Supplementary File. Table S1. Metabolic model.

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

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 6 8 6 4 2 (a) 2 C glucose 3 C glucose 2 3 4 5 6 7 OD6 Glucose Ethanol 35 3 25 2 5 5-5

Concentration (mm) Metabolites 24, 4 S3 5 (b) Figure S. Cont. 2 C glucose 3 C glucose 5 2 3 4 5 6 7 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-85.8.8 Alanine.6.4.6.4.2.2 5 5 2 25 5 5 2 25 Asp-57 Asp-85 Asp-59 Asp f32 Aspartate.8.6.4.8.6.4.8.6.4.8.6.4.2.2.2.2 5 5 2 25 5 5 2 25 5 5 2 25 5 5 2 25 Glu-57 Glu-85 Glu-59 Glutamate.8.6.4.8.6.4.8.6.4.2.2.2 5 5 2 25 5 5 2 25 5 5 2 25 Phe-85 Phe f32.8.8 Phenylalanine.6.4.2 5 5 2 25.6.4.2 5 5 2 25

Metabolites 24, 4 S4 Figure S2. Cont. Gly-57 Gly-85 Glycine.8.6.4.2 5 5 2 25.8.6.4.2 5 5 2 25 Leu-85 Leucine.8.6.4.2 5 5 2 25 Val-57 Val-85 Valine.8.6.4.2 5 5 2 25.8.6.4.2 5 5 2 25 Threonine Thr-57.8.6.4.2 5 5 2 25 Thr-85.8.6.4.2 5 5 2 25 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 74 75 77 F6P -> FBP 83 85 86 FBP <-> DHAP + GAP 83 85 86 DHAP <-> GAP 82 84 85 GAP <-> PGA 72 75 76 PGA <-> PEP 64 66 67 PEP -> Pyr 22 29 36 Pyr -> AcCoA 22 28 36 AcCoA + Oxa -> Cit 75 77 78 Cit ->αkg 44 5 58 αkg -> Suc 38 45 52 Suc <-> Fum 69 7 7 Fum <-> Mal 69 7 7 Mal <-> Oxa 48 53 57 Glyoxylate shunt 9 25 32 PEP + CO 2 <-> Oxa 29 34 4

Metabolites 24, 4 S5 Table S-. Cont. Reaction Lower Best fit Upper Mal -> Pyr 36 43 5 G6P -> 6PG 22 24 25 6PG -> Ru5P 8 2 24 Ru5P <-> R5P 2 Ru5P <-> Xu5P 8 2 R5P + Xu5P <-> S7P + GAP 5 6 7 GAP + S7P <-> E4P +F6P 5 6 7 E4P + Xu5P <-> GAP + F6P 3 4 5 ED pathway 3 6 Table S-2. Measured and estimated MID of PAAs_fullset. PAA_Ala-57 M.23.25 M+.228.29 M+2.6.6 M+3.398.46 PAA_Ala-85 M.248.249 M+.269.27 M+2.483.48 PAA_Asp-57 M.83.83 M+.8.78 M+2.245.245 M+3.276.277 M+4.25.26 PAA_Asp-85 M.2.2 M+.25.248 M+2.38.32 M+3.3.3 PAA_Asp-59 M.9.2 M+.247.248 M+2.32.32 M+3.33.3 PAA_Asp32 M.267.27 M+.29.287 M+2.443.443 PAA_Glu-57 M.33.3 M+.95.94 M+2.25.25 M+3.283.283 M+4.237.238 M+5.48.49

Metabolites 24, 4 S6 Table S-2. Cont. PAA_Glu-85 M.48.45 M+.38.39 M+2.294.299 M+3.3.298 M+4.22.29 PAA_Glu-59 M.49.45 M+.39.39 M+2.295.299 M+3.298.298 M+4.29.29 PAA_Phe-57 M.23.2 M+.45.43 M+2.82.8 M+3.2.8 M+4.39.36 M+5.7.72 M+6.43.47 M+7.23.25 M+8.86.87 M+9.67.74 PAA_Phe-85 M.26.2 M+.49.48 M+2.7.7 M+3.39.34 M+4.69.7 M+5.42.43 M+6.62.65 M+7.8.7 M+8.78.84 PAA_Phe32 M.424.42 M+.29.28 M+2.447.45 PAA_Gly-57 M.49.425 M+.3.23 M+2.45.452 PAA_Gly-85 M.48.48 M+.52.59 PAA_Ile-85 M.34.3 M+.92.94 M+2.99.25 M+3.283.283 M+4.239.238 M+5.53.49

Metabolites 24, 4 S7 Table S-2. Cont. PAA_Ile-59 M.34.3 M+.93.94 M+2.22.25 M+3.285.283 M+4.239.238 M+5.47.49 PAA_Leu-85 M.24.2 M+.84.85 M+2.86.9 M+3.295.296 M+4.248.25 M+5.62.58 PAA_Ser-57 M.89.89 M+.278.27 M+2.55.66 M+3.378.375 PAA_Ser-85 M.26.27 M+.366.367 M+2.429.426 PAA_Ser-59 M.28.27 M+.369.367 M+2.423.426 PAA_Val-57 M.59.54 M+.7.3 M+2.24.22 M+3.25.25 M+4.82.87 M+5.88.95 PAA_Val-85 M.67.62 M+.34.35 M+2.3.33 M+3.258.26 M+4.23.23 PAA_Tyr32 M.424.42 M+.29.28 M+2.448.45 PAA_Thr-57 M.82.83 M+.8.78 M+2.244.245 M+3.278.277 M+4.27.26 PAA_Thr-85 M.2.2 M+.25.248 M+2.323.32 M+3.35.3

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 69 74 8 F6P -> FBP 76 8 86 FBP <-> DHAP + GAP 76 8 86 DHAP <-> GAP 76 8 85 GAP <-> PGA 67 72 76 PGA <-> PEP 58 63 67 PEP -> Pyr 22 28 42 Pyr -> AcCoA 6 33 45 AcCoA + Oxa -> Cit 75 78 8 Cit ->αkg 37 49 6 αkg -> Suc 3 43 53 Suc <-> Fum 68 7 75 Fum <-> Mal 68 7 75 Mal <-> Oxa 46 56 68 Glyoxylate shunt 8 29 4 PEP + CO 2 <-> Oxa 2 32 43 Mal -> Pyr 3 44 56 G6P -> 6PG 9 25 29 6PG -> Ru5P 9 8 27 Ru5P <-> R5P 7 4 Ru5P <-> Xu5P 2 8 4 R5P + Xu5P <-> S7P + GAP 2 5 8 GAP+ S7P <-> E4P + F6P 2 5 8 E4P + Xu5P <-> GAP + F6P 3 6 ED pathway 7 4

Metabolites 24, 4 S9 Table S2-2. Measured and estimated MID of FAAs_fullset. FAA_Ala-57 M.27.25 M+.225.2 M+2.73.73 M+3.385.4 FAA_Ala-85 M.252.254 M+.27.267 M+2.478.479 FAA_Asp-57 M.72.79 M+.82.7 M+2.252.257 M+3.284.279 M+4.2.25 FAA_Asp-85 M.8.4 M+.256.247 M+2.33.33 M+3.36.39 FAA_Asp-59 M.7.4 M+.25.247 M+2.33.33 M+3.3.39 FAA_Asp32 M.254.26 M+.299.295 M+2.447.444 FAA_Glu-57 M.4.29 M+.94.93 M+2.27.24 M+3.288.285 M+4.244.24 M+5.52.48 FAA_Glu-85 M.28.43 M+.4.4 M+2.298.297 M+3.39.33 M+4.225.27 FAA_Glu-59 M.29.43 M+.4.4 M+2.299.297 M+3.37.33 M+4.223.27 FAA_Phe-85 M.2.2 M+.47.47 M+2.3.5 M+3.39.34

Metabolites 24, 4 S Table S2-2. Cont. M+4.7.73 M+5.48.49 M+6.64.66 M+7.23.6 M+8.76.79 FAA_Phe32 M.44.48 M+.38.43 M+2.449.439 FAA_Gly-57 M.425.42 M+.54.4 M+2.42.439 FAA_Gly-85 M.489.488 M+.5.52 FAA_Leu-85 M.27.2 M+.95.87 M+2.89.93 M+3.286.296 M+4.243.247 M+5.59.57 FAA_Val-57 M.54.55 M+.8. M+2.26.23 M+3.25.249 M+4.84.9 M+5.87.92 FAA_Val-85 M.67.64 M+.4.35 M+2.39.35 M+3.238.256 M+4.236.23 FAA_Tyr32 M.422.48 M+.36.43 M+2.442.439 FAA_Thr-57 M.79.79 M+.79.7 M+2.247.257 M+3.278.279 M+4.26.25 FAA_Thr-85 M.8.4 M+.253.247 M+2.324.33 M+3.35.39

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 55 89 99 F6P -> FBP 67 9 94 FBP <-> DHAP + GAP 67 9 94 DHAP <-> GAP 67 9 93 GAP <-> PGA 57 8 84 PGA <-> PEP 49 72 76 PEP -> Pyr 52 83 233 Pyr -> AcCoA 99 37 87 AcCoA + Oxa -> Cit 6 8 84 Cit ->αkg 6 49 84 αkg -> Suc 43 77 Suc <-> Fum 63 74 77 Fum <-> Mal 63 74 77 Mal <-> Oxa 23 5 54 Glyoxylate shunt 3 77 PEP + CO 2 <-> Oxa 6 4 5 Mal -> Pyr 96 G6P -> 6PG 43 6PG -> Ru5P 43 Ru5P <-> R5P 4 8 2 Ru5P <-> Xu5P 4 2 25 R5P + Xu5P <-> S7P + GAP 2 3 GAP + S7P <-> E4P + F6P 2 3 E4P + Xu5P <-> GAP + F6P 3 28 ED pathway 26

Metabolites 24, 4 S2 Table S3-2. Measured and estimated MID of FAAs_Glu+Asp+Ala. FAA_Asp-57 M.72.74 M+.82.74 M+2.252.253 M+3.284.285 M+4.2.23 FAA_Asp-85 M.8.8 M+.256.25 M+2.33.333 M+3.36.38 FAA_Asp-59 M.7.8 M+.25.25 M+2.33.333 M+3.3.38 FAA_Asp32 M.254.259 M+.299.294 M+2.447.447 FAA_Glu-57 M.4.26 M+.94.9 M+2.27.22 M+3.288.288 M+4.244.246 M+5.52.48 FAA_Glu-85 M.28.39 M+.4.36 M+2.298.295 M+3.39.3 M+4.225.22 FAA_Glu-59 M.29.39 M+.4.36 M+2.299.295 M+3.37.3 M+4.223.22

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 59 73 95 F6P -> FBP 72 85 93 FBP <-> DHAP + GAP 72 85 93 DHAP <-> GAP 7 84 92 GAP <-> PGA 62 75 83 PGA <-> PEP 53 67 74 PEP -> Pyr 8 88 222 Pyr -> AcCoA 3 43 79 AcCoA + Oxa -> Cit 7 75 83 Cit ->αkg 7 34 68 αkg -> Suc 28 62 Suc <-> Fum 64 69 77 Fum <-> Mal 64 69 77 Mal <-> Oxa 34 46 Glyoxylate shunt 4 72 PEP + CO2 <-> Oxa 54 24 52 Mal -> Pyr 77 G6P -> 6PG 4 26 4 6PG -> Ru5P 25 4 Ru5P <-> R5P 4 3 8 Ru5P <-> Xu5P 4 2 23 R5P + Xu5P <-> S7P + GAP 7 2 GAP + S7P <-> E4P + F6P 7 2 E4P + Xu5P <-> GAP + F6P 3 5 ED pathway 2

Metabolites 24, 4 S4 Table S4-2. Measured and estimated MID of FAAs_Glu+Asp+Ala. FAA_Asp-57 M.72.74 M+.82.74 M+2.252.253 M+3.284.285 M+4.2.23 FAA_Asp-85 M.8.8 M+.256.25 M+2.33.333 M+3.36.38 FAA_Asp-59 M.7.8 M+.25.25 M+2.33.333 M+3.3.38 FAA_Asp32 M.254.259 M+.299.294 M+2.447.447 FAA_Glu-57 M.4.26 M+.94.9 M+2.27.22 M+3.288.288 M+4.244.246 M+5.52.48 FAA_Glu-85 M.28.39 M+.4.36 M+2.298.295 M+3.39.3 M+4.225.22 FAA_Glu-59 M.29.39 M+.4.36 M+2.299.295 M+3.37.3 M+4.223.22

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 67 75 88 F6P -> FBP 76 84 9 FBP <-> DHAP + GAP 76 84 9 DHAP <-> GAP 75 83 89 GAP <-> PGA 65 74 8 PGA <-> PEP 57 65 72 PEP -> Pyr 23 4 65 Pyr -> AcCoA 25 4 62 AcCoA + Oxa -> Cit 73 77 83 Cit ->αkg 2 4 58 αkg -> Suc 4 34 52 Suc <-> Fum 67 7 77 Fum <-> Mal 67 7 77 Mal <-> Oxa 49 66 88 Glyoxylate shunt 22 36 54 PEP + CO 2 <-> Oxa 2 4 Mal -> Pyr 2 4 59 G6P -> 6PG 24 32 6PG -> Ru5P 2 32 Ru5P <-> R5P 4 5 Ru5P <-> Xu5P 4 9 7 R5P + Xu5P <-> S7P + GAP 6 GAP + S7P <-> E4P + F6P 6 E4P + Xu5P <-> GAP + F6P 3 4 8 ED pathway 3 6

Metabolites 24, 4 S6 Table S5-2. Measured and estimated MID of FAAs_Glu+Asp+Ala+Phe. FAA_Ala-57 M.27.22 M+.225.25 M+2.73.7 M+3.385.4 FAA_Ala-85 M.252.244 M+.27.277 M+2.478.479 FAA_Asp-57 M.72.77 M+.82.7 M+2.252.259 M+3.284.28 M+4.2.23 FAA_Asp-85 M.8. M+.256.249 M+2.33.33 M+3.36.38 FAA_Asp-59 M.7. M+.25.249 M+2.33.33 M+3.3.38 FAA_Asp32 M.254.26 M+.299.295 M+2.447.444 FAA_Glu-57 M.4.27 M+.94.92 M+2.27.23 M+3.288.286 M+4.244.244 M+5.52.48 FAA_Glu-85 M.28.4 M+.4.38 M+2.298.295 M+3.39.38 M+4.225.28 FAA_Glu-59 M.29.4 M+.4.38 M+2.299.295 M+3.37.38 M+4.223.28

Metabolites 24, 4 S7 Table S5-2. Cont. FAA_Phe-85 M.2.9 M+.47.47 M+2.3.4 M+3.39.36 M+4.7.72 M+5.48.49 M+6.64.65 M+7.23.9 M+8.76.79 FAA_Phe32 M.44.42 M+.38.4 M+2.449.447