure S9 Lichenicidin gene cluster. ....................................................................................................... 11 Figure S10 Cell envelope stress response. .............................................................................................. 12 Figure S11 Sporulation. .......................................................................................................................... 14 Figure S12 Iron starvation....................................................................................................................... 22 Figure S13 Heat shock response. ............................................................................................................ 23 Figure S14 Phosphate starvation response. ............................................................................................. 24 Figure S15 Putative Tat signal peptide of Subtilisin Carlsberg. ............................................................. 25 References ............................................................................................................................................... 26 R 200 I II III IV V 10 180 9 160 8 140 7 120 6 100 5 80 4 60 3 40 2 20 1 0 0 0 5 10 15 20 25 30 35 40 45 Time [h] pO2 [%] cAcetate [g/L] cGlucose [g/L] CO2 [%] feed [g/L] c + [g/10L] Glucose NH4 protease activity [%] I II III IV V M 180 9 160 8 140 7 120 6 100 5 80 4 60 3 40 2 20 1 0 0 0 5 10 15 20 25 30 35 40 45 Time [h] pO2 [%] CO2 [%] cGlucose [g/L] feedGlucose [g/L] protease activity [%] Figure S1 Protease production and process parameters. Process parameters are shown for fermentations R and M (please refer to Figure 1 for replicate L). Oxygen partial pressure pO2 [%], glucose concentration cGlucose [g/L], supplied glucose feedGlucose [g/L] and normalized protease activity [%] are displayed on the left y- axis, whereas acetate concentration cAcetate [g/L] (only for fermentation R), carbon dioxide content CO2 [%], and ammonium concentration cNH4+ [g/10L] (only for fermentation R) are scaled on the right y-axis. Process time t [h] is given on the x-axis. The sampling points I to V are indicated by light blue lines. 2 dipeptides/ oligopeptides oligopeptides dipeptides oligopeptides OppA_a AppA1_a DppE BLi00894 OppA_b AppA1_b OppD AppA1_c AppA1_d AppA1_e L-Hser L-Thr L-Ile L-Trp GcvT AroE_a LpdV_a AroE_b LpdV_b AroF PdhD_a PdhD_b GcvPA Gly SpeE L-Phe MtnN MtnD L-Met MetK L-Tyr L-Ser MtnN IlvC_a IlvC_b IlvC_c MetE IlvC_d IlvC_e IlvD Pyr YwaA CysK_a CysK_b Bcd_a Bcd_b LpdV_a LpdV_b L-Hcys L-Cys L-Glu -1.8 0 1.8 Figure S2 Proteome of the amino acid metabolism – Part I. Heat map representation of Z-score transformed protein spot volumes of proteins involved in amino acid transport and metabolism. In cases where a specific protein is assigned to more than one spot, the particular spots are indicated by an underscore, followed by an ordering letter. Statistically not significant values are indicated by light grey boxes. Pyr: Pyruvate. For the corresponding transcriptome data please refer to Figure 5. 3 L-Leu -1.8 0 1.8 Bcd_a Bcd_b LpdV_a LpdV_b Oxo IlvC_a Pyr IlvC_b IlvC_c IlvC_d IlvC_e IlvD YwaA L-Val Ald1 Bcd_a Ald2 Bcd_b LpdV_a LpdV_b L-Gln L-Ala GlnA_a L-Glu GlnA_b L-Cys L-Pro L-His DapA1_a DapA1_b YkuQ RocA_a RocA_b RocA_c YodQ L-Asp Asd RocF1 L-Hser L-Lys ArgB L-Arg ArgC SpeB L-Asn Figure S3 Proteome of the amino acid metabolism – Part II. Heat map representation of Z-score transformed protein spot volumes of proteins involved in amino acid metabolism. In cases where a specific protein is assigned to more than one spot, the particular spots are indicated by an underscore followed, by an ordering letter. Statistically not significant values are indicated by light grey boxes. Yellow frames indicate reactions with multiple assigned enzymes of which only one is strictly necessary. Pyr: Pyruvate, Oxo: 2-Oxoglutarate. For the corresponding transcriptome data please refer to Figure 6. 4 -1.8 0 1.8 (A) I II III IV V gltP proton/glutamate symport protein ycgF putative amino acid efflux protein yflA1 sodium/alanine symporter ycgO1 sodium/proline symporter yflA2 sodium/alanine symporter cstA putative peptide import permease dltB D-alanine transfer protein BLi02238 lysine exporter protein BLi03380 amino acid carrier protein alsT amino acid carrier protein rocE amino acid permease BLi02203 putative amino acid transporter blt spermidine-efflux transporter yclF putative oligopeptide transporter gltT proton/sodium-glutamate symport protein azlC putative branched-chain aminoacid transporter ycgO2 sodium/proline symporter yodF Na+/solute symporter trpP tryptophan transporter ybxG amino acid permease tcyP sodium/cystine symporter BLi00817 putative sodium-alanine symporter ybgF putative amino acid permease ydgF putative amino acid permease opuD glycine betaine transporter opuE sodium/proline symporter nrgA ammonium transporter BLi01175 ammonium transporter braB branched-chain amino acid transport system carrier protein 5 -1.8 0 1.8 (B) I II III IV V artR high affinity arginine ABC transporter ATP-binding protein artQ high affinity arginine ABC transporter permease artP high affinity arginine ABC transporter ATP-binding protein * BLi00894 dipeptide/oligopeptide ABC transporter solute-binding protein BLi00892 dipeptide/oligopeptide ABC transporter ATP-binding protein BLi00893 dipeptide/oligopeptide ABC transporter ATP-binding protein BLi00895 dipeptide/oligopeptide ABC transporter permease BLi00896 dipeptide/oligopeptide ABC transporter permease dppB dipeptide ABC transporter permease dppC dipeptide ABC transporter permease dppD dipeptide ABC transporter ATP-binding protein * dppE dipeptide ABC transporter dipeptide-binding protein ykfD putative dipeptide ABC transporter ATP-binding protein yckA putative amino acid ABC transporter permease protein yckB putative amino acid ABC transporter substrate-binding protein appD1 oligopeptide ABC transporter ATP-binding protein appF1 oligopeptide ABC transporter ATP-binding protein * appA1 oligopeptide ABC transporter substrate-binding protein appB1 oligopeptide ABC transporter permease appC1 oligopeptide ABC transporter permease * oppA oligopeptide ABC transporter oligopeptide-binding protein oppB oligopeptide ABC transporter permease oppC oligopeptide ABC transporter permease * oppD oligopeptide ABC transporter ATP-binding protein oppF oligopeptide ABC transporter ATP-binding protein tcyB L-cystine ABC transporter permease tcyC L-cystine ABC transporter ATP-binding protein tcyA L-cystine ABC transporter substrate-binding protein metQ2 methionine ABC transporter substrate-binding protein metP2 methionine ABC transporter permease metN2 methionine ABC transporter ATP-binding protein Figure S4 Amino acid transport. Heat map representation of Z-score transformed NPKM values. The depicted genes have been annotated as (A) amino acid or nitrogen transporters and (B) amino acid ABC transporter components. Please note that the figure does not give a complete list of genes involved in amino acid transport. Genes with an assigned antisense RNA [1] are marked in blue, asterisks indicate a detected protein spot for the respective gene (Additional File 2: Table S3) and statistically not significant values are marked by grey boxes. Transporter genes with high transcript abundances during the early stages of the fermentation process are for example encoding a tryptophan transporter (trp), cystine (tcyABC) and methionine (metNPQ) ABC transporters and diverse proteins for uptake of alanine or unspecific amino acids. Additionally, the transcript of the ammonium transporter NrgA, especially required for ammonium transport at low ammonium concentrations in B. subtilis [2], is highly abundant at sampling point I. The only operon besides the dipeptide ABC transporters mentioned in the main text with distinct transcript abundance at the later sampling points encodes a high-affinity arginine ABC transporter (artPQR). 6 PtsI_a PtsI_b Zwf_a Zwf_b Glucono- 6P- Glucose G6P Ribulose-5P lactone-6P Gluconate Pgi_a Pgi_b Pgi_c Pgi_d Arabino- F6P hexulose-6P PfkA Tkt Tkt Xylulose-5P Ribose-5P FBP Tkt Tkt FbaA Tkt Erythrose-4P Sedohep- YwjH_a tulose-7P TpiA YwjH_b DHAP GADP YwjH_c Tkt GapA BPG