Lecture #21. Exploring Network Func7ons

Transcription

1 Lecture #1 Exploring Network Func7ons

2 Outline Parsing te overall network Many simultaneous metabolic func7ons Core E. coli Model Op7mal ATP produc7on Op7mal NAD(P)H produc7on Op7mal syntesis of biosynte7c precursors Func7onal tes7ng sets

3 Wat to explore PARSING NETWORK FUNCTIONS

4 Four overall func7ons of cellular metabolism J. Teoret. Biol.165: (1993)

5 Core E. coli Metabolism

6 Consumption of ATP is represented troug a demand reaction: ATP + H O v ATPM! ADP + P i + H + (1) Te objective function for linear optimization is: Z = v ATPM () OPTIMAL ATP PRODUCTION

7 Op7mal aerobic ATP produc7on: core E. coli Flux map GLCpts (1) EX_glc(e) (-1) glc-d[e] EX_mal-L(e) (0) MALt_ (0) FUMt_ (0) fum[e] [e] mal-l[e] EX_fum(e) (0) [e] [e] THD (0) Te Objecve ATPS4r (13.5) PGK () ATPM (17.5) FRUpts (0) fru[e] EX_fru(e) (0) pep pyr p p o pyr g6p 6pgl G6PDHr (0) PGL (0) PGI (1) pep f6p FBP (0) PFK (1) TKT (0) o fdp FBA (1) dap g3p p co p 6pgc ru5p-d GND (0) RPI (0) RPE (0) xu5p-d r5p TKT1 (0) s7p g3p TALA (0) p NADTRHD () p o amp o ADK1 (0) ATPM (17.5) 0.5 o o q8 q8 ATPS4r (13.5) 4 [e] Ot (6) o[e] EX_o(e) (-6) CYTBD (1) [e] EX_(e) (0) PYK (1) SUCOAS () ATPM (17.5) Node Maps PFK (1) TPI (1) e4p f6p [e] GAPD () NADH16 (10) 13dpg FRD7 (0) 4 3 [e] PGK (-) q8 q8 EX_succ(e) (0) 3pg fum SUCDi () o SUCCt3 (0) succ [e] succ[e] SUCOAS (-) PGM (-) SUCCt_ (0) FUM () pg [e] mal-l AKGtr (0) EX_akg(e) (0) ENO () suc co ME1 (0) co akg[e] o pep o p MALS (0) [e] ac co MDH () AKGDH () PPC (0) ME (0) o co p amp p o co p glx EX_glu-L(e) (0) PPCK (0) n4 glu-l glu-l[e] akg PPS (0) p GLUDy (0) GLUtr (0) oaa co PYK (1) [e] p o co n4 ICL (0) n4 o GLUN (0) EX_pyr(e) (0) acald GLNS (0) PDH () ICDHyr () CS () acon-c icit p ac o pyr[e] pyr ACALD (0) PYRtr (0) PFL (0) cit [e] PTAr (0) for ACONTa () ACONTb () p GLNabc (0) EX_gln-L(e) (0) o GLUSy (0) gln-l LDH_D (0) actp eto ALCDx (0) gln-l[e] ACKr (0) o lac-d ac o co n4 ACtr (0) ATPS4r (3 x 13.5) CS () GAPD () MDH () PFK (1) ATPase leung protons in CYTBD ( x 1) NADH16 (4 x 10) PYK (1) Pumng protons out D-LACt (0) [e] lac-d[e] EX_lac-D(e) (0) FORt (0) FORti (0) o[e] [e] [e] [e] ACALDt (0) ETOHtr (0) HOt (-6) COt (-6) co[e] NH4t (0) n4[e] PItr (0) for[e] [e] acald[e] eto[e] ac[e] [e] EX_eto(e) (0) EX_for(e) (0) EX_ac(e) (0) EX_co(e) (6) EX_acald(e) (0) EX_o(e) (6) EX_n4(e) (0) EX_(e) (0) CYTBD ( x 1) NADH16 (3 x 10) [e] ATPS4r (4 x 13.5)

8 Te op7mal solu7on: te flux values, te reduced costs, and te sadow prices # Reaction Flux Reduced # Reaction Flux Reduced # Reaction Flux Reduced # Metabolite Sadow # Metabolite Sadow Name Value Cost Name Value Cost Name Value Cost Name Price Name Price 1 ACALD EX_lac_D(e) ME dpg[c] 9 37 gln-l[e] 11.5 ACALDt EX_mal_L(e) ME 0 0 pg[c] 8 38 glu-l[c] ACKr EX_n4(e) NADH pg[c] 8 39 glu-l[e] 1 4 ACONTa 0 36 EX_o(e) NADTRHD 0 4 6pgc[c] glx[c] ACONTb 0 37 EX_(e) NH4t pgl[c] o[c] 0 6 ACtr EX_pyr(e) Ot ac[c] o[e] 0 7 ADK EX_succ(e) PDH 0 7 ac[e] [c] AKGDH 0 40 FBA PFK acald[c] [e] 0 9 AKGtr FBP PFL acald[e] icit[c] ALCDx FORt PGI ac[c] lac-d[c] 8 11 ATPM FORti PGK acon-c[c] lac-d[e] ATPS4r FRD PGL actp[c] mal-l[c] Biomass FRUpts PGM [c] mal-l[e] COt FUM 0 78 PItr akg[c] [c] CS 0 47 FUMt_ PPC akg[e] [c] 0 16 CYTBD G6PDHr PPCK amp[c] - 5 p[c] D_LACt GAPD 0 81 PPS [c] 0 53 p[c] 0 18 ENO 0 50 GLCpts PTAr cit[c] n4[c] 0 19 ETOHtr GLNS PYK co[c] 0 55 n4[e] 0 0 EX_ac(e) GLNabc PYRtr co[e] 0 56 o[c] 0 1 EX_acald(e) GLUDy RPE [c] 0 57 o[e] 0 EX_akg(e) GLUN RPI 0 0 dap[c] oaa[c] 7 3 EX_co(e) GLUSy SUCCt_ e4p[c] pep[c] 8 4 EX_eto(e) GLUtr SUCCt eto[c] [c] EX_for(e) GND SUCDi 0 5 eto[e] [e] 0 6 EX_fru(e) HOt SUCOAS f6p[c] pyr[c] EX_fum(e) ICDHyr 0 91 TALA fdp[c] 0 63 pyr[e] EX_glc(e) ICL THD for[c] 0 64 q8[c] 0 9 EX_gln_L(e) LDH_D TKT for[e] 0 65 q8[c] EX_glu_L(e) MALS TKT fru[e] r5p[c] EX_(e) MALt_ TPI fum[c] ru5p-d[c] EX_o(e) MDH 0 3 fum[e] s7p[c] g3p[c] succ[c] g6p[c] succ[e] glc-d[e] suc[c] gln-l[c] xu5p-d[c]

9 Op7mal aerobic ATP produc7on: some caracteris7cs 13.5 ATP made by ATP syntase, 4 net made by substrate level posporyla7on for a total of 17.5 P/O ra7o is 5/4, 5 protons pumped out for eac electron pair, 4 protons pumped in for eac ATP made Proton transmembrane balancing determines ATP yield ATPS4r (13.5) PGK () PYK (1) SUCOAS () ATPM (17.5) PS4r (3 x 13.5) CS () GAPD () MDH () ATPM (17.5) PFK (1) CYTBD ( x 1) NADH16 (4 x 10) PYK (1) 4 protons needed to make 1 ATP, so [c] sadow price is PFK (1) CYTBD ( x 1) ATPS4r (4 x 13.5) ADH16 (3 x 10) [e]

10 Op7mal aerobic ATP produc7on: Sadow prices for te intermediates correspond to te ATP yield on tem Oter sadow prices demonstrate importance of protons: succ[c] SP = 8.75, succ[e] = 8.5, two protons pumped in wit every succinate Water is made some caracteris7cs Cells can produce about pw/µ 3 Te yield of 38 ATP/glucose seen in text books assumes a P/O ra7o of 3 and no systemic network constraints see analysis in yeast, PNAS 100: (003)

11 Te op7mal solu7on: some observa7ons # Reaction Flux Reduced # Reaction Flux Reduced # Reaction Flux Reduced # Metabolite Sadow # Metabolite Sadow Name Value Cost Name Value Cost Name Value Cost Name Price Name Price 1 ACALD EX_lac_D(e) ME dpg[c] 9 37 gln-l[e] 11.5 ACALDt EX_mal_L(e) ME 0 0 pg[c] 8 38 glu-l[c] ACKr EX_n4(e) NADH pg[c] 8 39 glu-l[e] 1 4 ACONTa 0 36 EX_o(e) NADTRHD 0 4 6pgc[c] glx[c] ACONTb 0 37 EX_(e) NH4t pgl[c] o[c] 0 6 ACtr EX_pyr(e) Ot ac[c] o[e] 0 7 ADK EX_succ(e) PDH 0 7 ac[e] [c] AKGDH 0 40 FBA PFK acald[c] [e] 0 9 AKGtr FBP PFL acald[e] icit[c] ALCDx FORt PGI ac[c] lac-d[c] 8 11 ATPM FORti PGK acon-c[c] lac-d[e] ATPS4r FRD PGL actp[c] mal-l[c] Biomass FRUpts PGM [c] mal-l[e] COt FUM 0 78 PItr akg[c] [c] CS 0 47 FUMt_ PPC akg[e] [c] 0 16 CYTBD G6PDHr PPCK amp[c] - 5 p[c] D_LACt GAPD 0 81 PPS [c] 0 53 p[c] 0 18 ENO 0 50 GLCpts PTAr cit[c] n4[c] 0 19 ETOHtr GLNS PYK co[c] 0 55 n4[e] 0 0 EX_ac(e) GLNabc PYRtr co[e] 0 56 o[c] 0 1 EX_acald(e) GLUDy RPE [c] 0 57 o[e] 0 EX_akg(e) GLUN RPI 0 0 dap[c] oaa[c] 7 3 EX_co(e) GLUSy SUCCt_ e4p[c] pep[c] 8 4 EX_eto(e) GLUtr SUCCt eto[c] [c] EX_for(e) GND SUCDi 0 5 eto[e] [e] 0 6 EX_fru(e) HOt SUCOAS f6p[c] pyr[c] EX_fum(e) ICDHyr 0 91 TALA fdp[c] 0 63 pyr[e] EX_glc(e) ICL THD for[c] 0 64 q8[c] 0 9 EX_gln_L(e) LDH_D TKT for[e] 0 65 q8[c] EX_glu_L(e) MALS TKT fru[e] r5p[c] EX_(e) MALt_ TPI fum[c] ru5p-d[c] EX_o(e) MDH 0 3 fum[e] s7p[c] g3p[c] succ[c] g6p[c] succ[e] glc-d[e] suc[c] gln-l[c] xu5p-d[c]

12 Systemic P/O in Yeast Results Te in silico model can be used to assess network properties suc as te P O ratioandenergymaintenancecostsandtocompute wole-cell functions. Te efficiency of aerobic resration is measured by te P O ratio.experimentalstudiesofisolated mitocondria ave sown tat S. cerevisiae lacks site I proton translocation (8). Consequently, estimation of te maximum teoretical or mecanistic yield of te ETS alone gives a P O ratio of 1.5 for oxidation of NADH in S. cerevisiae grown on glucose (8). However, based on experimental measurements, it as been determined tat te net in vivo P Oratiois 0.95 (8). Tis difference is generally attributed to te use of te mitocondrial transmembrane proton gradient needed to drive metabolite excange (suc as te proton-coupled translocation of pyruvate) across te inner mitocondrial membrane. In te reconstructed network, wic contains no proton leakage, 1.5 molecules of ATP are generated via te ETS. As complete oxidation of glucose leads to donation of 1 electron pairs (10 NADH and FADH )toteelectrontransportcain,tein silico P O ratiois1.04foroxidationofnadhandfadh during growt on glucose, i.e., , agreeing well wit te measured value witout including any proton leakage. Te network-based computation systemically accounts for all te steps required to import and export compounds from te mitocondria, computing a net overall P O ratio. Cells require energy for bot growt- and non-growtassociated activities (9). Te energy requirement for te formation of biomass as been measured experimentally for S. cerevisiae, andreportedvaluesrangefrom6.5to71.4mmolof ATP gdw (9, 30). A network-based calculation procedure of te growt-associated energy requirement as been developed (31), and wen applied to te reconstructed S. cerevisiae network, a value of 69. mmol of ATP gdw was computed (see Metods), wic falls in te range of experimentally determined values. Energy required for precursor metabolite formation and macromolecule polymerization can be calculated from te biosyntetic composition of te cell. Te model-based ATP requirement is entirely network-dependent and was derived from te in silico calculations. Te reconstructed metabolic network of S. cerevisiae can be PNAS 100: (003)

13 Op7mal anaerobic ATP produc7on: core E. coli ACKr (1) ATPM (.75) Flux map Te Objec7ve PGK () ATPS4r (0.5) GLCpts (1) EX_glc(e) (-1) glc-d[e] EX_mal-L(e) (0) EX_fum(e) (0) mal-l[e] MALt_ (0) FUMt_ (0) fum[e] [e] [e] [e] THD (0) PYK (1) PFK (1) A o amp pep pyr p p o p ATPS4r (-0.5) p co o 4 pyr [e] g6p 6pgl 6pgc ru5p-d ADK1 (0) ATPM (.75) FRUpts (0) G6PDHr (0) PGL (0) GND (0) p NADTRHD (0) p fru[e] PGI (1) RPI (0) EX_fru(e) (0) RPE (0) pep f6p xu5p-d r5p Ot (0) 0.5 TKT1 (0) FBP (0) o o[e] PFK (1) o EX_o(e) (0) TKT (0) o CYTBD (0) g3p fdp s7p [e] FBA (1) TALA (0) q8 q8 EX_(e) (3) dap g3p e4p f6p TPI (1) [e] GAPD () NADH16 (0) 13dpg FRD7 (0) 4 3 [e] PGK (-) q8 q8 EX_succ(e) (0) 3pg fum SUCDi (0) o SUCCt3 (0) succ [e] succ[e] SUCOAS (0) PGM (-) SUCCt_ (0) FUM (0) pg [e] mal-l AKGtr (0) EX_akg(e) (0) ENO () suc co co ME1 (0) akg[e] o pep o p MALS (0) [e] ac co MDH (0) AKGDH (0) PPC (0) ME (0) o co p amp p o co p glx EX_glu-L(e) (0) PPCK (0) n4 glu-l glu-l[e] akg PPS (0) p GLUDy (0) GLUtr (0) oaa co PYK (1) [e] p o co n4 n4 o ICL (0) GLUN (0) EX_pyr(e) (0) acald PDH (0) ICDHyr (0) GLNS (0) CS (0) acon-c icit p ac o pyr[e] pyr ACALD (-1) PYRtr (0) PFL () cit [e] PTAr (1) for ACONTa (0) ACONTb (0) p GLNabc (0) EX_gln-L(e) (0) o GLUSy (0) gln-l LDH_D (0) actp eto gln-l[e] ALCDx (-1) ACKr (-1) o lac-d ac o co n4 ACtr (-1) B ATPM (.75) GAPD () PFK (1) Node Maps ACALD (1) ACtr (1) ALCDx (1) ATPS4r (3 x 0.5) ETOHtr (1) PYK (1) D-LACt (0) FORt (0) FORti () [e] lac-d[e] EX_lac-D(e) (0) o[e] [e] [e] ACALDt (0) ETOHtr (-1) [e] HOt (1) COt (0) co[e] NH4t (0) n4[e] PItr (0) for[e] [e] acald[e] eto[e] ac[e] [e] EX_eto(e) (1) EX_for(e) () EX_ac(e) (1) EX_co(e) (0) EX_acald(e) (0) EX_o(e) (-1) EX_n4(e) (0) EX_(e) (0) ACtr (1) ATPS4r (4 x 0.5) [e] EX_(e) (3) ETOHtr (1)

14 Op7mal anaerobic ATP produc7on: some caracteris7cs.75 net ATP produced, all by substrate level posporyla7on net ATP produced in glycolysis, 1 by acetate kinase 4.75 net protons are produced 4 are secreted by etanol and acetate transporters 0.75 protons must be pumped out by ATP syntase, consuming 0.5 ATP

15 ATP produc7on from oter substrates: summary of te network s energy genera7on ability Metabolite Abbrev. Maximum Maximum Aerobic Yield Anaerobic Yield Glycolysis Glucose glc Fructose fru Pyruvate pyr Lactate lac-d Fermentation Etanol eto Acetaldeyde acald Acetate ac Amino acids Glutamine gln-l Glutamate glu-l TCA a-ketoglutarate akg Succinate succ Fumarate fum Malate mal -L

16 fru[e] pyr[e] [e] fru[e] pyr[e] [e] pyr o amp pep o pyr amp o pep o dap dap pep o [e] fdp pyr lac-d[e] pep o [e] fdp pyr lac-d[e] glc-d[e] g6p f6p g3p 3pg pyr 13dpg pg pep lac-d co [e] glc-d[e] g6p f6p g3p pyr 13dpg 3pg pg pep lac-d p co [e] co co o p o for p for 6pgl for[e] p 6pgl o co ac[e] ac o co for[e] ac[e] ac actp ac 6pgc actp ac [e] 6pgc p xu5p-d [e] s7p e4p p co co xu5p-d s7p e4p co co p ru5p-d p acald ru5p-d co p acald[e] acald co p acald[e] r5p g3p f6p [e] p r5p g3p f6p mal-l o eto eto[e] [e] p mal-l o eto mal-l[e] o [e] eto[e] oaa fum[e] fum mal-l[e] o [e] oaa [e] fum[e] fum [e] [e] [e] cit [e] [e] cit q8 q8 ac glx q8 o acon-c o ac glx q8 o acon-c o p o succ icit o[e] p o succ icit o[e] [e] p p p co [e] suc p co akg co co[e] suc p akg co co[e] p n4 co p n4 amp p p co p n4 n4[e] amp p p n4 n4[e] p n4 o p o glu-l gln-l n4 o o o glu-l gln-l o o o o o n4 o o o n4 o q8 q8 q8 q8 o[e] [e] [e] [e] [e] [e] [e] [e] [e] akg[e] glu-l[e] gln-l[e] o[e] [e] [e] [e] [e] [e] [e] [e] [e] glu-l[e] succ[e] akg[e] gln-l[e] succ[e] fru[e] pyr[e] fru[e] pyr[e] [e] [e] pyr amp o pep o pyr amp o pep o dap dap pep o [e] pep o fdp pyr lac-d[e] [e] fdp pyr lac-d[e] glc-d[e] g6p f6p g3p pyr 13dpg 3pg pg pep lac-d glc-d[e] g6p f6p g3p pyr 13dpg 3pg pg pep lac-d co [e] co [e] p co p co o o for for p 6pgl p 6pgl for[e] o co o co for[e] ac[e] ac ac[e] ac actp ac actp ac 6pgc 6pgc [e] [e] p xu5p-d p s7p e4p xu5p-d s7p e4p co co co co p ru5p-d p ru5p-d acald acald co p acald[e] co p acald[e] r5p g3p f6p r5p g3p f6p [e] p [e] mal-l p o eto eto[e] mal-l o eto eto[e] mal-l[e] o [e] oaa mal-l[e] o [e] fum[e] fum oaa fum[e] fum [e] [e] [e] [e] [e] [e] cit cit q8 q8 ac glx q8 o acon-c o ac glx q8 o acon-c o p o succ icit o[e] p o succ icit o[e] [e] p [e] p p co p co suc akg co suc co[e] akg co co[e] p p n4 n4 co co p amp p p amp p p p n4 n4[e] n4 n4[e] p p n4 o n4 o o glu-l gln-l o glu-l gln-l o o o o o o n4 o o o n4 o q8 q8 q8 q8 o[e] [e] [e] [e] [e] [e] [e] [e] [e] akg[e] glu-l[e] gln-l[e] o[e] [e] [e] [e] [e] [e] [e] [e] [e] akg[e] glu-l[e] gln-l[e] succ[e] succ[e] Lactate Malate EX_glc(e) (0) EX_mal-L(e) (0) EX_fum(e) (0) EX_glc(e) (0) EX_mal-L(e) (-1) EX_fum(e) (0) GLCpts (0) MALt_ (0) FUMt_ (0) THD (0) GLCpts (0) MALt_ (1) FUMt_ (0) THD (0) FRUpts (0) EX_fru(e) (0) FBP (0) PGI (0) PFK (0) G6PDHr (0) PGL (0) GND (0) RPE (0) TKT1 (0) TKT (0) FBA (0) TALA (0) RPI (0) NADTRHD (1) ADK1 (0) ATPM (7.75) 0.5 ATPS4r (6.75) 4 Ot (3) EX_o(e) (-3) CYTBD (6) FRUpts (0) EX_fru(e) (0) FBP (0) PGI (0) PFK (0) FBA (0) G6PDHr (0) PGL (0) TKT (0) GND (0) RPE (0) TKT1 (0) TALA (0) RPI (0) NADTRHD (1) ADK1 (0) ATPM (7.75) 0.5 ATPS4r (6.75) 4 Ot (3) EX_o(e) (-3) CYTBD (6) Aerobic EX_pyr(e) (0) PYRtr (0) PPS (0) TPI (0) GAPD (0) PGK (0) PGM (0) FUM (1) ENO (0) LDH_D (1) PYK (0) PPCK (0) PFL (0) PPC (0) PDH (1) PTAr (0) ACKr (0) ACtr (0) ME1 (0) ME (0) ACALD (0) ALCDx (0) MDH (1) CS (1) MALS (0) FRD7 (0) 4 SUCDi (1) ACONTa (1) ACONTb (1) ICL (0) ICDHyr (1) SUCCt3 (0) SUCOAS (-1) AKGDH (1) GLUSy (0) GLUDy (0) GLUN (0) GLNS (0) GLNabc (0) SUCCt_ (0) GLUtr (0) EX_(e) (-1) NADH16 (5) 3 EX_succ(e) (0) AKGtr (0) EX_akg(e) (0) EX_glu-L(e) (0) EX_gln-L(e) (0) EX_pyr(e) (0) PYRtr (0) PPS (0) TPI (0) GAPD (0) PGK (0) PGM (0) FUM (1) ENO (0) LDH_D (0) PYK (0) PPCK (0) PFL (0) PPC (0) PDH (1) PTAr (0) ACKr (0) ACtr (0) ME1 (1) ME (0) ACALD (0) ALCDx (0) MDH (1) CS (1) MALS (0) FRD7 (0) 4 SUCDi (1) ACONTa (1) ACONTb (1) ICL (0) ICDHyr (1) SUCCt3 (0) SUCOAS (-1) AKGDH (1) GLUSy (0) GLUDy (0) GLUN (0) GLNS (0) GLNabc (0) SUCCt_ (0) GLUtr (0) EX_(e) (-) NADH16 (5) 3 EX_succ(e) (0) AKGtr (0) EX_akg(e) (0) EX_glu-L(e) (0) EX_gln-L(e) (0) D-LACt (1) EX_lac-D(e) (-1) FORt (0) FORti (0) EX_for(e) (0) EX_ac(e) (0) ACALDt (0) ETOHtr (0) EX_eto(e) (0) EX_acald(e) (0) EX_(e) (0) HOt (-3) PItr (0) COt (-3) NH4t (0) EX_o(e) (3) EX_co(e) (3) EX_n4(e) (0) D-LACt (0) EX_lac-D(e) (0) FORt (0) FORti (0) EX_for(e) (0) EX_ac(e) (0) ACALDt (0) ETOHtr (0) EX_eto(e) (0) EX_acald(e) (0) EX_(e) (0) HOt (-3) PItr (0) COt (-4) NH4t (0) EX_o(e) (3) EX_co(e) (4) EX_n4(e) (0) GLCpts (0) EX_glc(e) (0) EX_mal-L(e) (0) EX_fum(e) (0) MALt_ (0) FUMt_ (0) THD (0) GLCpts (0) EX_glc(e) (0) EX_mal-L(e) (-1) EX_fum(e) (0) MALt_ (1) FUMt_ (0) THD (0) FRUpts (0) EX_fru(e) (0) FBP (0) PGI (0) PFK (0) G6PDHr (0) PGL (0) GND (0) RPE (0) TKT1 (0) TKT (0) FBA (0) TALA (0) RPI (0) NADTRHD (0) ADK1 (0) ATPM (0.375) 0.5 ATPS4r (-0.15) 4 Ot (0) EX_o(e) (0) CYTBD (0) FRUpts (0) EX_fru(e) (0) FBP (0) PGI (0) PFK (0) G6PDHr (0) PGL (0) GND (0) RPE (0) TKT1 (0) TKT (0) FBA (0) TALA (0) RPI (0) NADTRHD (0) ADK1 (0) ATPM (0.375) 0.5 ATPS4r (-0.15) 4 Ot (0) EX_o(e) (0) CYTBD (0) Anaerobic EX_pyr(e) (0) PYRtr (0) PPS (0) TPI (0) GAPD (0) PGK (0) PGM (0) FUM (0) ENO (0) LDH_D (1) PYK (0) PPCK (0) PFL (1) PPC (0) PDH (0) PTAr (0.5) ACKr (-0.5) ACtr (-0.5) ACALD (-0.5) ME1 (0) ME (0) ALCDx (-0.5) MDH (0) CS (0) MALS (0) FRD7 (0) 4 SUCDi (0) ACONTa (0) ACONTb (0) ICL (0) ICDHyr (0) SUCCt3 (0) SUCOAS (0) AKGDH (0) GLUSy (0) GLUDy (0) GLUN (0) GLNS (0) GLNabc (0) SUCCt_ (0) GLUtr (0) EX_(e) (0.5) NADH16 (0) 3 EX_succ(e) (0) AKGtr (0) EX_akg(e) (0) EX_glu-L(e) (0) EX_gln-L(e) (0) EX_pyr(e) (0) PYRtr (0) PPS (0) TPI (0) GAPD (0) PGK (0) PGM (0) FUM (0) ENO (0) LDH_D (0) PYK (0) PPCK (0) PFL (1) PPC (0) PDH (0) PTAr (0.5) ACKr (-0.5) ACtr (-0.5) ACALD (-0.5) ME1 (1) ME (0) ALCDx (-0.5) MDH (0) CS (0) MALS (0) FRD7 (0) 4 SUCDi (0) ACONTa (0) ACONTb (0) ICL (0) ICDHyr (0) SUCCt3 (0) SUCOAS (0) AKGDH (0) GLUSy (0) GLUDy (0) GLUN (0) GLNS (0) GLNabc (0) SUCCt_ (0) GLUtr (0) EX_(e) (-0.5) NADH16 (0) 3 EX_succ(e) (0) AKGtr (0) EX_akg(e) (0) EX_glu-L(e) (0) EX_gln-L(e) (0) D-LACt (1) EX_lac-D(e) (-1) FORt (0) FORti (1) EX_for(e) (1) EX_ac(e) (0.5) ACALDt (0) ETOHtr (-0.5) EX_eto(e) (0.5) EX_acald(e) (0) EX_(e) (0) HOt (0.5) PItr (0) COt (0) NH4t (0) EX_o(e) (-0.5) EX_co(e) (0) EX_n4(e) (0) D-LACt (0) EX_lac-D(e) (0) FORt (0) FORti (1) EX_for(e) (1) EX_ac(e) (0.5) ACALDt (0) ETOHtr (-0.5) EX_eto(e) (0.5) EX_acald(e) (0) EX_(e) (0) HOt (0.5) PItr (0) COt (-1) NH4t (0) EX_o(e) (-0.5) EX_co(e) (1) EX_n4(e) (0)

17 Consumption of redox potential (NADH or NADPH) is troug a demand reaction: NAD(P)H v NAD(P)H! NAD(P) + + H + + e (1) Te objective function ten becomes: Z = v NAD(P)H () representing a demand (DM) function on redox potential. OPTIMAL REDOX PRODUCTION

18 fru[e] pyr[e] [e] pyr pep amp o o dap pep o [e] fdp pyr lac-d[e] glc-d[e] g6p f6p g3p 13dpg 3pg pg pep pyr lac-d co [e] p co o for p 6pgl for[e] o co ac ac[e] actp ac 6pgc [e] p xu5p-d s7p e4p co co p ru5p-d acald co p acald[e] g3p f6p r5p [e] p mal-l o eto eto[e] mal-l[e] o [e] oaa fum[e] fum [e] [e] [e] cit q8 ac glx q8 o acon-c o p o succ icit o[e] [e] p p co suc akg co co[e] p co n4 p p p amp n4 n4[e] p n4 o o glu-l gln-l o o o o n4 o q8 q8 o[e] [e] [e] [e] [e] [e] [e] [e] [e] akg[e] glu-l[e] gln-l[e] succ[e] Op7mal aerobic NADH produc7on EX_glc(e) (-1) EX_mal-L(e) (0) EX_fum(e) (0) GLCpts (1) MALt_ (0) FUMt_ (0) THD (0) FRUpts (0) EX_fru(e) (0) PGI (1) G6PDHr (0) PGL (0) GND (0) RPE (0) RPI (0) NADTRHD () ADK1 (0) ATPM (0) ATPS4r (-4) 4 FBP (0) PFK (1) FBA (1) TKT (0) TKT1 (0) TALA (0) 0.5 Ot (1) EX_o(e) (-1) CYTBD () A EX_pyr(e) (0) PYRtr (0) PPS (0) TPI (1) GAPD () PGK (-) PGM (-) ENO () LDH_D (0) PYK (1) PPCK (0) PFL (0) PPC (0) ME1 (0) ME (0) FRD7 (0) 4 SUCDi () AKGDH () ICL (0) PDH () ICDHyr () CS () PTAr (0) ACKr (0) ACtr (0) DM_ (10) ACALD (0) ALCDx (0) MDH () FUM () MALS (0) ACONTa () ACONTb () SUCCt3 (0) SUCOAS (-) SUCCt_ (0) GLUSy (0) GLUDy (0) GLUN (0) AKGtr (0) GLNS (0) GLNabc (0) GLUtr (0) NADH16 (0) 3 EX_(e) (0) EX_succ(e) (0) EX_akg(e) (0) EX_glu-L(e) (0) EX_gln-L(e) (0) Te Objec7ve D-LACt (0) EX_lac-D(e) (0) FORt (0) FORti (0) EX_for(e) (0) EX_ac(e) (0) ACALDt (0) ETOHtr (0) EX_eto(e) (0) EX_acald(e) (0) EX_(e) (0) HOt (4) PItr (0) COt (-6) NH4t (0) EX_o(e) (-4) EX_co(e) (6) EX_n4(e) (0) AKGDH () GAPD () PGK () ATPS4r (4) MDH () DM_ (10) PYK (1) PFK (1) NADTRHD () SUCOAS () B PDH () CS () DM_ (10) ATPS4r (3 x 4) ATPS4r (4 x 4) GAPD () MDH () CYTBD ( x ) PYK (1) CYTBD ( x ) [e] EX_(e) (0) PFK (1)

19 Interpreta7on of te op7mal solu7on for aerobic NADH produc7on NADH yield is limited by proton transmembrane balancing and stoiciometry Transmembrane gradients are - 0 mv/ 7nm=300,000 V/cm Protons produced in metabolism must be pumped out by te ATP syntase, requiring ATP Glycolysis and te TCA cycle must be used to generate ATP, but some reducing power is used to reduce q8 by SUCDi If ATP is supplied ar7ficially, te flux distribu7on canges

20 Op7mal aerobic NADH produc7on: remove ATP constraints EX_glc(e) (-1) EX_mal-L(e) (0) EX_fum(e) (0) GLCpts (1) glc-d[e] MALt_ (0) [e] mal-l[e] fum[e] FUMt_ (0) [e] [e] THD (0) Te Objec7ve FRUpts (0) fru[e] EX_fru(e) (0) pep pyr p p o pyr g6p 6pgl G6PDHr (6) PGL (6) PGI (-5) pep f6p FBP (1) PFK (0) TKT () o fdp FBA (-1) p co p 6pgc ru5p-d GND (6) RPI (-) RPE (4) xu5p-d r5p TKT1 () s7p g3p TALA () p NADTRHD (1) p o amp o ATPM (-8) ADK1 (1) 0.5 o o ATPS4r (-6) 4 [e] Ot (0) o[e] EX_o(e) (0) CYTBD (0) [e] q8 A q8 dap g3p EX_(e) (4) e4p f6p TPI (-1) [e] GAPD (0) NADH16 (0) 13dpg FRD7 (0) 4 3 [e] PGK (0) q8 q8 DM_ (1) EX_succ(e) (0) 3pg fum SUCDi (0) o SUCCt3 (0) succ [e] succ[e] SUCOAS (0) PGM (0) SUCCt_ (0) FUM (0) pg [e] mal-l EX_akg(e) (0) AKGtr (0) ENO (0) suc co ME1 (0) akg[e] o pep co p MALS (0) [e] o ac PPC (0) co MDH (0) AKGDH (0) ME (0) o amp co p p o co p glx EX_glu-L(e) (0) glu-l glu-l[e] PPCK (0) akg n4 PPS (1) p GLUDy (0) GLUtr (0) oaa co PYK (0) p [e] o n4 co n4 o ICL (0) GLUN (0) EX_pyr(e) (0) acald GLNS (0) PDH (0) ICDHyr (0) CS (0) acon-c icit p pyr[e] pyr ac o PYRtr (0) ACALD (0) PFL (0) cit [e] PTAr (0) ACONTa (0) ACONTb (0) for p GLNabc (0) EX_gln-L(e) (0) o GLUSy (0) gln-l LDH_D (0) actp eto ALCDx (0) gln-l[e] ACKr (0) o lac-d ac o co n4 ACtr (0) D-LACt (0) [e] lac-d[e] EX_lac-D(e) (0) FORt (0) FORti (0) [e] for[e] ac[e] EX_for(e) (0) o[e] [e] ACALDt (0) ETOHtr (0) [e] HOt (6) COt (-6) co[e] NH4t (0) n4[e] PItr (0) [e] acald[e] eto[e] [e] EX_eto(e) (0) EX_ac(e) (0) EX_co(e) (6) EX_acald(e) (0) EX_o(e) (-6) EX_n4(e) (0) EX_(e) (0) ADK1 (1) NADTRHD (1) DM_ (1) ATPM (8) ATPS4r (6) PPS (1) B DM_ (1) Wat you see in textbooks!! G6PDHr (6) PGL (6) ATPM (8) ATPS4r (3 x 6) ATPS4r (4 x 6) EX_(e) (4) [e] PPS ( x 1)

21 Op7mal anaerobic NADH produc7on: EX_glc(e) (-1) EX_mal-L(e) (0) EX_fum(e) (0) GLCpts (1) glc-d[e] MALt_ (0) [e] mal-l[e] fum[e] FUMt_ (0) [e] [e] THD (0) FRUpts (0) fru[e] EX_fru(e) (0) pyr pep pep pyr p p o g6p 6pgl G6PDHr (1.5) PGL (1.5) PGI (-0.5) f6p p co p 6pgc ru5p-d GND (1.5) RPE (1) RPI (-0.5) p NADTRHD (3) p o amp o ATPM (0) ADK1 (0) ATPS4r (-3) 4 [e] FBP (0) PFK (0.5) o fdp FBA (0.5) TKT (0.5) xu5p-d s7p r5p TKT1 (0.5) g3p TALA (0.5) 0.5 o o Ot (0) o[e] EX_o(e) (0) CYTBD (0) [e] q8 Te Objec7ve A dap g3p e4p f6p TPI (0.5) GAPD (1.5) 13dpg FRD7 (0) 4 q8 EX_(e) (13.5) [e] PGK (-1.5) q8 q8 DM_ (6) EX_succ(e) (0) 3pg fum SUCDi (0) SUCCt3 (0) o succ [e] succ[e] SUCOAS (0) PGM (-1.5) SUCCt_ (0) FUM (0) pg [e] mal-l EX_akg(e) (0) AKGtr (0) ENO (1.5) suc co ME1 (0) akg[e] o pep co p MALS (0) [e] o ac PPC (0) co MDH (0) AKGDH (0) ME (0) o amp co p p o co p glx EX_glu-L(e) (0) glu-l glu-l[e] PPCK (0) akg n4 PPS (0) p GLUDy (0) GLUtr (0) oaa co PYK (0.5) p [e] o n4 co n4 PDH (1.5) o ICL (0) GLUN (0) EX_pyr(e) (0) acald ICDHyr (0) GLNS (0) CS (0) acon-c icit p pyr[e] pyr ac o PYRtr (0) ACALD (0) PFL (0) cit [e] PTAr (1.5) ACONTa (0) ACONTb (0) for p GLNabc (0) EX_gln-L(e) (0) o GLUSy (0) gln-l LDH_D (0) actp eto gln-l[e] ALCDx (0) ACKr (-1.5) o lac-d ac o co n4 ACtr (-1.5) NADH16 (0) 3 [e] D-LACt (0) [e] lac-d[e] EX_lac-D(e) (0) FORt (0) FORti (0) [e] for[e] ac[e] EX_for(e) (0) o[e] [e] ACALDt (0) ETOHtr (0) [e] HOt (3) COt (-3) co[e] NH4t (0) n4[e] PItr (0) [e] acald[e] eto[e] [e] EX_eto(e) (0) EX_ac(e) (1.5) EX_co(e) (3) EX_acald(e) (0) EX_o(e) (-3) EX_n4(e) (0) EX_(e) (0) GAPD (1.5) ACKr (1.5) ATPS4r (3) NADTRHD (3) PDH (1.5) DM_ (6) PGK (1.5) PYK (0.5) PFK (0.5) B DM_ (6) G6PDHr (1.5) ACtr (1.5) ACtr (1.5) GAPD (1.5) ATPS4r (3 x 3) EX_(e) (13.5) ATPS4r (4 x 3) [e] PFK (0.5) PYK (0.5) PGL (1.5)

22 Interpreta7on of te op7mal solu7on for anaerobic NADH produc7on Can not use te TCA cycle witout oxygen, so a different strategy is used Splits flux evenly between glycolysis and pentose pospate patway Generates 3 NADPH in PPP, ten converts tem to NADH wit te transydrogenase. Also makes 1.5 NADH in glycolysis and 1.5 by pyruvate deydrogenase. Needs ATP to pump out 9 protons. PPP doesn t produce ATP, so glycolysis must also be used even toug it doesn t yield as muc NADH

23 Cofactor Produc7on Rates Cofactor Yield PPS ATP sadow Constraint price Aerobic ATP % 0 Internal proton NADH % Energy and Stoiciometry NADPH % Energy and Stoiciometry Anaerobic ATP.750 0% 0 Internal proton NADH % Energy and Stoiciometry NADPH % Energy and Stoiciometry

24 Prelude to simula7ng growt BIOSYNTHETIC PRECURSORS

25 GROWTH REQUIREMENTS: Genome- scale A Cell wall Lids Purines Nucleosides Amino acids Pyrimidines Heme versus core αkg G6P, F6P models B OAA R5P, E4P AcCoA T3P, 3PG PEP, PYR 1 biosynte7c precursors

26 Aerobic Metabolite Yield Carbon ATP sadow Constraint conversion price 3PG 100% 0 PEP 100% 0 Pyr 100% 0 OA % 0 G6P % Energy F6P % Energy R5P % Energy E4P % Energy G3P % Energy AcCoA 66.67% 0 Stoiciometry akg % 0 Stoiciometry SuccCoA % 0

27 Anaerobic Metabolite Yield Carbon ATP sadow Constraint conversion price 3PG 1 50% 0 Stoiciometry PEP 1 50% 0 Stoiciometry Pyr 1 50% 0 Stoiciometry OA % 0 Stoiciometry G6P % Energy F6P % Energy R5P % 0.19 Energy E4P % 0.64 Energy G3P % Energy AcCoA % 0 Stoiciometry akg % 0 Stoiciometry SuccCoA % 0 Stoiciometry

28 Use of tests TO DEBUG AND VALIDATE NETWORKS

29 Network valida7on: te Tiele tests Test if network is mass- and carge balanced. Gap filling. Test for stoiciometrically balanced cycles. Test if biomass precursors can be produced in standard medium. Test if biomass precursors can be produced in oter growt media. Test if model can produce known secre7on products. Ceck for blocked reac7ons. Compute single gene dele7on penotypes. Test for known incapabilites of te organism. Compare predicted pysiological proper7es wit known proper7es. Test if te model can grow fast enoug. Test if te model grows too fast.

30 Network Valida7on: te Recon 1 tests 88 dis7nct uman metabolic func7ons were defined and simulated wit FBA to assess func7onality of Recon 1 Many are simple: melatonin trp- L

31 88 recon 1 tests

32 Gap- filling wen valida7on fails: see Biotecnol & Bioeng, 15;107:403-1 (010).

33 Summary Te capabili7es and caracteris7cs of networks can be explored using LP Objec7ve func7ons can be used to represent par7cular network proper7es of interest Sadow prices/reduced costs can be useful in interpre7ng te solu7on and governing constraints ATP and NADH yields can be studied in details Ability to make biosynte7c precursors can precede te study of growt Various substrates can easily be studied Explora7on can be in te form of func7onal and valida7on tests

34 THE END

35 SOME INTERESTING LITERATURE

36 Amino acid Yield Calcula7ons: defini7on of constraints

37 Amino acid Yield Calcula7ons:

38 Systemic P/O in Yeast Results Te in silico model can be used to assess network properties suc as te P O ratioandenergymaintenancecostsandtocompute wole-cell functions. Te efficiency of aerobic resration is measured by te P O ratio.experimentalstudiesofisolated mitocondria ave sown tat S. cerevisiae lacks site I proton translocation (8). Consequently, estimation of te maximum teoretical or mecanistic yield of te ETS alone gives a P O ratio of 1.5 for oxidation of NADH in S. cerevisiae grown on glucose (8). However, based on experimental measurements, it as been determined tat te net in vivo P Oratiois 0.95 (8). Tis difference is generally attributed to te use of te mitocondrial transmembrane proton gradient needed to drive metabolite excange (suc as te proton-coupled translocation of pyruvate) across te inner mitocondrial membrane. In te reconstructed network, wic contains no proton leakage, 1.5 molecules of ATP are generated via te ETS. As complete oxidation of glucose leads to donation of 1 electron pairs (10 NADH and FADH )toteelectrontransportcain,tein silico P O ratiois1.04foroxidationofnadhandfadh during growt on glucose, i.e., , agreeing well wit te measured value witout including any proton leakage. Te network-based computation systemically accounts for all te steps required to import and export compounds from te mitocondria, computing a net overall P O ratio. Cells require energy for bot growt- and non-growtassociated activities (9). Te energy requirement for te formation of biomass as been measured experimentally for S. cerevisiae, andreportedvaluesrangefrom6.5to71.4mmolof ATP gdw (9, 30). A network-based calculation procedure of te growt-associated energy requirement as been developed (31), and wen applied to te reconstructed S. cerevisiae network, a value of 69. mmol of ATP gdw was computed (see Metods), wic falls in te range of experimentally determined values. Energy required for precursor metabolite formation and macromolecule polymerization can be calculated from te biosyntetic composition of te cell. Te model-based ATP requirement is entirely network-dependent and was derived from te in silico calculations. Te reconstructed metabolic network of S. cerevisiae can be PNAS 100: (003)

39 Yields from iaf160 Substrate Glucose Xylose Glycerol Glucose Xylose Glycerol Aerobicit Anaerobic Anaerobic Anaerobic Aerobic Aerobic Aerobic product no. yof carbons Y p/s Y p/s Y p/s Y p/s Y p/s Y p/s Etanol 49%* 49%* 49% 49% 49% 54% D- Lactate 3 95%* 95%* 13% 95% 95% 97% Glycerol 3 37% 7% 75% 74% L- Alanine 3 95%* 76% 13% 95% 93% 96% L- Serine 3 47% 35% 6% 115% 114% 116% Pyruvate 3 71% 60% 6% 100% 99% 100% Fumarate 4 54% 40% 5% 110% 108% 117% L- Malate 4 63% 46% 5% 17% 15% 135% Succinate 4 93% 81% 1% 104% 101% 111% - Oxoglutarate 5 40% 3% 3% 98% 96% 101% L- Glutamate 5 44% 36% 3% 9% 90% 97%

40 APPENDIX: Old Core E. coli model Historical case: simpler maps, easier to interpret, from

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44

45

46

47

48

49

50

51 First report of equivalent op7mal solu7ons

52 Te end