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 Department of Bioproducts and Biosystems Engineering West Central Research and Outreach Center University of Minnesota a Dept. of Chemical Engineering and the Center for Biofilm Engineering, Montana State University, Bozeman MT. b Center for NMR Spectroscopy, Washington State University, Pulman WA. c Evironmental Biotechnology Consultants, Manhattan MT. Insight into lipid biogenesis during TAG accumulation using stable isotope tracers coupled with NMR spectroscopy and mass spectrometry
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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 Department of.
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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 EngineeringWest Central Research and Outreach Center
University of MinnesotaaDept. of Chemical Engineering and the Center for Biofilm Engineering, Montana State University, Bozeman MT.bCenter for NMR Spectroscopy, Washington State University, Pulman WA.cEvironmental Biotechnology Consultants, Manhattan MT.
Insight into lipid biogenesis during TAG accumulation using stable isotope tracers coupled with NMR spectroscopy and mass spectrometry
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
• Image from Schenk, P., et al. 2008. BioEnergy Research, 1(1), 20-43.
• 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
• Chlorophyll and carotenoid concentration remained stable• Suggests photosynthesis was maintained
0 5 10 15 200
2
4
6
8
10
12
14
Total Chlorophyll
Time (hr)
mg
/L
0 5 10 15 20 250
0.5
1
1.5
2
2.5
Carotenoid
Time(hr)
mg
/L
25 mM NaH13CO3 addition with N-deplete culturing
Quantification of the terminal methyl group
Quantification of the terminal methyl group
Total Correlation Spectroscopy (TOCSY)
• F1 decoupled TOCSY with 1D coupled spectrum• Separates along the diagonal
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
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
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
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
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
• PUFA signal• 3 signals make up the overall PUFA signal• One does not incorporate bicarbonate, one
~30%, the other ~65%
Total Correlation Spectroscopy (TOCSY)
• 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.
Summary – Key Points
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