Supplementary File. Table S1. Metabolic model.

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1 Supplementary File Table S. Metabolic model. Flux number Reaction Carbon transitions r Glucose --> G6P ABCDEF --> ABCDEF r2 G6P --> F6P ABCDEF --> ABCDEF r3 F6P --> G6P ABCDEF --> ABCDEF r4 F6P --> FBP ABCDEF --> ABCDEF r5 FBP --> DHAP + GAP ABCDEF --> CBA + DEF r6 DHAP + GAP --> FBP CBA + DEF --> ABCDEF r7 DHAP --> GAP ABC --> ABC r8 GAP --> DHAP ABC --> ABC r9 GAP --> PGA ABC --> ABC r PGA --> GAP ABC --> ABC r PGA --> PEP ABC --> ABC r2 PEP --> PGA ABC --> ABC r3 PEP --> Pyr ABC --> ABC r4 Pyr --> AcCoA + CO 2 _in ABC --> BC + A r5 AcCoA + Oxa --> IsoCit AB + CDEF --> FEDBAC r6 IsoCit -->αkg + CO 2 _in ABCDEF --> ABCDE + F r7 αkg --> Suc + CO 2 _in ABCDE --> BCDE + A r8 Suc --> Fum ABCD --> ABCD r9 Fum --> Suc ABCD --> ABCD r2 Fum --> Mal ABCD --> ABCD r2 Mal --> Fum ABCD --> ABCD r22 Mal --> Oxa ABCD --> ABCD r23 Oxa --> Mal ABCD --> ABCD r24 IsoCit + AcCoA --> Mal + Suc ABCDEF + GH --> ABHG + FCDE r25 PEP + CO2_in --> Oxa ABC + D --> ABCD r26 Oxa --> PEP + CO 2 _in ABCD --> ABC + D r27 Mal --> Pyr + CO 2 _in ABCD --> ABC + D r28 G6P --> 6PG ABCDEF --> ABCDEF r29 6PG --> Ru5P + CO 2 _in ABCDEF --> BCDEF + A r3 Ru5P --> R5P ABCDE --> ABCDE r3 R5P --> Ru5P ABCDE --> ABCDE r32 Ru5P --> Xu5P ABCDE --> ABCDE r33 Xu5P --> Ru5P ABCDE --> ABCDE r34 R5P + Xu5P --> S7P + GAP ABCDE + FGHIJ --> FGABCDE + HIJ r35 GAP + S7P --> Xu5P + R5P HIJ + FGABCDE --> FGHIJ + ABCDE r36 GAP + S7P --> F6P + E4P ABC + DEFGHIJ --> DEFABC + GHIJ r37 E4P + F6P --> S7P + GAP GHIJ + DEFABC --> DEFGHIJ + ABC r38 E4P + Xu5P --> F6P + GAP ABCD + EFGHI --> EFABCD + GHI r39 GAP + F6P --> Xu5P + E4P GHI + EFABCD --> EFGHI + ABCD r4 6PG --> Pyr + GAP ABCDEF --> ABC + DEF r4 PGA --> Ser ABC --> ABC r42 Ser --> Gly + THF_in ABC --> AB + C r43 Gly + THF_in --> Ser AB + C --> ABC

2 Concentration (mm) Metabolites 24, 4 S2 Table S. Cont. Flux number Reaction Carbon transitions r44 G6P --> [Biomass] r45 F6P --> [Biomass] r46 DHAP --> [Biomass] r47 Ser --> [Biomass] r48 Gly --> [Biomass] r49 PEP --> [Biomass] r5 Pyr --> [Biomass] r5 AcCoA --> [Biomass] r52 αkg --> [Biomass] r53 Oxa --> [Biomass] r54 R5P --> [Biomass] r55 E4P --> [Biomass] r56 Pyr --> [Lactate_ex] r57 Pyr --> AcCoA + [Formate_ex] ABC --> BC + A r58 AcCoA --> [Acetate_ex] R59 AcCoA --> [EtOH_ex] r6 CO2_in --> [CO2_ex] A --> A r6 CO2 --> CO2_in A --> A r62 THF_in --> [THF_ex] A --> A r63 THF --> THF_in A --> A Figure S. Fermentation profile. (a) The time course of optical density at 6 nm (Closed diamond) and the concentration of remained glucose (Closed square) and produced ethanol (Closed triangle) are shown. The concentration of glucose was zero constantly, indicating that glucose-limited chemostat culture was achieved. (b) The time course of organic acids concentration are shown. Closed circle, square, triangle and diamond indicates pyruvate, lactate, formate and acetate, respectively. The concentration was constant after 3 h from the start of culture, indicating that quasi metabolic steady state was assumable. OD (a) 2 C glucose 3 C glucose OD6 Glucose Ethanol

3 Concentration (mm) Metabolites 24, 4 S3 5 (b) Figure S. Cont. 2 C glucose 3 C glucose Pyruvate Lactate Formate Acetate Figure S2. Time course of 3 C enrichment of PAAs and FAAs. Fractional labeling of PAAs (Closed circle) and FAAs (Closed square) after the start of tracer feeding is shown in the figure. M-57 M-85 M-59 f32 Ala-57 Ala Alanine Asp-57 Asp-85 Asp-59 Asp f32 Aspartate Glu-57 Glu-85 Glu-59 Glutamate Phe-85 Phe f Phenylalanine

4 Metabolites 24, 4 S4 Figure S2. Cont. Gly-57 Gly-85 Glycine Leu-85 Leucine Val-57 Val-85 Valine Threonine Thr Thr Supplementary Data Results of metabolic flux analysis based on the PAAs_fullset Table S-. Estimated flux distribution and its 95% confidence interval. Reaction Lower Best fit Upper G6P <-> F6P F6P -> FBP FBP <-> DHAP + GAP DHAP <-> GAP GAP <-> PGA PGA <-> PEP PEP -> Pyr Pyr -> AcCoA AcCoA + Oxa -> Cit Cit ->αkg αkg -> Suc Suc <-> Fum Fum <-> Mal Mal <-> Oxa Glyoxylate shunt PEP + CO 2 <-> Oxa

5 Metabolites 24, 4 S5 Table S-. Cont. Reaction Lower Best fit Upper Mal -> Pyr G6P -> 6PG PG -> Ru5P Ru5P <-> R5P 2 Ru5P <-> Xu5P 8 2 R5P + Xu5P <-> S7P + GAP GAP + S7P <-> E4P +F6P E4P + Xu5P <-> GAP + F6P ED pathway 3 6 Table S-2. Measured and estimated MID of PAAs_fullset. PAA_Ala-57 M M M M PAA_Ala-85 M M M PAA_Asp-57 M M M M M PAA_Asp-85 M.2.2 M M M PAA_Asp-59 M.9.2 M M M PAA_Asp32 M M M PAA_Glu-57 M.33.3 M M M M M

6 Metabolites 24, 4 S6 Table S-2. Cont. PAA_Glu-85 M M M M M PAA_Glu-59 M M M M M PAA_Phe-57 M.23.2 M M M M M M M M M PAA_Phe-85 M.26.2 M M M M M M M M PAA_Phe32 M M M PAA_Gly-57 M M M PAA_Gly-85 M M PAA_Ile-85 M.34.3 M M M M M

7 Metabolites 24, 4 S7 Table S-2. Cont. PAA_Ile-59 M.34.3 M M M M M PAA_Leu-85 M.24.2 M M M M M PAA_Ser-57 M M M M PAA_Ser-85 M M M PAA_Ser-59 M M M PAA_Val-57 M M+.7.3 M M M M PAA_Val-85 M M M M M PAA_Tyr32 M M M PAA_Thr-57 M M M M M PAA_Thr-85 M.2.2 M M M

8 Metabolites 24, 4 S8 Supplementary Data 2 Results of metabolic flux analysis based on the FAAs_fullset Table S2-. Estimated flux distribution and its 95% confidence interval. Reaction Lower Best fit Upper G6P <-> F6P F6P -> FBP FBP <-> DHAP + GAP DHAP <-> GAP GAP <-> PGA PGA <-> PEP PEP -> Pyr Pyr -> AcCoA AcCoA + Oxa -> Cit Cit ->αkg αkg -> Suc Suc <-> Fum Fum <-> Mal Mal <-> Oxa Glyoxylate shunt PEP + CO 2 <-> Oxa Mal -> Pyr G6P -> 6PG PG -> Ru5P Ru5P <-> R5P 7 4 Ru5P <-> Xu5P R5P + Xu5P <-> S7P + GAP GAP+ S7P <-> E4P + F6P E4P + Xu5P <-> GAP + F6P 3 6 ED pathway 7 4

9 Metabolites 24, 4 S9 Table S2-2. Measured and estimated MID of FAAs_fullset. FAA_Ala-57 M M M M FAA_Ala-85 M M M FAA_Asp-57 M M M M M FAA_Asp-85 M.8.4 M M M FAA_Asp-59 M.7.4 M M M FAA_Asp32 M M M FAA_Glu-57 M.4.29 M M M M M FAA_Glu-85 M M+.4.4 M M M FAA_Glu-59 M M+.4.4 M M M FAA_Phe-85 M.2.2 M M M

10 Metabolites 24, 4 S Table S2-2. Cont. M M M M M FAA_Phe32 M M M FAA_Gly-57 M M M FAA_Gly-85 M M FAA_Leu-85 M.27.2 M M M M M FAA_Val-57 M M+.8. M M M M FAA_Val-85 M M M M M FAA_Tyr32 M M M FAA_Thr-57 M M M M M FAA_Thr-85 M.8.4 M M M

11 Metabolites 24, 4 S Supplementary Data 3 Results of metabolic flux analysis using FAAs_Glu+Asp Table S3-. Estimated flux distribution and its 95% confidence interval. Reaction Lower Best fit Upper G6P <-> F6P F6P -> FBP FBP <-> DHAP + GAP DHAP <-> GAP GAP <-> PGA PGA <-> PEP PEP -> Pyr Pyr -> AcCoA AcCoA + Oxa -> Cit Cit ->αkg αkg -> Suc Suc <-> Fum Fum <-> Mal Mal <-> Oxa Glyoxylate shunt 3 77 PEP + CO 2 <-> Oxa Mal -> Pyr 96 G6P -> 6PG 43 6PG -> Ru5P 43 Ru5P <-> R5P Ru5P <-> Xu5P R5P + Xu5P <-> S7P + GAP 2 3 GAP + S7P <-> E4P + F6P 2 3 E4P + Xu5P <-> GAP + F6P 3 28 ED pathway 26

12 Metabolites 24, 4 S2 Table S3-2. Measured and estimated MID of FAAs_Glu+Asp+Ala. FAA_Asp-57 M M M M M FAA_Asp-85 M.8.8 M M M FAA_Asp-59 M.7.8 M M M FAA_Asp32 M M M FAA_Glu-57 M.4.26 M M M M M FAA_Glu-85 M M M M M FAA_Glu-59 M M M M M

13 Metabolites 24, 4 S3 Supplementary Data 4 Results of metabolic flux analysis using FAAs_Glu+Asp+Ala Table S4-. Estimated flux distribution and its 95% confidence interval. Reaction Lower Best fit Upper G6P <-> F6P F6P -> FBP FBP <-> DHAP + GAP DHAP <-> GAP GAP <-> PGA PGA <-> PEP PEP -> Pyr Pyr -> AcCoA AcCoA + Oxa -> Cit Cit ->αkg αkg -> Suc Suc <-> Fum Fum <-> Mal Mal <-> Oxa Glyoxylate shunt 4 72 PEP + CO2 <-> Oxa Mal -> Pyr 77 G6P -> 6PG PG -> Ru5P 25 4 Ru5P <-> R5P Ru5P <-> Xu5P R5P + Xu5P <-> S7P + GAP 7 2 GAP + S7P <-> E4P + F6P 7 2 E4P + Xu5P <-> GAP + F6P 3 5 ED pathway 2

14 Metabolites 24, 4 S4 Table S4-2. Measured and estimated MID of FAAs_Glu+Asp+Ala. FAA_Asp-57 M M M M M FAA_Asp-85 M.8.8 M M M FAA_Asp-59 M.7.8 M M M FAA_Asp32 M M M FAA_Glu-57 M.4.26 M M M M M FAA_Glu-85 M M M M M FAA_Glu-59 M M M M M

15 Metabolites 24, 4 S5 Supplementary Data 5 Results of metabolic flux analysis using FAAs_Glu+Asp+Ala+Phe Table S5-. Estimated flux distribution and its 95% confidence interval. Reaction Lower Best fit Upper G6P <-> F6P F6P -> FBP FBP <-> DHAP + GAP DHAP <-> GAP GAP <-> PGA PGA <-> PEP PEP -> Pyr Pyr -> AcCoA AcCoA + Oxa -> Cit Cit ->αkg αkg -> Suc Suc <-> Fum Fum <-> Mal Mal <-> Oxa Glyoxylate shunt PEP + CO 2 <-> Oxa 2 4 Mal -> Pyr G6P -> 6PG PG -> Ru5P 2 32 Ru5P <-> R5P 4 5 Ru5P <-> Xu5P R5P + Xu5P <-> S7P + GAP 6 GAP + S7P <-> E4P + F6P 6 E4P + Xu5P <-> GAP + F6P ED pathway 3 6

16 Metabolites 24, 4 S6 Table S5-2. Measured and estimated MID of FAAs_Glu+Asp+Ala+Phe. FAA_Ala-57 M M M M FAA_Ala-85 M M M FAA_Asp-57 M M M M M FAA_Asp-85 M.8. M M M FAA_Asp-59 M.7. M M M FAA_Asp32 M M M FAA_Glu-57 M.4.27 M M M M M FAA_Glu-85 M.28.4 M M M M FAA_Glu-59 M.29.4 M M M M

17 Metabolites 24, 4 S7 Table S5-2. Cont. FAA_Phe-85 M.2.9 M M M M M M M M FAA_Phe32 M M M

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