1. Using LCMS to investigate fatty acid oxidation in cyanobacteria
George Taylor

2. Cyanobacteria Microscopic, unicellular
Ancient – fossils found from ~2800 MYA*
Ancestors of chloroplasts in modern plants
Photosynthetic
Metabolically diverse
Stahl , 2008
microscopic and unicellular, although some exist in filamentous forms as you can see in the picture
very old organisms, been around since at least 2.8bn ya. are thought to be responsible for producing an oxidising atmosphere required for life today
related to chloroplasts that are found in modern plants, evolutionary biologists know this by looking at similarities between choloplast and cyano DNA sequences
and like chloroplasts, are the reaction centres where photosynthesis occurs – the fixing of atmospheric co2 using light into complex organic molecules
they are also very metabolically diverse – some can fix nitrogen into ammonia/glutamate/organic compounds, some produce nasty neurotoxins, and some….
*Olson, 2006

3. Why are some cyanobacteria interesting from a biofuels perspective?

4. Why is fatty acid oxidation interesting from a biofuels perspective?
CO2
respiration
photosynthesis
Glycerate-3-phosphate
glycolysis
Acetyl-Coenzyme A
β-oxidation
Fatty acid biosynthesis
Fatty acids / fatty acyl-ACPs / acyl-CoAs
Acyl-ACP reductase + aldehyde decarbonylase*
heptadecane
*Schirmer et al. 2010

5. Appears to be lacking in cyanobacteria!
β-oxidation
Major fatty acid degradative pathway
Appears to be lacking in cyanobacteria!

6. Hypothesis – Cyanobacteria do not have the β-oxidation pathway
Testing the hypothesis:
Looking for homology between known β-oxidation enzymes and unknown cyanobacterial protein sequences using basic bioinformatics tools
Detection of the substrates of β-oxidation; acyl-CoAs
Assay of the rate-limiting enzyme of β-oxidation; acyl-CoA dehydrogenase
Metabolite tracing – feeding 3H/14C labeled fatty acids to cyanobacteria

7. Detection of Acyl-CoAs using LCMS-QQQ
Acyl-CoAs are the substrates of β-oxidation
palmitoyl-CoA (16:0-CoA)
adenosine 3,5 diphosphate/ 4-phosphopantothenic acid/ beta alanine/ beta mercapto ethyl amine/ R group

8. Extraction and sample preparation
3 cyanobacterial strains and E. coli (positive control) were harvested at an OD of 4 by centrifugation, homogenised and extracted in acetonitirile/isopropanol/KH2PO4 at pH 6.7
Acyl-CoAs are acidified and enriched by SPE using a 2-(2-pyridyl)ethyl silica gel column, eluted at pH 7, dried and resuspended in water
Minkler et al. 1999

9. Method Development
Standards of palmitoyl-CoA (16:0-CoA), palmitoleoyl-CoA (16:1-CoA) and stearoyl-CoA (18:0-CoA) were used at a concentration of 75 μM in water
HPLC:
Acyl-CoAs eluted isocratically on a 30 mm x 2mm reverse phase column (3.5 μm particle size). Mobile phase is 55% ACN and 45% 10 mM ammonium acetate in water. Run time 3 min. Wash and re-equilibration time 7 min to eliminate carryover contamination
MS-QQQ:
Acyl-CoAs are ionised by ESI (positive polarity).
Veld et al. 2009

10. MS2 Scans of standards Precursor masses: 16:1-CoA 1004.5 m/z

11. Product Ion Scans 16:0-CoA 1006.5 m/z M+H Product ion = 499.9 m/z M+HQ1
Collision cell (Q2) Q3
Detector
Ion source
Precursor masses:
16:1-CoA
16:0-CoA
18:0-CoA
Fragmentation:
135 V
Product ion selection and detection
16:0-CoA m/z M+H
Product ion = m/z M+H

12. Product Ion Scans
From this, multiple reaction monitors can be set up for a range of chain length acyl-CoAs on the instrument
Precursor masses:
16:1-CoA
16:0-CoA
18:0-CoA
Product masses:
16:1-CoA m/z
16:0-CoA m/z
18:0-CoA m/z

13. Calculating MRMs Compound Precursor m/z Product m/z 18:0-CoA 1034.5
527.3 =
= 555.3
20:0-CoA

14. -CoAs instrument is set-up to detect
methyl-16:0-CoA
4:0-CoA
22:1-CoA
18:3-CoA
14:0-CoA
propanoyl-CoA
20:0-CoA
18:2-CoA
β-hydroxy-14:0-CoA
malonyl-CoA
20:5-CoA
18:1-CoA
12:0-CoA
acetyl-CoA
20:3-CoA
16:0-CoA
10:0-CoA

15. A wide range of –CoAs are detected in extracts of E. coli
Compound
nmol acyl-CoA / 1 x 107 cells
18:0-CoA
0.293
12:0-CoA
0.314
18:1-CoA
0.746
10:0-CoA
0.25
16:0-CoA
2.67
8:0-CoA
0.352
16:1-CoA
0.584
6:0-CoA
0.081
16:2-CoA
0.125
4:0-CoA
0.903
Me-16:0-CoA
0.154
acetyl-CoA
2.46
14:0-CoA
1.03
propionyl-CoA
1.84
β-OH-14:0-CoA
0.529
malonyl-CoA
2.29

16. Long chain Acyl-CoAs cannot be detected in cyanobacteria
Acetyl-CoA (0.774 nmol/1x107 cells)

17. Method has also been set up to detect and quantify Carnitines
palmitoyl carnitine

18. Acknowledgements Nick Smirnoff Rob Lee Christoph Edner Hannah Florance
Mezzanine Lab
Shell Global Solutions