Using LCMS to investigate fatty acid oxidation in cyanobacteria George Taylor

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

Why are some cyanobacteria interesting from a biofuels perspective?

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

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

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

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

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

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

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

Product Ion Scans 16:0-CoA 1006.5 m/z M+H Product ion = 499.9 m/z M+H Q1 Collision cell (Q2) Q3 Detector Ion source Precursor masses: 16:1-CoA 1004.5 16:0-CoA 1006.5 18:0-CoA 1034.5 Fragmentation: 135 V Product ion selection and detection 16:0-CoA 1006.5 m/z M+H Product ion = 499.9 m/z M+H

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 1004.5 16:0-CoA 1006.5 18:0-CoA 1034.5 Product masses: 16:1-CoA 497.4 m/z 16:0-CoA 499.5 m/z 18:0-CoA 527.3 m/z

Calculating MRMs Compound Precursor m/z Product m/z 18:0-CoA 1034.5 527.3 1034.5 + 28 = 1062.5 527.3 + 28 = 555.3 20:0-CoA

-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

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

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

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

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