Synthetic biology and energy from the sun future is bright for food, feed & fuels
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This document discusses using synthetic biology and photosynthesis to produce sustainable food, feed, and fuels from sunlight. It focuses on engineering cyanobacteria, which are photosynthetic bacteria, to more efficiently produce useful compounds. Specifically, it describes work done engineering the cyanobacterium Synechococcus elongatus UTEX 2973, the fastest growing cyanobacterium, to produce biofuels and chemicals through its metabolic pathways directly from carbon dioxide and sunlight. It also discusses the goal of endowing photosynthetic organisms with the ability to fix nitrogen from the atmosphere.
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Synthetic biology and energy from the sun future is bright for food, feed & fuels
- 1. Otago Nov 22, 2017 Himadri Pakrasi InCEES.wustl.edu Synthetic Biology and Energy from the Sun: Future is Bright for Food, Feed and Fuels https://sites.wustl.edu/photosynthbio/
- 2. Otago Nov 22, 2017 Food, Feed and Fuels An eternal nexus Food and Feed productions need many resources, including ENERGY
- 3. Otago Nov 22, 2017 How Much Energy Do We Use?
- 4. Otago Nov 22, 2017 Nate Lewis, I-CARES, 2008
- 5. Otago Nov 22, 2017 Nate Lewis, I-CARES, 2008
- 6. Otago Nov 22, 2017 From where will we get so much ENERGY? The SUN
- 7. Otago Nov 22, 2017 Nate Lewis, I-CARES, 2008
- 8. Otago Nov 22, 2017 CO2 + H2O Sugar + O2 light CarbonDioxide Oxygen Global Oxygenic Photosynthesis Thronton, 2004
- 9. Otago Nov 22, 2017 Photosynthesis • Brought oxygen to the biosphere • Changed the planet’s atmosphere • Has produced almost all food and feed that sustain our lives • Helped generate energy-rich fossil fuels such as petroleum and coal (over millennia) that have so far been critical in accelerating the pace of development of modern human societies
- 10. Otago Nov 22, 2017 What is the role of Photosynthesis today in food, fuel and chemical production? • Multi step process • Photosynthesis produces sugar (carbohydrate) and other energy rich molecules • These products are used in subsequent processes to produce food, fuel and chemicals Example • Beer production: Plants (autotrophs) make sugar that is then used by yeast (heterotroph) to make alcohol
- 11. Otago Nov 22, 2017 Can we make this a one step process that is more efficient? • Efficiency is key to future success. • We will have less agricultural land, water and other resources per capita when the world’s population reaches 10 billion. • Conventional Agriculture will fail to meet the planet’s need by 2050. • We need a step change in Agriculture.
- 12. Otago Nov 22, 2017 National Geographic, October 2007
- 13. Otago Nov 22, 2017 National Geographic, October 2007, pg. 58-59
- 14. Otago Nov 22, 2017 What are Cyanobacteria? CO2 Nutrients O2 Berla, 2014
- 15. Otago Nov 22, 2017 Cyanobacteria are •oxygenic photosynthetic prokaryotes •the progenitors of chloroplasts •most abundant fossils in precambrian rocks •found in most diverse ecological niches in the modern world - ocean, fresh water lake, soil, hot spring, deserts, Antarctica …… •the only known bacteria with circadian clocks
- 16. Otago Nov 22, 2017 Cyanobacteria • Fresh or salt water • Plankton blooms or dense mats • Unicellular or filamentous • Key contributors to global biological carbon capture and utilization AnabaenaSynechocystis Synechococcus ~2 µm ~2-3 µm ~1.5-2 µm
- 17. Otago Nov 22, 2017 Synthetic (Engineering) Biology • Aided by recent advances in Reading and Writing DNA • Quantitative Approaches: Principles of Physics, Chemistry and Engineering applied to Biology • Enormous potential to develop bio-based economies • Will provide solutions necessary to emerging needs in a world challenged with food and energy impoverishment over this century • New tools and applications being developed to meet such global challenges
- 18. Otago Nov 22, 2017 Cell Factories for Biomanufacturing • Heterotrophic Cells § Bacteria (E. coli, B. subtilis, Lactobacilli….) § Yeast (Saccharomyces cerevisiae, Pichia pastoris....) § Fungal & Insect cells § Mammalian cells • Autotrophic cells § Photosynthetic microbes (Cyanobacteria, Algae) § Plant Cells
- 19. Otago Nov 22, 2017 Why Engineer Cyanobacteria? CO2 Image: Forbes Magazine Berla, 2014
- 20. Otago Nov 22, 2017 Calvin cycle Photosynthesis N2 fixation N2 carbohydrates Lipids Biopolymers CO2 H2O Nutrients Isoprenes PUFA Biodiesel Amino Acids Nucleic Acids Vitamins Hydrogen Alkanes Pigments Useful Compounds from Cyanobacterial Cell Factories Modified from Rosenberg et al., 2008
- 21. Otago Nov 22, 2017 • Three widely used model strains – all unicellular and reasonably fast growing •Synechocystis sp. PCC 6803 •Synechococcus sp. PCC 7942 •Synechococcus sp. PCC 7002 • Naturally Transformable • Large number of knockout and knock-in mutants • Recent use as chassis for photoautotrophic production of various biofuels or their precursor molecules
- 22. Otago Nov 22, 2017 • Unicellular cyanobacterium • Both photoautotrophic and heterotrophic growth • Natural transformation • First photosynthetic organism with a completely sequenced genome (1996) • 3.6 Mbp genome + 7 plasmids • Large collection of multiomics level datasets • Many genome scale models Synechocystis 6803
- 23. Otago Nov 22, 2017 Cyanobacterial Systems in the Era of Genomics • Genomes of more than 250 cyanobacterial strains have been sequenced • Provide blueprints for the diverse metabolic potentials of these oxygenic phototrophs
- 24. Otago Nov 22, 2017 Synechococcus elongatus UTEX 2973 The Fastest Growing Cyanobacterium (to date) Yu, J., Liberton, M., Cliften, P. F., Head, R. D., Jacobs, J. M., Smith, R. D., Koppenaal, D. W., Brand, J. J. and Pakrasi, H. B. (2015) DOI: 10.1038/srep08132
- 25. Otago Nov 22, 2017 Growth comparison with other strains Rapid Growth of Synechococcus 2973 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 4 8 12 16 OD730 Time (h) UTEX 2973 PCC 7002 PCC 7942 PCC 6803 0h 7h 16h 2973 7942 B C T OD730 PCC 1.2 0.8 0.4 0 0 4UTEX 0 0.2 0 2 4 6 8 1 Time (h) Yu et al, 2015 Just with light and CO2, Synechococcus 2973 can double in 1.5 hours, comparable to industrial yeast grown with sugar and nutrients.
- 26. Otago Nov 22, 2017 Synechococcus PCC 6301 Synechococcus UTEX 2973 Synechococcus PCC 7942 inversion region Genome nucleotide differences big deletion piece big insertion piece !! Synechococcus! UTEX!2973*! Synechococcus! PCC!7942*! Synechococcus! PCC!6301*! Genome!size! (bp)! 2,690,418) 2,695,903) 2,696,255) GC!content! 55.4%) 55.4%) 55.5%) Protein!coding! genes! 2,645) 2,661) 2,525) rRNA!operons! 6) 6) 6) tRNA!genes! 44) 44) 45) Yu et al, 2015 Genome Sequence of Synechococcus 2973 is >99% identical to that of Synechococcus elongatus PCC 7942 Genome Sequence and Annotation
- 27. Otago Nov 22, 2017 • Tri-parental conjugation with E. coli cells • Efficiency: ~3 X 10-5 • Exconjugant colonies in 2 to 3 days • Rapid complete segregation of desired mutations • Facilitates construction of a comprehensive library of single-gene knockout mutants Genetic Manipulation of Synechococcus 2973 Knock-in Yu et al, 2015
- 28. Otago Nov 22, 2017 Phycobilisomes: light harvesting antenna protein complex in cyanobacteria • Reduction in antenna size is expected to enhance photosynthetic efficiency and productivity of cyanobacteria • A PBS absorbs light energy and transfers to Photosystems (shown by arrows) • Phycobiliproteins assemble to form massive complexes (~7-15 MDa) • Accounts for more than 30% of total cellular protein Govindjee, Shevela D. (2011). Front Plant Sci 2: Article 28 PBS PSII
- 29. Otago Nov 22, 2017 PBSs possess tremendous plasticity N repletion -N Re-synthesis of phycobiliproteins with addition of N N depletion +N Bleaching due to degradation of phycobiliproteins Decrease in phycobilisome absorption peak (620 nm) during N starvation • Acclimate to changes in environment e.g., nutrient availability and light quality • During Nitrogen (or Sulfur) starvation, cells undergo bleaching (PBS degradation) • Degradation of PBSs is reflected in a decrease in 620 nm absorption • Process is reversible after readdition of Nitrate (or Sulfate)
- 30. Otago Nov 22, 2017 Genetic Manipulation of Synechococcus 2973 Knock out Nbl: non bleaching Yu et al, 2015
- 31. Otago Nov 22, 2017 Yu et al, 2015 Genetic Manipulation of Synechococcus 2973 Nbl: non bleaching +N -N
- 32. Otago Nov 22, 2017 Recent Developments • Efficient CRISPR mediated genome editing system developed for diverse cyanobacterial species • Development of a highly efficient transposase for cyanobacteria • Construction of a genome wide inactivation library • Stable and rapid growth in 100 liter advanced photobioreactors
- 33. Otago Nov 22, 2017 An efficient photosynthetic cell factory
- 34. Otago Nov 22, 2017 Synechococcus 2973 has remarkably fast growth property Lin, P-C, Unpublished
- 35. Otago Nov 22, 2017 Synechococcus 2973 Synechococcus 7942 High CO2 High Light Yu et al, 2015
- 36. Otago Nov 22, 2017 21-fold Synechococcus 2973 accumulates glycogen as storage of excess carbon Lin, P-C, Unpublished
- 37. Otago Nov 22, 2017 Engineer Synechococcus 2973 to produce high value chemicals 0 2 4 6 8 2973 7942 7002 6803 Productivity (mg/L/D) 2973 79421 70022 68033 Limonene 1Wang et al., 2016; 2Davies et al., 2014; 3Lin et al., 2017 (in review) Lin, P-C, Unpublished
- 38. Otago Nov 22, 2017 Knoot, Ungerer, Pakrasi, JBC, 2017 Photosynthetic Metabolism and Green Chemicals Production in Cyanobacteria
- 39. Otago Nov 22, 2017 Designing Nitrogen Fixing Ability in Oxygenic Photosynthetic Cells
- 40. Otago Nov 22, 2017 • Nitrogen is an essential nutrient for plant growth • A long term goal of this NITROGEN program is to endow plant cells with nitrogen fixing ability • Photosynthesis in plants produces oxygen, a potent inhibitor of nitrogenase • The overall goal of our project is to use Systems and Synthetic Biology approaches to develop the design principles to establish nitrogen fixing ability in the unicellular cyanobacterium, Synechocystis 6803 • The knowledgebase created in this project will be directly applicable to engineer nitrogen-fixing crop plants
- 41. Otago Nov 22, 2017 Achieving nitrogen fixation in crop plants Cyanothece 51142 Diazotrophic Synechocystis 6803 Non-diazotrophic Synechocystis 6803 Diazotrophic Nitrogenase Nif cluster Design Principles N2 Fixing N2 fixing module Engineer N2 fixing symbiosis
- 42. Otago Nov 22, 2017 Cyanobacterial strains ~2 µm Synechocystis 6803 Non-diazotrophic ~4 µm Cyanothece 51142 Diazotrophic ~2 µm Anabaena Diazotrophic
- 43. Otago Nov 22, 2017 Cyanobacterial strains ~2 µm Synechocystis 6803 Non-diazotrophic ~4 µm Cyanothece 51142 Diazotrophic Phylogenetically Closely Related Our goal is to establish in Synechocystis, the nitrogen fixation circuitry, normally present in Cyanothece
- 44. Otago Nov 22, 2017 LIGHT DARK Photosynthesis Nitrogen Fixation Glycogen granules Cyanophycin granules Cyanothece diurnal cycle Photosynthesis and nitrogen fixation are separated temporally in the same cell
- 45. Otago Nov 22, 2017 •Photosynthesis results in O2 production during the day. •High rates of respiration generate low intracellular oxygen tension during early night hours. •Oxygen-sensitive enzymes such as nitrogenase can then function in the same cell. In CyanotheceCyanothece diurnal cycle
- 46. Otago Nov 22, 2017 Expression profiles of 35 genes involved in nitrogen fixation in Cyanothece 51142 Stöckel et al., PNAS 2008 samples collected for analysis
- 47. Otago Nov 22, 2017 NifS 400 aa Cysteine desulfurase (EC 2.8.1.7) NifU 293 aa Iron-sulfur cluster assembly protein, Formation of [4Fe-4S] NifB 490 aa Functional protein for the formation of large Fe-S core NifV 377 aa Homocitrate synthase (EC 2.3.3.14) NifE 472 aa NifN 454 aa NifZ 114 aa Nitrogenase MoFe maturation protein Nitrogenase FeMo-cofactor scaffold and assembly protein Necessory cofactor NifH 327 aa Nitrogenase iron protein NifD 480 aa Nitrogenase molybdenum-iron protein alpha chain NifK 511 aa Nitrogenase molybdenum-iron protein beta chain Structural Protein Cyanothece 51142 has the largest contiguous nif cluster
- 48. Otago Nov 22, 2017 NifO 142 aa Putative nitrogenase-associated protein NifT 66 aa Nitrogen fixation protein NifW 116 aa Nitrogenase stabilizing/protective protein CysE2 236 aa Serine O-acetyltransferase HesA 271 aa Putative molybdenum cofactor biosynthesis HesB 169 aa Probable iron binding protein cce_0556 120 aa [4Fe-4S] ferredoxin cce_0571 113 aa [2Fe-2S] ferredoxin FdxB 114 aa [4Fe-4S] ferredoxin FeoB2 487 aa Ferrous iron transport protein B FeoA2 84 aa Ferrous iron transport protein A ModB 651 aa Putative molybdate ABC transporter, permease protein Accessory Protein NifX 137 aa Negative regulation of nif operon in response to NH4 + and O2 Regulatory protein Cyanothece 51142 has the largest contiguous nif cluster
- 49. Otago Nov 22, 2017 cce_0546 198 aa Hypothetical protein cce_0550 79 aa Hypothetical protein cce_0551 32 aa Hypothetical protein cce_0552 94 aa Hypothetical protein cce_0555 66 aa Hypothetical protein cce_0562 102 aa Hypothetical protein cce_0566 171 aa Hypothetical protein cce_0567 78 aa Hypothetical protein cce_0572 142 aa Hypothetical protein cce_0573 117 aa Hypothetical protein cce_0574 208 aa GTP-binding protein, HSR1-related cce_0577 270 aa Hypothetical protein Others NifJ 1210 aa Pyruvate oxidoreductase Related protein not in the cluster Cyanothece 51142 has the largest contiguous nif cluster
- 50. Otago Nov 22, 2017 Yeast Bacteria Structural Cofactor Accessory Regulatory Other Yeast Bacteria Syn Nif1 39,620 bp Introduction of Cyanothece 51142 nif gene cluster in Synechocystis 6803
- 51. Structural Cofactor Accessory Other Syn-Nif1 39,620 bp 35 genes in nif-cluster DNA assembler Stably replicating for three years Conjugation Synechocystis 6803 ~2 µm
- 52. nifH nifD nifK nifT nifZ nifV nifB nifS nifU nifE nifN nifX nifW hesA hesB cysE2 feoB2 feoA2 m odB Syn-Nif1 (35 genes) Syn-Nif2 (24 genes) Transcription of 35 nif-genes in Syn-Nif1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 0 1 2 3 4 5 6 7 8 9 10 Day Wild type T2379 T2397 OD730nmOD730nm Syn-Nif1 Syn-Nif2 BG11 Light/Dark cycles M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 nifH nifD nifK nifT nifZ nifV nifB nifS nifU cysE2 M 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 nifE nifN nifX nifW hesA hesB feoB2 feoA2 m odB Growth under a diurnal condition
- 53. 0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 Wild-type T2379 T2397 Nitrogenaseactivity(nmolC2H4/mgpeotein/h) Syn-Nif1 Syn-Nif2 nifH nifD nifK nifT nifZ nifV nifB nifS nifU nifE nifN nifX nifW hesA hesB cysE2 feoB2 feoA2 m odB Syn-Nif1 Syn-Nif2 Nitrogenase activity Synechocystis 6803 can now fix N2!!
- 54. Otago Nov 22, 2017 A Scenario in the Future (2050) • The available “land now must provide not only food but also the biofuels and other petrochemicals that were once cheaply available from underground. • One solution is engineered cyanobacteria, which can grow on marginal lands and use brackish or ocean water unsuitable for most conventional crops, possibly reclaiming land recently lost to desertification. • And they can do so at photosynthetic efficiencies far higher than corn, switchgrass, and other favorite crops. • Creative food technology might make cyanobacteria-derived cuisine taste the same as chicken or beef (or even better!), with consequent fifteen-fold reductions of energy use relative to animals.” From: George M. Church. “Regenesis” (2012)
- 55. Otago Nov 22, 2017 Pakrasi Lab, 2017 Department of Energy, National Science Foundation
- 56. Otago Nov 22, 2017 Thank you!
- 57. Otago Nov 22, 2017