GE nome -scale M etabolic RE construction and analysis of Cyanobacteria:

1. GEnome-scale Metabolic REconstructionand analysis of Cyanobacteria: A systems biology approach towards full exploitation of their biotechnological applications Juan Nogales Enrique Departament of EnvironmentalBiologyCentro de Investigaciones Biológicas (CSIC), Madrid, Spain jnogales@cib.csic.es

2. What is a GEnome-scale Metabolic REconstruction? “A GEMRE is a stequiometric representation of the metabolic capabilities of a given organism at genome-scale, which can be further translated to a mathematical format allowing the computation of its phenotype from its genotype” BiochemicalRepresentation StequiometricRepresentation gene HEX1 transcript HEX1 PGI protein PFK reaction MathematicalRepresentation TPI GADP PGK ENO Reed JL et al, Nature Reviews Genetics: 2006

3. Phylogeny of COnstraintsBased Reconstruction and Analysis Methods

4. Applications of GEMs McCloskey et al, MSB: 2013

5. Addressing the 1,2 propanediol overproduction in Synechocystis mgsA gldA kan dkgB Eric Knight IcelandUniversity DkgB MgsA GldA Synechocystissp. PCC6803 DHAP Methylglyoxal Acetol R-1,2-PD • Multiple problems found during the engineering and fermentation processes • Lowgeneticstability of thesyntheticpathway • Thefermentationprocesslostefficiency at longterm • Verylowyield (≈ µg/L) gldA mgsA mgsA gldA dkgB dkgB Synechocystissp. PCC6803 Genome-ScaleModelReconstruction and Analysis

6. Genome-ScaleMetabolicReconstruction of Synechocytissp. PCC 6803

7. ModelValidation 54.5 mmol.gDW-1.h-1 13.14 - 17.14 µE.m2.s-1 Cell mass = 0.5 pg Cell diameter = 1.75 µm Photosynthesis efficiency = 4.6-6 % 15 µE.m2.s-1

8. Carbon Flux DistributionValidation Photoautotrophic τ=0.96 Mixotrophicτ=0.92 Heterotrophic τ=0.89

9. The photosynthetic metabolism... so simple? E. coli NADPH ATP Pi + ADP H+ NADP + H+ FNR H+ Fdox Fdrd ATPase PSI PSII CYT BF PQ PC CytC PQH2 H2O O2 + H+

10. Extracellular OM Periplasm H2O O2 O2 H2O PC CytC CM PQ PQH2 CYT BF CYO CO2 + H2O HCO3 + H+ Cyd BD NDH-1 NDH-2 SDH NADP NADPH H+ NADPH NADP PQH2 PQH2 H+ Pi + ADP NDH-14 Succ Fum ATP H+ NADH NAD H+ H+ H2ase Cytoplasm O2 H2 NADH NAD MEHLER NADPH H2O NDH-2 CO2 + H2O HCO3 + H+ ATP Pi + ADP H+ Succ NADP + H+ NADPH NADP FNR H+ H+ Fum FQR NDH-13 H+ H+ Fdox Fdrd PQ PQ ATPase ATPase Cyd BD TM SDH CYO NDH-1 PSI PSII CYT BF PQ H2O PC CytC PQH2 O2 H2O O2 + H+ Thylakoid H2O O2

11. The photosynthetic metabolism... so simple? so complex !!!.

12. iJN678 as computationaltoolforstudyingthephotoautotrophicmetabolism

13. iJN678 as computationaltoolforstudyingthephotoautotrophicmetabolism • Defining a key photosynthetic parameter: • Optimalphotosynthesisoperates at ATP/NADPH ≈ 1.5 • LEF provides a ATP/NADPH = 1.28

14. Autotrophicgrowth as a function of Ci and light availability * 9 AEF pathways - 5 CEF pathways - 2 PCEF pathways - 2 NADPH consuming pathways * 2 Metabolic pathways - Photorespiration - NO3 reduction 10-3 NH4 Flux (mmol.gDW-1.h-1) Light input (mmol.gDW-1.h-1)

15. Quantification and classification of alternativephotosyntheticpathways. Nogales J., et al, PNAS: 2012

16. Photosyntheticrobustness at work Photorespiratory 2-phosphoglycolate metabolism and photoreduction of O2 cooperate in high-light acclimation of Synechocystis sp. strain PCC 6803. Hackenberg et al. Planta. 2009 Sep;230(4):625-37 Double KO Mehler KO Wild type PHOTOR KO Double KO Wild type Mehler KO PHOTOR KO

17. Defining additional emergent properties of photosynthetic networks Essential Synthetic Lethal Reduced metabolic robustness

18. Impact of the photosynthetic systems properties on biotechnology mgsA gldA kan dkgB Eric Knight IcelandUniversity DkgB MgsA GldA High Photosynthetic Robustness DHAP Methylglyoxal Acetol R-1,2-PD Low Metabolic Robustness • Multiple problems found during the engineering and fermentation processes • Lowgeneticstability of thesyntheticpathway • Thefermentationprocesslostefficiency at longterm • Verylowyield (≈ µg/L)

19. Computational design of growth-coupled overproducer strains Wild type Mutant A Mutant B Mutant C Nogales et al., Bioengineered 4:3, 1–6; May/June 2013 • Multiple problems found during the engineering and fermentation processes • Lowgeneticstability of thesyntheticpathway • Thefermentationprocesslostefficiency at longterm • Verylowyield (≈ µg/L)

20. Computational design of growth-coupled overproducer strains: Autotrophic conditions

21. Computational design of growth-coupled overproducer strains: Heterotrophic conditions

22. Computational design of growth-coupled overproducer strains: Mixotrophic conditions

23. Summary • Multiple problems found during the engineering and fermentation processes • Lowgeneticstability of thesyntheticpathway • Thefermentationprocesslostefficiency at longterm • Verylowyield (≈ µg/L) Computational evaluation of Synechococcus sp. PCC 7002 metabolism for chemical production. Vu et al, Biotechnol J. May 2013, 8(5):619-30

24. GEnome-scale Metabolic REconstructionand analysis of Cyanobacteria: A systems biology approach towards full exploitation of their biotechnological applications Juan Nogales Enrique Departament of EnvironmentalBiologyCentro de Investigaciones Biológicas (CSIC), Madrid, Spain jnogales@cib.csic.es

25. Thanks Prof. Bernhard O. Palsson Dr. InesThiele Dr. SteinGudmundsson