13. PG3 + ATP + NADH => GA3P + ADP + Pi + NAD +
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1 Additional file Stochiometric model for P. pastoris containing some additional reactions from the 13 C model (section 1.2) Methanol metabolism 1. Metoh => Form 2. Form => FOR + NADH 3. FOR + NAD + => NADH + CO 2 4. Xul5P + FOR + ATP => ADP + GA3P per + DHA 5. GA3P per => GA3P 6. DHA => GA3P Glycolysis and gluconeogenesis pathways 7. Glc ext + 2 ATP <=> Glc6P + 2 ADP 8. Glc6P <=> Fru6P 9. Fru6P + 1 ATP => FBP + 1ADP 10. FBP => Fru6P + Pi 11. FBP <=> DHAP + GA3P 12. GA3P + ADP + Pi + NAD + => PG3 + ATP + NADH 13. PG3 + ATP + NADH => GA3P + ADP + Pi + NAD PG3 <=> Pep 15. Pep + ADP => Pyr + ATP 16. Pyr + NAD + => ACCoA mit + CO 2 + NADH 17. Pyr NAD + => ACCoA cyt + CO 2 + NADH
2 18. Pyr + CO 2 + ADP => OAA + ATP Glc6P + UTP + H 2 O => H + +2 Pi + UDP + T6P 20. T6P + H 2 O => Pi + Treh 21. Treh + H 2 O => 2 Glc6P Pentose phosphate pathway 22. Glc6P + 2 NADP + => Rul5P + 2 NADPH + CO Rul5P <=> Rib5P 24. Rul5P <=> Xul5P 25. Rib5P + Xul5P <=> Sed7P + GA3P 26. Sed7P + GA3P <=> Fru6P + E4P 27. Xul5P + E4P <=> Fru6P + GA3P TCA cycle 28. ACCoA mit + OAA => CIT 29. CIT => ICIT 30. ICIT + NAD + => αkg + CO 2 + NADH 31. αkg + NAD + => SUCCoA + CO 2 + NADH 32. SUCCoA + Pi + ADP => SUCC + ATP 33. SUCC + ATP => SUCCoA + ADP + Pi 34. SUCC + NAD + => FUM + NADH 35. FUM + H 2 O <=> MAL 36. MAL + NAD + <=> OAA + NADH 37. Asp + 2 ATP + H 2 O=> FUM + 2 ADP + 2Pi
3 Biosynthesis of amino acids Serine family 38. PG3 + Glut + NAD + => Ser + αkg + NADH +Pi 39. Ser + THF => Gly + MetTHF 40. Ser + ACCoA + H 2 S => Cys Alanine family 41. Pyr + NADPH => Ala + NADP Pyr + Glut => αkg Pyr + NADPH => Kval + CO 2 + NADP Kval + Glut => Val + αkg 45. Kval + ACCoA mit + Glut + NAD + => Leu + αkg + NADH + CO 2 Histidine family 46. Rib5P + ATP => PRPP + AMP 47. PRPP + ATP + Gln + 2 NAD + => His + αkg + Pi + 2 NADH Aspartic family 48. OAA + Glut => Asp + αkg 49. Asp + Gln + ATP => Asn + Glut + AMP 50. Asp + ATP + 2 NADPH => Ser + ADP + Pi + 2 NADP Thr + NADPH + Glut + Pyr => Ile + αkg + NH 4 + CO 2 + NADP ACCoA + Ser + H 2 S + MTHF => Met + THF
4 Aromatic family Pep + E4P + ATP + NADPH => CHOR + ADP + 4 Pi 54. CHOR + Glut => Phe + αkg + CO CHOR + Glut => Tyr + αkg + NADH + CO CHOR + Gln + PRPP + Ser => Trp + Glut + Pyr + GA3P + CO 2 Glutamic family 57. αkg +NH 4 + NADPH => Glut + NADP + H 2 O 58. Glut + ATP + NH 4 => Gln + ADP + Pi 59. Glut + ATP + 2 NADPH => Pro + ADP + Pi 60. Gln + CO ATP => CaP + Glut + 2 ADP + Pi 61. Glut + ACCoA mit + 4 ATP + NADPH + CaP + Asp => Arg + αkg + 4 ADP + FUM + 5 Pi Glut + ACCoA mit + 3 ATP + 2 NADPH + 2 NAD + => Lys + αkg + CO NADH + 2 NADP + Biosynthesis and interconversion of one-carbon units 63. DHF + NADPH => THF + NADP Gly + THF + NAD + => MTHF + NH NADH + CO MTHF + NADH => THF + NAD + Transport reactions 66. ACCoA cyt => ACCoA mit 67. NH 4 + ext + ATP => NH 4 + cyt + ADP + Pi
5 68. SO 4-2 ext + ATP => SO 4-2 cyt + ADP + Pi 69. Metal ext + ATP => Metal cyt + ADP + Pi Respiratory chain 70. NADH O 2 => NAD + + H 2 O 71. ADP + Pi => ATP Biomass synthesis 1. Protein synthesis (Composition derived from the measured amino acid composition [21]. The energy needed to biosynthesize 1 C-mol of protein was derived from the synthesis of each amino acid and the protein polymerization value taken from [50]) Pyr NAD NADPH ATP Rib5P E4P OAA αkg mit ACCoA cyt => 1 C-mol Protein GA3P NADH NADP ADP Pi CO 2 2. Carbohydrate synthesis (Composition derived [51]) Glc6P Fru6P ATP => 1 C-mol Carbohydrate ADP 3. Lipids synthesis (derived from the mean lipid composition from [30]) Glc6P Pyr GA3P CO ACCoA mit ACCoA cyt NADH NADPH ATP O 2 => 1 C-mol Lipid NAD NADP ADP Pi
6 4. RNA synthesis (derived from the RNA composition [57]) Pyr CO Rib5P NAD NADPH ATP OAA=> 1 C-mol RNA Pi ADP NADP NADH 5. DNA synthesis (composition derived from [57] and the DNA polymerization [51]) Pyr NAD NADPH ATP CO Rib5P OAA => 1 C-mol DNA Pi ADP NADH NADP +
7 1.2 Reactions and atom transitions network used in 13 C-MFA, following the notation of [65]. Name feedglcb: Reaction FullyGlu > Glu ext feedglcc: CGlu > Glu ext uptglc: Glu ext > Glu int feedmeohb: MetohL > Metoh ext #a > #a uptmeoh: Metoh ext > Metoh int #a > #a upt1: Glu int > Glc6P upt2: Metoh int > Form #a > #a TRE1: Glc6P > T6P TRE2: T6P > Treh TRE3: Treh > Glu int emp1: Glc6P <> Fru6P
8 emp2: Fru6P > FBP emp2b FBP > Fru6P emp3: FBP <> DHAP + GA3P #abcdef > #cba + #def emp4: DHAP <> GA3P emp5: GA3P <> PG3 emp6: PG3 <> PG2 emp7: PG2 <> Pep #abc > # abc emp8: Pep > Pyr emp9: Pyr > ACCoA cyt + CO2 #abc > #bc + #a emp10: Pyr + CO2 > OAA mit #abc + #d > #abcd emp11: Pyr > Pyrt emp11a: Pyr > Pyr mit emp11b: Pyr mit > Pyrt emp11c: NPyr > Pyr mit
9 #ABC > #ABC emp11d: Pyr mit > Pyr ext #ABC > #ABC emp12: ACCoA cyt > ACCoA mit #AB > #AB ppp1: Glc6P > CO2 + Rul5P #abcdef > #a + #bcdef ppp2: Rul5P <> Xul5P #abcde > #abcde ppp3: Rul5P <> Rib5P #abcde > #abcde ppp4: Xul5P + E4P <> GA3P + Fru6P #ABCDE + #abcd > #CDE + #ABabcd ppp5: Xul5P + Rib5P <> Sed7P + GA3P #abcde + #ABCDE > #ABabcde + #CDE ppp6: GA3P + Sed7P <> E4P + Fru6P #ABC + #abcdefg > #defg + #abcabc TCA1: Pyr mit > ACCoA mit + CO2 #ABC > #BC + #A TCA2: ACCoA mit + OAA > CIT mit #AB + #abcd > #dcbaba TCA3: CIT mit > αkg + CO 2 #ABCDEF > #ABCEF + #D TCA4: αkg > SUCC + CO 2 #ABCDE > #BCDE + #A TCA4B: αkg > SUCC + CO 2 #ABCDE > #EDCB + #A TCA5: SUCC <> FUM
10 #ABCD > #ABCD TCA5B: SUCC <> FUM #ABCD > #DCBA TCA6: FUM <> MAL #ABCD > #ABCD TCA7: MAL <> OAA #ABCD > #ABCD TCA8: Asp > FUM #ABCD > #ABCD Met1: Form > CO 2 #A > #A Met2: Xul5P + Form > DHA + GA3P per #ABCDE + #F > #FAB + #CDE Met2B: Xul5P + Form > DHA + GA3P per #ABCDE + #F > #ABF + #CDE Met3: DHA > DHAP #ABC > #ABC Met4: GA3P per > GA3P #ABC > #ABC BIO1: Glc6P > Glc6P bio #ABCDEF > #ABCDEF BIO2: Fru6P > Fru6P bio #ABCDEF > #ABCDEF BIO3: ACCoA cyt > ACCoA bio #BC > #BC BIO4: OAA > OAA bio #ABCD > #ABCD BIO5: E4P > E4P bio
11 #ABCD > #ABCD BIO6: Rib5P > Rib5P bio #ABCDE > #ABCDE BIO7: GA3P > GA3P bio #ABC > #ABC BIO8: αkg mit > αkg bio #ABCDE > #ABCDE BIO9: Pyrt > Pyr bio #ABC > #ABC CO2out1: CO 2 > CO 2 _ext #A > #A aa_ala: Pyr <> Ala #ABC > #ABC aa_glu: αkg mit <> Glut #ABCDE > #ABCDE aa_asp: OAA <> Asp #ABCD > #ABCD
12 1.3 Reaction network used for ananet analysis Abbreviation Reaction HXK PGI PFK FB FBA TPI GAPDH PGK GPM ENO PYK G6PDH 6PGDH RPI RPE TK(1)+TA TK(3) TA TK(1) G3PDH PYRCK TPP TreP Glc int + ATP > Glc6P + ADP Glc6P <> Fru6P ATP + Fru6P > ADP + FBP + h FBP + h2o > Fru6P + Pi FBP <> DHAP + GA3P DHAP <> GA3P GA3P + NAD + pi <> h + NADH + 13dpg 13dpg + ADP <> PG3 + ATP PG3 <> PG2 PG2 <> h2o + Pep ADP + h + Pep > ATP + Pyr Glc6P + NADP > PG6 + h + NADPH PG6 + NADP + h2o > Rul5p + NADPH + h + CO 2 tot Rib5p <> Rul5p Rul5p <> Xul5p Rib5p + Xul5p <> Fru6P + E4P E4P + Sed7P <> Fru6P + Rib5p GA3P + Sed7P <> Fru6P + E4P Rib5p+ Xul5p <> GA3P + Sed7P DHAP + NADH <> glyc3p + NAD Pyr + ATP + CO 2 tot <> OAA + ADP + Pi + h2o (2) Glc6P + utp + h2o > Treh + ppi + udp + Pi Treh + h2o > (2) Glu int
13 PDC DHAK PMI CAT MET Pyr > ACALD + CO 2 tot DHA + ATP <> DHAP + ADP Fru6P <> Man6p Metoh + (0.5) o2 + (2) NAD > CO2tot + (2) NADH Metoh + Xul5p + (0.5) o2 > DHA + GA3P per
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