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Overview

Insight into lipid biogenesis during TAG accumulation using stable isotope tracers coupled with NMR spectroscopy and mass spectrometry. Robert D. Gardner , Gregory L. Helms b , William C. Hiscox b , Egan J. Lohman a , Brent M. Peyton a , Robin Gerlach a , and Keith E. Cooksey c

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Overview

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  1. Insight into lipid biogenesis during TAG accumulation using stable isotope tracers coupled with NMR spectroscopy and mass spectrometry Robert D. Gardner, Gregory L. Helmsb, William C. Hiscoxb, Egan J. Lohmana, Brent M. Peytona, Robin Gerlacha, and Keith E. Cookseyc Department of Bioproducts and Biosystems Engineering West Central Research and Outreach Center University of Minnesota aDept. of Chemical Engineering and the Center for Biofilm Engineering, Montana State University, Bozeman MT. bCenter for NMR Spectroscopy, Washington State University, Pulman WA. cEvironmentalBiotechnology Consultants, Manhattan MT.

  2. Overview 1) Bicarbonate-Enhanced Growth and Bicarbonate-Induced TAG Accumulation (See poster 204 – Brent Peyton) i) Background and nitrogen dependency 2) NMR and MS to monitor inorganic carbon fixation (See poster 123 – Greg Helms) i) NMR for real time analysis ii) Verified using MS techniques on the molecular ion

  3. Photosynthesis for Biofuels • Algae are biocatalysts that convert renewable sunlight into biofuels and chemical substrates • Strain dependent characteristics during lipid biogenesis • De novo biosynthesis vs. C-recycling in the cell • Image from Schenk, P., et al.2008. BioEnergyResearch, 1(1), 20-43.

  4. Algal Carbon Concentrating Mechanisms Carbon utilization • Ribulose 1,5-bisphosphate carboxylaseoxygenase (Rubisco) • Relatively low affinity for CO2 • Carbonic anhydrase • Reversibly convert CO2 ↔ HCO3 • Extra & intracellular • HCO3 Transporters • Plasmamembrane and Chloroplast membrane bound • C4 carbon utilization • Theorized in diatoms • Reversibly convert CO2 ↔ Oxaloacetate • PEPcarboxylase and PEPcarboxykinase • Images from Moroney 2007, Roberts 2007

  5. Bicarbonate – Enhanced Growth • Optimized Growth Scenario • (5 mM Bicarbonate) • 50 mM Bicarbonate stops (4 d)cellular replication • Desired cell density can beachieved by “tweaking” initialnutrient concentrations • Specific Growth Rate(µ) • Increased by 69% • Biomass Productivity (g L-1 Day-1: DCW)Increased by 27% 50mM NaHCO3 • Lohman E, Gardner R, et al. 2013. Biotechnology for Biofuels (manuscript in review).

  6. An Optimized DIC Regime – Enhanced Growth • Optimized Growth Scenario • Optimal system had significantly higher chlorophyll content • More photosyntheticallyactive • More efficient DIC fixation • Lohman E, Gardner R, et al. 2013. Biotechnology for Biofuels (manuscript in review).

  7. Bicarbonate Addition at Medium N-Depletion Scenedesmus sp. WC-1 ½ time required Scenedesmus sp. C. reinhardtii Air • Addition of HCO3- stops cellular cycle and induces TAG accumulation in green algae. • Addition of HCO3-does not stops cellular cycle but induces TAG accumulation in diatoms. • Confirmed on over 20 green algae and diatom strains, both freshwater and marine. 5% CO2 HCO3 • Gardner R, et al. 2013. Journal of Applied Phycology. 24(5): 1311-1320 • Gardner R, et al. 2013. Biotechnology and Bioengineering 110(1): 87-96

  8. Advanced NMR Method Development 1H NMR Detection and Quantitation of TAGs in Live Algal Cells Directly sample culture Monitor TAG accumulation Quantitate TAG content • Davey et al. Algal Research 2012 doi:10.1016/j.algal.2012.07.003

  9. NMR Metabolite Signals • NMR Metabolite Signals • Sucrose (Carbohydrates) • Pyruvate • Allylic (MUFA) • Alpha C • Beta C • CH2 (Bulk lipid) • Choline (3 signals) • Double Allylic (PUFA) • Glyceraldehyde • Glycerol (CH2 and CH) • MAG • DAG • TAG • Methyl C • Olefin • Omega-3 Image from Schuhmann, et al. 2011. Biofuels, 3(1), 71-86.

  10. Experimental Outline – 24 hr lighting • Forward Experiment– de novo synthesis • Growth in 5 mM12C (DIC) • NaH13CO3 addition at N-limitation • Monitored (48 hrs) • 13C-incorperation and labeling with 1H HR-MAS NMR • Medium 13C concentration and speciation with 13C NMR • Chlorophyll and carotenoid concentration • Dry cell weight change • Reverse Experiment – recycling C • NaH13CO3 labeled biomass • NaH12CO3 at N-limitation • Monitored (24 hrs) • 13C-recycling with 1H HR-MAS NMR • Dry cell weight change • GC-FID and GC-MS analysis

  11. Experimental Results (GC analysis)

  12. Chlorophyll & Carotenoid • Chlorophyll and carotenoid concentration remained stable • Suggests photosynthesis was maintained

  13. 25 mM NaH13CO3 addition with N-deplete culturing

  14. Quantification of the terminal methyl group

  15. Quantification of the terminal methyl group

  16. Total Correlation Spectroscopy (TOCSY) • F1 decoupled TOCSY with 1D coupled spectrum • Separates along the diagonal

  17. Sucrose and Bulk Lipid • Sucrose (>80% de novo synthesis at 24 hrs) • Rapid DIC incorporation (within 15 min) • Metabolic switch (steady-state) after 10 hrs • Bulk CH2 (>70% de novo synthesis at 24 hrs) • DIC incorporation and recycled biomass for 8 hrs, after which increased rate of DIC incorporation

  18. Allylic (MUFA) and Double Allylic (PUFA) • MUFA (Allylic) (>60% de novo synthesis at 24 hrs) • Initial recycling • High DIC incorporation after 12 hrs • PUFA (double allylic) (>60% recycled C at 24 hrs) • High initial and continued incorporation of recycled carbon • De novo synthesis using DIC after 6 hrs

  19. Omega-3 • Omega-3 (>80% recycled C at 24 hrs) • Initial C-recycling and unobservable de novo synthesis from DIC • C-recycling continues and de novo synthesis begins after 10 hrs

  20. Mass Spectrometry Confirmation of NMR Findings • Determined molecular ion values based on unlabeled standard or unlabeled extractant • Fill in A-matrix diagonal and correct M+1 and M+2 values below the diagonal to correct for natural abundance of 13C. • Inherently corrects for MS ionization efficiency

  21. Molecular Ion Analysis (GC-MS) • High bicarbonate incorporation into C16:0 and C18:1 (primary FAs that increased) • High bicarbonate incorporation into C18:3 (6,9,12), de novo synthesis • High Recycling in C18:3 (9,12,15), membrane lipid reallocation

  22. Total Correlation Spectroscopy (TOCSY) • PUFA signal • 3 signals make up the overall PUFA signal • One does not incorporate bicarbonate, one ~30%, the other ~65%

  23. Summary – Key Points • NMR can be used as a metabolic microscope to track carbon from bicarbonate to TAG. • Carbon rates of incorporation are being processed. • Initial carbon source is identified (i.e., bicarbonate, CO2, or biomass). • Fundamental and controversial questions are being answered (i.e., de novo or carbon recycling in fatty acid synthesis). • Additional deconvolution and fluxomic developments are in process.

  24. Acknowledgements U of MN BBE Dept. & WCROC • Greg Helms & Bill Hiscox (WSU) Collaborators • University of Minnesota • Montana State University • Washington State University Contributors & MSU Biofuels Group Members • Brent Peyton (Peyton Lab Group) • Robin Gerlach *Environmental and Biofilm Mass Spectrometry Facility • Keith Cooksey Funding • NSF IGERT Program in Geobiological Systems • Church & Dwight Co., Inc. • US DoE/DoD

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