1. NREL is a national laboratory of the U.S. Department of Energy, Office of Energy Efficiency and Renewable Energy, operated by the Alliance for Sustainable Energy, LLC. Metabolic Network Plasticity in Ethylene-producing Cyanobacteria Algae Biomass Summit Jianping Yu October 2, 2014

2. 2 Metabolic network plasticity vs rigidity A rigid or “hard-wired” metabolic network has limited flux A flexible metabolic network has expandable flux A B C E D 1 2 3 4 5 7 6 8 9 10 F A B C E D 1 2 3 4 5 7 6 8 9 F

3. 3 Cyanobacteria can produce ethylene CO 2 in air Ethylene Measured by GC Ethylene- forming enzyme Ungerer et al 2012, Energy and Environmental Science

4. 4 Ethylene production in Synechocystis

5. 5 Up to 10% of fixed carbons go to Ethylene Growth rates are the same for all these strains!

6. 6 Kinetic 13 C-Metabolic flux analysis M0 M1 M2 M3 m/z Measured Simulated A B C E D Isotopic substrate Cell culture Flux-dependent metabolite isotope patterns Metabolic modeling Fitting Feeding MS measurement over time Comparison Best-fit fluxes Simulation 1 2 3 4 5 7 6 8 9 10

7. 7 Strains: Synechocystis OD 730 ~1.5 Medium: liquid BG11 20ml Light Intensity: 80 µE Culture conditions: shaking at 200rpm at 30 o C in 5% CO 2 growth chamber Cell Harvest: filtering with 0.8µM nylon membrane Quenching: -80 o C methanol Extraction: 50% cold methanol Post processing: evaporated with speed vacuum and re-dissolve in 200 µl 50% cold methanol; the supernatant is ready for LC-MS analysis Kinetic labeling

8. 8 Metabolic flux analysis using 13 C labeling CO 2 Acetyl CoA Citrate Isocitrate Malate Fumarate Succinyl semialdehyde 2-oxoglutarate Fructose-6p Sedoheptulose-7p Pentose-5p Glucose Glucose-6p Fructose- bp Glyceraldehyde-3p (Dihydroxyacetone-p) Phosphoglycerate Ribulose-bp Erythrose-4p Sedoheptulose-bp CO 2 Succinate Oxaloacetate phosphoenolpyruvate pyruvate CO 2 Glutamate Aspartate CO 2 Time (min) Relative Fraction

9. 9 TCA cycle is bifurcated in WT Citrate Isocitrate 2-oxoglutarate Succinate Fumarate Malate Oxaloacetate Phosphoenolpyruvate Pyruvate Acetyl CoA Ethylene Citrate Isocitrate 2-oxoglutarate Succinate Fumarate Malate Oxaloacetate Phosphoenolpyruvate Pyruvate Acetyl CoA

10. 10 Mapping TCA cycle flux using labeled glutamate NH 4 + Glutamate Glutamine 2-oxoglutarate NH 4 + GDH GOGAT GS U- 13 C- 15 N-glutamate GT Succinate Ethylene Efe Malate TCA cycle GS/GOGAT cycle

11. 11 TCA cycle topology is changed WTJU547 Succinate is derived from Glu by Efe Malate is also derived from Glu

12. 12 Central carbon metabolism in WT G6P Ru5P FBP DHAP GAP PGA RuBP E4P SBP S7P CO 2 F6P CIT ICT 2OG SUC FUM MAL OAA SSA PEP Pyr AcCoA Glycogen CO 2 X5P R5P CO 2 100 50 20 ≤5 RBC 135.2 ±17.2 RBC 135.2 ±17.2 PGI 27.1±5.5 PGI 27.1±5.5 G6PD 24.2±5.5 G6PD 24.2±5.5 PFK 65.5±29.3 PFK 65.5±29.3 PK 13.2 ±4.1 PK 13.2 ±4.1 ENO 23.4 ± 2.1 ENO 23.4 ± 2.1 GAPDH 244.0 ± 10.9 GAPDH 244.0 ± 10.9 FBA 65.5±29.3 FBA 65.5±29.3 PPE 75.5 ± 0.5 PPE 75.5 ± 0.5 PPI 35.4 ± 0.2 PPI 35.4 ± 0.2 PRK 135.2 ± 5.5 PRK 135.2 ± 5.5 TKT 75.5 ± 0.5 TKT 75.5 ± 0.5 TKT 38.4 ± 0.2 TKT 38.4 ± 0.2 TKT 37.2 ± 0.2 TKT 37.2 ± 0.2 TAL 0.0 ± 25.5 TAL 0.0 ± 25.5 TAL 0.0 ± 25.5 TAL 0.0 ± 25.5 SBA 37.2 ± 26.8 SBA 37.2 ± 26.8 PEPC 7.8 ± 0.3 PEPC 7.8 ± 0.3 ME 1.5 ± 0.3 ME 1.5 ± 0.3 PDH 11.8 ± 0.3 PDH 11.8 ± 0.3 CS 3.0 ± 0.3 CS 3.0 ± 0.3 ACO 3.0 ± 0.3 ACO 3.0 ± 0.3 ICTDH 3.0 ± 0.3 ICTDH 3.0 ± 0.3 SDH 0.0 ± 0.3 SDH 0.0 ± 0.3 FUS 1.7 ± 0.3 FUS 1.7 ± 0.3 MDH 0.2 ± 4.6 MDH 0.2 ± 4.6 SSADH -0.4 ± 0.2 SSADH -0.4 ± 0.2 SBPS 37.2 ± 26.8 SBPS 37.2 ± 26.8 TPI 102.6 ± 11.3 TPI 102.6 ± 11.3 A B FBP S7P RuBP SUC G6P PGA PEP 2OG OAA* F6P SBP 2OGDH 0.0 ± 1.5 2OGDH 0.0 ± 1.5 INCA

13. 13 Central carbon metabolism in JU547 G6P Ru5P FBP DHAP GAP PGA RuBP E4P SBP S7P CO 2 F6P CIT ICT 2OG SUC FUM MAL OAA SSA PEP Pyr AcCoA Glycogen CO 2 X5P R5P CO 2 Ethylene CO 2 F6P FBP S7P SBP RuBP SUC G6P PGAPEP2OG MAL 100 50 20 ≤5 RBC 137.6±0.6 RBC 137.6±0.6 PGI 4.1±0.7 PGI 4.1±0.7 G6PD 1.5±0.7 G6PD 1.5±0.7 PFK 50.0±1.2 PFK 50.0±1.2 PK 9.1 ±1.3 PK 9.1 ±1.3 ENO 33.2 ± 0.2 ENO 33.2 ± 0.2 GAPDH 239.4 ± 1.4 GAPDH 239.4 ± 1.4 FBA 50.0±1.2 FBA 50.0±1.2 PPE 92.2 ± 0.2 PPE 92.2 ± 0.2 PPI 44.0 ± 0.1 PPI 44.0 ± 0.1 PRK 137.6 ± 0.7 PRK 137.6 ± 0.7 TKT 92.2 ± 0.2 TKT 92.2 ± 0.2 TKT 46.6 ± 0.1 TKT 46.6 ± 0.1 TKT 45.5 ± 0.1 TKT 45.5 ± 0.1 TAL 0.6 TAL 0.6 TAL 0.6 TAL 0.6 SBA 46.1 ± 0.2 SBA 46.1 ± 0.2 PEPC 21.8 ± 0.7 PEPC 21.8 ± 0.7 ME 7.6 ± 1.3 ME 7.6 ± 1.3 PDH 17.7 ± 0.2 PDH 17.7 ± 0.2 CS 9.8 ± 0.2 CS 9.8 ± 0.2 ACO 9.8 ± 0.2 ACO 9.8 ± 0.2 ICTDH 9.8 ± 0.2 ICTDH 9.8 ± 0.2 Efe 2.5 ± 0.0 Efe 2.5 ± 0.0 SDH 2.1 ± 0.2 SDH 2.1 ± 0.2 FUS 3.6 ± 0.2 FUS 3.6 ± 0.2 MDH 7.6 ± 1.3 MDH 7.6 ± 1.3 SSADH -0.4 ± 0.2 SSADH -0.4 ± 0.2 SBPS 46.1 ± 0.2 SBPS 46.1 ± 0.2 TPI 96.2 ± 0.6 TPI 96.2 ± 0.6 A B

14. 14 Flux rates in TCA cycle

15. 15 Fluxes in amphibolic reactions and TCA cycle are increased Citrate Isocitrate 2-oxoglutarate Succinate Fumarate Malate Oxaloacetate Phosphoenolpyruvate Pyruvate Acetyl CoA Ethylene JU547 Citrate Isocitrate 2-oxoglutarate Succinate Fumarate Malate Oxaloacetate Phosphoenolpyruvate Pyruvate Acetyl CoA WT

16. 16 Carbon is redistributed toward TCA metabolites G6P Ru5P FBP DHAP GAP PGA RuBP E4P SBP S7P CO 2 F6P CIT ICT 2OG SUC FUM MAL OAA SSA PEP Pyr AcCoA Glycogen CO 2 X5P R5P CO 2 Ethylene CO 2 100 50 20 ≤5 RBC 137.6±0.6 RBC 137.6±0.6 PGI 4.1±0.7 PGI 4.1±0.7 G6PD 1.5±0.7 G6PD 1.5±0.7 PFK 50.0±1.2 PFK 50.0±1.2 PK 9.1 ±1.3 PK 9.1 ±1.3 ENO 33.2 ± 0.2 ENO 33.2 ± 0.2 GAPDH 239.4 ± 1.4 GAPDH 239.4 ± 1.4 FBA 50.0±1.2 FBA 50.0±1.2 PPE 92.2 ± 0.2 PPE 92.2 ± 0.2 PPI 44.0 ± 0.1 PPI 44.0 ± 0.1 PRK 137.6 ± 0.7 PRK 137.6 ± 0.7 TKT 92.2 ± 0.2 TKT 92.2 ± 0.2 TKT 46.6 ± 0.1 TKT 46.6 ± 0.1 TKT 45.5 ± 0.1 TKT 45.5 ± 0.1 TAL 0.6 TAL 0.6 TAL 0.6 TAL 0.6 SBA 46.1 ± 0.2 SBA 46.1 ± 0.2 PEPC 21.8 ± 0.7 PEPC 21.8 ± 0.7 ME 7.6 ± 1.3 ME 7.6 ± 1.3 PDH 17.7 ± 0.2 PDH 17.7 ± 0.2 CS 9.8 ± 0.2 CS 9.8 ± 0.2 ACO 9.8 ± 0.2 ACO 9.8 ± 0.2 ICTDH 9.8 ± 0.2 ICTDH 9.8 ± 0.2 Efe 2.5 ± 0.0 Efe 2.5 ± 0.0 SDH 2.1 ± 0.2 SDH 2.1 ± 0.2 FUS 3.6 ± 0.2 FUS 3.6 ± 0.2 MDH 7.6 ± 1.3 MDH 7.6 ± 1.3 SSADH -0.4 ± 0.2 SSADH -0.4 ± 0.2 SBPS 46.1 ± 0.2 SBPS 46.1 ± 0.2 TPI 96.2 ± 0.6 TPI 96.2 ± 0.6 Decreased pool sizes for sugar phosphates Increased pool sizes for Suc, Fum, Mal Decreased pool size for 2OG

17. 17 Ethylene production increases energy demand

18. 18 Ethylene production stimulates photosynthesis Light ReactionCarbon Metabolism

19. 19 Conclusions Metabolic network in a cyanobacterium is very flexible TCA cycle topology can change in response to an engineered ethylene pathway Amphibolic reactions and TCA cycle fluxes can be increased to accommodate this engineered pathway Carbon can be pulled from elsewhere to accommodate an engineered pathway Photosynthesis can run faster to meet the increased demand

20. 20 Metabolic network in a cyanobacterium is very flexible TCA cycle topology can change in response to an engineered ethylene pathway Amphibolic reactions and TCA cycle fluxes can be increased to accommodate this engineered pathway Carbon can be pulled from elsewhere to accommodate an engineered pathway Photosynthesis can run faster to meet the increased demand Source-sink relationship in photosynthesis Sink expansion can stimulate photosynthesis, so that loss of carbon from an engineered pathway is fully compensated without slowing down cell growth. Conclusions

21. 21 Conclusions Metabolic network in a cyanobacterium is very flexible TCA cycle topology can change in response to an engineered ethylene pathway Amphibolic reactions and TCA cycle fluxes can be increased to accommodate this engineered pathway Carbon can be pulled from elsewhere to accommodate an engineered pathway Photosynthesis can run faster to meet the increased demand Source-sink relationship in photosynthesis Sink expansion can stimulate photosynthesis, so that loss of carbon from an engineered pathway is fully compensated without slowing down cell growth. How much is the unrealized potential in photosynthesis? Can we find the trigger to unleash that potential and increase algal productivity?

22. 22 Acknowledgements Wei Xiong Justin Ungerer Pin-Ching Maness John Morgan Funding from DOE BETO DOE BER NREL