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1 Relative abundance (%) Relative abundance (%) Relative abundance (%) Relative abundance (%) 100% 80% 60% 40% F6P / G6P M+0 M+1 M+2 M+3 M+4 M+5 M+6 100% 80% 60% 40% 2PG / 3PG M+0 M+1 M+2 M+3 20% 20% 0% Time (min) 0% Time (min) 100% 80% PEP M+0 M+1 100% 80% Pyruvate M+0 M+1 M+2 M+3 60% M+2 M+3 60% 40% 40% 20% 20% 0% Time (min) 0% Time (min) Supplementary Figure S1. 13 C-incorporation in M. alcaliphilum 20Z. Measurement of 13 C incorporation into fructose-6-phosphate and glucose-6-phosphate (F6P/G6P); 2- phosphoglycerate and 3-phosphoglycerate (2PG/3PG); phosphoenolpyruvate (PEP); and pyruvate. M+n indicates the number of 13 C-labels. All experiments were performed in triplicate. Error bars represent standard deviation. 1
2 Supplementary Figure S2. l3 C-pyruvate labeling patterns in Methylomonas sp. LW13. Intracellular pyruvate was resolved by multiple reactions monitoring scan mode on mass spectrometry. Green: doubly and triply labeled 13 C-pyruvate; Red: 13 C- pyruvate labeled in position [1]; Blue 13 C-pyruvate labeled in position [3]. 2
3 Supplementary Figure S3. M. alcaliphilum 20Z metabolism. Predicted pathways for mixed acid fermentation and H 2 production. 3
4 Embden Meyerhof Parnas Primary Oxidation Supplementary Table S1. Gene expression profile in methane-grown cells of M. alcaliphilum 20Z. Locus tag Gene Description Aerobic Bioreactor (mean RPKM*) Fold change (Aerobic vs. low O 2 ) MALCv4_0514 pmoc particulate methane monooxygenase, C subunit MALCv4_0515 pmoa particulate methane monooxygenase, A subunit MALCv4_0516 pmob particulate methane monooxygenase, B subunit MALCv4_3445 mxai methanol dehydrogenase, small subunit MALCv4_3448 mxaf methanol dehydrogenase, large subunit MALCv4_2428 fae formaldehyde-activating enzyme MALCv4_1882 fdh1a tungsten-containing NAD-dependent formate dehydrogenase, alpha subunit MALCv4_2229 nqre NADH-quinone reductase subunit E MALCv4_2230 nqrd NADH-quinone reductase subunit D MALCv4_2231 nqrc NADH-quinone reductase subunit C MALCv4_2232 nqrb NADH-quinone reductase subunit B MALCv4_2233 nqra NADH-quinone reductase subunit A MALCv4_0104 pgi glucose-6-phosphate isomerase MALCv4_3302 pfp PPi-dependent phosphofructotransferase MALCv4_3947 fbaa fructose-bisphosphate aldolase MALCv4_1811 tpia triosephosphate isomerase MALCv4_3079 gap glyceraldehyde 3-phosphate dehydrogenase MALCv4_3549 pgk phosphoglycerate kinase MALCv4_1633 pgm3 2,3-bisphosphoglycerate-independent phosphoglycerate mutase MALCv4_1462 eno enolase MALCv4_3080 pyka pyruvate kinase II EDD MALCv4_1362 eda 2-dehydro-3-deoxy-phosphogluconate aldolase MALCv4_1363 edd 6-phosphogluconate dehydratase
5 H2- production Mixed Acid Fermentation /O2-limitation MALCv4_0534 ldh lactate dehydrogenase MALCv4_2852 xfp D-fructose 6-phosphate phosphoketolase MALCv4_2572 xfp D-xylulose 5-phosphate MALCv4_2342 pta phosphate acetyltransferase MALCv4_2853 acka acetate kinase MALCv4_0240 putative kinase MALCv4_2679 sdhb succinate dehydrogenase (ubiquinone), Fe-S protein MALCv4_2680 sdha succinate dehydrogenase, flavoprotein subunit MALCv4_2681 sdhd succinate dehydrogenase, hydrophobic membrane anchor protein MALCv4_2682 sdhc succinate dehydrogenase cytochrome b556 subunit MALCv4_0281 fumc fumarate hydratase class II (fumarase C) MALCv4_1122 sfca NAD-dependent malic enzyme MALCv4_3220 mdh malate dehydrogenase MALCv4_0310 acna aconitate hydratase (aconitase) MALCv4_1360 glta citrate synthase MALCv4_3024 glta citrate synthase MALCv4_3025 acnb aconitate hydratase MALCv4_3844 icd NADP-dependent isocitrate dehydrogenase MALCv4_0453 phba 3-hydroxyacyl-CoA dehydrogenase NAD-binding MALCv4_0454 phbb 3-ketoacyl-CoA thiolase MALCv4_2316 bht bacteriohemerythrin MALCv4_1307 hoxf NAD-reducing hydrogenase hoxs subunit alpha MALCv4_1306 hoxg NAD-reducing hydrogenase, subunit G, iron-sulfur binding MALCv4_1305 hoxy NAD-reducing hydrogenase hoxs subunit delta MALCv4_1304 hoxh NAD-reducing hydrogenase hoxs subunit beta *RPKM, reads per kilobase of coding sequence per million reads mapped. 5
6 Supplementary Table S2. Effect of various metabolites on the activity of PK from M. alcaliphilum 20Z. Effectors Concentration, mm Relative activity, %* Control (no effector) Ribose-1-phosphate 5 92 Ribose-5-phosphate Ribulose-5-phosphate Glucose-1-phosphate 5 67 Glucose-6-phosphate Fructose-1,6-bisphosphate Fructose -6-phosphate Fructose -1-phosphate Erythrose-4-phosphate Malate 1 82 Citrate Serine phosphoglycerate 2 75 ATP PPi 2 14 Pi 6 11 AMP * All experiments were performed in triplicates. The standard deviation of the relative activities is 5%. 6
7 Supplementary Table S3. Intracellular pool of key metabolites in M. alcaliphilum 20Z. Metabolite Relative abundance (%) Ribulose-5-phosphate / Ribose-5-phosphate 1.20%±0.28% Fructose-1, 6-bisphosphate 1.60%±0.42% Fructose-6-phosphate 3.39%±0.31% Glucose-6-phosphate 2.59%±0.18% Glyceraldehyde-3-phosphate / Dihydroxyacetone 2.60%±0.54% 6-Phosphogluconic acid 0.21%± 0.04% 2-dehydro-3-deoxy-phosphogluconate 0.004%±0.003% Phosphoglycerate 5.65%±1.12% Phosphoenolpyruvate 4.28%±1.26% Pyruvate 6.43%±1.82% Acetyl-CoA 0.41%±0.12% Succinate 1.08%±0.33% Malate 2.55%±0.62% Fumarate 0.27%±0.07% Citrate 1.28%±0.32% Alanine 4.08%±1.34% Glycerate 0.64%±0.11% Glycine 7.00%±1.40% Serine 1.40%±0.27% Aspartate 16.04%±2.71% Glutamate 26.37%±4.12% Glutamine 10.93%±2.13% 7
8 Supplementary Table S4. Accumulation of extracellular metabolites (mm) from 13 C- methane in low O 2 closed vial incubations. Initial concentration After 12 h incubation with 13 C-methane 12 C-Formate 0.88± ± C-Formate 0.01± ± C-Succinate ± ± C-Succinate ND ND 12 C-Acetate 0.026± ± C-Acetate ND 0.01± C-Lactate ± C-Lactate ND ND ND, not detected 8
9 Mixed Acid Fermentation /O2- limitation / H2- production Embden Meyerhof Parnas Supplementary Table S5. Core functional enzymes shared among Type I gammaproteobacterial methanotrophs. EDD Gene Description 20Z 1 5G 2 BG8 3 A45 4 AML 5 LW14 6 IMV 7 MC09 8 SV96 9 eda 2-dehydro-3-deoxy-phosphogluconate aldolase X X X X X X X X X gnd 6-phosphogluconate dehydrogenase, decarboxylating X X X X X X X X X edd 6-phosphogluconate dehydratase X X X X X X X X X pgi glucose-6-phosphate isomerase X X X X X X X X X PPi-dependent phosphofructotransferase (PPidependent pfp phosphofructokinase) X X X X X X X X X pfk ATP-dependent phosphofructokinase X fbaa fructose-bisphosphatealdolase X X X X X X X X X tpia triosephosphate isomerase X X X X X X X X X gap glyceraldehyde 3-phosphate dehydrogenase X X X X X X X X X pgk phosphoglycerate kinase X X X X X X X X X pgm phosphoglyceratemutase X X X X X X X X X eno enolase X X X X X X X X X pyka pyruvate kinase II X X X X X X X X X ldh lactate/malate dehydrogenase X X X X X X phba 3-hydroxyacyl-CoA dehydrogenase NAD-binding X X X X X X X phbb 3-ketoacyl-CoA thiolase X X X X X X X X pta phosphate acetyltransferase X X X acka acetate kinase X X X X X X X X X xfp D-fructose 6-phosphate phosphoketolase X X X X X X D-xylulose 5-phosphate/D-fructose 6-phosphate xfp phosphoketolase X X X X X X X X X hoxf NAD-reducing hydrogenase hoxs subunit alpha X X X X X X X X X NADH:ubiquinoneoxidoreductase, subunit G, ironsulfur hoxg binding X X X X X X X X X hoxy NAD-reducing hydrogenase hoxs subunit delta X X X X X X X X X 9
10 hoxh NAD-reducing hydrogenase hoxs subunit beta X X X X X X X X X sdhb succinate dehydrogenase (ubiquinone), Fe-S protein X X X X X X X X X sdha succinate dehydrogenase, flavoprotein subunit X X X X X X X X X succinate dehydrogenase, hydrophobic membrane anchor protein X X X X X X X X X sdhc succinate dehydrogenase cytochrome b556 subunit X X X X X X X X X fumc fumarate hydratase class II (fumarase C) X X X X X X X X sfca NAD(P)-dependent malic enzyme X X X X X X mdh malate dehydrogenase X X X X X X X X X bacteriohemerythrin X X X X X X X X X 1. Methylomicrobium alcaliphilum 20Z; 2. Methylomicrobium buryatense 5G; 3. Methylomicrobium album BG8; 4. Methylobacter marinus A45; 5. Methylosarcina fibrata AML C10; 6. Methylosarcina lacus LW14; 7. Methylobacter luteus IMV B 3098T; 8. Methylomonas methanica MC09; 9. Methylobacter tundripaludum SV96; 10. O 2 limitation 10
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