Swammerdam Institute Klaas J. Hellingwerf On the use and optimization of Synechocystis PCC6803 as a bio-solar cell factory in a bio-based economy
Acknowledgements: S.A. Angermayr, P. Savakis, Dr. A. de Almeida, O. Borirak, R.M. Schuurmans, P. van Alphen, Dr. A. Van der Woude, & Dr. V. Puthan Veetil. 31-10-2014 PHYCONET Cambridge 2
Contents: Solar biofuel production: From one generation to the next Cell factories for photo-fermentation The 4 th generation started wit ethanol Optimization of product formation with MCA The PEP bypass CO 2 as the ultimate substrate in biotechnology 31-10-2014 PHYCONET Cambridge 3
What is needed for sustainable production with solar energy? EET Antenna e - Reaction Center e - Catalytic Site 2 e - R (H +, CO2 ) P (H 2, MeOH) For any large-scale process, H 2 O is the only candidate electron donor Catalytic Site 1 (fuels, chemicals, ingredients) R (H 2 O, HA) P (O 2, A, H + ) Use the auto-regenerative capacity of living organisms A solution for solar fuel with as few conversions as possible (0.33 4 = 0.01!) 31-10-2014 PHYCONET Cambridge 4
1 st Generation: Sugar cane and ethanol: drink the best and drive the rest 2 nd Generation: produce ethanol, butanol, etc., through fermentation of (ligno)cellulosic waste 3 rd Generation: Produce biodiesel from triglycerides, extracted from plants, or from algae grown in mass culture All three approaches, however: (i) do not comply with the minimal # of conversions requirement, and (ii) create significant problems with respect to (soil) mineral balance and have (iii) a high water requirement 31-10-2014 PHYCONET Cambridge 5
Fourth generation type of process: Cyanobacterial cell factories 31-10-2014 PHYCONET Cambridge 6
As the engineering progresses: Wild type: CO 2 biomass First mutant: CO 2 biomass product After several rounds: CO 2 Definition cell factory: CO 2 partitioning > 50 % biomass product CO 2 + H 2 O C 2 H 6 O + O 2 (catalyst) 31-10-2014 PHYCONET Cambridge 7
Cyanobacterial metabolism: gluc/glycogen fatty acids/phb new cells 31-10-2014 8
Coupling to cyanobacterial metabolism: CO 2 Ethylene 31-10-2014 PHYCONET Cambridge 9
Making a cyanobacterial cell factory for ethanol from Synechocystis: 1] The proper molecular biology should be applied (transformability, polyploidic character, etc.). 2] Knowledge about promoters, RBSs, enzymes, cofactors, etc. should be available 31-10-2014 PHYCONET Cambridge 10
Ethanol productie in stram SAA012 CH 3 CH 2 OH CO 2 partitioning to ethanol: 60 en 70 % 31-10-2014 PHYCONET Cambridge 11
Analysis of ethanol production: Rate of CO 2 assimilation: sink-effect 31-10-2014 PHYCONET Cambridge 12
Production of Lactic acid: CO 2 Lactic acid 31-10-2014 PHYCONET Cambridge 13
Lactate Production 31-10-2014 14 PHYCONET Cambridge
Does product inhibition play a role? Solvent IC50 (mm) ( LogP o/w ) range (mm) Ethanol 868-0,4 0-1000 Lactate 382-0,79 0-1000 meso-bu-diol 376-0,59 0-500 Acetoine 95-0,66 0-200 2-butanol 48 0,66 0-100 acetaldehyde 37-0,03 0-50 31-10-2014 PHYCONET Cambridge 15
Co-factor specificity: L-lactate production +/- Transhydrogenase starting OD 730 =0.1; BG-11 + 50mM NaCO 3 + 20ug/ml km; 30 C; 120rpm; ~25µE. Transhydrogenase only mutant 31-10-2014 PHYCONET Cambridge 16
Using a codon-optimized LDH from L. lactis 31-10-2014 PHYCONET Cambridge 17
Thermodynamics of product formation: Lactic acid: pyr + NADH lactic acid + NAD + ΔG 0 = -23,8 kj/mol (ΔG 0 values from the classical review of Thauer et al, 1977, Energy conservation in chemotrophic anaerobic bacteria. Bacteriological Reviews 41: 100-180). Keq = 10 23,8/5,7 = 10 4,17 Keq = 1,5.10 4. The following relation holds: Keq = [lact]/[pyr].[nad + ]/NADH] (green = 1) If we assume [pyr] in = 50 µm [lact] < 750 mm; however, at 1 µm [pyr] in [lact]<15 mm! An NADPH-specific pyruvate reductase would help increase the maximally achievable [lactate]!! 31-10-2014 PHYCONET Cambridge 18
Schematic representation of a cyanobacterial cell factory for lactic acid thylakoids H 2 O NADPH (+ ATP) + O 2 Calvin CO 2 pyruvate lactate cycle V1 V2a V2b LDH cells Control of LDH over lactic acid production: δ ln (J lact )/ δ ln (C LDH ) 31-10-2014 PHYCONET Cambridge 19
Sensitivity analysis of the solar-cell factory Angermayr & Hellingwerf (2013) J Phys Chem B. Mar 29. [Epub ahead of print] 31-10-2014 PHYCONET Cambridge 20
Update on control over L-lactate production: Concl: partition coëfficiënt > 50 %; sink-effect observable 31-10-2014 PHYCONET Cambridge 21
Other products formed from CO 2 with cyanobacteria: hydrogen, ethanol, ethylene, propanol, acetone, acetoine, meso-butanediol, S,S-butanediol, iso-butyraldehyde, n- butanol, iso-butanol, L-lactic acid, D- lactic acid, glucose, sucrose, isoprene, long-chain alkanes, long-chain alkenes, long-chain fatty acids, long-chain fatty alcohols, etc.,... CO 2 can replace the sugar one would use in E. coli! 31-10-2014 PHYCONET Cambridge 22
select a desired product. General approach: search the nearest metabolite in the metabolic network of Synechocystis. add phosphatase, reductase, hydrolase(s), etc., to connect the two. try to achieve an equal distribution of control in: (a) from CO 2 to metabolite and (b) metabolite to product. assure that product can properly leave the cells. Example: Jacobsen JH & Frigaard NU (2014) Engineering of photosynthetic mannitol biosynthesis from CO 2 in a cyanobacterium. Metab Eng. 21: 60-70. 31-10-2014 PHYCONET Cambridge 23
Scale-up: will 2D or 3D win? Natural photosynthesis in a bio-based economy: A field of photovoltaic cells driving LEDs in a 3D reactor? 31-10-2014 PHYCONET Cambridge 24
Conclusions: Cyanobacteria can be engineered with base-pair precision We and many others can now make a wide range of products Synechocystis is plugbug for CO 2 For selected products > 70 % carbon partitioning is achievable This approach does not compete with food supply; does not create a minerals problem, and has a limited water requirement For maximal efficiency in this approach it will be necessary to make designer organisms. 31-10-2014 PHYCONET Cambridge 25