Supplementary Information SUPPLEMENTARY … · SUPPLEMENTARY INFORMATION 2 | RESEARCH Supplementary Information 3 Fig. S2 Figure S2: Carbon Rearrangement Networks Used in Various

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SUPPLEMENTARY INFORMATIONdoi:10.1038/nature12575

Supplementary Information

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Fig. S1

Figure S1. Detailed Structure of Non-Oxidative Glycolysis (NOG). a) The phosphoketolase can either react directly with F6P or X5P. In the latter case, F6P input is first converted to X5P via transketolase. b) An example of carbon rearrangement networks involving SBP. The carbon color scheme illustrates carbon rearrangement. Hydroxyl groups are not shown. The red arrows indicate irreversible reactions. Metabolite abbreviations: D-sedoheptulose 7-phosphate (S7P), D-sedoheptulose 1,7-bisphosphate (SBP), D-fructose 6-phosphate (F6P), fructose 1,6-bisphosphate (FBP), D-xylulose 5-phosphate (X5P), D-ribose 5-phosphate (R5P), D-ribulose 5-phosphate (Ru5P), D-erythrose 4-phosphate (E4P), D-glyceraldehyde 3-phosphate (G3P), dihydroxyacetone phosphate (DHAP), acetyl-phosphate (AcP). Enzyme abbreviations are 1a:F6P phosphoketolase (Fpk), 1b: X5P phosphoketolase (Xpk), 2: transaldolase (Tal),3:Transketolase (Tkt),4:ribose-5-phosphate isomerase (Rpi),5:ribulose-5-phosphate epimerase (Rpe),6:Triose phosphate isomerase (Tpi),7:Fructose 1,6 bisphosphate aldolase (Fba),8: Fructose 1,6 bisphosphatase (Fbp), 9: Sedoheptulose-1,7-bisphosphate aldolase (Sba), 10:Sedoheptulose-bisphosphatase (Sbp).

AcP E4P

F6P

E4P X5P

F6P G3P

AcP G3P

Phosphoketolase system

1a1b

3

a.

or

DHAP

X5P E4P

F6P G3P

SBP

E4P X5P

G3P F6PS7P

R5P X5PRu5PX5P

3

3

3

10

9

5

E4P

4

6

b. Carbon rearrangement involving SBP

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RESEARCHSupplementary Information

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Fig. S2

Figure S2: Carbon Rearrangement Networks Used in Various Pathways. The simplified carbon rearrangement diagram show the overall carbon flow. Only the carbon numbers are shown. Isomerases and phosphatases are excluded, FA is fructose-1,6-bisphosphate aldolase, SA is sedoheptulose-1,7-bisphosphate aldolase TK is transketolase, TA is transaldolase. These carbon rearrangement schemes contain the well-known transketolase, transaldolase, and aldolase reactions that catalyze two and three carbon transfers. The highly active isomerases (triose phosphate isomerase, ribose-5-phospahte isomerase, and ribulose-phosphate-3-epimerase) quickly interconvert similar intermediates to allow carbon rearrangement to proceed. Kinase and phosphatases add or remove phosphates where needed. a) Carbon rearrangement used in the RuMP pathway, b) carbon rearrangement used in the PPP pathway, c) carbon rearrangement in the CBB pathway, and d) FBP-dependent carbon rearrangement in NOG to convert three E4P to two F6P. These examples illustrate that carbon rearrangements are commonly used in metabolism.


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Fig. S3

Figure S3. NOG with Pentose and Triose Sugar Phosphates. NOG can take any sugar as input molecules, as long as it can be converted to sugar phosphates that are present in the carbon rearrangement network. The pathways using R5P (A) and G3P (B) are shown. These pathways use F/Xpk dual activity, though similar pathways can be drawn using Fpk only or Xpk only. Carbon rearrangement can convert any sugar phosphate (triose to sedoheptulose) to stoichiometric amounts of F6P. Here the conversion of a ribose and triose are illustrated. Abbreviations and enzyme numbers are defined in Figure 1 legend.



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Fig. S4

Figure S4: SDS-PAGE of HIS-tagged NOG proteins. SDS-PAGE denaturing gels of purified Rpe, Rpi, Tkt, Tal, F/Xpk, Ack, and Fbp. Sizes corresponded within theoretical values. These proteins were design with N-terminal polyhistidine tags and crude extracts were purified using a one-step affinity chromatography procedure.

Fig. S5

Figure S5: Expression of pIB4 in JCL118. Expression of F/Xpk and Fbp from JCL118 with pIB4. Lanes 1: Low Range Ladder, 2: Un-induced Crude Extract, 3: Induced Crude Extract, 4 and 5 are duplicates of 2 and 3. Lanes 6 through 9 are HIS-tag purified elutions of Lanes 2 through 5.


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Fig. S6




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Figure S6: In Vitro Enzyme Assay Schemes. A series of enzyme assays were performed to confirm the activity of purified NOG enzymes. For some enzymes (Ack, F/Xpk, and Fbp), both a colorimetric and a UV based assays were performed. Specific activities of all enzymes are listed in the Supplementary Table S1. In all enzyme-coupled assays, the key enzyme of interest was limiting by while the rest were added in excess. Glk (baker’s yeast), Zwf (baker’s yeast), Pfk (from Bacillus stearothermophilus), Tpi (rabbit muscle), Gpd (rabbit muscle), and Pgi (baker’s yeast) were purchased from Sigma-Alrich (St. Louis, MO).



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Table S1: Calculation of Specific Activities of HIS-tag Purified Enzymes. One unit of enzyme activity (U) is defined as conversion of 1 micro mole of substrate per minute.

Assay Other Enzymes Added

Substrate Protein (mg) Units (µmole/min)

Specific Activity (µmole/min/mg protein)

Fbp – UV Pgi, Zwf 2 mM F6P 5.0*10-3 0.020 4.1 Xpk – UV Rpi, Rpe, Ack, Glk,

Zwf 10 mM R5P 2.5*10-3 0.00885 3.5

Xpk – Color Rpi, Rpe 10 mM R5P 2.85*10-2 0.1437 2.5 Fpk – UV Ack, Glk, Zwf 5 mM F6P 7.5*10-3 0.0053 0.71 Tkt – UV Rpe, Rpi, Tpi, Gpd 5 mM R5P 9.2*10-4 0.013 14.0 Tal – UV Rpe, Rpi, Tkt, Pgi,

Zwf 2 mM R5P 1.2*10-3 0.022 18

Ack – UV Glk, Zwf 5 mM AcP 8.5*10-5 0.065 764 Ack – Color Glk 5 mM AcP 1.3*10-4 0.27 1030



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Figure S7



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Figure S7 Various glucose-degrading (glycolytic) pathways. A) The Embden–Meyerhof–Parnas (EMP). In the most common glycolytic pathway, glucose is oxidized to two pyruvates which can then form two lactates. B) The Entner–Doudoroff (ED) pathway overlaps somewhat with the lower EMP pathway but differs in the initial utilization of glucose. C) The Phosphoketolase (PK) Pathway. The PK pathway produces a mixture of ethanol, lactate, and CO2. It utilizes a X5P-specific phosphoketolase unlike the Bifid Shunt and only produces 1 net ATP. D) The Bifid Shunt is specific to Bifidobacteria which contain a dual F6P/X5P phosphoketolase. This pathway produces a mixture of 1.5 acetate and lactate with a relatively high net of 2.5 ATP/glucose. Both the PK and Bifid Shunt are oxidative and non-cyclical. E) The Wood-Ljungdahl pathway (W-L) is used by some acetogens to produce a third AcCoA from wasted CO2 released by the oxidative decarboxylation of pyruvate. This complex pathway is highly-oxygen sensitive but can produce the highest net 4 ATP/glucose F) NOG contains well-established enzymes found in three distinct pathways: the pentose phosphate pathway (PPP), gluconeogenesis, and the phosphoketolase pathway1,2The NOG employs fewer enzymes which are oxygen-tolerant and can produce three acetates from glucose with a net of 2 ATP/glucose.


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Table S2. Overall reactions and Properties of Various Glycolytic Pathways

Name Products from Glucose Net ATP Net Redox O2 Sensitive? # Enzymes EMP 2 lactate 2 0 No 11

ED 2 lactate 1 0 No 11 PKP lactate + ethanol + CO2 1 0 No 15 B-S 1.5 acetate + lactate 2.5 0 No 14

EMP-WL 3 acetate 4 0 Yes >20

NOG* 3 acetate 2 0 No 11 *Theoretical conversion.

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Table S3: Summary of Properties of NOG enzymes. One unit of enzyme activity (U) is defined as conversion of 1 micro mole of substrate per minute.

Name # Abbrev. EC# Source

Specific Activity (U/mg) Km Ref

F6P-Phosphoketolase 1a HIS-Fpk 4.1.2.22 B. adolescentis 0.5 N/A This work X5P-Phosphoketoalse 1b HIS-Xpk 4.1.2.9 B. adolescentis 3.5 N/A This work F6P-Phosphoketolase 1a Fpk 4.1.2.22 B. breve 14 9.6 3 X5P-Phosphoketoalse 1b Xpk 4.1.2.9 B. breve 29 N/A 3

Transaldolase B 2 Tal

2.2.1.2 E. coli 60 E4P-0.09 4 2.2.1.2 E. coli 60 F6P-1.2 4

HIS-Tal 2.2.1.2 E. coli 18 N/A This work

Transketolase A 3 Tkt

2.2.1.1 E. coli 50-110 G3P-2.1 5 2.2.1.1 E. coli

E4P-0.09 5

2.2.1.1 E. coli

R5P-1.4 5 2.2.1.1 E. coli

X5P-0.16 5

2.2.1.1 E. coli

F6P-1.1 5 2.2.1.1 E. coli

S7P-4.0 5

HIS-tkt 2.2.1.1 E. coli 14 N/A This work

Ribose-5-phosphate isomerase 4

Rpi 5.3.1.6 E. coli N/A R5P-3.1 6 5.3.1.6 E. coli 135 N/A 7 5.3.1.6 Yeast 2429 R5P-1.6 8

HIS-Rpi 5.3.1.6 E. coli >1000 N/A This work

Ribulose-3-phosphate epimerase 5

Rpe 5.1.3.1 E. coli N/A Ru5P-1.6 9 5.1.3.1 Rice 16000 N/A 10 5.1.3.1 Spinach 17000 Ru5P-0.22 11

HIS-Rpe 5.1.3.1 E. coli >1000 N/A This work

Triose Phosphate Isomerase 6 Tpi

5.3.1.1 O. cuniculus - liver 6400 G3P-0.42 12 5.3.1.1 O. cuniculus - liver

DHAP-0.75 12

5.3.1.1 O. cuniculus - muscle 7800 N/A 13

Fructose 1,6 bisphosphate Aldolase 7 Fba

4.1.2.13 O. cuniculus - muscle 14 N/A 14 4.1.2.13 O. cuniculus - muscle 11 N/A 15 4.1.2.13 O. cuniculus - liver 12 N/A 16 4.1.2.13 O. cuniculus - muscle 16 N/A 17 4.1.2.13 O. cuniculus - muscle N/A FBP-0.0013 18

Fructose 1,6 Bisphosphatase 8

Fbp 3.1.3.11 E. coli 24.2 FBP-0.015 19 3.1.3.11 E. coli 35 FBP-0.0017

HIS-Fbp 3.1.3.11 E. coli 4.1 N/A This work



RESEARCH Supplementary Information

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Table S4. Properties of Wood-Ljungdahl and EMP Enzymes. One unit of enzyme activity (U) is defined as conversion of 1 micro mole of substrate per minute.

Name Abbrev. Specific Activity (U/mg)

O2 Sensitive Ref

Formate dehydrogenase (NADPH) Fdh 1050* Very 20 Formyl-THF-Synthetase Fts 780 N/A 21

230 N/A 22 Methenyl-THF Cyclohydrolase Mtc 310 N/A 23

Methylene-THF Dehydrogenase (NADPH) Mtd 200 N/A 23 Methylene-THF Reductase Mtr 139 Very 24

Methyltransferase Mtf 150 N/A 25 CO Dehydrogenase/Acetyl-CoA Synthase Complex Cod 0.75* Very 26

0.82* Very 27 Glyceraldehyde-3-Phosphate Dehydrogenase Gap 205 N/A 28

Phosphoglycerate kinase Pyk 800 N/A 28 Phosphoglycerate mutase Gpm 2000 N/A 28

Enolase Eno 160 N/A 28 Pyruvate Kinase Pyk 280 N/A 28

Pyruvate Dehydrogenase Complex Pdh 1791 N/A 29 Phosphotransacetylase Pta >1000 N/A

*Assayed in the reverse direction

Figure S8. Overlapping enzymes in the two aceotogenic pathways: NOG and EMP+ WL .



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Table S5. Kinetic parameters of various F6P and X5P Phosphoketolases

Organism Km Specific Activity (U/mg) Ref

Bifidobacterium adolescentis N/A X5P-3.5 This work Bifidobacterium adolescentis N/A F6P-0.7 This work

Bifidobacterium longum BB 536 F6P-26 N/A 30 Bifidobacterium dentium F6P-23 N/A 30

Bifidobacterium animalis. lactis F6P-11.5 N/A 30 Bifidobacterium globosum F6P-12.5 N/A 30

Bifidobacterium breve st. 203 X5P-N/A X5P-29.0 3 F6P-9.7 F6P-14.0 3

Bifidobacterium globosum RU 230 F6P-1.4 F6P-24 31 Bifidobacterium dentium B 764 F6P-39 F6P-30 31

Lactobacillus paraplantarum F6P-5.1 F6P-147.3 32 Lactobacillus pentosus N/A X5P-4.5 33

Lactobacillus plantarum X5P-3.6 X5P-4.1 34 F6P-24 F6P-1.8 34

Bifidobacterium animalis. lactis X5P-45 X5P-27 35 F6P-10 F6P-5.2 35

Acetobacter xylinum F6P-2.5 F6P-0.267 36




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Table S6: Comparison of acetyl-CoA synthesis using CBB+NOG and CBB+EMP pathways. The CBB+NOG pathway requires 2 CO2 turnover by Rubisco and 6 ATP, while the CBB+EMP pathway requires 3 CO2 turnover and 7 ATP.


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Table S7. Primers used to Clone NOG enzymes and Create pIB4.

N-Terminal HIS-Tagged Template Primers

pQE9-HIS-Fxpk B. adolescentis

206

207

TTTTATTTGATGCCTCTAGATCACTCGTTATCGCCAGCGG

AAAACCTGTATTTTCAGGGAATGACGAGTCCTGTTATTGGCACC

pQE9-HIS-Tkt E. coli (JCL16)

235

236

AAAACCTGTATTTTCAGGGAATGTCCTCACGTAAAGAGCTTGC

TTTTATTTGATGCCTCTAGATTACAGCAGTTCTTTTGCTTTCGC

pQE9-HIS-Tal E. coli (JCL16)

233

234

AAAACCTGTATTTTCAGGGAATGACGGACAAATTGACCTCCCTT

TTTTATTTGATGCCTCTAGATTACAGCAGATCGCCGATCATTTTTTC

pQE9-HIS-Rpe E. coli (JCL16)

202

204

AAAACCTGTATTTTCAGGGAATGAAACAGTATTTGATTGCCCCCTC

TTTTATTTGATGCCTCTAGATTATTCATGACTTACCTTTGCCAGTTC

pQE9-HIS-Rpi E. coli (JCL16)

199

201

AAAACCTGTATTTTCAGGGAATGACGCAGGATGAATTGAAAAAAGC

TTTTATTTGATGCCTCTAGATCATTTCACAATGGTTTTGACACCG

pQE9-HIS-Ack E. coli (JCL16)

229

230

AAAACCTGTATTTTCAGGGAATGTCGAGTAAGTTAGTACTGGTTC

TTTTATTTGATGCCTCTAGATCAGGCAGTCAGGCGGC

pQE9-HIS-Fbp E. coli (JCL16)

211

213

AAAACCTGTATTTTCAGGGAATGAAAACGTTAGGTGAATTTATTGTCG

TTTTATTTGATGCCTCTAGATTACGCGTCCGGGAACTCAC

Vector Backbone pQE9 (Qiagen)

94

005F

TCTAGAGGCATCAAATAAAACGAAAGGC

TCCCTGAAAATACAGGTTTT

pIB4:

Vector Backbone pZE12-luc

316

005F

CTGGCGATAACGAGTGATCTAGAGGCATCAAATAAAACGAAAGGC

TCCCTGAAAATACAGGTTTT

Fbp pQE9-HIS-Fbp

211

314

AAAACCTGTATTTTCAGGGAATGAAAACGTTAGGTGAATTTATTGTCG

TCTCATAGTTAATTTCTCCTCTTTAATTTACGCGTCCGGGAACTCAC

Fpk pQE9-HIS-Fxpk

315

206

CGTAAATTAAAGAGGAGAAATTAACTATGAGAGGATCGCATCACCATCAC

TTTTATTTGATGCCTCTAGATCACTCGTTATCGCCAGCGG



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Table S8. Strains and Plasmids Used

Strains

JCL16 BW25113/F’ [traD36, proAB+, lacIq, ZΔM15 (TetR)]

37

JCL166 JCL16 , but ΔadhE, ΔldhA, ΔfrdBC 37

JCL166/pIB4 JCL166 with pIB4 This work

JCL118 JCL16 , but ΔadhE, ΔldhA, ΔfrdBC, ΔpflB This work

JCL118/pIB4 JCL118 with pIB4 This work

Plasmids

pZE12-luc ColE1 ori; AmpR;PLlacO1::luc(PP) 38

pIB4 ColE1 ori; AmpR;PLlacO1::fbp(EC)-fxpk(BA) This work

pQE9 ColE1 ori; AmpR; PT5lacO:: Qiagen (Chatsworth, CA)

pQE9-HIS-F/Xpk From pQE9, PT5lacO::fxpk(BA) This work

pQE9-HIS-Ack From pQE9, PT5lacO::ackA(EC) This work

pQE9-HIS-Tkt From pQE9, PT5lacO::tktA(EC) This work

pQE9-HIS-Tal From pQE9, PT5lacO::talB(EC) This work

pQE9-HIS-Fbp From pQE9, PT5lacO::fbp(EC) This work

pQE9-HIS-Rpe From pQE9, PT5lacO::rpe(EC) This work

pQE9-HIS-Rpi From pQE9, PT5lacO::rpiA(EC) This work EC denotes Escherichia coli. PP denotes Photinus pyralis. BA denotes Bifidobacterium adolescentis.



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Supplementary Information SUPPLEMENTARY … · SUPPLEMENTARY INFORMATION 2 | RESEARCH Supplementary Information 3 Fig. S2 Figure S2: Carbon Rearrangement Networks Used in Various